Murumbula Shedo

THE PHARMACOLOGICAL ACTIVITY OF RHOICISSUS
TRIDENTATA SUBSP CUNEIFOUA IN RELATION TO

PARTURITION
10mm
Lynn Coleen Katsoulis

A thesis submitted to the Faculty of Health Sciences, University of the
Witwatersrand, in fulfilment of the requirements for the degree
of
Doctor of Philosophy
Johannesburg, 2000
d e c l a r a t io n
I, Lynn Coleen Katsoulis declare that this thesis is my own work. It is being
submitted for the degree of Doctor of Philosophy to the University of the
Witwatersrand, Johannesburg. It has not been submitted before for any
degree or examination at this or any other University.
day of , 2000
This thesis is dedicated to my loving husband, Terry.
His unending encouragement and support, gave me the confidence to apply

to university and also carried me through to completing this thesis.
May my precious children achieve far more.

PUBLICATIONS AND PRESENTATIONS
1.1. Publications
L.C. Katsoulis, D.J.H. Veale and I. Havlik (1999) The pharmacological action
of Rhoicissus tndentata on isolated rat uterus and ileum. Phytotherapy
Research (in press).
L.C. Katsoulis (1999) Rhoicissus tndentata subsp cuneifolia: the effect of

geographical distribution and plant storage on rat uterine contractile
activity. South African Journal of Botany 65(4):
D.J.H. Veale, I. Havlik, L.C. Katsoulis, T. Kaido, N.S. Arangies, D.W. Oliver, T.
Dekker, K.B. Brookes & O.V. Doudoukina (1998) The
pharmacological assessment of herbal oxytocics used in South
African traditional medicine. Biomarkers and Environment 2(3):42-45
K.B. Brookes, O.V. Doudoukina, LC. Katsoulis & D.J.H. Veale (1999)
Uteroactive traditional medicines: Combretum kraussii. South African
Journal of Chemistry (In press)
L.C. Katsoulis, D.J.H. Veale & I. Havlik (2000) Seasonal variation in uterotonic
activity of Rhoicissus tndentata extracts. South African Medical
Journal (In Press).
1.2. Oral preceritfiiions

L.C. Katsoulis, D.J.H. Veale and I Havlik (1997) Seasonal and distributional
variation in the contractile activity of Rhoicissus tndentata subsp
cuneifolia on isolated rat uterus and ileum. South African
Pharmacology Society Annual Conference. Drakensberg
8. Brookes, 0. Doudoukina, D.J.H. Veale and L.C. Katsoulis (1997) Isolation

of compounds from C. kraussi, uteroactive traditional medicine. Joint
annual symposium of the South African Societies for Microbiology and
Biochemistry.
v
LC. Katsoulis, D.J.H. Veale and I. Havlik (1998) Variation in the contractile
response to Rhoicissus tridentata subsp cuneifolia with different plant
parts used from different seasons - ecological implications. South
African Association of Botanists 24th Annual conference. Cape Town
LC. Katsoulis, I. Havlik (1999) The Pharmacological Action of Rhoicissus

tridentata on isolated rat uterus and ileum. University of the
Witwatersrand Medical School Research Day. Johannesburg
B. Brookes, 0. Doudoukina, LC. Katsoulis and D.J.H. Veale (2000)

Uteroactive components in traditional medicine: isihlambezo. Natural
Products Symposium.
LC. Katsoulis (2000) Plants used in pregnancy-related remedies in the

Gauteng. Indigenous Plant Use Forum. Nelspruit
1.3. Poster presentations

LC. Katsoulis, D.J.H. Veale, N. Butkow and I. Havlik (1996) Preliminary
studies on the effects of Rhoicissus tridentata subsp cuneifolia on
isolated rat uterus and ileum. The First international Conference on
Pharmaceutical and Pharmacological Sciences (South Africa).
Midrand
B. Brookes, O. Doudoukina, D.J.H. Veale and LC. Katsoulis (1996)

Biologically active components from plants used in traditional
pregnancy related herbal medicine or "isihlambezo”. 33rd convention
of the South African Chemistry institute.
LC. Katsoulis (1999) The necessity of standardising herbal remedies: An

example using Rhoicissus tridentata. Herbal Medicine into the Next
Millennium. Conference Proceedings. Lismore, Australia
L.C. Katsoulis, D.J.H Veale, I. Havlik (1999) The Pharmacological Action of
Rhoicissus tridentata on isolated rat uterus and ileum. The Indigenous
Plant Use Forum. Richards Bay, South Africa.
Award
Outstanding Research from South African Pharmacology Society Annual 1997
ABSTRACT
Decoctions and infusions of Rhoicissus tridentata subsp. cuneifolia (Vitaceae)
roots and Hgnotubers are widely used as traditional medicine by South African
women during pregnancy and childbirth. Pharmacological studies using isolated rat
uterus and ileum were done to determine whether there is any pharmacological
grounding for the use of the remedies to induce labour. Initial studies showed a large
variation in the contractile activity of the extracts, so investigations were done to
determine whether the contractile activity varied according to the season in which
plant material was harvested, the location in which the plant grew, or the length of
storage of harvested plant material.
Plant material harvested from Umlazi (KwaZulu-Natal) and the Suikerbos Nature
Reserve (Gauteng) was used for studies on the mechanism of contractile activity.
Seasonal variation was investigated by harvested three plants from Suikerbosrand
for two years at approximately three monthly intervals. The distributional effect was
done using material harvested from around South Africa. Material from
Suikerbosrand was stored for either three months or a year to determine whether
storage altered the contractile activity. After each harvesting, the different parts of the
plants were separated, dried, milled and boiled for approximately an hour. The
solutions were allowed to settle overnight at 4°C, after which the supernatant was
siphoned off, then frozen and lyophilised. All lyophilised end products were kept
frozen until use.
Oestrogenized virgin Sprague-Dawley rats euthanazed with CO2. Uterine and ileal
tissue was dissected out and mounted in 50 ml organ baths containing Tyrode
solution, aerated with 5% CO2 in O2. After a resting period the organs were
challenged with cumulative doses of reference agonist or herbal extract, or
pretreated with the herbal extract before adding the reference drugs.
The pharmacological action of an aqueous extract of R. tridentata subsp.

cuneifolia was investigated using isolated rat uterus and ileum. The results
showed that the extract directly stimulates concentration dependent contractions
of uterus and ileum. Preincubation of the organs with the plant extract had no
effect on the maximal uterine response to the cumulative addition of acetylcholine
or oxytocin, and slightly depressed the response to serotonin and noradrenaline.
The maximal ileal response to acetylcholine was depressed where the response to
viii
maximal concentrations of serotonin was unchanged. Pretreatment with atropine
and indomethacin both blocked the initial response to the Rhoicissus extract which
indicates that the muscarinic receptors and prostaglandin synthesis could be
involved in the contractile response to the extract. Methysergide and prazosin had
no effect on the direct action of the extract which infers that serotonin receptors
and a-adrenoceptors do not play a role in mediating the smooth muscle response
to the plant extract.
Cellular toxicity of R. tridentata extracts from Umlazi and Suikerbosrand was
determined using M Tf assays, and enzyme immunoassays were used to determine
the effect of the extracts on cellular prostaglandin E2 production.
Results from the pharmacological studies indicate that the contractions of isolated rat
uterus and ileum seem to be mediated by muscarinic receptors and the synthesis of
cyclooxygenase products. The contractility of the plant extract appears to be
mediated predominantly by muscarinic M4 receptors. The other muscarinic receptor
subtypes play less of a role. The contractions seem to be devoid of serotonergic,
adrenergic, histaminergic or nicotinic activity.
Studies on the variation in contractile activity suggest that the activity of the extract
does vary according to the season or location of material harvesting. The extracts
form material harvested during summer or autumn was more active than material
harvested during winter or spring. The lignotubers yielded the most active extracts.
Extracts of plants from most geographic areas stimulated contractions although
the response varied in magnitude, however, the extract of a plant from Mondeor,
acted in an opposite manner, inhibiting acetylcholine induced contractions. Storing
dried plant material did not alter the activity of the extracts.
The plant extracts, up tv a concentration of 1mg/ml, appear not to be toxic to

human kidney epithelial ns>H, human hepatoma cells, human histiocytoma cells or
mouse leydig cells. The production of PGE2 by human histiocytoma cells was
stimulated by 1mg/ml R. tridentata extract confirming that contractions are
possible mediated by the synthesis of cyclooxygenase products.
ACKNOWLEDGMENTS
• To my mother for her patience and dedication in teaching me Std 8-10
Mathematics after I had completed matric which enabled me to get university
excemption. Without her, the road leading to this thesis would never have
begun.
• To my father, Harry, for supporting whatever I chose to do.
• Joy Veale for all the pioneering work she has done in the field of traditional
remedies used in pregnancy which opened the path for me. Thank you also
for supervising the project before it was upgraded to a PhD and for reading
all the write-ups so meticulously.
• Dr Neil Butkow for believing in me and supporting me to do a Degree in

Pharmacology, and for supervising the project before it was upgraded to a
PhD.
• Dr James Keegan for supervising this project once it was upgraded to a PhD,
and for all the encouragement, helpful advice and direction in the completion
of this thesis, as well as for his thorough checking of the final transcript.
• Prof. Ivan Havlik for heading up a successful department during turbulent

times, and for supervising my project once it had been upgraded to a PhD.
• Mr. Jacob Modebedi for his help with injecting the rats and for keeping the
isolated organ equipment clean and in a working order.
• Norman Arrangies for helping with the administration of ordering materials

and repairing equipment.
• Mr. Joshua for milling dried plant material and for keeping the glassware
clean.
e Inorganic Ion analyses were done by the Johannesburg General Hospital

Laboratories.
x
The FRD provided persona! financial support during the first year of this
degree.
Financial support from the University Research Committee was used to buy
most antagonists used in this study.
University grants provided financial support for the purchase of the

prostaglandin ELISA kits.
Helene Plein who was a wonderful colleague, and provided much needed
motivation throughout the course of this research.
Most importantly, to The Almighty, who made all this possible.

TABLE OF CONTENTS
Page number
DECLARATION ............................................................................................. iii
DEDICATION ................................................................................................ iv
PUBLICATIONS AND PRESENTATIONS ..................................................... v
ABSTRACT .................................................................................................. viii
ACKNOWLEDGMENTS ................................................................................. x
TABLE OF CONTENTS ................................................................................ xii
LIST OF FIGURES ....................................................................................... xix
LIST OF TABLES ...................................................................................... xxviii
NOMENCLATURE ...................................................................................... xxxi
CHAPTER 1: INTRODUCTION
1.1. Traditional and herbal medicine........................................................ 1
1.1.1. Traditional medicine in South Africa............................................ 1
1.1.2. Types of healers........................................................................... 2
1.1.3. Registration of traditional healers in South Africa........................ 2
1.1.4. Reports of adverse effects of African traditional medicines 4
1.1.5. Advantages of traditional health care systems............................ 5
1.1.6. Integration of traditional healers into primary health care............ 6
1.2. Researching South African Traditional Medicinal Plants............... 8
1.3. Pregnancy Related Traditional Remedies........................ 9
1.3.1. South African pregnancy related traditional remedies................ 9
1.3.2. Classification of pregnancy related traditional remedies............. 9
1.3.3. Composition of pregnancy related traditional remedies............ 10
1.3.4. Transplacental transfer of drugs............................................ ....11
1.3.5. Altered maternal pharmacokinetics during pregnancy............... 12
1.3.6. Altered pharmacokinetics in the foetus and neonate................. 14
1.3.7. Teratogenicity............................................................................ 14
1.3.8. Lactation.................................................................................... 16
1.3.9. Pregnancy-related remedy usage.............................................. 16
1.3.10.Traditional Birth Attendants (TBA)............................................. 17
1.3.11. Adverse effects of pregnancy-related traditional remedies 19
1.3.12. Westerners' approach to pregnancy....................................... 21
1.4. Herbal M edicine................................................................................. 23
1.4.1. Secondary plant metabolites...................................................... 23
1.5. Shortcomings of Traditional Herbal Medicine................................ 25
1.5.1. Lack of evidence..........................................................................25
1.5.2. Availability and trade of medicinal plant material........................ 25
1.5.3. Standardisation of herbal products...............................................26
1.5.4. Identification and contamination of plant material......................... 27
1.5.5. Temporal variation in constituents of medicinal plants...............27
1.5.6. Spatial variation.......................................................................... 28
1.5.7. Stability of plant constituents...................................................... 29
1.6. Rhoicissus tridentaia...................................................................... 29
1.6.1. Classification...............................................................................29
1.6.2. Characteristics of Rhoicissus tridentaia subsp cuneifolia 31
1.6.3. Zulu usage and preparation of remedies containing Rhoicissus ..32
1.6.4. Scientific findings on R. revoilii and R tridentaia ......................32
1.6.5. Toxicity of R tridentata............................................................... 32
1.6 .6 . Chemistry of R tridentata and R revoilii.....................................35
1.6.7. Identification of pharmacologically active components..................35
1.7. Physiology of Labour...................................................................... 36
1.7.1. Myometrial contraction ............................................................... 37
1.7.2. Oxytocin...................................................................................... 39
1.7.3. Prostaglandins and myometrial contraction................................40
1.7.4. Prostaglandins as abortifacients.................................................. 44
1.7.5. Control of the onset of labour....................................................... 44
1.7.6. Gustaviib hypothesis on the onset of labour .............................46
1.7.7. Other physiological effects of prostaglandins............................. 48
1.8. Muscarinic Receptors..................................................................... 49
1.8.1. The autonomic nervous system.................................................. 49
1.8.2. Muscarinic receptors and labour.................................................50
1.8.3. Subtypes of muscarinic receptors ....................................... 50
1.9. A im s......................................................................... 54
xiii
CHAPTER 2: METHODS
2.1. Interviews........................................................................................... 56
2.2. Harvesting regimens for Rhoicissus tridentata used fo r ail
stu d ie s.............................................................................................. 56
2 .2 . 1 . different receptor systems.........................................................56
2.2.2. receptor subtype.........................................................................57
2.2.3. seasonal variation .....................................................................57
2.2.4. different plant parts..................................................................... 57
2.2.5. distributional variation...............................................................57
2.2.6. effect of storage ............................................... 58
2.3. R. tridentata subsp. cuneifolia decoction preparation ................ 58
2.4. Animals and Isolated Organ Preparations...................................... 61
2.5. Agonists and antagonists.................................................................62
2.6. Analysis o f Inorganic Ion C ontent....................................................63
2.7. Prostaglandin Syntiiesis in Cultured C e lls ..................................... 63
2.8. C ytotoxicity........................................................ 66
2.8.1. Cytotoxicity using the MTT assay method................................ 6 6
CHAPTER 3: Results section 1

3. Results from Open Ended Interviews..................................................... 6 8
3.1. Training of traditional healers........................................................ 6 8
3.2. Preparation of remedies containingRhoicissus tridentata............. 69
3.3. Dangers of herbal remedies during pregnancy............................ 73
3.4. Duration of therapy........................................................................ 73
3.5. Plants prescribed by the traditional healers in pregnancy related
remedies .....................................................................................73
3.6. Toxicity of Rhoicissus tridentata .................................................. 74
3.7. Previous documentation ofuse of prescribed plants during
pregnancy....................................................................................80
3.8. Literature citings of plants used in pregnancy related remedies ...84
xiv
4. Pharmacological action of Rhoicissus tridentata on isolated rat uterus
and ile u m ................................................................................................. 89
4.1. Inorganic Ion Concentrations........................................................... 89
4.2. Comparison Between the Efficacy and Potency o f the Different
Reference D rugs............................................................................... 89
4.3. Pharmacology of Rhoicissus tridentata subsp. cuneifolia on
isolated Rat Uterine and Ileal Smooth M uscle............................... 91
4.3.1. Extracts used for the investigations of pharmacological action .. 91
4.3.2. The contractile action of R. tridentata subsp cuneifolia ............ 92
4.4. involvement of Different Receptor Systems in the Contractile
Response to the Rhoicissus Extract .............................................. 95
4.4.1. Oxytocic receptor system ........................................................ 96
4.4.2. Prostaglandin synthesis .......................................................... 97
4.4.3. Adrenergic receptor system .................................................... 98
4.4.4. Muscarinic receptor system ...................................................100
4.4.5. Serotonergic receptor system ................................................104

4.4.6. Ganglion and second messenger blockers ............................107
4.4.7. Summary of results of non-specific antagonists..... ................. 108
4.4.8. Prostaglandin synthesis inhibitors ......................................... 110
4.4.9. a-adrenergicantagonists ....................................................... I l l
4.4.10. Muscarinic receptor antagonists ......................................... 112
4.4.11. Serotonin receptor antagonists ...................................... 113
4.4.12. The effect of R. tridentata on the basal tone of theuterus ....114

5. Temporal and Spatial Variation in the Extent of Contractile Response
Induced by R. tridentata ..................................................................... 115
5.1. Seasonal variation in the contractile activity of extracts from different
plant parts of Rhoicissus tridentata ................................................... 115
5.2. Variation in the contractile activity of extracts from different plant parts of
Rhoicissus tridentata ......................................................................... 118
5.3. Box and whisker plots of range of seasonal data............................... 120
xv
5.4. Seasonal variation in the increase in basal tone of isolated rat uterus. 121
5.5. Distributional variation .........................................................................123
5.6. The effect of storing dried plant material on the contractile activity 125

6. Results Obtained from Cell Culture Experiments
6.1. Effect of R. tridentata extracts on Prostaglandin Synthesis by

Histiocytoma Cells ........................................................................ 126
6.1.1. Prostaglandin synthesis overtim e.......................................... 126
6.1.2. Dose-response curves of theeffect of R. tridentata on
prostaglandin synthesis by histiocytomacells...........................127
6.2. Toxicity A ssa ys............................ 128

6.2.1. Parameters for the MTT Assays.................................................128
6.2.2. Reason for method modifications.............................................. 130
6.2.3. MTT assay results from different cell lines................................ 132
6.2.4. Kidney epithelial cells................................................................ 134
6.2.5. Hepatoma cells..................................................... 135
6.2.6. Mouse leydig cells.....................................................................136
6.2.7. Human histiocytoma cells................ ... .................................. 137
6.2.8. Seasonal differences............................................................... 138
6.2.9. Plant parts used....................................................................... 138
6.3. Relationship Between Contractile Activity and the Effect on
Cellular V ia b ility.............................................................................. 139
CHAPTER 7: DISCUSSION AND CONCLUSIONS

7.1. Interviews with traditional healers ................................................. 140
7.1.1. Training of traditional healers...............................................140
7.1.2. Plants used during pregnancy.............................................. 142
7.2. Pharmacological action on isolated organ s.................................. 143
7.2.1. Inorganic ion concentrations.................................................143
7.2.2. Reference drugs...................................................................144
7.2.3. Direct activity........................................................................144
xvi
7.2.4. Oxytocic receptor system.................................................... 145
7.2.5. Prostaglandin synthesis....................................................... 146
7.2.6. Adrenergic receptor system................................................. 147
7.2.7. Muscarinic receptor system............................................... 148
7.2.8. Muscarinic receptor subtypesand theuterine response 149
7.2.9. Serotonergic receptors........................................................ 151
7.2.10. Histamine receptors............................................................. 151
7.2.11. Ganglionic receptors............................................................ 151
7.2.12. Second messengers............................................................ 152
7.2.13. Blocking of the acion of the plantextract by atropine and
indomethacin..................... 153
7.2.14. Increase in the baseline of uterus following theinitial dose of R.
tridentata..................................................................... ....... 155
7.3. Variation in contractile a ctivity................................................ 157
7.3.1. Seasonal Variation............................................................... 157
7.3.2. Plant part used..................................................................... 158
7.3.3. Relevance to conservation.................................................... 158
7.4. Distributional variation in contractile a ctivity.........................159
7.4.1. Clinical relevance of variation incontractile activity 160
7.5. The effect of storage on contractile a c tiv ity ........................... 161
7.6. The effect of R. tridentata extracts on prostaglandin synthesis in
human histiocytoma c e ll......................................................... 162
7.7. Effect of R. tridentata on cell culture su rviva l............................. 163
7.7.1. Toxicity on different cell lines ...... 163
7.7.2. Seasonal differences in theeffect of the plantextracts on
cellular viability.............................................................. 164
7.7.3. The effect of extracts from different plant parts oncellular
viability...........................................................................164
7.7.4. Correlation between contractile activity and cytotoxicity 165
7.8. CONCLUSIONS
7.8.1. Ingredients used in pregnancy-related remedies.............166
7.8.2. Contractile activity of R. tridentata ......................................... 166
7.8.3. Pharmacological activity of R. tridentata.........................166
xvii
7.8.4. Variation in the contractile activity of R. tridentata ................ 166
7.8.5. Cytotoxicity of aqueous extracts of R. tridentata.................... 167
7.8.6. The effect of aqueous extracts of R. tn'dentata on prostaglandin
synthesis by human histiocytoma cells...................................167
7.9. Recommendations fo r further w o rk ................... 168
7.9.1. Herbal remedies used during pregnancy................................168
7.9.2. Absorption of active components from gastrointestinal tract... 168
7.9.3. Pharmacological action of Rhoicissus tridentata....................168
7.9.4. Screening of plants used during pregnancy.......................... 169
7.9.5. Variation in the contractile activity of Rhoicissus tridentata... 170
7.9.5. Long term studies on the clinical impact of traditional remedies
used during pregnancy...........................................................170
REFERENCES........................................................................................... .172
APPENDICES............................................................................................. 193
A.1. Tyrode solution fo rm u la ................................................................. 193
A.2. Details of cell cultures u s e d ............................................................193
A.3. Results o f statistical analyses o f all d a ta ................................ 195
A.3.1 Effect of the antagonists on the direct activity of the Rhoicissus
extracts ...................... 195
A.3.2 Seasonal differences in contractile activity.............................195
A.3.3 Contractile activity of extracts from different plant parts 196
A.3.4 Cytotoxicity of R, tridentata on different cell lines ,97
A.3.5 Seasonal differences in cytotoxicity........................................197
A.3.6 Cytotoxicity of extracts from different plant parts................... 197
A.4. Layout of microtitre plates fo r prostaglandin assays.................. 201
A.4.1 Prostaglandin synthesis over tim e..................................... . 201
A.4.2 Dose response experiment............................................ 201
A.4.3 Effect of indomethacin and hydrocortisone............................ 202
A.5. Raw data collected from open-ended interviews with traditional
healers ....................................................................................... 2 0 2
A.6 . Raw data from all experiments...................... 212
REPRINTS OF PUBLICATIONS
xviii
LIST OF FIGURES
Figure 1.1. Comparison of leaf shape between R. tridentaia subspecies

a) tridentata and b) cuneifolia (Urton et al. 1986)......................................... 30
Figure 1.2. Distribution of a) Rhoicissus tridentata subsp, cuneifolia and b) R.

tridentata subsp. tridentata (Urton etai. 1986)............................................... 31
Figure 1.3. Compounds isolated from Rhoicissus tridentata subsp.

cuneifolia....................................................................................................... 35
Figure 1.4. Regulation of uterine activity during pregnancy and labour.

Question marks indicate a possible influence........................... 36
Figure 1.5. Pharmacology of the smooth muscle c ell....................... 38
Figure 1.6. Metabolic pathway of phospholipids in the cascade of

prostaglandin synthesis (1 = reductase, 2 = isomerase, 3 = oxidation)......... 40
Figure 1.7. A scheme for the involvement of phospholipase containing

lysosomes in the synthesis of prostaglandins................................................ 47
Figure 1.8. Contractile mechanism for muscarinic receptor subtypes in

smooth muscle. All the mechanisms may not exist simultaneously in the same
smooth muscle cell, but rather to a varying extent in different cells depending
upon the expression of the various signaling proteins (Ehlert eta/. 1997)..... 53
Figure 2.1. Aerial parts of Rhoicissus tridentata subsp. cuneifolia harvested

from Suikerbosrand........................................................................................ 59
Figure 2.2. Rhoicissus tridentata subsp. cuneifolia growing at

Suikerbosrand Nature Reserve, a) overall growth habit b) leaf structure c and
d) underground parts showing a small lignotuber.......................................... 60
xix
Figure 2.3. The principle of the prostaglandin E2 enzyme immunoassay. The
wells are precoated with goat anti-mouse immunoglobulin. The wells are then
treated with PGE2 antibodies which bind to the immunoglobulins. Once the
unknown samples are added to the wells either PGE2 or PGE2 ~peroxidase
binds to the PGE2 -antibodies. The wells are then rinsed thoroughly and the
3,3’5,5tetramethylbenzidine (TMB) is added which is converted into a yellow
product by the peroxidase, and can be read on the multiwell scanning
spectrophotometer. That is, the intensity of the colour is inversely proportional
to the amount of PGE2 present in the well..................................................... 65
Figure 3.1. Plant matter for sale at the Faraday Herbal Market,
Johannesburg .......................................................................................... 70
Figure 3.2. Plant vendors selling their merchandise at Faraday Herbal

Market...........................................................................................................71
Figure 3.3. A plant vendor and traditional healer. The mixture (front left) is his
personal Imbiza"remedy which is sold in the dry form wrapped in newspaper,
or as a bottled decoction (behind the dry mix)............................................... 72
Figure 3.4. Rhoicissus lignotuoers for sale.................................................. 72
Figure 3.5. The number of plants prescribed by different traditional healers in

pregnancy related remedies. The colours indicate the proportion of plants
either documented or not documented in the scientific literature for use during
pregnancy......................................................................................................80
Figure 4.1. Comparison of the isolated rat a) uterus and b) ileum response
to the cumulative addition of the different reference agonists used in this
study. All values were calculated relative to the organ b maximal response to
acetylcholine. Each point represents the mean with the error bars representing
the standard error of the mean....................................................................... 90
xx
Figure 4.2. Direct action of R.tridenfata subsp. cuneifolia aqueous root
extracts on isolated rat a) uterus and b) ileum. All values were calculated
relative to the maximal response obtained from a prior cumulative dose
response curve to acetylcholine alone. Each point represents the mean
response from seven rats with the SEM......................................................... 93
Figure 4,3. Box and whisker plot showing the range of maximal contractions
stimulated by 1.3 mg/ml R tridentata harvested from Umlazi. The box extends
from the 25th to the 75s1 percentile, with the horozontal line at the median. The
whiskers show the range of the data.............................................................. 93
Figure 4.4. Tracing of the a) uterine and b) ileal contractions caused by (A)
cumulative additions of acetylcholine, (B) 1.3 mg/ml R. tridentata extract (Rh)
followed by cumulative additions of acetylcholine. The arrows indicate when
doses of exponentially increasing concentrations of acetylcholine were added
to the organ bath. The baths were rinsed between tests to allow the organs to
relax................................................................................................................ 94
Figure 4.5. The isolated uterine response to oxytocin alone (+) compared to
when the organs were pretreated with 1.3 mg/ml Rhoicissus extract for 5
minutes before the cumulative addition of oxytocin (# )................................. 96
Figure 4.6. The contractile response of isolated rat a) uterus and b) ileum to
acetylcholine (+) when pretreated with 5 (iM indomethacin for 15 minutes (v),
1.3 mg/ml R. tridentata extract for 5 minutes (e), and indomethacin followed
by the plant extract before the cumulative additions of acetylcholine (v )...... 97
Figure 4.7. The isolated a) uterine response to noradrenaline when

pretreated with 2 . 7 propanolol for 5 minutes then 1.3 mg/ml Rhoicissus
extract for 5 minutes (e), compared to the response to noradrenaline when

pretreated with propanolol only (+), propanolol and 2\M prazosin (V) or
prazosin followed by the Rhoicissus extract (V)............................................ 98
xxi
Figure 4.8. The isolated rat uterine response to noradrenaline when
pretreated with 2.7 pM propanolol for 5 minutes followed by 1.3 mg/ml
Rhoicissus aqueous extract for 5 minutes (@), compared to the response to
noradrenaline when pretreated with propanolol only (+), propanolol and 2pM
yohimbine (V) or yohimbine followed by the Rhoicissus extract (T)............ 99
Figure 4.9. The a) uterine and b) ileal response to acetylcholine (+)

compared to when the organs were pretreated with 1.3 mg/ml R. tridentata
root extract ( • ) for 5 minutes before the addition of acetylcholine. The

response of isolated rat uterus and ileum to acetylcholine when pretreated
with 4x1 O^M atropine for 5 minutes (v) or atropine for 5 minutes followed by
the plant extract for another 5 minutes before the cumulative addition of
acetylcholine (▼)........................................................................................ 101
Figure 4.10. The effect of antagonists on muscarinic subtypes on the

isolated rat uterine response to acetylcholine alone or after pretreatment with
the R. tridentata extract............................................................................... 103
Figure 4.11. The response of isolated rat a) utorine and b) ileal muscle to
serotonin (+) when pretreated with 1.3 mg/ml R. tndentata extract (®). The
contractile response of isolated rat uterus and ileum to serotonin when
pretreated with 1pM methysergide for 5 minutes (v), and methysergide

followed by the plant extract then cumulative additions of serotonin (V ).... 104
Figure 4.12. The effect of ketanserin, a 5-HTi receptor subtype antagonist, on

the isolated rat a) uterine and b) ileal response to serotonin with or without the
pretreatment of the R. tridentata extract....................................................... 105
Figure 4.13. The effect of tropisetron, a 5 -HT2 receptor subtype antagonist,

on the isolated rat a) uterine and b) ileal response to serotonin with or without
the pretreatment of the R. tridentata extract............................... 106
xxii
Figure 4.14. Bar graphs showing the maximal uterine responses to 1.3mg/ml
R. tridentata extract after the organs had been pretreated with the antagonists
below the x-axis........................................................................................... 107
Figure 4.15. The bar graphs showing the mean maximal uterine responses
to 1.3mg/ml R. tridentata extract after the organs had been pretreated with the
antagonists below the x-axis. The responses to the extract alone is charted
next to the response to the extract after pretreatment with the relevant
antagonist. The stars represent significant differences from the contractile
response to R. tridentata extract alone (*** = p< 0.001)...............................108
Figure 4.16. Bar graphs illustrating the maximal ileal response to 1.3 mg/ml
aqueous extracts of R. tridentata after the organs have been incubated with
the antagonists below the x-axis. The stars represent significant differences
from the contractile response to R. tridentata extract alone..........................109
Figure 4.17. The bar graphs showing the mean maximal uterine responses to
1.3mg/m! R. tridentata extract after the organs had been pretreated by various
cyclooxygenase inhibitors. The error bars represent the standard deviation of
the mean. The stars represent significant differences from the contractile
response to R. tridentata extract alone (*** = p< 0.001, ** = p< 0.01)......... 110
Figure 4.18. The bar graphs show the mean maximal uterine responses to
1.3mg/ml R. tridentata extract after the organs had been pretreated by
prazosin and yohimbine. The error bars represent the standard deviation of
response to R. tridentata extract alone (*** = p< 0.001, ** = p< 0.01)......... 111
Figure 4.19. The bar graphs show the mean maximal uterine responses to
1.3mg/m! R. tridentata extract after the organs had been pretreated by various
muscarinic antagonists. The stars represent significant differences from the
contractile response to R. tridentata extract alone (*** - p < 0.001,
* = p<0.05)................................................................................................... 112
Figure 4.20. Bar graphs showing the mean maximal uterine responses to
1.3 mg/ml R. tridentata extract after the organs had been pretreated by various
serotonin antagonists. The stars represent significant differences from the
contractile response to R. tridentata extract alone (* - p< 0.05)................. 113
Figure 4.21. The correlation between the contractions of the isolated rat
uterus stimulated by 1.3 mg/ml R. tridentata extract and the extent to which
the baseline was raised after the organ was rinsed and allowed to return to its
resting state.................................................................................................. 114
Figure 5.1. Dose response curves of isolated rat uterus when pre-treated with
1.3 mg/ml aqueous extracts of Rhoicissus tridentata showing the seasonal
variation in the contractile response to different plant parts....................... .117
Figure 5.2. The effect of extracts of different parts of the plants har vested
during the four seasons................................................................................ 119
Figure 5.3. A box and whisker plot of the maximal direct contractile
response to 1.3 mg/m! tuber extracts from R. tridentata harvested during
different seasons. The box extends from the 25*40 the 75* 1 percentile, with
the horozontal line at the median. The whiskers show the range of the data.
The colour of the stars indicates the season significantly different from the
data set........................................................................................................ 1 2 0
Figure 5.4. Variation in the increase in baseline once the organs had been
rinsed at least five times after a test challenge which included 1.3 mg/ml R.
tridentata. The stars represent significant differences where the colour of the
stars or the letter before the stars indicate from which that specific data set is
different........................................................................................................ 1 2 2
Figure 5.5. The contractile response of isolated uterine smooth muscle to

aqueous extracts of R. tridentata plants harvested from different localities
around South Africa. The organs were incubated with 1.3 mg/ml crude
aqueous extracts for 5 minutes before the cumulative addition of
acetylcholine........................................ 124
xxiv
Figure 5.6. Uterine contractile response to R. tridentata extracts from plant
material that had been stored for 1 month (A) compared to extracts from plant
material that had been stored for 12 months ('V). The organs were incubated
with 1.3 mg/ml of plant extract before the cumulative addition of acetylcholine.
..................................................................................................................... 125
Figure 6.1. Prostaglandin synthesis over time by histiocytoma cells

stimulated with 1 mg/ml Rhoicissus exiract................................................... 126
Figure 6.2. Dose response curve of intracellular, extracellular and total

prostaglandin concentrations after histiocytoma cells were incubated with
varying concentrations of 4 different Rhoicissus extracts for 20 minutes...... 127
Figure 6.3. The optical densities at different wavelengths corresponding to

different concentrations of histiocytoma cells per well................................. 128
Figure 6.4. The absorbance spectra from wells with different numbers of
histiocytoma cells.......................................................................................... 129
Figure 6.5. The absorbance spectra from wells containing 13 500

histiocytoma cells per well, and treated with different concentrations of R.
tridentata exiract........................................................................................... 129
Figure 6 .6 . Dose response curves of the results from the MTT assays using
different calculations to analyze the results. The top graph represents results
calculated from the absorbance at 540 nm, where the absorbance from the test
wells was calculated as a percentage of the control. The midd'e graph
represents the absorbance of the results, where either the absorbance at
620nm or 405nm is subtracted from the absorbance at 540nm. The bottom
graph displays the results In the middle graph as a percentage of the
controls.......................................................................................................... 131
xxv
Figure 6.7. Dose response curves showing the cellular viability after the cells
were incubated with varying concentrations of Rhoicissus extracts for 24 hours.
The graph represents the mean results from extracts tested in the number of
wells given in the legends. Significant differences between the absorbance of
test wells and control wells are shown in later graphs...................................133
Figure 6 .8 . Dose response curve of cell viability after being incubated with
varying concentrations of Rhoicissus extract for 24 hours. The graph
represents the results from 16 plant extracts tested in 12 wells each. No
significant differences between the absorbance of test and control wells
occurred....................................................................................................... 134
Figure 6.9. The effect of Rhoicissus extracts from a) different seasons and
b) different plant parts on the viability of human kidney epithelial cells. Stars
indicate significant differences between the extracts................................... 134
Figure 6.10. The cellular viability of human hepatoma cells after being
incubated with aqueous extracts of Rhoicissus for 24 hours. The curves
represent results from 8 plant extracts tested in 6 wells each, stars represent
significant differences between the absorbance of test and control wells. ..135
Figure 6.11. Comparisons between the effect of Rhoicissus extracts from

different a) seasons and different b) plant parts on the viability of human
hepatoma cells after being exposed to the extracts for 24 hours. There were
no significant differences.............................................................................. 135
Figure 6.12. Results from the MTT assay testing the effect of Rhoicissus
aqueous extracts on the viability of mouse leydig cells after being exposed to
the extracts for 24 hours. Each point represents the mean of 8 plant extracts
each tested in 1 2 wells................................................................................. 136

different a) seasons and different b) plant parts on the viability of mouse leydig
cells after being exposed to the plant extracts for 24 hours.......................... 136
xxvi
aqueous extracts on the viability of human histioctytoma cells after being
exposed to the extracts for 24 hours. Each point represents the mean of 16
plant extracts each tested in 8 wells............................................................ 137

histiocytoma cells after being exposed to the plant extracts for 24 hours.... 137
Figure 6.16. Correlation between the direct contractile activity of 3.1 mg/ml
on isolated rat uterine tissue and the viability of all cell types tested after
exposure to 1mg/ml R. tridentata extract for 24 hours................................139
Figure 7.1. Summary of the contractile mechanism of R. tridentata aqueous

extracts in isolated smooth muscle. Muscarinic receptors are represented by
blue pentagons, where prostanoid receptors are represented by violet
octagons....................................................................................................... 156
xxvii
LIST OF TABLES
Table 1.1 Medicinal usage of the Rhoicissus genus by other African tribal
groups. Indications related to reproduction are coloured blue.......................33
Table 1,2 Diversity of prostaglandin (PG) receptors that affect smooth muscle
tone (Campbell & Halushka 1995)................................................................... 42
Table 1.3 Typical locations of the various cholinergic receptor subtypes and
the second messengers mediating the cellular response to each subtype... 52
Table 2.1 Collection details of plants harvested to determine the effect of

distribution on the contractile activity of aqueous extracts..............................58
Table 2.2 Reference drugs used to elucidate the pharmacological action of

the R. tridentata extract on isolated rat uterus and ileum............................... 62
Table 3.1 Ingredients prescribed by the traditional healers interviewed for

use during pregnancy, or pregnancy related indications. Superscripts after the
botanical names refer to whether the plants are reported to be toxic (T) or to
be used in pregnancy related remedies (P) in the scientific literature 75
Table 3.2 Plants prescribed by traditional healers in pregnancy related

remedies tabulated according to indications, and the number of healers using
the plant for the specific indication................................................................... 81
Table 3.3 Documented pregnancy related uses of plants prescribed by the

traditional healers interviewed................ 84
Table 3.4 Potential toxic effects of plants prescribed by the traditional healers
interviewed in pregnancy related traditional remedies.................................... 87
Table 5.1 Statistical differences between the contractile activity of extracts

from Rhoicissus from different plant parts harvested in different seasons.... 120
xxviii
Table A.1 P-values from student-t tests done comparing the contractile
response of isolated rat uterus to the Rhoicissus extract alone as
compared to the response to the extract after the organs had been treated
with an antagonist................................................................................... 195
Table A.2 P-values from student-t tests comparing the seasonal differences
in the direct activity of Rhoicissus extracts on isolated rat uterus 195
Table A.3 P-values from student-t tests comparing the isolated rat uterine
maximal contractile response to acetylcholine after the organs had been
pretreated with 1.3mg/ml of various Rhoicissus extracts for 5 minutes... 196
Table A.4 P-values from student-t tests comparing the direct contractile
activity of Rhoicissus extracts from different plant parts harvested in
different seasons on isolated rat uterus.................................................. 196
Table A.5 P-values from student-t tests comparing the absorbance at 540nm
from the MTT assays performed using different concentrations of
Rhoicissus extract on the four cell lines.................................................. 197
from the MTT assays performed using Rhoicissus extracts from dormant
(D) and growing (G) seasons on the four cell lines. Highlighted blocks
show P-values less than 0.05................................................................. 197
from the MTT assays performed using Rhoicissus extracts from tubers (T)
and stems (S) on hepatoma cells........................................................... 197
from the MTT assays performed using Rhoicissus extracts from different
plant parts on the Graham cell line. Highlighted blocks show P-values less
than 0.05................................................................................................. 198
X XIX
from the MTT assays performed using Phoicissus extracts from different
plant parts on leydig cells. Highlighted blocks show P-values less than
0.05............................ 199
Table A.10 P-values from student-t tests comparing the absorbance at

