university of kwazulu-natal faculty of science and agriculture school ...

UNIVERSITY OF KWAZULU-NATAL 

FACULTY OF SCIENCE AND AGRICULTURE 

SCHOOL OF CHEMISTRY 

THE ISOLATION, STRUCTURE ELUCIDATION AND 

BIOLOGICAL TESTING OF COMPOUNDS FROM 

PLECTRANTHUS HADIENSIS 

BY 

SHIKSHA DUKHEA 

2010 

SUPERVISOR: DR. N.A. KOORBANALLY 

CO-SUPERVISOR: DR. B. MOODLEY

THE ISOLATION, STRUCTURE ELUCIDATION AND 

BIOLOGICAL TESTING OF COMPOUNDS FROM 

PLECTRANTHUS HADIENSIS 

BY 

SHIKSHA DUKHEA 

Submitted in partial fulfilment of the requirements for the degree 

of Master of Science in the School of Chemistry, Faculty of Science 

and Agriculture, University of KwaZulu-Natal. 

Supervisor: Dr N. A. Koorbanally 

Co-Supervisor: Dr. B. Moodley 

ii

Preface 

The experimental work described in this dissertation was carried out from March 2007 to 

January 2009 in the School of Chemistry, Howard College and Westville campuses, Durban, 

under the supervision of Dr. N.A. Koorbanally and Dr. B. Moodley. 

This study represents original work by the author and has not been submitted in any other form 

to another university. Where use was made of work of others it has been duly acknowledged in 

the text. 

I hereby certify that the above statement is correct 

SIGNED: _______________________ 

Shiksha Dukhea 

B-Tech (DIT) 

SIGNED: _______________________ 

Dr. N.A. Koorbanally 

Ph.D (Natal) 

SIGNED: _______________________ 

Dr. B. Moodley 

Ph.D (UKZN) 

iii

I, SHIKSHA DUKHEA, declare that 

Declaration 

1. The research reported in this thesis, except where otherwise indicated, is my original 

research. 

2. This thesis has not been submitted for any degree or examination at any other 

university. 

3. This thesis does not contain other persons’ data, pictures, graphs or other information, 

unless specifically acknowledged as being sourced from other persons. 

4. This thesis does not contain other persons’ writing, unless specifically acknowledged as 

being sourced from other researchers. Where other written sources have been quoted, 

then: 

a) Their words have been re-written but the general information attributed to them 

has been referenced 

b) Where their exact words have been used, then their writing has been placed in 

italics and inside quotation marks, and referenced. 

5. This thesis does not contain text, graphics or tables copied and pasted from the Internet, 

unless specifically acknowledged, and the source being detailed in the thesis and in the 

References section. 

Signed:…………………………………………………….. 

iv

Acknowledgements 

First and foremost I would like to thank Mother Saraswathi for her guidance and for granting 

me with wisdom, patience and faith. 

Thank you to my supervisor, Dr. N.A. Koorbanally and co-supervisor, Dr. B. Moodey, for 

guidance, clarity and support. Thank you to Prof. N. Crouch from the South African National 

Botanical Institute (SANBI), for the collection of the plant material as well as for your advice, 

insight and availability when assistance was needed. Thank you to Mr. D. Jagjivan for help 

with the NMR, Mrs. A. Naidoo for her assistance and training with the HPLC and Ms. S. 

Misthry for her technical support during her tenure at the university. 

My gratitude is also extended to all my colleagues who have helped me throughout my studies 

by offering intellectual support, empathy and sincerity: Erick Korir, Kaalin Gopaul, Gugulethu 

Ndlovu, Vusi Mchunu, Siyabonga Gumede, Joyce Kiplimo, Hamisu Ibrahim, Thrineshan 

Moodley, Maya Makatini and Edith Sabata. You all are very much appreciated and will never 

be forgotten. 

Words cannot express how grateful I am for the support and words of encouragement that my 

family has showered me with. Thank you Mum and Dad, for your understanding and 

unwavering faith in me and to my sister, Karishma for your positive and caring nature. To my 

fiancé, Akash Rampersad, I thank you for always being there for me and to my friends, Nitesh 

and Swasthie Doolabh, thank you for always believing in me. 

My final words of gratitude are to the National Research Foundation (NRF) for their financial 

support and the Council for Science and Industrial Research (CSIR) in Pretoria, for the efficient 

analysis of isolated compounds for biological activity as well as a special thank you to Dr. N. 

Moodley (CSIR) for his assistance with the interpretation of the anticancer assay results. 

v

List of Abbreviations 

NMR - nuclear magnetic resonance 

1 H NMR - proton nuclear magnetic resonance 

13 C NMR - carbon nuclear magnetic resonance 

DEPT - distortionless enhancement by polarization transfer 

HSQC - heteronuclear single quantum coherence 

HMBC - heteronuclear multiple bond coherence 

COSY - correlation nuclear magnetic spectroscopy 

NOESY - nuclear overhauser effect spectroscopy 

J - coupling constant 

s - singlet 

d - doublet 

dd - doublet of doublets 

t - triplet 

brs - broad singlet 

m - multiplet 

td - triplet of doublets 

TLC - thin layer chromatography 

IR - infrared spectroscopy 

UV - ultra-violet 

CDCl3 - deuterated chloroform 

CD3OD - deuterated methanol 

DMSO - dimethylsulphoxide 

m/z - mass-to-charge ratio 

[M] + - molecular ion peak 

IPP - isopentenyl diphosphate 

GPP - geranyl pyrophosphate 

FPP - farnesyl pyrophosphate 

DMAPP - dimethylallyl diphosphate 

vi

GC-MS - gas chromatography-mass spectrometry 

LC-MS - liquid chromatography-mass spectrometry 

Hz - Hertz 

W½ - half-width 

GI50 - growth inhibition - the concentration at which the growth 

of the cell is inhibited by 50% 

TGI - total growth inhibition 

LC50 - lethal dose concentration - concentration at which 50% of 

the cells are killed 

LC100 - lethal dose concentration - concentration at which 100% 

of the cells are killed 

RPMI - Roswell Park Memorial Institute 

ATCC - American Type Culture Collection 

SRB - sulforhodamine B 

MIC - minimum inhibitory activity 

TCA - trichloroacetic acid 

MRSA - methicillin resistant Staphylococcus aureus 

VRE - vancomycin-resistant Enterococcus faecalis 

I.U.P.A.C - international union of pure and applied chemistry 

∆ - delta (to indicate the position of the double bond) 

vii

List of Tables 

Table 1: Summary of the medicinal uses of various Plectranthus species 21 

Table 2: Summary of uses of various Plectranthus species 23 

Table 3: Antibacterial activity of Plectranthus and Coleus extracts 28 

Table 4: Antifungal activity of Plectranthus and Coleus extracts 32 

Table 5a: Antiparasitic, anti-inflammatory, antioxidant, antitumour and insect antifeedant activity of 

polar extracts from seven Plectranthus species 

Table 5b: Other important inhibitory activity of polar extracts from Plectranthus and Coleus species 37 

Table 6a: Royleanone-type abietanes with an isopropyl side chain at C-13, isolated from Plectranthus 

and Coleus species 

Table 6b: Royleanone-type abietane dimers isolated from Plectranthus and Coleus species 44 

Table 6c: Royleanone-type abietanes with a linear side chain at C-13 isolated from Plectranthus and 

Coleus species 

Table 7: Spirocoleons isolated from Plectranthus and Coleus species 49 

Table 8: Vinylogous quinones isolated from Plectranthus species 56 

Table 9a: Coleon-type abietanes isolated from Plectranthus and Coleus species 60 

Table 9b: Coleon-type abietanes with an olefinic bond at ∆ 5 isolated from Plectranthus and Coleus 

species 

Table 9c: Coleon-type abietanes with double bonds within ring A and B isolated from Plectranthus 

species 

Table 10: Miscellaneous abietanes isolated from Plectranthus and Coleus species 69 

Table 11: Antibacterial and antifungal activity of compounds extracted from Plectranthus species 72 

Table 12: Antibacterial and antifungal activity of abietanes isolated from Salvia species 74 

Table 13: Inhibitory activity of abietanes isolated from Plectranthus and Coleus extracts 76 

Table 14: Pharmacological activity of abietanes isolated from other plant species 78 

Table 15: 1 H NMR data for compound I compared with three reference compounds (CDCl3, 400MHz) 109 

Table 16: 13 C NMR data for compound I compared with three reference compounds (CDCl3, 400MHz) 110 

Table 17: 1 H NMR data for compound II compared with three reference compounds (CDCl3, 400MHz) 111 

Table 18: 13 C NMR data for compound II compared with one reference compound (CDCl3) 112 

Page 

34 

40 

46 

64 

68 

viii

Table 19: NMR data of compound III (CDCl3) and its acetylated equivalent 116 

Table 20: 1 H NMR data for compound III (CDCl3) compared with two similar compounds 117 

Table 21: 13 C NMR data for compound III (CDCl3) compared with two similar compounds 118 

Table 22: NMR data of compound IV (CD3OD) compared with reference compound 124 

Table 23: Comparison of 13 C NMR data of compound IV (CD3OD) with two similar compounds 126 

Table 24: Comparison of 1 H NMR data of compound IV (600MHz, CD3OD) with three reference 

compounds 

Table 25: 1 H and 13 C NMR data of stigmasterol (V) compared with reference data (CDCl3, 400MHz) 130 

Table 26: 1 H and 13 C NMR data of lupeol (VI) compared with reference data (CDCl3, 400MHz) 131 

Table 27: Antibacterial activity of compounds I to IV 144 

Table 28: Growth inhibition values for compounds I-IV against TK-10, UACC-62 and MCF-7 cell lines 152 

Table 29: GI50 values (uM) of compounds I and II and their 7α-isomers 152 

List of Figures 

Figure 1a: Acyclic (class 1), monocyclic (class 2) and bicyclic (class 3) diterpenes 2 

Figure 1b: Tricyclic (Class 4) diterpenes 3 

Figure 1c: Tetracyclic (Class 5) diterpenes 3 

Figure 1d: Macrocyclic (Class 6) diterepenes 4 

Figure 2: Formation of cassane type diterpenoids 4 

Figure 3: Numbering system for abietanoids 5 

Figure 4: Representative structures of the different types of abietane diterpenoids 6 

Figure 5: Examples of the use of the prefix “nor”, “abeo” and “seco” 7 

Figure 6: IPP derived from mevalonic acid (MVA) 8 

Figure 7: IPP derived from deoxyxylulose phosphate (DXP) 9 

Figure 8: Derivation of GGPP from DMAPP 10 

Page 

127 

ix

Figure 9: Formation of ring A from GGPP 10 

Figure 10: Biosynthetic pathway for bi-, tri- and tetracyclic diterpenes 11 

Figure 11: Biosynthesis of beyerane, trachylobane, atisane and aconane diterpenes 12 

Figure 12: Synthesis of abietadiene 13 

Figure 13: Plectranthus hadiensis 16 

Figure 14: Illustration of the 2 clades within the Plectranthus species, categorized according to their 

phylogeny 

Figure 15: Plectranthus hadiensis in bloom 94 

Figure 16: Chair conformation of compound I showing the relationship between H-5, H-6 and H-7 107 

Figure 17: Fragment of Ring C 114 

Figure 18: NOESY correlations for compound III 115 

Figure 19: NOESY correlations for compound IV 124 

Figure 20: Dose response curve for 7β-acetoxy-6β-hydroxyroyleanone (I) against TK-10, UACC-62 

and MCF-7 cell lines 

Figure 21: Dose response curve for 7β,6β-dihydroxyroyleanone (II) against TK-10, UACC-62 and 

MCF-7 cell lines 

Figure 22: Dose response curve for ent-pimara-8(14),15-diene-3β,11α-diol (III) against TK-10, 

UACC-62 and MCF-7 cell lines 

Figure 23: Dose response curve for 2α,3α,19α-trihydroxyurs-12-en-28-oic acid (IV) against TK-10, 


List of Spectra 

Compound I, 7β-acetoxy-6β-hydroxyroyleanone 161 

Spectrum 1a: 1 H NMR spectrum of compound I 162 

Spectrum 1b: 13 C NMR spectrum of compound I 163 

Spectrum 1c: Expanded 13 C NMR spectrum of compound I 164 

Page 

18 

150 

150 

151 

151 

x

Spectrum 1d: Expanded DEPT spectrum of compound I 165 

Spectrum 1e: HSQC spectrum of compound I 166 

Spectrum 1f: Expanded HSQC spectrum of compound I 167 

Spectrum 1g: HMBC spectrum of compound I 168 

Spectrum 1h: Expanded HMBC spectrum of compound I 169 

Spectrum 1i: COSY spectrum of compound I 170 

Spectrum 1j: NOESY spectrum of compound I 171 

Spectrum 1k: IR spectrum of compound I 172 

Spectrum 1l: UV spectrum of compound I 173 

Spectrum 1m: GC-MS spectrum of compound I 174 

Compound II, 6β, β, β,7β-dihydroxyroyleanone β, 

175 

Spectrum 2a: 1 H NMR spectrum of compound II 176 

Spectrum 2b: 13 C NMR spectrum of compound II 177 

Spectrum 2c: Expanded 13 C NMR spectrum of compound II 178 

Spectrum 2d: Expanded DEPT spectrum of compound II 179 

Spectrum 2e: HSQC spectrum of compound II 180 

Spectrum 2f: Expanded HSQC spectrum of compound II 181 

Spectrum 2g: HMBC spectrum of compound II 182 

Spectrum 2h: Expanded HMBC spectrum of compound II 183 

Spectrum 2i: COSY spectrum of compound II 184 

Spectrum 2j: NOESY spectrum of compound II 185 

Spectrum 2k: IR spectrum of compound II 186 

Spectrum 2l: UV spectrum of compound II 187 

Spectrum 2m: LC-MS spectrum of compound II 188 

Compound III, ent-pimara-8(14),15-diene-3β,11α-diol 189 

Spectrum 3a: 1 H NMR spectrum of compound III 190 

Page 

xi

Spectrum 3b: 13 C NMR spectrum of compound III 191 

Spectrum 3c: Expanded DEPT spectrum of compound III 192 

Spectrum 3d: HSQC spectrum of compound III 193 

Spectrum 3e: Expanded HSQC spectrum of compound III 194 

Spectrum 3f: HMBC spectrum of compound III 195 

Spectrum 3g: Expanded HMBC spectrum of compound III 196 

Spectrum 3h: COSY spectrum of compound III 197 

Spectrum 3i: NOESY spectrum of compound III 198 

Spectrum 3j: IR spectrum of compound III 199 

Spectrum 3k: UV spectrum of compound III 200 

Spectrum 3l: GC-MS spectrum of compound III 201 

Compound IV, euscaphic acid 202 

Spectrum 4a: 1 H NMR spectrum of compound IV 203 

Spectrum 4b: 13 C NMR spectrum of compound IV 204 

Spectrum 4c: Expanded 13 C NMR spectrum of compound IV 205 

Spectrum 4d: DEPT spectrum of compound IV 206 

Spectrum 4e: Expanded DEPT spectrum of compound IV 207 

Spectrum 4f: HSQC spectrum of compound IV 208 

Spectrum 4g: Expanded HSQC spectrum of compound IV 209 

Spectrum 4h: HMBC spectrum of compound IV 210 

Spectrum 4i: Expanded HMBC spectrum of compound IV 211 

Spectrum 4j: COSY spectrum of compound IV 212 

Spectrum 4k: NOESY spectrum of compound IV 213 

Spectrum 4l: IR spectrum of compound IV 214 

Spectrum 4m: UV spectrum of compound IV 215 

Spectrum 4n: GC-MS spectrum of compound IV 216 

Page 

xii

Compound V, stigmasterol 217 

Spectrum 5a: 1 H NMR spectrum of compound V 218 

Spectrum 5b: Expanded 1 H NMR spectrum of compound V 219 

Spectrum 5c: 13 C NMR spectrum of compound V 220 

Spectrum 5d: Expanded 13 C NMR spectrum of compound V 221 

Spectrum 5e: IR spectrum of compound V 222 

Compound VI, lupeol 223 

Spectrum 6a: 1 H NMR spectrum of compound VI 224 

Spectrum 6b: Expanded 1 H NMR spectrum of compound VI 225 

Spectrum 6c: 13 C NMR spectrum of compound VI 226 

Spectrum 6d: Expanded 13 C NMR spectrum of compound VI 227 

Spectrum 6e: IR spectrum of compound VI 228 

Page 

xiii

Abstract 

Three diterpenes of the abietane class, 7β-acetoxy-6β-hydroxyroyleanone (I), 6β,7β- 

dihydroxyroyleanone (II) and ent-pimara-8(14),15-diene-3β,11α-diol (III) and three 

triterpenes, 2α,3α,19α-trihydroxyurs-12-en-28-oic acid (IV), stigmasterol (V) and lupeol (VI) 

were isolated from the stem and leaf material of Plectranthus hadiensis. The structures of the 

compounds were elucidated using 2D NMR spectroscopy and Mass spectrometry. All six 

compounds have been isolated previously, but this is the first occurrence of compounds III-VI 

in Plectranthus hadiensis. This is also the first report of the isolation of a pimarene from 

Plectranthus, which provides a biochemical link to other genera in the family Lamiaceae where 

this class of compounds exist. 

Compounds I to IV were tested for their antibacterial activity against Enterococcus faecalis and 

Pseudomonas aeruginosa as well as their anticancer activity against breast (MCF-7), renal (TK- 

10) and melanoma (UACC-62) cell lines. Compounds I and II exhibited good antibacterial 

activity against Enterococcus faecalis and Pseudomonas aeruginosa and although the ent- 

pimara-8(14),15-diene-3β,11α-diol (III), was inactive against E. faecalis, it was very active 

against P. aeruginosa. Compound IV, the triterpenoid, was structurally different to I-III and 

did not show any anti-bacterial activity. Compounds I-III were weakly active toward the 

cancerous renal (TK-10), melanoma (UACC-62) and breast (MCF-7) cell lines, while IV was 

inactive in all of the cell lines. 

xiv

2 

19 

1 

4 

18 

O 

20 

5 

Structures of compounds isolated from Plectranthus hadiensis 

9 

10 

11 

6 7 

OH 

OH 

12 

14 

8 

R 

R 

13 

16 

(I) OC(O)CH3 

(II) OH 

HO 

3 

15 

O 

19 

5 

10 

6 

17 

7 

21 

HO 

18 

14 

2 

3 

20 

1 

HO 

20 11 

9 

4 5 6 7 

10 8 

18 19 

22 

29 

16 

23 

12 

13 

17 

14 

15 

16 

HO 

HO 

1 

4 

25 26 

9 

5 

10 

6 7 

8 

23 24 

(III) (IV) 

28 

27 

25 

26 

HO 

24 23 

(V) (VI) 

3 

10 

2 

30 

25 26 

20 

29 

27 

18 

12 

29 

19 

27 

17 

18 

14 

OH 

15 

17 

22 

28 

30 

21 

22 

COOH 

xv

Table of Contents 

Page 

Preface ....................................................................................................................................... iii 

Declaration ................................................................................................................................. iv 

Acknowledgements ....................................................................................................................... v 

List of Abbreviations ................................................................................................................... vi 

List of Tables ............................................................................................................................ viii 

List of Figures ............................................................................................................................. ix 

List of Spectra .............................................................................................................................. x 

Abstract ..................................................................................................................................... xiv 

Chapter 1 Introduction to diterpenoids ...................................................................................1 

1.1 Classification of diterpenoids ............................................................................................1 

1.2 Classification of the abietane diterpenoids ........................................................................5 

1.3 Biosynthesis .......................................................................................................................7 

References ..................................................................................................................................14 

Chapter 2 An introduction to the Plectranthus in South Africa ..........................................15 

2.1 Phylogeny, occurrence and description ...........................................................................15 

2.2 Ethnobotanical uses .........................................................................................................19 

2.3 Pharmacological/biological uses of the plant extract .......................................................25 

2.4 The phytochemistry of Plectranthus ................................................................................39 

2.5 Biological activity of the phytochemical constituents of Plectranthus ...........................71 

References ..................................................................................................................................80 

Chapter 3 Extractives from Plectranthus hadiensis ..............................................................94 

3.1 Introduction ......................................................................................................................94 

3.2 Foreword to Experimental ...............................................................................................97 

3.3 Experimental ....................................................................................................................99 

xvi

3.4 Results and Discussion of the compounds isolated .......................................................102 

3.4.1 Structural elucidation of Compounds I and II.........................................................104 

3.4.2 Structure elucidation of Compound III ...................................................................112 

3.4.3 Structure elucidation of Compound IV ...................................................................120 

3.4.4 Structure elucidation of Compounds V and VI.......................................................128 

Physical data for compounds I-VI .......................................................................................133 

References ................................................................................................................................136 

Chapter 4 Antibacterial and anticancer activity of the compounds isolated ...................141 

4.1 Antibacterial testing .......................................................................................................143 

4.1.1 Experimental ...........................................................................................................143 

4.1.2 Results and discussion ............................................................................................144 

4.2 Anti-cancer screening ....................................................................................................146 

4.2.1 Experimental ...........................................................................................................147 

4.2.2 Results and discussion ............................................................................................148 

References ................................................................................................................................153 

Chapter 5 Conclusion ............................................................................................................156 

References ................................................................................................................................158 

Page 

xvii

Chapter 1 Introduction to diterpenoids 

1.1 Classification of diterpenoids 

Diterpenes consist of four isoprene units (C20) and are by far the most diverse group of 

compounds. They are placed into different classes (termed acyclic, monocyclic, bicyclic, 

tricyclic, tetracyclic and macrocyclic) based on the number of carbocyclic rings in the 

structure (figures 1a-d). 

The acyclic and monocyclic compounds (figure 1a) do not have much variation in that 

they are either straight chain structures or contain one ring and a side chain respectively, 

such as phytane (1) and vitamin A (2). 

Bicyclic compounds belonging to Class 3 (figure 1a), contain two six-membered 

carbocyclic rings with alkyl groups attached at either C-8 or C-9 or both. Labdanes and 

clerodanes are differentiated because of the different positions of the methyl groups. In 

clerodanes these methyl groups occur at C-4, C-5, C-8, C-9 and C-13 while in labdanes 

there are two methyl groups at C-4 and one each at C-8, C-10 and C-13. 

Class 4 diterpenes (figure 1b) are the tricyclic diterpenoids and have three six-membered 

rings but differ in the substitution pattern on these rings. Abietane and totarane 

diterpenoids are fairly similar in structure, the only difference being the position of the 

isopropyl group in ring C, which is situated at C-13 in abietanes and C-14 for totaranes 

due to different alkyl shifts, abietane being the precursor to totarane (Dewick, 2002; 

Nakanishi, 1974). 

The structural difference between pimarane and cassane type diterpenes is the position of 

the methyl group (CH3-17) on ring C, which occurs at C-13 in pimaranes and C-14 in 

cassanes, pimarane being the precursor to the cassanes (Nakanishi, 1974) and occurs by a 

methyl shift from C-13 in the pimaranes to C-14 in the cassanes when a carbocation at C- 

14 is created by a hydride shift in the sandaracopimarenyl cation (Figure 2). Taxane type 

1

compounds are also classified as tricyclic, but have a different biosynthetic pathway 

(Dewick, 2002). 