540nm from the MTT assays performed using Phoicissus extracts from
different plant parts on histiocytoma cells. Highlighted blocks show P-
values less than 0.05.............................................................................. 209
xxx
NOMENCLATURE
liM - micromolar
5-HT - serotonin
AC - adenylyl cyclase
ACh - acetylcholine
ATP - adenosine triphosphate
Ca2-1" - calcium ions
cAMP - cyclic adenosine monophosphate
carbogen - 5% carbon dioxide, 95% oxygen
CNS - central nervous system
COX - cyclooxygenase
COX-1- cyclooxygenase isoform 1
COX-2 - cyclooxygenase isoform 2
DAG - diacylglycerol
DMSO - dimethylsulphoxide
EDRF - endothelium-derived relaxing factor
IL-1 alpha - interleukin-1 alpha
IPs - inositol-1,4,5-frisphosphate
K*" - potassium ions
Ml - muscarinic subtype 1 acetylcholine receptors
M2 - muscarinic subtype 2 acetylcholine receptors
mM - millimolar
mRNA - messenger ribonucleic acid
MTT - 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
NA - noradrenaline
Na+ - sodium ions
nM - nanomolar
NO - nitric oxide
PBS - Dulbecco phosphate buffer
PG - prostaglandin
PGE - prostaglandin E
xxxi
PGF2 a - prostaglandin F2„
PGI2 - prostacyclin
PKC - protein kinase C
PLA2 - phospholipase Pq
PLC-p - phospholipase C-p
STD - sexually transmitted diseases
TBA - traditional birth attendant
tbsp - tablespoon
tsp - teaspoon
TXA2 - thromboxane A
xxxii
CHAPTER 1: INTRODUCTION
1.1. Traditional and Herbal Medicine

The medical use of herbs is deeply rooted in the history and folklore of virtually
all cultures (Garty 1993; Dubick 1986). Even though modern medicine has
dominated the medical field for most of this century, recently there has been an
exponential increase in the sale of herbs around the world (Rosti et a/. 1994),
Herbal medicine is the most common form of all alternative treatments in
western countries (Angell & Kassirer 1998), which are used predominantly by
the more affluent and more highly educated portion of the population, between
the ages of 25-49 (Eisenberg et a!. 1993; Biackmore 1999). This return to herbal
remedies is probably due to the misconception that because herbs are 'natural'
they are safe as well as the efficacy of herbal remedies in the treatment of
simple and uncomplicated illnesses (Dubick M.A. 1986; Pillans 1994). This
misconception is coupled with dissatisfaction with modem health care, rising
costs (Taylor 1996) and the often hurried and impersonal care delivered by
conventional physicians (Angell & Kassirer 1998). The siv ation in third world
countries is very different to that of developing countries. Many communities
still do not have the privilege of access to evidence based medicine and
therefore have no choice but to rely on traditional medicines. Traditional
medicine is ;he principle and often the only form of health care for many
Africans (Pate! 1993), especially in the rural areas.
1.1.1. Traditional medicine in South Africa

Traditional practitioners are widely spread throughout South Africa. It is
estimated that approximately 200 000 traditional healers practice in South
Africa compared with 25 000 doctors of modern medicine (Abdool Karim et al.
1992). In sub-Saharan Africa the ratio of traditional healers to people is
estimated at 1:100-1000, whereas the ratio of medically qualified doctors to
people is estimated at 1:10 000-100 000 (Unterhalter 1982). In South Africa,
66% of the population lives in a rural setting yet only 20% of doctors are
located in rural areas (Unterhalter 1982). This low doctor-patient ratio in rural
black communities means that the traditional healer is often "ie first, nearest
1
or even only contact for the rural black African to medical care (Buchmann et
al. 1989). As many as 80% of black South Africans visit a traditional healer
before visiting an allopathic doctor (Gumede 1990). Traditional healers are
also very popular because they are culturally familiar, speak the same
language and provide culturally familiar ways of explaining the cause of ill-
health and its relationship to the people's social and supernatural worlds
(Chipfakacha 1994).
The theory underlying traditional medicine in the many black ethnic groups in
South Africa is essentially similar (Kale 1995). Most blacks are superstitious,
with deep-rooted traditions which make no provision for modern medicine and
its derivatives. Scientific medicine is and has always been a foreign cultural idea.
For example, the “germ" theory is not understood or appreciated. To the African
diseases fall into three major categories: (i) those due to magic or evil spirits; (ii)
those due to conditions for which causes have been empirically determined; and
(ii) those due to psychological phenomena (Chipfakacha 1994).
1.1.2. Types of healers

inyangas are herbalists who possess extensive knowledge about curative herbs
and medicines of animal origin. Ninety percent of inyangas are male.
Isangomas are diviners who determine the cause of illness by using ancestral
spirits, and 90% are female.
Umthandazi are faith healers who are professed Christians belonging to one of
the many independent African churches. They heal by prayer, using holy water
or ash, or by touching the patient (Conco 1972; Kale 1995).
Traditional birth attendants (TBA) are not healers, but deliver babies bom in their
villiage. They are usually elderly woman who are respected in society for their
skills. If a complication occurs, the birth attendant seeks the advice of an
inyanga (Kale 1995).
1.1.3. Registration of traditional healers in South Africa

In South Africa there are no recognised institutes for the training and
registering of traditional healers, thus no standardisation between traditional
2
healers exists. There have been attempts by national traditional healer
associations to set examinations testing the competence of healers with the
aim of setting up a registry for traditional healers. This would help regulate the
standards of healers, as minimum qualifications and levels of training could
be stipulated (Schuster Campbell 1998; Hutchings 1999). However, this
process is still in its initial stages. Currently there is no way of measuring the
competence of a traditional healer. A registry for traditional healers in
Zimbabwe was set up in 1980, yet a survey conducted in 1995 showed that
nearly half of traditional healers in the suburbs of Harare were not registered
with any formal organisation (Winston ef a/. 1995). It appears that incentives
would have to be offered, or the registration and training would have to benefit
the healer before the general level of competence of healers could be raised.
At the moment, the traditional healer in most parts of central, southern and
eastern Africa practices his art in an empirical manner, relying on personal
insight and experience for success (Harries & Cullinan 1994). The thought of
testing the efficacy of a certain remedy in sufficient numbers of patients and
controls is a completely foreign concept to most traditional healers (Healers.
Pers. comm.) Since there is no reference against which to compare a healer,
the opportunities for irresponsible quackery and reckless profiteering are
completely open (Bodenstein 1977). Even if traditional healers have scientific
training, or have the will to learn more, very little work has been done on the
toxicity and pharmacological activity of the South African flora (Huxtable
1990). More importantly, there is no institute in this country providing sound
training in herbalism as is the case in the United States, Europe, China and
India.
Traditional healers have become more acceptable since the change of

government in 1994. This is manifest in the agreements by Medical Aid
Societies such as the NBC Medical Aid Society which has agreed with the
African National Healer’s Association to honour claims for its members who
have consulted sangomas (Kanhema 1993).
3
1.1.4. Reports of adverse effects of African traditional medicines
Many adverse effects of African traditional remedies have been reported in
the literature. A few of these reports are as follows. Traditional medicines
caused 51.7% (31/60) of fatal poisonings admitted to Ga-Rankuwa Hospital
during the period from 1981 to 1985 (Joubert 1990). Acute dichromate caused
7 cases of poisoning after the use of traditional purgatives obtained from local
inyanga. Symptoms of poisoning included renal failure and abnormal liver
function tests and fatalities (Wood et al. 1990; Michie et al. 1991). Potassium
dichromate used as enemas caused patients to suffer from severe colonic
complications (Dunn et al. 1991). Inappropriate use of traditional medicines
resulted in numerous fatalities, invariably in children (Bye & Dutton 1991).
Cardiac glycosides were present in 44% of autopsies where deaths were
presumed to have been caused by traditional remedies, where the most
common clinical presentation was gastrointestinal irritation which occurred in
54% of the patients (McVann etal. 1992). Traditional eye medicines are used
frequently in African traditional medicine, which have been found to be used
by 33% of the patients presenting with corneal ulcers (Courtright et al. 1994;
Lewallen & Courtright 1995). The risk of oesophageal cancer associated with
regular consumption of Solanum nigrum as a food is higher than the risk
associated with smoking (relative-risks 3.6 and 2.6 respectively). Even though
the above severe and fatal complications due to traditional medicines have
been described in the literature, the overall incidence of herbal poisonings in
South Africa is not known (Kale 1995).
An indirect, and presumably more common problem caused by traditional

medicine is the delayed presentation of patients to clinics or hospitals. The
reason black patients present to western doctors late is thought to be due to
initial recourse to traditional healers coupled with inadequate or expensive
orthodox medical care (Onuba 1992). Unfortunately in many cases, valuable
time is lost while a certain disease is in its initial stages and still curable. A
patient waits until the disease is severe, often until irreversible damage has
been caused before turning to orthodox medicine. Often no help can be given
at this stage. This in turn decreases the faith black patients have in orthodox
medicine, making it appear as if orthodox medicine is as effective as
4
traditional medicines. Therefore it is a vicious circle. Two examples of this
scenario are given below.
Two children were taken to traditional healers for apparently minor ailments,
one with a lesion that had developed around the point of entry of a thorn in the
hand and the other with a lesion in the knee, it took close on a year for each
of the children to be presented to hospitals by which time secondary
conditions had developed and both lost the use of a limb. Over the year, the
child with a thorn in the hand developed chronic osteomyelitis which then
progressed to a point of irreversible damage, requiring removal of the radius
resulting in the loss of use of his right hand. The other child presented with a
malignant lesion requiring an above-knee amputation of the left leg
(Chipfakacha 1994). In both cases the limbs could have been saved if the
children had been referred to orthodox medicine early in the progression of
their diseases.
Closer collaboration between traditional healers and western doctors together

with basic medical training of traditional healers enabling them to recognise
when their medical system will fail, could avoid the perpetual repetition of
cases such as described above.
1.1.5. Advantages of traditional health care systems

Despite many shortcomings c " traditional and herbal medicines, African
traditional medicine offers several advantages compared to conventional
orthodox medicine. These include: (i) the whole family is involved in the
treatment. The focus of attention is not only the patient but also the reaction of
the family and close relatives; (ii) traditional healing has a closeness, a shared
view, informality, and the use of everyday language in the consultations; and
(Hi) traditional healing both reinforces and articulates the values of the
community to which the healer belongs (Chipfakacha 1994). Modern doctors
in contrast, are separated from their patients by social class, language,
economic position, specialised education and cultural background. Most
physicians usually interrupt their patients only 18 seconds after they begin
5
describing their problem, while traditional healers depend on good
communication (McKenzie 1994).
Many traditional healers only charge a patient once the patient has been
healed (McKenzie 1994), even if the treatment involved weeks to months of
daily consultations (Healers, Pers. comm.). However, the fees may sometimes
be exorbitant, for instance a cow (Kale 1995). This system seems to be
changing as many traditional healers adopt the western practitioners’ custom
of charging per consultation (Healers, Pers. comm.).
1.1.6. Integration of traditional healers into primary health care

Since the Alma Ata conference held by the WHO/UNICEF there has been a
growing impetus towards either integrated systems or the beneficial co
existence of traditional and modern medical systems (Pretorius 1991). Closer
collaboration between the two medical systems has been proposed in many
countries such as Zimbabwe, Malawi, Swaziland, Tanzania and Mali
(Freeman & Motsei 1992; Ghana et al. 1994; Berger et al. 1994; Mbura er al.
1985). It is believed that all interested groups stand to gain from such a
liaison: the authorities responsible for health care delivery, the western-trained
health care workers, the traditional healers and the users of these services
(Pretorius 1991).
Providing traditional healers are educated to recognize illnesses which they

cannot and should not treat (e.g. tuberculosis and eye disease) traditional
healers fulfil three of Morrell's (1986) four primary health care objectives,
being that primary health care: (i) should be acceptable to the people; (ii)
should be accessible to the people; and (Hi) should identify those medical
needs of the population which can be prevented, modified or treated.
Incorporating traditional healers into modern health services would fulfil
Morrell's fourth objective of primary health care, that primary health care
should make maximum use of the available manpower and resources to meet
the medical needs of the people (Chipfakacha 1994).
6
With regard to the developing world, doctors should reconsider the flaw in the
attitudes that systems developed in the West are inherently preferable to the
local alternatives (Patel 1993), Western medicine should not ignore, devalue
or ban traditional practice. To do so would jeopardise black South Africans
suffering from depression, mental handicaps or AIDS as well as any other
poor person for whom the traditional healer is often the only and best relief.
Traditional remedies are also believed to be effective in diarrhoea, headaches,
other pains, and for sedating a patient. It is thought, that traditional health care
is more cost effective than official health care for some health problems such
as psychiatric disorders (Patel 1993), and the success in treating psychological
disorders is well known and often recognised (Kale 1995).
Edwards (1986) investigated the diagnosis and treatment of a group of

psychiatric patients by Zulu healers and modern clinical psychologists. The
results indicated that while traditional and modern healers worked from
different theoretical orientations, there was significant agreement in the
diagnosis and treatment by the healers and psychologists. The patients
perceived both traditional and modern practitioners as being more or less
equally helpful.
Western medicine should embrace, train and collaborate with traditional

healers, while encouraging traditional healers to administer safe, hygienic
treatments for conditions that are more amenable to the healers' type of
practice. Dialogue should be conducted on an equal footing as the sooner
some mutual understanding is achieved the better (Harries & Culling 1994).
Incorporation of traditional healers into the modern health care system would
help identify diseases before they reach an advanced stage, Before the
patient becomes disabled or dies because of the loss of valuable time.
Traditional healers should be encouraged to refer patients they are incapable
of curing or treating (Chipfakacha 1994). However, Chief Zungu, leader of the
Traditional Healers Association of South Africa, argues that traditional healers
have been referring patients to mainstream doctors and hospitals for years,
yet physicians will never refer a patient to a traditional healer. He states that
this is a very unfair relationship (McKenzie 1994).
7
Skepticism about the possibility of western medicine being able to collaborate
with any type of traditional healer has been expressed by Kiernan (1978). In
his opinion, medical doctors could collaborate with sangomas as they are
competent in the physical and social realms and therefore their practices are
complementary to modern medicine. The same does not apply to inyangas as
he feels that inyangas practice in opposition to modern medicine and
therefore collaboration with inyangas would not be possible or desirable.
1.2. Researching South African Traditional Medicinal Plants

Traditional healers claim that when they undergo training, ancestral spirits are
their principal teachers (Gumede 1990; Schuster Campbell 1998). Thus there
are no standard treatment regimens for specific indications. A wide variety of
plants are prescribed for the same condition by different practitioners making
research on the impact of traditional medicine an almost impossible task as
there are insufficient resources and manpower to determine the efficacy and
safety of every plant used by traditional healers. Over a thousand species are
used by the Zulu people alone (Hutchings et al. 1996). Part of the solution to this
problem lies in collaboration between traditional healers and researchers. This
process would be more likely to succeed if traditional healers could be
persuaded to cooperate with orthodox physicians. Traditional healers need to
identify the plants most commonly used and those that pose the greatest risk to
the patients. Then scientific researchers need to focus their research on this
selected group of plants. Results should then be simplified and made accessible
to traditional healers.
Previous work has shown that traditional healers are not merely custodians of
tradition, but are innovative and willing to learn from their Western counterparts
(Simon 1991; Green et a!. 1995). Traditional healers are therefore likely to heed
advice given to them. However, healers seem more likely to accept positive
findings than negative ones, as was apparent when results on the toxicity of
impila (Callilepis laureola) were presented to some traditional healers (Hutchings
1999).
8
1.3. Pregnancy Related Traditional Remedies
Pregnancy-related traditional and herbal remedies are used in many countries
throughout Africa (Mbura etal. 1985), as well as other countries such as China,
Japan, India, Melanesia, Hawaii, North and South America (Varga & Veale
1997). In rural South Africa, up to 75% of pregnant Zulu women visit traditional
healers to request remedies specifically for pregnancy (Morris & Mdlalose 1991).
There is also a renewed interest in these remedies in the United States and
Europe (Bunce 1987; Ehudin-Pagano etal. 1987).
1.3.1. South African pregnancy related traditional remedies

Fertility is a dominant theme in the culture of black South Africans as it ensures
the preservation and propagation of the tribe. A large family is regarded as
insurance against hunger and want in old age, with childbearing and infant care
being two sacred duties of women in African society (Gumede 1978).
Barrenness is regarded as a disgrace, as procreation is expected to
automatically follow marriage. Barrenness has been identified as a cause of
emotional stress in black patients living according to black customs and may
lead to typical clinical presentation of stress (Ramasuvha 1982). Girls as young
as 16 years visit doctors complaining of sterility, as the boyfriend wants proof of
womanhood (namely a baby) before marriage (Gumede 1978). Pregnancy is
therefore an event of great importance, which is manifested in the number of
traditional remedies used for pregnancy related indications, and the number of
customs and taboos that have been developed around pregnancy.
African folklore teaches that many of the complications during pregnancy are
caused by spells cast by the "abathakathi" (sorcerers). The role of the African
traditional healer is to diagnose the source of the problem and to prescribe a
remedy to ensure the well-being of the foetus (Harries & Cullinan 1994; Conco
1972).
1.3.2. Classification of pregnancy related traditional remedies

Four groups of pregnancy related remedies are prescribed. The first group is
isihlambezo which comprises the most common antenatal traditional remedies.
9
These remedies are prescribed to enhance foetal growth, strengthen the mother
and the foetus, aid the normal physiological functioning of the mother, ensure
an easy and complication free c iivery, speed up delivery, produce a clean baby
with no vemix, and remove excess fluid from the abdomen (Gerstner 1941;
Gumede 1978; Gumede 1990; Morris & Mdlalose 1991; Varga & Veale 1997).
The routine use of these remedies usually commences from the 7th lunar month
and continues through to delivery (Morris & Mdlalose 1991; Veale et al. 1992).
Imbiza is another group of remedies which have cleansing properties (Varga &
Veale 1997). These are not specific to pregnancy, but are commonly prescribed
to pregnant women at any stage of pregnancy. The third group of remedies is
known as umsekelo (Bryant 1966; Veale et al. 1992). These remedies are used
specifically to prevent miscarriages and premature deliveries. The last group,
known as either imbelekisane or inembe is used specifically to induce or
augment labour. Components of these remedies are thought to have strong
uterotonic properties (Conco 1972; Gumede 1990; Varga & Veale 1997;
Gumede 1978). Some healers use an additional type of remedy. After the birth,
the mother is given an infusion (e.g. ugobho - Gunnera perpensa) to aid the
involution and healing of the uterus (Larsen etal. 1983).
1.3.3. Composition o f pregnancy related traditional remedies

A literature review compiled by Veale et al, (1992) revealed that 57 plant species
were reported to be used in herbal remedies during pregnancy and childbirth, of
which Rhoicissus tridentata is one of the plants prescribed. Of the 57 plant
species, 16 (28%) are known to be potentially toxic, and four (7%) are known to
have caused fatal human poisonings.
Most traditional healers mix two or three components in one decoction (Varga &
Veale 1997). The mixing of 57 different ingredients in a variety of permutations
yields a tremendous diversity between different kinds of isihlambezo, inembe,
imbelekisane or imbiza, yet most research done on these remedies treats
pregnancy-related remedies as an homogenous group (Bullough 1981, Larsen
etal. 1983, Mbura at al. 1985, Mitri etal. 1987; Morris & Mdlalose 1991). This is
no different to treating all scientific medicines as homogenous. If specific
components of remedies are not identified the results have very little
10
applicability, as the different components of the remedies are chemically diverse
and will therefore have diverse pharmacological effects. The pharmacology and
chemistry of each plant needs to be investigated individually before the effect of
these remedies on pregnancy can be elucidated enabling predictions to be
made.
1.3.4. Transplacental transfer of drugs

The placenta is an organ with many physiological functions, several involving
exchange of various endogenous and exogenous substances between the
maternal and foetal circulation (Bourget et al. 1995). Most drugs taken by
pregnant woman can cross the placenta and expose the developing embryo and
foetus to their pharmacological and teratogenic effects (Keren 1998). The
potential toxicity to the foetus is greater than to the mother due to the increased
blood-brain-barrier permeability and the poorer liver enzyme conjugating
function in the foetus (Norman 1992). Critical factors affecting the intensity of
placental drug transfer and drug effects on the foetus (Seller 1965; Norman
1992) include the following: (i) the physicochemical properties of the drug; (ii) the
duration of exposure to the drug; (Hi) distribution characteristics in different foetal
tissues; (iv) the effects of the drugs used in combination; (v) the surface area of
the exchange membrane; (vi) the thickness of the endothelio-syncytial
membrane; (vii) the maternal blood flow and hydrostatic pressure in the
intravillous chamber; (viii) the blood pressure in foetal capillaries; and (ix) the
difference between maternal and foetal osmotic pressures. As term approaches,
foetal-maternal exchanges intensify, in part because of thinning of the
membranes.
Passive transfer is the predominant form of exchange, however, active

transport, facilitated diffussion, transfer by molecular binding, endocytosis and,
passage by effraction also occur. The placenta contains microsomal enzymes
which metabolise many exogenous substances. The result is to produce more
polar metabolites that are more readily excreted. This process usually results in
detoxification activity, however, theoretically the possibility remains that
metabolism could produce a substance that is more foetotoxic than the parent
compound (Bourget etal. 1995).
11
A pH gradient could affect the transplacental passive transport of drugs since
only non-ionised or slightly ionised compounds cross membranes in this manner
(Reynolds & Knott 1990). It has been established that if the pH of foetal plasma
falls, the foeto-maternal ratio of basic compounds increases (Biehl et al. 1978;
Kennedy et al. 1979). This so-called ion-trapping effect is believed to be more
pronounced in the case of strong rather than weak bases (Reynolds & Knott
1990).
1.3.5. Altered maternal pharmacokinetics during pregnancy

Plasma protein binding of drugs may fall during pregnancy because of a
oestrogen induced fall in plasma albumin concentrations by up to 20%.
Therefore, there may be a fall in the bound fraction of drugs normally extensively
bound to plasma albumin. This will normally lead to increased clearance of the
drug, even though the concentration of the free drug does not fall to the same
extent (Grahame-Smith & Aronson 1992; Norman 1992).
Metabolism of drugs is also altered during pregnancy. The functions of different

enzymes are affected differently during pregnancy. During late stages of
pregnancy, the conjugation of drugs with glucuronic acid is reduced to about half
the normal level. This is probably due to the high levels of progesterone and
pregnanediol, which are known to inhibit glucuronyi transferases in vitro
(Robson & Stacey 1968). Certain oxidative metabolic transformations of drugs
are also inhibited, where the activity of mono-oxygenase is increased (Norman
1992). That is, compounds that undergo conjugation with glucorinic acid or
oxidation will be metabolised at a reduced rate leading to increased plasma
concentrations of these compounds, in tuin causing more of the compound to
pass into the foetal circulation.
Excretion. Renal blood flow doubles by the third trimester, and the glomerular
filtration rate increases by about 70%. Thus drugs which are mainly eliminated
by renal excretion will be cleared more rapidly. In contrast the clearance of
drugs with a high hepatic extraction ratio show no change in the clearance rate.
12
This is because there is no change in the blood flow to the liver during
pregnancy (Norman 1992).
Nutrition is also an important factor in drug metabolism. Dingell et al. (1966)

showed that rats maintained on a diet deficient in protein aiid calcium have
lowered capacities to metabolise drugs, both by oxidative and reductive
pathways, due to decreased activity of the microsomal enzymes of the liver.
Animals on diets deficient in protein and magnesium have been shown to be
more prone to acetylsalicylic acid toxicity (West 1964).
The increased plasma concentrations of effected compounds during pregnancy

could be further accentuated if the mother has a nutrient deficient diet. The diet
of the majority of subeconomic and/or rural black South Africans has been
shown to consist of mainly carbohydrates and is deficient in protein, as well as
certain vitamins and minerals (Goetzee 1974; Clarke & Ngobese 1975;
Bembridge 1987). This is especially apparent in communities with insufficient
land to cultivate enough food and who rely on bought refined meals rather than
eating whole grains grown themselves. There is also a cu?tom of discouraging
pregnant women from consuming milk and meat from the family herd and from
eating eggs and fish during pregnancy (Larsen et al. 1983; Sindiga 1995).
Therefore, herbal remedies metabolised by microsomal enzymes have the
potential of being more toxic during pregnancy.
The complex interactions influencing the pharmacokinetics of drugs during

pregnancy, with either possible increases or decreases in plasma levels of
drugs, have led to the general rule in modem medicine to avoid drugs as far as
possible during pregnancy. If drugs cannot be avoided the drug concentration
should be kept in the lower third of the therapeutic range. This rule should be
extended to the use of herbal remedies during pregnancy, however, the use of
herbs during pregnancy is so deeply entrenched in black African culture, that it
is unlikely that caution will be exercised for many years to come.
13
1.3.6. Altered pharmacokinetics in the foetus and neonate
In general, foetal and newborn animals have a limited capacity to metabolise
drugs. The microsomal enzymes of the liver which oxidatively metabolise drugs,
including cytochrome P-450, are more or less absent in the foetus, but increase
rapidly after birth, reaching adult levels at about 30 days in rats and 8 weeks in
humans.
Reactions which are diminished or impaired in foetuses and new-born infants

are: the reduction of nitro and azo compounds; the conjugation of drugs and
their metabolites; conjugation with glucuronic acid, glycine and glutathione
(mercapturic acids). Acetylation and sulphate reactions however occur at normal
adult levels. This lowered rate of drug metabolism usually leads to a decreased
ability to deactivate drugs, and consequently to prolonged pharmacological
activity and increased toxicity. On the other hand, when metabolism is
responsible for converting an inactive prodrug into an active drug, the effects are
reversed - diminished pharmacological activity and lowered toxicity would be
expected (Robson and Stacey 1968).
Evaluation of foetal effects and efficacy of modem drugs during pregnancy is a

fielo ihat has been minimally investigated (Bourget et al. 1995). This is even
more apparent with investigations on the safety and efficacy of herbal remedies
during pregnancy. No clinical studies on the safety of herbal remedies during
pregnancy could be located. The only data on the use of herbs during
pregnancy are in the m of case reports or animal studies (Keeler & Crowe
1984; Panteref a/. K o, Lemonica & Alvarenga 1994).
1.3.7. Teratogenicity
The thalidomide disaster in the early 1960‘s where there was a dramatic
increase in the incidence of a rare birth defect including phocomelia, a condition
involving the shortening or complete absence of limbs, horrified the western
world, highlighting the dangers of ingesting medicines during pregnancy. It is
estimated that 10 000 children were born with birth defects because of maternal
exposure to this one agent (Berkowitz & Katzung 1998). Other allopathic drugs
have been identified to possess teratogenic properties which include cytotoxic
14
agents, retinoic acid, phenytoin, carbamazepine, sodium valproate, warfarin,
gentamicin and lithium (Norman 1992). Million of dollars are spent by
pharmaceutical companies to determine the safety of drugs during pregnancy. It
is unknown whether spending the same amount researching the safety of herbal
remedies during pregnancy would also yield results showing that certain herbs
are teratogenic. Interviews with traditional birth attendants showed that they are
aware that alcohol and cigarettes should be avoided during pregnancy (Sindiga
1995), but no mention is made of possible dangers of traditional herbal
remedies.
Organogenesis of the foetus occurs within the first trimester of pregnancy,

therefore the greatest risk of causing major congenital malformation is during the
first trimester. However, malformations in sexual differentiation, and neurological
development may occur if a harmful compound is ingested during the second or
third trimester.
Undoubtedly teratogenic compounds do occur in higher plants, but no plant

species has been shown as having been responsible for congenital
malformations in humans. However, experiments on laboratory animals, where
large doses of plants not usually experienced by humans were incorporated into
the food source, have demonstrated that the potential for certain plants to be
teratogenic exists (Evans 1996). Some plant species or genera used in South
African traditional medicines that have been shown to have caused
malformations in laboratory animals include: Senecio (Asteraceae), Indigofera
spicata (Solanaceae), Lobelia (Campanulaceae), Mimosa (Mimosaceae),
Lupinus sericeus (Fabaceae), Conium maculatum (Umbelliferae), Veratrum
califomicum (Liliaceae) (Evans 1996). Cinnamonium zeylanicum has been
shown to be embryofetotoxic (Lemonica & Macedo 1994).
Despite all the potential dangers listed above associated with drug ingestion
during pregnancy, less than one percent of congenital malformations are
thought to be caused by the use of modern drugs during pregnancy (Norman
1992).
15
1.3.8. Lactation
Most drugs pass into breastmilk. As mentioned above the hepatic metabolism of
many drugs is reduced in neonates making neonates particularly prone to toxic
effects of herbs ingested by lactating mothers.
Two breastfed neonates were admitted to hosp I in Italy after their mothers
drank copious amounts (more than 21 per day) of a herbal mixture to stimulate
lactation. The remedies contained liquorice, fennel, anise and Gaiega officinalis.
The neonates showed neurological symptoms of hypotonia, lethargy, emesis,
weak cry, poor sucking and torpid reactions to painful stimuli. Once the teas
were discontinued the condition of the neonates improved within 24-36 hrs.
Essential oils (anetholes and related compounds) within the anise and fennel
were suspected to have been the causative agents in the poisonings (Rosti et al.
1994).
Two species of Senecio containing pyrrolizidine alkaloids were linked to fatal

hepatic veno-occlusive disease in a newborn infant of r woman, who again was
drinking copious amounts of a herbal tea as a lactagogue (Roulet 1988). The
symptoms of pyrrolizidine poisoning mimic Reye's syndrome (Fox et al. 1978).
1.3.9. Pregnancy-related remedy usage

South African traditional healers generally do not administer isihlambezo
during the first trimester. The use of isihlambezo normally commences from
the beginning of the third month, or whenever the patient consults the healer.
Treatment continues on a daily basis, or whenever needed, until the end of
the eighth month or until birth (Varga & Veaie 1997), Morris & Mdlalose (1991)
found that up to 75% of rural Zulu women use traditional remedies specifically
for pregnancy. In Tanzania, where approximately 50% of the women use
traditional remedies during pregnancy, remedies are used throughout
pregnancy, with the highest usage rate being during the first trimestei,
followed by usage during labour. The usage rate increases with age,
indicating that young people are being swayed towards accepting western
culture and abandoning traditional practices as "uncivilized”. It is the older
women who tend to keep cultural habits including traditional medicines (Mbura
etal. 1985).
16
1.3.10. Traditional Birth Attendants (TBA)
Traditional birth attendants are characteristic of most developing countries. They
are usually older women who are respected in their communities for their skills
for assisting during confinement. The conditions for becoming a traditional birth
attendant usually include having at least two children and an apprenticeship
lasting for anytime up to 15-20 years. Birth attendants do not charge for their
services but may accept gifts (Kale 1995).
60-80% of all babies in the developing world are delivered at home by either
trained or untrained birth attendants (Islam et a!. 1982b; Gosminsky 1983;
Bullough 1981; Fajemilehin 1995; Wollast et al. 1993). Home delivery is
especially common in rural areas where long-held superstitions are observed.
Tetanus is the most common danger associated with home deliveries, which
accounts for 20-40% of neonatal deaths in developing countries. Neonatal
tetanus is mainly associated with the use of unsterile instruments for cutting
the umbilical cord, unsanitary surroundings and the prevalent practice of
dressing the umbilical stump with substances such as cow-dung, herbs, clay
crash (Islamefa/. 1982a; Buchmanne<a/. 1989; Fajemilehin 1995).
South Africa has a lower rate of home deliveries than that reported from other
developing countries. Buchmann et al. (1989) reported that 46% of woman in
the Mosveld health ward (rural northern Kwazulu-Natal) delivered at home, of
which only 47% were attended by traditional birth attendants. These lower
proportions of home-oillis are despite this region having a particularly high
density of traditional healers (BuJ'.man, Pers. comm.). The reasons given for
homo deliveries were predominantly shortage of transport and sudden or
unexpected labour. Of the women who ir 'entionally gave birth at home 94%
wem multiparas (Buchman etal. 1989).
Larsen et al. (1983) reported the following practices of TBAs after close
interactions with 5 Zulu traditional birth attendants.
Antenatal care: No physical examinations are done on pregnant women. The
TBA serves more as an educator. Pregnant women are advised to avoid
eating eggs and meat or milk from the family herd. The TBA may recommend
17
or prescribe isihlambezo. Pregnant woman are advised to be as active as
possible, with no tradition that heavy manual labour may possibly be harmful.
TBA were shown to have little knowledge about complications such as
eclampsia or antepartum haemorrhage.
Labour: TBAs are usually only called late in the first stags of labour. If labour
is prolonged (primigravida >24hrs and multigravida >12 hrs), the woman in
labour is given imbelekisane. TBAs were shown to be unable to detect foetal
distress, meconium staining or postpartum haemorrhage.
Care of the neonate: Care is taken to keep the neonate warm. Once the cord
is cut with a razor blade the cord stump is treated with various traditional
remedies to help it dry out quickly. Colostrum is regarded with the same
revulsion as pus. Therefore, babies are only put to the breast after 12-24
hours while the colostrum is expressed and thrown away. During this time
babies are given sugar water. Enemas are also very commonly used in the
first week if the baby does not pass a stool or cries persistently or is restless
with colic. Tachypnoea is treated with traditional medicines. If there is no
improvement the baby is referred to the nearest clinic. Jaundiced and vomiting
neonatal infants are usually referred immediately.
The TBAs were found to be eager to learn and to assist in the better care of
their people. This is similar to what was found with traditional healers as
mentioned earlier.
As with other traditional ' -alth workers there have been suggestions and
attempts to incorporate TBAs into the primary health care system. TBAs
would have to be trained tu identify high-risk mothers and to refer these
mothers to adequately staffed and equipped health facilities. This would
reduce birth-related injuries or illnesses and improve these mothers’ chances
of survival. By TBAs encouraging birth spacing, breast feeding, sanitary
practices and appropriate weaning and feeding practices, infant deaths can be
reduced (Sindiga 1995).
Wollast et at. (1993) reported the results of giving 280 TBAs such training.
The TBAs were given a one month training course, during which they
18
attended about 20 deliveries in prenatal clinics, followed by a two week
refresher course in the second year. The training focused on sanitary
practices, simple obstetrical manipulations, and recognizing the criteria for
referral and evacuation in the course of the pregnancy and delivery. The
results showed that the training was successful, and the traditional birth
attendants were able to recognise high-risk patients who they did refer to
clinics. Evacuation criteria were also heeded. However, the training focused
on antenatal care neglecting postpartum care. This led to a high maternal
mortality (27/6129) which occurred postpartum, and was thought to have been
caused mainly by delayed evacuation and/or the referral centers being
insufficiently equipped to receive these cases. Even though the program was
a success, they concluded that the training courses needed to be carefully
constructed.
1.3.11. Adverse effects of pregnancy-related traditional remedies

Anecdotal observations on the effects of traditional remedies used to induce or
augment labour (Baragwanath Nursing Sisters, Pers. Comm.) indicate that the
risks are similar to the risks of labour induction using oxytocin. These risks
include foetal distress, cephaiopelvic disproportion, uterine hyperstimulation and
low Apgar scores (Liston & Campbell 1974; Knutzen et al. 1977; Davies et at.
1973). Frequent contractions of the uterus in response to oxytocin stimulation
can interfere with the uterine blood flow and so produce distress in the foetus
(Liston & Campbell 1974). Labour induction using oxytocin has been linked to
increased risk of neonatal jaundice (Davies et al. 1973), which has also been
shown to occur with the use of Chinese herbal remedies during pregnancy. Two
commonly used Chinese herbal medicines, Coptis chinsnsus ("chuen-lin") and
Artemisia scoparia ("yin-chen”) have been shown tc displace bilirubin from
serum proteins and this increases the risk of hyperbilirubinaemia (Chan 1994).
Oxytocin has little to no effect on the nondilated uneffaced cervix and does not
alter cervical ripening. Stimulation of myometrial activity while the cervix is
undilated causes’ increased intrauterine pressure increasing stress on the
foetus, while simultaneously decreasing cervical compliance (Olah et al. 1993).
It is assumed that certain herbal oxytocics may have the potential to cause
19
similar effects. In extreme cases the increased uterine pressure may cause
rupturing of the uterus. A study on maternal deaths occurring in all ethnic groups
throughout South Africa between 1980 and 1982 revealed that 2.3% were
caused by uterine rupture. Of the overall maternal deaths, 96% of these
foetuses died (Boes 1987a & b). In the central region of Malawi in 1975, 105
cases of uterus ruptures were reported of which 20 were reported fatal. It is not
known whether herbal remedies contributed towards these figures or not. The
use of herbal remedies was implicated in the rupturing of some uteri in
Cameroon (Nasah & Drouin 1978).
Deaths from acute renal failure associated with the use of herbal medicine have
been reported in Zambian pregnant women (Loventhal et al. 1974). Accidental
poisonings with herbal remedies caused 15/109 maternal deaths in Malawi
(Bullough 1981). Fatal herbal poisonings are commonly caused by hepatorenal
failure(Huxtable 1990; Pillans 1994; Bodenstein 1977; Wainwright e/a/. 1977).
Fatal oesophageal strictures in Nigeria have also been associated with the use
of herbal remedies (Mbura et al. 1985). Certain childhood disorders such as
congenital malformations, malnutrition and tumours were also thought to be
caused by toxic or carcinogenic constituents of herbal remedies taken during
pregnancy (Shoental 1972).
The use of isihlambezo during pregnancy has been associated with premature
delivery and growth retarded babies. However, a small sample size was used
in this study which prevented any conclusions being made (Morris & Mdlalose,
1991). The ingestion of large quantities of imbelekisane was implicated in
several cases of unexplained foetal death resulting from premature labour
(Broster 1981). Mitri et ah (1987) found that women who had recently taken
isihlambezo had an increased incidence of foetal meconium passage, which in
turn was correlated to a significant increase in the incidence of caesarean
section and decreased Apgar scores. An increased incidence of meconium
passage in foetuses of drug-dependent mothers has been ascribed to the
intestinal hyperperistalsis effect of drug withdrawal (Ostrea et al. 1982). The
pathophysiology of meconium passage may reflect: i) a response to foetal
hypoxemia, which could cause contractions in foetal gut, peristalsis and anal
20
sphincter relaxation; ii) an increased vagal activity from in utero stresses (eg.
cord compression) without concomitant hypoxemia; iii) a normal physiological
activity related to foetal gut maturity; or iv) a combination of the above (Houlihan
& Knuppel *994). Bofh i) and ii) are caused by uterine hypertonia, which has
been shown to be ,uced in vitro by the ingestion of imbelekisane infusions
(Larsen et ai. 1W3) and the components of pregnancy-related remedies
Triumfetta rhomboidea, Gunners perpensa, Clivia miniata, Pentanisia
prunelloides and Agapanthus africanus (Larsen et al. 1983; Veale et at. 1989;
Osore 1982, Kaido et al. 1997). The latter threes plant infusions also stimulate
ileal contractions (Veale et al. 1989; Kaido et al. 1997). Provided that the
active constituent(s) of these plant extracts pass into foetal circulation, the
possibility exists that these plants may increase the incidence of meconium
staining through increased intestinal peristalsis caused by direct stimulation of
intestinal muscle.
Despite the potential hazardous effects of isihlambezo and imbelekisane,

together with their wide usage, there is considerable lack of research in this
area. Note that no plant species were identified in any of the clinical studies
represented above. Even though some pharmacological studies have been
done, very few active ingredients of the plants utilised have been identified, and
the pharmacological actions of most remedies are generally unknown. Since the
molecular size and lypophilicity of the components are unknown, it is
impossible to postulate which of the compounds are absorbed, what their
volumes of distribution would be, or which are likely to cross the placenta and
enter foetal circulation.
Even though there is this risk of teratogenicity, the effects of herbal remedies on
the uterus and labour dominate the literature, and will be the focus of this thesis.
1.3.12. Westerners' approach to pregnancy

In western civilization, despite the advancement of medical science,
pregnancy is shrouded by "wives' tales" and traditions passed down from
previous generations. From personal experience, the community treats a
pregnant woman with far more concern and interest than they treat a non
21
pregnant woman. Throughout her pregnancy she is told about traditional
practices on how to determine whether she is carrying a Lay or a girl, how to
overcome nausea or discomfort, how to ensure ti 3 well-being of the baby, or
is told about a remedy or practice which will ease labour. Once the child is
born, more emphasis is placed on orthodox medical treatment of the baby.
Medical training does not immunise one against irrationalism. The magical
and religious handling of sickness for centuries has not vanished in the west,
in the presence of sickness, suffering, crippling and death, religion must
speak, often in the form of alternative or complementary medicine (Levin
1996). With the renewed interest in traditional or alternative health care in the
West has come an increase in the formalised use of herbal remedies during
pregnancy. The Journal of Nurse-Midwifery provides recipes for the preparation
of herbal remedies for use during pregnancy, giving indications very similar to
those of isihlambezo. They are intended to enhance maternal well-being and to
ease labour (Bunce 1987; Ehudin-Pagano et a!. 1987). Books such as Roberts
(1986) are also written for the general public specifically on the use of herbs
during pregnancy.
Herbs used in western herbalism considered to be unsafe in the first two

trimesters because of abortifacient action, but are used during the third
trimester, especially from week 36 onwards to increase uterine tone and to
regulate contractions at birth include (Grieve 1971; Mills 1988; McIntyre 1994):
Acorus calamus (Calamus) Foeniculum vulgare (Fennel)
Alchemilla vulgaris (Lady's mantle) Glycyrrhiza glabra (Licorice)
Artemisia spp. (Wormwood) Hydrastis canadensis (Goldenseal)
Capsicum frutescens (Cayenne) Linum usitatissimum (Flax seed)
Cassia acutifolia (Senna) Mentha pulegium (Pennyroyal)
Caulophyllum thalictroides (Blue Mitchella repens (Partridge Berry)
Cohosh) Passiflora incarnata (Passion Flower)
Chrysanthemum parthenium Phytolacca arnericana (Poke root)
(Feverfew) Podophyllum peltatum (American
Cimicifuga racemosa (Biack Cohosh) Mandrake, Mayapple)
Dryopteris filix-mas (Male fern) Prunus serotina (Wild Cherry)
22
Rheum spp. (Rhubarb) Thuja occidentalis (Thuja)
Rubus ideeus (Red Raspberry leaf) Thymus vulgaris (Thyme)
Salvia officinalis (Sage) Vinca rosea (Periwinkle)
Sanguinaria canadensis (Bloodroot) Viscum album (Mistletoe)
Tanacetum vulgare (Tansy)
No clinical trials on these plants in relation to labour have been reported in the
literature. A case report by Jones & Lawson (1998) showed that maternal
consumption of Caulophyllum thalictroides was implicated as the cause of
acute nonfatal neonatal myocardial infarction associated with profound
congestive heart failure and shock. The authors confirmed that C. thalictroides
was the causative agent using laboratory assays of the vasoactive glycoside
extracted from the plant, which was shown to have toxic effects on the
myocardium in the laboratory animals.
1.4. Herbal Medicine
1.4.1. Secondary plant metabolites

Secondary metabolites are thought to be the compounds possessing
pharmacological activity within the plant. Even though they have been the
subject of investigation for most of the past century, there is no clear definition
of the term secondary metabolite or for their role within plants. It is still difficult
to separate primary metabolites from secondary metabolites especially when
considering several plant honnones and storage compounds. (Mothes 1980).
The middle of this century saw a tremendous increase in the interest in
secondary metabolites as the pharmaceutical industry became interested in
secondary metabolites as a source of compounds in their search for
antibiotics. This was initiated by the discovery of penicillin and by the
investigation of cancer-inhibiting substances. The search led to the discovery
and structure elucidation of nearly 800 alkaloids of the tryptamine-
monoterpene type from the Gentianales alone (Mothes 1980). However, these
23
screening programs led to disappointment, despite impressive
pharmacological histories of certain groups of compounds (Gordon 1994).
Today the focus of drug discovery is on chemically synthesising compounds
and on the genetic basis of many diseases.
Plants synthesise a greater array of secondary compounds than animals.

Insects and molluscs produce more secondary metabolites than higher
animals but this is still much lower than that found in plants. The reason for
the greater array of secondary compounds in plants (as opposed to animals)
is thought to be the inability of plants to move and their lack of excretory
organs. The first theory leads to the supposition that secondary metabolites
confer an ecological advantage to the plant, such as defence from attack from
micro-organisms and/or animals. This is related to the irritant, toxic or
unpalatable characteristics of several secondary metabolites (Bell 1980). For
example, physiologically active compounds such as histamine, acetylcholine
and 5-hydroxytryptamine are present in the stinging cells of the European
stinging nettle (Urtica dioica). Another possible role of secondary metabolites
is that they may increase the ability of one plant species to compete with
another species in a given environment (Bell 1980). For these reasons, there
has been speculation that plants under stress, both from unfavourable
environmental conditions and herbivores, will produce more secondary
metabolites than unstressed plants.
Apart from a source of pharmaceutical compounds, secondary metabolites

are also useful as phylogenetic tools. The discipline of chemotaxonomy was
developed in the 1960's and is still being used to clarify the taxonomy of
plants. Since chemical compounds are genetically coded only certain species
or even races of a plant species synthesise specific secondary metabolites.
Secondary metabolites tend to vary qualitatively between taxa whereas
primary metabolites vary quantitatively and are subject to both environmental
and genetic control (Bell 1980).
24
1.5. Shortcomings of Traditional Herbal Medicine
1.5.1. Lack of evidence
Alternative medicine as a whole has received much criticism from the western
medical fraternity for their lack of hard evidence on the efficacy of their remedies
and medical practices. (Pantowitz 1996, Levin 1996). It is this lack of evidence
which drives the wedge between orthodox medical practitioners and alternative
or traditional medicines (Richards 1996). Alternative medicine relies on
anecdotes and theories. On the whole, claims and anecdotes about alternative
therapies are published in books and magazines for the general public rather
than peer-reviewed scientific journals (Angell & Kassirer 1998).
Modern practitioners are not always justified > their accusations against
alternative therapies, especially in the case of herbal medicines, as many clinical
trials have proven the efficacy of herbal remedies. Orthodox physicians however
are unaware of these studies (Biumenthal 1998).
Until the 20th century, most remedies worldwide were botanicals. A few were
found through trial and error to be helpful. Therapeutic successes with
botanicals came at great human cost. The indications for using given botanicals
were ill defined, dosage was arbitrary because the concentrations of the active
ingredients were unknown, and all manner of contaminants were often present.
Many of the remedies simply did not work, and some were harmful or even
deadly (Angell & Kassirer 1998). This is no different to the stage at which
traditional herbal medicine in South Africa is today. Unfortunately, no clinical
studies have been done on South African herbals to determine which species
are toxic, or which are effective. We only have case reports on the adverse
effects of certain species.
1.5.2. Availability and trade of medicinal plant material

In urban areas such as Johannesburg, there are very few areas where
medicinal plants can be harvested. Even though considerable work is being
done to encourage traditional healers to grow their own medicinal plants
25
(Mander etal. 1998), it is not yet the custom of traditional healers to grow their
own plants. There are myths such as the planting of a plant which has been
collected from the wild next to one’s house attracts lightning to strike the
house.
Traditional healers within and around Johannesburg rely on periodic visits to

rural areas to harvest plant material, or plant collectors harvest traditionally
used plants from all around South Africa and its neighbouring countries and
transport them to the cities. The collectors then sell the plant material to herb
vendors in urban Herbal Markets who in turn sell the plants to traditional
healers or the end user (Cunningham 1988). It is very seldom that the end
user of the traditional remedy knows where the plant grew or how long the
plant has been stored (Healers, Pers, comm).
1.5.3. Standardisation of herbal products

A problem facing the herbal industry world-wide is that the potency of different
plants of the same species may be highly variable, as the concentration of the
pharmacologically active principles in plants varies according to the growth
environment, the part used, time of harvesting, conditions and length of storage
(Huxtabie 1990). In the United States, analyses of ginseng products showed
that the concentration of the active ingredient in each pill varied by as much as a
factor of 10 among brands that were labelled as containing the same amount.
Some brands contained none at all (Angell & Kassirer 1998). Germany,
Europe’s leading importer of herbal medicinal products has prepared
monographs defining quality standards and potency tests for over 350 single
plant drugs to monitor these herbal products. Known as the Commission E
monographs, they include descriptions of uses, contraindications, side effects
and dosages (Taylor 1996). Herbal products produced by reputable herbal
companies undergo rigorous processes of standardisation (Mills 1999), based
on criteria stipulated in the Commission E.
26
However, in Africa and other developing countries herbal products are seldom
standardised, since herb vendors in urban areas sell plants from a wide variety
of localities. This causes a problem as the plants will vary genetically and
phenotypically and traditional healers have no way of determining the
concentration of the active ingredients with the plants they dispense. This poses
a risk of accidentally overdosing patients. In rural areas, traditional healers
usually harvest their plants from the same location. Therefore, they are not
exposed to as high a risk of variation in the potency and efficacy of the plants as
there will be an inherent form of standardisation in the growth environment or
race of the plant. However, the plant constituents may vary qualitatively and/or
quantitatively overtime as the seasons change.
1.5.4. Identification and contamination of plant material

Another problem with herbal medicine is the misidentification of plant material.
The possibility exists of a pharmacologically inert plant being substituted by a
highly toxic plant (Huxtable 1990). For example, the confusion between comfrey
and digitalis has been documented (Roulet ef a/. 1988). In South African urban
areas this is highly possible as collectors sell plant material to traditional healers
without diagnostic leaves or flowers, or the material is chopped or ground before
being sold. This makes it difficult for healers to verify the identity of plant
material, especially to a species level.
There have been warnings that herbal products sold in western countries
containing Plantain have been contaminated with Digitalis lanata cardiac
glycosides (Slifman et at. 1998). Lead poisoning has also been reported after
ingestion of Indian diabetic herbal remedy (Beige! et al, 1998). Similar
contaminations are highly possible in the unregulated and largely primitive plant
markets throughout South Africa.
1.5.5. Temporal variation in constituents of medicinal plants

Seasonal variation in chemical constituents and biological activity of plants is
a well documented phenomenon (Bos et al. 1998; Dolling et al. 1994;
Brackenbury & Appleton 1997). The levels of solaneso! and castaprenols in
horsechestnut leaves (Aescuius hippocastrum) are 5 and 80 times greater in
27
October than they are in April (Threlfall 1980). The expression r f genes
coding for chemical constituents of plants are under the control of various
factors, such as the developmental stage of the organism, duration and
intensity of light, nutrient supply, triggering of internal signals and sequential
expression of genes coding for secondary metabolites (Luckner 1980). All the
above factors vary temporally which in turn cause seasonal fluctuations in the
levels of both primary and secondary metabolites within plant tissues.
1.5.6. Spatial variation

As with the temporal variation in levels of chemical constituents within plants,
factors affecting the nature and quality of secondary metabolites can be
affected by temperature, rainfall, aspect, length of day, light quality and
altitude (Evans 1996). These factors all vary spatially, whether it be on a micro
or macro level.
Soil types also vary considerably from one location to another. The different
particle sizes of the soils impact on the water content of the soils (Raven et at.
1986). The neighbouring plant species composition also vary which impact on
the competitive impact on the plant and on the amount and composition of the
micro- and macronutrients recycled to the soil. The pH of the soils also differ
which impacts on the availability of the various nutrient elements within the
soil (Taiz & Zeiger 1991). The microorganisms, (bacteria, fungi and protozoa)
also differ both in species composition and number. All these factors alter the
water and nutrient availability to the plant in a certain locality and therefore
a,’ter the chemical composition of the individual plant. This in turn alters the
eoncentraiio/is of primary and secondary metabolites within the plants to an
extent which is dependant on the homeo?*3tic mechanisms of the specific
synthetic pathway of the relevant chemical compound within the plant.
Different populations of a particular plant species may differ genetically. This

has been shown in the Tea Tree oil plants. The plant populations in and
around Bailina (New South Wales, Australia) were shown to produce tea tree
oil of a higher quality and quantity than anywhere else in Australia. These
28
differences are caused by genetic differences in the different chemical races
of the plant (Leach and Bell 1999).
1.5.7. Stability of plant constituents

The marketing system of African medicinal plants in urban areas requires the
healers to store plant material for considerable lengths of time under
uncontrolled conditions. Once harvested, the plant material is subject to
bacterial and fungal degradation. For practical reasons linked to the expense
of packaging, plant material sold in South African herbal markets is normally
sold as whole plant parts, and is not ground before selling. Therefore, the
water content of the material is relatively high increasing the possibility that
the plant material is colonised by microorganisms. The plant material is not
only prone to being degraded by the microbe, but the possibility also exists of
the microbe excreting pharmacologically active exudates into the plant
material. The conditions in which the plant material is stored is also not
conducive to protecting the stability of the chemical constituents of the plant.
Plant material was often seen stored in direct sunlight, or close to roadsides.
Not only do the warm conditions, particularly in the humid environment of
Kwazuiu-Natal, favour the growth of bacteria and fungi, but the storage of
plants on roadsides, increases the possibility that the plant material is
contaminated with heavy metals, such as lead, or other pollutants from the
road.
1.6. Rhoicissus tridentata

1.6.1. Classification
Rhoicissus tridentata is a polymorphic species that has undergone numerous
nomenclature revisions over the last two centuries. Originally the species was
divided and classified as a number of species in the genus Rhus. The species
were later reclassified in the genus Cissus. Rhoicissus was classified as a
separate genus by Planchon (1887), but again there was confusion on the
delineation between species. Later Wild and Drummond (1963) combined
three different species, R. cuneifolia, R. erythrodes and R. cirrhiflora and
classified them as R. tridentata. Their work only dealt with species collected
north of the Zambezi river and thus did not consider species such as
29
R. paucifiora and R. dimidiata. Urton et al. (1986) classified all the above
mentioned Rhoicissus species as Rhoicissus tridentata, but divided the
species into two subspecies, being subspecies tridentata and subspecies
cuneifolia. The subspecies are divided according to their leaflet margins. If the
leaflets have no dentations or crenations on the leaflet margins, or if the
number of dentations or crenations is four or less they are classified as
subspecies tridentata. If the leaflets have more than four dentations or
crenations they are classified as subspecies cuneifolia (Figure 1.1 a & b). The
subspecies cuneifolia is more prevalent having a wider distribution, extending
from the eastern Cape in the south to the Northern limits of South Africa,
where subspecies tridentata is limited from Riversdale in the west to Port St
Johns in the east and extending inland into the Karoo (Figure 1.2 a & b).
4 0 mm
40mm
Figure 1.1. Comparison of leaf shape between R. tridentata subspecies a)

tridentata and b) cuneifolia (Urton et al. 1986).
30
W|
tr
29* ur 32*
Figure 1.2. Distribution of a) Rhoicissus tridentata subsp. cuneirolia and b) R.

tridentata subsp. tridentata (Urton et a/. 1986).
1.6.2. Characteristics of Rhoicissus tridentata subsp cuneifolla

Rhoicissus tridentata (L.f.) Wild & Drum, subsp cuneifolia (Eckl & Zeyh). N.R.
Urton, is a deciduous shrubby creeper in the family Vitaceae, commonly
known as wild grape (Smith 1966) or isiNwazi (Zulu) (Gerstner 1941). It has
branches spreading outwards from a thick woody base. The leaves are
trifoliate with wedge-shaped leaflets, each having a serrated margin. The plant
bears small inconspicuous green flowers followed by small grape-like bunches
of small brownish red berries. Lignotubers ranging in size from 5 to 30 cm in
diameter are attached to the roots. If damaged the tubers reveal a blood red
colour beneath the brown epidermis (Urton et al. 1986; van Wyk 1997).
The overall form of the plant varies according to its habitat. If growing in forests,
it forms a woody climber of up to 10m or more. In high bush it scrambles freely
31
over other plants. In open grassveld it occurs as an erect shrub of up to 2m or
more (Urton etal. 1986).
1.6.3. Zulu usage and preparation of remedies containing Rhoicissus

Decoctions and infusions of Rhoicissus tridentata roots or lignotubers are
taken orally or as enemas in many South African traditional herbal remedies.
Varga & Veale (1997) found that Rhoicissus tridentata is commonly used by
Zulu women in pregnancy related remedies in the Durban region (Kwazulu-
Natal). Decoctions are also used as enemas to relieve painful menstruation or
taken orally to treat impotence and infertility (Watt & Breyer-Brandwijk 1962).
The roots or tubers a;e used for stomach ailments, kidney and bladder
complaints (Bryant 1966; Pujol 1990; Hutchings 1996). The tubers are also
used for cattle diseases (Pujol 1990). The usage of Rhoicissus spp. by other
African tribal groups or countries is given in Table 1.1.
The focus of this thesis is on the use of R. tridentata subsp. cuneifolia during
pregnancy
1.6.4. Scientific findings on R. revoilii and R. tridentata

R. revoilii has been found to display antifungal activity inhibiting trichophyton,
mentagrophytes and Candida albicans (Sawhney 1978b), but was found to be
inactive against the bacteria Neisseria gonorrhoea (Sawhney 1978a). R.
tridentata harvested in Rwanda was found to lack in vitro acaricidal activity
against the insects Appendiculatus and Rhipicephalus (Van Puyvelde 1985).
1.6.5. Toxicity of R. tridentata

R. tridentata has I w i reported to be toxic, and has been implicated in a
number of fatalities causad by CNS depression followed by respiratory failure
(Watt & Breyer-Brandwijk 1962; Brandt & Muller 1995). Deaths of swine from
ingestion of tubers have been reported in Tanzania and the toxicity of the
tubers has been confirmed in rabbits (Hutchings 1996). No differentiation
between the two subspecies were made in these reports.
32
1.1. Medicinal usage of the Rhoicissus genus by other African tribal groups. Indications related to reproduction are
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1.6.6. Chemistry of R. tridentata and R. revoilii
Chhabra et al. (1984) found R. tridentata leaves to contain anthocyanins,
coumarins, essential oils, flavanoids, saponins, sterols and triterpenes.
Whereas R. revoilii root bark was found to contain anthocyanins, coumarins,
flavanoids, quinones, sterols and tannins.
1.6.7. Identification of pharmacologically active components

Isolation of the uterotonic components of R. tridentata subsp. cuneifolia has
been done by Bridget Brookes (Chemistry Department, Mangosuthu
Technikon, Durban) and myself. B. Brookes fractionated extracts from the
plant, and the pharmacological activity of the fractions were tested by myself.
Currently, the active fractions are being characterised and the results will form
part of her PhD thesis.
Preliminary findings suggest that (3-sitosterol (24p-ethylcholest-5-en-3|3-ol), p-

sitosterol-3-O-jB-D-gycoside, imberbic acid (1-a,3-p-dihydroxyolean-12-en-29-
oic acid) as well as an unidentified sugar appear to account for most of the
contractile activity. The structures are given below (Figure 1.3).
.coo
OH
HO
0-sitosterol
(24p-ethylcholest-5-en-3p-ol)
imberbic acid
(1-a,3-p-dihydroxyolean-12-en-29-oic acid)
on
OH
P-sitosterol-3-O-p-D-glycoside
Figure 1.3. Compounds isolated from Rhoicissus tridentata subsp.

cuneifolia.
35
1.7. Physiology of Labour
Labour is the physiological process by which a foetus is expelled from the
uterus to the outside world. Labour is divided into three phases according to
uterine activity. Phase 0 is the quiescent stage during which there is minimal
uterine activity, mainly mild uncoordinated Braxten-Hicks contractions.
Activation of uterine activity occurs during Phase 1 causing a linear increase
in uterine activity, followed by an exponential increase in uterine activity during
phase 2. The third phase is characterised by a decrease in uterine activity,
during which time the placenta is discharged and involution of the uterus takes
place. Each stage of labour is subject to different set of chemical controls
(Figure 1.4).
£L
Phase 0 Phase 1 Phase 2 Phase 3

Quiescence Activation Stimulation involution
Progesterone Oestrogen Prostaglandins Oxytocin
Prostacyclin Prostaglandins Oxytocin Thrombin ?
Relaxin CRH
Nitric oxide Progesterone ?
PHRH
CRH ?; HPL?
Figure 1.4. Regulation of uterine activity during pregnancy and labour.

Question marks indicate a possible influence. PHRH = Parathyroid hormone-
related hormone; CRH = Corticotropin releasing hormone; HPL = human placental
lactogen (Norwitz et al. 1999).
36
1.7.1. Myometrial contraction
Uterine smooth muscle shows a high degree of spontaneous contractility which
increases greatly at puberty and varies thereafter with the ovulatory cycle, stage
of gestation, degree of stretch and region of the uterus. Waves of depolarization
of the myometrial cell membrane, with superimposed spike activity are
associated with contraction. Cell-to-cell spread of excitation occurs, but electrical
conduction is slow and decremental in nature. Low-resistant contacts (gap
junctions) greatly facilitate the spread of excitation (Graves 1995).
The uterine contraction is initiated by either neurotransmitter or hormonal

stimulation resulting in the influx of Na+, depolarisation of the membrane and an
action potential. The action potential leads to a rise in intracellular calcium
which in turn causes the actin and myosin filaments to interact by means of an
ATP-dependent mechanism resulting in myometrial contraction (Steer &
Johnson 1998), In smooth muscle, contraction results from the relatively slow
process of phosphorylation of light chains of myosin, a reaction that is catalysed
by myosin light-chain kinase, a calcium and calmodulin-dependant enzyme
(Graves 1995).
Oxytocin and prostaglandins Eg and F%, are the most important compounds
controlling contractions during laboui. Both prostaglandins and oxytocin
stimulate contractions by activating excitatory membrane bound receptors. The
two compounds interact in a complementary and sometimes synergistic manner
(Graves 1995).
The uterus is also innervated with both parasympathetic and sympathetic

neurones. The former by way of the pelvic nerve and the latter by way of the
postganglionic fibres from the inferior mesenteric ganglia. Both can elicit
increased activity in the mature human uterus, but denervation causes little
change in uterine motor activity. Adrenergic innervation includes both excitatory
a-receptors and inhibitory (preceptors (Bulbring & Tomita 1987). The relative
dominance of these receptors is altered by hormonal status (Steer & Johnson
1998). Muscarinic receptors are also expressed on the surface of myometrial
cells which when stimulated cause contraction of myometrial cells (Ehlert et al.
37
1997). Other receptors mediating myometrial or other smooth muscle
contractions include oxytocic, prostanoid, serotonergic and histaminergic
receptors (Biattner et al. 1978; Graves 1995; Steer & Johnson 1998) (Figure
1.5).
receptor
Figure 1.5. Pharmacology of the smooth muscle cell.
38
1.7.2. Oxytocin
Oxytocin is a neurohypophyseal hormone. Oxytocin is synthesised in the
supraoptic and paraventricular nuclei of the hypothalamus, and is stored in
nerve endings in the neurohypophysis (Graves 1995). Oxytocin is released from
the posterior pituitary in a pulsatile manner. The pulse frequency increases as
labour advances (Fuchs et al. 1991). Oxytocin release is stimulated by reflex
sensory stimuli from the cervix, vagina and breasts, and is inhibited by ovarian
relaxin. The secretion of oxytocin is also suppressed by ethanol which is the
basis for the use of ethanol as a tocolytic agent in the past (Graves 1995). Other
possible sources of oxytocin are the foetus and the placenta itself. However,
whether or not oxytocin is released from these sites in amounts sufficient to yield
a contractile response is still uncertain (Bossmar 1998).
The sensitivity of the uterus to oxytocin is dependent on both gestational age

and the level of spontaneous uterine activity (Caldeyro-Barcia & Peiros 1959,
Theobald et al. 1969). In early to mid-pregnancy the uterus is insensitive to
oxytocin, but from week 30 of gestation onwards the sensitivity to oxytocin
increases markedly until week 40 when the uterus displays the maximal levels
of sensitivity to the hormone (Seitchik & Castillo 1983). The increased sensitivity
of the myometrium and decidua to oxytocin is caused by an increase in the
number of oxytocin receptors (Forsling 1979). Prolonged exposure to oxytocin
causes a decrease in the myometrial content of oxytocin receptors, and a
subsequent decrease in the uterine sensitivity to the peptide (Phaneuf et al.
1994). This correlates with the clinical observations that oxytocin is a less potent
stimulant in later stages of delivery than at the beginning of labour (Bossmar et
al. 1994). Oxytocin is present in both maternal and foetal circulation during late
gestation (Forsling 1979). However, parturition may occur in the absence of
oxytocin, although labour may be prolonged under these circumstances. In
addition, mechanical stimulation of the foetal membranes or cervix can induce
labour with little change in plasma levels of oxytocin. While maternal oxytocin
may not be the sole trigger for the onset of labour, it can be considered to play a
vital facilitatory role in parturition (Graves 1995).
39
1.7.3. Prostaglandins and myometrial contraction
Prostaglandins are autacoids derived from membrane phospholipids (Figure
1.5). They can be considered to be local hormones since, with a few exceptions,
they exert their effects and are inactivated in the tissues or organs in which they
are synthesised. Prostaglandins are extremely prevalent and have been
detected in almost every tissue and body fluid. Their production increases in
response to diverse stimuli and they produce a wide array of effects on the
different biological systems. With regard to myometrial contractility and
parturition, prostaglandins E2 and F2a are considered to be the important
prostaglandins (Graves 1995).
Membrane phospholipids
Diacylglycero!
Arachidonic acid
12-HPETE Prostag andin Gz
1
12-HETE v
Prostaglandin Hz
13,14-dihydro-
iS-keto-PGEz
13,14-dihydro-
15-keto-PGF2a
Figure 1.6. Metabolic pathway of phospholipids in the cascade of
prostaglandin synthesis (1 = reductase, 2 = isomerase, 3 = oxidation).
40
The specific activity of phospholipases that catalyse the rate limiting step in the
formation of prostaglandins increases in human amnion late in gestation. The
foetal membrane also possesses large amounts of cyclooxygenase and
phospholipids that contain arachidonic acid (Figure 1.5). The level of PGE2 and
P G F 2a increases in the maternal uterine vein with the onset of labour which has
lead to the hypothesis that prostaglandins are involved in the onset of labour
(Davidson et al. 1987). It is not certain whether the increased levels of
prostaglandins during labour is a major determinant in the onset of labour, or
whether they only sen/e to sustain contractions that have been initiated by
oxytocin (Cambell & Halushka 1995). The formation of prostaglandins by the
amnion may increase progressively during the later stages of pregnancy as a
result of the accumulation of substances derived from the foetus, especially
platelet activating factor (PAF) (Graves 1995). These increased levels of
prostaglandins may be sufficient to stimulate contractions during labour in the
absence of any change in the number of prostaglandin receptors expressed
(Carsten & Miller 1987). In addition to causing the elaboration of prostaglandins,
PAF can initiate uterine contractility directly. SincePAF-hydrolase in the
maternal plasma declines during the last trimester, it is possible that the
amounts of PAF and prostaglandinsthat reach the myometrium may be
sufficient to initiate labour (Angle & Johnston 1989).
The contractile effects of PGE2 and F2a are mediated through their ability to
increase intracellular calcium. This intracellular increase in calcium is
mediated by both ceil surface and sarcoplasmic reticulum receptors (Kurachi
et al. 1989). Table 1.2 shows the diversity of prostaglandin receptors to which
the above two prostaglandins bind thus affecting smooth muscle tone. The
affinity of the PGE receptor seems to be 10-20 times that of the PGF2areceptor
(Carsten & Miller 1987). Prostaglandins together with oestrogens enhance the
formation of gap junctions between myometrial cells (Garfield & Hayashi 1981;
Mackenzie & Garfield 1985) which appear around the time of onset of labour
(Beyer et al. 1989). Gap junctions connect the interiors of neighbouring cells
providing low resistance connections between cells allowing the rapid
41
propagation of electrical activity throughout the uterus. This enables the
myometrium to contract synchronously (Bleasdale 1990).
Table 1.2 Diversity of prostaglandin (PG) receptors that affect smooth muscle
tone (Campbell & Halushka 1995).
PG RECEPTOR SMOOTH SECOND
PROSTAGLANDIN
SUBTYPE MUSCLE TONE MESSENGER
PGE, PGFgct EPi + IP3/DAG/Ca2+
PGE EP2 - CAMP (T)
PGE ep 3 + CAMP (4-/T)
p g f 2« FP + IP3/DAG/Ca2+
The expression of the various prostaglandin receptors differs between pregnant

on nonpregnant uteri. The expression of relaxant prostaglandin E receptor
subtype two (EP(2)) and contractile prostaglandin F receptor (FP) mRNA in
the rat uterus is changed during various states of pregnancy and is regulated
by steroid hormones, thus, steroid hormones regulate uterine activity during
pregnancy and labour. The expression of EP(2) receptors in the rat uterus is
three times higher during pregnancy than in the nonpregnant state and
decreases during labour at term where it is a third that of the nonpregnant
state. The expression of FP receptors in rat uterus increases three fold during
pregnancy and reaches maximal levels during labour when the expression is
5 times higher than in the nonpregnant uterus (Dong & Yallampalli 2000).
The local instillation of prostaglandins can induce cervical ripening at doses that
do not affect uterine motility. They can also produce softening of the cervix late
in the first trimester of pregnancy, by which time the major structural changes in
the cervical collagen have occurred. This has been shown by administering
intravaginal misoprostol (PGEi analogue) which causes ripening of the cervix
and induces labour at term (Fletcher et al. 1993).
Other metabolites of arachidonic acid are prostacyclin (PGh) and

thromboxane Ag (TXAg). In relation to parturition, PGIg is largely confined to
42
the uterine, umbilical and foetal vasculature, where it may serve to ensure an
adequate flow of blood and a patent ductus arteriosus (Grave 1995). PGh
may have a direct effect on uterine smooth muscle whereas there is no
evidence that TXA2 is involved in myometrial activity. Thromboxane and
prostacyclin have potent platelet aggregatory and antiaggregatory effects
respectively. These two autocoids may be of more importance in the control of
factors, such as haemostasis after delivery of the placenta, than in stimulating
uterine contractility during labour (Vane 1978). Low dose aspirin (50 mg/day)
strongly inhibits maternal TXA2 but not PGh production and thus shifts the
balance between PGI2/TXA2 to the dominance of the vasodilatory, anti
aggregatory side. This is manifested by improved foetal haemodynamic
performance and reduces the need of intensive neonatal care (Viinlkka et al.
1993).
Women who ingest analgesic doses of aspirin (a cycloxygenase inhibitor)

towards the end of pregnancy have pregnancies prolonged by about 7 days
compared to controls (Lewis & Schulman 1973). This prolongation of
pregnancy also occurs with other prostaglandin synthesis inhibitors, such as
indomethacin. However, these drugs should not be used routinely because of
the possible adverse effects, notably postpartum haemorrhage and
intrauterine closure of the ductus arteriosus (Creasy 1987; Insel 1998).
Foetal membranes are involved in the synthesis and metabolism of

prostaglandins in late pregnancy (Schwarz et al. 1975a; Schwarz et al.
1975b). The enzymes involved in degrading prostaglandins are more active in
the chorion than in the amnion (Duchesne et al. 1978). The metabolic rates of
prostaglandins are not different before or after the onset of labour (Keirse et
al. 1976).
Foetal membranes are the source of the primary prostaglandins PGE2 and
PGFza (Keirse et al. 1977). The amnion has the greatest capacity to
synthesize prostaglandins and it increases markedly towards term (Okazaki et
al, 1981). The synthesis of cycloxygenase-2 (COX-2) increases markedly with
43
advancing gestation (Fuchs et a!. 1999) and the onset of labour (Smieja et al.
1993; Slater et al. 1994; Dong et al. 1996), whereas only low concentrations
of cycloxygenase-1 (COX-1) mRNA are present in myometnum at term
(Fuchs et al. 1999). Endogenous oxytocin is a major factor in the induction of
COX-2 expression and PGFza release at term and during parturition1 (Fuchs
et al. 1999). The availability of arachidonic acid in the foetal membranes does
not seem to change with the onset of labour (Aitkin et al. 1990). It is unclear
whether these prostaglandins have access to the myometrium, or whether
they are metabolized as they cross the chorion and amnion (Nackla et al.
1986). It appears that some cells lack prostaglandin dehydrogenase, and thus
allow the transfer of prostaglandins to the myometrium (Chailis et al. 1991).
1.7.4. Prostaglandins as abortifacients

Therapeutic uses of prostaglandins in relation to gestation and parturition
include the use of prostaglandins as abortifacients and cervical ripening agents.
Abortions during the first trimester are usually accomplished by means of
suction curettage or the use of the contraversial drug, RU 486 (mifepristone, a
progesterone antagonist), together with a prostaglandin. The combination
causes an abortion in 99% of the cases. Misoprostol (PGEi analogue) together
with methotrexate (antifolate drug) given in early pregnancy induce abortions in
96% of cases (Hausknecht 1995).
Beyond the first few weeks of the second trimester vaginal suppositories of
dinoprostone (PGE2 analogue) are effective. In other circumstances, such as
following membrane rupturing without the elimination of the uterine contents,
carboprost (15-methyl PGF2«) has been effective (Graves 1995).
1.7.5. Control of the onset of kbour

Labour is defined as a increase in myometrial activity, or more precisely, a
switch in the pattern of myometrial contractility from irregular contractions
(long-lasting, low frequency activity) to regular contractions (high-intensity.
1 Experiments done pn cows, PGE2 concentrations were not measured.
44
high frequency activity), resulting in effacement and dilatation of the uterine
cervix (Nathanielsz et a/. 1997). In normal labour, there seems to be a time-
dependent relation between the biochemical, changes in the connective tissue
in the cervix that usually occur before the spontaneous rupture of the foetal
membranes (Duff et a!. 1984). Considerable evidence suggests that the foetus
controls the timing of labour and thus its birth, but exactly how is still unknown
(Norwitz et a!. 1999).
Throughout pregnancy contraction of the uterus needs to be inhibited to

prevent the detachment of the placenta from the uterine wall. In contrast, at
the onset of labour the uterus needs to become sensitive to contraction
inducing stimuli, .i.e. the uterus needs to undergo numerous biochemical and
physiological changes towards the end of pregnancy.
Labour at term is thought to result from a decreas. in factors which inhibit

myometrial activity throughout pregnancy (Lopez Bernal et a/. 1995). This is
demonstrated by removing quiescent myometrial tissue obtained from a
uterus at term and placing it in an isotonic solution. The tissue will contract
vigorously and spontaneously without added stimuli (Lopez Bernal et al. 1995;
Garrioch 1978). In vivo, however, it is likely that both the removal of inhibitory
influences together with an increase in uterine stimulation are important
(Norwitz etal. 1999).
In sheep, parturition is initiated by a surge of cortisol secretion from the foetal

adrenal cortex, which acts on placental enzymes to activate the biosynthetic
pathway causing progesterone to be converted to oestrogens. Thus, placental
secretion of progesterone falls and oestrogen secretion rises as progesterone
is increasingly metabolized to oestrogens. This rapid change in steroid
hormone ratios stimulates the release of prostaglandins from both the
placenta and the myometrium, and increases the sensitivity of the
myometrium to oxytocin which initiates uterine contractions that become
powerful enough to expel the foetus, provided that softening and dilatation of
the cervix have occurred (Steer & Johnson 1998).
The situation is much less clear in humans. We are still not certain whether
the human foetal adrenal gland plays any role in the initiation of labour.
Experiments suggest that increasing cortisol secretion plays a role in initiating
labour. The exact mechanisms are unclear as the biosynthetic pathways for
oestrogen synthesis in sheep and humans are very different. In sheep
oestrogen synthesis is predominantly a placental event whereas in the human
not only the placenta but also the foetal and maternal adrenal cortexes are
involved (Steer & Johnson 1998).
Oestrogen concentrations in maternal plasma and amniotic fluid increase

during the later stages of pregnancy, especially in the final two or three
weeks. Progesterone concentrations may decrease simultaneously. A
progesterone-binding protein accumulates in foetal membranes that may
further decrease the effective concentrations of progesterone in these
structures. The progressive dominance by oestrogen is thought to be
responsible for the increases in myometrial excitability (owing to increases in
slow Na+ channels and gap junctions), the myometrial sensitivity to oxytocin,
and the capacity to elaborate prostaglandins in the foetal membranes, The
changing ratios of progesterone and oestrogen have also been thought to be
responsible for the ripening of the uterine cervix, which is partly caused by a
progressive decrease in the collagen content. These alterations are thought to
be crucial in preparation for the softening, dilatation, and effacement that
occur during normal labour and delivery (Graves 1995).
1.7.6. Gustavii's hypothesis on the onset of labour