Within the class 5 diterpenoids (figure 1c), the kauranes, beyeranes and atisanes are all 

derived from the sandaracopimarenyl cation. This is shown later in figures 10 and 11. 

They are therefore similar in structure, the kauranes having a methylene group at C-16, 

the beyeranes having a methyl group at C-17 and the methylene bridge occurring 

between C-12 and C-8 in the atisanes. The gibberellins are similar to the kauranes, the 

difference being the five-membered ring in gibberellins arising from the biosynthesis 

(Figure 10). Phorbols and ginkolides follow different biosynthetic pathways (Dewick, 

2002). 

The macrocyclic compounds (figure 1d), the trachylobanes and the aconanes, are similar 

to the atisanes from which they are derived. This is shown later in figure 11. 

O 

16 

17 18 19 20 

H 

6 

1 

A 

5 

1 

10 

5 

9 8 

A B 

4 

12 

13 

20 

17 

11 

15 

16 

(1) phytane 

7 

9 13 

(2) Vitamin A 

OH 

OH O 

18 19 

OH 

(3) 15-hydroxy-3-cleroden-2-one (clerodane) (4) Forskolin (labdane) 

Figure 1a: Acyclic (class 1), monocyclic (class 2) and bicyclic (class 3) diterpenes 

Continued on next page…. 

4 

10 

3 

OH 

OH 

8 

1 

13 

O 

OAc 

2

HO 

19 

19 

20 

10 

A H B 

4 6 

H 

COOH 

OH 

O 

20 

13 

12 

11 

C 14 

1 

10 

A 

5 

4 

9 

B 

8 

7 

OH 

18 

17 

15 

O 

OH 

11 

C D 16 

15 

17 

AcO 

O 

19 

10 

H 

4 6 

(11) Steviol (kaurane) (12) 2-acetoxy-1,15-beyeradiene-3,12-dione 

(beyerane) 


16 

3 

11 

20 

10 

5 

18 19 

H 

18 

9 

20 

H 

Figure 1c: Tetracyclic (Class 5) diterpenes 

13 

11 

O 

15 

OH 

17 

17 

15 

16 

16 

O 

O 

1 

O 

OH 

20 

(5) Horminone (abietane) (6) Totarol (totarane) (7) Phytocassane E (cassane) 

3 

1 

19 18 

OH 

20 

H 

H 

12 

13 

14 

17 

15 

16 

(8) 3β-dihydroxy-8(14),15-pimaradiene 

(pimarane) 

6 

9 

AcO 

18 

14 

AcO OAc 

H 

17 

11 

A 15 

1 

20 

B 

19 

8 

C 

4 

H 

H 

16 

OAc 

3 

18 

1 

20 

19 

1 

H 

3 5 

12 

11 13 

15 

10 

H 

8 

12 

H 

14 

3 

14 

HO O 

19 18 

(9) Taxusin (taxane) (10) 3β,12-dihydroxy-13-methyl- 

5,8,11,13-podocarpatetraen-7-one 

(podocarpane) 

Figure 1b: Tricyclic (Class 4) diterpenes 

20 

OH 

17 

(13) Ent-16α-hydroxyatisane-3-one 

(atisane) 

8 

12 

8 

OH 

15 

17 

14 

15 

16

1 

H 

CO2H 19 

20 

CHO 

18 10 

5 

H 

12 

14 

15 

CO2H 7 

OH 

17 

16 

O 

1 

A 

HO 

HO 

H 

20 

5 

14 

B 

OH 

OH 

11 

C D 

10 

19 

18 

8 

H 

H 

(14) GA19 (gibberellin) 

CH2OH 17 

(15) phorbol (16) Bilobalide (ginkgolide) 

H 

H 

13 

17 

14 

H 

sandaracopimarenyl cation 

8 

16 

Figure 1c: Tetracyclic (Class 5) diterpenes 

12 17 

12 

1 

A 

HO 

HOOC 

11 

20 

E 

13 

C 14 

15 

10 

H 

B 

H 

17 

1 

11 

4 

19 18 

14 

9 

7 

(17) hydroxytrachylobanic acid (trachylobane) (18) aconane 

Figure 1d: Macrocyclic (Class 6) diterepenes 

H 

Figure 2: Formation of cassane type diterpenoids 

H 

O 

O 

O 

H 

H 

16 

15 

H 

O 

HO 

H 

O 

cassane type diterpenoid 

OH 

4 

O

1.2 Classification of the abietane diterpenoids 

Abietane diterpenoids consist of approximately twenty carbon atoms and commonly have 

three tertiary methyl groups (two at C-4 and one at C-10), and one isopropyl group at C- 

13. Their numbering system is based on the nomenclature of natural product hydrides as 

recommended by the I.U.P.A.C. in 1993 (Figure 3). These abietanes can be further 

classified into eight types based on structural differences through reductions (addition of 

hydrogen across a double bond) or oxidations such as hydroxylations and dehydrations as 

well as cyclisations between different oxygenated groups. 

20 

13 

12 

11 

C 

14 

1 

2 10 

A 

3 5 

4 

9 

B 

6 

8 

7 

19 

18 

Figure 3: Numbering system for abietanoids 

Acidic abietanes (e.g. abietic acid (19), figure 4) are characterised by the presence of a 

carboxylic acid in the compound. In levopimaric acid (20) there is a double bond in ring 

C (figure 4). In ferruginol (21) and carnosol (22) type abietanes, ring C is phenolic in 

character. Due to the presence of a lactone ring which extends across ring B from C-7 to 

C-10, carnosol type abietanes are differentiated from ferruginol type abietanes. 

Callicarpone (23) abietanes are based on the epoxide ring at ∆ 12 and the presence of a 

hydroxy isopropyl group attached to C-15 as well as α,β-unsaturated ketone groups in 

rings B and C. The royleanones (24) have a benzoquinone ring C. Tanshinone (25) 

abietanes consist of highly oxidised rings where two of the rings, A and B are aromatic, 

ring C contains two adjacent α,β-unsaturated systems and a fourth ring, a furan ring is 

present as ring D. Fichtelite (26) closely resembles the abietane skeletal structure and 

17 

15 

16 

5

may be referred to as a norabietane due to the absence of the methyl group at C-4. Also 

present in the structure is a double bond at ∆ 4 . 

19 

9 

H 

H 

H 

COOH 

H 

COOH 

H 

(19) abietic acid (20) levopimaric acid (21) ferruginol 

HO 

O 

10 

O 

H 

O 

OH 

7 

H 

O 

H 

α 

9 

β 8 

(22) carnosol (23) callicarpone (24) royleanone 

O 

O 

16 

15 

(25) tanshinone (26) dl-fichtelite 

H 

Figure 4: Representative structures of the different types of abietane diterpenoids 

(Nakanishi et al., 1974) 

The terms “nor”, “abeo” and “seco” are used widely in the abietanes. The use of “nor” 

occurs when a methyl group is eliminated from the structure. The number preceding 

“nor” refers to the carbon atom which has been eliminated. The prefix “abeo” is used 

12 

H 

O 

O 

15 

OH 

20 

O 

10 

7 

OH 

OH 

O 

6

when a methyl group migrates to an adjacent carbon atom and “seco” refers to a bond 

that has been broken on the skeletal structure. The numbers preceding “abeo” refers to 

the carbon atoms involved in the migration e.g. (10→5) abeo indicates that a methyl has 

migrated from C-10 to C-5 and the numbers preceding “seco” refers to the carbon atoms 

in which the bond has been broken e.g. in 4,5-seco the bond has been broken between C- 

4 and C-5. Figure 5 includes examples of these. 

1 

2 10 

3 

4 

19 

20 

13 

12 

11 

9 

8 

5 

6 

7 

14 

16 

15 

17 

20 

4 5 

(a) 18-nor (b) 4,5-seco-20(10→5)abeoabietane 

Figure 5: Examples of the use of the prefix “nor”, “abeo” and “seco” 

1.3 Biosynthesis 

The acyclic diterpenes (Class 1), for example phytane (1) (Figure 1a) is formed by the 

addition of four molecules of IPP (isopentenyl diphosphate). The formation of IPP can 

follow either one of two biosynthetic pathways, the methylerythritol phosphate pathway 

(MEP), also known as the 1-deoxy-D-xylulose (DOX) pathway and the mevalonic acid 

(MVA) pathway. The MVA pathway is the only pathway used by animals, while both 

pathways are present in plants. The enzymes involved in the MVA pathway are found in 

the cytosol while those in the MEP or DOX pathway are found in the chloroplast of the 

plant. 

10 

7

In the mevalonic acid pathway, the biosynthesis starts with the Claisen condensation 

reaction where two molecules of acetyl-CoA react to produce acetoacetyl-CoA which 

reacts further with an additional molecule of acetyl-CoA in a stereospecific aldol reaction 

producing HMG-CoA (β-hydroxy-β-methylglutaryl-CoA) (figure 6). The carbonyl 

group in HMG-CoA is then reduced with NADPH to the primary alcohol producing 

mevaldic acid hemithioacetal, then mevaldic acid and further reduction to mevalonic acid 

(MVA). The addition of two ATP molecules leads to the sequential phosphorylation of 

MVA to mevalonic acid diphosphate and a further molecule of ATP results in the release 

of CO2 producing isopentenyl disphosphate (IPP) which is subsequently isomerised to 

DMAPP in a reversible reaction. The forward reaction is favoured due to the 

electrophilic nature of DMAPP. 

H 

O 

1 

HO2C 2 

SCoA 

O 

6 

H 

O 

3 

SCoA 

OH 

4 

mevalonic acid 

(MVA) 

2 x ATP 

OH 

OH 

NADPH 

O OPP 

5 

Claisen 

reaction 

mevalonic acid diphosphate 

O 

H 

O 

OH P O ADP 

OH 

HO 2C 

-CO 2 

O 

SCoA 

acetoacetyl-CoA 

O 

-H + 

CoA 

OH 

mevaldic acid 

H 

4 

5 

3 

H R 

2 

O 

Linkage 

Hydrolysis of 

acetyl-CoA 

H S 

isopentenyl PP 

(IPP) 

1 

-H + 

OPP 

HO 2C 

HO 2C 

isomerase 

HMG-CoA 

reductase 

OH 

O 

HMG-CoA 

OH 

NADPH 

OH 

H 

mevaldic acid 

hemithioacetal 

dimethylallyl PP 

(DMAPP) 

Figure 6: IPP derived from mevalonic acid (MVA), (reproduced from Dewick, 2002) 

SCoA 

SCoA 

OPP 

EnzSH 

8

In the MEP pathway, 1-deoxy-D-xylulose-5-phosphate is derived from pyruvic acid. 

Thiamine diphosphate bonds to the pyruvic acid, which leads to decarboxylation 

producing a TPP/pyruvate enamine which then reacts with D-glyceraldehyde, followed 

by the loss of TPP to yield 1-deoxy-D-xylulose 5-P (DXP). DXP undergoes a pinnacol 

like rearrangement followed by a reduction to produce 2-methyl-D-erythritol 4-P. This is 

then transformed into 4-(CDP)-2-methyl-D-erythritol due to its reaction with cytidine 

triphosphate (CTP) and then phosphorylated with ATP. Cyclisation involving the 

oxygen of the terminal phosphate group with the phosphorus of the cytidine bound 

phosphate results in a phosphoanhydride. Opening of the ring and subsequent reduction 

and dehydration may lead to IPP, however these latter steps still need to be determined. 

9

N 

thiamine diphosphate (TPP) 

O 

OH 

NH 2 

N 

OH 

OH 

B 

OP 

H 

N S 

OPP 

OH 

O OH 

STEPS TO BE DETERMINED 

-H 2O 

OPP 

OPP 

NADPH 

OH 

IPP DMAPP 

H 

N 

H 

O 

O 

OP 

OPP 

OPP 

NH 2 

N 

OH 

H 3C 

TPP anion 

OP 

1-deoxy-D-xylulose 5-P (DXP) 

+ TPP anion regenerated 

NADPH 

O 

C 

H 

N S 

OH 

CO 2H 

pyruvic acid 

OH 

OH 

OPP 

OP 

2-methyl-D-erythritol 4-P 

H OH 

O 

H3C C 

H + 

O OH 

O 

P 

O O 

P 

O OH 

OH 

H OH 

N 

OH 

N S 

CTP 

2-methyl-D-erythritol-2,4-cyclophosphate 

NH 2 

N 

-H + 

OP 

OH 

OH 

H 3C 

HO O 

C 

C 

N S 

OH 

OH 

OH 

O 

O 

H 

OPP 

H 

H3C OH 

C 

N S 

O 

H 

OH 

TPP/pyruvate-derived enamine 

O 

O P OH 

OH 

O 

O 

P 

OH 

O 

P 

OH 

O 

O 

O 

4-(CDP)-2-methyl-D-erythritol 

OP 

D-glyceraldehyde 3-P 

P O CH2 O 

OH 

ATP 

O 

NH 2 

N 

HO OH 

P O CH2 O 

OH 

N 

N 

NH 2 

HO OH 

Figure 7: IPP derived from deoxyxylulose phosphate (DXP), (reproduced from 

Dewick, 2002) 

DMAPP contains a diphosphate anion which acts as a good leaving group resulting in the 

isoprene unit being electrophilic. DMAPP yields an allylic cation by means of a SN1 

reaction. IPP then adds to this allylic cation and with a stereospecific loss of a proton 

(HR) which then forms geranyl pyrophosphate (GPP). Addition of two further IPP 

molecules result in the formation of first farnesyl diphosphate (FPP) and then 

geranylgeranyl diphosphate (GGPP), the precursor to diterpenoid biosynthesis (figure 8). 

N 

O 

O 

10

DMAPP 

IPP 

OPP 

S N1 

geranyl PP (GPP) 

farnesyl PP 

(FPP) 

resonance-stabilised allylic cation 

OPP 

OPP 

IPP 

H R H S 

OPP 

geranylgeranyl PP 

(GGPP) 

Figure 8: Derivation of GGPP from DMAPP (reproduced from Dewick, 2002) 

Monocyclic diterpenes e.g. vitamin A (2) are formed via the first cyclisation step after the 

formation of GGPP, which is initiated by the protonation of the isopropylidene unit in 

GGPP. 

H 

GGPP 

H 

OPP 

1 st 

cyclisation 

A 

IPP 

H 

monocyclic type diterpene 

Figure 9: Formation of ring A from GGPP 

A series of cyclisations as in figure 10 yields the bicyclic intermediate, copalyl 

diphosphate. The subsequent loss of pyrophosphate and cyclisation of copalyl PP results 

in the formation of the tricyclic diterpenes as in the sandaracopimarenyl cation 

(intermediate I). Attack by the double bond on the carbocation in the 

sandaracopimarenyl cation results in a fourth ring (intermediate II). Stabilisation of the 

cation in intermediate II, by forming a tertiary cation followed by loss of a proton result 

OPP 

OPP 

OPP 

11

in the kauranes. From the kauranes, loss of a hydride ion in ring B followed by ring 

contraction and further oxidations result in the gibberellins (figure 10) (Dewick, 2002). 

H 

4 

H 

COOH 

GGPP 

H 

7 

- H 

H 

ent-7-hydoxykaurenoic acid 

H 

H 

CO2H H 

OH 

O 

H 

OPP 

sequence of 

oxidation 

reactions 

ring 

contraction 

W-M 1,2-alkyl 

shift 

O 

4 

A H B 

H 

H 

copalyl PP 

H 7 

ent-kaurene 

GA 12-aldehyde 

D 

O 

OPP 

-H 

sequence of oxidation 

reactions, lactone 

formation 

H 

H 

13 

C 


(Intermediate I) 

HO 

H 

H 

O 

C O 

4 

12 

10 9 

8 

17 

16 

15 

COOH 

gibberellic acid 

12 

13 

15 

H 

1,2-hydride 

migration 

16 

OH 

17 

H 

W-M 1,2-alkyl 

shift 

H 

H 

H 

13 

15 

(Intermediate II) 

Figure 10: Biosynthetic pathway for bi-, tri- and tetracyclic diterpenes (reproduced 

partially from Dewick (2002), Nakanishi (1974) and Hanson (1968)) 

Loss of a proton at C-16 (in intermediate I) with retention of the methyl at C-13 would 

lead to the beyeranes (figure 11). Formation of a carbocation at C-12 from intermediate II 

followed by an alkyl migration of the methylene bridge gives rise to the atisane type 

compounds (Figure 11). The macrocyclic type compounds, the trachylobanes and 

aconanes form pentacyclic and tetracyclic diterpenes respectively. The bond between C- 

12 and C-15 is formed with loss of a proton producing the trachylobanes and 

rearrangement of the 8,9 bond to the 9,16 position with loss of the C-17 methylene group 

(Hanson, 1968) result in the aconanes (Figure 11). 

H 

12

H 

1,3 H + migration 

H 

H 

12 

13 

14 

D 

H 

16 

15 

Intermediate II 

H 

H 

D 

16 

15 

13 

C 


H 

H 

H 

8 

(Intermediate I) 

-H (17) 

19 

16 

4 

17 

15 

-H + (16) 

H H 

HO 

16 

14 

H H 

12 

CH 2OH 

15 

13 

17 

13 

12 17 

15 

9,16-bond formation 

8 

14 

1,2 H + loss of CH2 11 

20 H 

9 

shift 

18 

Atisirene 

(atisane type compound) 

16 

Beyerol 

(beyerane type diterpene) 

CH 2OH 

15 

-H (12) 

12,15-bond 

formation 

19 

4 

20 

18 

19 

4 

18 

20 

aconane type compound 

12 

17 

15 13 

16 

9 

8 

trachylobane type diterpene 

15 

16 

9 

8 

Figure 11: Biosynthesis of beyerane, trachylobane, atisane and aconane diterpenes 

(Manitto, 1981) 

The loss of a proton at C-14 in the sandaracopimarenyl cation leads to the formation of a 

double bond between C-8 and C-14 after which a methyl migration from C-17 to C-15 

yields an abietenyl cation, which loses a proton at C-7 to produce abietadiene (figure 12). 

H 

H 

8 

14 

16 

H 

17 

H 

8 

sandaracopimarenyl cation (-)-sandaracopimaradiene 

H 

15 

17 

14 

H 

H 

H 

7 

H 

16 

15 

abietenyl cation O (-)-abietadiene 

Figure 12: Synthesis of abietadiene (reproduced from Dewick, 2002) 

H 

H 

13

References 

Abad, A., Agullo, C., Cunat, A.C., de Alfonso Marzal, I., Navarro, I. and Gris, A. (2006) 

A unified synthetic approach to trachylobane-, beyerane-, atisane- and kaurane-type 

diterpenes, Tetrahedron, 62, 3266-3283 

Dev, S. (1985) CRC handbook of terpenoids, Diterpenoids, CRC Press, pp 37, 353-361 

Dewick, P.M. (2002) Medicinal natural products: a biosynthetic approach, Wiley, pp 170, 

171, 192, 204, 208, 210, 212 

Hanson, J.R. (1968) The tetracyclic diterpenes, Pergamon, Volume 9, pp 114-120 

Manitto, P. (1981) Biosynthesis of Natural Products, Ellis Horwood Limited, pp 258-262 

Nakanishi, K. (1974) Natural products chemistry, Academic Press, Inc., Volume 1, pp 

186-188, 218-230, 267-270 

Newman, A.A. (1972) Chemistry of Terpenes and Terpenoids, Academic Press, pp 155- 

191 

Panico, R., Powell, W.H. and Richer, J.C. (1993) International Union of Pure and 

Applied Chemistry, Organic Chemistry Division, Commission of Nomenclature of 

Organic Chemistry, A Guide to IUPAC Nomenclature of Organic Compounds 

(Recommendations 1993), Blackwell Scientific publications, pp 55-56 

Rigandy, J. and Klesney, S.P. (1979) International Union of Pure and Applied Chemistry, 

Organic Chemistry Division, Commission of Nomenclature of Organic Chemistry, 

Nomenclature of Organic Chemistry, Sections A-H, Pergamon Press, pp 491-511 

Templeton, W. (1969) An introduction to the chemistry of the terpenoids and steroids, 

Butterworths, pp 111-121 

14

Chapter 2 An introduction to the Plectranthus in South 

Africa 

2.1 Phylogeny, occurrence and description 

The genus Plectranthus is one of twenty-five genera in the subfamily Nepetoideae of the 

family, Lamiaceae. Plectranthus is an “Old World” genus belonging to the Mint/Sage 

family. It contains about three-hundred species found in tropical Africa, Asia, India, 

Madagascar, Australia and a few Pacific islands (Rijo et al., 2007; Lukhoba et al., 2006; 

van Jaarsveld, 2006). The name Plectranthus literally means ‘spur flower’ due to the 

characteristic spurred corolla tube present in the first Plectranthus species that was 

discovered. This physical attribute however, is not consistent throughout the genus and 

may easily be confused for belonging to the Solenostemon or Thorncroftia species. 

Within Africa, Plectranthus can be found in the southern part for example, Namibia, 

Swaziland, Lesotho and South Africa. It grows abundantly in eight out of the nine 

provinces within South Africa, with the largest concentration being found in the north- 

eastern part of the Eastern Cape and southern KwaZulu-Natal which has as much as 

thirty-six species. Plectranthus is desirable as a garden plant because it suppresses weed 

growth and prevents erosion (van Jaarsveld, 1988) and is simple and inexpensive to 

maintain. It is also of ornamental interest and is cultivated for their attractive foliage. 

They are however susceptible to attack by the eel-worm which, if not treated 

appropriately can lead to complete deterioration of the plant (van Jaarsveld, 1988). 

They are usually found growing in shade under trees where the soil is rich in humus and 

well drained. However, not all Plectranthus species thrive under these conditions. 

Species with succulent leaves prefer growth in drier regions such as the dry bushveld or 

in rockeries (Figure 13). Even though Plectranthus species perish or wilt under extreme 

weather conditions such as frost and heat, they recover relatively quickly after a shower 

of rain or sprout again in spring (van Jaarsveld, 1988). 

15

The flower colour varies among the species in Plectranthus, either being white, blue or 

mauve to pink. At the end of February, the plants begin to develop flowerbuds and reach 

full bloom between March and April. While some species of Plectranthus grow as 

upright shrubs to a height of approximately 1.5 meters, for example, ecklonii, fruticosus 

and hadiensis, others occur as groundcover plants, varying between 10 to 30 centimetres 

in height, for example madagascariensis and saccatus. Plectranthus hadiensis can be 

easily identified because of its large, hairy leaves. 

Figure 13: Plectranthus hadiensis (picture courtesy of Prof. N. Crouch) 

Species of Plectranthus are used as ornamental, economic and medicinal plants. 

The phylogeny of Plectranthus was well documented by Paton et al. (2004) and Lukhoba 

et al. (2006) based on its DNA sequence and augmented morphological data of the genus. 