Decidual cells which degenerate in late pregnancy contain lysosomes
containing phospholipase A2, and oestrogens can labilir ' tl , ' .uranec of
46
these lysosomes (Gustavii 1975). It is thought that the rising oestrogen levels
coupled with the falling progesterone concentrations at the end of pregnancy
act as lysosomal labilizers (Gustavii 1975). The released phospholipase Az
acts on the membrane phospholipids causing the release of arachidonic acid,
which in turn gets converted to primary PGE2 and PGFa* (Figure 1.6 ).
This hypothesis also explains the mechanism of midtrimester abortion

following intramniotic injection of hypertonic saline which stimulates the
release of prostaglandins into the amniotic fluid. Missed abortions are also
explained by Gustavii's hypothesis, as the maintained high levels of
progesterone and falling oestrogen levels delays the degeneration of decidual
cells, and thus suppresses prostaglandin synthesis (Gustavii 1975).
lysosomal labilizers
lysosome
phospholipids arachidonic acid
V
prostaglandins
Figure 1.7. A scheme for the involvement of phospholipase containing

lysosomes in the synthesis of prostaglandins.
47
1.7.7. Other physiological effects of prostaglandins
Cyclooxygenase (COX) exists in two isoforms: constitutive COX-1 and
inducible COX-2. These enzymes catalyze the same reaction but differ in
terms of regulation of expression. The constitutive isoform COX-1 is
responsible for the production of PGs involved in prostanoid-mediated
physiological functions such as gastric cytoprotection, maintenance of renal
homeostasis, and maintenance of normal platelet functions. The second
isoform, COX-2, has been demonstrated to be highly expressed in response
to inflammatory or mitogenic stimuli. Thus, it is proposed that COX-2 is
responsible for the production of PGs associated with inflammatory conditions
(Chan etal. 1999). Both COX-1 and COX-2 have been found to be expressed
in the following human tissues: lung, uterus, testis, brain, pancreas, kidney,
liver, thymus, prostate, mammary gland, stomach and small intestine (0 Neill
& Hutchinson 1993).
a) Reproduction. Seminal fluid has very high concentrations of

prostaglandins of which substantial amounts are absorbed by the
vagina following coitus. It is thought that the prostaglandins may play
a role in conception through actions on the cervix, uterine body,
fallopian tubes and semen transport. However, their precise role still
remains obscure (Campbell & Halushka 1995).
b) Menstruation. Menstruation is coupled with disruption of uterine
membranes, the release of arachidonate and the stimulation of
prostaglandin synthesis. Concentrations of prostaglandins in
menstrual fluid are elevated, which are thought to contract uterine and
gastrointestinal smooth muscle and sensitise afferent pain fibres and
thereby contribute towards the symptoms of primary dysmenorrhoea.
This is the reason why cyclooxygenase inhibitors are more effective
relievers of these symptoms than narcotic analgesics (Ylikokala &
Dawood 1978).
c) Vascular and pulmonary smooth muscle. Locally generated PGEz and
PGIz modulate vascular tone. PGIz seems to counteract the effects of
circulating vasoconstrictor autacoids, to maintain blood flow to vital
organs. PGEz and PGIa play a role in the hypotension related to septic
48
shock. PGE2 causes bronchodilation whereas PGF2a, TXAz and
leukotriene C4 cause bronchocontriction. All these autocoids are
released in allergic constriction of the airways (Campbell & Halushka
1995).
d) Kidney. Prostaglandins modulate renal blood flow and may serve to
regulate urine formation by both renovascular and tubular effects. The
elaboration of PGE2 and PGb is increased by factors that reduce
renal blood flow, such as stimulation of sympathetic nerves and
angiotensin.
e) Inflammatory and immune response. Prostaglandins and leukotrienes
are released by a host of mechanical, thermal, chemical, bacterial,
and other insults. They contribute importantly to the genesis of the
signs and symptoms of inflammation. They serve as potent
chemoattractants for polymorphonuclear leukocytes and can promote
exudation of plasma by mobilizing this source of additional
inflammatory mediators. PGE2 and PGI2 do not appear to have a
direct effect on vascular permeability, but they enhance oedema
formation and leukocyte infiltration by promoting blood flow in the
inflamed region. They also potentiate the pain producing activity of
bradykinin and other autacoids (Campbell & Halushka 1995).
1.8. Muscarinic Receptors
1.8.1. The autonomic nervous system

The autonomic nervous system regulates the activities of structures that are
not under voluntary control and that are functions below the level of
consciousness, Thus, respiration, circulation, digestion, body temperature,
metabolism, sweating and the secretions of certain endocrine glands are
regulated in part or entirely by the autonomic nervous system. The
sympathetic and parasympathetic nervous systems have contrasting functions
in regulating the internal environment.
The sympathetic system, and its associated adrenal medulla are not essential
to life in a controlled environment. It is continuously active but the degree of
49
activity varies from moment to moment and from organ to organ. The
sympathetic system acts as a unit during rage and fright when the
sympathetically innervated structures throughout the body are affected
simultaneously. Heart rate is accelerated, blood pressure rises, red blood cells
are poured into circulation from the spleen, blood flow is shifted from the skin
and splanchnic region to the skeletal muscles, blood glucose rises, the
bronchioles and pupils dilate, and the whole system is better prepared for
“fight and flight". Adrenaline and noradrenaline function as the sympathetic
hormone and neurotransmitter, respectively. The adrenergic receptors
mediate the transmission of adrenergic sympathetic impulses.
The parasympathetic system is organised mainly for discrete and localised
discharge and is concerned primarily with conservation of energy and
maintenance of organ function during periods of minimal activity. It slows the
heart rate, lowers the blood pressure, stimulates gastrointestinal movements
and secretions, aids absorption of nutrients, protects the retina from excessive
light, and empties the urinary bladder and rectum. All these effects are
mediated by muscarinic receptors which are activated by the endogenous
neurotransmitter acetylcholine.
Muscarinic receptors maintain a contractile state of smooth muscle of the

ciliary muscle, iris sphincter, bronchi, bladder and gastrointestinal tract, and
cause relaxation of the esophageal sphincter, penile blood vessels and other
organs (Ehlert etal. 1997).
1.8.2. Muscarinic receptors and labour

Even though muscarinic receptors mediate contractions of equal magnitude to
oxytocin (see Figure 4.1a), muscarinic receptors are not thought to play a role
in the onset or progression of labour and are never included in any review of
the physiology or management of labour. The only role they could play in
parturition is the enhancement of Ca2+~induced myometrial contraction,
producing additional contractile force with the available amount of intracellular
calcium (Izumi et al. 1996) which may enhance the expelling forces of
oxytocin and prostaglandins.
50
Muscarinic receptors may play a role in mediating intrauterine emptying of the
foetal rectum, if exogenous muscarinic agonists are administered to the
mother and they pass into either foetal circulation or sufficiently high levels in
the amniotic fluid to enable stimulation of intestinal muscarinic receptors. This
would increase the possibility of intrauterine meconium staining of the liquor.
No experimental or clinical evidence could be found in the literature proving
this.
1.8.3. Subtypes of muscarinic receptors

Traditionally muscarinic receptors were classified primarily on the basis of the
effect and relative potencies of selective agonists and antagonists. The effects
of acetylcholine that are mimicked by the plant alkaloid muscarine, and
selectively antagonised by atropine were termed muscarinic effects and were
found to be mediated by muscarinic receptors. Other effects of acetylcholine
are mimicked by nicotine, but are not antagonised by atropine but rather by
tubocurarine. These were termed nicotinic effects and were found to be
mediated by nicotinic receptors (Ross 1995).
As the diversity and selectivity of drugs increased, it became apparent that

multiple subtypes of receptors exist within this system. Molecular cloning as a
tool has revealed further differences between closely related subtypes of
receptors. The use of pharmacological methods to distinguish one subtype of
muscarinic receptor from another has identified four subtypes (M 1 -M 4 ),
whereas five subtypes can be differentiated using molecular cloning (mi-ms)

(Ehlert etal. 1997).
More than one subtype of muscarinic receptor is expressed in the smooth

muscle of various tissues. Typical locations of receptor subtypes and their
responses are given in table 1.3. Competitive binding studies demonstrated
the abundance of M2 receptors in most smooth muscle. In most smooth
muscle, including guinea pig uterus and longitudinal rat and guinea pig ileum,
M2 receptors account for 80% of the total receptor density (Ehlert et al. 1997).
Hybridization techniques on whole guinea pig ileum detected M2 mRNA, a
51
small amount of Ms mRNA and a trace amount of Mi mRNA (Maeda et al.
1988).
Despite Ms receptors being the dominant population, contractions have been

shown to be mediated predominantly by the Ms receptor population. Ms
muscarinic receptors induce a contractile response through stimulation of
phosphoinositide hydrolysis. In guinea pig ileum, muscarinic receptors inhibit
voltage dependent calcium currents. Muscarinic ms and ms receptor subtypes
were found to be expressed on intestinal smooth muscle. The ms receptors
induce smooth muscle contraction and secretion from exocrine glands
(Bernstein & Haga 1990). The antagonism of Ms receptors causes an
increase in heart rate whereas antagonising Ms had no effect on heart rate
(Lin etal. 1997).
Table 1.3 Typical locations of the various cholinergic receptor subtypes and
the second messengers mediating the cellular response to each subtype.
Receptor name Typical Ideations Result of ligand binding
Muscarinic Mi CNS neurons Formation of IPs and DAG
Sympathetic postganglionic Increased intracellular Ca2+
neurons
Some presynaptic sites
Muscarinic Ms Myocardium Opening K+ channels
Smooth muscle Inhibition of adenylyl cyclase
Some presynaptic sites
Muscarinic Ms Exocrine glands Formation of IPs and DAG
Smooth muscle and Increased intracellular Ca2+
endothelium
Muscarinic M4 Opening K* channels
Inhibition of adenylyl cyclase
Muscarinic Ms
Nicotinic Nn Postganglionic neurones Opening of Na+, K*
Some presynaptic cholinergic channels, depolarization
terminals
52
m a mpLoifm#mpKc
Contraction depolarization
A
# cAMP
Figure 1.8 Contractile mechanisms for muscarinic receptor subtypes in smooth muscle. All the mechanisms
may not exist simultaneously in the same smooth muscle cell, but rather to a varying extent in different cells
depending upon the expression of the various signaling pnteins (Ehlert et al. 1997). The second
messengers for Mi and M4 subtypes are similar to those of M3 and M2 respectively.
Abbreviations: PLC-p = phospholipase C-P; IP3 = inositol-1 ,4,5-frisphosphate; DAG = diacylglycerol;
PKC = protein kinase C; AC = adenylyl cyclase; p = a subtype of the p-adrenergic receptor
family; cAMP = cyclic AMP.
1. M3 receptors stimulate phosphoinositide hydrolysis.

2. In the colon and stomach, muscarinic receptors potentiate the voltage dependent calcium current
through activation of PKC.
3. Muscarinic receptors stimulate a non-selective cation conductance (mainly sodium) that triggers
depolarization and an influx of calcium through voltage dependent calcium channels. This
conductance is likely to be mediated by the M2 receptor and is dependent on calcium to varying
degrees in different types of smooth muscle.
4. M2 muscarinic receptors inhibit adenylyl cyclase activity and inhibit the increase in cAMP elicited
by forskolin, the p-adrenergic receptor and other receptors that signal through Gs.
5. p-adrenergic receptors, forskolin and other agents cause relaxation through cAMP, and the M2
receptor can oppose this relaxation by inhibiting the increase in cAMP.
6. Many types of smooth muscle cells contain calcium-activated potassium channels that act to
restore membrane potential following depolarization and an influx of calcium.
7. p-adrenergic receptors may have an alternaiate, non-cAMP-dependent mechanism for causing
relaxation in the trachea.
8. Muscarinic roceptors suppress the calcium-activated potassium conductance. This conductance
is pertussis toxin-sensitive; consequently, we suggest that it is mediated through the M2 receptor
by signaling through a G protein of the Gi and Go family.
9. In the guinea pig ileum, muscarinic receptors inhibit the voltage dependent calcium current.
53
1.9. A im s
1. To document the ingredients and the various methods of preparations of

pregnancy-related remedies in Gauteng.
South African traditional healers prepare almost all their remedies using tap
water. Pregnancy-related remedies containing Rhoicissus are usually boiled in
water. The preparation of extracts used in this study will all be prepared
mimicking the preparation techniques of the traditional healers as the aim of the
study is to determine the pharmacological activity of the extracts used by local
pregnant women.
2. Pharmacology of crude aqueous decoctions of Rhoicissus tridentata subsp.

cunetfolia.
a. To determine the mechanism of contractile activity of R. tridentata subsp.

cuneifolia decoctions on isolated rat uterus and ileum.
b. To determine whether the plant extracts are cytotoxic on mammary cells,

hepatocytes and fibroblasts.
c. To investigate the effect of the R. tridentata subsp. cuneifolia decoctions

on prostaglandin synthesis in cultured cells.
d. To determine whether the contractile response to R. tridentata subsp.

cuneifolia decoctions exhibits seasonal variation.
e. To determine whether the contractile response to R, tridentata subsp.

cuneifolia decoctions exhibits distributional variation.
f. To determine whether the uterotonic activity of decoctions of different plant

parts of R. tridentata subsp, cuneifolia varies.
g. To determine whether the activity of the extracts is altered by storing dried

R. tridentata subsp. cuneifolia material for a year as is common practice
amongst traditional healers.
54
Rationale for this research
» The pharmacological assays of R. tridentata subsp. cuneifolia will provide

scientific evidence to determine whether the use of R. tridentata subsp.
cuneifolia has the potential to induce or augment labour, and wheth~ ' the
plant has the potential to cause birth complications caused by \ jrina
hypertonia.
• Once the receptor system involved in mediating the contractile response is

identified potential adverse effects of R. tridentata subsp. cuneifolia
remedies may be postulated.
• The results on possible sources of variation in the effect of the R. tridentata

subsp. cuneifolia extracts will indicate whether the plant remedies need to
be standardised and will give an indication of when the plant is the most
potent.
Information gathered will be published in refereed scientific journals and

simplified versions will be published in the “Indigenous Plant Use Forum
newsletter” which is circulated to traditional healers and other interested parties
throughout the country.
55
CHAPTER 2: METHODS
2.1. Interviews
Three traditional healer associations in Johannesburg were consulted. After

much persistence and patience, names of individual healers were obtained
from the presidents of the associations. Healers were interviewed either at
their own homes or consulting rooms, or they were brought to the Medical
School premises where they were interviewed.
Traditional haalers were interviewed using open ended questions to determine

where they were trained, how long they were practising, and what plants they
prescribe 'J r what indications during pregnancy. In order to make our
conversations a two-way flow of information, any questions the healers had
about basic biology were answered and time was taken to inform them about
findings from this study.
Traditional healers used vernacular names (usually Zulu) of the plants. The
corresponding botanical names were determined from the following texts:
Gerstner (1941), Watt & Breyer-Brandwyk (1962), Hutchings (1996), Van Wyk
(1997).
2.2. Harvesting regimens for Rhoicfesus tridentata used for

all studies
2.2.1. Material for investigations into the role of different receptor systems in
mediating the contractile response to R. tridentata decoctions.
R. tridentata subsp. cuneifolia roots were collected from Treasure Beach
Grasslands, Umlazi, Kwazulu-Natal in July and October 1995. The roots were
harvested by Bridget Brookes (a colleague from Mangosuthu Technikon who
worked on the chemistry of Rhoicissus) and identified by Geoff Nichols
(Horticulturist from Durban Municipality). (Voucher s. admen number -
086931, C.E. Moss Herbarium). Details on the location are given in Table 2.1.
56
2.2.2. Material for receptor subtype investigations.
Lignotubers harvested in Autumn (April 1997) from Suikerbosrand Nature
Reserve, 60 km south of Johannesburg, were used for the investigation into
the involvement of the various receptor subtypes in the mediation of the
contractile response to aqueous extracts of R. tndentaia subsp. cuneifolia.
2.2.3. Material to investigate seasonal variation in contractile activity.

R. tridentata subsp. cuneifolia plant material was collected from Suikerbos
Nature Reserve. The plants were collected within a thicket on a North East
facing slope of the grassland. Three plants approximately 10m apart were
harvested every three months from May 1996 to April 1998. Plants which
were sufficiently far apart to be different individuals were chosen to allow a
replicate of plants. The identity of the plants was validated by L. Katsoulis and
voucher specimens were lodged in the C.E. Moss Herbarium. Voucher
specimen numbers 086928, 0B2929 and 082930.
2.2.4. Material ror investigations into the contractile activity of extracts from
different Plant parts.
The same plants harvested for the study of seasonal variation, from Suikerbos
Nature Reserve, were used to determine whether there was any variation in
response to different parts of the plant.
2.2.5. Material to investigate the distributional variation in contractile activity.

Lignotubers are interspersed along the roots which usually grow within
crevices between rocks making them very difficult to locate. Stems, as
opposed to roots or leaves, were shown to have the uterotonic activity most
similar to that of the lignotubers. Therefore, stems were chosen as the plant
part to be Investigated for distributional variation in uterotonic activity.
Plant material was always collected during summer. The plant material was
harvested from around the south-eastern part of South Africa, from a variety
of different habitats (Table 2.1),
57
Table 2.1 Collection details of plants harvested to determine the effect of
distribution on the contractile activity of aqueous extracts.
LOCATION PROVINCE VEGETATION SEASON PART Altitude MAR*
TYPE* (m)+ (mm)+
Suikerbosrand Gauteng Mixed grassveld Autumn stems 1541 698
Nature
Reserve
Mondeor Gauteng Sour grassveld Autumn stems 1541 698
Magaliesburg Gauteng Bushveld gorge Summer stems 1157 659
Mountains
Umlazi Kwazulu- Coast-belt scrub Summer roots 8 1022
Natal forest
Durban Kwazulu- Coast-belt scrub Summer roots 8 1022
Natal forest
Morgenzon Mpumalanga Climax forest in Autumn stems 1525 905.8
Bos sourveld gorge
' Vegetation classification according to Acocks 1988

* Supplied by the South African Weather Bureau
# MAR = mean annual rainfall
2.2.6. Harvesting of material to investigate the effect of storage on the

contractile activity of the decoctions.
Stems and lignotubers of R. tridentata subsp. cuneifolia collected from
Suikerbos Nature Reserve were used to test whether storing the plant
material altered the response to aqueous extracts of the plant.
2.3. R. tridentata subsp. cuneifolia decoction preparation

All plant material harvested was prepared in the same manner. Different plant
parts were separated, washed and dried overnight at approximately 60°C.
Woody material (roots, branches and lignotubers) was cut into small pieces
approximately 1cm3 using an electrical band saw. The material was milled in a
rotomill and sieved through a 1mm mesh. The milled sample was then boiled
in round bottom flasks fitted with a condenser. The sample was boiled at a
58
ratio of about 100g plant material per litre distilled water for an hour mimicking
the traditional healer’s preparation methods. The solids were allowed to settle
in measuring flasks overnight at 4°C. The supernatant was siphoned off,
frozen and lyophilised. Filters were not used as they blocked within a couple
of minutes, whereas the particulate matter precipitated out sufficiently if left to
stand overnight. All lyophilised products were stored at -20°C until use. The
mean percentage yield of lyophilate as a percentage of dry weight was
13.3% ±7.5%.
1 mm
10mm
Figure 2.1. Aerial parts of Rhoicissus tridentata subsp. cuneifolia harvested

from Suikerbosrand.
59
Figure 2.2 Rhoicissus tridentata subsp cuneifolia growing at Suikerbosrand Nature
Reserve, a) overall growth habit b) leaf stucture c) & d) underground parts showing a
small lignotuber.
60
2.4. Animals and isolated organ preparations
Virgin Sprague-Dawiey rats weighing about 250g were oestrogenized by
injection with stilboestroi (10|ag/100g) (Maybaker (SA)) i.p. 24 hours before
being euthanased with CO2. Portions of uterus, approximately 1.5cm long,
were dissected from the central portion of each uterine horn. The uterine
portions were trimmed of fat and connective tissue, opened longitudinally then
mounted in 50 ml organ baths containing Tyrode’s solution (136 mM NaCI,
2.68 mM KCI, 1.80 mM CaCI2, I.OSmM MgCI2, 11.9mM NaHCOs, 0.42 mM
NaHgPCk 5.55mM glucose). Tyrode’s buffers the pH at approximately pH 7.
The baths were aerated with 5% CO2 in O2 and the temperature maintained at
26°C to decrease spontaneous contractility (Kumagai et al. 1952).
Two 1.5cm sections of ileum, approximately 4cm from the ileocaecal junction,
were dissected out of the same rat the uteruv> was obtained from. The tissues
were trimmed of connective tissue, then mounted in the organ baths as for the
uterus. The bath temperature was maintained at 37°C.
Both the organs were allowed to equilibrated suspended in the organ baths
filled with Tyrode solution for at least 30 min, during which time the baths were
rinsed frequently. Isotonic contractions of the organs were measured against
1g resistance and recorded electronically using potentiometer recorders. The
organs were challenged with cumulative doses of reference agonists or herbal
extract, or pretreated 5 min before adding the reference drug with either an
antagonist, the herbal extract, or both, according to Van Rossum (1963).
Standard curves using the relevant reference agonist alone were run at the
start of each experiment and between each test challenge. Repeat
experiments were carried out using tissues from different rats.
The response to each concentration of drug plotted by the potentiometer

recorder was measured using a ruler, then expressed as a percentage of the
maximal response to the reference drug control curve run prior to the test
curve. The graphs were drawn using GraphPad Prism®, and the curves
converged using the variable slope sigmoidal dose-response or Boltzmann
61
sigmoidal equations. Student-t tests were used to analyse all data for
significant differences using Systat®.
2.5. Agonists and antagonists
Table 2.2 Reference drugs used to elucidate the pharmacological action of

the R. tridentata extract on isolated rat uterus and ileum.
Chemical used Pharmacological action Source
acetylcholine chloride muscarinic receptor agonist Sigma
atropine sulphate muscarinic receptor antagonist BDH
pirenzepine 2HCI Mi-receptor antagonist RBI
gallamine triethiodide Mz-receptor antagonist RBI
4-DAMP mustard HCI IVb-receptor antagonist RBI
tropicamide IVU-receptor antagonist RBI
oxytocin (Syntocinon) oxytocin receptor agonist Sandoz
noradrenaline bitartrate adrenoceptor agonist Sigma
propanolol Pa-adrenoceptor antagonist Sigma
prazosin ai-adrenoceptor antagonist RBI
yohimbine cte-adrenoceptor antagonist RBI
serotonin creatinine 5-HT receptor agonist Sigma
sulphate
methysergide maleate 5-HT receptor antagonist RBI
ketanserin 5-HT, receptor antagonist RBI
tropisetron 5-HT2 receptor antagonist RBI
indomethacin cyclooxygenase inhibitor Sigma
NS-398 COX-2 inhibitor RBI
pyrilamine maleate Hi histamine receptor antagonist RBI
hexamethonium ganglion blocker RBI
dichloride
mecamylamine HCI nicotinic antagonist RBI
cholera toxin Ga-protein inhibitor RBI
pertussis toxin islet-activating protein RBI
62
2.6, Analysis of Inorganic Ion Content
The concentration of potassium, sodium and calcium ions of the plant extracts
was determined using ion selective electrodes in a Beckman CX3 automatic
analyzer.
2.7. Prostaglandin Synthesis in Cultured Ceils

Choice of cell type: The results from the organ bath studies indicated that
products of cyclooxygenase, such as prostaglandins, are involved in the
mediation of the smooth muscle contractile response to the R. tridentata
extracts. Cellular studies were done to confirm that the plant extract stimulates
the synthesis of prostaglandin Ez which is one of the two prostaglandins that
cause myometrial contractions.
This investigation could have been done by either exposing the plant extract
to cyclooxygenase itself, to test whether the plant has any direct action on the
enzyme, or by using cells that produce prostaglandins. Cellular studies were
chosen to determine whether the plant extract was able to stimulate cellular
production of prostaglandins, either by penetrating the cells or activating an
extracellular receptor.
Ideally uterine cells should have been used for these studies. Considering this
study of cellular production of prostaglandin Ez was a small adjunct to the
project, together with the high cost of quantifying prostaglandin levels, a
human lung histiocytoma cell line previously shown to produce prostaglandins
was purchased, rather than using a uterine cell line that at that stage had not
been shown to produce prostaglandins.
Molecular studies have shown that COX-2 mRNA expression is higher than
COX-1 mRNA expression during parturition (Dong et al. 1996; Fuchs et at.
1999). Similarly COX-2 mRNA has been found to be overexpressed in human
non-small cell lung cancers (Ochiai et al. 1999). Therefore, human lung
histiocytoma cells (giant cell tumour cells), shown to produce prostaglandin Ez
(ATCC catalogue number TIB-223), were used to determine whether the
63
Rhoicissus extract increases the production of PGEg. As only
radioimmunoassays were available to detect PGFaa levels, and as I was
pregnant at the time, no PGFza levels were measured.
Plant extracts from material harvested from both Suikerbosrand and Durban
displaying low, medium or high levels of contractile activity were used in both
experiments outlined below.
Cells were plated out into 96 well microtitre plates at a concentration of 13 500
cells per well, where they were exposed to the plant extract. The plates were
incubated at 36°C and aerated with carbogen. The concentrations of
prostaglandins in each well were determined by transferring 50p.l cell media to
plates coated with goat anti-mouse IgG against PGEa provided in the “Biotrak
prostaglandin Eg enzymeimmunoassay system" (Code: RPN 222).
Instructions provided were followed to determine the levels of PGEg in each
well (Figure 2.3). Standard curves were used to extrapolate PGE2 levels. The
layouts of the microtitre plates are given in appendix A4.
The first experiment done was to determine the time frame in which
prostaglandins are detectable. Cells were exposed to 1mg/ml plant extract for
5,10,15, 30, 60,120 and 180 minutes.
Once the optimal time span for the experiments was determined dose
response curves were constructed after cells in 96-well microtitre plates were
exposed to 6 concentrations of Rhoicissus extract (O.lpg/ml -1 mg/ml).
An outline of the method used to detect PGEg levels: 100pL assay buffer was
pipetted into the non-specific binding (NSB) wells, and 50pL assay buffer was
pipetted into the zero standard wells. 50pL of each standard (2.5, 5, 10, 20,
40, 80, 160 and 320pg/50pl) and unknown sample was added to the relevant
wells. Diluted antibodies (50p!) were added to all wells except the blank (B)
and NSB wells. Diluted conjugate (50pl) was added to all wells except the
blank. The plate was then covered and incubated at room temperature for 1
64
hour on a microtitre plate shaker. All the wells were then washed four times
with SOOpL washing buffer. Residual wash buffer was removed by blotting the
plate on tissue paper. Enzyme substrate (150uL) was added to all wells. The
plate was covered and shaken on a microtitre plate shaker for 30 minutes at
room temperature. The absorbance of the blue colour that developed was
read at 620nm. A confirmation reading was taken after the reaction was halted
by adding 100pL of 1M sulphuric acid to each well, which converts the colour
to yellow with proportionally similar optical densities. The plates were shaken
for 30 seconds and the absorbance was reread at 450nm within 10 minutes of
adding the sulphuric acid.
Goat Mouse PGE2 - peroxidase TMB

anti anti-
mouse PGE2
Incubate 1 hr Incubate 30 min

< > < >
Figure 2.3. The principle of the prostaglandin E2 enzyme immunoassay. The

wells are precoated with goat anti-mouse immunoglobulin. The wells are then
treated with PGE2 antibodies which bind to the immunoglobulins. Once the
unknown samples are added to the wells either PGE2 or PGE2-peroxidase
binds to the PGE2-antibodies. The wells are then rinsed thoroughly and the
3,3',5,5'tetramethylbenzidine (TMB) is added which is converted into a blue
product by the peroxidase, and can be read on the multiwell scanning
spectrophotometer. That is, the intensity of the colour is inversely proportional
to the amount of PGE2 present in the well.
65
2.8. Cytotoxicity
The toxicity of the Rhoicissus extracts should have been tested doing LD50
tests using either rats or mice. Unfortunately the application to do this was
turned down by the Animal Ethics Screening Committee of the University. A
second resort was to test the effect of the plant extracts on either mammalian
cells or on crustaceans, such as brine shrimps. These latter methods could
not be used to show the respiratory depressing effect the Rhoicissus has
been reported to have (Brandt & Muller 1995).
2.8.1. Cytotoxicity using the MTT assay method.

The effect of the plant extracts on the viability of four mammalian cell lines
was determined using MTT (tetrazolium) assays according to Mosmann
(1983). The assay is based on the principle that colourless tetrazolium rings
are cleaved by various dehydrogenase enzymes in active mitochondria
yeilding coloured lipophilic formazan that can be measured using a scanning
multiwell spectrophotometer. Since only active mitochondria are able to
cleave the tetrazolium salt the reaction only occurs in living, metabolically
active cells and the amount of formazan generated is directly proportional to
the active cell number (Mosmann 1983). Mosmann developed the technique
to avoid any washing steps to cut down on running times. He added
isopropanol to the culture media in the final step of the experiment to dissolve
the formazan crystals. In preliminary assays this was problematic as rigorous
mixing was required and the results were highly variable. Therefore, the
crystals were dissolved with DMSO according to Lopez de Cerrain et al
(1996), where all the media was removed before adding the organic solvent
(DMSO) to dissolve the crystals. This was more time consuming but the
formazan crystals dissolved more consistently. Accordingly, the absorbance
was read at 540nm only. The absorbance at 620nm was not subtracted from
the 540nm reading as unlike Mosmann's method, the growth media had been
removed from the wells, and did not have to be accounted for.
The cytotoxicity of Rhoicissus extracts was determined using four cell lines: a)
Graham's cells (human kidney epithelial cells), b) human hepatocytoma cells.
66
c) human histiocytoma cells and d) mouse leydig cells. Growth media used for
the different cell lines are given in Appendix A.2. Single cell suspensions
were obtained by trypsinization of the confluent cells. The trypsin was
inactivated by adding growth media containing 5% Fetal Calf Serum. The
viable cells were quantified using a haernocytometer after staining a sample of
the cell suspension with trypan blue in saline. Viable cells exclude trypan blue
by means of an energy dependent mechanism, so unstained cells were
presumed to be viable. Cells were only used if the percentage viability was
above 90%. The cell concentration was corrected to 0.15 million cells/ml,
before plating 90pL into each well of 96-well microtitre plates yielding 13 500
cells per well. The cells were then incubated at 37°C and aerated with
carbogen for 24 hours, allowing the cells to stabilize before adding the plant
extracts.
Ultracentrifuged plant extract (10pL) which had been filtered through a 2pm
microfilter was added to six test wells for each concentration of plant extract.
Cells were exposed to extract concentrations ranging from 0.1pg/ml to
1Qmg/mi. Equal volumes of the relevant culture media were added to six
untreated wells. The cells were then incubated for a further 24 hours. 10pL
5mg/ml MTT (3-(4,5,-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
(Sigma) in PBS (Dulbecco phosphate buffer) was added to each well and
incubated as above, for a further 4 hours. The growth media was then
removed using a multichannel pippette and replaced with 100 pL pure DMSO
to solubilize the MTT-formazan product (Lopez de Cerain et at. 1996). The
plates were left for an hour to allow the MTT to solubilize. The plates were
then shaken by the microplate reader (Labsystems, EMS Reader MF) and the
absorbance was read at 540nm. Each experiment was done duplicate.
The viability of the cells exposed to each dilution of plant extract was
calculated relative to the survival of untreated cells. The absorbance of treated
wells was divided by the absorbance of the untreated well in the same column
and expressed as a percentage viability.
67
CHAPTER 3: Results Section 1
3. Resuits from Open Ended Interviews

No firm conclusions can be made from results presented in this section as
only six traditional healers were extensively interviewed, and a further eight
healers were casually questioned about certain aspects relating to plant
remedies.
Making contact with traditional healers was extremely difficult and time
consuming. Only a small percentage of arrangements were honoured by the
traditional healers. Appointments were usually made at venues deep in crime
ridden areas. On numerous occasions the journey to the particular venue was
made only to find that the traditional healer was not there. The frustration in
trying to meet with traditional healers and the risk on my life caused me to
stop trying to increase the sample size.
Traditional healers were contacted through the presidents of three traditional

healer associations in Johannesburg or Soweto (Traditional Healers’
Association; African Skilled Herbalists Association; Traditional Healers
Organisation). Using this method of contacting healers allows one to speak to
a healer having the confidence of the healer from the beginning of an
interview. Healers contacted in this manner were willing to discuss any
information relevant to the study. None of the healers appeared to be
protecting age-old secrets as is the common perception people have of
healers. Each interviewing session was treated as a two way learning
process. More than half our time together was always spent teaching them
about basic medical principles. All healers spoken to were very aware of their
inadequate training and lack of knowledge, and each healer had questions
about some medical Issue. Indications from all the healers were that they
were willing to learn and wanted more rigorous methods of training.
3.1. Training of traditional healers

While interviewing traditional healers, ten were asked about their levels of
education and about the training procedure they went through to become
68
traditional healers. None of the healers went through years of rigorous training
and apprenticeship as is the general perception of how traditional healers are
trained. Out of ten traditional healers, 5 had not completed primary education
(grade 7), three had completed matric (grade 12) of which two had worked as
clerks for a number of >ears, and one worked as a journalist for over 15 years.
The remaining two traditional healers had tertiary education qualifying as
midwives who had worked in hospitals for a number of years. The two healers
who previously worked as clerks in banks left their professions to become
traditional healers after the change of government. Both said that perceptions
on traditional medicines had changed since the change of government in
1994, making traditional healers far more acceptable. This change caused
them to follow previous callings to become traditional healers.
The duration of training varied from 6 months to 3 years. Each traditional

healer spoke about ancestral spirits as being their primary teachers on the
medical use of plants. Their living tutors "served more as mentors and taught
them how to divine the spirits and how to interpret dreams. Three of the
healers were trained in Soweto, whereas the remaining healers went to a rural
setting to undergo initiation and to receive part of their training.
3.2. Preparation of remedies containing Rhoicissus tridentata

Fourteen traditional healers were asked how they prepare Rhoicissus
containing reme\ ' for any indication (not specific to pregnancy). All ground
dried tubers o r . _ws of Rhoicissus by hand and then prepared the decoction
by boiling the sample in water for between thirty minutes and an hour. There
was a difference between the manner in which they dispensed the remedies.
Four of the traditional healers prepared the decoctions mixing three to four
different ingredients chosen according to the specific complaints of the
patient. These remedies were sold as bottled decoctions (Figure 3.3). The rest
of the healers sold mixtures of dried ingredients (usually wrapped in
newspaper) and gave instructions on how the remedy should be prepared.
The dosages they prescribed differed. Most commonly measurements were in
handfuls. None of the healers used standard measuring items such as cups,
bottle tops, or scales. When the suggestion was made that they should use a
scale, most healers were accepting of the concept, but none of them started
69
Some healers instructed patients to drink a set dose per day, where others told the
patients to drink the decoction as required. No concerns were voiced about possible
contamination of the decoctions or about fermentation or degradation of the "muties"
over time.
Figure 3.1 Plant matter for sale at the Faraday Herbal Market, Johannesburg.
70
Figure 3.2 Plant vendors selling their merchandise at Faraday Herbal Market.
Figure 3.3 A plant vendor and traditional healer. The mixture (front left) is his
personal "imbiza" remedy which he sells either as a dry mixture wrapped in
newspaper or as a bottled decoction (behind the dried mix).
Figure 3.4 Rhoicissus lignotubers for sale.
72
3.3. Dangers of herbal remedies during pregnancy
None of the healers questioned with respect to the possible dangers of
prescribing herbal remedies during pregnancy were aware of possibly
teratogenic effects of the remedies. Healers were aware that the imbelekisane
remedies were potentially dangerous and should be used with care.
3.4. Duration of therapy

Remedies were usually prescribed for varying durations starting from the first
through to the third trimesters. The remedies were usually prescribed for a
certain complaint of the patient, such as backache, fatigue or fear of a
recurrent miscarriage. Thus the healers would prescribe a relevant
isihlambezo for the particular indication. The duration of therapy usually lasted
until the particular complaint dissipated. When the remedy was being taken to
ensure an easy delivery, the remedy was taken from the beginning of the third
trimester until delivery. Some traditional healers differentiate between
imbelekisane and isihlambezo, while others do not.
3.5. Plants prescribed by the traditional healers in pregnancy related

remedies.
The six traditional healers that were extensively interviewed prescribed
between 1 and 16 plants in their pregnancy related remedies. A total of 48
species were prescribed (Table 3.1, Page 75), of which 14 (29%) were listed
in the literature r view on pregnancy related remedies compiled by Veale et
al. (1992). A further 11 species (total of 52%) were documented in other
literature sources (Table 3.3, Page 84). Of the plants prescribed by each
healer, 50 to 100% have previously been documented to be used during
pregnancy (Figure 3.5, Page 80). Of the 48 plant species prescribed, 18
species (37.5%) have been reported to be potentially poisonous, and 10
(20.8%) have been reported to have caused fatal poisonings in either
livestock or humans (Table 3.4, Page 87).
73
3.6. Toxicity of Rhoicissus tndentata
Despite reports in the literature implicating R. tndentata in fatal poisonings (Watt
& Breyer-Brandwijk, 1962; Brandt & Muller, 1995), none of 14 traditional
healers questioned about the safety of Isinwasi” were aware of any toxic effects
of the plant. None of the traditional healers were aware of seasonal or
distributional differences in the effects of the plant. All material obtained by the
healers from plant collectors were treated as being the same, and were
dispensed in equal quantities.
74
Table 3.1 Ingredients prescribed by the traditional healers interviewed for use
during pregnancy, or pregnancy related indications. Superscripts after the botanical
names refer to whether the plants are reported to be toxic (T) or to be used in
pregnancy related remedies (P) in the scientific literature.
PP=plant part used; TH = name of traditional healer.
Vernacular Botanical Use PR* TH*

ALLIACEAE
ubani Agapanthus africanus isihlambezo bb 2
(L)HoffmgJ'p
ishaqa Tulbaghia violacea Harv.T imbiza rt 6
AMARYLLIDACEAE
umayime Clivia miniata RegelT,p isihlambezo - reduces pain rt 2;4
- eases delivery bb
- revives nerves and veins
of reproductive system
umsekelo
impompo Scadoxus puniceus (L.) imbiza - cleansing bb 4;6
Friis & Nordal1, p labour induction if overdue
ANACARDIACEAE
marula Sclerocarya hinrea (A. isihlambezo bk 5
Rich.) Hochst. subsp
caffra (Sond.) Kokwaro'3
ASPARAGACEAE
isigobo Protasparagus africanus isihlambezo bb 2
(Lam.) Oberm. - menstrual pains
- stomach pains
- skin rash
- infertility
- mental retardation
- swollen nerves in lung
- cleanses blood
ASTERACEAE
iphungula Berkeya rhapontica (DC.) isihlambezo - cleansing rt 2
Hutch. & Burtt Davy agent
subsp. aristosa R ssl. - eases backache
var. aristosa - easy delivery
uqadolo Bidens pilosa L. isihlambezo - stops bladder, rt 2
amalenjane kidney and back pains
- limits amniotic fluid
75
Vernacular Botanical Use PP* TH*
- heals sores in bladder
- giv<:s blood more tonic
- maternal strength
impila Callilepis laureola T' p isihlambezo - easy delivery rt 2;4
- maternal and foetal health
- calm down restless child
- bone fusion
- wound healing
- embalming
- exorcism (steam)
- hayfever (steam)
impepo Helichrysum decorum isihlambezo - headache It 4
DC.
uhlunguhlungu Vernonia hirsute imbiza - cleansing rt 4
(DC.)Sch. Bip.
V^neocorymbosa Hilliard fertility - opens tubes and
cleanses womb
Brachylaena ellyptica
(Thunb.) DC. p
CAESALPINACEAE
mosethla Peltophorum africanum bk 4
Sond.
CAMPANULACEAE
amazombe Roella glomerate A. DC. It 2
CELASTRACEAE
ingwavuma Cassine transvaalensis infant - helps growth bk 2
(Burtt Davy) Codd
CONVOLVULACEAE
uboqom Convolvulus sp p isihlambezo rt 6
CUGURBITACEAE
intshungu Momordica foetida isihlambezo - reduces rt 2
Schumach. p amniotic fluid
- revives reproductive
system
CYPERACEAE
ibhuma Cyperus sp isihlambezo - foetal strength 2
DRACAENACEAE
mogaga Sanseviera hyacynthoides infertility caused by STD bb 5
(L) Drucep
76
“ —— 1 — ------- ------- r ' pp*
Vernacular Botanical ] Use TH*
EUPMORBIACEAE
umahlabekufeni Croton grattisimus Burch, imbiza - cleansing V/d 4
var. gratissimu$r
HALORAGACEAE
ugobo Gunnera perpensa L p isihlambezo - general rt 12;
isihlambezo - backache or 6
tiredness
imbiza - cleansing
fertility - if menstruate too
often or if has STD
HYACiNTHACEAE
ugibisila Bowiea volubifis Harv. ex imbiza - cleansing bb 4
Hook. f . T' p
umathunga Eucomis autumnalis fertility - open tubes bb 3;4
(mill.) Chitt. subsp. sores in uterus
clavata (Bak.) imbiza - cleansing
Reyneke1, p
E. autumnalis (mill.) Chitt.
subsp. autumnalis T' p
inguduza Scilla natatensis imbiza - cleansing bb 3:4
Planch.T,p - sores in uterus
umahlokotoza Urgineajohysodes (Jacq.) fertility bb 3
HYPOXIDACEAE
modi Hypoxis acuminata isihlambezo bb 7
ilabatekha Hypoxis colchicifolia isihlambezo bb 1;5
B ak/ isihlambezo - for headache
H. hemerocailiclea Fisch. fertility - if menstruate too
& C.A. May. often, or has STD
LORANTHACEAE
iphakama Tieghemia quinquenervia isihlambezo 2
(Hochst.) Bailie
MIMOSACEAE
intolwane Elephantorrhiza fertility - opens tubes bk 3;6
umdabu elephantina (Burch.) imbiza - cleansing rt
Skeels T| p isihlambezo - loose muscles
- easy delivery rt 5
E.burkeiT,p
- maternal strength
- urinate (keeps system
77
going)
6 months after birth - takes wd
away clots 3
MYRSINACEAE
umaguqu Maesa alnifolia Harv. isihlambezo - to settle 4;3
foetus if too high or
M. lanceolata ForsskJ
breech
ORCHIDACEAE
imfe-yenkawu Ansiella africana Lindl. isihlambezo 2
amabelejonosi Eulophia angolensis isihlambezo - reduces pain 2
(Reichb. f.) Summerh.
E. cucullata (Afzel. ex
Swartz) Steud. p
Especiosa (R. Br. ex
Lindl.) H.Boi.
E. streptopetala Lindl.
PAPILIONACEAE
umsintsi Erythrina caffra Thunb. imbiza - cleansing wd 4
RU3IACEAE
morokolopuli Kohautia amatymbica isihlambezo rt 5
Eckl. & Zeyh. p umsekelo
menstrual pains
icimamlilo Pentanisia prunelloides isihlambezo - kills pain bb 1
(Eckl. & Zeyh.) Walp.p
STERCULIACEAE
patengaka Hermannia depressa N.E. isihlambezo - backache 5
Br.
TILIACEAE
ihlalanyathi Grewia caffra Meisn. isihlambezo - makes baby bk 2
move
G. occidentalis L r' p strengthens nerves of
reproductive system ..
VITACEAE
isinwazi Rboicissus digitata (Lf) isihlambezo - strengthens rt 2;4
Gilg & Brandtp reproductive system bb
-protects mother and foetus
R. tomentosa (Lam.) Wild
from illness
& Drum.7, p
78
R. tridentata (Lf.) Wild & umsekelo
Drum, subsp. cuneifolia
(Eckl. &Zeyh.) N.R.
UrtonT' p
mbola Parinari curatellifolia fertility - open tubes bb 3
sehlulamanye Schrebera saundersiae 4
sekaname Homeria pallida fertility bb 3,5
NON-BOTANICAL INGREDIENTS
iqaku leufene Baboon urine isihiambezo - helps uterus 1;2
nempila - cystitis
skhupha chicken egg yolk isihiambezo - maternal and 1
sequanda foetal strength
qanda lentshe ground ostrich eggshell isihiambezo - maternal and 1;3
foetal strength
mtsansi we horse placenta isihiambezo - easy delivery 1
hashi contractions, ease pain
liquid soap & water imbiza 1
(sunlight soap)
liquid mercury7 imbelekisane 1
LP records imbelekisane 4,7,
8
UNIDENTIFIED INGREDIENTS
dumalala Not identified infant - makes baby sleep It 2
isobo Not identified imbiza - if not menstruating rt 6
mokgalakane Not identified fertility 3
ndodaemadevu Not identified imbiza - if not menstruating rt 6
udabulibayi Not identified isihiambezo 3
udonuzibovu Not identified imbiza - sores in uterus bb 3
ukunyakaza Not identified isihiambezo - strengthens 2
nerves
umeluze Not identified imbiza - cleansing bb 4
umsongelo Not identified imbiza rt 6
Key: bb = bulb; bk=bark; rt=root; vvd=branch; lt=leaves and twigs
*TH = Traditional healers or midwives: 1 - Nomsa; 2 - Bouylix; 3 - Martha;
4 - Fiki; 5 - Moseki; 6 - Laymond; 7 - Baragwanath Nursing Sisters,
8 - Johannesburg General Hospital Nursing Sisters
79
3.7. Previous documentation o f use of prescribed plants during
pregnancy
The number of plants prescribed by each healer ranged between one species
through to sixteen (Figure 3.2). The proportion of plants prescribed by the
various healers which are documented to be used during pregnancy varies
from 50-100%.
MSB Documented
BBsesE N o t documented
Nomsa Bouylix Martha Fiki Moseki Laymond

Figure 3.5. The number of plants prescribed by different traditional healers in
pregnancy related remedies. The colours indicate the proportion of plants
either documented or not documented in the scientific literature for use during
pregnancy.
80
Table 3.2 Plants prescribed by traditional healers in pregnancy related
remedies tabulated according to indications, and the number of healers using
the plant for the specific indication.
Indication Plant species No.
Imbiza
cleansing Protasparagus africanus 1
Berkeya rhapontica 1
Bowiea volubilis 1
Croton grattisimus 1
Etephantorrhiza elephantine 1
Erythrina caffra 1
Gunnera perpensa 3
Scadoxus puniceus 3
Scilla natalensis 2
Tulbaghia violacea 1
Vernonia spp. /Brachylaena Bllyptica 1
Isihlambezo
menstrual pains Protasparagus africanus 1
Kohautia amatymbica 1
stomach pains Protasparagus africanus 1
skin rash Protasparagus africanus 1
cystitis baboon urine 2
maternal/foetal chicken egg yolk 1
strength grcund ostrich eggshell 2
Bidens pilosa 1
CalUlepis laureola 2
Cyperus sp. 1
Etephantorrhiza elephantina / E. burkei 1
Gunnera perpensa 3
to ease labour Horse placenta 1

Berkeya rhapontica 1
Callilepis laureola 2
Clivia miniata 2
Etephantorrhiza elephantina / £ burkei 1
Eulophia cucullata 1
Pentanisia prunelbides 1
81
avoid repeated Clivia miniata 2
miscarriage Kohautia amatyrnbica 1
Rhoicissus digitata / R. tomentosa/R. tridentata 2
to ease back pain Berkeya rhapontica 1
Bidens pilosa 1
Gunnera perpensa 3
Hermannia depressa 1
headache Hypoxis colchicifolia / H. hemerocallidea 2
diuresis Elephantorrhiza elephantina / E. burkei 1
to strengthen nerves Clivia miniata 2
of reproductive Grewia occidentalis 2
system Momordica foetida 1
to make baby move Grewia occidentalis 1
to make baby drop or Maesa ainifolia / M.lanceoiata 2
turn if breech
to reduce amniotic Momordica foetida 1
fluid
general / unspecified Agapanthus africanus 1
Convuivuius sp. 1
Gunnera perpensa 3
Hypoxis colchicifolia / H. hemerocallidea 2
Roella glomerata 1
Sclerocarya birrea subsp calfra 1
Schrebera saudersiae 1
Tieghemia quinquenervia 1
Imbelikesane
(labour induction) baboon urine 2
Horse placenta 1
Scadoxus puniceus 2
Fertility
unspecified Protasparagus africanus 1
Homeria pallida 2
Urginea physodes 1
to open tubes Elephantorrhiza elephantina 1
Eucomis autumnalis 2
Parinari curatellifolia 1
Vernonia neocorymbosa 1
STD related Gunnera perpensa 3
Sanseviera hyacinthoides 1
Hypoxis colchicifolia /H . hemerocallidea 2
82
frequent menstruation Gunnera perpensa 3
Hypoxis colchicifolia / H. hemerocallidea 2
Paediatrics
calm restless child Callilepis iaureoia 1
to make baby sleep not identified 1
to help infant growth Maytenus heterophylla / 1
Cassine transvaalensis
83
3.8. Literature citings o f plants used in pregnancy related remedies
The use of pregnancy related remedies is extensively reported in the literature.
Table 3.3 gives leferences for citings of the various ingredients prescribed by
the interviewed traditional healers and Table 3,4 gives the documented toxic
effects of the plants.
Table 3.3 Documented pregnancy related uses of plants prescribed by the

traditional healers interviewed.
Ingredient Use / Effect Reference
Agapanthus afhcanus isihlambezo; inembe 11

expulsion of placenta 6
stimulate contractions of isolated
uterus and ileum
agonise muscarinic receptors and
12
promote prostaglandin synthesis
Bowiea volubilis bulbs - facilitate delivery 3

procure abortions 13
isihlambezo, inembe 11
Brachylaena ellyptica procure abortions 2
inembe 3; 11
Callilepis lavreola isihlambezo, inembe 3; 4
infertility 5; 11
Clivia miniata induce / facilitate birth 3; 11
inembe, isihlambezo 5
uterotonic on isolated rat uterus 10
Convulvufus sp unspecified medicine during 13
pregnancy in Lesotho.
(C. sagittatus)
Cyperus sp inembe 11
Elephantorrhiza womb cleansers soon after birth 9
elephantine
E.burkei
84
ingredient Use / Effect Reference
Eucomis autumnalis prevent premature birth (Lesotho) 13
Eulophia cucullata impotence 1
barrenness 5
Grewia occidentalis facilitate or procure delivery 1
spasmolytic on in vitro guinea-pig 5
ileum and spasmogenic on rat and
11
rabbit duodenum.
isihlambezo, inembe
Gunnera perpensa isihlambezo, inembe 1
cystitis 11
facilitate expulsion of the placenta in 2
women and animals
11
uterotonic on isolated rat uterus
6
Hypoxis colchicifolia impotency and barrenness 1
Kohautia amatymbica sterility (Lesotho) 5
Momordica foetida abortifacient and ecbolic (Uganda) 13
antinicotinic and antimuscarinic action
in vitro
5
Pentanisia prunelloides facilitate placental expulsion 13
, isihlambezo, inembe 3
stimulate contractions of isolated rat 6
uterus and ileum
11
Rhoicissus digitata isihlambezo, inembe 5
R. tomentosa dysmenorrhoea 11
R. tridentata
Sanseviera prevent miscarriage (Western Cape) 13
hyacynthoides
ease birth pains (Tswana) 5
inhibits uterine contractions stimulated
by oxytocin and serotonin in vitro
8
no effect on isolated ileum
85
Ingredient Use / Effect Reference
Scadoxus puniceus ensure safe delivery 3

Scilla nalalensis isihlambezo, inembe 3; 11
Sclerocarya birrea subsp isihlambezo, inembe 5
caffra
menorrhagia (Zimbabwe) 11
infertility (Vhavenda)
Urginea physodes isihlambezo, inembe 11; 2
Vemonia neocorymbosa isihlambezo, inembe 3
menstrual irregularities 11
abortifacient 13
Baboon urine isihlambezo 7
oxytocic in vitro
horse placenta oxytocic in vitro 14

liquid mercury
* References:
1. Bryant 1966 8. Neuwinger 1996

2. Gerstner 1939 9. Pujol 1990
3. Gerstner 1941 10. Veale et al. 1989
4. Gumede 1990 11. Veale etal. 1992
5. Hutchings etal. 1996 12. Veale etal. 1999
6. Kaido etal. 1997 13. Watt & Breyer-Brandwijk 1962
7. Morris & Mdlalose 1991 14. Onaguluchi & Ghasi 1996
86
Table 3.4 Potential toxic effects of plants prescribed by the traditional healers
interviewed in pregnancy related traditional remedies3.
ingredient Symptoms of poisoning Toxic principle Ref

Agapanthus africanus Haemolytic anaemia 5; 6
Sap causes severe mouth
ulceration
Bowiea volubilis Haemolysis, severe vomiting bufadienolides, 2; 4;
and diarrhoea, salivation, cardiac 6; 7
cardiac failure, death. glycosides
Caltilepis laumola Confusion, vomiting,, diarrhoea, atractyloside 3; 5;
convulsions, hypoglycaemia, 9
hepatic centrilobar necrosis,
and renal tubular necrosis,
death.
CIMa miniata Salivation, vomiting, diarrhoea, lycorine 4; 5
CNS depression
Croton grattisimus Bark reputed to be highly toxic; diterpenoids 4; 6
Skin and mucosal irritation.
Elephantorrhiza Oral administration: constipation 3; 6
elephantine
Parental administration:
E.burkei pulmonary oedema, loss of
appetite, diarrhoea,
exhaustion, liver degeneration,
death.
Erythrina caffra Highly toxic erysovine, 4
erythraline
(Erythrina
alkaloids)
Eucomis autumnalis Death in sheep haemolytic 6,7
saponins
Abdominal pain, diarrhoea, renal
failure
Hypoxis colchicikJ!* Corms reputed to be highly toxic 3
Maesa lanceolate Reported to be toxic 6
Momordica sp. Fruit caused death of dogs and 8
sheep.
Violent diarrhoea and vomiting
Protasparagus africanus No reports
P. exuvialis High fever, confusion 8
87
Rhoicissus tomentosa Severe colic and childhood 3; 6
diarrhoea.
CNS depression leading to
R. thdentata
respiratory arrest.
Sanseviera sp. Leaves act as neuromuscular 8
blockers
Scadoxus puniceus Dizziness, visual disturbances, lycorine. 3; 5;
CNS excitation or depression, haemanthidine 7; 8
death.
Scilla natalensis Death of experimental sheep 5; 6;
with dyspnoea, weak quickened 7
pulse.
Tulbaghia violacea Abdominal pain, gastro-enteritis, 1;3;
acute inflammation, intestinal 6
mucosa sloughing, inhibition of
intestinal peristalsis, miosis,
death.
U ^ e a physodes 3g/kg bulbs fed to sheep caused cardiac 3; 4
death within 22 hours. glycosides
eg. scillaren A
Vemonia neocorymbosa Fatal if used incorrectly 3
(according to the Vhavenda)
Vemonia sp. Used as arrow poison by Ndorba 8
Decreases heart rate and
causes CNS paralysis
Based on:
1- Burton 1990 6- Watt and Breyer-Brandwijk 1962
2- Gerstner 1941 7- Hutchings & Terblanche 1989
3- Hutchings ef al. 1996 8- Neuwinger 1996
4- Van Wyk et al. 1997 9- Watson et al. 1979
5- Veale et al. 1992
88
4. PHARMACOLOGICAL ACTION OF RHOICISSUS TRIDENTATA ON

ISOLATED RAT UTERUS AND ILEUM
4.1. Inorganic Ion Concentrations.
The concentrations of K \ Ca+ and Na+ in the maximal doses of the R.
tridentata extract (from roots harvested in Umlazi) added to the organ baths
were 0.96 mmol/L, 0.15 mmol/L and 0.70 mmoi/L respectively. That is a
collective osmolarity of 1.81 mmol/L. The maximal volume of plant extract
added was 1.3ml in 50ml Tyrode. Therefore, the plant extract would not have
affected the tonicity of the Tyrode solution surrounding the organs.
4.2. Comparison Between the Efficacy and Potency of the Different
Reference Drugs.
The contractions caused by the various reference drugs were expressed
relative to the contractions stimulated by acetylcholine on the same piece of
tissue. All concentrations stated refer to final concentrations in the organ baths
in all the in vitro experiments.
In the uterus (Figure 4.1a), acetylcholine and oxytocin exhibited similar
eficacies, followed by serotonin then noradrenaline. The maximal contractions
induced by noradrenaline were only about 28% of those caused by
acetylcholine. This is the reason why (later in this chapter) the plant extract
appears to be more effective when compared to contractions stimulated by
noradrenaline. In the ileum, the maximal contractions stimulated by serotonin
were half the size of those stimulated by acetylcholine (Figure 4.1b).
89
Oxytocin was the most potent agonist in the oestrogenised uterus, followed by
serotonin, acetylcholine and then noradrenaline (Figure 4.1a), whereas in the
ileum acetylcholine was more potent than serotonin (Figure 4.1b).
100-1 a) uterus
oxytocin (n=lo) ♦
serotonin (n=iO)d
® noradrenaline (n=4)
* acetylcholine (n=i6)
i ; i i i i i i i n i
-12 "11 "10 "9 "8 “7 "6 -5 "4 -3 -2
Log agonist concentration (M)
b) ileum
d serotonin (n=ii)
a acetylcholine (n=H)
t i i r i i i ; f j
-12 "11 "10 -9 -8 -7 -6 -5 -4 -3 -2
Log agonist concentration (M)
Figure 4.1 Comparison of the isolated rat, a) uterus and b) ileum responses
to the cumulative addition of the different reference agonists used in this
study. All values were calculated relative to the organ’s maximal response to
acetylcholine. Each point represents the mean with the error bars representing
the standard error of the mean.
90
4.3. THE PHARMACOLOGY OF RHOICISSUS TRIDENTATA SUBSP.
CUNEIFOUA ON ISOLATED RAT UTERINE AND ILEAL SMOOOTK
MUSCLE
4.3.1. Extracts used for the investigations of pharmacological action

Crude aqueous decoctions of R. tridentata subsp. cuneifolia were used for this
study to determine the pharmacological action of the extracts ingested by
women treated by local traditional healers. Organic solvents and various
fractionation methods were avoided as these are not used by local healers.
Aqueous extracts of R. tridentata subsp. cuneifolia roots harvested in either

Durban or Umiazi were used for the followin'] investigations into the
pharmacological action of the plant extracts presented in this chapter:
Cumulative additions of R. tridentata (Figure 4.2)
Oxytocin (Figure 4.5)
Noradrenaline and prazosin (Figure 4.7)
Acetylcholine and atropine (Figure 4.9)
Once the stock of the above extracts was depleted, all the other studies were
done using aqueous extracts from tubers harvested during summer and
autumn from Suikerbosrand. These extracts displayed the same
pharmacological action as the roots harvested in KwaZulu-Natal. The following
studies were done using extracts from tubers harvested from Suikerbosrand:
Indomethacin (Figure 4.6)
Noradrenaline and yohimbine (Figure 4.8)
Muscarinic subtype selective antagonists (Figure 4.10)
Serotonin and methysergide (Figure 4.11)
Serotonin and ketanserin (Figure 4.12)
Serotonin and tropisetron (Figure 4.13)
Hexamethonium, mecamylaine (Figure 4.14)
Cholera toxin, pertussis toxin (Figure 4.14)
pyrilamins (Figure 4.15)
NS-398 (Figure 4.17)
91
4.3,2. The contractile action of R. tridentaia subsp cuneifolia
The Rhoicissus extract stimulated concentration dependent contractions in
both isolated rat uterus and ileum (Figure 4.2). In the doses given, the ileal
dose response curve was sigmoidal, whereas the uterine curve was not.
Higher doses could not have been given as explained later.
The crude aqueous extracts of R. tridentata at a bath concentraction of

1.3mg/ml exhibited a wide range in the size of contractions in the different
uteri and the ileui used (Figure 4.3). The range in the uterine and ileal
responses, relative to acetylcholine, varied from 0-85% and 0-95%
respectively.
It is not possible to determine the ICm of the extract in the uterus since a
maximal response was not obtained. The ICso for the ileum was 0.8ug/ml.
Figure 4.4 shows the resulting contractions following the addition of

accumulating doses of acetylcholine to the baths containing the isolated
uterus (a) and ileum (b). After washing out the baths, plant extract was added
to the baths, exposing the organs to 1.3 mg/ml plant extract. Both the uterine
and ileal muscle contracted to about 50% of the maximum effect of
acetylcholine. Without washing, accumulating doses of acetylcholine were
added to the baths. In the case of the uterus, there was a slight increase in the
contraction, but the increase in contraction was more marked in the case of
the ileum. On washing out the baths, it was found that in the case of the ileum,
the organ returned to its original length, however, the uterus did not relax fully
irrespective of repeated washouts.
92
4 0 -i 4 0 -,
a) uterus b) ileum
30- 30-
§1
g-o 20- 20 -
P
| g 10-
m to
10-
- 8.6 -7 .6 - 6.6 -5 .6 -4 .6 -3 .6 - 8 .6 -7 .6 - 6.6 -5 .6 -4 .6 -3 .6
Log Rhoicissus extract concentration (g/ml) Log Rho/c/ssus extract concentration (g/ml)
Figure 4.2 Direct action of R.tridentata subsp. cuneifolia aqueous root

extracts on isolated rat a) uterus and b) ileum. All values were calculated
relative to the maximal response obtained from a prior cumulative dose
response curve to acetylcholine alone. Each point represents the mean
response from seven rats with the SEM.
100-1
<D 80-
ii 60-
11
If (C
40
20 -
uterus ileum
Figure 4.3 Box and whisker plot showing the range of maximal contractions
stimulated by 1.3 mg/ml R. tridentata roots. The box extends from the 25th to
the 75th percentile, with the horizontal line at the median. The whiskers show
the range of the data.
93
a) uterus
Rh
b) ileum ^pl/ ^
Rh
><AMl4-uaJU*-e-u.
40 min
Figure 4.4 Tracing of the a) uterine and b) ileal contractions caused by (A)
cumulative additions of acetylcholine, (B) 1.3 mg/ml R tridentata Umlazi root
extract (Rh) followed by cumulative additions of acetylcholine. The arrows
( \ j / ) indicate when doses of exponentially increasing concentrations of
acetylcholine were added to the organ bath (0.32ng - 3mg/ml). The baths were
rinsed between tests to allow the organs to relax ( I).
94
4.4. INVOLVEMENT OF DIFFERENT RECEPTOR SYSTEMS IN THE
CONTRACTILE RESPONSE TO THE RHOICISSUS EXTRACT
All the dose response curves in the following section are presented in the
same format. Each point represents the mean with the error bars representing
the standard errors of the mean. The black curves represent the contractile
response to the agonist alone and serve as references. The green curves
represent the contractile response to R. tridentata root aqueous extracts (with
no washing out of the organ baths) followed by the cumulative addition of the
reference agonist. The red curves represent the response to the antagonist
followed by the reference agonist that indicates to what extent the receptors
are blocked at the concentration of antagonist used. The blue curves
represent responses to the plant extract and reference agonist after the
relevant receptors have been blocked by an antagonist. These curves indicate
whether the contractile activity of the plant extract is altered if the relevant
receptors are blocked. The sample size is given in the legends on the graphs
for the different curves.
95
4.4.1, OXYTOCIC RECEPTOR SYSTEM
The effect of the Rhoicissus extract on the uterine response to oxytocin.
The direct contractile action of a final concentration of 1.3 mg/ml crude extract
was 28.5% ± 14 (95% Cl) of the maximal response to oxytocin (Figure 4.5),
The extract had no effect on the maximal response to oxytocin. No oxytocic
antagonists were available.
lo o - uterus
80-
+ Oxytocin (n=7)
• Rh & Oxytocin (n=7)
Rh -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1
Log roytocin concentration (iU/ml)
Figure 4.5 The isolated uterine response to oxytocin alone (+) compared to
when the organs were pretreated with a final concentration of 1.3 mg/ml
Rhoicissus extract for 5 minutes before the cumulative addition of oxytocin.
96
4.4.2. Prostaglandin synthesis
The effect of indomethacin on the response to the cumulative addition of

acetylcholine was negligible. Preincubation with 5 \M indomethacin for 15 min
significantly reduced the initial activity of 1.3 mg/ml plant extract in both the
uterus and ileum (p<0.001), without affecting the organs' response to the
acetylcholine following the plant extract (Figure 4.6).
loo-) a) uterus
+ ACh (n= 1 2 )
® Rh & ACh (n= 1 2 )
v Ind & ACh (n= 1 2 )
v Ind. Rh & ACh (n = i 2 )
Rh -9 -8 -7 -6 "5 -4 **3
+ ACh (n=6 )
• Rh & ACh (n=8)
v Ind & ACh (n=6 )
r Ind, Rh & ACh (n= 6 )
~p I—,-------- 1-------- r------- r-------- 1--------- 1--------- r
Rh -9 -8 -7 -6 -5 -4 -3
Log acetylcholine concentration (M)
Figure 4.6 The contractile response of isolated rat a) uterus and b) ileum to
acetylcholine (+) when pretreated with 5 pM indomethacin for 15 minutes (v),
1.3 mg/ml R. tridentata extract for 5 minutes (e), and indomethacin followed
by the plant extract before the cumulative additions of acetylcholine (v).
97
4.4-3. Adrenergic Receptor System
The effect of the plant extract on the a-receptor response to

noradrenaline.
The direct action of the plant extract increased the initial response to
noradrenaline in the uterus once the p-adrenergic receptors had been blocked
by 2.7\M propanolol. The direct action of the plant extract was 54.9% ± 5.5
(95% Cl) of the maximal response to noradrenaline. The extract did not alter
the response to maximal concentrations of noradrenaline (Figure 4.7).
Prazosin, an -adrenoceptor antagonist, did not alter the uterine response to
the Rhoicissus extract, even though the same dose of prazosin shifted the
dose response curve to noradrenaline to the right. Yohimbine, an az-
adrenoceptor antagonist, at a concentration which shifted the dose response
cun/p to noradrenaline to the right, also did not alter the contractile activity of
the plant extract (Figure 4.8).
uterus
[ + Prop & N A (n = 1 2 )
® Prop, R h & N A ( n = 7 )
v Prop, Prazosin & N A <n=7 )
v Prop, Prazosin, R h & N A (n= 6 )
if l— i------------ 1------------ 1------------ 1------------ 1------------i
Rh -9 -8 -7 "6 -5 -4
Log noradrenaline concentration (M)
Figure 4.7 The isolated a) utetine response to noradrenaline when
pretreated with 2 . 7 propanolol for 5 minutes then 1.3 mg/ml Rhoicissus
extract for 5 minutes (e), compared to the response to noradrenaline when
pretreated with propanolol only (+), propanolol and 2jiM prazosin (V) or
prazosin followed by the Rhoicissus extract (v).
98
uterus
+ Prop& NA fn=9)
• Prop, Rh & NA (n=7)
v Prop, Yohimbine & NA (n=9)
v Prop, Yohimbine, Rh £ NA (n= 6 )
Rh "9 ™8 -7 -6 -5 -4
Log noradrenaline concentration (M)
Figure 4.8 The isolated rat uterine response to noradrenaline when

pretreated with 2.7 nM propanolol for 5 minutes followed by 1.3 mg/ml
Rhoicissus aqueous extract for 5 minutes (e), compared to the response to
noradrenaline when pretreated with propanolol only (+), propanolol and 2\M
yohimbine (V) or yohimbine followed by the Rhoicissus extract (▼).
99
4.4.4. Muscarinic receptor system
The effect of the Ritoicissus extract on the isolated uterus and ileum
response to acetylcholine.
Pretreatment of the organs with 1,3 mg/ml plant extract directly stimulated
contractions 18.3% + 4.6 (95% Cl) of the maximal contractions stimulated by
acetylcholine in the uterus and 14.3 ± 4.3 (95% Cl) in the ileum (Figure 4.9).
The extract did not affect the uterine response to cumulative addition of
acetylcholine, whereas the maximal response was inhibited in the ileum.
Preincubation for 5 minutes with 0.4nM atropine, a non-specific muscarinic
antagonist, shifted the acetylcholine curve to the right without blocking the
response to maximal concentrations of acetylcholine, and completely blocked
the direct action of the Rhuicissus in both the uterus and the ileum.
100
100- a) uterus
S 80-
60-
1 1
i t "20 -
+ ACh (n=10)
a m
Rh & ACh (n=15)
0J v -4—8y--vj-'1 Atr & ACh (n=8)
v Atr, Rh & ACh (n=14)
■H N - ~T~ —i -
Rh -9 -6 -5 -4 -3 -2
100
b) ileum
-
80-
$
II|f 60-
40-
20 -
+ ACh (n=9)
S CO Rh & ACh (n=9)
E
V - 9 - V -V - Atr & ACh (n=6)
Atr, Rh & ACh (n=?)
if M 1------- 1— r
Rh -9 -8 -7 -5 -3
Figure 4.9 The a) uterine and b) ileal response to acetylcholine (+)

compared to when the organs were pretreated with 1.3 mg/ml R. tridentata
root extract (®) for 5 minutes before the addition of acetylcholine. The
response of isolated rat uterus and ileum to acetylcholine when pretreated
with 4x1 O^M atropine for 5 minutes (v ) or atropine for 5 minutes followed by
the plant extract for another 5 minutes before the cumulative addition of
acetylcholine (y).
101
The effect of muscarinic subtype specific antagonists on the uterine
response to the extract and acetylcholine.
Pretreating the isolated rat uterus with the muscarinic antagonists pirenzepine
(1.5gM), tropicamide (4.5|iM) and 4-DAMP (2nM) which have affinities
selective for muscarinic receptor subtypes Mi, M3 and M4 respectively, all
shifted the dose response curve to acetylcholine to the right (Figure 4.10 a, b
& d). Gallamine a M2 muscarinic receptor antagonist did not alter the
uterine response to acetylcholine (Figure 4.10c). Pirenzepine and Gallamine
did not alter the contractile activity of the plant extract, whereas tropicamide
completely blocked the uterine response to Rhoicissus. 4-DAMP inhibited the
uterine response to the Rhoicissus extract, and suppressed the uterine
response to maximal concentrations of acetylcholine in the presence of the
plant extract.
102
100- a) M-i
II|! 80-
6 0 -
4 0 -
20-
+ A C h ( n = 8)
R h & A C h ( n = io )
Pirenzepine & A C h ( n = 8)
®w
Pirenzepine, R h & A C h ( n = 8)
100- b) M 2
80-
Is 6 0-
If
n 4 0 -
20 -
0-1
“iH
¥ ip
-
,_____________ v
y * e
v Gallamine
Gallamine, R h
A C h std
R h & A C h (n = 1 7 )
( n = 6)
& A C h ( n = 6)
& A C h ( n = 6)
Rh -8 -6 -3
1 00 - C) M 3
h
m^
#a <0
8 20-
8 0-
6 0-
4°-
®
+ A C h S t d ( n = 6)
R h & A C h (n = 1 7 )
4 - D A M P & A C h ( n = 6)
4 - D A M P , R h & A C h ( n = 6)
100- d) M4
Hit
8 0-
6 0 -
4 0 -
+ ACh std (n=8 )
20- I f f H iy Rh & ACh (n=17)
E v Tropicamide & ACh (n=6 )
0-
Tropicamide, Rh & ACh (n=6 )
tI hr —r— 1
Rh 9 -8 -7 -6 -5 -4 -3 -2
Figure 4.10 The effect of antagonists on muscarinic subtypes on the isolated

rat uterine response to acetylcholine alone or after pretreatment with the
R. tridentata extract.
103
4.4.5. Serotonergic receptor system
The effect of the plant extract on the response of the isolated organs to
serotonin
The direct activity of the plant extract increased the initial response to
serotonin in the uterus and ileum (Figure 4.11). The mean direct contractile
activity of the Rhoicissus extract was 22% in the uterus and 18% in the ileum.
The maximal response to serotonin was unchanged by the extract in the
uterus but depressed in the ileum. Methysergide, a non-specific serotonin
receptor antagonist, had no effect on the uterine response to the Rhoicissus
extract, but increased the ileal response.
100 a) uterus
i ,
ro in
80-
60-
40-
+ 5 - H T ( n = 8)
% 20- e R h & 5 - H T ( n = 8)
E v M e t h & 5 - H T ( n - 6)
# 0 v M e th , R h & 5 - H T ( n = 6)
100 b) ileum
I 80
60-
I £
a $ 40
E
5 H T ( n = 10 )
20-j
& 5 H T ( n = 6)
I 0U & 5 H T ( n = 6)
M e t h , R h & 5 H T (n = G )
Tt t- —T—
Rh -9 -8 -7 ~6 -5 -4
Log 5-HT concentration (M)

Figure 4.11 The response of isolated rat a) uterine and b) ileal muscle to
serotonin (+) when pretreated with 1.3 mg/ml R. tridentata extract (©). The
contractile response of the isolated rat uterus and ileum to serotonin when
pretreated with 1p.M methysergide for 5 minutes (v), or methysergide followed
by the plant extract and then cumulative additions of serotonin (v).
104
The effect of serotonin receptor subtype antagonists on the uterine and
ileal response to the plant extract and serotonin.
As with the methysergide response, ketanserin and tropisetron, antagonists
specific for serotonin receptor subtypes 5-HTi and 5 -HT2 respectively, had no
effect on the uterine response to the R. tridentata extract at concentrations
which shifted the dose response curves to serotonin to the right (Figure 4.12a
& 4.13a). However, both antagonists increased the direct ileal response to the
extract (Figures 4.12b & 4.13b).
100 - a) uterus
80-
I
IS
a
E 10
60-
40-
20- + 5-HT (n=12)

I QJ
• Rh & 5-HT (n=8)
v Ket & 5-HT (n=7)
Ket, Rh & 5-HT (n=6)
-ri H 1 —r
Rh -9 -6 -5 -4
1 0 0 -I b) ileum
L
80-
60-
(0 10
40-
E 5-HT (n=8)
20-
& 5HT (n=6)
0J & 5HT (n=6)
£
Ket, Rh & 5HT (n=5)
Tl 1 —r -
Rh -9 -7 -6 -5 -4
Figure 4.12 The effect of ketanserin, a 5-HTi receptor subtype antagonist,

the pretreatment of the R. tridentata extract.
105
a) uterus
y + 5-HT (n=10)
d. * Rh & 5 -H ! (n=3)
v Tropisetron & 5-HT (n=6)
v Tropisetron, Rh & 5-HT (n=6)
"h •-------1---------1---------1--------- 1——— i---- — i
Rh -9 -8 -7 -6 -5 -4
100 -
b) ileum
80-
8
60-
I 40
(0 LO
E + 5-HT (n=10)
20
I “ Rh & £i$! i (n=6)
• Tropisetron & 5HT (n=5)
it ~T~ —r-
Tropisetron.
r T
Rh & 5HT (n=6)
Rh -9 -8 -7 -6 -5 -4
Figure 4.13 The effect of tropisetron, a 5-HTz receptor subtype antagonist,
the pretreatment of the R. tridentata extract.
106
Bar Graphs
The following bar graphs represent the direct response of the isolated organs
to the plant extract alone, or to the plant extract after the organ had been
exposed to an antagonist, i.e. the bars represent the first point on the dose
response curves above. The error bars in all the bar charts represent the
standard deviation from the mean, unlike earlier graphs which gave the
standard error of the mean.
4.4.6. Ganglion and second messenger blockers