This information was presented in the form of a cladogram (figure 14) which divided the 

Plectranthus genus into two clades or groups. Clade 1 contains Plectranthus species 

formally known as Coleus and are grouped together based on their 

ethnobotanical/medicinal uses with Clade 1b containing a greater number of medicinally 

active species than Clade 1a. The groups in bold in figure 14 all have reported medicinal 

uses. Of the three subclades, subclade 1b seems to be the most widely used medicinally, 

16

followed by subclade 2 and lastly subclade 1a where only groups 2 and 8 are used 

medicinally. Species within Clade 1 are also sources of food and are used as food 

flavourants, fodder for domestic animals, ornamental displays in homes and gardens as 

well as other uses such as building material (Lukhoba et al., 2006). For example, the 

wood from Plectranthus insignis can be used to build huts or temporary houses (Cheek et 

al., 2000; Lukhoba et al., 2006). 

There are further species that fit into clades 1 and 2 but could not be placed into any of 

the subclades as they were morphologically different species within these subclades. 

These are listed as unplaced groups A-E in each clade. 

Plectranthus is a synonym for Coleus (Lukhoba et al., 2006) and therefore in searching 

the literature for phytochemical reviews of Plectranthus, one has to also consider the 

Coleus species. Plectranthus amboinicus itself has synonymns of Plectranthus 

aromaticus Roxb., Coleus aromaticus Benth. and Coleus amboinicus Lour. (Lukhoba et 

al., 2006). 

Beside morphological characteristics and DNA sequencing, it would also be useful to 

have chemotaxonomic data linking the different species together and hence, 

phytochemical studies on the different species of Plectranthus is important to build up a 

database of the secondary metabolites linking these species together. 

17

Clade 1 unplaced groups 

Group A: Plectranthus melleri (P. luteus) 

P. esculentus 

Group B: P. edulis, P. punctatus 

Group C: P. lactiflorus, P. stachyoides 

Group D: P. gracillimus 

Group E: P. mollis, P beddomei 

1 

1a 

1b 

Group 1: Pycnostachys 

Group 2: Plectranthus sylvestris, P. alpinus, 

P. defoliatus, P. insignis, P. decurrens 

Group 3: P. robustus, P. baumii, P. foliatus, 

P. katangensis, P. insolitus 

Group 4: P. thysoideus, P. daviesii, P. sereti 

Group 5: P. glabratus, P. parishii 

Group 6: Anisochilus, Leocus africanum 

Group 7: P. helfleri, P. albicalyx 

Group 8: P. hadiensis, P. grandidentatus, 

P. madagascariensis, P. argentatus, 

P. parvifolius, P. congestus 

P. graveolens, P. asirensis 

Group 9: P. calycinus, P. rehmanii 

Group 1: Plectranthus igniarus 

Group 2: P. tetensis, P. barbatus, P. kivuensis, 

P. lanuginosus, P. caninus 

Group 3: P. coeruleus 

Group 4: P. fredericii, P. hereroensis 

Group 5: P. bojeri, P. rotundifolius, P. vettiveroides, 

P. occidentalis, Plectranthus sp. aff 

occidentalis, P. variifolius 

Group 6: P. buchananii 

Group 7: P. montanus, P. prostratus, 

P. pseudomarruboides, P. lanceolatus 

Group 8: P. aegyptiacus, P amboinicus 

Clade 2 unplaced groups Group 1: Tetradenia 

Group 2: Thomcroftia 

Group A: Plectranthus adenophorus, 

Group 3: Plectranthus elegans, P. fruticosus, 

P. modestum 

P. ciliatus, P. oertendahlii, P. saccatus 

Group B: P. longipes 

Group C: P. gracilis 

2 

P.ambiguus, P. zuluensis, P. parvus 

P. mandalensis, P. eckonliii, P. verticillatus, 

Group D: P. pulcherissima 

P. grallatus 

Group E: P. pubescens, P. radiatus 

P. viphyensis 

Group 4: Aeollanthus 

Group 5: Capitanopsis, Dauphinea, Madlabium 

Group 6: Plectranthus glandulosus, P. longipes 

P. laxiflorus, P. kamerunensis, P. stolzii 

Key 

represents groups cited as being useful represents groups with no/few cited uses Groups shown in bold have recorded medicinal uses 

Figure 14: Illustration of the 2 clades within the Plectranthus species, categorized 

according to their phylogeny (reproduced from Lukhoba et al., 2006) 

18

2.2 Ethnobotanical uses 

A comprehensive review was done by Lukhoba et al. (2006), which contains 

ethnomedicinal information of a variety of Plectranthus species between 1934 and 2005. 

This is summarised in tables 1 and 2 with additional information added for published 

work between 2006 and 2010. 

Table 1 is an inventory of Plectranthus species that are used by traditional healers to help 

alleviate and/or heal skin, digestive, respiratory, muscular-skeletal and genito-urinary 

conditions as well as infections, fever and pain, while table 2 contains an inventory of 

species used to treat heart, circulatory and blood disorders, ailments affecting the sensory 

and nervous system, treatment of poisonous substances in the body, inflammation and 

medical conditions which could not be assigned to any of the other categories, labelled as 

‘unspecific’. 

A total of twenty-one Plectranthus species are used for digestive conditions, nineteen 

used for skin conditions and sixteen and fifteen species respectively used for respiratory 

conditions and infections and fever (Table 1). This in itself is evidence of the genus’ 

widespread use ethnomedicinally. 

The conditions listed in table 2 are not treated by as many species as are those listed in 

table 1 with the highest being eight species used for nervous conditions, followed by six 

species to treat sensory conditions, five species for heart, circulatory and blood disorders 

and four species each for treatment against poisons and inflammation. 

Plectranthus barbatus and Plectranthus amboinicus have proven to be the most widely 

used species, being used in all of the medical conditions listed in Tables 1 and 2 with 

Plectranthus laxiflorus being used in all conditions in table 1 and three of the categories 

as reflected in Table 2. 

It must be noted that digestive conditions as categorised by Lukhoba et al. (2006) contain 

nausea, vomiting and diarrhoea and that “pain” can be associated with a number of 

19

different medical conditions and in many cases, the treatment of inflammation will also 

result in the relief of pain. Respiratory and genito-urinary conditions could also lead to 

infection and fever. It is thus unclear from the information presented in Lukhoba et al. 

(2006) or the papers cited therein whether or not the plant extracts are treating a certain 

condition or the symptoms arising from these conditions as pain and fever are the 

symptoms of a wide variety of diseases. The same can be said about nausea, vomiting 

and diarrhoea. They are the symptoms of a wide variety of viral and bacterial infections. 

Examples of heart, circulatory and blood disorders are congestive heart failure, 

hypertension and angina. Epilepsy, convulsions and depression are related to the nervous 

system while ear and eye problems are grouped as ‘sensory’ conditions. Poisons 

treatment refers to the treatment of a person who has been bitten or stung by an insect or 

animal which posesses venom. 

Even though in many instances, these ethnomedicinal uses are unsubstantiated and 

unclear, they can be useful leads in the testing of plant extracts or compounds isolated 

from them. 

20

Table 1: Summary of the medicinal uses of various Plectranthus species (Lukhoba et al., 

2006*) 

Plectranthus 

species 

Skin 

conditions 

Digestive 

conditions 

Medical conditions 

Respiratory 

conditions 

Infections 

and fever 

Genitourinary 

conditions 

Pain 

Muscular 

–skeletal 

conditions 

P. aegypticus 

(Forssk.) C. Chr 

 

P. alpinus (Vatke) 

O. Ryding 

 

P. ambiguus (Bolus) 

Codd 

 

P. amboinicus 

(Lour.) Spreng 

 

P. asirensis J.R.I 

Wood 

 

P. barbatus Andr. 

P. beddomei Raiz. 

P. bojeri (Benth.) 

Hedge 

 

P. caninus Roth 

P. coeruleus 

(Gurke) Agnew 

 

P. coleoides 2 

P. congestus R.Br. 

P. decurrens 

(Gurke) J.K. Morton 

 

P. defoliatus Hochst. 

Ex Benth 

 

P. eckonlii Benth 

P. edulis (Vatke) 

Agnew 

 

P. elegans Britten 

P. esculentus 

N.E.Br. 

 

P. fruticosus L’Her. 

P. glandulosus 

Hook.f. 

P. hadienis (Forssk.) 

 

Schweinf. Ex 

Spreng. 

+ + + 

P. hereroensis Engl. 

P. ignarius 

(Schweinf.) Agnew 

 

P. insignis Hook.f. 

P. kamerunensis 

(Gurke) 

 

P. lactiflorus 

(Vatke) Agnew 

 

P. lanceolatus Bojer 

ex Benth 

Continued on next page....... 

 

21

Plectranthus 

species 

Skin 

conditions 

Digestive 

conditions 


Respiratory 

conditions 

Infections 

and fever 

Genitourinary 

conditions 

Pain 

Muscular 

–skeletal 

conditions 

P. lanuginosus 

(Benth.) Agnew 

 

P. laxiflorus Benth. 

P. longipes Baker 

P. madagascariensis 

Benth. 

 

P. mandalensis 

Baker 

 

P. melleri Baker 

P. mollis (Aiton) 

Spreng. 

1 

P. montanus Benth. 

P. parviflorus 

(Poir.) Henckel 

 

P. prostratus Gurke 

P. 

 

pseudomarrubiodes 

Willemse 

 

P. pubescens Baker 

P. punctatus L’Her 

P. rogosus 3 

P. stachyoides Oliv. 

P. stolzii Gilli 

P. sylvestris Gurke 

P. tetensis (Bak.) 

Agnew 

P. vettiveroides 

 

(K.C. Jacob) H.I 

Maass 

 

Total 19 21 16 15 7 10 6 

* Where data was not taken from Lukhoba et al. (2006) the references are denoted by superscripts in the table 

and the references are given below 

+ No mention in the references cited within Lukhoba et al. (2006) to these conditions, however Lukhoba et al. 

(2006) lists these as being prevalent 

1 Ayyanar and Ignacimuthu, 2005 

2 Ignacimuthu et al., 2006 

3 Khan et al., 2007 

22

Table 2: Summary of uses of various Plectranthus species (Lukhoba et al., 2006) 

Plectranthus 

species 

Heart, 

circulatory 

and blood 

Nervous Sensory 


Poisons 

Treatment 

Inflammation Unspecific 

P. aegypticus 

(Forssk.) C. Chr. 

 

P. alpinus (Vatke) 

O. Ryding 

 

P. amboinicus 

(Lour.) Spreng 

 

P. barbatus Andr. 

P. bojeri (Benth.) 

Hedge 

 

P. congestus R.Br. 

P. edulis (Vatke) 

Agnew 

 

P. fruticosus 

L’Her. 

 

P. glandulosus 

Hook.f. 

 

P. grallatus Briq. 

P. grandidentatus 

Gurke 

P. hadienis 

 

(Forssk.) 

Schweinf. Ex 

Spreng. 

+ 

P. ignarius 

(Schweinf.) 

Agnew 

P. kivuensis 

 

(Lebrun & Touss.) 

R.H. Willemse 

 

P. laxiflorus 

Benth. 

 

P. longipes Baker 

P. 

 

madagascariensis 

Benth. 

 

P. mandalensis 

Baker 

 

P. mollis (Aiton) 

Spreng 

 

P. montanus 

Benth. 

 

P. occidentalis 

B.J. Pollard 

 

P. pubescens 

Baker 

 

P. punctatus 

L’Her 

Continued on next page..... 

 

23

Plectranthus 

species 

Heart, 

circulatory 

and blood 

Nervous Sensory 


Poisons 

Treatment 

Inflammation Unspecific 

P. sp. aff. 

occidentalis 

 

P. stolzii Gilli 

P. vettiveroides 

 

(K.C. Jacob) H.I 

Maass 

P. viphyensis 

 

Brummit & J.H. 

Seyani 

 

Total 5 8 6 4 4 18 

+ No mention in the references cited within Lukhoba et al. (2006) to these conditions, however Lukhoba et 

al. (2006) lists these as being prevalent 

The modes of administration of the plant extracts differ according to the ailment being 

treated. The treatment of skin conditions such as burns, wounds, allergies and insect 

bites are treated by applying ground plant material on the affected area whilst bathing in 

herbal extracts may help alleviate the irritation associated with measles and chicken pox. 

Digestive and respiratory conditions are treated by drinking teas, infusions or decoctions 

to alleviate constipation, indigestion and dyspepsia as well as asthma, bronchitis and 

other respiratory conditions and the inhalation of steam or smoke is used for the treatment 

of colds and influenza. Enemas and injections are also used as a means of administering 

plant extracts to the patient (Gurib-Fakim, 2006). For instance, the leaf infusion of 

Plectranthus defoliatus Hochst. ex Benth. is either drunk or admininstered as an enema 

(Neuwinger, 2000). With regard to injections being administered, no information is 

given as to how the extracts are injected and whether they use conventional needles and 

syringes or home-made equivalents as there is doubt as to where and how traditional 

healers would get access to needles and syringes. Furthermore, there is doubt as to 

whether traditional healers are trained to administer injections, and the improper use of 

needles and syringes, for example the repeated use of these could do the patient more 

harm than the illness itself. 

24

2.3 Pharmacological/biological uses of the plant extract 

Extracts of Plectranthus species have shown to be active in a wide range of bioassays. 

These include antibacterial, antifungal, antiparastitic, anti-inflammatory, antioxidant, 

antitumour and insect antifeedant activity. Of these, they find most widespread activity 

in antibacterial and antifungal assays. References are given in the subchapters that 

follow. 

Methods used for the determination of antibacterial activity 

The two most common qualitative methods for testing antimicrobial activity of extracts 

are performed by either using the agar well assay or the disc diffusion assay. In the agar 

well method, wells or holes are made into the solidified, sterile agar plate once the test 

inoculum has been evenly distributed on the agar surface. These wells are then filled 

with solutions of the plant extracts and test controls. After incubation, the antimicrobial 

activity is determined by measuring the diameters of zones of inhibition in milimeters. 

In the disc diffusion assay, filter paper discs are dissolved in solutions of plant extracts 

and placed onto the inoculated plate. Once incubation of the plate is complete the 

antimicrobial activity is determined by measuring the diameters of zones of inhibition in 

milimeters. Wells or holes are not required for this method of testing. Since the unit of 

measurement is the same for both test methods, results can be compared. 

The bioautography agar overlay method is derived from the disc diffusion assay/method 

and uses thin layer chromatography (TLC) plates instead of filter paper. In this method 

the plant extract or compound of interest is adsorbed onto the TLC plate followed by the 

introduction of a thin layer of inoculum onto the very same plate. The plant extract or 

compound being evaluated then diffuses from the TLC/adsorbent into the inoculum. 

After incubation of the TLC plate, the Rf values of the observed spots are recorded and 

zones of inhibition are measured in mm. 

All three methods described above, are performed in triplicate to ensure reproducible 

results and are measured against control standards. 

25

Use of the minimum inhibitory concentration (MIC) assay allows one to determine the 

lowest concentration at which an extract will be active. This is normally represented in 

µg/ml and is carried out by serial dilutions of the plant extracts or compounds under 

study. 

Antibacterial activity of Plectranthus and Coleus plant extracts 

It is observed from Table 3 that the Plectranthus species are active against a variety of 

bacterial strains but being more active toward Staphylococcus and Bacillus species with 

sixteen of the twenty-one Plectranthus species being active against Staphylococcus 

aureus and Bacillus subtilis listed in table 3, below. Although the Pseudomonas bacterial 

strain was also a popular choice for antibacterial testing, only eight Plectranthus species 

was active against this pathogen. 

The leaves, roots and aerial parts were the most common parts of Plectranthus which 

have been tested for antibacterial activity with there being one report on the activity of 

the stems and the flowers against bacteria (Rabe and van Staden, 1998; Matu and van 

Staden, 2003). In one study, the root and flower extracts displayed higher antibacterial 

activity than the leaf extracts (Rabe and van Staden, 1998; Matu and van Staden, 2003; 

Mothana et al., 2008) suggesting that further antimicrobial studies of the root and flower 

extracts in Plectranthus should also be carried out. 

The solvents used to prepare the extracts for bioassays, were predominantly polar with 

there being just two instances where hexane was used as the solvent medium. Ideally, a 

high activity of the water extracts is desired as water is readily available, inexpensive and 

safe for human consumption. An alternative to using solvents to prepare plant extracts 

for bioassays, is the hydrodistillation of the plant or certain parts of the plant producing 

an essential oil (Ascensao et al., 1998; Alsufyani et al., (see footnote X under table 3); 

Marwah et al., 2007; Oliveira et al., 2007; Vagionas et al., 2007). This method however, 

excludes the secondary metabolites which are not volatile at the temperatures being used. 

The polar extracts are more active than the non-polar extracts at inhibiting the growth of 

bacteria (Matu and van Staden, 2003). 

26

P. barbatus and P. madagascariensis were the two most commonly tested plant species, 

with extracts from both plants being more susceptible to gram-positive bacteria 

(Staphylococcus aureus and Bacillus subtilis) than gram-negative bacteria (Ascensao et 

al., 1998; Rabe and van Staden, 1998; Matu and van Staden, 2003; Wellsow et al., 2006; 

Kisangau et al., 2007; Figueiredo et al., 2010). Coleus kilimandschari was found to be 

active against a wide variety of gram-negative and -positive bacteria (Vagionas et al., 

2007). 

27

Plectranthus 

species 

Coleus 

kilimandschari 

P. aff. puberulentus 

Table 3: Antibacterial activity of Plectranthus and Coleus extracts 

Extraction 

medium and 

plant 

part/s used 

80% methanol lv 

acetone lv 

Antibacterial activity Reference 

Bacillus cereus, Enterobacter 

cloacae, Escherichia coli, 

Klebsiella pneumoneae, 

Mycobacterium fortuitum, 

Proteus vulgaris, 

Pseudomonas aeruginosa, 

Salmonella typhimurium, 

Staphylococcus aureus, 

Streptococcus pyogenes 

Bacillus subtilis, 

Pseudomonas syringae 

P. argentatus B. subtilis 

P. barbatus 

petroleum ether lv , 

dichloromethane lv 

and water lv 

water w 

hexane rt and 

methanol rt 

hexane s , methanol s 

and water s 

methanol lv 

S. aureus, B. subtilis, E. coli, 

P. aeruginosa 

Cariogenic bacteria 

(Streptococcus sobrinus and 

Streptococcus mutans) 

B. subtilis, S. aureus, 

Micrococcus luteus 

P. ciliatus 

(3 different collections) 

methanol lv 

P. ciliatus 

S. aureus, B. subtilis 

S. aureus, B. subtilis, 

(1 collection) 

M. luteus 

P. cylindraceus oil a 

E. coli, P. aeruginosa, K. 

pneumoniae, S. aureus, B. 

subtilis, Salmonella 

choleraesuis 

water 

P. eckonlii 

w Cariogenic bacteria (S. 

sobrinus and S. mutans) 

methanol lv S. aureus, B. subtilis 

methanol f 


M. luteus, Staphylococcus 

epidermis 


Cos et al., 2002 

Wellsow et al., 

2006 

Kisangau et al., 

2007 

Figueiredo et al., 

2010 

Matu and van 

Staden, 2003 

Rabe and van 

Staden, 1998 

Marwah et al., 

2007 


2010 


Staden, 1998 

28

Plectranthus species 

P. forsteri 

‘marginatus’ 

P. fruticosus 


Extraction 

medium and plant 

part/s used 

P. fruticosus methanol f 

P. hadiensis 


acetone lv B. subtilis, P. syringae 

methanol lv S. aureus, B. subtilis 

water rt 

methanol rt+lv and 

water lv 


M. luteus 

multi-resistant strains of S. 

epidermidis, 

Staphylococcus 

haemolyticus and S. 

aureus, the North German 

epidemic strain 


Micrococcus flavus and 

multi-resistant strains of S. 

epidermidis, S. 

haemolyticus and S. 

aureus, the North German 

epidemic strain 

hexane a 8 bacillus formulations 

dichloromethane a 

P. hereroensis acetone rt 

P. laxiflorus essential oil a 




P. oribiensis 

P. oribiensis 


B. subtilis, Xanthomonas 

campestris 

S. aureus, Vibrio cholera, 

Streptococcus faecalis 

S. aureus, S. epidermis, E. 

coli, E. cloacae, K. 

pneumoniae, P. aeruginosa 

acetone lv B. subtilis, P. syringae 

essential oil a 

methanol lv 

B. subtilis, *Micrococcus 

sp., S. aureus, Yersinia 

enterocolitica 


M. luteus, S. epidermis 






2006 


Staden, 1998 

Mothana et al., 

2008 

Laing et al., 2006 

Batista et al., 1994 

Vagionas et al., 

2007 


2006 

Ascensao et al., 

1998 


Staden, 1998 

29


Extraction 

medium and plant 

part/s used 

P. ornatus essential oil lv 


S. aureus, S. pyogenes, 

E. coli, S. typhimurium 

P. petiolaris methanol lv S. epidermis, B. subtilis 

P. puberulentus acetone lv P. syringae 

P. rubropunctatus 

*Plectranthus sp. 

(3 hybrids) 


(1 hybrid) 


(1 hybrid) 


S. epidermis 

S. aureus, B. subtilis 

methanol lv S. aureus, B. subtilis, 

M. luteus 


S. epidermis 

P. strigosus 



P. tenuiflorus essential oil lv 

S. aureus, S. pyogenes, 

E. coli, K. pneumoniae, 

P. aeruginosa 

P. verticillatus methanol lv S. aureus, B. subtilis, 

S. epidermis 

Oliveira et al., 2007 


Staden, 1998 


2006 


Staden, 1998 

x Alsufyani et al., 


Staden, 1998 

NB. A species of Coleus is also included in this table because of the uncertain relationship to the 


* Unknown species 

x 

available electronically on http://www.kau.edu.sa, date accessed 27/09/2010; the date of the publication 

is not apparent. 

Key: lv: leaves, rt: roots, s: stems, a: aerial parts, w: whole plant, f: flowers 

Table 4 contains antifungal activity of eight Plectranthus species and one Coleus species. 

All of the species in table 4 were active against at least one Candida species with six 

Plectranthus species being active against Candida albicans, the most widely tested 

fungal pathogen. P. amboinicus and P. barbatus were the two most tested species for 

antifungal activity from all the Plectranthus species. 

From the studies reviewed, hydrodistillation seemed to be the most popular method of 

extraction, followed by solvent extraction with water, methanol and more uncommonly 

solvents such as ether, hexane and dichloromethane. 

30

There were two comparative studies on the solvent extracts with regard to antifungal 

activity (Laing et al., 2006; Kisangau et al., 2007). In one study, the ether extract showed 

higher activity than the dichloromethane and aqueous extracts while in the other study, 

the hexane extract was more effective than the dichloromethane extract. This indicates 

that future studies on ether and hexane extracts need to be conducted since these extracts 

were shown to be active. 