Hexamethonium (1.4pM) a ganglionic blocker, the nicotinic antagonist
mecamylamine (2pM) and cholera toxin (1pM) a Ga-protein inhibitor all did not
alter the response to the extract. Pertussis toxin (50pM) slightly inhibited the
response to the extract but not significantly (Figure 4.14). Micromolar
concentrations of these antagonists have been shown to block contractile
responses mediated by stimulation of nicotinic receptors in many studies using
isolated organs (Bartho etal. 1987; Okamura etal. 1995; Hopkins etal. 1996).
50-
uterus
40-
30-
20-
10-
0-
extract alone hexamethonium m ecam ylam ine cholera toxin pertussis toxin
(n=20) (n=14) (n=10) (n=15) (n=7)
Figure 4.14 Bar graphs showing the maximal uterine responses to 1.3mg/ml
R. tridentata extract after the organs had been pretreated with the antagonists
below the x-axis.
107
4.4.7. Summary of results of non-specific antagonists
The direct contractile response of the isolated rat uterus to the R. tridentata
extract was blocked by both the muscarinic blocker atropine (0.4nM) and the
cyclooxygenase inhibitor indomethacin (5|j,M). The 5-HT antagonist methysergidf
(Ij-iM), the ai-adrenoceptor blocker prazosin (2(.iM), and the histamine antagonist
pyrilamine (8nM) all had no effect on the direct activity of the extract (Figure 4,15).
These antagonists have been shown to block the reference agonists in figures 4.6
to 4.9.
60
a) uterus n=7
s 55 H
n=15
1 50-
1
0
45-
40
35-1
n=7
n=7
n=8 n=7
I 30-
25-
n=7
I
1
20-
15-
f
I 10-
5- ***
n=9 I ***
n=7
0-1
Atropine Methysergide Prazosin Pyrilamine Indomethacin
(muscarinic) (serotonergic) (a-adrenergic) (histaminergic) (PG synthesis)
Figure 4.15 The bar graphs showing the mean maximal uterine responses to
1.3mg/ml f t tridentata extract after the organs had been pretreated with the
antagonists given below the x-axis. The response to the extract alone is charted
next to the response to the extract after pretreatment with the relevant antagonist.
The stars represent significant differences from the contractile response to f t
tridentata extract alone (*** = p< 0.001).
108
As with the uterus, the direct activity of the R. tridentata extract on the isolated
rat ileum was blocked by both the muscarinic blocker atropine (0.4nM) and the
cyclooxygenase inhibitor indomethacin (5pM) but not the 5-HT antagonist
methysergide (IpM). (Figure 4.16).
60
b) ileum
55-
I n=7
50- n=16
1
45-
n=7
tto 40-
S 35'
8 30-
I 25
20-
15-
10-
*** ***
5 n=‘l7 n=7
0-1
Atropine M ethysergide Indomethacin
(muscarinic) (serotonergic) (PG synthesis)
Figure 4.16 Bar graphs showing the mean maximal ileal responses to
1.3mg/ml R, tridentata extract after the organs had been pretreated with the
antagonists below the x-axis. The stars represent significant differences from
the contractile response to R. tridentata extract alone (*** = p< 0.001).
109
4.4.8. Prostaglandin synthesis inhibitors
indomethacin, which inhibits both COX-1 and COX-2, almost completely
blocked the contractile activity of the R. tridentata extract, whereas '-M NS-
398, a COX-2 selective inhibitor, reduced the contractile ac ,ty of the extract
(p<0.01).
501 uterus
to o
18
ffl js>
***
extract only indomethacin NS-398 NS-398

1.3mg/ml 5pM 0.i|iM 1hM
(n=15) (n=7) (n=7) (n=9)
Figure 4.17 Bar graphs showing the mean maximal uterine responses to
1.3mg/ml R. tridentata extract after the organs had been pretreated by various
cyclooxygenase inhibitors. The error bars represent the standard deviation
from the mean. The stars represent significant differences from the contractile
response to R. tridentata extract alone (*** = p< 0.001, **= p<0.01).
110
4.4.9, a-adrenoceptor antagonists
Pretreatment of uterine tissue with the a-adrenoceptor antagonists, prazosin

and yohimbine did not alter the response to the Rhoicissus extract.
120l a) uterus n=6 n=6

110 -
isII ioo4
90
80-
70-
60-
n=7
(0 T 3 50-
E s 40-
I8 30
E 204
10
0^
Rh only Prazosin Yohombine
tti V2
Figure 4.18 The bar graphs show the mean maximal uterine responses to
1.3mg/ml R. tridentata extract after the organs had been pretreated by
prazosin and yohimbine. The error bars represent the standard deviation from
response to R. tridentata extract alone (*** = p< 0.001, **= p<0.01).
111
4.4.10. Muscarinic receptor antagonists
Pretreatment of uterine tissue with the non-specific muscarinic antagonist,

atropine, blocked the uterine response to the R. tridentata extract, whereas
the antagonists on muscarinic subtypes Mi and M2, pirenzepine and galiamine
respectively, did not inhibit the uterine response to the extract. 4-DAMP, an
irreversible muscarinic antagonist specific for M3 receptors, reduced the
contractile response to the Rhoicissus extract. Tropicamide, a M4 receptor
antagonist blocked the contractile action of the plant extract.
70 uterus n= 8
S 604 n= 6
$ n=17
50
II 40
if
f0 10
E
as
30
20-
10- ***
n=8
***
n= 6
Rh only Atropine Pirenzepine Galiamine 4-D A M P Tropicamide

Mi Mg M3 M4
Figure 4.19 The bar graphs show the mean maximal uterine responses to
1.3mg/ml R tridentata extract after the organs had been pretrested by various
muscarinic antagonists. The stars represent significant differences from the
contractile response to R. tndentata extract alone (*** = p< 0.001, *= p<0.05).
112
4.4.11. Serotonin receptor antagonists
Pretreatment of both uterine and ileal tissue with the serotonergic receptor
antagonists, methysergide, ketanserin and tropisetron did not alter the
response to the Rhoicissus extract.
70-
a) uterus n= 6
n=7
60-
%
hl! 50-
40-
30-|
20 -
10-J
Rh only M eth ysergid e Ketanserin Tropisetron

5-H T 5-H T 1 5-H T 2
120
110 -
b) ileum
s 100 -
i 90
IsI
i
80
704
60
50
40-
n= 6
I 30-
20
104
0
Rh only M eth ys erg id e Ketanserin Tropisetron
5 -H T 5-H T, 5-H T 2
Figure 4.20 Bar graphs showing the mean maximal responses to 1.3mg/ml
R. tiidentata extract after the organs had been pretreated with various
serotonin antagonists. The stars represent significant differences from the
contractile response to R. tridf .ntata extract alone (* = p< 0.05).
113
4.4.12. The effect of R. tridentata on the basal tone of the uterus
As mentioned at the start of this chapter, Rhoicissus extracts increase the
basal tone of the uterus. This effect was apparent after the organs had been
rinsed at the end of a test challenge that included incubating the uterus with
the plant extract. The plant did not alter the basal tone of the ileum. The extent
to which the basal tone is increased was not correlated to the extent to which
the extract stimulated contractions, as shown in figure 4.21.
100 -
0>
© £
80- r2 = o.19
n
Q.CSO n = 243
60
-5
s e 404
++
eg -= 20
c
o
o
o 10 20 30 40 50
Increase in baseline
% maximal response to acetylcholine
Figure 4.21 The correlation between the contractions of the isolated rat
uterus stimulated by 1.3 mg/ml R, tridentata extract and the extent to which
the baseline was raised after the organ had been rinsed and allowed to return
to its resting state.
114
CHAPTER 5: Results Section 3
5. TEMPORAL AND SPATIAL VARIATION IN THE EXTENT OF

CONTRACTILE RESPONSE INDUCED BY R. TRIDENTATA
Investigations on the factors influencing the contractile activity were done

using uterine tissue. The same maximum dose of R. tridentata extract
(1.3mg/ml) was given in ail cases. Results obtained from extracts made from
Rhoicissus plant material harvested during different seasons indicated that the
contractile activity of the plant extracts is affected by the timing of harvesting.
The results are arranged on the axes from two different perspectives. The first
set of graphs (Figure 5.1) illustrate the extreme reactions from different parts
of the plant showing the effect of seasonal differences in the timing of
harvesting. The second set of graphs (Figure 5.2) is drawn from the same
data, but the data are arranged according to seasons to show the differences
resulting from the use of different plant parts.
5.1. Seasonal variation in the contractile activity of extracts from

different plant parts of Rhoicissus tridentata.
Tuber extracts: The extracts responsible for stimulating the largest

contractions were obtained from ne tubers harvested in summer and autumn
whereas the extracts from tubers harvested during winter or spring displayed
the lowest direct activity (Figures 5.1). At a 95% confidence level, the summer
tubers were more active than the spring and winter tubers, and the autumn
tubers were more active than the winter tubers, The uterine response to
maximal concentrations of acetylcholine was not significantly increased by the
summer, autumn and winter tuber extracts. The spring tuber extracts, however,
increased the maximal responses significantly (13% increase, P<0.05).
115
Root extracts: The extracts of roots harvested in summer stimulated the
largest contractions (27.2% relative to acetylcholine), whereas the extracts
from roots harvested in autumn and spring stimulated the smallest contractions
(6.6% and 5.5% relative to acetylcholine) (Figures 5.1). This is different from
the pattern seen in the tuber extracts, where the autumn tubers yielded the
most active extracts. The winter extracts gave a response between that of the
summer and spring extracts. At a 95% confidence level, the contractions
stimulated by the summer root extracts were significantly higher than the those
stimulated by the spring and autumn root extracts, and the results from the
winter extracts were higher than those from the autumn extracts. The maximal
uterine response to acetylcholine was not increased by any roots extract, but
the autumn root extract depressed the maximal response by 15% (P < 0.01).
Stem extracts: Summer stem extracts elicited the greatest contractile

response (36.5%) whereas the smallest response was to the spring extract
(8.7%) (Figure 5.1). The autumn and winter extracts gave similar initial
responses which were midway between the responses to the summer and
spring extracts, The only significant differences were between the summer and
spring extracts (P < 0.001). The summer stem extract was the only stem
extract to increase the maximal response to acetylcholine (112% relative to
acetylcholine) (P< 0.05).
Leaf extracts: Since the plant is deciduous, leaves were only available in
summer and autumn. Extracts from these two harvests directly stimulated
contractions equal in size (summer: 18.1%; winter: 18.4%), but the effects on
the maximal response to acetylcholine differed. The summer extract increased
the Maximal response to acetylcholine (106.3%) whereas the autumn extracts
inhibited the maximal response (88.6%; P<0.05) (Figure 5.1).
116
Tubers
s 1 00 -
8
c <D 80
60-
40 a Summer ( n = 4 )
O e Autumn ( n = 12)
CO 20-
vWinter ( n = i 9 )
* Spring ( n = 6)
+ acetylcholine ( n = l 6 )
100 - Roots
80-
ll
to o 6 0-
55
4 0 - Summer ( n = 4 )
I tCO v Winter ( n = 1 9 )
20 -
E ♦ Spring ( n = l 4 )
» Autumn ( n = l 3 )
+ acetylcholine ( n = i 2 )
100 Stems
8 80
11 60
I I
4 0 - Summer (n = 1 3 )
I f
Autumn ( n = 1 7 )
I s 20 Winter ( n = l 9)
♦ Spring ( n = i s )
0J
acetylcholine (n=l6)
-rl I— r —r- —r— —r-
Rh -9 -8 -7 -6 -4 -3
100 - Leaves
80-
II 60-
4 0-
20 A Summer ( n = i 4 )
• Autumn ( n = 22)
0-I + + —K"
+ acetylcholine ( n = l 6)
Hr —r— — r—
Rh -6 -4
Figure 5.1. Dose response curves of isolated rat uterus when pre-treated
with 1.3 mg/ml aqueous extracts of Rhoicissus tridentata showing the seasonal
variation in the contractile response to different plant parts.
117
5.2, Variation in the contractile activity o f extracts from different plant
parts of Rhoicissus tridentata.
Spring extracts: Extracts from all parts of the plants harvested during spring
displayed similar direct contractile activity (Figure 5.2). The stem and root
extracts did not alter the maximal response to acetylcholine whereas the tuber
extracts increased the maximal response (113.1%; P<0.05).
Summer extracts: The tuber extracts were the most potent, followed by the
stems, and then the roots, with the leaf extracts being the least potent (Figure
5.2). The leaf extracts were significantly less active than the tuber and stem
extracts (P< 0.05). Stem and leaf extracts increased the response to maximal
concentrations of acetylcholine (P<0.05).
Autumn extracts: The most potent extract again was obtained from the
tubers and the least potent extract was obtained from the roots (Figure 5.2).
The direct contractile response to the stem, leaf and fruit extracts was similar,
which was midway between the initial responses to the tuber and root
extracts. The tuber extracts were significantly more active than the root
(99.9% confidence level), stem and leaf extracts (95% confidence level), and
the stems were more active than the roots (95% confidence level). The
maximal response to acetylcholine was unchanged by the tuber and stem
extracts but depressed by the root and leaf extracts (95% confidence level).
Winter extracts: As in the case of the response to the spring extracts, the
initial responses to the stem, root and tuber extracts were similar. Root extracts
depressed the maximal response to acetylcholine slightly (P<0.01).
118
100- Spring
80-
I I 60
Is
40-
IS tm 20 -
v roots ( n = 1 4 )
tubers ( n = 6 )
stems ( n = i s )
acetylcholine ( n = i 6 )
Rh -9
100 - Summer
80-
ll 60-
I I
40
i t « tuber ( n = 6 )
'§ CO » stems ( n = i 3 )
20 -
V roots ( n = 1 4 )
a leaves ( n = i 4 )
acetylcholine ( n = i s )
100
Autumn
2
<n 80-
ll 60-
s i , ■ tubers ( n = i 2)
* fruit (n=5)
p 40 stems ( n = i ? )
8 to 20-
leaves ( n = 22)
0J » roots (n=i3)
acetylcholine ( n = i s )
A n
100 -
Winter
80-
$
II-
60-
o stems ( n = l 9 )
% to 20
V roots ( n = 1 9 )
• tuber ( n = is)
acetylcholine ( n = i 6 )
Figure 5.2. The effect of extracts of different parts of the plants harvested
during the four seasons.
119
5.3. Box and whisker plots o f range o f seasonal data
The data range of the direct contractile activity stimulated by the tuber extracts
is shown in figure 5.3. These graphs illustrate the wide range of responses to a
single extract. The response to the crude autumn tuber extract ranged from
13.5% to 93,5% of the maximal response to pure acetylcholine.
100 -,T u b e r s 100- R o o ts
OO
SI
g*
75-
50-
T IS
1 ™
75-
50-
a s
25 T II
^ &
$
25-
ZLT
Spring Summer Autumn Winter Spring Summer Autumn Winter
<n=8) (n=6) (n-12) (n=16) (n=14) (n=14) (n=i3) (n=l9)
100 Stems 100-1 Leaves

a
II
F ru it
I ”
•Is
® <D
75-
50-
II
c ®
II
75-
50
25-
T ^ a
25-
Spring Summer Autumn Winter Summer Autumn Fruit

(n=15) (n=l3) (n=17) (n=19) (n=14) (-1=22) (n=5)
Figure 5.3. Box and whisker plots of the maximal direct contractile responses to
1.3 mg/ml extracts from R tridentata harvested during different seasons. The box
extends from the 25th to the 75th percentile, with the horizontal line at the median.
The whiskers show the range of the data. The colour of the stars indicates the
season significantly different from the data set.
Table 5.1 Statistical differences between the contractile activity of extracts

from Rhoicissus from different plant parts harvested in different seasons.
Tubers Roots
spring and summer ** spring and summer **
spring and autumn *** summer and autumn *#
summer and winter *** autumn and winter

autumn and winter ** Stems
spring and summer
Key: * = p<Q,05, ** = p<0.01, *** = p<0.001
5.4. Seasonal variation in the increase in basal tone of isolated rat
uterus.
It was shown in chapter 3 that there was no correlation between the
contractile response to a Rhoicissus extract and the extent to which the basal
tone of the uterus was increased. On the other hand Figure 5.4 shows that
there are significant differences in the increase in basal tone between extracts
from plant material harvested during different seasons. When all part plants
are meaned the winter and summer extracts increase the basal tone to a
higher extent than ttn spring extract do (P<0.001), and the winter extracts
more than the autumn extracts (P < 0.05).
121
tubers
Summet
roots tubers
Autumn
roots stems tubers (eaves fruit

18-]
16 Winter
14-
12-
J if f 10 -
-IS 8-
IP 6-
4
2-
0
roots stems tubers
Mean of all plant parts

1
ii
.E th
8 8
II
Spring Summer Autumn Winter
Figure 5.4. Variation in the increase in baseline (mean lowest point) once
the organs had been rinsed at least five times after a test challenge which
included 1.3 mg/ml R. tridentata. The stars represent significant differences
•where the colour of the stars indicates the data set from which that specific
data set is different (e.g. the data from the summer and autumn extracts are significantly
different from the data from spring extracts).
122
5.5. Distributional variation
The results show that there is a variation in the pharmacological response to
aqueous extracts from R.tridentata stems and roots harvested from different
localities (Figure 5.5). All the extracts stimulated contractions, but the
magnitude of these contractions varied. The effect on the maximal response
to acetylcholine is where the interesting differences occurred. The extract from
the plant harvested in Mondeor was the only extract to shift the dose response
curve to the right and inhibit the response to maxima! concentrations of
acetylcholine, i.e. most of the extracts from R. tridentata appeared to act as
agonists whereas the extract from Mondeor had results similar to those
expected from an antagonist. The Mondeor extract seemed to compete with
acetylcholine in a non-competitive manner. This inhibition of acetylcholine was
negated by rinsing the bath, suggesting that the inhibition is reversible,
The possibility of misidentification of the Mondeor sample was investigated by

rechecking the herbarium specimens. The identification appeared to be
correct. The only apparent morphological difference was that the stem lengths
between the leaf axils were shorter than in other specimens, giving the
specimen a much denser appearance.
Extracts from R. tridentata roots and stems harvested from Suikerbosrand

during summer were previously shown to have similar contractile activity
(Figure 5.2). The root extract from the plant harvested at Umlazi gave a
response similar to that of extracts from stems harvested at Suikerbosrand.
However, the roots harvested near Durban yielded an extract that stimulated
contractions significantly higher than the contractions stimulated by the
Suikerbosrand stem extracts.
123
1 2 0 -1
Stems
100 -
8 80-
I I
60-
If
I I
8 ro
E
40-
20- x
Magaliesburg (n=5)
Morgenzon Bos (n= 6 )
a Suikerbosrand ( n = i 2 )
0J + -K " + ♦ Mondeor (n=5)
+ ACh, no extract (n=8)
t—r - -r- i
Rh -9 -8 -6 -4

120-,
Roots
100 -
b 80-
60-
it
— si
E
40-
20 -
a
® Durban (n = 8 )
Suikerbosrand (n=12)
v Umlazi (n=5)
0
+ ACh, no extract (n = 8 )
Rh -9 8 ■7 -6 5 4 ■3
Figure 5.5. The contractile response of isolated uterine smooth muscle to

aqueous extracts of R. tridentata stems and roots harvested from different
localities around South Africa. The tissues were incubated with 1.3 mg/ml
crude aqueous extracts for 5 minutes before the cumulative addition of
acetylcholine.
124
m
5.6, The effect of storing dried plant material
The contractile responses •) Rhoicissus stem and tuber extracts that had
been stored for either one or twelve months yielded identical contractile
responses from isolated rat uterus (Figure 5.6). The extracts stored for one
month or twelve months directly stimulated uterine contractions 27% ± 4 and
26% ± 5 (95% Cl) respectively, relative to the maximal contractions to
acetylcholine. Neither of the extracts altered the response to maximal
concentrations of acetylcholine.
100
80
jz ^
ll 60-
40-
i f
ffl (0 20
+ acetylcholine (n=26)
0J
a stored 1month (n=24)
stored 12 months (n=20)
Ti H r T — i— r
Rh -9 -8 -7 -6 -5 -4 -3
Figure 5.6. Uterine contractile response to R. tridentata extracts from plant

material that had been stored for 1 month (A) compared to extracts from plant
material that had been stored for 12 months (▼). The tissues were incubated
with 1.3 mg/ml of plant extract before the cumulative addition of acetylcholine.
125
RESULTS OBTAINED FROM CELL CULTURE EXPERIMENTS
6.1. Effect of R. tridentata Extracts on Prostaglandin Synthesis in

Histiocytoma Cell
As mentioned earlier, it would have been preferable to use a uterine cell line for these
following experiments, but no uterine cell line was available that had been shown to
produce prostaglandins at that time.
Extensive literature searches and discussions with scientists from all over the world
could not reveal in what time span prostaglandins were synthesized. Therefore, the
first step was to determine what incubation time should be used to detect the
maximum levels of prostaglandins. Cells were incubated with 1 mg/ml of plant extract,
compared to 1.3 mg/ml used in the isolated organ experiments.
6.1.1. Prostaglandin synthesis over time

Once challenged with R. tridentata extract, the histiocytoma cells start excreting
prostaglandins into the cell medium almost immediately. The levels of extracellular
prostaglandin Ea reach a maximum at 15 to 20 minutes, after which there is a rapid
drop in the extracellular concentrations of the prostaglandins (Figure 6.1).
60n
3 50-
Q.
c 40-
3 0 -
20 -
10 -
0 30 60 90 120 150 180
Time (minutes)
Figure 6.1. Prostaglandin Eg synthesis over time by histiocytoma cells stimulated
with 1 mg/ml Rhoicissus extract.
126
6,1.2. Dose-response curves of the effect o f R. tridentata on
prostaglandin synthesis by histiocytoma cells
R tridentata extracts only stimulated minimal amounts of prostaglandin Ea
synthesis at plant extract concentrations of 0.1 p.g/ml to 0.1 mg/ml, whereas
1mg/ml caused a 7.8 fold increase in the amount of extracellular prostaglandins
compared to untreated cells (Figure 6.2). This significant increase in
prostaglandin synthesis was not evident in intracellular prostaglandin E2.
300-i
Extracellular
275- Intracellular
250- ■v- Total
| 225-
g 200-
I 175-
B 150-
§ 125-
§ 100-
g 75-
50- .—-v-
25-
-7 -6 5 -4 ■3
Log Rhoicissus extract concentration (g/ml)
Figure 6.2. Dose response curve of intracellular, extracellular and total

prostaglandin concentrations after histiocytoma cells were incubated with varying
concentrations of four different Rhoicissus extracts for 20 minutes.
127
6.2. Toxicity assays
6.2.1. Parameters fo r the NITT Assays
Different numbers of human histiocytoma cells were incubated with MTT for four
hours, without the addition of any plant extract to determine what would be the
optimal number of cells to have per well. Figure 6.3 shows that the absorbance at
three wavelengths, 405nm, 540nm and 620nm, all increased exponentially
between 690 and 5 500 cells per well. The absorbance tapers off, increasing
linearly at a cell concentration above 5 500. The vertical dotted line marks 13 500
cells per well which is the concentration of cells used for the MTT assays for all
cell lines.
2.5-1
2. 0 -
<o
o 1.5-
c
$0
_Q
10 1 .0 -
L=
0.5-
o.oJ
♦ 405nm
a 540nm
v 620nm
0 5000 10000 15000 20000 25000
NumL 3r of cells per well
Figure 6.3. The optical density at different wavelengths corresponding to
different concentrations of histiocytoma cells per well.
The absorbance spectrum from 340nm to 690nm for the different concentrations
of histiocytoma cells per well, which have been incubated with MTT for four hours,
is shown in Figure 6.4. The absorbance either peaks or is close to the peak at
540nm for all cell concentrations. Similarly, when 13 500 cells per well are treated
128
with varying concentrations of R. tridentata extract for 24 hours, followed by
treatment with MTT for 4 hours, the absorbance also peaks at around 540nm, as
indicated by the vertical dotted line (Figure 6.5). Based on the above results, the
absorbance was read at 540 nm for all the following MTT assays.
Number of cells/ well
2.5-1
— 22050
2.0 - 11025
■5512
0 1.5-
2756
1
■1378
t 1-0H
689
3
< 0.54
0.0J
1--------- ;------,--------;--------1------- 1------- 1
340 390 440 490 540 590 640 690
Wavelength (nm)
Figure 6.4. The absorbance spectra from wells with different numbers of
histiocytoma cells.
1.50-1
1 .2 5 -
1x100 mg/ml
0) 1.0 0 - — 1x10-img/ml
<2 — 1x10-2mg/mli
ra 0 .7 5 4
JQ — 1x10-3mg/m|
8 0 ,5 0 -
— 1x10-4mg/mll
< 0 .2 5 4 cells alone
0.00-J blanks
340 390 440 490 540 590 640 690
Wavelength (nm)
Figure 6.5. The absorbance spectra from wells containing 13 500 histiocytoma
cells and treated with different concentrations of R. tridentata extract.
129
6.2.2. Reason for method modifications
Mossman et at. (1983) developed the MTT assay for the rapid screening of large
numbers of samples. They analyzed their results by subtracting the absorbance at
620nm from the absorbance read at 570nm. The absorbance at 620nm
represents the absorbance of the culture media and the sample being tested. The
assay was designed to save time by avoiding the removal of media, and therefore
they had to account for the background absorbance. In preliminary assays for this
study, it was found that removing the culture medium and dissolving the formazan
crystals with DMSO yielded far more consistent results, and avoided the step of
having to mix the solutions within the wells using a multichannel pipette. Since, in
this modified method, all the culture media and plant extract are being removed it
was no longer necessary to account for the absorbance caused by these two
components. It was therefore decided to represent cellular viability as the
absorbance obtained from the test wells in relation to the absorbance obtained
from the control wells. The results using the different calculations are given in
Figure 6.6. The results from the highest concentrations of plant extract were
discarded, as a large proportion of the plant extract lysed the cells as the
osmolarity was too high, which accounts for the discrepancies in the results using
the different calculations at a plant concentration of Log-2 g/ml (0.01 g/ml).
130
• 5 4 0 nm (test-blank/control-blank)
0-L-1—| H]— —t--------r--------j-------- 1-------- r~
C ontrols -7 -6 -5 -4 -3 -2
1 .00-1
0)
0
c
1o 0 .5 0 -
JS
<
540-620nm
540-405nm
o.oo
C on trols -7 ■6 5 •4 3 2
100 -
! 75-
50-
25-
0-
I
-2 5 -
-50
-75
-100
-1 2 5 -
-1 5 0 -
t - 540-620nm / contro s
< -175-
200 - t- h h-r ■540-405nm / contro s
- —r ~r
Controls -7 -6 -5 -4 -3 -2
Log Rhoicissus concentration (g/ml)

Figure 6.6. Dose response curves of the results from the M IT assays using
different calculations to analyze the results. The top graph represents results
calculated from the absorbance at 540 nm, where the absorbance from the test
wells was calculated as a percentage of the control. The middle graph represents
the absorbance of the results, where either the absorbance at 620nm or 405nm is
subtracted from the absorbance at 540nm. The bottom graph displays the results
in the middle graph as a percentage of the controls.
The curves in the following section represent the results from MTT assays used to
measure the cellular viability after the four different cell types were exposed to a
range of concentrations of aqueous extracts of R tridentata harvested at
Suikerbosrand and Durban. The samples were chosen to represent a wide range
of contractile activities. The points on the graphs represent the mean and the error
bars represent the standard errors of the mean. Stars on the graphs indicate
significant differences between that value and the control (* = p < 0.05;
**= PO.Oi; *** = P<0.001).
6.2.3. MTT assay results from different cell lines

The results from the cytotoxicity assays using R. tridentata extracts from all plant
parts and different seasons, on the four cell lines show that the extracts do not
reduce the cellular viability to below 90% relative to the controls in any of the cell
lines. On the contrary, the absorbance at 540nm for most of the assays on human
kidney, human hepatoma and mouse leydig cells, was above that of the controls
for all concentrations of the plant extracts (Figure 6.7). Significant differences
between the absorbance of the test wells and that of the controls wells were only
evident at the higher concentrations of plant extract in the hepatoma, leydig and
histiocytoma cells, but not in the kidney epithelial cells. In the histiocytoma cells,
the absorbance of the wells with lower concentrations of plant extract was below
that of the control wells. However, the decreases were minimal, the mean
absorbance was always above 90% of that of the control wells. The results from
student-t tests are given in the appendices (Table A.4).
132
140-i
Ley dig cells (192 wells)
01 130-
2 Hepatoma cells (48 wells)
8 izoH —<*—Kidney cells (192 welis)
110 - —v - Hstiocytoma cells (128 wells)
S 100-
■i
90-
o
80 -H i—r I
Control -7 -6 -5 -4 -3
Log Rhoicissus extract concentration (mg/ml)
Figure 6.7. Dose response curves showing the cellular viability after the cells
were incubated with varying concentrations of Rhoicissus extracts for 24 hours,
The graph represents the mean results from extracts tested in the number of wells
given in the legends. Significant differences between the absorbance of test wells
and control wells are shown in later graphs.
133
6.2.4. Kidney epithelial cells
120- ]
'to'
1c 110 -
o
o
o
*
& 90-
n
"I 80-
m
O
Controls -7 •6 •5 -4 •3
Figure 6.8. Dose response curve of cell viability after being incubated with
varying concentrations of Rhoicissus extracts for 24 hours. The graph represents
the mean results from extracts of 16 different plant samples tested in 12 wells
each. No significant differences between the absorbance of test wells and control
wells occurred.
120-
2 100'- —- - - | iio-
i 1H 1 100- ■*5c
% 90-
90-
60- tubers
70- roots
70- - s ~ Growing = stems
Dormant °
leaves
■7 -6 -5 -4 ■3 Controls -7 -6 ■5 -4 ■3
Log R hoicissus extract concentration (mg/ml) Log R hoicissus extract concentration (mg/ml)
Figure 6.9. The effect of Rhoicissus extracts from a) different seasons and b)
different plant parts on the viability of human kidney epithelial cells. Stars indicate
significant differences between the extracts.
134
6.2.5. Hepatoma cells
120-]
2 110-
C
o
*0 100-
&
i- 90-
JD
.2
> 80-
0)
o
70- -4 1—r I 1 —
i
Controls -6 --5
5 -4 -3
Figure 6.10. The cellular viability of human hepatoma cells after being incubated
with aqueous extracts of Rhoicissus for 24 hours. The cu ves represent results
from 8 plant extracts tested in 6 wells each. Stars represent significant
differences between the absorbance of test and control wells.
130
- ® - Growing - Tubers
120 - — Dormant I" 120-1 - Stems
c
110 - 8 no
100 - £ 100 -
4 ^
90- 90-|
80- <D 80-

o
70 -*-i— I t—r- 7 0 — I I—i------------ 1--------------1------------- 1------------- r -
Controls -7 -6 ■5 -4 •3 Controls -7 -6 -5 -4 -3
hepatoma cells after being exposed to the extracts for 24 hours. There were no
significant differences.
135
6,2.6. Mouse leydig cells
140-i
(ft 130-
Q
O 120 -
O
110 -
100
Si
90-
I
<D 80-
o
Controls -7 -6 -5 -4 -3
Figure 6,12. Results from the MTT assay testing the effect of Rhoicissus
aqueous extracts on the viability of mouse leydig cells after being exposed to the
extracts for 24 hours. Each point represents the mean of extracts from 16 plant
samples each tested in 12 wells.
Dormant roots
leaves
Growing
tuber
- 140- ■S 140 stems
£ 120-
g 100-
80 60
Controls -7 -6 -S -4 -3 Controls -7 -6 -5 -4 -3

different a) seasons anc different b) plant parts on the viability of mouse leydig
cells after being exposed to the plant extracts for 24 hours.
136
6.2.7. Human histiocytoma cells
120-1
110 -
C
8
•5 100 -
% 90-
•55
I
O
§
Q.
80-
60
Controls 7 ■6 ■5 -4 ■3
aqueous extracts on the viability of human histioctytoma cells after being
exposed to the extracts for 24 hours. Each point represents the mean of extracts
from 16 plant samples each tested in 8 wells.
150-i —® - Dormant tubers

2" 140- — Growing roots
stems
1 130- leaves
! 120
£ 110
•t ioo4
0) 90
*o
To 80
70
O
60
Controls -7 -6 -5 -4 -3 Controls -7 -6 -5

histiocytoma cells after being exposed to the plant extracts for 24 hours.
137
6.2.8. Seasonal differences
The cellular viability of the hepatoma and leydig cell lines after being exposed to
R tridentata extracts obtained from material harvested during either the dormant
or growing seasons yielded similar results (Figures 6.11a & 6.13a), The only
differences in the kidney epithelial and histiocytoma cells occurred at the higher
concentrations of plant extract (Figures 6.9a & 6.15a). At the highest
concentrations of plant extract, extracts obtained from dormant plant material yield
a lower absorbance than extracts obtained from growing plant material. However,
the differences are negligible. Results from the statistical analyses are given in the
appendices (Table A.5).
6.2.9. Plant parts used
There were no significant differences between the results from tuber extracts and
those from stem extracts in the hepatoma cell line (Figure 6.11b; Table A6). At a
confidence level of 99%, there were no differences between the cellular viability
after leydig cells had been exposed to a range of extract concentrations from
different parts of R. tridentata (Figure 6.13b; Table A.8). Minor differences in the
viability of kidney epithelial and histiocytoma cells occurred after exposure to
exposed to extracts from different parts of R. tridentata. The viability of kidney
epithelial cells after being exposed to root extracts was about 10% lower than the
Viability after being exposed to either tuber or stem extracts (1 mg/ml; P< 0.001;
figure 6.6b; Table A 7). The viability of hfeiiocytoma cells was slightly lower after
exposure to stem extracts than was the case after exposure to either tuber or
root extracts (1 mg/ml; PO.OOI ; Figure 6.15b; Table A.9).
138
6.3. Relationship Between Contractile Activity and Effect of Cellular
Viability
The following graph shows the relationship between the contractile activity and
the toxicity of R, tridentata extracts. The data from the previous experiments were
analysed to determine whether the contractile activity was correlated to the
cellular toxicity of the extracts.
There is a very poor correlation between the direct contractile activity of R.
tridentata extracts on isolated rat uterine tissue and the effect of the same extract
on cellular viability in all cell types tested (Figure 6.16).
SO-,
"cT
X Grahams (r2=o.o44)
+ Leydig (1-2=0 .21 4)
i 40-
x +++ + X Hepatoma (r2=0.029)
•S'S + Histiocytomas (r2=o.l84)
i= 2 30-
O <D
es +
C D. 20- -T
02 y x NxX-— +
i 10-
+
X X + - +
&
0- i
1 iI "Ii
0 100 200 300
Cell viability (% controls)
Figure 8.16. Correlation between the direct contractile activity of 1.3 mg/ml R.
tridentata extract on isolated rat uterine tissue and the viability of all cell types
tested after exposure to 1mg/ml R. tridentata extract for 24 hours.
139
CHAPTER 7: DISCUSSION AND CONCLUSIONS
7.1. Interviews with traditional healers
7.1.1. Training of traditional healers

With Gauteng being a center of immigration from rural areas all over Southern
Africa, one would expect the traditional medicines used to represent a mixture
of plants used by the different cultures. However, most of the healers spoken
to were of Zulu origin and cited Zulu names for the plants. This could be a
feature of the Faraday market (Central Johannesburg) and could indicate that
the traditional healer associations contacted were biased to Zulu origins. This
does not necessarily indicate that there are more Zulu remedies than
remedies from other cultures. Anecdotally, the general perception in Gauteng
is that most traditional remedies are of Zulu origin.
When describing the methods of training to become an inyanga, all traditional

healers described how the mentor would teach the pupil how to interpret
dreams. None of the healers were taught about what plant to use for specific
indications. The trainee would then report back to the mentor, telling the
mentor details of dreams which included what plants should be used for a
particular ailing patient. Some mentors would write down the plant names
together with the relevant indication without ever teaching the trainee healer
about the medicinal properties of the plants. The transfer of knowledge
appeared not to follow a logical direction. Two of the healers spoke about their
training with deep dissatisfaction and discontent. They would have preferred
more rigorous, thorough and perhaps more academic training. None of the
healers underwent long periods of apprenticeship, contrary to the common
perception of how traditional healers are trained. The minimum training period
was three months and the maximum was three years. These results do not
mean that no traditional healers ever undergo several years of training.
Reports in the literature about traditional healers undergoing years of
thorough training are numerous (Schuster Campbell 1998), but these healers
are usually located in rural areas (Gericke 1999). However, these results do
140
show that there are many inadequately trained traditional healers within urban
areas who are prescribing plants containing pharmacological compounds that
are potentially very toxic.
Most healers and plant vendors spoken to were very keen for their plants to
be tested in the laboratory to determine whether their plants are effective for a
specific indication. On the whole, they were pleased to hear that “isinwazi”
(Rhoicissus spp.) does cause contractions of isolated uterine tissue. This
willingness of traditional healers to accept positive results is consistent with
previous reports (Simon 1991; Green et al. 1995). However, when told that
the plant had on occasions caused fatal poisonings, or that the contractile
activity of the species was highly variable, they either paid little attention or
openly showed their disapproval of what they were being told. This opposition
to negative implications of traditional medicines is consistent with the
approach of traditional healers when told of the number of childhood deaths
caused by “impila" (Callilepis laureola) (Hutchings 1999).
The traditional healers’ ignorance of the toxic potential of R. tridentata despite

reports implicating the plant in fatal poisonings (Watt & Breyer-Brandwijk 1962;
Brandt & Muller 1995) suggests that perhaps only certain plant samples are
toxic. The possibility exists that the depressing effects of the plant extracts on
the CNS fluctuate seasonally, similar to the fluctuations seen in the contractile
action. This would explain why the traditional healers are generally unaware of
the toxic potential of Rhoicissus tridentata. Another possibility is that the plant
only contains sufficiently high concentrations of the toxic compound(s) when
harvested from a specific location. Further investigation should be done to
determine whether the poisonings that have occurred are clustered around the
harvesting of plant material during a certain season or from a specific location.
(Unfortunately clinical records of the poisonings reported by Brandt & Muller
1995 could not be located by the staff at Garankua hospital.)
The toxicity of R. tridentata may be disguised by the healers using the plant in
mixtures of two or three ingredients. The other remedy ingredients may act as
antidotes to the toxic effects of Rhoicissus, or Rhoicissus may be diluted, thus
141
avoiding toxic effects. The combination of factors may explain why the healers
don't recognise Rhoicissus as the poisonous ingredient.
7.1.2. Plants used during pregnancy