The leaves are the most popular part of the plant used to make extracts for antifungal 

activity. Other studies mention aerial parts being used for extraction but these parts are 

not specified and could be the leaves, flowers, seeds or fruit. There is also one report on 

the roots being used. There is thus a need for more studies on the roots and stem material 

to be carried out for antifungal activity as not many studies have been done on these plant 

parts. 

There is also a need for comparative studies to be done with the different plant parts as 

well as with the solvent used for extraction. This would then provide a much clearer 

indication of which plant part as well as which solvent is best suited for antifungal 

activity and would be a good guide for phytochemists to use when choosing a suitable 

plant part to study. 

31

Plectranthus 

species 

Coleus 


P. amboinicus 

P. barbatus 

Table 4: Antifungal activity of Plectranthus and Coleus extracts 

Extraction 


plant 

part/s used 

80% methanol lv 

essential oil lv 

Antifungal activity Reference 

Candida albicans, 

Microsporum canis, 

Trichophyton rubrum, 

Epidermophyton floccosum 

Aspergillus flavus, Aspergillus 

niger, Aspergillus ochraceus, 

Aspergillus oryzae, Candida 

versatilis, Fusarium 

moniliforme, *Penicillium sp., 

Saccharomyces cerevisiae, in 

stored food commodities 

methanol lv Candida krusei 


C. albicans, Candida 

tropicalis, Candida 

guilliermondii, Candida krusei 

methanol lv Candida krusei 

80% methanol rt+lv C. albicans 

petroleum ether lv , 


and water lv 

P. cylindraceus oil a 

C. albicans 

C. albicans, M. canis, 

Microsporum gypseum, 

T. rubrum, Trichophyton 

mentagrophytes 

Inhibits fungal spore 

germination and growth of 

*Bipolaris sp., Alternaria 

alternate, Fusarium 

oxysporum, Curvularia lunata, 

Stemphyllum solani 

P. grandis methanol lv Candida krusei 



Murthy et al., 

2009 

Tempone et al., 

2008 

de Oliveira et 

al., 2007 


2008 

Runyoro et al., 

2006 

Kisangau et al., 

2007 

Marwah et al., 

2007 


2008 

32

Plectranthus 

species 

P. hadiensis 

P. laxiflorus 

Extraction 


plant 

part/s used 

hexane a 

dichloromethane a 

Antifungal activity Reference 

Sclerotinia sclerotiorum, 

Rhizoctonia solani, *Candida 

species 

S. sclerotiorum, *Candida 

species 

essential oil a C. albicans, C. tropicalis, 

C. glabrata 

P. neochilus methanol lv Candida krusei 

P. tenuiflorus essential oil lv C. albicans 

Laing et al., 

2006 


2007 


2008 

x 

Alsufyani et 

al., 

NB. A species of Coleus is also included in this table because of the uncertain relationship to the 



x 

available electronically on http://www.kau.edu.sa, date accessed 27/09/2010; the date of the publication 

is not apparent. 


Besides posessing antifungal and antibacterial activity, Plectranthus and Coleus species 

also have anti-tumor, antiparasitic, antioxidant, antifeedant and anti-inflammatory 

properties, as shown in Table 5a. 

P. amboinicus appears to be the most commonly used Plectranthus species, being active 

against a wide variety of pharmacological activities. P. barbatus was also a popular plant 

species used, being active in three of the five pharmacological activities contained in 

table 5a. Other Plectranthus species are used less frequently. 

The leaves seem to be the most popular plant part used to inhibit the growth of parasites, 

to act as an antioxidant and antifeedant as well as for the treatment of inflammation and 

tumors. There has been only two reports on root extracts being used and just one report 

on the stem being used. More research on the root, stem and aerial parts is required as it 

is still unclear as to which plant part is the most active. 

33

Methanol, ethanol and water seem to be used more frequently than hexane and acetone 

for making extracts to be tested in these bioassays. There has been one report where 

hexane and acetone extracts were used. This may limit the studies to polar extracts and 

more studies using hexane, dichloromethane and acetone is needed to ascertain whether 

the non-polar fractions are active in these same bioassays. 

Table 5a: Antiparasitic, anti-inflammatory, antioxidant, antitumour and insect antifeedant 

activity of polar extracts from seven Plectranthus species 

Plectranthus 

species 

P. amboinicus 

Solvent of 

Extraction 

aqueous lv 

methanol lv 

P. barbatus methanol lv 

P. cylindraceus essential oil a 

P. grandis methanol lv 

a, lv 

P. marruboides essential oil 

P. neochilus methanol lv 

P. punctatus 

aqueous lv and 

80% 

methanol lv 


Pharmacological details Reference 

Antiparastic activity 

inhibits growth of 

Plasmodium berghei yoelii 

antileishmanial activity 

against Leishmania 

chagasi and Leishmania 

amazonensis 




amazonensis 

nematicidal activity against 

Meloidogyne javanica 




amazonensis 

toxic fumigant against 

Anopheles gambiae 




amazonensis 

ovicidal and larvicidal 

activity against 

Haemonchus contortus 

Periyanayagam et 

al., 2008 


2008 


2008 

Onifade et al., 

2008 


2008 

Omolo et al., 2005 


2008 

Tadesse et al., 

2009 

34

Plectranthus 

species 

Coleus 



Extraction 

80% 

methanol lv 


Antiviral activity 

Antiviral activity against 

DNA-virus Herpes simplex 

virus type 1 

Antioxidant Activity 

P. amboinicus essential oil lv antiradical activity 

P. barbatus 

P. eckonlii 

P. fruticosus 

P. hadiensis 

aqueous lv 

methanol rt+lv 

and water rt+lv 

P. lanuginosus aqueous lv 

antioxidant activity 

confirmed by DPPH and βcarotene/linoleic 

acid test 

assays 

antiradical activity 



acid test 

assays 

P. laxiflorus essential oil a antiradical activity 

P. verticillatus aqueous lv 

P. amboinicus 

P. barbatus 

Continued on next page.... 

70% ethanol lv 



acid test 

assays 

Anti-inflammatory activity 


Murthy et al., 

2009 

Fale et al., 2009 

Mothana et al., 

2008 



2007 


Gurgel et al., 2009 

composition anti-inflammatory Wong et al., 2009 

hexane rt, s+lv , 

water rt, s+lv and 

rt, s+lv 

methanol 

Matu and van 

Staden, 2003 

35

Plectranthus 

species 

P. amboinicus 

P. aff. 

puberulentus 

P. forsteri 


P. puberulentus 

P. saccatus 

P. zuluensis 


Extraction 

70% ethanol lv 

leaf juice 

composition 

(orally, 

topically or 

injections) 

acetone lv 


Anti-tumor activity 

anti-tumor (Sarcoma-180 

ascite and Ehrlich ascite 

carcinoma) activity in mice 

Inhibits growth of 

carcinoma (HepG2, Huh7) 

and melanoma (Bowes) 

cells 

Insect-antifeedant activity 

antifeedant activity against 

S. littoralis 

Gurgel et al., 2009 

Cheng-Yu et al., 

2006 


2006 

NB. The term ‘mixture’ refers to the testing of a composition comprising of a Plectranthus plant extract 

with either another plant extract/essential oil (not necessarily belonging to the Plectranthus species) or an 

appropriate pharmaceutical drug. 


Plectranthus and Coleus species act as abortificients (Almeida and Lemonica, 2000), 

prevent the formation of plaque build-up on teeth (Figueiredo et al., 2010), are used 

topically as a barrier against harmful UV rays (Chen et al., 2009), act as a hair growth 

stimulant (Kiyoshi et al., 2005) and inhibitor (Kazuo et al., 2006), are used in the 

treatment of rheumatoid arthritis (Rey-Yuh et al., 2008; Jui-Yu et al., 2009), act as a 

scorpion venom antidote (Uawonggul et al., 2006) and are used to treat Alzheimers 

disease (Fale et al., 2009) as well as many other ailments, disorders and diseases (table 

5b). 

P. amboinicus and P. barbatus seem to be the most commonly tested species each having 

widespread application. Once again the leaves appear to be the most popular plant part 

used. There has been just one study on the root extract. The stems and roots need to be 

studied further. 

36

There are plant extracts in table 5b which have been tested as mixtures either in 

combination with other plant extracts or pharmaceutical agents, which highlights the 

synergistic effect of various Plectranthus and Coleus species. 

Table 5b: Other important inhibitory activity of polar extracts from Plectranthus and 

Coleus species 

Plectranthus 

species 

C. barbatus 

C. foskohlii 

*Coleus 

species 

P. amboinicus 



Extraction 

Other inhibitory activity 

Other Activities Reference 

70% ethanol lv anti-implantation effects 

in rats 

Almeida and 

Lemonica, 2000 

mixture prevents foul breath Sonoko et al., 2005 

prevention of progression 

of diabetic complications 

and skin aging 

Masayuki et al., 

2007 

mixture 

antispasmodic effect on 

respiratory tract smooth 

muscle 

Rongqiu et al., 

2005 

treat all forms of obesity 

and associated metabolic 

syndromes 

Marcin et al., 2005; 

Han et al., 2005 

hair growth stimulant Kiyoshi et al., 2005 

body hair growth inhibitor Kazuo et al., 2006 

food mixture 

improves bowel 

movement 

antiasthmatic, cough- 

Motoyuki et al., 

2007 

composition 

relieving,phlegmexpelling, treats acute and 

chronic bronchitis 

Jian et al., 2006 

food/cosmetic promotes longevity and 

compostions, increases bone mass and Sayuri et al. 2008 

powder decreases body weight 

mixture 

remedy for menopause 

disorder 

Chiyoko et al., 

2005 

aqueous lv 

acts as scorpion venom 

antidote against 

Heterometrus laoticus 

Uawonggul et al., 

2006 

mixture treat rheumatoid arthritis Jui-Yu et al., 2009 

essential oil mixture 

relaxing uterus and 

alleviating dysmenorrhea 

Jiali et al., 2007 

37

Plectranthus 

species 

P. amboinicus 

P. barbatus 

P. eckonlii 

P. fruticosus 


Extraction 

composition 

essential oil mixture 

(applied on belly) 

composition (taken 

orally or applied 

topically) 

aqueous lv 


treatment of skin disorders 

and healing of wounds 

especially in diabetic 

patients 

Relieves menstrual pain 

by relaxing the uterus, 

tested in mice 

Treat rheumatoid arthritis 

(tested in animals) 

helps in treatment of 

Alzheimers disease 

water lv prevents dental caries 

70:30 

ethanol:propylene 

glycol rt 

aqueous lv 

used topically as a 

mixture for UV 

protection. Tested (in 

vitro) on human and 

guinea pig skin 



waterlv prevents dental caries 

aqueous lv 

essential oil (diethyl 

ether extract) 


P. grandis leaves 

P. lanuginosus aqueous lv 

*Plectranthus 

species 

hot water 

P. striatus mixture 

Continued on next page.... 



antifertility activity by 

inhibiting implantations, 

in rats 

embryotoxic properties, in 

rats 

possesses gastroprotective 

properties 



increases blood vessel 

elasticity in stroke prone 

rats 

promotes function of 

gallbladder 

Rey-Yuh et al., 

2007 

Chia-Li et al., 2007 

Rey-Yuh et al., 

2008 



2010 

Chen et al., 2009 



2010 


Chamorro et al., 

1991 

Pages et al., 1988 

Rodrigeus et al., 

2010 


Tetsuji et al., 2007 

Yaoliang et al., 

2006 

38

Plectranthus 

species 


Extraction 

P. ternifolius mixture 

P. verticillatus aqueouslv 


treat liver function 

impairment 



Guo et al., 2006 


NB. Coleus species are also included in this table because of the uncertain relationship to the Plectranthus 

species 



2.4 The phytochemistry of Plectranthus 

The main phytochemical constituents of Plectranthus are diterpenoids, with about one- 

hundred and forty-seven of these compounds being isolated from the coloured leaf-glands 

of Plectranthus species, the majority of which were highly modified abietanoids. 

Essential oils, monoterpenoids, sesquitepenoids and phenolics have also been isolated 

from Plectranthus species (Abdel-Mogib et al., 2002; Lukhoba et al., 2006). 

Compounds 27 to 174 are grouped into five categories, the royleoanone-type abietanes 

(compounds 27 to 69), the spirocoleons (compounds 70 to 108), vinylogous quinones 

(compounds 109 to 130), coleon-type abietanes (compounds 131 to 167) and the 

miscellaneous abietanes (compounds 168 to 174) which do not resemble any of the 

abietanes in the previous four categories. 

Twenty royleanone type abietanes with the presence of a hydroxybenzoquinone or p- 

quinoid ring C system and an isopropyl group at C-13 have been isolated from eleven 

known Plectranthus species and three Coleus species (Table 6a). These compounds all 

have methyl groups at C-4 and C-10 with the exception of compound 42 where the 

methyl group at C-10 is oxidized to an alcohol. Compound 45 is notably different from 

compounds 27 to 46, as the substituent at C-12 is an acetyl group. At C-6 and C-7 of 

compounds 27 to 42, there are either hydroxy, aldehyde or acetyl groups attached to these 

positions, with the substituent at C-6 being in the beta position. In compounds 43 to 46, 

39

variation is observed within ring B with a double bond being present either at the ∆ 5 or ∆ 6 

positions. Compound 46 has an epoxide ring in place of the olefinic bond at ∆ 8 . 

Table 6a: Royleanone-type abietanes with an isopropyl side chain at C-13, isolated from 

Plectranthus and Coleus species 

Compound Name Synonym 

27 

28 

29 

30 

31 

32 

Continued on next page….. 

12-hydroxy-8,12abietadiene-11,14-dione 

7α,12-dihydroxy-8,12abietadiene-11,14-dione 

7β,12-dihydroxy-8,12abietadiene-11,14-dione 

7-formyl-12-hydroxy- 

8,12-abietadiene-11,14dione 

7α-acetoxy-12-hydroxy- 

8,12-abietadiene-11,14dione 

6β,12-dihydroxy-8,12abietadiene-11,14-dione 

royleanone 

horminone 

taxoquinone 

7-Oformylhorminone 

7αacetoxyroyleanone 

6βhydroxyroyleanone 

Isolated from 

Plectranthus (P) or 

Coleus (C) species 

P. grandidentatus; 

C. carnosus; 

*Plectranthus 

species 

P. hereroensis; 


C. carnosus; 

*Plectranthus 

species; 

P. sanguineus 

*Plectranthus 

species 

P. sanguineus 

Reference 

Gaspar-Marques et al., 

2006; 

Cerqueira et al., 2004; 

Yoshizaki et al., 1979; 

Hensch et al., 1975 


2006; 

Batista et al., 1994; 


Hensch et al., 1975; 

Matloubi-Moghadam et 

al., 1987 



al., 1987 

C. carnosus Yoshizaki et al., 1979 


C. carnosus; 

P. sanguineus 


2006; 



al., 1987 

40


33 

34 

35 

36 

37 

38 

Continued on next page…… 

6β,7α,12-trihydroxy- 

8,12-abietadiene-11,14dione 

6β,7β,12-trihydroxy- 

8,12-abietadiene-11,14- 

dione 

7α-acyloxy-6β,12dihydroxy-8,12abietadiene-11,14-dione 

7α-acetoxy-6β,12dihydroxy-8,12abietadiene-11,14-dione 

7β-acetoxy-6β,12dihydroxy-8,12abietadiene-11,14-dione 

7α-formyloxy-6β,12dihydroxy-8,12abietadiene-11,14-dione 

6β,7αdihydroxyroyleanone 

6β,7βdihydroxyroyleanone 

7α-acyloxy-6βhydroxyroyleanone 

7α-acetoxy-6βhydroxyroyleanone 

7β-acetoxy-6βhydroxyroyleanone 

7α-formyloxy-6β- 

hydroxyroyleanone 

Isolated from 




C. carnosus; 

P. argentatus; 

*Plectranthus 

species; 

P. sanguineus; 

P. edulis; 

P. hadiensis; 

P. fasciculatus 

Reference 


2006; 


2002; 



Alder et al., 1984a; 



al., 1987; 

Kunzle et al., 1987; 

Laing et al., 2006; 

Rasikari, 2007 

C. zeylanicus Mehrota et al., 1989 

P. grandidentatus 


C. carnosus; 


*Plectranthus 

species; 

C. zeylanicus; 


P. hadiensis; 

P. actites 



2002 

Teixera et al., 1997; 


2006; 


2002; 





Mehrotra et al., 1989; 


al., 1987; 

van Zyl et al., 2008; 


C. zeylanicus Mehrotra et al., 1989 

P. myrianthus; 


P. hadiensis; 

P. sanguineus 

Miyase et al., 1977a; 


van Zyl et al., 2008; 


al., 1987 

41


39 

40 

41 

42 

43 

44 

45 

46 

* unknown species 

6β-formyloxy-7α,12dihydroxy-8,12- 

abietadiene-11,14-dione 

12-hydroxy-8,12abietadiene-7,11,14- 

trione 

6β,12-dihydroxy-8,12abietadiene-7,11,14- 

trione 

7α-acetoxy-6β,12,20trihydroxy-8,12abietadiene-11,14-dione 

12-hydroxy-6,8,12abietatriene-11,14-dione 

6,12-dihydroxy-5,8,12abietatriene-7,11,14trione 

12,16-diacetoxy-6hydroxy-5,8,12abietatriene-7,11,14- 

trione 

8α,9α-epoxy-6,12dihydroxy-5,12abietadiene-7,11,14trione 

6β-formyloxy-7αhydroxyroyleanone 

7-oxoroyleanone 

Isolated from 



Reference 

P. argentatus Alder et al., 1984a 

*Plectranthus 

species 

5,6-dihydrocoleon U P. sanguineus 

7α-acetoxy-6β,20dihydroxyroyleanone 

6,7dehydroroyleanone 

Coleon U quinone 



al., 1987 

C. carnosus Yoshizaki et al., 1979 


C. carnosus; 

*Plectranthus 

species; 

P. graveolens 

P. forsteri 

‘marginatus’; 

C. xanthanthus; 


P. sanguineus 


2006; 




Wellsow et al., 2006; 

Mei et al., 2002; 



al., 1987 

Xanthanthusin H C. xanthanthus Mei et al. 2002 

8α,9α-epoxycoleon 

U quinone 

C. xanthanthus; 


P. sanguineus 

Mei et al. 2002; 



al., 1987 

42

4 

20 

10 

O 

H 

19 18 

6 7 

R 1 

OH 

C 

13 

R 2 

O 

R1 R2 

(27) H H 

(28) H α-OH 

(29) H β-OH 

(30) H α−ΟCΗ(Ο) 

(31) H α-OC(O)CH3 

(32) OH H 

(33) OH α-OH 

(34) OH β-OH 

(35) OH Fatty acid ester 

(36) OH α-OC(O)CH3 

(37) OH β-OC(O)CH3 

(38) OH α-OCH(O) 

(39) OCH(O) α-OH 

3 

1 

20 

O 

H 

19 18 

10 

6 

(43) 

OH 

12 

7 

O 

3 

3 

1 

20 

O 

H 

19 18 

1 

20 

O 

H 

19 18 

R 

12 

7 

OH 

R 

(40) H 

(41) OH 

12 

OH 

7 

R 1 

O 

R 2 

R1 R2 

(44) OH CH3 

(45) OC(O)CH3 CH2OC(O)CH3 

O 

O 

O 

O 

H 

19 18 

OH 

OH 

12 

HOH2C 1 

O 

6 7 

20 

11 13 

10 8 

4 

O 

(42) 

OH 

OCCH 3 

Seven compounds 47 to 53 consisting of two abietane compounds joined either by 

oxygen or directly by carbon atoms from C-7 in one abietane to C-11' and C-12' (as in 

compound 47) or to C-7' and C-14' (as in compound 51) in the other have also been 

isolated from Plectranthus species. These have been isolated from P. grandidentatus, P. 

sanguineus, P. myrinathus and C. carnosus with P. grandidentatus yielding all seven 

5 

O 

OH 

(46) 

8 

O 

O 

O 

43

compounds (Table 6b). Depending on the orientation at C-7 and how this carbon atom 

joins to the next abietane, isomers result as in compounds 47 and 48; 49 and 50; and 51 

and 52. Compound 53 is a dimer joined at C-7 of both compounds. These compounds 

are difficult to name systematically and therefore only the common names are given in 

Table 6b. 

Table 6b: Royleanone-type abietane dimers isolated from Plectranthus and Coleus 

species 

Compound Common name 

47 Grandidone A 

Isolated from 

Plectranthus (P) 

or Coleus (C) 

species 


P. sanguineus 

48 7-epigrandidone A P. grandidentatus; 

49 Grandidone B P. myrianthus; 

50 7-epigrandidone B 

C. carnosus; 

P. sanguineus 

51 Grandidone D P. grandidentatus; 

52 7-epigrandidone D 

53 Grandidone C 

2 

19 

4 

10 

O 

H 

18 

2' 

19' 

6 7 

O 

4' 

9 

11 

OH 

14 

β 

O 

O 

α 

12' 

O 

11' 

9' 

7' 

16 

O 

17 

16' 

OH 


C. carnosus 

17' 

18' 

OH 

ΟΗ 

(47) (48) 

Ο 

Η 

Ο 

7 

ΟΗ 

Ο 

β 

α 

Reference 



2002 

Uchida et al., 1981; 

Matloubi-Moghadam et al., 

1987 

Ο 

Ο 

Uchida et al., 1981 

Ο 

ΟΗ 

44

19 

O 

20 

9 

11 

4 6 7 

OH 

O 

O 

H 

H 

18 

O 

O 

7' 

6' 

9' 

4' 

14' 

17' 

12' 

20' O 

OH 

16' 

14 

16 

O 

17 

O 

H 

O 

OH 

O 

O 

O 

OH 

OH 

O 

H 

H 

O 

O 

(49) (50) 

O 

H 

O 

OH 

O 

O 

O 

H 

O 

O 

O 

OH 

(51) (52) (53) 

Seventeen compounds with linear side-chains at C-13 instead of the isopropyl group, 

compounds 54 to 69 were isolated from P. hereroensis, P. edulis, P. lanuginosus, P. 

sanguineus and C. coerulescens (Table 6c). P. lanuginosus yielded the highest number 

of compounds compared to the other species which yielded less than five compounds. 

The side chains are either saturated as in 54, contain a double bond as in compounds 55- 

63, an acetyl group as in compound 64 or an alcohol as in compounds 65, 68 and 69. 

Compounds 57 and 61 and 65-69 have functional groups at C-3 with 66-69 being abeo- 

abietanes where a methyl from C-4 has migrated to C-3. 