Insufficient traditional healers were interviewed to determine conclusively how
many plants are being used during pregnancy by black women in Gauteng.
However, the results do give an indication of which plants are being used.
The traditional healers interviewed mentioned 46 ingredients which could be

identified using published lists of plant names (Gerstner 1941; Watt & Breyer
Brandwijk 1962; Hutchings et al. 1C96). A further 9 ingredients were not included
in any of the lists. 40 ingredients were plants, 4 were animal products and 2 were
non-organic (Table 3.1; Page 75). Of the identified plant species, 23 were
documented in the literature as being used in pregnancy-related remedies (Table
3.3; Page 84), 19 were documented to be toxic, of which 12 have been reported
to have caused fatal poisonings to either humans, dogs or sheep (Table 3.4;
Page 87).
Eleven plants were being used in general “cleansing" remedies which is

consistent with the perception that it is the African traditional healers' role to
diagnose the source of a problem occurring during pregnancy and that the
foetus needs to be protected from harm while in the womb. These remedies
are used for both spiritual and physical catharsis. There were many specific
indications mentioned by the traditional healers (Table 3.2; Page 81). The
highest number of plants were used for aiding maternal and foetal strength
throughout pregnancy and for easing labour.
Rhoicissus tridentata and Clivia rmxafawere both mentioned as being used to

prevent miscarriages, yet both have been shown to cause uterine contractions
in vitro. Therefore, these two plants are more likely to increase the chances of
a spontaneous abortion than to prevent it.
Most citings in South African literature on the use of plants during pregnancy
do not stipulate specific indications for using herbal remedies during
142
pregnancy. However, during interviews traditional healers seldom used the
terms isihfambezo or imbelekisane. They spoke of specific pregnancy-related
indications for each plant. This is consistent with Mbura et a!. (1985) who
interviewed Tanzanian women in antenatal clinics on the reasons why they
used traditional remedies during pregnancy. They also listed the use of plants
according to specific pregnancy-related complaints.
Callilepis laureola was reported to be used during pregnancy to enhance foetal

and maternal health as well as to ease labour i.e. the plant may be prescribed
at any stage of gestation. The plant is also used specifically to calm a restless
child, yet C. laureola has been implicated in many fatal childhood poisonings
in South Africa (Stewart et al. 1998). The toxic principles are atractylosides
which often kill the patient within 24 hours, often before they are able to reach
a hospital. Atractylosides cause hypoglycaemia which in turn inhibits oxidative
phosphorylation. The symptoms of poisoning usually include centrilobar
hepatic necrosis, acute renal failure, disturbed consciousness and convulsions
(Steenkamp etal. 1999).
The prevalence of remedies used specifically for children could reflect the
morbidity and mortality patterns typical of a poverty-stricken population where
the main burden of disease is on children within the first 5 years of life
(Unterhalter 1982).
7,2. Pharmacological action of R. tridentata on isolated organs
7.2.1. Inorganic ion concentrations

The ion content of the plant extract once diluted in the Tyrode solution in the
organ baths was too low to have any direct effect on the isolated smooth
muscle preparation. This indicates that the contractions were stimulated by
pharmacologically active ingredients) within the plant extract, and not the
action of ions on the cell membranes.
143
7.2.2. Reference drugs
Uterus: Oxytocin was the most potent and efficacious agonist on the
oestrogenised non-pregnant isolated uterus. The efficacy of acetylcholine was
equal to that of oxytocin, but the potency was approximately three orders of
magnitude lower. Adrenaline had only half the efficacy of acetylcholine and an
even lower potency (Figure 4.1a; Page 90).
Ileum: The efficacy of acetylcholine was double that of serotonin while the
potency on a molecular concentration was a hundred times that of serotonin
(Figure 4.1b; Page 90).
7.2.3. Direct activity
The direct activity of the Rhoicissus extract (Figures 4.2a & b; Page 93) shows
that the plant is able to stimulate concentration dependent contractions of both
isolated uterus and ileum in vitro. The ileal response was sigmoidal and
reached a maximal effect plateau at 7.9 ng/ml, whereas the uterine response
was hyperbolic, i.e. the uterine response to the doses given was increasing
exponentially at a plant concentration three orders of magnitude higher than
that yielding the maximum ileal response. The Tyrode solution was virtually
opaque when the highest concentrations of the plant extract were used. To
add even higher concentrations of plant extract would not only have depleted
the stocks of plant material but would have also changed the ionic
concentrations of the Tyrode solution.
The crude extract stimulated contractions of approximately 30% of the

maximal contractions stimulated by acetylcholine in both the uterus and ileum.
Figure 4.3 (Page 93) shows that there was a wide range in the contractile
response to the plant extract. The wide range in responses seems to be
particular to R. tridentata, as this was also evident while doing antimalaria!
tests on the plant extracts (data not included). Simultaneous dosing of two
isolated uterus or ileum preparations, made from the same animal, in different
organ baths alongside one another with the same extract could yield
significantly different responses. One preparation could not respond at all
144
whereas the other could contract 40% or more relative to the maximal
response to acetylcholine. The maximum responses to the Rhoicissus extract
in the isolated rat uterus and ileum were 81.7% and 98,1% of the maximal
response to acetylcholine, respectively. These are exceptionally high
responses considering that the plant extracts were crude aqueous extracts.
The crude aqueous extract of R. tridentata directly stimulates contractions of

both isolated uterus and ileum, yet a permanent increase in the baseline is
only seen in the uterus (Figures 4.4a & b; Page 94). The mechanism by which
this occurs is not known.
The results obtained from inese in vitro experiments cannot be directly applied
to the clinical setting as it is not known whether the active component(s) in the
plant extract is(are) absorbed from the gastrointestinal lumen and whether or
not the compound(s) are resistant to first pass metabolism. However, these
data provide pharmacological evidence that should the active ingredients in
the plant extracts be absorbed, then they will be able to stimulate contractions
of ileal and uterine smooth muscle, and therefore there is the potential that
they are able to stimulate contractions in vivo.
Assuming that the active component(s) of the plant is(are) able to stimulate
contractions in vivo, the plant extract would increase uterine contractions, and
thus augment labour and increase the risk of birth complications associated
with uterine hypertonia, such as foetal distress. The extract can nlso
stimulates ileal contractions and therefore has the potential of stimulating the
foetal intestinal wall, directly increasing the incidence of meconium staining.
7.2.4. Oxytocic receptor system

The plant extract did not alter the response of isolated uterus to oxytocin,
suggesting that the plant extract does not possess antagonistic or non
competitive agonist activity on oxytocin receptors (Figure 4.5; Page 96). No
deductions can be made on whether the plant extract is able to competitively
agonise the oxytocin receptors or not, as no oxytocin antagonists were
available. Atosiban is an oxytocin antagonist that could be used in the
145
laboratory (Bossmar 1998). A source for this compound, however, could not
be located.
7.2.5. Prostaglandin synthesis

The direct action of the plant extract was significantly inhibited by
pretreatment of the organs with the cyclooxygenase inhibitor indomethacin in
both the uterus and the ileum (Figure 4.6a & b, Figure 4.17; Pages 97 & 110).
This suggests that products of cyclooxygenase, such as prostaglandins or
thromboxane A2, may be involved in the contractile response to the plant
extract.
As mentioned in the introduction, prostaglandins are thought to play a vital

role in the onset and progress of labour. Prostaglandins increase the
formation of gap junctions facilitating rapid transfer of action potentials
between cells, and directly stimulate uterine contractions. If the Rhoicissus
extract is able to induce prostaglandin synthesis in vivo, the plant extract has
the potential of contributing towards the or set of labour and of augmenting
contractions during labour.
COX-1 and COX-2 proteins are both expressed in rat uteri. They are primarily
localised to epithelial cells of the endometrium and smooth muscle cells in the
circular layers of the myometrium (Dong et at. 1996). NS-398 has been shown
to prevent the augmentation of prostaglandins produced by IL-1 alpha in
oestrogenised rat uterus (Franchi et al. 1998). Indomethacin inhibits COX-1
and COX-2 activity to the same degree (Futaki et al. 1994), whereas NS-398
is a COX-2 specific inhibitor (Futaki et al. 1994). At a concentration of 0.1 pM,
NS-398 did not alter the activity of the crude Rhoicissus extract whereas 1pM
inhibited the contractile response (Figure 4.17; Page 110). These data
suggest that R. tridentata promotes the synthesis of prostaglandins through
COX-1 and COX-2.
An increase in plasma levels of prostaglandin synthesis has been found to

occur when other labour inducing agents have been used. Orally administered
castor oil (60ml) which was found to induce labour in 75% of patients with
146
prematurely ruptured membranes, as opposed to 58% of controls. After
administration of the castor oil in these patients, it was found that there were
increased levels of circulating prostaglandin E (Davis 1984). Agapanthus
africanus leaves also stimulate uterine contractions by promoting
prostaglandin synthesis (Veale etal. 1999).
7,2.6. Adrenergic receptor system

The adrenergic system is not thought to play a crucial role in the progression
of labour, but rather in the inhibition of labour. (3-receptors mediate myometrial
relaxation. This aspect of p-receptor activity is utilised therapeutically in the
treatment of premature labour.
Noradrenaline had less than half the efficacy of acetylcholine causing the R.
tridentata extract to appear more active than when compared to acetylcholine
or oxytocin (Figure 4.1a; Page 90 & Figure 4.7; Page 98). The contractile
response of the uterus to the plant extract when compared to acetylcholine is
18% ± 4 (SEM) whereas when it is compared to noradrenaline the response is
65% ± 8 (SEM). If the response to the extract relative to noradrenaline is
corrected according to the response of noradrenaline relative to acetylcholine,
the response to the plant extract works out to be 17 ± 2 (SEM) which is the
same as the response when compared to acetylcholine.
The response to the plant extract after blocking the (3-adrenoceptors with
propanolol is the same as without propanolol (graph not given). This suggests
that the plant extract does not stimulate the (3-adrenoceptors. The plant
extract did not alter the isolated uterus response to noradrenaline once the (3-
adrenoceptors were inactivated by propanolol, leading to the inference that
the plant extract has neither agonistic nor antagonistic activity on j3-adrenergic
receptors.
Pretreating the uterine tissue with the ai-adrenoceptor antagonist prazosin

(2nM) or the az-adrenoceptor antagonist yohimbine (2|iM) both successfully
antagonised the a-adrenoceptors as is evident in the shift of the uterine dose
147
response curves of noradrenaline to the right, and the suppression of the
maximal response to noradrenaline (Figure 4.7 & 4.8; Pages 98 & 99). These
doses of prazosin and yohimbine did not alter the organs' response to the
plant extract alone or the dose response curve to noradrenaline once the
tissue had been pretreated with the plant extract. These data suggest that the
plant extract does not have any agonistic activity on either of the a-
adrenoceptor subtypes.
7.2,7. Muscarinic receptor system

Uterus and ileum: The contractile response to the plant extract of both uterus
and ileum was completely blocked by pretreatment of the organs with the
muscarinic antagonist atropine (40nM) (Figure 4.9a & b; Page 101). The same
concentration of atropine shifted the dose-response curve to acetylcholine to
the right, without significantly reducing the maximal response to acetylcholine.
These results suggesting that muscarinic receptors are involved in mediating
the contractile response to the plant extract.
Preincubation of the organs with both atropine and the plant extract caused
further inhibition of the response to acetylcholine, shifting the dose response
curve further to the right. In both organs the response to maximal doses of
acetylcholine was suppressed by pretreatment with atropine (40nM) and the
plant extract (1.3 mg/ml).
Muscarinic innervation is of greater clinical significance in the intestinal wall

than in the uterus. Figures 4.2b & 4.4b (Pages 93 & 94) show that the
aqueous extracts of R. tridentata stimulate contractions of the isolated rat
ileum. The contractions appear to be mediated by muscarinic receptors
(Figure 4.9b; Page 101). Provided that the active component of the plant
extract which stimulates muscle contraction is absorbed into systemic
circulation in a pharmacologically active form, and is able to pass into foetal
circulation, Rhoicissus extracts have the potential of directly stimulating
muscarinic receptors in the foetus, which in turn may increase the incidence of
foetal meconium staining. This provides possible pharmacological evidence
148
for the correlation drawn by Mitri et a/. (1987) that ingestion of herbal
remedies towards the end of gestation increases the incidence of foetal
meconium staining.
Muscarinic receptors are present in many different smooth muscle tissues.

Again, assuming that the active component(s) of the plant is (are) absorbed
into systemic circulation and are able to withstand first-pass metabolism, the
plant extract may stimulate these muscarinic receptors, resulting in many
effects not related to parturition, such as, decreased heart rate, increased
salivation, abdominal cramps, diarrhoea, urinary urgency and miosis.
7.2.8. Muscarinic receptor subtypes and the uterine response

The Mi, Ms and M4 muscarinic receptor antagonists, pirenzepine (1.5gM), 4-
DAMP (2nM) and tropicamide (4.5|iM), respectively, all shifted the uterine
dose-response curves to acetylcholine to the right, whereas the M2 receptor
antagonist gallamine (1.5|j.M) had no effect on the uterine response to
acetylcholine (Figures 4.10 a-d; Page 103). These results suggest that
contractions in rat uterus are mediated by Mi, M3 and M4 receptors.
Muscarinic receptor populations in different animals

A first glance at the above results could lead to the conclusion that they are
incorrect, as since M2 receptors account for 80% of the total receptor density
in most smooth muscle (Ehlert et al. 1997), one would expect the M2
receptors to mediate uterine contractions. However, the prevalence and
efficacy of different muscarinic receptors differ in different animal species as
well as in the different smooth muscles within the species.
No reports on the muscarinic receptor subtype populations of rat uterus could

be found, but in rat and pig ileum, these three receptor subtypes were found
to be present as minor receptor populations (Ehlert et al. 1997). In guinea-pig
ileum, M3 receptors were found to be the principle receptors mediating
149
contractions (Boxall et ai. 1998). Mi receptors were detectable, but only in
trace amounts and WUwere not detected at all (Maeda etal. 1988).
In many smooth muscle tissues a minor Ms muscarinic receptor population

mediates contraction, despite IVh outnumbering Ms receptors by a factor of
about four (Boxall etal. 1998; Ehlert et al. 1999). Pretreating the organs with
gallamine only, blocks Mz receptors but leaves Ms receptors unopposed. In
order to test the role of Mz receptors, the Ms receptors have to be inactivated
with a Ms antagonist such as 4-DAMP together with histamine and a relaxing
agent such as isoproterenol orforskolin (Ehlert etal. 1999).
Interpretation of results
Pretreatment with pirenzepine and gallamine (Mi and Mz antagonists
respectively) did not alter the uterine response to the R. tridentata extract,
even though pirenzepine antagonised acetylcholine. These results suggest
that in the case of pirenzepine, the active component(s) of the extract has
(have) a very low affinity for the Mi receptors, or that the intrinsic activity is
low.
The uterine response to the extract in the presence of gallamine is unaltered.

Despite the Ma receptor population being the most dense, M2 receptors
appear not play a prominent role in mediating the contractile response to the
extract.
Pretreatment with 4-DAMP (2nM; Ms antagonist) partially blocked the

contractile activity of the extract whereas tropicamide (4.5|aM; M4 antagonist)
completely blocked the contractile activity of the f t tridentata extract. Both
antagonists caused a suppression of the maximal response of the uterus to
acetylcholine in the presence of the extract. These results suggest that the
contractile response to the extract is mediated by both M3 and M4 receptors.
150
No investigations on the involvement of receptor subtypes in the ileum were
done.
7.2.9, Serotonergic receptors

Serotonin receptors are also not important in labour, but are important in the
contraction of the intestinal wall. Pretreating the organs with 1.3 mg/ml plant
extract does not alter the uterine or ileal response to serotonin (Figure 4.11;
Page 104). This suggests that the plant extract does not have an affinity for
serotonin receptors.
The serotonin antagonist, methysergide, at a concentration that shifts the

serotonin dose-response curve to the right, does not alter the uterine or ileal
response to the plant extract alone, or to serotonin after pretreatment with the
plant extract. These results suggest that the contractile response of the
organs to the plant extract does not involve serotonergic receptors.
This is confirmed by data represented in figures 4.12 and 4.13 (Pages 105 &
106) which show that ketanserin and tropisetron, which are antagonists on 5-
HTi and 5-HT2 receptors respectively, do not alter the response of the uterus
or ileum to the plant extract.
7.2.10, Histamine receptors

Pyrilamine (8pM) did not alter the direct contractile response of the isolated
rat uterus to the aqueous R. tridentata extract (Figure 4.15; Page 108). This
suggests that histamine receptors are not involved in the contractile response
to the extract. No experiments on ileum were done.
7.2.11, Ganglionic receptors

Hexamethonium and mecamylamine (in micromolar concentrations) have
been shown to block contractile responses mediated by stimulation of nicotinic
receptors in many studies using isolated organs (Bartho et at. 1987; Okamura
et al. 1995; Hopkins et al. 1998). In this study the ganglionic blocker,
151
hexamethonium (1.4pM) and the non-competitive nicotinic antagonist,
mecamylamine (2 ^M) both did not alter the contractile activity of the plant
extract on the uterine smooth muscle (Figure 4.14; Page 107). These results
indicate that the uterine contractile activity of the plant extract does not involve
nicotinic receptors.
7.2.12. Second messengers

The islet-activating protein, pertussis toxin (50|iM) inhibited the direct uterine
contractile activity of the plant extract from 30.3% to 18.7% of the maximal
response to acetylcholine (Figure 4.14; Paje 107). That is a 38% reduction in
the contractile activity. However, this reduction in activity was not significant.
These results are surprising as tropicamide completely blocked the contractile
response suggesting that M4 receptors are involved in the mediation of the
contractile response, yet M4 receptors mediate pertussis toxin sensitive
inhibition of adenylate cyclase and subsequent accumulation of cAMP (Ehlert
etal. 1997). However, the contractile activity of the plant extract also seems to
be mediated by M3 receptors which stimulate contractions by potently
activating phosphatidyl inositol hydrolysis and stimulating large, rapid and
transient chloride currents by a pertussis toxin insensitive G protein pathway
(Lechleiter et al. 1991; Ehlert et a!. 1997).
PGE2 and PGFza have also been shown to inhibit adenylate cyclase through a
pertussis sensitive mechanism in the pregnant rat myometrium (Goureau et
al. 1990).
These data suggest that adenylate cyclase is not a major mediator of the
contractile action of the R. tridentata extract suggesting that the M3 receptors
mediate a large portion of the response to the plant extract. The pertussis
toxin induced reduction in the contractile activity of the extract may indicate
the proportion of the response which is mediated through either M2 or M4
receptors or through the activation of prostanoid receptors.
152
Pertussis toxin acts by inhibiting the inhibitory nucleotide regulatory protein
mediating the signal transmission to adenylate cyclase, whereas cholera toxin
inhibits the stimulatory nucleotide regulatory protein on the same pathway
(Lux & Schultz 1986), known as the Get protein. Cholera toxin (I^M ) also did
not alter the uterine response to the plant extract. These results suggest that
the Ga protein is not involved in the mediation of the contractile response to
the extract.
7.2.13. Blocking of the action of the plant extract by atropine and

indomethacin
The results from ihe isolated organ studies indicate that the plant extract
stimulates smooth muscle contractions through two mechanisms: the
activation of m u c l inic receptors and through promoting the synthesis of
cyclooxygenase products. The involvement of these seemingly independent
mechanisms of contraction has also been found to mediate smooth muscle
contractility in response to Agapanthus africanus aqueous extracts (Veale et
al. 1999) as well as to venom from the soldierfish (Gymnapistes marmoratus)
(Hopkins et a!. 1997).
Tobacco smoking by pregnant women increases the frequency of

spontaneous abortions and preterm births. The mechanism by which this is
thought to occur is also similar to the pharmacological activity of R. tridentata
extracts. Nicotine and its major metabolite cotinine both activate PLAa,
thereby increasiLg the production of PGE& Nicotine also activates nicotinic
receptors in the placenta causing the release of acetylcholine which in turn
activates muscarinic receptors (Sastry et al. 1999).
Myometrium: PGF2a, PGEa, and muscarinic agonists all enhance Ca2+-

induced contraction of myometrium in late gestation (Izumi et al. 1996). These
contractile elements produce additional contractile force with the same
amount of intracellular calcium, thus providing additional expelling forces for
delivery of a foetus (Izumi et al. 1996). The activation of IVfc receptors also
153
Pertussis toxin acts by inhibiting the inhibitory nucleotide regulatory protein
mediating the signal transmission to adenylate cyclase, whereas cholera toxin
inhibits the stimulatory nucleotide regulatory protein on the same pathway
(Lux & Schultz 1986), known as the Ga protein. Cholera toxin (1|J.M) also did
not alter the uterine response to the plant extract. These results suggest that
the Ga protein is not involved in the mediation of the contractile response to
the extract.
7.2.13. Blocking of the action of the plant extract by atropine and

indomethacin
The results from the isolated organ studies indicate that the plant extract
stimulates smooth muscle contractions through two mechanisms: the
activation of muscarinic receptors and through promoting the synthesis of
cyclooxygenase products. The involvement of these seemingly independent
mechanisms of contraction has also been found to mediate smooth muscle
contractility in response tc Agapanthus africanus aqueous extracts (Veale et
af. 1999) as well as to venom from the soldierfish (Gymnapistes marmoratus)
(Hopkins etal. 1997).
Tobacco smoking by pregnant women increases the frequency of

spontaneous abortions and preterm births. The mechanism by which this is
thought to occur is also similar to the pharmacological activity of R. tridentata
extracts. Nicotine and its major metabolite cotinine both activate PLAz,
thereby increasing the production of PGE2. Nicotine also activates nicotinic
receptors in the placenta causing the release of acetylcholine which in turn
activates muscarinic receptors (Sastry et al. 1999).
Myometrium: PGF2a, PGE2s and muscarinic agonists all enhance Ca2+-

induced contraction of myometrium in late gestation (Izumi et al. 1996). These
contractile elements produce additional contractile force with the same
amount of intracellular calcium, thus providing additional expelling forces for
delivery of a foetus (Izumi et al. 1996). The activation of M2 receptors also
153
inhibits the relaxant effects of (3-adrenoceptor activation (Ehlert et a/. 1999)
which would otherwise impair labour.
Endothelial cells of blood vessels: In addition to increasing uterine

contractility, activation of muscarinic receptors of endothelial cells results in
the release of endothelium-derived relaxing factor (EDRF), and possibly the
smooth muscle relaxing factor, nitric oxide (NO) (Sastry et at. 1999).
Therefore, activation of muscarinic receptors counteracts the vasoconstrictive
effects of PGEg through the release of EDRF / NO and therefore counteracts
the effects of PGE2 on the placental vascular resistance which would usually
decrease foetal blood flow through the placenta, i.e. R. tridentata would
produce opposing effects on the placental vasculature. The potentiation of
prostaglandin synthesis would increase vascular resistance, whereas
activation of the muscarinic receptors would decrease vascular resistance.
Gastrointestinal tract: As mentioned earlier, muscarinic agonists have the

potential of directly stimulating intrauterine meconium passage through
stimulating foetal intestinal motility. Prostanoids have also been found to be
diarrhoeagenic (Weiler et at. 1990) by increasing the fluid content of the
intestines (Keith et at. 1992) and by a receptor mediated increase in smooth
muscle tone (Campbell & Halushka 1995). That is, R. tridentata theoretically
has the potential of increasing meconium staining of the liquor by two
independent mechanisms.
Further links between muscarinic receptors and prostaglandins have been

identified. PGIz (prostacyclin) facilitates acetylcholine release from intramural
nerves in the guinea-pig ileum, possibly by increasing the excitability of
cholinergic cell bodies (Gaion & Trento 1983). However, this is a pertussis
toxin sensitive mechanism. Contractile responses to Mi and IVU receptors in
the iris sphincter have been found to involve the formation of prostanoids, that
partially inhibit the contractile response. It is thought that this is a possible
mechanism in other types of smooth muscle (Ehlert 199-7).
154
One possible mechanism by which the plant extract is acting is by stimulating
the production of cyciooxygenase products, which in turn stimulate the release
of acetylcholine from the nerve endings which then stimulates the
postsynaptic muscarinic receptors. The reverse interaction is also possible.
This would explain why indomethacin and atropine both completely block the
action of the plant extract, and why either doesnt only partially block the
contractile activity.
A method to test this hypothesis is to block the release of acetylcholine from

nerve terminals using tetrodotoxin. Owing to local legislation, tetradotoxin was
unavailable and this experiment could not be conducted.
Figure 7.1 (Page 156) illustrates the proposed mechanisms by which the
Rhoicissus extract is stimulating contraction of the isolated rat uterus and
ileum.
7.2.14. Increase in the baseline o f uterus following the initial dose of R.

tridentata
Figure 4.21 (Page 114) shows the relationship between the contractile response
of 243 test challenges on the uterus with Rhoicissus aqueous extracts, plotted
against the extent to which the same challenge caused an increase in the basal
length of the uterine tissue, following repeated washing out of the Tyrode
solution. The graph shows that there is no correlation between the contractile
response to the extract and the extent to which the baseline is raised. The r2 is
0.19 indicating that there is no direct relationship between the two responses of
the uterus to the plant extract.
155
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7.3. Variation in contractile activity
7.3.1. Seasonal Variation

Figure 5.1 (Page 117) illustrates how the various parts of the plants harvested
in winter and spring all yield aqueous extracts with low levels of contractile
activity. On the contrary, there is considerable variation between the extracts
from the various plant parts harvested during summer and autumn. During
summer the tubers yield the most active aqueous extracts, followed by the
stems then roots and leaves. The autumn plants exhibit a similar pattern of
variation except that the roots are the least active. The autumn and summer
tubers are 4 and 5 times more active than the winter and spring extracts. The
stems display similar seasonal variability.
The root and stem extracts show a similar pattern of seasonal variation in
uterotonic activity to that of the tuber extracts. That is, the activity of the extracts
increases when the plants are harvested after the first rains. The activity
increases throughout the growing (or rainy) season reaching a peak in autumn.
The activity of the extracts then falls as the plants lose their leaves and become
dormant during winter. The rise in activity as the rains start follows the statement
of Birch (1960) that an improved uptake of minerals from the soil follows the
mineralization flush after the first rains. This usually leads to a sharp rise in the
nitrogen content within the plant.
The variability in the contractile activity of the extracts could be caused by i)

altered concentrations of the active principle(s), ii) a change in the quality or
nature of the active principle(s), or in) changes in the production of compounds
such as saponins which may alter the extraction of the pharmacologically active
component(s).
157
The extent to which the extracts increased the basal tone of the uterus did not
change significantly between extracts from different plant parts (Figure 5.4;
Page 122). After pooling all the results according to season, extracts from
plant material harvested during summer and winter increased the basal length
more than extracts from plant material harvested during spring (p<0.001).
7.3.2. Plant part used

All parts of the plant harvested in summer and autumn are able to directly
stimulate contractions of isolated uterus. The traditional healers usually use the
lignotubers, which yield the most uterotonically active extracts. The activity of the
tuber extracts is the highest during the growing season and decreases during
the seasons when the plant is dormant. The stems are the next most active
parts of the plant, followed by either the roots or leaves, depending on the
season.
7.3.3. Relevance to conservation

The stem extracts yield a similar response to the most active part, the
lignotubers. This is very significant from an environmental perspective, as
harvesting stems for the trade in traditional remedies would have a much lower
impact on the plant populations of Rhoioissus tridentata than harvesting the
lignotubers and roots. With the current use of the lignotubers, the plant has to be
uprooted for lignotubers to be harvested resulting in a decrease in the
population size. Unfortunately, lignotubers are far easier to transport and to
market compared to the stems and branches which have to be packaged.
Secondly, the stems and branches and are not as easily identified. Therefore, it
is unlikely that herb vendors would revert to selling stems rather than tubers.
This could only be implemented if the herbal market could advance to a stage of
selling remedies packaged in boxes, or as bottled liquid remedies.
158
7.4. Distributional variation in contractile activity
The results show that there is variation in the pharmacological response to
aqueous extracts from R.tridentata plants harvested from different localities.
All the extracts stimulated uterine contractions and either slightly increased or
inhibited the maximal response to acetylcholine (Figure 5.5; Page 124). The
extract from the plant harvested in Mondeor was the exception, in that it
stimulated minimal uterine contractions but significantly inhibited the maximal
response. That is, this is the only extract that competed with acetylcholine in a
non-competitive manner.
Turning to the activity of plant parts from Suikerbosrand, the root extracts
were less active than the extracts from lignotubers or stems (Figure 5.2; Page
119). In the investigation of distributional variation, the extract from the roots
harvested from Umlazi gave a response similar to that of the extracts from
roots harvested from Suikerbosrand, whereas the roots harvested from
Durban yielded an extract with an unexpectedly high level of activity.
Even though these results have illustrated the variation with distribution, no
conclusions can be made about what factors may be responsible for this
variation. Soil samples, the mean rainfall, temperature and the rate of
photosynthesis prior to harvesting were not obtained. Further studies using a
controlled environment, such as in a phytotron, would be necessary to
ascertain which factors influence the composition of compounds within the
plant that are extracted when making the decoctions.
Further taxonomic studies should be done to validate the deliniation of the

subspecies proposed by Urton et al (1986). The occurrence of certain
secondary metabolites is sometimes restricted to a few species or even to a
single chemical race, as they are sometimes formed at certain stages of the
individual's development (so-called idiophase) (Mothes 1980).
159
Chemotaxonomic studies could test whether there are chemical components
which could be used to characterise further deliniations at the species or
subspecies level within the Rhoicissus genus. Perhaps the difference in
contractile response is a manifestation of taxonomic differences.
There is also the possibility that the contractile component of the plant
extracts are only produced when R. tridentata is colonised by a fungus which
is restricted to certain regions. This has been shown to occur with compounds
such as phytoalexins, which are only produced under a stimulus such as the
invasion of the plant tissue by a fungus (Bell 1980).
7.4.1. Clinical relevance of variation in contractile activity

The data presented have highlighted two independent variations in the
contractile activity of the plant extracts, viz. temporal and distributional variation.
These variations highlight a serious flaw in the use of traditional herbal
remedies. Collectors and vendors have no way of determining the chemical
composition of the plant material harvested. Nine traditional healers in and
around Johannesburg were questioned on whether they were aware of any
seasonal variation in the efficacy of R. tridentata, and none of them seemed
aware of the concept.
The distributional variation in contractile activity appears to be more significant

than seasonal variation as it was only the extent of the response that varied
seasonally, whereas the type of response varied distributionally, i.e. the plant
extracts acted either agonistically or antagonistically. The traditional healers
have no way of determining what the effect of a specific R. tridentata sample will
be. This is extremely worrying, especially considering that the crude extracts
yielded contractile responses of up to 98.1% of the maximal response to
acetylcholine. The variability in pharmacological activity is more than likely the
reason why traditional healers have not identified R. tridentata as a potentially
toxic plant. One can assume that the plant is more often not toxic.
160
The variability in the pharmacological activity of Rhoicissus also reiterates the
urgent need to establish standardisation criteria for herbal remedies, particularly
southern African traditional remedies. The results also support the suggestion
made by Tobler (1994) that there should be a holistic approach to
standardising the production of medicinal plants. That is, the entire production
of medicinal plants must be controlled, starting from the cultivation, harvesting
and plant part selection, and continuing through the entire process of remedy
formulation.
7.5. The effect of storage on contractile activity

The results of investigations into the effect of storing dried plant material
indicate that the active components of the plant that are extracted by water
are stable at room temperature and away from sunlight for at least a year
(Figure 5.6; Page 125). This is relevant to the traditional healer, as dried plant
material is stored by medicinal plant vendors and by the traditional healers
themselves.
161
7.6. The effect of R. tridentata extracts on prostaglandin
synthesis in human histiocytoma cells
The extracellular concentrations of PGEz in the histiocytoma cell cultures

increased almost immediately once the cells were exposed to the plant
extracts, reaching a peak at approximately 15 to 20 minutes (Figure 6.1; Page
126). This is similar to what is seen in the isolated organs, where the initial
responses to the plant occur almost immediately after dosing and the
response increases continuously, reaching a maximum response in 15 to 20
minutes. The sudden drop in the amounts of extracellular PGEz suggests that
the cells produce enzymes to metabolise prostaglandins that rapidly degrade
extracellular prostaglandins.
The sudden increase in the amounts of PGE2 once the plant extract
concentration is increased above 0.1 mg/ml (Figure 6.2; Page 127) is also
consistent with results obtained from the contractile response of the isolated
rat ileum where the response increases exponentially (Figure 4.2b; Page 93).
However, the isolated uterus responded to lower concentrations of the R.
tridentata extract. The ileum and cells responded to 2.5jag/ml and 1mg/ml
respectively.
These results confirm the results obtained from the isolated organs, indicating
that aqueous R. tridentata extracts stimulate the production of
cyclooxygenase products, in particular prostaglandin Eg. There still is the
possibility that other cyclooxygenase products are also increased by the plant
extracts.
162
7.7. Effect of R. tridentata on cell culture survival
7.7.1, Toxicity on different ceil lines

To test the toxicity of the plant extracts LD50 tests should ideally have been
done. However, since the Animal Ethics Committee of the University of
Witwatersrand does not allow LD50 tests to be done, MTT assays on a variety
of cell lines were done to test whether the plant extracts affect the viability of
cells. Results from these studies give no indication on whether the plant
extracts are able to cause respiratory depression or cardiac function for
example.
The plant extracts did not significantly reduce the absorbance of all the cell
lines consistently, but rather there was a consistent increase in the
absorbance of test wells relative to the control wells. These results suggest
that the plant extracts, in the concentrations teste i, <r e not toxic on any of the
cell lines used, being human kidney epithelial cells, mouse leydig cells, human
hepatoma cells and human histiocytoma cells. The first two are non-
cancerous lines whereas the latter two are cancerous lines.
The increased reduction of the tetrazolium salt (MTT) by the cells to form the
formazan product does not necessarily indicate that there is an increase in the
number of viable cells in the test wells relative to the control wells. The
increase in the formation of the formazan product could indicate that the plant
extracts activate the mitochondria, or the mitochondrial enzymes directly.
Mosmann et ai. (1983) found that metabolically active cells produce more
formazan than resting cells. There is also the possibility of a direct interaction
between the plant extract and the MTT. For future experiments it is suggested
that the cell media is removed and the cells washed with PBS before the
addition of MTT, to cancel out any possibility of this interaction.
163
7.7.2. Seasonal differences in the effect o f the plant extracts on celiuiar
viability
The effect of the plant extract from plant material harvested in either dormant
or growing seasons did not show a great degree of variation in th r .feet on
cellular viability. The only differences that were found were in the assays on
the kidney epithelial cells and the histiocytoma cells (Figure 6.9 & 6.15; Pages
134 & 137). In the kidney cells, 10mg/ml plant extracts from dormant plant
material reduced the cellular viability of the treated cells by approximately
10% relative to the controls, whereas the same extract concentration from
plant material harvested during growing seasons did not alter the cellular
viability. In the histiocytoma cells, concentrations of up to O.lgg/ml of extracts
from dormant plant material reduced the cellular viability to approximately
90% relative to the controls, whereas extracts from plant material harvested
during the growing seasons did not alter the cellular viability.
These results suggest that there are only minor differences in the cytotoxic
effects of extracts from different seasons, unlike the contractile activity of the
plant extracts which appear to vary seasonally. Extracts from plant material
harvested from dormant plants are slightly more cytotoxic to kidnsy epithelial
and histiocytoma cells than extracts from plant material harvested while
growing.
7.7.3, The effect of extracts from different plant parts on cellular viability
There were only minor differences in the effect of extracts from different plant
parts on the cellular viability of the four cell lines on which the plant extracts
were tested, suggesting that there are no consistent differences in the
cytotoxic effects of extracts from different plant parts (Figure 6.9, 6.11, 6.13 &
6.15; Pages 134-137).
164
These results are not consistent with results obtained by Martina Geheeb-
Keller (Pers. comm.; unpublished results) who found that aqueous root
extracts from R. tridentata reduced the cellular viability of a HepG2 cell line
(human hepatoma cells), whereas extracts from any other plant part did not
alter cellular viability.
7.7,4. Correlation between contractile activity and cytotoxicity
When the contractile activity on isolated rat uterus of each extract was plotted
against the effect of the extract on cellular viability, there was a very poor
correlation between the two variables (Figure 6.16; Page 139).
165
7.8. CONCLUSIONS
7.8.1. ingredients used in pregnancy-related remedies

The traditional healers interviewed used a total of 46 ingredients in their
pregnancy-related remedies. 40 of these were plant material, 4 were animal
products and 2 were non-organic. 19 of these plant species have been
reported to be toxic, of which 12 have caused fatal poisonings. The traditional
healers interviewed all had very limited training as herbalists, but were all
willing and open for further training or opportunities to increase their
knowledge.
7.8.2. Contractile activity of R. tridentata

Rhoicissus tridentata was found to directly stimulate concentration dependant
contractions of both isolated rat uterus and ileum. The baseline length of the
uterus was increased to varying degrees after exposure to the plant extract,
whereas the Ileal baseline length remained unchanged. The extent to which
the uterine basal contraction was increased was not a function of the original
contractile response.
7.8.3. Pharmacological activity of R. tridentata

The contractile response in the isolated rat uterus and ileum appeared to be
mediated through muscarinic receptors and through the stimulation of
prostaglandin synthesis, and did not involve serotonergic receptor activity.
The role of oxytocin receptors in the contractile response to the aqueous
extract of R. tridentata was inconclusive. The uterine response also did not
involve either adrenergic or histamine receptors. Furthermore, the contractile
response appeared to be pertussis toxin insensitive, i.e. it did not involve
adenylate cyclase, or nicotinic receptors.
7.8.4. Variation in the contractile activity of R. tridentata

The contractile activity of R. tridentata varies seasonally. Extracts of the plant
are v ;t active when prepared from plant material harvested during summer
and autumn, and least active during winter and spring. The activity of extracts
166
from all plant parts varies in a similar pattern. Generally, the lignotubers yield
the most active extracts followed by the stems, roots and leaves. The
contractile response is influenced by the locality where the plant was grown.
All but one extract tested stimulated uterine contractions to varying degrees
whereas one plant yielded extracts that antagoniseo the activity of
acetylcholine. Storing dry plant material for a year does not effect the
contractile activity of decoctions made from the plant material.
7.8.5. Cytotoxicity of aqueous extracts of R. tridentata

Aqueous extracts of R. tridentata appeared not to reduce cellular viability of
human kidney epithelial cells, human hepatoma cells, human histiocytoma
cells and mouse leydig cells. The effect of the extracts on the cells did not
appear to display the seasonal variation evident in the contractile activity,
neither were there marked differences in the cellular responses to different
plant parts.
7.8.6. The effect of aqueous extracts of R. tridentata on prostaglandin

synthesis by human histiocytoma cells
Aqueous extracts from R. tridentata increased extracellular levels of
prostaglandin Eg in cultures of human histiocytoma cells. The PGEz levels
peaked at approximately 20 minutes following exposure of the cells to R.
tridentata extract.
167
7.9. Recommendations for further work
7.9.1. Herbal remedies used during pregnancy