O 

O 

H 

O 

O 

O 

OH 

O 

H 

O 

O 

7 

OH 

H 

H 

7' 

OH 

O 

O 

H 

O 

O 

45

Table 6c: Royleanone-type abietanes with a linear side chain at C-13 isolated from 

Plectranthus and Coleus species 


54 

55 

56 

57 

58 

59 

60 

61 

62 

63 

64 


16-acetoxy-7α,12-dihydroxy- 

8,12-abietadiene-11,14-dione 

7α,12-dihydroxy- 

17(15→16)-abeoabieta- 

8,12,16-triene-11,14-dione 

6β,7α,12-trihydroxy- 


8,12,16-triene-11,14-dione 

3α-formyloxy-6β,7α,12trihydroxy-17(15→16)abeoabieta-8,12,16-triene- 

11,14-dione 

19-formyloxy-6β,7α,12trihydroxy-17(15→16)abeoabieta-8,12,16-triene- 

11,14-dione 

7α-ethoxy-6β,12,19trihydroxy-17(15→16)abeoabieta-8,12,16-triene- 

11,14-dione 

12-hydroxy-17(15→16)abeoabieta-6,8,12,16- 

tetraene-11,14-dione 

3α-formyloxy-12-hydroxy- 


6,8,12,16-tetraene-11,14- 

dione 

12,19-dihydroxy- 


6,8,12,16-tetraene-11,14- 

dione 

12-hydroxy-19-formyloxy- 


6,8,12,16-tetraene-11,14dione 

16-acetoxy-7α,12-dihydroxy- 

8,12-abietadiene-11,14-dione 

6β,7α-dihydroxy(allyl) 

royleanone 

Lanugone D 

Lanugone E 

Lanugone A 

Lanugone B 

Lanugone C 

Isolated from 

Plectranthus 

(P) or Coleus 

(C) species 

P. hereroensis 


P. sanguineus 

Reference 

Gaspar-Marques et 

al., 2006; 



al., 2006; 


Matloubi- 

Moghadam et al., 

1987 

P. edulis Kunzle et al., 1987 

P. lanuginosus Schmid et al., 1982 

P. edulis; 



Schmid et al., 1982 



P. hereroensis Batista et al., 1995 

46


65 

66 

67 

68 

69 

19 18 

OH 

O 11 

15 

OCCH3 20 

1 

10 

13 

14 

O 

16 

H 

17(1516)abeo-3α,18diacetoxy-6β,7α,12,16tetrahydroxy-8,12- 

abietadiene-11,14-dione 

3β-acetoxy-6β,7α,12trihydroxy-17(15→16),18(4→3)bisabeo-abieta- 

4(18),8,12,16-tetraene- 

OH 

11,14-dione 

17(1516),19α(43)bisabeo-6β,7α,12trihydroxy-4(18),8,12,16- 

abietatetraene-11,14-dione 

17(1516),19β(43)bisabeo-6β,7α,12,16tetrahydroxy-4(18),8,12- 

abietatriene-11,14-dione 

17(1516),19β(43)bisabeo-6β,12,16trihydroxy-7α-methoxy- 

4(18),8,12-abietatriene- 

11,14-dione 

O 

R 1 

1 

20 

O 

H 

19 18 

17(1516),19(43)bisabeo-6β,7α,16trihydroxyroyleanone 

17(1516),19(43)bisabeo-6β,16-dihydroxy- 

7α-methoxyroyleanone 

10 

6 

R 2 

OH 

11 13 

14 

OH 

(54) R1 R2 

(55) H H 

(56) H OH 

(57) OCH(O) OH 

15 

O 

16 

Isolated from 


or Coleus (C) 

species 

R 1O 

CH 2 

19 

4 

O 

H 

11 

OH 

7 

OH 

13 

14 

O 

OR 2 

R1 R2 

(58) CH(O) H 

(59) H C2H5 

47 

Reference 

C. coerulescens Grob et al., 1978 

P. grandidentatus Gaspar-Marques 

et al., 2006 

P. edulis 

Kunzle et al., 

1987 


15 

16

R 1 

R2CH2 19 

O 

H 3CCO 

19 

O 

H 

6 

12 

OH 

14 

8 

O 

R1 R2 

(60) H H 

(61) OCH(O) H 

(62) H OH 

(63) H OCH(O) 

3 

1 

4 

18 

O 

H 

10 

6 7 

OH 

OH 

12 

16 

OH 

O 

15 

19 

O 

OH 

1 O 

H 

3 

7 

20 

O 

OH 

17 

16 

O 

OCCH 3 

O 

H 3CCO 

3 

H3CCOH2C 18 

O 

(64) (65) 

1 O 

H 

6 7 

OH 

OH 

OR 1 

(66) R1 R2 

(67) H CH2CH=CH2 

(68) H CH2CH(OH)CH3 

(69) CH3 CH2CH(OH)CH3 

18 

Thirty-eight compounds 70-108, commonly referred to as spirocoleons were isolated 

from P. edulis (eleven compounds), C. garckeanus (nine compounds), P. lanuginosus and 

C. coerulescens (seven compounds), C. barbatus (three compounds), P. grandis and C. 

somaliensis (two compounds) and P. barbatus (one compound) (Table 7). These 

compounds are refered to as spirocoleons as C-13 is the common carbon atom which 

links the cyclopropyl ring to the coleon-type abietane. The cyclopropyl bridge is 

represented as being either in the α or β position with the methyl group at C-15 being 

oriented such that the molecule is either R or S with respect to C-15. In compounds 71- 

82, R1 to R4 have either hydrogen, acetyl or formyloxy groups. The difference between 

72 and 83 is the orientation/stereochemistry of the methyl group at C-15. Oxidised 

R 2 

1 

20 

O 

H 

12 

6 7 

OH 

OH 

OH 

48 

O 

16 

OH


functional groups occur at C-6 and C-7, with C-6 being predominantly hydroxyl. 

Compound 100 is the only compound which has an acetyl group present at C-17. 

Table 7: Spirocoleons isolated from Plectranthus and Coleus species 


70 

71 

72 

73 

74 

75 

76 

77 

78 

79 

80 

*cannot be conventionally named due to 

the linkage between C-1 and C-11 

(15S)-6β,7α,12α-trihydroxy- 

13β,16-cycloabieta-8-ene-11,14- 

dione 

(15S)-7α-formyloxy-6β,12αdihydroxy-13β,16-cycloabieta-8- 

ene-11,14-dione 

(15S)-7α-formyloxy-6β,12α,19trihydroxy-13β,16-cycloabieta-8- 


(15S)-19-formyloxy-6β,7α,12αtrihydroxy-13β,16-cycloabieta-8- 

ene-11,14-dione 

(15S)-7α,19-bis(formyloxy)- 

6β,12α-dihydroxy-13β,16- 

cycloabieta-8-ene-11,14-dione 

(15S)-3α-formyloxy-6β,7α,12αtrihydroxy-13β,16-cycloabieta-8- 

ene-11,14-dione 

(15S)-3α,7α-bis(formyloxy)- 


cycloabieta-8-ene-11,14-dione 

(15S)-3α-formyloxy-19-acetoxy- 

6β,7α,12α-trihydroxy-13β,16- 

cycloabieta-8-ene-11,14-dione 

(15S)-3α-formyloxy-6β-acetoxy- 

7α,12α-dihydroxy-13β,16- 


(15S)-3α-acetoxy-6β,7α,12αtrihydroxy-13β,16-cycloabieta-8ene-11,14-dione 

Barbatusin 

Lanugone F 

Lanugone G 

Lanugone H 

Lanugone I 

Lanugone J 

3-O-desacetyl-3-Oformyl 

coleon Y 

6,12-bis(Odesacetyl) 

coleon R 

Isolated from 

Plectranthus 

(P) or Coleus 

(C) species 

C. barbatus; 

P. grandis 


Reference 

Zelnik et al., 1977; 

Rodrigues et al., 

2010 

Schmid et al., 1982; 

Kunzle et al., 1987 






49


81 

82 

83 

84 

85 

86 

87 

88 

89 

90 

91 

92 

(15S)-3α,6β-diacetoxy-7α,12αdihydroxy-13β,16-cycloabieta- 


8-ene-11,14-dione 

(15S)-3α,19-diacetoxy- 

6β,7α,12α-trihydroxy-13β,16- 


(15R)-7α-formyloxy-6β,12αdihydroxy-13β,16-cycloabieta- 


(15R)-7α-acetoxy-6β,12αdihydroxy-13β,16-cycloabieta- 


(15R)-3α,7α-bis(formyloxy)- 



(15R)-3α-formyloxy-6β,12αdiacetoxy-7α-hydroxy-13β,16- 


(15S)-6β,12α-acetoxy-7αhydroxy-13β,16-cycloabieta-8- 

ene-3,11,14-trione 

(15S)-6β,12α-acetoxy-3,7αdihydroxy-13β,16-cycloabieta- 


(15S)-19(43)-abeo-7αacetoxy-6β,12α-dihydroxy- 

13β,16-cycloabieta-3,8-diene- 

11,14-dione 

(15R)-19(43)-abeo-6βacetoxy-7α,12α-dihydroxy- 

13β,16-cycloabieta-3,8-diene- 

2,11,14-trione 

(15S)-19α(43)-abeo- 

6β,7α,12α-trihydroxy-13β,16cycloabieta-4(18),8-diene- 

11,14-dione 

(15S)-19β(43)-abeo- 

6β,7α,12α-trihydroxy-13β,16cycloabieta-4(18),8-diene- 

11,14-dione 

12-O-desacetyl 

coleon R 

Coleon Y 

Lanugone K’ 

Lanugone K 

Barbatusin 

3β-hydroxy-3deoxybarbatusin 

Isolated from 



Reference 




C. barbatus; 



Zelnik et al., 1977; 


2010 


2010 

Coleon O C. coerulescens Grob et al., 1978 

Plectrin P. barbatus Kubo et al., 1984 

Coleon J 

7,12-bis(Odesacetyl) 

coleon N 

C. somaliensis; 

P. edulis 

Moir et al., 1973b; 



50


93 

94 

95 

96 

97 

98 

99 

100 

101 

102 

103 

(15S)-19α(43)-abeo-7αacetoxy-6β,12α-dihydroxy-13β,16-cycloabieta-4(18),8- 

diene-11,14-dione 

(15S)-19β(43)-abeo-7αacetoxy-6β,12α-dihydroxy-13β,16-cycloabieta-4(18),8- 


(15S)-19α(43)-abeo-12αacetoxy-6β,7α-dihydroxy-13β,16-cycloabieta-4(18),8- 


(15S)-19α(43)-abeo-7αformyloxy-6β,12α-dihydroxy-13β,16-cycloabieta-4(18),8- 


(15S)-19α(43)-abeo-7αformyloxy-12α-acetoxy-6βhydroxy-13β,16-cycloabieta- 

4(18),8-diene-11,14-dione 

(15S)-19α(43)-abeo-7α,12αdiacetoxy-6β-hydroxy-13β,16cycloabieta-4(18),8-diene- 

11,14-dione 

(15S)-3α,7α,19-triacetoxy- 

6β,12β-dihydroxy-13α,16- 


(15S)-6β,17-diacetoxy-7α,12βdihydroxy-13α,16-cycloabieta- 


(15S)-6β,12α,19-triacetoxy-7αhydroxy-13α,16-cycloabieta-8- 


(15S)-7α,19-diacetoxy-6β,12αdihydroxy-13α,16-cycloabieta- 


(15S)-7α,19-diacetoxy-6β,12βdihydroxy-13α,16-cycloabieta- 



Coleon G 

12-O-desacetylcoleon N 

12-O-desacetyl-7-Oacetyl-3β,19-diacetyloxy- 

coleon Q 

12-O-desacetyl-6-Oacetyl-17-acetyloxy 

coleon P 

6-O-acetyl-19-acetyloxycoleon 

Q 

12-O-desacetyl-7-Oacetyl-19-acetyloxy- 

coleon Q 

12-O-desacety-7-Oacetyl-19-acetyloxycoleon 

P 

Isolated from 

Plectranthus 

(P) or Coleus 

(C) species 

C. 

somaliensis; 

P. edulis 

C. 

coerulescens 

P. edulis 

P. edulis 

C. garckeanus 

Reference 

Moir et al., 1973b; 


1987 

Grob et al., 1978 


1987 


1987 

Miyase et al., 

1979 

51


104 

105 

106 

107 

108 


13α,16-cycloabieta-3,8-diene- 

11,14-dione 

(15S)-19(43)-abeo-7α,19diacetoxy-6β,12α-dihydroxy- 

13α,16-cycloabieta-3,8-diene- 

11,14-dione 

(15S)-19β(43)-abeo-3αacetoxy-6β,7α,12α-trihydroxy-13α,16-cycloabieta-4(18),8- 


(15S)-19β(43)-abeo-7α,3αdiacetoxy-6β,12α-dihydroxy-13α,16-cycloabieta-4(18),8- 



13α,16-cycloabieta-3(19),4(18), 

8-triene-11,14-dione 

Isolated from 

Plectranthus 

(P) or Coleus 

(C) species 

Coleon O C. garckeanus 

19-acetyloxy coleon O 

7-desoxy-12-Odesacetyl-3-acetyloxy 

coleon N 

Coleon Z 

C. garckeanus 

P. edulis 

C. 

garckeanus; 

P. edulis 

Reference 


1979 


1979 


1987 


1979; 


1987 

52

O 

R 1 

3 

1 

OH 

H 

O 

H 

12 

OH 

6 7 

OCCH 3 

O 

O 

OCCH 3 

13 

OH 

O 

CH 3 

15 

H 

R 1 

R2CH2 19 

O 

H 

6 7 

OR 3 

(70) R1 R2 R3 R4 

(71) H H H H 

(72) H H H CH(O) 

(73) H OH H CH(O) 

(74) H OCH(O) H H 

(75) H OCH(O) H CH(O) 

(76) OCH(O) H H H 

(77) OCH(O) H H CH(O) 

(78) OCH(O) OC(O)CH3 H H 

(79) OCH(O) H C(O)CH3 H 

(80) OC(O)CH3 H H H 

(81) OC(O)CH3 H C(O)CH3 H 

(82) OC(O)CH3 OC(O)CH3 H H 

11 

6 7 

OR 2 

OR 4 

OR 3 

O 

H 

CH 3 

R1 R2 R3 R4 

(83) H H CH(O) H 

(84) H H C(O)CH3 H 

(85) OCH(O) H CH(O) H 

(86) OCH(O) C(O)CH3 H C(O)CH3 

13 

15 

R 

O 

H 

11 

6 7 

OH 

OCCH 3 

O 

O 

13 

OR 4 

OCCH 3 

14 

OH 

R 

(87) =O 

(88) β-OH 

O 

O 

CH 3 

15 

CH 3 

15 

H 

H 

53

R 1 

19 

3 

3 

1 

18 

1 

18 

20 

20 

O 

H 

O 

H 

6 7 

OH 

6 7 

OH 

OH 

OR 3 

16 

OR 2 

16 

13 

O 

CH 3 

R1 R2 R3 

(91) α-CH3 H H 

(92) β-CH3 H H 

(93) α-CH3 C(O)CH3 H 

(94) β-CH3 C(O)CH3 H 

(95) α−CH3 H C(O)CH3 

(96) α−CH3 CH(O) H 

(97) α−CH3 CH(O) C(O)CH3 

(98) α−CH3 C(O)CH3 C(O)CH3 

O 

OCCH 3 

O 

CH 3 

H 

H 

O 

19 

O 

CH 3CO 

3 

CH 3COCH 2 

O 

3 

1 

18 

20 

1 

O 

H 

20 

O 

H 

6 7 

OH 

OCCH 3 

(89) (90) 

11 

O 

12 

6 7 

OH 

(99) 

14 

OH 

16 

OH 

13 

O 

O 

OCCH 3 

O 

H 

H 

CH 3 

CH 3 

54

H 

3 

R 1CH 2 

1 

20 

O 

H 

OR 2 

R 4 

6 7 

13 

OR 3 

O 

H 

15 

CH2R5 17 

R1 R2 R3 R4 R5 

(100) H C(O)CH3 H β-OH OC(O)CH3 

(101) OC(O)CH3 C(O)CH3 H α- H 

(102) OC(O)CH3 H C(O)CH3 

OC(O)CH3 

α-OH H 

(103) OC(O)CH3 H C(O)CH3 β-OH H 

19 

H 3CCO 

O 

3 

1 

18 

20 

O 

H 

6 7 

OH 

OH 

OR 

R 

(106) H 

(107) C(O)CH3 

O 

H 

CH 3 

19 

3 

1 

18 

RCH 2 

20 

O 

H 

19 

6 7 

3 

OH 

1 

20 

OH O 

(108) 

O 

6 7 

OH 

O 

OCCH 3 

H 

18 

OH O 

(104) 

R 

H 

(105) OC(O)CH3 

O 

OCCH 3 

Twenty-two quinone methides, 109-130 were isolated from ten Plectranthus species 

(Table 8). Eight compounds were isolated from P. strigosus and six from P. lanuginosus 

and P. parviflorus while the other species yielded less than four compounds each. 

Compounds 109-120 closely resemble benzoquinones, but there is only one carbonyl 

present in ring C, located at C-12 instead of C-11 and C-14 and has double bonds at ∆ 7 , 

C-9(11) and ∆ 13 . Compounds 121-130 have an additional double bond in ring B at ∆ 5 . 

Yet again variation of the isopropyl group at C-13 is observed in compounds 109-117. In 

compound 118 the isopropyl group has cyclised onto ring C, resulting in a partially 

H 

CH 3 

55 

H 

CH 3

educed furan ring. The linear side chains which have replaced the isopropyl group are 

either saturated as in 112, oxidized to a double bond as in compound 116 or contain 

methoxy groups (114) and hydroxy groups (111, 112, 115, 117, 119-122). The tertiary 

methyl group usually present at C-19, has been oxidized to a formyloxy (-OCHO) group 

in compounds 114 and 117. In abietanes 126-130 the methyl group at position 19 has 

been modified to include either an aromatic group as in 126-129 or a prenylated group as 

in 130. Compound 122 has an acetyl group present at C-3 as well as a hydroxy group at 

C-15 while compounds 123-125 have aromatic substituents at C-2. 


Table 8: Vinylogous quinones isolated from Plectranthus species 


109 

110 

111 

112 

113 

114 

115 

116 

11-hydroxy-7,9(11),13abietatriene-12-one11,14-dihydroxy-7,9(11),13abietatriene-6,12-dione 

11,14,16-trihydroxy- 

7,9(11),13-abietatriene-6,12dione 

11,14,16-trihydroxy- 


7,9(11),13-triene-6,12-dione 

16-acetoxy-11,14-dihydroxy- 


7,9(11),13-triene-6,12-dione 

19-formyloxy-6β,11,14trihydroxy-16-methoxy- 


7,9(11),13-triene-12-one 

6β,19-epoxy-11,14dihydroxy-13-(2hydroxypropyl)-7,9(11),13abietatriene-12-one13-allyl-6β,19-epoxy-11,14dihydroxy-7,9(11),13abietatriene-12-one 

14hydroxytaxodione 

Lanugone O 

Lanugone N 

Lanugone L 

Lanugone M 

Isolated from 


species 

Reference 

P. elegans Dellar et al., 1996 

P. grandidentatus Uchida et al., 1981 

P. lanuginosus; 

P. edulis 

Schmid et al., 

1982; 



56


117 

118 

119 

120 

121 

122 

123 

124 

125 

126 

127 


19-formyloxy-11,14,16trihydroxy-17(15→16)abeoabieta-7,9(11),13-triene- 

6,12-dione 

(15S)-14,16-epoxy-11hydroxy-7,9(11),13abietatriene-6,12-dione 

*Too complex to name 

systematically 




5,7,9(11),13-abietatetraene- 

12-one 

3β-acetoxy-11,15-dihydroxy- 

5,7,9(11),13-abietatetraene- 

12-one 

2-(3-hydroxybenzoyl)-11hydroxy-5,7,9(11),13abietatetraene-12-one 

2-(3,4-dihydroxybenzoyl)- 

11-hydroxy-5,7,9(11),13abietatetraene-12-one; 

2-(4-hydroxy-3methoxybenzoyl)-11hydroxy-5,7,9(11),13abietatetraene-12-one 

19-(3-methyl-2-butenoyl)-11hydroxy-5,7,9(11),13abietatetraene-12-one 

19-(4-hydroxybenzoyl)-11hydroxy-5,7,9(11),13abietatetraene-12-one 

Lanugone P 

Lanugone Q 

Nilgherron A 

Nilgherron B 

Fuerstione 

3β- 

Acetoxyfuerstione 

Parvifloron D 

Parviflorone F 

Isolated from 


species 

Reference 


P. nilgherricus 

P. parviflorus; 

P. strigosus; 

P. eckonlii; 

P. lucidus 

P. nummularius; 


P. strigosus; 

P. eckonlii 


1977b 

Ruedi et al., 1978; 

Alder et al., 1984b; 

van Zyl et al., 

2008; Nyila et al., 

2009 

Narukawa et al. 

2001; 




2008; 

Nyila et al., 2009 

Parviflorone G P. strigosus Alder et al., 1984b 

Parviflorone A 

Parviflorone C 


P. strigosus; 

P. purpuratus 

subsp. purpuratus; 

P. lucidus 


P. strigosus; 

P. purpuratus 

subsp. tongaensis 



van Zyl et al., 2008 

Ruedi et al., 1978 

57


HCO 

O 

128 

129 

130 

H 

19 

HO 

20 

10 

18 

19-(3,4-dihydroxybenzoyl)- 

11-hydroxy-5,7,9(11),13abietatetraene-12-one 

19-(4-hydroxy-3methoxybenzoyl)-11hydroxy-5,7,9(11),13- 

abietatetraene-12-one 

19-(3-methyl-2-butenoyl)- 

2,11-dihydroxy-5,7,9(11),13abietatetraene-12-one 

HO 

9 

O 

12 

7 

13 

3 

1 

Parviflorone E 

Parviflorone B 

Isolated from 


species 

P. nummularius; 


P. strigosus; 

P. purpuratus 



P. strigosus 

Reference 


2001; 



van Zyl et al., 2008 



Parviflorone H P. strigosus Alder et al., 1984b 

HO 

20 

H 

10 

11 

6 

O 

O R 

(109) R 

(110) CH3 

(111) CH2(OH) 

11 

6 

H 

CH2 OH 

19 

O 

14 

OH 

16 

OCH 3 

HO 

H 

O 

11 12 

14 

OH 

14 

OH 

(114) R 

(115) CH2CH(OH)CH3 

(116) CH2-CH=CH2 

19 

6 

O 

R 

O 

HCO 

3 

1 

HO 

20 

H 

10 

11 

6 

O 

O 

R 

(112) H 

(113) C(O)CH3 

HO 

11 

CH2 19 

H 

O 

(117) 

OH OR 14 

O 

7 

58 

16 

16 

14 OH 

OH

R 

2 

HO 

5 

11 

9 

HO 

9 

H O 

O 

H 

C 

7 

O 

11 13 

13 

OH 

O 

7 

14 

O 

15 

16 

17 

R 

HO 

O 

H 

9 

O 

O 

11 13 

5 7 

OH 

(118) R 

(119) H 

(120) OC(O)CH3 

2 

COCH 2 

19 

(123) R = OC OH 

O 

O 

(124) R = OC OH 

OH 

(125) R = OC OH 

OH 

5 

(130) 

11 

9 

O 

7 

O 

13 

OCH 3 

RCH 2 

19 

OH 

OH 

5 

OH 

11 

9 

O 

7 

R 

13 

3 

HO 

5 

11 

9 

O 

7 

13 

14 

15 

R 

(121) H 

(122) OC(O)CH3 

59 

OH 

(126) R = OC(O)CH=(CH 3) 2 

O 

(127) R = OC OH 

(128) R = 

O 

OC OH 

O 

OH 

(129) R = OC OH 

OCH 3

Thirty-six compounds (131-167) having a coleon-type structure were isolated from 

eleven Plectranthus and ten Coleus species, where most of the compounds have been 

isolated from P. edulis and C. coerulescens (Table 9a-c). The coleon-type compounds 

differ from the royleanones in that ring C is aromatic in coleon-type compounds as 

apposed to having a hydroxybenzoquinone or p-quinoid ring system. 

Compounds 131-148 (Table 9a) differ in ring B with regard to positions 6 and 7 as they 

both could be saturated as in 131, or oxidized, with either a hydroxy or carbonyl group 

(132-135) or two carbonyl groups (136-145). Alternatively either C-6 or C-7 could be 

oxidized as in 132-134. Compounds 138 to 141 are the only compounds where the 

methyl group/s at C-15 have been oxidized to an alcohol or acetyl group, while 134 is the 

only compound where the methyl group at C-20 has been oxidized to an alcohol. 

Plectranthol B (131), has an aromatic substituent at C-19 with a prenyl group occurring at 

C-12. In compounds 146 to 148, an oxygen bridge is formed between C-20 and either C- 

6 or C-7. Compound 147 has a methoxy group present at C-20 while 148 has a carbonyl 

function at C-7. 

Table 9a: Coleon-type abietanes isolated from Plectranthus and Coleus species 


131 

132 

133 

134 

135 


12-O-(3-methyl-2-butenoyl)- 

19-O-(3,4dihydroxybenzoyl)-11hydroxy-8,11,13-abietatriene7α,11-dihydroxy-12- 

methoxy-8,11,13-abietatriene 

11,12-dihydroxy-8,11,13abietatriene-7-one 

11,12,20-trihydroxy-8,11,13abietatriene-7-one 

6β,11,12,14-tetrahydroxy- 

8,11,13-abietatriene-7-one 

Isolated from 

Plectranthus 

(P) or Coleus 

(C) species 

Plectranthol B P. nummularius 

11,20dihydroxysugiol 

11hydroxysugiol 

5,6dihydrocoleon 

U 

Reference 


2001 

P. elegans Dellar et al., 1996 

P. cyaneus 

P. cyaneus; 

C. forskohlii 

P. argentatus 

Horvath et al., 

2004 


2004; 

Lingling et al., 

2005 

Alder et al., 

1984a 

60


136 

137 

138 

139 

140 

141 

142 

143 

144 

145 

146 


11,12,14-trihydroxy-8,11,13abietatriene-6,7-dione 

14-formyloxy-11,12dihydroxy-8,11,13abietatriene-6,7-dione 

11,12,14,16-tetrahydroxy- 

8,11,13-abietatriene-6,7dione 

16-acetoxy-11,12,14trihydroxy-8,11,13- 


3α-acetoxy-11,12,14,16tetrahydroxy-8,11,13- 


16,17-diacetoxy-3α,11,12,14tetrahydroxy-8,11,13- 


16-acetoxy-11,12,14,17tetrahydroxy-8,11,13- 


16,17-diacetoxy-11,12,14trihydroxy-8,11,13abietatriene-6,7-dione11,12,16-trihydroxy-8,11,13- 

abietatriene-6,7-dione 

3β,11,12,14-tetrahydroxy- 

8,11,13-abietatriene-6,7- 

dione 

7β,20β-epoxy-11,12dihydroxy-8,11,13abietatriene 

Coleon V 

14-O-formylcoleon 

-V 

Coleon D 

16-Oacetylcoleon 

D 

Coleon I 

Coleon K 

Coleon X 

16-O-acetyl 

coleon X 

Isolated from 

Plectranthus 

(P) or Coleus 

(C) species 


C. carnosus; 

C. 

coerulescens; 

P. sanguineus 

P. myrianthus 

C. aquaticus; 

C. coerulescens 

C. 

coerulescens; 

P. edulis 

Reference 


1977a; 

Yoshizaki et al., 

1979; 

Grob et al., 1978; 

Matloubi- 


1987 


1977a 



Schmid et al., 

1982; 


1987 



1987 

C. somaliensis Moir et al., 1973a 

C. garckeanus 

P. edulis 

Coleon T P. caninus 

20deoxocarnosol 

C. barbatus 


1979 


1987 

Arihara et al., 

1977 

Kelecom et al., 

1984 

61


147 

148 

HO 

HO 

7β,20β-epoxy-20S-methoxy- 

11,12-dihydroxy-8,11,13abietatriene 

6β,20β-epoxy-6α,11,12trihydroxy-8,11,13abietatriene-7-one 

19 

O 

O 

19 

HO 11 

R 

20 

10 

H 

A 

OH 

11 

B 

O 

C 

12 

8 

O 

13 

17 

15 

16 

18 

1 

HO 

(131) (132) 

OH 

12 

8 

7 

O 

15 

14 

18 

R 

(133) H 

(134) OH 

Isolated from 

Plectranthus 

(P) or Coleus 

(C) species 

3 

1 

20 

19 

HO 

19 18 

H 

10 

11 

6 7 

OH 

(135) 

OCH 3 

OH 

12 

8 

H 

OH 

14 

OH 

O 

Reference 

Esquirolin D C. esquirolii Li et al., 1991 

Carnosolone 

P. cyaneus; 

C. carnosus 


2004; 


1979 

62

R 1 

3 

1 

HO 

H 

19 18 

11 

OH 

6 7 

8 

O 

12 

O 

14 

16 

R 3 

15 

OR 2 

R1 R2 R3 R4 

(136) H H CH3 CH3 

(137) H CHO CH3 CH3 

(138) H H CH2(OH) CH3 

(139) H H CH2OC(O)CH3 CH3 

(140) OC(O)CH3 H CH2(OH) CH3 

(141) OH H CH2OC(O)CH3 CH2OC(O)CH3 

19 

HO 

20 

10 

18 

H 

R 1 

1 

HO 

19 18 

11 

6 7 

O 

OH 

12 

8 

O 

13 

R 3 

R 2 

R 4 

17 

R1 R2 R3 

(144) H H CH2CH(OH)CH3 

(145) OH OH CH(CH3)2 

OH 

OH 

3 

1 

OH 

11 

19 18 O 

H 

7 

OH 

12 

8 

O 

R 

(142) H 

(143) C(O)CH3 

11 

12 15 

HO 

CH3O 11 

12 15 

HO 

20 

11 

12 15 

14 

14 

14 

O 

6 

8 

7 

19 

10 

H 

18 

O 

6 

OH 

7 

19 

10 

H 

18 

O 

OH 

O 

(146) (147) (148) 

OH 

O 

16 

CH2OCCH3 CH2OR 17 

14 

OH 

63

In compounds 149-165 (Table 9b), an olefinic bond is present at C-5 while oxygenated 

substituents occur at C-11, C-12 and C-14. With the exception of compound 157, 

compounds 153-165 have either one or both of the isopropyl methyl groups at C-15, 

being oxidized to an acetyl and/or alcohol function. Compounds 152 and 159 have a 

hydroxy and acetoxy group present at C-3 respectively while compounds 160 and 161 

have an acetyl group at C-2. Abietanes 163 and 164 have a formyloxy (-OCHO) group at 

C-19 while the isopropyl group at C-13 has been replaced by a linear, 3-carbon side- 

chain containing either a double bond or alcohol in compounds 162-164. 

Table 9b: Coleon-type abietanes with an olefinic bond at ∆ 5 isolated from Plectranthus 

and Coleus species 


149 

150 

151 

152 



5,8,11,13-abietatetraene-7one 

14-acetoxy-6,11,12trihydroxy-5,8,11,13- 

abietatetraene-7-one 



3β,6,11,12,14pentahydroxy-5,8,11,13abietatetraene-7-one 

Coleon U 

14-O-acetylcoleon 

U 

Coleon U 11 

acetate 

Isolated from 


or Coleus (C) 

species 


P. 

grandidentatus; 

P. forsteri 

’marginatus’; 

C. carnosus; 


P. edulis; 

P. fasciculatus 

P. 

grandidentatus 

Coleon S P. caninus 

Reference 

Wellsow et al., 2006; 


al., 2006; 


al., 2002; 

Cerqueira et al., 

2004; 

Miyase et al., 1977a; 


1979; 

Matloubi-Moghadam 

et al., 1987; 


Coutinho et al., 

2009; 


Rijo et al., 2007 

C. xanthanthus Mei et al. 2002 

Arihara et al., 1977: 

64


153 

154 

155 

156 

157 

158 

159 

160 

161 

162 


6,11,12,14,16pentahydroxy-5,8,11,13abietatetraene-7-one 

16-acetoxy-6,11,12,14tetrahydroxy-5,8,11,13- 


16-acetoxy-6,11,12,14,17pentahydroxy-5,8,11,13- 


16,17-diacetoxy- 


5,8,11,13-abietatetraene-7- 

one 



11,16-diacetoxy-6,12,14trihydroxy-5,8,11,13- 


3α-acetoxy-6,11,12,14,16pentahydroxy-5,8,11,13- 


2α,17-diacetoxy- 

6,11,12,14,16pentahydroxy-5,8,11,13- 


2α,6,11,12,16,17hexacetoxy-14-hydroxy-5,8,11,13-abietatetraene-7- 

one 

6,11,12,14,16pentahydroxy-17(15→16)abeoabieta-5,8,11,13tetraene-7-one 

Coleon C 

16-O-acetyl 

coleon C 

Coleon W 

16-O-acetyl 

coleon W 

11-acetoxy-coleon 

U 

11,16-diacetoxycoleon 

U 

Isolated from 


or Coleus (C) 

species 

C. aquaticus, 

C. coerulescens; 

P. edulis; 

C. forskohlii 


P. edulis 



C. garckeanus 

Reference 

Ruedi et al.,1971; 


Schmid et al., 1982; 


Yanwen et al., 2007; 

Xiu et al., 2008 




Miyase et al., 1977a, 

Miyase et al., 1979 

C. garckeanus Miyase et al., 1979 

C. xanthanthus Mei et al., 2000 

Coleon H C. somaliensis Moir et al., 1973a 

C. blumei Ragasa et al., 2001 


65


163 

164 

165 

R 1 

3 

19 

19-formyloxy-6,11,12,14tetrahydroxy-17(15→16)abeoabieta-5,8,11,13,16 

1 

R 2O 

5 

18 

pentaene-7-one 

19-formyloxy- 

6,11,12,14,16pentahydroxy-17(15→16)abeoabieta-5,8,11,13- 

tetraene-7-one 

16-acetyl-11,12,14trihydroxy-5,8,11,13abietatetraene-7-one(6,18lactone) 

11 

6 

OH 

OH 

12 

8 

O 

14 

15 

OR 3 

R1 R2 R3 

(149) H H H 

(150) H H C(O)CH3 

(151) H C(O)CH3 H 

(152) OH H H 

Lanugone R 

Lanugone S 

R 1 

19 

1 

R 2O 

3 5 

Isolated from 


or Coleus (C) 

species 

18 

11 

6 

OH 

OH 

12 

8 

O 

16 

R 3 

R 4 

17 

14 

OH 

Reference 



R1 R2 R3 R4 

(153) H H CH2OH CH3 

(154) H H CH2OC(O)CH3 CH3 

(155) H H CH2OC(O)CH3 CH2OH 

(156) H H CH2OC(O)CH3 CH2OC(O)CH3 

(157) H C(O)CH3 CH3 CH3 

(158) H C(O)CH3 CH2OC(O)CH3 CH3 

(159) OC(O)CH3 H CH2(OH) CH3 

66

O 

CH 3CO 

3 

2 

1 

O 

OH 

5 

18 

OR 

5 

11 

6 

OR 

OR 

12 

8 

14 

OH 

O 

R 

(160) H 

(161) C(O)CH3 

11 

9 

O 

6 

7 

OH 

12 

8 

(165) 

14 

OH 

O 

CH 2OR 

16 

CH2OCCH3 O 

CH 2OCCH 3 

O 

3 

1 

OH 

5 

11 

9 

R 1CH 2 OH 

7 

OH 

12 

8 

R 2 

14 

OH 

R1 R2 

(162) H CH2CH(OH)CH3 

(163) OCH(O) CH2CH=CH2 

(164) OCH(O) CH2CH(OH)CH3 

Compounds 166 and 167 (Table 9c) have double bonds within rings A and B. In 

compound 166, the double bonds are located at C-1(10) and ∆ 6 respectively while the 

methyl group is no longer at C-10 but at C-5. In 167 the double bonds occur at ∆ 3 and ∆ 5 

and the methyl group at C-19 has migrated from C-4 to C-3. The linear, 3-carbon side- 

chain in this compound has been oxidized to an alcohol. In compound 166, the 

substituent at C-19 is aromatic. 

O 

67

Table 9c: Coleon-type abietanes with double bonds within ring A and B isolated from 



HO 

HO 

166 

167 

3' 

4' 

2' 

5' 

19-O-(3’,4’dihydroxybenzoyl)-11,12dihydroxy-20(10→5)abeoabieta- 

1(10),6,8,11,13-pentene 

19(43),17(1516)bisabeoabieta- 


3,5,8,11,13-pentene-2,7dione 

1' 

6' 

O 

O 

Miscellaneous constituents 

1 

OH 

10 

11 

A B 

5 

4 

20 

19 

6 

OH 

C 

12 

8 

Plectranthol 

A 

Isolated 

from 

Plectranthus 

(P) species 

P. 

nummularius 

Plectrinone A P. barbatus 

13 

Reference 

Narukawa et al., 

2001 

Schultz et al., 

2007 

hydroxybenzoquinone structure. Compound 173 is similar to the royleanones but B ring 

68 

19 

O 

2 

3 

4 

HO 

10 

5 

(166) (167) 

11 

6 

7 

OH 

12 

8 

13 

14 OH 

OH 

O 

15 16 17 

Eight compounds 168-174 (table 10) do not resemble the abietanes 27-167 as they 

contain carbocyclic and heterocyclic rings which are fused differently from the other 

abietanes. Compounds 168 to 171 contain a highly conjugated system with ring A being 

aromatic and ring B resembling the hydroxybenzoquinone system. An oxidised furan 

ring also forms a part of structures 168 to 171. In compound 172 rings A and B are 

linked by a carbon-to-carbon bond. Ring A resembles that of the normal ring A in 

abietanes, with methyl groups at C-4 and C-6 and a ketone at C-5, and ring B has the

has been expanded by an extra oxygen atom forming a seven membered ring with 

carbonyl groups flanking the oxygen at either side. Compound 174 has four rings fused 

together including an aromatic and lactone ring. Both compounds 173 and 174 have 

highly conjugated systems. 

Table 10: Miscellaneous abietanes isolated from Plectranthus and Coleus species 

Compound Name 

168 

169 

170 

171 

172 

173 

174 

(4R,19S)-7,12,19αtrihydroxy-1,5(10),6,8,12- 

abietapentene-11,14-dione 

(4R,19R)-7,12,19βtrihydroxy-1,5(10),6,8,12abietapentene-11,14-dione 


1,5(10),6,8,12-abietapentene- 

11,14,19-trione 

16-acetoxy-7,12-dihydroxy- 

1,5(10),6,8,12-abietapentene- 

11,14,19-trione 







Common 

name 

Coleon A 

(19S) 

Coleon A 

(19R) 

Coleon A 

lactone 

Xanthanthusin 

E 

Isolated from 


species 

P. aff puberulentus 

P. puberulentus; 

P. edulis 

P. edulis 

Sanguinon A P. sanguineus 

Edulon A P. edulis 

Reference 


2006 


2006; 


1987 


1987 

C. xanthanthus Mei et al., 2002 

Matloubi- 


1987 

Buchbauer et al., 

1978; 


1987 

69

1 

18 

3 

20 

4 

O 

5 

19 

R 1 R2 

11 

9 

10 

A 

O 

6 7 

OH R 3 

B 

8 

14 

OH 

R1 R2 R3 

(168) H OH CH3 

(169) OH H CH3 

O 

H 

O 

OH 

O 

O 

O 

15 17 

O 

1 

HO 

18 

3 

20 

O 

4 

O 

11 

9 

10 

5 

19 

A 

O 

OH R 

B 

6 7 

R 

8 

14 

OH 

(170) CH3 

(171) CH2OC(O)CH3 

O 

OH 

(173) (174) 

O 

O 

OH 

O 

15 17 

O 

CH 2OCCH 3 

O 

4 5 

6 

O 

(172) 

OH 

COOH 

70 

O

2.5 Biological activity of the phytochemical constituents of Plectranthus 

The antibacterial and antifungal activities of abietanes isolated from Plectranthus species 

are listed in table 11. Of the one-hundred and fifty abietanes isolated from Plectranthus 

and Coleus species, only twenty are reported as having showed antimicrobial activity. 

These twenty abietanes were isolated from eight Plectranthus species with horminone 

(28), 7α-acetoxy-6β-hydroxyroyleanone (36), 7α,12-dihydroxy-17(15→16)-abeoabieta- 

8,12,16-triene-11,14-dione (55), 16-acetoxy-7α,12-dihydroxy-8,12-abietadiene-11,14- 

dione (54) and Coleon U (149) being isolated from more than one species. 

Of all the abietanes isolated from Plectranthus and Coleus species the highest 

antibacterial and antifungal activities were shown by the royleanone and coleon type 

compounds. These were compounds 28, 36, 38 (7α-formyloxy-6β-hydroxyroyleanone), 

44 (coleon U quinone) and 149. All these compounds are structurally related and have 

the same basic skeleton with different functional groups located in the molecules. 

However, coleon U has an aromatic ring and the others have a hydroxyquinoid ring C. 

Bacillus subtilis and Staphylococcus aureus were the most commonly tested bacterial 

strains while the anti-fungal pathogens tested were Cladosporium cucumerinum, 

Rhizoctonia solani, Sclerotinia sclerotiorum, Pythium ultimum and Candida albicans. 

Acetone was the most frequently used solvent for extraction, while dichloromethane, 

chloroform and ethyl acetate were reportedly used only once for the extraction of 

abietanes from the plant material. 

Of the eight antimicrobial studies carried out on abietanes, two were done on the roots 

only and two on the entire plant. The rest of the studies were carried out on the aerial 

parts of the plant and the leaves (aerial parts in many instances mean leaves and flowers). 

Comparative studies need to be done on the different plant parts, to ascertain where the 

largest concentration of abietane diterpenoids exist. 

71

The majority of the abietanes isolated remain untested against microbes (bacteria and 

fungi) and there is thus a need to carry out antimicrobial assays on the remainder of the 

abietanes. 

It was found that the coleon-type compound (where ring C is aromatic) are more active 

than royleanone-type compounds (where ring C has a 12-hydroxy-p-benzoquinone 

moeity) (Gaspar-Marques et al., 2006; Wellsow et al., 2006). 

It has been observed that the oxidation pattern at the C-6 and C-7 positions in compounds 

having a royleanone-type structure plays a role in the antibacterial activity of the 

compound with compounds where C-7 is oxidised being more active than those oxidised 

at C-6 or not oxidised at all (Teixeira et al., 1997; Gaspar-Marques et al., 2006). Another 

important observation is that the presence of an isopropyl group at C-13 in royleanone- 

type compounds results in better activity than those compounds having an oxidised 

isopropyl group or allylic group at the same position (Gaspar-Marques et al., 2006; 

Batista et al., 1995; Batista et al., 1994). 

Table 11: Antibacterial and antifungal activity of compounds extracted from Plectranthus 

species 

Plectranthus 

species 

P. aff. 

puberulentus 

Plant 

part 

Compound 

Antimicrobial 

Activity 

acetone lv (168), (169) B. subtilis 

P. ecklonii ethyl acetate w (123), (124) 

P. elegans chloroform a (109), (132) 


Drug resistant 

Mycobacterium 

tuberculosis 

Listeria 

monocytogenes 

B. subtilis, S. aureus, 

Streptomyces scabies, 

P. aeruginosa, 

Pseudomonas 

syringae, Erwinia 

carotovora, 

*Cladosporium 

cucumerinum 

Reference 

Wellsow et 

al., 2006 

Nyila et al., 

2009 

Dellar et al., 

1996 

72

Plectranthus 

species 

P. forsteri 


P. 


Plant 

part 

Compound 

acetone lv (44), (149) 

acetone w (36) 

acetone a+rt 

P. hadiensis dichloromethane a 



acetone a+rt 

acetone rt 

(27), (28), (32), 

(33), (36), (43), 

(149) 

x (36), (38) 

x (33) 

(28), (54), (55), 

(64) 

(55) 

(28), (54) 

Antimicrobial 

Activity 


Pseudomonas 

syringae 

Staphylococcus 

aureus, 

Vibrio cholera 

Six strains of 

methicillin resistant 

S. aureus, two strains 

of vancomycinresistant 

E. faecalis 

B. subtilis, 

Xanthomonas 

campestris, 

*Rhizoctonia solani, 

*Sclerotinia 

sclerotiorum, 

*Pythium ultimum, 

*unknown Candida 

species 

*S. sclerotiorum, 

*unknown Candida 

species 

Six strains of 

methicillin resistant 

S. aureus, two strains 

of vancomycinresistant 

E. faecalis 

S. aureus, 

V. cholerae 

S. aureus, V. cholera, 

*Candida albicans, 

P. aeruginosa, 

Escherichia coli, 

Shigella dysenteriae, 

Salmonella 

typhimurium, 

Steptococcus faecalis 

Reference 

Wellsow et 

al., 2006 

Teixera et 

al., 1997 

Gaspar- 

Marques et 

al., 2006 

Laing et al., 

2006 

Gaspar- 

Marques et 

al., 2006 

Batista et 

al., 1995 

Batista et 

al., 1994 

73

Plectranthus 

species 

Plant 

part 

Compound 

P. puberulentus acetone lv (170) 

Antimicrobial 

Activity 


Pseudomonas 

syringae 

Reference 

Wellsow et 

al., 2006 

* denotes anti-fungal pathogens 

x Laing et al. (2006) reported the isolation of 7α-acetoxy-6-hydroxyroyleanone and 7α,6-dihydroxyroyleanone 

without including the stereochemistry in the name. It is thus assumed that these compounds are 7α-acetoxy-6βhydroxyroyleanone 

(36) and 7α,6β-dihydroxyroyleanone (33), respectively 


Three abietanes have also been found in Salvia species where antimicrobial studies were 

carried out (Table 12). All three of these abietanes have a royleanone-type structure. 

Compounds 28 (horminone) and 31 (7α-acetoxyroyleanone) exhibited similar and in 

some cases stronger antibacterial activity to commonly used antibiotics such as 

cefoperazone, amicain, kanamycin and vancomycin (Ulubelen et al., 2001; 2003). 