Further interviews on both pregnant women and traditional healers in Gauteng
need to be done before we shall have a complete list of plants being used
during pregnancy by local women. An accurate reflection on what plants are
being used would be very useful if clinical studies on the impact of different
herbal remedies were ever to be done.
Interviews of traditional healers should be more structured, asking what they

would consider to be isihlambezo, inembe, imbiza, imbelikesane or umsekelo.
Traditional healers should be asked to fill in questionaires on what plants they
would prescribe for the common complaints during pregnancy.
In certain areas, especially the more affluent areas, alternative medicines

have become popular and acceptable to both patients and midwives. This
means that patients would admit to using herbal remedies. Case control
studies could easily be done on women who are using popular herbal
products (as listed on page 22) to determine whether there are any
differences in the birth outcomes or labour durations of these births.
7.9.2. Absorption o f active components from gastrointestinal tract

Animal experiments should be done, where plasma concentrations of the
pharmacologically active ingredients of R tridentata are measured following
the administration of the active components. These experiments could identify
which components are absorbed and whether the active components are able
to withstand first pass metabolism.
7.9.3. Pharmacological action o f Rhoicissus tridentata

In vivo pharmacological investigations on the plant in relation to pregnancy
are not justified until there is conclusive proof that this plant is causing an
impact in the clinical setting.
168
Further in vitro tests should be done to determine whether an oxytocin
antagonist such as atosiban or tetrodotoxin alters the uterine response to the
plant extracts, as well as tests on possible mechanisms of respiratory
depression. Opioid receptors have been shown to be present on ileal tissue.
Therefore isolated ileum preparations can be used to determine whether the
plant is able to agonise opioid receptors. Isolated ileal muscle needs to be
electrically stimulated, and then the effects of the plants need to be tested,
using opioid receptor agonists as references and testing whether opioid
antagonists such as naloxone alter the dose response curves to the plant
extract.
Further work on the relationship between prostaglandin synthesis and

muscarinic receptor activation will clarify the mechanism of the contractile
activity of Rhoicissus. Either, these are two independent systems mediating
contractions or possibly the one process stimulates the other.
Results from investigations done by Ribeiro et al. (1999) suggest that there is
an interaction among epidermal growth factor, nitric oxide (NO) and PGs and
that in this interrelationship are involved COX-II and iNOS. This mechanism
might be important during implantation and labor. Chaud et al. (1998) also
found NO to be involved in the synthesis of PGs in the uterus. They suggest
that NO is an important intermediate in the stimulation of PG synthesis caused
by platelet activating factor.
Clearly, PG synthesis in the uterus is a complex process. At this stage it is

unclear how the Rhoicissus extracts interact in this process.
7.9.4. Screening of plants used during pregnancy

A large scale screening project on the plants listed in the review of plants
documented to be used in pregnancy-related remedies, and the plants listed
in table 3.1, should be done to determine which of the plants are uterotonic
169
and which are not. The plants which are uterotonic should be tested for
muscarinic activity and the effect of cyclooxygenase on the direct activity of
the plant. These effects may possibly be a common mechanism of action
among many of the uterotonic plants.
7.9.5. Variation in the contractile activity of Rhoicissus tridentata

Inconclusive findings from work done for this thesis indicate that the
contractile activity of R. tridentata extracts varies depending on the plant size
or age. Investigations could be done to determine whether this is possibly a
further factor affecting the variation in contractile activity.
The possibilities of Rhoicissus tridentata ever being marketed in a controlled

market are very small. Not only is the possibility of controlling the herbal
market small, but Rhoicissus tridentata has been reported to be a toxic plant
and if the market is controlled, this species should not be included as a
permissible plant. Therefore, further studies on which environmental factors
affect the composition of active components of the plant would not be applied,
and would therefore be of academic value only. The results from this study
simply highlight that the contractile activity of the species is highly variable,
and measures should be taken to minimise the factors which affect the
variablity as far as possible.
7.9.6. Long term studies on the clinical impact of traditional remedies

used during pregnancy
The question of the impact of traditional medicine on the outcome of
pregnancy can be investigated in two ways, by analysing the pharmacological
action of individual plant species, or by doing clinical investigations. However,
in vitro pharmacological studies on the extracts only provide indirect evidence
and always raise the question of whether the active components are absorbed
or not. Clinical evidence gives direct evidence negating questions of whether
compounds are absorbed and whether they can withstand first pass
metabolism. Since many patients are taking these plants, clinical studies on
the effects of the plants would be possible.
170
The use of traditional remedies is regarded by black South Africans as very
private. Medical professionals also often frown on the use of traditional
remedies. Therefore, it is ineffective to question women in a clinical setting on
whether they use traditional remedies or not. If they are willing to divulge their
use of traditional remedies it is very seldom that they are aware of what plant
species they used, or which plant species were used to mix up a concoction of
a number of plant species.
Therefore, it would be necessary to devise another mechanism to determine

what plant species was ingested. This could be done with chemical analyses
of urine. Foukaridis ef a/. (1994) demonstrated that this is possible using
HPLC diode array. With the advancement of chemical analytical equipment,
equipment such as HPLC-MS would give a fingerprint for each plant species
overcoming the obstacle of conditions within the column affecting the elution
time of different substances.
Clinical investigations using the urine of children suspected of ingesting impila

{Callilepis laureola) have shown that this method is possible (Steenkamp et ai.
1999). HPLC and GC-MS were used for these studies.
Clinical studies using urine analytical techniques would identify which plants
increase the incidence of adverse birth outcomes, and which possibly improve
birth outcome, and would determine whether pregnancy-related remedies do
impact on the clinical outcome of birth.
171
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A. APPENDICES
A.1. Tyrode solution formula
Stock Solution 1
NaCI lOOOg
KCI 25g
CaCl2.2H20 33g
MgCl2.6H20 27g
Stock Solution 2
NaHCOa 252g
NaH2P0 4 12.5g
Add 800 ml of solution 1 and 400 ml of solution 2 to approximately 10 L of

distilled water. Add 30 g glucose and make up to 20 L.
Stilboestrol used to oestrogenise rats:

Stilboestrol in arachis oil (0.2mg/ml)
dose 0.15ml/300g animal weight
A.2. Details of cell cultures used in project

a) Graham cells
ATCC number: CRL-1573.1

Organism: human
Designation: 293/SF
Tissue: kidney; transformed with adenovirus 5 DNA
Age / Stage: embryo
Morphology: epithelial
Gender: male
Tumorigenic: yes, in nude mice
Growth properties: monolayer
Receptors expressed vitronectin
Culture medium
HAM FI 0 90%
Foetal bovine: 10%
b) Human fibrous histiocytoma cells
ATCC number: TIB-223

Designation: GCT (giant cell tumor)
Tissue: histiocytoma, fibrous, from metastatic site, lung
Age: 29 years
Gender: male
193
Tumorigenic: yes
Growth properties: adherent
Products: prostaglandin Ez
plasminogen activator
colony stimulating activity
erythroid enhancing activity
Receptors expressed: luteinizing hormone

epidermal growth factor
androgen
oestrogen
progesterone
Culture medium
McCoy’s 5a medium with 1.5 mM L-glutamine 90%
Foetal bovine serum 10%
c) Mouse Testis (leydig) cells
ATCC number: CRL-1714

Designation: TM3
Strain: BALB/c nu/+
Tissue: normal; testis; leydig cells
Age: 11-13 days
Gender: male
Morphology: epithelial
Tumorigenic: no
Growth properties: adherent
Products: prostaglandin F2a
Receptors expressed: luteinizing hormone
epidermal growth factor
androgen
oestrogen
progesterone
Cell Culture Medium (Modified Dulbecco’s medium )
DMEM w/HEPES (lOmM) 100 ml

L-Glutamine (lOOx) 1 ml
Solution 1 1 ml
Non-essential amino ucids (100x) 1 ml
NCTC 135 10 ml
Foetal bovine Serum 12 ml
Gentamicin 1.4 ml
Solution 1 (100x)
1320 mg oxalacetic acid (100 mM, MW 132)
80 mg crystalline bovine insulin (20 units/ml, 25 units/mg)
Add Na pyruvate 550 mg (50 mM, F W 110)
194
Mix the oxalacetic acid and bovine insulin together, stir at 37°C. Add
the Na pyruvate. Bring up to 100 ml with distilled water. Stir at 37°C
until solution dissolves. Filter, aliquot and store frozen.
A.3. Resuits of statistical analyses of all data
A.3.1 Effect of the antagonists on the direct activity o f the Rhofcissus

extracts
Table A.1 P-values from student-t tests done comparing the contractile
response of isolated rat uterus to the Rhoicissus extract alone as
compared to the response to the extract after the organs had been treated
with an antagonist.___________________________________
uterus ileum
Rh extract vs Indomethacin
NS-398 0.687
NS-398 2 ;0:oo3 ;H
vs Prazosin 0.343
Yohomibine 0.882
vs Atropine
Pirenzepine 0.054
Gallamine 0.939
4-DAMP
Tropicamide ;p.oog_
vs Methysergide 0.920 0.066
Tropisetron 0.145 0.022
Ketanserin 0.690 0.022
Hexamethonium 0.440
Mecamylamine 0.928
Cholera toxin 0.767
Pertussis toxin 0.094
°yrilamine 0.794
A.3.2 Seasonal differences in contractile activity
Table A.2 P-values from student-t tests comparing the seasonal differences
in the direct contractile activity of Rhoicissus extracts on isolated rat
uterus.
tubers roots stems leaves
spring vs summer =0.000 0 00 6_ 0.000
spring vs autumn 10.007 # .7 6 7 0.266
summer vs autumn 0.704 0.060 0.591
spring vs winter 0.765 0.077 0.058
summer vs winter 1-0.000. # .1 9 0 0.138
autumn vs winter ^ # 0 # 0,541^ T ! 0.554
195
Table A.3 P-values from student-t tests comparing the isolated rat uterine
maximal contractile response to acetylcholine after the organs had been
pretreated with 1.3mg/mi of various Rhoicissus extracts for 5 minutes.
tubers roots stems leaves
DF P value DF P value DF P value DF P value
summer vs autumn 5 0.478 12 0.061 11 0.082 13 0 003
summer vs winter 5 0.382 13 0.302 11 10-921
autumn vs winter 11 0.495 12 0.102 12 0.615
summer vs spring 5 0.132 13 0.903 11 :Q.042. _
autumn vs spring 5 0.076 12 0.104 12 0.533
winter vs spring 5 0.134 13 0.464 14 0.912
summer vs control 5 0.054 13 0.709 11 M m 13
autumn vs control 11 0.039 12 m m # 12 0.405 21 10,021. _
winter vs control 17 0.715 18 0 008 _ 18 0.411
spring vs control 5 0.033 13 0.617 14 0.933
A.3.3 Contractile activity o f extracts from different plant parts
Table A.4 P-values from student-t tests comparing the direct contractile
activity of Rhoiciseus extracts from different plant parts harvested in
different seasons on isolated rat uterus.
Seasons spring summer autumn winter
tubers vs stems 0.680 0.407 0.050 ; 0.044
tubers vs roots 0.287 0.187 0.000 0.213
stems vs roots 0 02J :0.309 .0.019 0.371
tubers vs leaves 0 007 ' o,giz: '
stems vs leaves u0.021 . . #0.665
roots vs leaves 0.090 0.093
196
A.3.4 Cytotoxicity o f R. tridentata on different ceil lines
from the MTT assays performed using different concentrations of
Rhoicissus extract on the four cell lines.____________________________
Hepatoma Leydig Histiocytoma Graham
_______________________________ _________ (DF=127) (DF=144)
0.01 g/mi vs controls 0.259 mm# a m m m
1mg/ml vs controls m m 0.083 0.001 0.134
0.1 mg/ml vs controls 0.645 0.095 0.000 0.081
0.01 mg/ml vs controls 0.306 0.002 0.317
lug/ml vs controls 0.872 0.756 0.000 0.687
0. lug/ml vs controls 0.775 0.053 0.562
A.3.5 Seasonal differences in cytotoxicity
from the MTT assays performed using Rhoicissus extracts from dcrmant
(D) and growing (G) seasons on the four cell lines. Highlighted blocks
show P-values less than 0.05. ___ __________________________
Hepatoma Ley/dig Histiocytoma Graham
(DF=71) (DF=77)
D controls vs G controls 0.948 0.092 0.959 0.299
D 0.01 g/ml vs G 0.01 g/ml 0.135 0.522 0.465
D 1mg/ml vs G 1mg/ml 0.396 0.619 0.694 0 .0 1 1 ;
D 0.1 mg/m! vs G 0.1 mg/ml 0.943 0.350 0.616 0.618
D 0.01 mg/ml vs G 0.91 mg/ml 0.597 0.066 0.969 0.109
D 1jig/mi vs G 1ng/ml 0.596 0.945 om 0.566
D 0.1ng/ml vs GO. lug/ml 0.318 0.475 0.000 0.836
A.3.6 Cytotoxicity of extracts from different plant parts
from the MTT assays performed using Rhoicissus extracts from tubers (T)
and stems (S) on the hepatoma cells._______________
p-value
T controls vs S controls 0.274
T 0.01 g/ml vs S 0.01 g/ml 0.678
T 1mg/ml vs S Img/ml 0.302
T 0.1 mg/ml vs S 0.1 mg/ml 0.579
T 0.01 mg/ml vs S 0.01 mg/ml 0.184
T l^g/ml vs S 1iig/ml 0.623
T 0. lug/ml vs S 0. lug/ml 0.080
197
plant parts on the Graham cell line. Highlighted blocks show P-values less
than 0.05.
Tuber controls vs Root controls 0.217

Tuber 0.01 g/ml vs Root 0.01 g/ml 0.429
Tuber 1mg/ml vs Root 1mg/ml w m
Tuber 0.1 mg/m! vs Root 0.1 mg/ml 0.040
Tuber 0.01 mg/ml vs Root 0.01 mg/ml 0.678
Tuber1ug/ml vs Root 1ug/ml 0.685
Tuber 0.1ug/ml vs Root 0.1 ug/ml 0.792
Tuber controls vs Stem controls 0.830
Tuber 0.01 g/ml vs Stem 0.01 g/ml 0.453
Tuber 1mg/ml vs Stem 1mg/ml 0.701
Tuber 0.1 mg/ml vs Stem 0.1 mg/ml 0.099
Tuber1ug/ml vs Stem 1ug/ml 0.313
Tuber Q.1ug/ml vs Stem 0.1 ug/ml 0.877
Tuber controls vs Leaves controls 0.802
Tuber 0.01 g/ml vs Leaves 0.01 g/ml 0.000
Tuber 1mg/ml vs Leaves 1mg/ml 0.458
Tuber 0.1 mg/ml vs Leaves 0.1 mg/ml 0.074
Tuber 0.01 mg/ml vs Leaves 0.01 mg/m! 0.206
Tuber1ug/ml vs Leaves 1ug/ml 0.255
Tuber 0.1ug/ml vs Leaves 0.1 ug/ml 0.174
Root controls vs Stem controls 0.131
Root 0.01 g/ml vs Stem 0.01 g/ml 0.014
Root 1mg/ml vs Stem 1mg/ml &m '
Root 0.1 mg/ml vs Stem 0.1 mg/ml 0.774
Root 1ug/ml vs Stem 1ug/ml 0.092
Root 0.1 ug/ml vs Stem 0.1 ug/ml 0.606
Root controls vs Leaves controls 0.263
Root 0.01 g/ml vs Leaves 0.01 g/ml 0.001!
Root 1mg/ml vs Leaves 1mg/ml 0.081
Root 0.1 mg/ml vs Leaves 0.1 mg/ml 0.374
Root 1ug/ml vs Leaves 1ug/ml 0.378
Root 0.1 ug/ml vs Leaves 0.1 ug/ml 0.423
Stem controls vs Leaves controls 0.445
f tern 0.01 g/ml vs Leaves 0.01 g/ml 0.671
Stem 1mg/ml vs Leaves 1mg/ml 0.535
Stem 0.1 mg/ml vs Leaves 0.1 mg/ml 0.932
Stem 1ug/ml vs Leaves 1ug/ml 0.467
Stem 0.1 ug/ml vs Leaves 0.1 ug/ml 0.664
198
plant parts on leydig cells. Highlighted blocks show P-values less than
0.05.
Tuber 0.01 g/ml vs Root 0.01 g/ml 0.814
Tuber 1mg/ml vs Root 1mg/ml 0.222
Tuber lug/ml vs Root 1ug/ml 0.026
Tuber 0. lug/ml vs Root 0.1 ug/ml 0.315
Tuber 0.01 g/ml vs Stem 0.01 g/ml 0.208
Tuber 1mg/ml vs Stem 1mg/ml 0.666
Tuber lug/ml vs Stem 1ug/ml 0.006
Tuber 0.1ug/ml vs Stem 0.1 ug/ml 0.147
Tuber 0.1 mg/mi vs Leaves 0.1 mg/ml 0.143
Tuber lug/ml vs Leaves lug/ml o-oog
Tuber 0. lug/ml vs Leaves 0.1 ug/ml 0.696
Root 1mg/ml vs Stem 1mg/ml 0.892
Root 0.01 mg/ml vs Stem 0.01 mg/ml m m
Root 0.1 ug/ml vs Stem 0.1 ug/ml 0.018
Root 0.01 g/ml vs Leaves 0.01 g/ml 0.371
Root 1mg/ml vs Leaves 1mg/ml &m a
Root 0.1 mg/ml vs Leaves 0,1 mg/ml 0.137
Root 1ug/ml vs Leaves 1ug/ml 0.975
Stem 0.01 g/ml vs Leaves 0.01 g/ml 0.335
Stem 1mg/ml vs Leaves 1mg/ml poW
Stem 0.1 mg/ml vs Leaves 0.1 mg/ml o.m z
Stem lug/ml vs Leaves 1ug/ml 0.707
199
Table A.10 P-values from student-t tests comparing the absorbance at
540nm from the MTT assays performed using Rhoicissus extracts from
different plant pan's on histiocytoma cells. Highlighted blocks show P-
values less than 0.0'".

Tubrr 0.01 g/ml vs Root 0.01 g/ml 0.134
Tuber 1mg/ml vs Root 1mg/ml m m
Tuber 0.1 mg/ml vs Root 0.1 mg/ml 0,050
Tuber1ug/ml vs Root 1ug/ml 0.064
Tuber 0. lug/ml vs Root 0.1 ug/ml 0.678
Tuber 0.01 g/ml vs Stem 0.01 g/ml
Tuber 1mg/ml vs Stem 1mg/ml 0.000:
Tuber 0.1 mg/ml vs Stem 0.1 mg/ml 0.001!
Tuber lug/ml vs Stem 1ug/ml 0.581
Tuber 0. lug/ml vs Stem 0.1 ug/ml 0.697
Tuber lug/ml vs Leaves 1ug/ml 0.189
Tuber 0.1ug/ml vs Leaves 0.1 ug/ml 0.258
Root 1mg/ml vs Stem 1mg/ml b.ooti
Root 0. lug/ml vs Stem 0.1 ug/ml 0.501
Root 0.01 g/ml vs Leaves 0.01 g/ml 0.502
Root 1mg/ml vs Leaves 1mg/ml 0.475
Root lug/ml vs Leaves 1ug/ml 0,398
Stem 0.01 g/ml vs Leaves 0.01 g/ml 0.397
Stem 1mg/ml vs Leaves 1mg/ml 0383
Stem 0.1 mg/ml vs Leaves 0.1 mg/ml 0,090
Stem 0.01 mg/ml vs Leaves 0.D1 mg/ml 0.816
Stem 1ug/ml vs Leaves 1ug/ml 0,301
200
A.4. Layout of microtitre plates for prostaglandin assays
A.4.1 Prostaglandin synthesis over time
intracellular extracellular
A .
r r
1 2 3 4 5 6 7 8 9 10 11 12
A ® ® ® 0 0 0 0 0
Time (hours)
0 0 0 0 0 0
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Standards and
H© © © © © © © © © © © © blanks
A.4.2 Dose-response experiment
Extract 1 Extract 2 Extract 3

>____ ■*> *------- - 'v—
1 2 3 4 5 6 7 8 9 10 11 12
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B 0 0 0 0 0 0 0 0 0 0 0 0
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> plant extracts
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201
A.4.3 Effect of indomethacin and hydrocortisone
indomethacin Prednisolone NS-398

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202
A.S. Raw data collected from open-ended interviews with traditional
healers
Notes taken from interviews with six of the traditional healers are given below.
In most cases the terms used by the traditional healers are given.
NOMSA
Sangoma
Ex nursing sister, midwife
Trained under Mr Nkosi, Deepdale, Swaziland who now has a shop in Mayi
Mayi Bazaar, Jeppe Street, Johannesburg
Pregnancy
■ 6-7 months
Labatega (1/2 bulb) - make way for baby
Gobho (1/2 bulb)
Baboon urine (5 fingernail size) - good for uterus and cystitis
Boil mixture for ~ 15 minutes, and give the patient the prepared extract.
Drink % glass/day
a Last two weeks of 8th month

Qarda lentshe (ground ostrich shell) ~2cm2 -strengthens mother and
foetus
Skhupha seguarda (chicken egg yolk)
Mercury (small droplet) - cause contractions
1 tsp of remedy once a week
■ 9th month
Sunlight soap and water enema once a week
Labour (once bleeding has started)

As for 8th month plus
Mtsansi we hashi (horse placenta; paperlike; 1cm2) - easy delivery, helps
contractions & baby, eases
pain
Mix all together, 1 tbsp.
Postnatal
Labatega (1/2 bulb) - clears womb
Cimamlilo (1/2 bulb) -kills pain
Usually sends patients to hospital to deliver, but delivered three babies at

home as there was not enough time to get to a hospital.
203
BOUYUX
Trained in Soweto
Makes all medicines in the same way as described below.
Normally single plants used.
Dosage: normally 1 handful of plant material, powdered.
Brought to the boil in water
Patients given liquid extract and takes (1/2 glass/day (after straining); babies 1
tsp/day).
No set isihlambezo regime - plants prescribed according to patient’s needs.
Pregnancy
u 2-2 months after menstruation stops
Ubani (Agapanthus africanus) 1 handful; enema - strengthens foetus
Iqaku lenifene nempila (baboon urine)
Impila (Callilepis laureoia)
■ 3-7 months after menstruation stops

Amabelejongosi (Ussochilus arenarius) - reduces pain
Ugadolo/Amaleyane (Bidens pilosa) roots - stops pain from bladder,
kidneys
- helps in limiting arnniotic fluid
- kills seres in bladder
- gives blood more tonic
- strengthens body
Ibhuma (Cyperus sp / Typha spp) - strengthens foetus
■ 6 months to delivery
Iphengulula (Iphingula - Berkheya rhapontica) roots - cleanses bladder
- cleanses kidneys
- helps ease backache
- helps baby’s growth
- easy delivery
Mix with ostrich shell; nutrients for baby growth; 3 x/day
Medicines for baby

Impila
Ingwavuma (bark) - helps baby’s growth
Dumalala - pinch of ground twigs and leaves placed in babies bath water
- makes baby sleep
Other plants used

■ If no movement of baby:
Hlanyathi ihlalanyathi (Grew/a occidentalis) bark
204
- helps baby move
- strengthens nerves and veins
of reproduction system
Intshingu (Momordica foetida) roots - takes out unneeded amniotic
fluid
- revives female reproduction
system
Amazombe (Gynandropsis gynandra) leaves
(G. pentaphylla)
Ukunyakaza - gives strength to all the
nerves and veins of the body
Isinwasi (Rhoicissus cuneifolia or Cissus quandrangularis) roots

- strengthens female
reproductive system
Umayime (Clivia miniata) roots - reduces pain allowing easy
delivery (use from 3-9 month)
- revives nerves and veins of
reproductive system
- snake antivenom
Iblucu (roots)
Ipahatana
Ghobo (isighobo - Asparagus sp) - menstrual pains
- stomach pains
- skin rash
- infertility
- mental retardation
- swollen nerves in lungs
- cleanses blood
1 pinch ashed roots; lick off palm of hand
Infe-yentawu (Ansiella humilis)
Causes of birth complications according to Bouvlix

- early induction of labour using isihlambezo
(this may cause extreme pains and rupturing of the womb)
- protein in urine causes high blood pressure
- traces of blood in urine, caused by bad insertion of catheter
- umbilical cord may wrap around babies neck, and if it tightens it may
suffocate or strangle the baby.
205
MARTHA
Zulu Chemist along Louis Botha; grandparents (deceased) taught her.
Isihiambezo
■ 1-7 months
powered roots; % handful in 1 litre water
Boil 30 minutes, sisve, give extract; % cup, 2 x/day
Strengthens baby, makes baby move, takes away pains
■ 8-9 months
ground bulb; 1 litre water; 2 flat tbsp.
Boil 30 minutes; sieve; give extract; Vz cup 3 x1 day
- easy delivery, opens cervix
- makes breech baby turn
Labour induction
Ostrich egg - % tsp powdered
Burnt record - 1 tsp powdered ash
Mix in % cup water and drink (warm)
Baby will be born within about 3 hours
Fertility
■ For woman who has periods but cannot fall pregnant
Mahlokoloze (% bulb)
Sekaname (% bulb)
Mokgaiakane (1 cm3)
Mix in 2 litres water, simmer for 3 hours, sieve, % cup 2 x/day
Drink a total of 5 litres
■ Blocked tubes
Mathunga (Vz bulb)
Mbola (1/2 bulb)
Itolwane (~ 15 x 3cm bark)
Mix in 2 litres water, simmer 1 hour; make up to ~ 1 litre; % cup 1 x /day
* Sores in uterus (drop; VD)

Inguduza (5cm3 bulb)
Udonguzibovu (1/2 bulb)
Umathunga (1/2 bulb)
2 litres water; simmer 1 hour; enema; 1 cup once a week
Remedy to take awav clots six months after birth

Umdabu (1 handful, branch)
Itolwane (1 handful, branch)
Udabulibayi - chemist, 1 pinch
1 % litres; boil 30 minutes; Vs. cup 2 x/day - only 1 litre
Clean womb-bleeding
206
FIKl
Trained in 1980 in Meadowlands ~ 1 year.

Worked as nurse assistant in Johannesburg General, then assistant for a
gynaecologist in Hillbrow. Currently in “River” training.
Now waiting for spirits to call. They are asked for their cow which means she
has probably almost finished.
Infertility
isinwazi (tuber) - cleansing
Boil 30cm, in 1 litre % cup/day (1st day full cup)
Husband and wife drink 1 litre in total, “like DNC, settling womb”
Mpopo (1 bulb) - cleansing

Boil 30cm in 1 litre. Both drink 1 cup 1st day, then % cup/day, till litre
finished. Take off tops, leave bottoms on while they drink, helps
conception.
If don't conceive, throw bones to find out reason.
Reasons eg. - use ‘prevent’ earlier (contraception not favoured)
Spirits muj- want something e.g. want the new bride presented to the
ancestvv’s grave.
Remedies used during pregnancy
■ 3 - 8 months
isinwazi - as before; no danger
Lady takes as she feels, max % cup/ day
She may miss a day
Protects baby and mother from other illnesses (makes them healthy)
» 8 - 9 months
Nothing
" Once deep into labour, just before going to hospital

Sehlula ananye ~ level tsp
isinwazi pinch
Put powder above lighted charcoal
Kneel with it between legs
Won’t give any other medicines during pregnancy, even if ill.
The only one is:

Impepe (grass)
Light it and breath fumes (same as if not pregnant) for headache
Bile - common cause of headaches and stomach aches.
Remedies for baby

Impila - use if child is restless - or add to imbiza
1 tsp boiled in cup of water - calms the child.
207
If woman previously miscarried
Umayime, isinwazi and unagugu are mixed together
Umaguqu - to settle child if too high or breech.
Womb cleansing remedy

Hlunguhlung umathung
- burn to reduce potency
- roll ash in vaseline - small amounts
- place in vagina - opens tubes and clears womb, then give imbiza
MOSEKI
Traditional healer for + 30 years

Trained in Bekkersdal.
infertility
» Infertility caused by VD
Ghobo (1 flat tsp)
Sekaname (% bulb)
Magaga (% bulb)
Labeteka (% bulb)
Boil 1 hour in 2 litres of water, make up to 2 litres.
% cup after breakfast, lunch and supper. First day 1 cup.
When pain gone % cup 2 x /day.
■ Menstruate too often

Labetega
Gobo
14 cup 3 x / d a y
Also massage as womb may not be in good condition
Pregnancy
Don't give medicines routinely. Usually only massage.
Isihlambezo:
Intolani (1 dsp roots)
Marula (1 dsp bark)
Boil 1 hour in 2 litres water, Vz cup 3 x / day
Backache:
patheyengaka
Menstrual pains & during pregnancy if miscarried before

Morokalapudi (1 tsp roots)
Boil 1 hour in 2 litres; Vz cup 3 x I day.
Ntolwani - makes woman urinate, keeps system going; loosens muscles

allowing an easy delivery;maternal strength
208
Morning sickness
1 green apple in morning
Mageuo (soft porridge)
2 cups water.
Overdue pregnancy - massage to induce labour.
LAYMOKD
Zulu
Has shop in Soweto
Inyanga and sangoma but practices as an inyanga.
Previously journalist for World News
Used to dig out plants for his grandfather
Trained under 2 people - 1 inyanga and 1 sangoma.
Imbiza
Umdabou (1 dsp root) boil in % litre and cool
Use as an enema or drink % cup 3 x day - no side effects
Ishaqa (1 dsp root in % litre)
Umsongelo (1 dsp root in % litre)
If not menstruating - imbiza

Gobo (1 dsp root)
Ndoda emadevu (1 dsp root)
Boil, or just pour on hot water
Pregnancy - isihlambezo
First finds out how mother-to-be feels and diagnoses according to symptoms
Isihlambezo - used to make new baby grow perfectly
Start giving from 1st month
* If woman feeling well

Ubogo 2 dsp / 1(root)
Boil/pour on hot water, % cup 3 x /day.
Drinks it when babies not moving or feels she needs it.
Eaten as porridge.
■ Backache or tired
Ugobho (root) grows in wet area 2 dsp/litre
■ Headache
Labatega -1 dsp/litre % cup 3 / day.
If labour overdue
Impompo 1 tsp Vz litre
Induces vomiting. Drinks 2 sp / hr x Spyt. Giyes birth same day without
fail.
209
A.6. Harvesting details
Rh A roots
Harvesting'. Umlazi grasslands, 5/2/96 (from Bridget)
Sample prep: Came as a shredded sample which I milled and boiled in

distilled water for approximately an hour, filtered through itself and then
lyophilized.
Organ Response: variable. Four responses

initial augmentation, no suppression of the maximal response
initial augmentation, suppression of the max
no initial augmentation, no supression of the max
no initial suppression, suppression of the max
Did many runs as results were variable. The high n value elicited the different
responses to the same samples run on different organs simultaneously.
That is with all variables held constant four distinct responses were
evident.
This could be explained by there being different compounds in the extract
which all have similar intrinsic rates of activity and dissociation constants.
The response of a particular dosing would be dependant on the order in
which the different compounds bound to the receptors or which compound
dominated the dose. The second option is unlikely as the lyophilized
matter was mixed in relatively large quantities and then decanted into
eppindorff tubes to ensure all constituents of the extract were randomly
distributed through different dosing fractions.
Rh B: Autumn, 2/5/96, Suikerbosrand, in a thicket. (1st patch)

No tuber could be found while digging.
Sample prep: Dried sample away from direct light for two weeks. Cut the large
portions into pieces about 1cm3 then milled the sample through rotomill
in the pharmacy department.
Rh C: spring, bone dry. 2/9/96, Suikerbosrand, in a thicket. (1st patch)

There were no leaves on the plant yet, just a few emerging leaves.
Rh D: spring, bone dry. 2/9/96, Suikerbosrand, in a thicket. (2nd patch)

Plant was approximately 10m away from Rh B and C.
No tuber was found. There were no leaves on the plant yet, just a few
emerging leaves.
Rh E: early summer, before first rains. 3/11/96, Suikerbosrand, in a thicket

(1st patch).
Rh F: mid summer, after a very wet period. 3/12/96, Suikerbosrand, in a
thicket (1st patch).
Rh G: mid summer, after a very wet period. 3/12/96, Suikerbosrand in a

thicket (2nd patch).
210
Rh H: Tonquani Gorge, Magaliesburg Mountains, Halfway up side of gorge in
dry, humus rich soil below a dense canopy.
Rh I: Umlazi, Durban, 10/96 (collected by Bridget). May be the same as Rh A
Rh J: Garden in Mondeor, 3/97 (Leonie's Garden).
Rh K: Suikerbosrand, late summer - autumn, after a very wet period, 1/4/97

(1st patch).
Rh L: Suikerbosrand, late summer - autumn, after a very wet period, 1/4/97

(2nd patch).
Rh M: Suikerbosrand, late summer - autumn, after a very wet period, 1/4/97

(3rd patch).
Rh N: Mt Sheba, 12/6/97 - winter, but plant still growing. The plantwas

growing in a fairly sunny part of the gorge, in the crevices ofalarge
rock close to the stream at the bottom of the gorge.
Rh Q: Suikerbosrand, mid-winter, 1st patch (1st patch)
Rh P: Suikerbosrand, mid-winter, 1st patch (2nd patch)
Rh Q: Suikerbosrand, mid-winter, 1st patch (3rd patch)
211
A.7. Raw data from all experiments
Raw data are presented in the following order:

(Excel w a s used to tabulate the data, which does not have the facility to number pages)
Isolated organ experiments with the following reference drugs:

(Data from uterine tissue is always given before data from ileal tissue)
Experiments testing the mechanism of action:

All Reference drugs
Oxytocin
Indomethacin
Atropine
Pirenzepine
Gallamine
4-DAMP
Tropicamide
Prazosin
Yohimbine
Methysergide
Ketanserin
Tropisetron
All bar graphs
Correlation between baseline and contraction
Experiments testing the variation in contractile response:

ACh standards
Different plant parts and seasons
Plant extract alone - box & whisker plots
Stability of harvested plant material
MTT Assays:
Graham’s cells (Kidney epithelial cells)

Leydig cells
Hepatocyoma cells
Histiocytoma cells
212
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Author Katsoulis L C
Name of thesis The Parmacological Activity Of Rhoicissus Tridentata Subsp Cuneifolia In Relation To Parturition Katsoulis L
C 2000
PUBLISHER:
University of the Witwatersrand, Johannesburg
©2013
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THE PHARMACOLOGICAL ACTIVITY OF RHOICISSUS

TRIDENTATA SUBSP CUNEIFOUA IN RELATION TO

Lynn Coleen Katsoulis

His unending encouragement and support, gave me the confidence to apply

May my precious children achieve far more.

L.C. Katsoulis (1999) Rhoicissus tndentata subsp cuneifolia: the effect of

1.2. Oral preceritfiiions

8. Brookes, 0. Doudoukina, D.J.H. Veale and L.C. Katsoulis (1997) Isolation

LC. Katsoulis, I. Havlik (1999) The Pharmacological Action of Rhoicissus

B. Brookes, 0. Doudoukina, LC. Katsoulis and D.J.H. Veale (2000)

LC. Katsoulis (2000) Plants used in pregnancy-related remedies in the

1.3. Poster presentations

B. Brookes, O. Doudoukina, D.J.H. Veale and LC. Katsoulis (1996)

LC. Katsoulis (1999) The necessity of standardising herbal remedies: An

The pharmacological action of an aqueous extract of R. tridentata subsp.

The plant extracts, up tv a concentration of 1mg/ml, appear not to be toxic to

• To my father, Harry, for supporting whatever I chose to do.

• Dr Neil Butkow for believing in me and supporting me to do a Degree in

• Prof. Ivan Havlik for heading up a successful department during turbulent

• Norman Arrangies for helping with the administration of ordering materials

e Inorganic Ion analyses were done by the Johannesburg General Hospital

University grants provided financial support for the purchase of the

Most importantly, to The Almighty, who made all this possible.

2.8.1. Cytotoxicity using the MTT assay method................................ 6 6

CHAPTER 3: Results section 1

4.4.4. Muscarinic receptor system ...................................................100

4.4.5. Serotonergic receptor system ................................................104

CHAPTER 5: Results section 3

CHAPTER 6: Results section 4

6.1. Effect of R. tridentata extracts on Prostaglandin Synthesis by

6.2. Toxicity A ssa ys............................ 128

CHAPTER 7: DISCUSSION AND CONCLUSIONS

Figure 1.1. Comparison of leaf shape between R. tridentaia subspecies

Figure 1.2. Distribution of a) Rhoicissus tridentata subsp, cuneifolia and b) R.

Figure 1.3. Compounds isolated from Rhoicissus tridentata subsp.

Figure 1.4. Regulation of uterine activity during pregnancy and labour.

Figure 1.5. Pharmacology of the smooth muscle c ell....................... 38

Figure 1.6. Metabolic pathway of phospholipids in the cascade of

Figure 1.7. A scheme for the involvement of phospholipase containing

Figure 1.8. Contractile mechanism for muscarinic receptor subtypes in

Figure 2.1. Aerial parts of Rhoicissus tridentata subsp. cuneifolia harvested

Figure 2.2. Rhoicissus tridentata subsp. cuneifolia growing at

Figure 3.2. Plant vendors selling their merchandise at Faraday Herbal

Figure 3.4. Rhoicissus lignotuoers for sale.................................................. 72

Figure 3.5. The number of plants prescribed by different traditional healers in

by the plant extract before the cumulative additions of acetylcholine (v )...... 97

Figure 4.7. The isolated a) uterine response to noradrenaline when

extract for 5 minutes (e), compared to the response to noradrenaline when

Rhoicissus aqueous extract for 5 minutes (@), compared to the response to

Figure 4.9. The a) uterine and b) ileal response to acetylcholine (+)

root extract ( • ) for 5 minutes before the addition of acetylcholine. The

acetylcholine (▼)........................................................................................ 101

Figure 4.10. The effect of antagonists on muscarinic subtypes on the

pretreated with 1pM methysergide for 5 minutes (v), and methysergide

Figure 4.12. The effect of ketanserin, a 5-HTi receptor subtype antagonist, on

Figure 4.13. The effect of tropisetron, a 5 -HT2 receptor subtype antagonist,

Figure 5.5. The contractile response of isolated uterine smooth muscle to

Figure 6.1. Prostaglandin synthesis over time by histiocytoma cells

Figure 6.2. Dose response curve of intracellular, extracellular and total

Figure 6.3. The optical densities at different wavelengths corresponding to

Figure 6.5. The absorbance spectra from wells containing 13 500

Figure 6.11. Comparisons between the effect of Rhoicissus extracts from

Figure 6.13. Comparisons between the effect of Rhoicissus extracts from