Table 12: Antibacterial and antifungal activity of abietanes isolated from Salvia species 

Plant species Compound Activity Reference 

Salvia sclarea (40) S. aureus, *C. albicans 

Ulubelen et al., 

1994 

Salvia 

blepharochlaena; 

Salvia 

amplexicaulis; 

Salvia eriophora 

* denotes anti-fungal pathogens 

(28) 

(31) 

S. aureus, S. epidermis, B. 

subtilis, E. faecalis Kolak et al., 

2001; 

S. aureus, S. epidermis, B. 

subtilis 


2001; 2003 

Table 13 lists twenty-seven abietanes with antioxidant, anticancer, antifeedant and other 

activity which have been isolated from thirteen Plectranthus species and two Coleus 

species. 

Compounds 36 (7α-acetoxy-6β-hydroxyroyleanone), 123 and 124 commonly known as 

Parvifloron D and F respectively, have been isolated the most and have exhibited both 

74

anticancer (Cerqueira et al., 2004; Nyila et al., 2009; Gaspar-Marques et al., 2002) and 

antimalarial activity (van Zyl et al., 2008). Compound 124 also has antioxidant activity 

(Narukawa et al., 2001). Compound 36 has a royleanone-type structure while the other 

two compounds (123-124) have a vinylogous quinone-type structure. In the three studies 

that were done on coleon-type and royleanone-type compounds (Mei et al., 2002; 

Cerqueira et al., 2004; Gaspar-Marques et al., 2002), it was found that the coleon-type 

compounds were more effective as anticancer agents than the royleanone-type 

compounds. 

When Kabouche et al. (2007) tested the antioxidant activity of royleanone and coleon- 

type compounds, they found that the presence of a carbonyl group at C-7 led to higher 

activity as apposed to compounds having no substituent at this position or having a 

substituent other than a carbonyl at C-7. When the hydroxyl group at C-12 is replaced by 

a methoxy group as in 176, the activity is increased as well (Kabouche et al., 2007). In 

studies where coleon (compounds where ring C is aromatic) and royleanone-type (where 

ring C has a 12-hydroxy-p-benzoquinone moeity) compounds were isolated, the coleon- 

type compounds proved to be more active than the royleanone-type compounds 

(Kabouche et al., 2007; Cerqueira et al., 2004; Mei et al., 2002; Gaspar-Marques et al., 

2002; Narukawa et al., 2001). However there has been one instance were a royleanone- 

type diterpene (Coleon U quinone (44)) showed potent antifeedant activity whereas the 

coleon-type diterpene (coleon U (149)) displayed no activity as an antifeedant (Wellsow 

et al., 2006). 

O 

R 

12 

O 

OCCH 3 

O 

R 

(175) OH 

(176) OCH3 

75

The leaves seem to be the most frequently used plant part. There has been three instances 

where compounds from the entire plant has been tested but only one report on abietanes 

being isolated from the roots of Plectranthus species. 

In ten of the eleven studies on Plectranthus species, polar solvents (ethyl acetate, 

dichloromethane, acetone, ether and water) were used for extraction with there being just 

one report of hexane and dichloromethane being used as a solvent for extraction. This is 

probably because abietanes are best isolated with a polar solvent medium as many of the 

abietanes are highly functionalised with carbonyl and hydroxyl groups. 

Table 13: Inhibitory activity of abietanes isolated from Plectranthus and Coleus extracts 

Plectranthus 

species 

C. forskohlii 

Plant 

part 

NS 

taken orally or 

applied topically 

C. xanthanthus 70% acetone a 

Compound Other Activity Reference 

Anticancer activity 

(153) 

(44), (45),(46), 

(151), (172) 


P. 


P. 



acetone w (149) 

acetone extract w 

acetone w 

(35), (36), (47), 

(149) 

(36), (149) 

(33), (35), (36), 

(47), (149) 

anti-tumor activity 

(in vivo and in vitro) 

inhibits growth and 

proliferation of tumors 

cytotoxic against K562 

human leukemia cells 

active against vero cell 

line 

promising anti-cancer 

drug 

antiproliferative activity 

against human 

lymphocytes 

antiproliferative activity 

against the expression of 

CD69 by T- and Bmouse 

lymphocytes and 

induces lymphocyte 

apoptosis 

anti-tumor activity 

(breast, lung, renal, 

melanoma and CNS) 

Xiu et al., 2008 

Yanwen et al., 

2007 

Mei et al., 2002 

Nyila et al., 

2009 

Coutinho et al., 

2009 

Cerqueira et al., 

2004 

Gaspar-Marques 

et al., 2002 

76

Plectranthus 

species 

Plant 

part 

P. nummularis acetone lv 

Compound Other Activity Reference 

Antioxidant activity 

(124), (128), 

(131), (168) 

Antifeedant activity 


P. aff. 

puberulentus 

acetone lv (168), (169) 

antifeedent activity 

against S. littoralis 

P. barbatus ether lv insect antifeedent 

(90) 

activity against S. 

gramium and 

P. gossypiella 

P. forsteri 

‘marginatus’ acetone lv 

P. puberulentus 

(44) 

(170) 

antifeedent activity 

against S. littoralis 

Antimalarial activity 

P. ecklonii dichloromethane lv (123), (124) 

P. hadiensis dichloromethane lv (38), (36) 

P. lucidus 

P. purpuratus 

subsp. purpuratus 

P. purpuratus 



(123), (126) 

(126) 

(127), (128) 

P. barbatus water lv (169) 

Other activity 


P. grandis hexane lv (70), (87) 

P. hereroensis acetone rt (64) 

antimalarial activity; 

inhibits β-haematin 

formation 



formation 



formation 

antisecretory and 

antiulcer activities 

inhibits tyrosinase 

activity 

gastroprotective 

properties, in mice 

antiviral activity 

against Herpes virus ie. 

Herpes simplex type II 

Narukawa et al., 

2001 


2006 

Kubo et al., 1984 


2006 


2008 


2008 


2008 

Schultz et al., 

2007 

Nyila et al., 2009 


2010 

Batista et al., 

1995 

NB. Coleus species are also included in this table because of the uncertain relationship to the Plectranthus species 

Key: lv: leaves, rt: roots, s: stems, a: aerial parts, w: whole plant, f: flowers, NS: not specified 

77

Table 14 contains six abietanes isolated from five known Salvia species, one unknown 

Salvia species and one Lepechinia species. These compounds have been shown to 

possess anticancer, antioxidant or cardiovascular activity with compounds 27 

(royleanone) having antitumor and antioxidant activity and 28 (horminone) having 

antitumor, anticancer and antioxidant activity. 

The royleanone-type abietanes (27, 28, 32 and 44) have shown to exhibit anticancer, 

antioxidant and cardiovascular activity while the coleon-type compounds (134 and 146) 

have proved to be active only against cancer cell lines. 

Table 14: Pharmacological activity of abietanes isolated from other plant species 

Plant species Compound Activity Reference 

Salvia pachyphylla (146) 

Active against human 

cancer cell lines 

Guerrero et al., 

2006 

Salvia hypargeia (134) 

Active against ovarian 

cancer cell line (A2780) 

Topcu et al., 2008 

*Salvia species 

(27), (28), 

(32) 

Antitumor 

Topcu and Goren, 

2007 

Lepechinia bullata (28), (43) Anticancer 

Jonathan et al., 

1989 

Kolak et al., 2001; 

Salvia amplexicaulis 

Cardiovascular activity Ulubelen et al., 

Salvia amplexicaulis; 

Salvia eriophora 

(44) 

Antihypertensive 

2003 

Kolak et al., 2001; 


2002 

Salvia barrelieri 

(27), (28), 

(31) 

Antioxidant activity 

Kabouche et al., 

2007 

* unknown species 

The distinguishing feature of an abietanoid structure which determines the activity of the 

compound, is the oxidation pattern within ring B and ring C. It has been observed that 

the oxidation patterns at C-6, C-7 and C-12 influence the activity of the abietanoid. 

Compounds having a coleon-type structure (where ring C is aromatic) have proven to be 

more active than royleanone-type compounds (where ring C has a 12-hydroxy-p- 

benzoquinone moeity) (Gaspar-Marques et al., 2006; Wellsow et al., 2006). 

78

Two compounds, 7α-acetoxy-6β-hydroxyroyleanone (36) and coleon U (149) have 

shown to have the widest range of biological activity with compound 149 always being 

more active than compound 36 and occasionally having even stronger activity than the 

well known drugs used as controls. 

Although much has been done on testing the abietanes for biological activity, there are 

still many abietanes which have not been tested for any biological or pharmacological 

activity. These assays will prove useful in determining the structure-activity relationships 

of the abietanes. 

79

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93

Chapter 3 Extractives from Plectranthus hadiensis 

3.1 Introduction 

Plectranthus hadiensis is commonly known as the ‘hairy spurflower’ or ‘imbozisa’ by the 

Zulus. This plant is a shrubby perennial which grows to a height of 1.5 metres and 

flowers between March and June every year, peaking in May (van Jaarsveld, 2006). The 

flower blossoms are purple in colour (figure 15) and are pleasantly aromatic. 

Figure 15: Plectranthus hadiensis in bloom (picture courtesy of Prof. N. Crouch) 

P. hadiensis favours dry conditions for growth and can be found growing between the 

Kwa-Zulu Natal coastline and Gauteng, South Africa. The cuttings of this plant are used 

for propogation purposes (van Jaarsveld, 2006). 

Ethnobotanically, P. hadiensis was reported by Lukhoba et al. (2006) as having been 

known to treat certain skin conditions, diarrhoea in South India and Sri Lanka (Mehrotra 

et al., 1989) respiratory conditions and inflammation (loc cit Sivarajan and Balachandran, 

1986; Neuwinger, 2000; Hutchings et al, 1996). However the literature cited in Lukhoba 

et al. (2006) does not make mention of these reports. 

94

There have been only four biological studies on Plectranthus hadiensis where the 

antiplasmodial, insect-antifeedant, antifungal, antiradical and antibacterial properties of 

this plant extract and the isolated compounds were evaluated for activity (van Zyl et al., 

2008; Laing et al., 2006; Mothana et al., 2008; Wellsow et al., 2006). The acetone 

extract of this plant was found to be inactive as an insect-antifeedant (Wellsow et al., 

2006). The hexane extract showed good activity against Sclerotinia sclerotiorum, 

Rhizoctonia solani and an unknown Candida species while the dichloromethane extract 

showed moderate activity against bacterial strains, B. subtilis and X. campestris and the 

fungal microorganism, Sclerotinia sclerotiorum as well as an unknown Candida species 

(Laing et al., 2006). 

Three royleanones 33, 36 and 38 were isolated previously from the dichloromethane 

extract of the aerial parts of P. hadienis (van Zyl et al., 2008) and the isomers of 33 and 

36, 34 and 37 respectively, were isolated from the ethanol extract of Coleus zeylanicus (a 

synonym for P. hadiensis) (Mehrotra et al., 1989). Compounds 33 and 36 exhibited 

anticancer activity against breast, lung, renal, melanoma and central nervous system cell 

lines (MCF-7, NCI-H460, TK-10, UACC-62 and SF-268, respectively) with both 

compounds proving to be less potent than the positive control, Cyclosporin A (Cerqueira 

et al., 2004; Gaspar-Marques et al., 2002). These three compounds (33, 36 and 38) have 

also proven to be active against six strains of methicillin resistant Staphylococcus aureus, 

two strains of vancomycin-resistant Enterococcus faecalis, Bacillus subtilis, Vibrio 

cholera and Xanthomonas campestris (Laing et al., 2006; Teixera et al., 1997; Gaspar- 

Marques et al., 2006). Compounds 36 and 38 (van Zyl et al., 2008) exhibited 

antimalarial activity with 38 displaying synergistic properties when combined with 

quinine (Tables 11 and 13 on pages 72 and 76, respectively). There were no reports of 

biological activity in compounds 34 and 37. 

95

4 

20 

10 

O 

H 

19 18 

6 7 

OH 

OH 

C 

13 

R 

O 

R 

(33) α-OH 

(34) β-OH 

(36) α-OC(O)CH3 

(37) β-OC(O)CH3 

(38) α-OCH(O) 

The purpose of this study was to perform phytochemical analyses on the stem and leaf 

material of Plectranthus hadiensis in the hope of isolating abietane diterpenes to test 

against Enterococcus faecalis and Pseudomonas aeruginosa bacterial microorganisms 

and breast (MCF-7), renal (TK-10) and melanoma (UACC-62) cancer cell lines. Since 

there has been no reports of compounds 34 and 37 possessing any biological activity, it 

was decided to evaluate these compounds for antibacterial and anticancer activity. We 

hope to compare our activity against E. faecalis with that reported in the literature and to 

report for the first time the antibacterial activity. We also hope to compare the anticancer 

activity of known compounds from P. hadiensis with that in the literature and report on 

the anticancer activity of any new compounds isolated from P. hadiensis. 

Research has been conducted previously on the aerial parts, roots and leaves (Mothana et 

al., 2008; Laing et al., 2006), but there has been no phytochemical reports on the stem 

material. Since the aerial parts, roots and leaves of P. hadiensis were found to possess 

antiradical, antibacterial and antifungal activity (Mothana et al., 2008; Laing et al., 2006), 

this plant made an interesting phytochemical and pharmacological subject. 

96

3.2 Foreword to Experimental 

Nuclear Magnetic Resonance (NMR) Spectroscopy 

The NMR spectra ( 1 H, 13 C, DEPT, HETCOR, COSY, NOESY, HSQC and HMBC) were 

recorded on either the Bruker 400MHz or 600MHz NMR spectrometer at the University 

of Kwa-Zulu Natal, Westville campus. TMS was used as an internal standard and 

chemical shifts were given as δ (ppm), and the coupling constants (J) were reported in 

Hz. In the case of the 400 MHz NMR instrument, the operating frequency for the 1 H 

NMR spectra was 400 MHz and for the 13 C NMR spectra was 100 MHz and in the 600 

MHz NMR instrument, the operating frequencies were 600 and 150 MHz respectively for 

the 1 H and 13 C NMR spectra. 

General chromatography 

Column chromatography was carried out with silica gel (Merck 60, 0.040-0.063 mm) as 

the stationary phase. All crude extracts were separated on 4.5 cm diameter columns with 

step gradients of binary solvent systems and 100 ml fractions were collected with ten 

fractions for each step of the gradient. Elution of the columns was initiated with the least 

polar organic solvent, hexane, followed by dichloromethane, ethyl acetate and methanol 

in a stepwise gradient until the column had been eluted with 100% methanol. Thin layer 

chromatography (TLC) was done concurrently with column chromatography in order to 

monitor the elution of compounds. Merck 20 x 20 cm silica gel 60 F254 aluminium plates 

were used and visualized with anisaldehyde spray reagent [anisaldehyde: concentrated 

hydrochloric acid: aqueous methanol (1:2:97)] by spraying the plates and then heating. 

UV light (254 and 366 nm) was also used to detect compounds with conjugated double 

bonds. 

Similar fractions were combined and purified on columns of varying diameter (1.5-2.5 

cm) depending on the amount of the material to be purified and are referred to as 2cm 

columns (meaning 2cm diameter columns) in the text that follows. With the exception of 

97

a few cases (specifically stated in the experimental) all purifications were carried out on 

2cm diameter columns collecting 50 ml fractions. Where 3cm and 1cm columns were 

used, 100 ml fractions and 10 ml fractions were collected respectively. Any deviations to 

these conditions are mentioned specifically in the experimental. 

Infrared Spectroscopy (IR Spectroscopy) 

Infrared spectra were recorded on a PerkinElmer TM Spectrum 100. The sample was 

dissolved in either dichloromethane or methanol before being loaded onto the surface of 

potassium bromide discs. 

Ultraviolet Absorption Spectroscopy (UV) 

Ultraviolet absorption spectra were recorded with a PerkinElmer TM Lambda 35 UV/VIS 

spectrophotometer. Samples were dissolved in either dichloromethane or methanol and 

made to concentrations between 0.0052g/100ml and 1.2g/100ml. 

Optical Rotation 

Optical rotations were performed with a PerkinElmer TM , Model 341 Polarimeter. The 

samples were dissolved in dichloromethane or methanol and were measured at room 

temperature. Concentrations were calculated in g/100mL. 

Gas Chromatography-Mass Spectrometry (GC-MS) 

The mass spectra were registered on an Agilent MS 5973 instrument which was coupled 

to an Agilent GC 6890 instrument equipped with a DB-5MS (30m x 0.25 id, 0.25 µm 

stationary phase) column. Helium (at 0.7ml/min) was used as the carrier gas. Optimal 

results required that the oven temperature be maintained at 200 o C for one minute 

followed by 5 

98 

o C increments in temperature every minute until an oven temperature of

280 o C was attained. This temperature was then held constant for a further eight minutes. 

The MS was operated in the EI mode at 70eV, in m/z range 100-500. 

Liquid Chromatography-Mass Spectrometry (LC-MS) 

The mass spectra were recorded on an Agilent 1100 Series LC/MSD Trap instrument 

operating in the negative ion mode and equipped with an electrospray interface. Helium 

(at 9ml/min) was used as the carrier gas. Separation was achieved using a mobile phase 

consisting of 95:5 acetonitrile:water containing 0.1% TFA at a flow rate of 0.3ml/min. 

The mass spectrometer was run in a partial scan mode range m/z 100-500. 

Melting Point Determination 

Melting points were determined on a Kofler micro hotstage melting point apparatus and 

are uncorrected. Compounds for melting point determination were recrystallised from 

dichloromethane or methanol before determining the melting point. 

General extraction procedure 

Plants were air dried, the stems milled and the leaves ground. Using a soxhlet, the plant 

material was extracted successively using hexane, dichloromethane, ethyl acetate and 

methanol. The plant material was extracted for forty-eight hours each with each of the 

four solvents and the extracts concentrated using a Buchi rotary evaporator. Crude 

extracts were stored in the fridge to prevent degradation of compounds and fungal 

contamination until they were ready to be purified further. 

3.3 Experimental 

Leaf and stem materials of Plectranthus hadiensis were collected in May 2007 in Kloof, 

Kwa-Zulu Natal by Professor Neil Crouch. A voucher specimen (N. Crouch 1126, NH) 

was retained at the National Botanic Institute, Berea Road, Durban, South Africa. 

99

The leaves (184.63g) were air-dried, shredded in a domestic blender and extracted using 

the general extraction procedure above. Concentration of the extracts resulted in masses 

of 9.42 g, 1.72 g, 5.67 g and 17.86 g respectively for hexane, dichloromethane, ethyl 

acetate and methanol. The stems (511.29g) were milled and extracted as above, yielding 

extracts of 3.96 g, 1.39 g, 0.98 g and 9.32 g from hexane through to methanol 

respectively. 

Separation of the leaf extract 

The hexane extract (9.42g) was eluted with a step gradient of hexane:dichloromethane 

(3:2; 1:1; 2:3; 1:4; 0:1), dichloromethane:ethyl acetate (4:1; 3:2; 1:1; 2:3; 1:4; 0:1) and 

ethyl acetate:methanol (4:1; 1:1; 0:1). TLC and NMR analysis of the crude fractions 

indicated that fractions 20-22, 23-40, 61-66 and 67-71, each contained compounds that 

showed characteristic 1 H NMR resonances for either abietane diterpenes or sterols and 

were chosen to be purified further. 

Fractions 20-22 was purified with 80% dichloromethane in hexane on a 2cm diameter 

column collecting 20 ml fractions, where fractions 11-12 contained stigmasterol (V). 

This compound was identified by its 1 H NMR spectrum and by comparison with an 

authentic sample. 

Fractions 23-40, eluted with 60% dichloromethane in hexane and needed no further 

purification (compound I). 

Fractions 61-66 was first purified with ethyl acetate:dichloromethane (20:80) on a 3cm 

column collecting 50 ml fractions. Fractions 8-15 of this column was further purified 

with ethyl acetate:hexane (60:40) on a 2cm column collecting 20 ml fractions where the 

pure compound III eluted in fractions 6-8. 

100

Fraction 67-71 was purified with 20% ethyl acetate in dichloromethane on a 2 cm 

diameter column collecting 20 ml fractions where fraction 25-42 contained the pure 

compound III. 

The dichloromethane, ethyl acetate and methanol extracts were chromatographed with 

step gradients from hexane through to methanol but did not result in the isolation of any 

compounds that could be identified. 

Separation of the stem extract 

The hexane extract (3.96g) was eluted with dichloromethane:hexane (1:4; 2:3; 3:2; 4:1; 

1:0), ethyl acetate:dichloromethane (1:4; 2:3; 3:2; 4:1; 1:0) and ethyl acetate:methanol 

(4:1; 1:1; 0:1). Two fractions of interest were obtained; fractions 13-16 and 17-23. 

Fraction 13-16 was purified further on a 2 cm diameter column with 20% 

dichloromethane in hexane collecting 20 ml fractions. Fraction 18-20 of this column 

was further purified with 20% ethyl acetate in hexane as above where fraction 6 

contained more stigmasterol (V). 

Fractions 17-23 of the crude column was purified on a 3cm column with 

dichloromethane:hexane (3:7 for fractions 1-20 and 7:3 for fractions 21-40), where 

fractions 27-30 was purified with 30% ethyl acetate in hexane and then fraction 6 of this 

column was purified with 20% ethyl acetate in hexane where fraction 10-11 resulted in 

the pure compound II and fraction 12 contained lupeol (VI). The purifications were done 

in 2cm columns, collecting 20 ml fractions. Lupeol was identified from its 1 H NMR 

spectrum and in comparison with an authentic sample contained in the laboratory. 

The dichloromethane extract (1.39g) was eluted with dichloromethane:hexane (2:3; 3:2; 

4:1; 1:0), ethyl acetate:dichloromethane (1:4; 2:3; 3:2; 4:1; 1:0) and ethyl 

acetate:methanol (4:1; 1:1; 0:1) on a 3 cm column collecting 100 ml fractions at each 

stage. Fraction 53-60 was purified with ethyl acetate:dichloromethane (1:1) on a 3cm 

column collecting 50 ml fractions, where fractions 4-6 yielded compound IV. 

101

The ethyl acetate extract (0.98g) was eluted with dichloromethane:hexane (1:4; 2:3; 3:2; 

4:1; 1:0), ethyl acetate:dichloromethane (1:4; 2:3; 3:2; 4:1; 1:0) and ethyl acetate: 

methanol (4:1; 1:1; 0:1). The 1 H NMR spectrum of the combined fractions 27-31 

indicated the presence of stigmasterol (V). 

The methanol extract was chromatographed with a step gradient of 10%-40% methanol in 

dichloromethane but no compounds were isolated for which structural elucidation could 

be carried out. NMR spectroscopy of the crude extracts showed mainly the presence of 

sugars. 

3.4 Results and Discussion of the compounds isolated 

We have isolated the two previously found royleanone compounds (7β-acetoxy-6β- 

hydroxyroyleoanone (I or compound 37 as listed in table 6a) and 6β,7β- 

dihydroxyroyleaonone (II or compound 34 as listed in table 6a) from the hexane extract 

of the leaves and the hexane extract of the stem of P. hadiensis, respectively. This was 

the first occurrence of compound III (ent-pimara-8(14),15-diene-3β,11α-diol) in 

Plectranthus, which was isolated from the hexane extract of the leaves. Compound III 

was found previously in Erythroxylum cuneatum which belongs to the Erythroxylaceae 

family (Ansell, 1989). Further to this, three triterpenoids (IV-VI) were isolated from the 

dichloromethane extract of the stem. All six compounds isolated from P. hadiensis are 

known compounds. 

102

2 

19 

1 

4 

18 

O 

20 

9 

10 

5 

11 

6 7 

OH 

OH 

12 

14 

8 

R 

R 

13 

16 

(I) OC(O)CH3 

(II) OH 

HO 

3 

15 

O 

19 

5 

10 

6 

17 

7 

21 

HO 

18 

14 

2 

3 

20 

1 

HO 

20 11 

9 

4 5 6 7 

10 8 

18 19 

22 

29 

16 

23 

12 

13 

17 

14 

15 

16 

HO 

HO 

1 

4 

25 26 

9 

5 

10 

6 7 

8 

23 24 

(III) (IV) 

28 

27 

25 

26 

HO 

24 23 

(V) (VI) 

3 

10 

2 

30 

25 26 

20 

29 

27 

18 

12 

29 

19 

27 

17 

18 

14 

OH 

15 

17 

22 

28 

30 

103 

21 

22 

COOH

3.4.1 Structural elucidation of Compounds I and II 

2 

19 

1 

4 

O 

20 

18 

9 

10 

5 

11 

6 7 

OH 

OH 

12 

14 

8 

R 

13 

16 

15 

O 

17 

I 

II 

R 

OC(O)CH 3 

Compound I is a yellow crystalline compound soluble in dichloromethane and methanol 

with a melting point of 197 °C and an optical rotation of [α] 20 D +29° (c 0.0052g/100ml, 

CHCl3) [Mehrotra et al., 1989, optical rotation = +23°]. The mass spectrum showed a 

molecular ion peak at m/z 390 corresponding to a molecular formula of C22H30O6 and 

fragment ion peaks at m/z 372 (M + - H2O) and 357 (M + - H2O - CH3). The IR spectrum 

showed carbonyl stretching bands at 1734 cm -1 , O-H stretching bands at 3377 cm -1 , 

alkene stretching bands at 1641 cm -1 and C-H bending vibrations of the isopropyl group 

at 1375 cm -1 . The UV spectrum showed a maximum absorbance at a wavelength of 

275nm (log ε = 1.61). 

The 1 H NMR spectrum of compound I showed deshielded methine resonances at δH 5.64 

(H-7) and δΗ 4.30 (H-6) as well as a methine resonance of an isopropyl group (H-15) at 

δΗ 3.14. One of the protons of the methylene group at C-1 resonating at δΗ 2.62 could 

also be clearly seen as well as three methyl group resonances at δH 0.92 (3H-18ax), 1.62 

(3H-20eq) and 2.02 (OC(O)CH3). Three other methyl groups were also present in the 1 H 

NMR spectrum, but were not clearly distinguishingable as they overlapped with each 

other. These were the methyl resonances at positions 16, 17 and 19 which occurred at 

δH 1.18 and δH 1.23. 

OH 

104

The 13 C NMR spectrum showed the presence of twenty-two carbon resonances of which 

three were attributed to carbonyl groups (C-11, 14 and the acetyl carbonyl), four to 

substituted olefinic carbon atoms (C-8, 9, 12 and 13) (indicating two double bonds in the 

structure), two to quaternary carbon resonances (C-4 and 10), four to methine (C-5,6,7 

and 15), three to methylene (C-1, 2 and 3) and six to methyl (C-16, 17, 18, 19, 20 and the 

acetyl methyl carbon). The methine and methyl resonances were identified using both 

the DEPT and the HSQC spectra. It must be noted that the C-18 methyl carbon 

resonance is more deshielded than the methine carbon resonance of C-15. This can be 

be distinguished clearly in the HSQC spectrum with δC 24.17 (C-15) showing a 

correlation to the methine proton resonance at δH 3.14 (H-15) and δC 33.68 (C-18) 

showing a correlation to the methyl proton resonance at δH 0.92 (3H-18ax). 

The resonances at δH 4.30 and δH 5.64 were both coupled in the COSY spectrum. Since 

both these resonances were deshielded, both these groups had an oxygen substituent 

attached to them. The H-7 resonance showed HMBC correlations to six carbon 

resonances (δC 49.75, 67.05, 137.08, 149.90, 169.61 and 185.76), two of which are 

aliphatic, two are olefinic and two are carbonyl. The aliphatic carbon resonance at δC 

49.75 also shows HMBC correlations to three methyl resonances and was therefore 

attributed to C-5, with the three methyl resonances at δH 0.92, δH 1.23 and δH 1.62 being 

3H-18, 3H-19 and 3H-20. Both the olefinic carbon resonances which showed HMBC 

correlations to H-7 were fully substituted and were therefore attributed to C-8 (δC 137.08) 

and C-9 (δC 149.90), which could be distinguished because the resonance at δC 137.08 

showed an additional HMBC correlation to H-6. Of the two carbonyl resonances which 

showed HMBC correlations to H-7, one was assigned to the acetyl carbonyl at δC 169.61 

as this is coupled to the acetyl methyl group at δH 2.02 in the HMBC spectrum. The 

other carbonyl group at δC 185.76 was assigned to C-14. 

The C-14 carbonyl group showed HMBC correlations to the methine proton resonance at 

δH 3.14 (H-15). This resonance was the methine resonance of the isopropyl group and 

showed COSY correlations to the equivalent methyl resonances at δH 1.18. The H-15 

105

esonance showed HMBC correlations to the other two olefinic resonances at δC 150.90 

and δC 124.67 as well as the carbonyl resonance at δC 185.76. These resonances were 

therefore assigned to C-12 (δC 150.9), C-13 (δC 124.67) and C-14 (δC 185.76). The C-12 

and C-13 resonances were distinguished based on chemical shift as the more deshielded 

resonance of C-12 at δC 150.90 was assigned to the olefinic carbon where the hydroxyl 

group is situated. The hydroxyl group proton resonance at δH 7.21 showed HMBC 

correlations to both C-12 and C-13 and to the other carbonyl resonance at δC 183.28, 

which was then assigned to C-11. 

One of the 2H-1 resonances was present at δH 2.62 and the other overlapped with the 3H- 

16 and 3H-17 methyl resonances at δH 1.18, determined by the HSQC correlation with C- 

1. The other two non equivalent methylene resonances at C-2 and C-3 were assigned due 

to the C-3 carbon resonance at δC 42.27 showing a HMBC correlation with the two 

methyl resonances at δH 1.23 (3H-19ax) and δH 0.92 (3H-18eq). The C-2 carbon 

resonance was assigned to the remaining methylene resonance. The resonance at δH 1.62 

was assigned to 3H-20 since C-5 showed correlations to all three methyl resonances, 3H- 

18eq, 3H-19ax and 3H-20ax. 

Of the remaining two quaternary resonances at δC 33.54 and δC 38.63, the resonance at δC 

38.63 was assigned to C-4 based on a HMBC correlation with H-6. 

The stereochemistry of H-5, H-6 and H-7 were ascertained from coupling constants and 

using a molecular model to determine their dihedral angles and assuming the H-5 proton 

to be axial. Since the H-5, H-6 and H-7 proton resonances were all slightly broadened 

singlets, we were looking for a model where the dihedral angles between H-5 and H-6, 

and H-6 and H-7 were close to 90°. This was possible if the H-5 proton was axial 

(alpha), H-6 was equatorial (alpha) and H-7 was axial (alpha). This indicates that the 

hydroxyl group at C-6 is axial (beta) and that the acetoxy group at C-7 is equatorial (beta) 

(Figure 16). This also supports the downfield chemical shift of δH 1.62 of 3H-20 as the 

106

1,3-diaxial interaction between 3H-20 and the 6β-hydroxy group results in a 

paramagnetic shift of the methyl resonance, supporting this assignment. 

3 

2 

4 

1 

19 

18 

5 

HO 

20 

H ax 

O 

10 

OH 

6 

12 

16 17 

11 

H 

9 

15 

13 

7 

H 

14 

8 

O 

O 

OCCH 3 

Figure 16: Chair conformation of compound I showing the relationship between H- 

5, H-6 and H-7 

There have been two reports of compound I being isolated previously (Mehrotra et al., 

1989; Rasikari, 2007). The NMR data compares well with literature (Rasikari, 2007), 

however the C-4 and C-10 resonances are switched around, but we have assigned C-4 

based on an HMBC correlation with H-6 and are confident in our assignment. With 

regard to the stereochemistry of the molecule, in Rasikari (2007) it is important to point 

out that the stereochemistry in the name of the compound does not correspond with the 

structure. We think that the compound was incorrectly named as 7α-acetoxy instead of 

7β-acetoxy as a coupling constant of 3.5 Hz is used as a basis for this assignment. 

Mehrotra et al. (1989) is also used as a comparison in the identification in Rasikari et al. 

(2007). However, Mehrotra et al. (1989) observed a JH-6, H-7 of 3.5 Hz, close enough to 

that observed by Rasikari (2007) of JH-6, H-7 of 2.0 Hz, and concluded a 6β-axial, 7β- 

equatorial configuration. The structure of the molecule in Rasikari (2007) is however 

correctly depicted as the 6β-ax, 7β-eq form. 

While the 1 H NMR data also compares well to that in Mehrotra et al. (1989), the carbon 

resonances of C-6, 13, 15-17, 19 and 20 all differ by 1-3 ppm and the carbon resonance 

of C-18 is very different (33.68 as opposed to 21.00 in the literature). The carbonyl 

107

esonance at δC 185.76 of C-14 is also different to that of 179.00 and that of the acetyl 

carbon at δC 169.61 is 3ppm higher than the acetyl carbon at 166.00. The C-2 carbon 

resonance in compound I is also 5ppm lower than that cited in Mehrotra et al. (1989). 

Although Hensch et al. (1975) report the 7α-axial acetoxy isomer, the coupling constant 

JH-6, H-7 is reported to be 2.0 Hz and we think that this is the 7β-equatorial acetoxy isomer. 

The NMR data is therefore used for comparison. The NMR data for compound I 

compares well with that in Hensch et al. (1975). 

Compound II is a yellow compound soluble in dichloromethane and methanol with a 

melting point of 125 °C and an optical rotation of [α] 20 D -47° (c 0.0063g/100ml, CHCl3). 

The GC-MS did not show a molecular ion peak at m/z 348 but the LC-MS showed a peak 

at m/z 347 in the negative ion mode which supports the molecular formula of C20H28O5 

with a molecular mass of 348 amu. The UV spectrum showed a maximum absorbance at 

a wavelength of 192nm (log ε = 2.73). 

The 1 H and 13 C NMR data for compound II is similar to that of compound I, with a few 

notable differences. The H-7 resonance moved upfield from δH 5.64 to δH 4.54. The 

methyl acetyl resonance at δH 2.02 was also absent and a new one-proton resonance 

appeared as a broad singlet at δH 3.00. Another broad singlet could now be distinguished 

at δH 1.70. In the 13 C NMR spectrum of compound II, the acetyl carbonyl resonance was 

absent as well as the acetyl methyl resonance. Furthermore, the carbonyl stretching band 

in the IR spectrum was also absent. All these changes were consistent with the acetyl 

group at C-7 being replaced by a hydroxyl group. 

The broad singlet resonance at δH 3.00 was attributed to the hydroxyl group proton at C-7 

because of a COSY correlation to H-7. This proton resonance did not correlate to any of 

the carbon resonances in the HSQC spectrum. The resonance at δH 1.70 also did not 

correlate to any of the carbon resonances and was attributed to the hydroxyl group at C-6. 

This resonance could have also been present in compound I, but could not be 

108

distinguished from the other resonances that occurred along with it. The C-9 carbon 

resonance showed a HMBC correlation with H-7 and CH3-20 and the C-8 carbon 

resonance showed a HMBC correlation to the H-7 resonance as well as the H-6 

resonance. These correlations confirmed the assignments of H-6 and H-7 as well as C-8 

and C-9. 

The H-5, H-6 and H-7 resonances had the same slightly broadened singlet profile as that 

of compound I and therefore the same stereochemistry would be prevalent here as well 

with a 6β-ax, 7β-eq-dihydroxy form. The paramagnetic shift for the 3H-20 resonance is 

also observed in compound II as well as for 3H-19β-ax methyl group which is more 

deshielded at δH 1.62 than that of 3H-18 at δH 1.06. 

Compound II was isolated previously from Mehrotra et al. (1989). Rasikari (2007) and 

Hensch et al. (1975) report the 7α-axial-hydroxy isomer. However, the coupling 

constants of JH-6, H-7 in both Rasikari (2007) and Hensch et al. (1975) are 2.0 Hz and 

therefore we think that these compounds may also be the 7β-equatorial-hydroxy and not 

the 7α-axial-hydroxy isomer. The NMR data of all three references are thus used for 

comparison. The 1 H NMR data compare well, with a consistent 6-7 ppm difference for 

Hensch et al. (1975). The 13 C NMR data compared well with that of Rasikari (2007). No 

13 C NMR data is given in both Mehrotra et al. (1989) and Hensch et al. (1975). 

Table 15: 1 H NMR data for compound I compared with three reference compounds 

(CDCl3, 400MHz) 

Position Moiety 

Compound I 

2.62, d 

(J = 12.72 Hz) 


(500MHz) 

2.65, dt 

(J = 12.8 Hz) 

1 CH2 

1.18 1.22, m 

2 CH2 

1.57 

1.83 

1.59, dt 

(J = 13.4, 3.5 Hz) 

1.85, qt 

(J = 13.4, 3.5 Hz) 


(Mehrotra. et al., 

1989) 

(80MHz) 

(Hensch et al., 1975) 

(60MHz) 

2.64, m 

109

Position Moiety Compound I 

3 CH2 

5 CH 

6 

1.49 


(500MHz) 

1.49, dt 

(J = 12.8 Hz) 

1.18 1.24, m 

1.32, s 

(w½ = 3.6 Hz) 

1.35, s 

CH 4.30, s 4.33, s 

(Mehrotra. et al., 

1989) 

(80MHz) 

4.25, dd 

(J = 3.5, 2.0 Hz) 


(60MHz) 

4.34, m 

-OH 2.46, s 

7 CH 5.64, s 

5.62, d 

(J = 3.5 Hz) 

5.70, d 

(J = 2 Hz) 

12 -OH 7.21, s 7.20 7.20, s 7.25, s 

15 CH 3.14, sep 

3.18, hept 

(J = 7.1 Hz) 

3.15, sep 

(J = 7 Hz) 

3.16, m 

(J = 7 Hz) 

16 

17 

CH3 

CH3 

1.18, d 

(J = 7.08 Hz) 

1.24, d 

(J = 7.1 Hz) 

1.15, d 

(J = 7 Hz) 

1.20, d 

(J = 7 Hz) 

18 CH3 0.92, s 0.96, s 0.90, s 0.88,s 

19 CH3 1.23, s 1.24, s 1.20, s 1.24, s 

20 CH3 1.62, s 1.63, s 1.60, s 1.62, s 

7- 

OCOCH3 

2.02, s 2.05, s 2.00, s 2.02, s 

Table 16: 13 C NMR data for compound I compared with three reference compounds 



(Rasikari., 2007) 

(125MHz) 

(Mehrotra. et al., 1989) 

(20MHz) 


(60MHz) 

1 CH2 38.35 38.6 38.50 38.7 

2 CH2 18.97 19.2 24.00 24.2 

3 CH2 42.27 42.5 42.50 42.5 

4 C 38.63 33.9 39.00 38.4 

5 CH 49.75 50.0 49.50 49.9 

6 CH 67.05 67.3 68.00 67.2 

7 CH 68.74 69.0 70.00 68.9 

8 C=C 137.08 137.3 137.50 137.3 

9 C=C 149.90 150.1 150.50 150.1 

10 C 33.54 38.9 34.50 33.6 

11 C=O 183.28 183.5 183.50 185.9 

12 C=C 150.90 151.1 151.00 151.1 

13 C=C 124.67 124.9 126.00 124.8 

14 C=O 185.76 186.0 179.00 183.6 

15 CH 24.17 24.4 23.00 23.8 


110


(Rasikari., 2007) 

(125MHz) 

(Mehrotra. et al., 1989) 

(20MHz) 


(60MHz) 

16 CH3 19.71 19.9 22.00 *19.7 

17 CH3 19.85 20.1 22.50 *19.8 

18 CH3 33.68 33.7 21.00 

# 33.7 

19 CH3 23.82 24.1 20.00 *19.1 

20 CH3 21.51 21.8 23.50 *21.5 

7- 

OCOCH3 

20.95 21.1 20.50 *20.9 

7- 

OCOCH3 

169.61 169.8 166.00 169.9 

* Resonance for each methyl group not specified 

# Resonance was assigned to C-19 by Hensch et al. (1975) but we suspect that it is the resonance for C-18 

Table 17: 1 H NMR data for compound II compared with three reference compounds 


Position Moiety Compound II 

1 CH2 

2 CH2 

3 CH2 

5 CH 

6 

7 

2.61, d 

(J = 3.08 Hz) 

1.17 


(500 MHz) 

2.59, dt 

(J = 10.2, 3.5 Hz) 

1.19, td 

(J = 12.7, 3.6 Hz) 

1.57 1.61, m 

1.85 

1.27 

1.48 

1.46, s 

(w½ = 4.4 Hz) 

1.82, qt 

(J = 13.7, 3.6 Hz) 

1.27, td 

(J = 13.3, 4.0 Hz) 

1.48, dt 

(J = 13.1, 2.6 Hz) 

1.45, s 


(60MHz) 

2.53, m 

(Mehrotra et al., 1989) 

(80MHz) 

CH 4.48, s 4.45, s 4.39, m 4.50, m 

-OH 1.70, brs 

CH 4.54, s 

4.50, d 

(J = 1.8 Hz) 

4.43, d 

(J = 2 Hz) 

4.50, m 

-OH 3.00, brs 

12 -OH 7.34, s 7.25, s 

15 CH 3.18, sep 

3.17, hept 

(J = 7.1 Hz) 

3.10, m 

(J = 7 Hz) 

3.15, sep 

(J = 7 Hz) 

16 CH3 1.24, d 

1.21, d 

1.14, d 

1.30, d 

17 CH3 (J = 6.92 Hz) (J = 7.1 Hz) 

(J = 7 Hz) 

(J = 7 Hz) 

18 CH3 1.06, s 1.04, s 0.99, s 1.05, s 

19 CH3 1.27, s 1.26, s 1.23, s 1.20, s 

20 CH3 1.62, s 1.60, s 1.56, s 1.65, s 

111

Table 18: 13 C NMR data for compound II compared with one reference compound 

(CDCl3) 


Compound II 

(400MHz) 


(125 MHz) 

1 CH2 38.43 38.8 

2 CH2 19.03 19.3 

3 CH2 42.31 42.6 

4 C 33.75 34.0 

5 CH 49.50 49.8 

6 CH 69.31 69.6 

7 CH 69.13 69.4 

8 C=C 140.94 141.2 

9 C=C 147.54 147.7 

10 C 38.56 38.8 

11 C=O 183.44 183.7 

12 C=C 151.20 151.4 

13 C=C 124.26 124.5 

14 C=O 189.14 189.4 

15 CH 24.02 24.3 

16 CH3 19.86 20.0 

17 CH3 19.80 20.0 

18 CH3 33.52 33.7 

19 CH3 24.27 24.5 

20 CH3 21.62 21.9 

3.4.2 Structure elucidation of Compound III 

HO 

2 

3 

1 

HO 

20 11 

9 

4 5 6 7 

10 8 

18 19 

12 

13 

17 

14 

15 

Ha 

16 

Hb 

Compound III is a colourless compound soluble in both dichloromethane and methanol 

with a melting point of 89°C and an optical rotation of [α] 20 D -79° (c 0.0082g/100ml, 

CHCl3). The mass spectrum showed a molecular ion peak at m/z 304 corresponding to a 

112

molecular formula of C20H32O2 and fragment ion peaks at m/z 286 and 268. These 

fragment ion peaks are brought about when compound III loses one and two water 

molecules consecutively. The IR spectrum showed O-H stretching bands at 3396 cm -1 

and C-H stretching bands at 2930 cm -1 . The UV spectrum showed a maximum 

absorbance at a wavelength of 228 nm (log ε = 1.70). 

The 1 H NMR spectrum indicated the presence of four tertiary methyl groups with 

resonances at δΗ 1.01, 0.99, 0.78 and 0.75, two double bonds with four olefinic proton 

resonances at δH 5.72, δH 5.20, δH 4.90 and δH 4.88 and two oxygenated methine groups 

with resonances at δΗ 3.26 and δΗ 3.85. The resonances at δH 5.72 (dd, J = 17.21, 10.57 

Hz), δH 4.90 (dd, J = 10.57, 1.16 Hz) and δH 4.88 (dd, J = 17.21, 1.16 Hz) was typical of 

a vinyl group (H-15, H-16a and H-16b, respectively) (Lyder et al., 1998). A deshielded 

methine resonance at δΗ 3.25 is typical for compounds with a hydroxyl group at the 3- 

position (H-3, dd, J = 3.84, 10.84 Hz) (Kalauni et al., 2005). 

The 13 C NMR spectrum showed the presence of twenty carbon resonances typical of a 

diterpenoid of which four were olefinic 146.18 (CH=), δC 137.46 (C), 127.64 (CH=) and 

112.69 (CH2) indicating two double bonds, three were quaternary (δC 39.11, 39.05 and 

39.57), four were aliphatic methine carbon groups, five were methylene and four were 

methyl. 

The interesting point about this molecule is that the methyl resonance at δH 1.01 showed 

HMBC correlations to the vinylic carbon resonance (C-15), the olefinic resonance at δC 

127.64 (C-14) and a methylene resonance at δC 45.85 (C-12). Since this methyl group 

was aliphatic, indicated by its chemical shift, the two double bonds could not be 

conjugated and had to be separated by an aliphatic carbon. Ring C and its constituents 

could then be constructed as in figure 17 and the methyl proton resonance was assigned 

to 3H-17. 

113

The equatorial methyl group at C-4, usually resonates at approximately δΗ 28 and the 

axial methyl group at this position resonates at approximately δΗ 15 (Ansell, 1989; Kang 

et al., 2005; Meragelman et al., 2003). This was used to assign the resonances at 

δΗ 28.43 and δΗ 15.59 to 3H-18βax and 3H-19αeq, respectively. In the HMBC spectrum, 

3H-18βax and 3H-19αeq showed correlations to C-3 as well as a quartenary carbon at δC 

39.11 (C-4) and an aliphatic methine at δC 53.99 (C-5). The C-5 resonance in turn 

showed a HMBC correlation to another methyl group at δH 0.75 which could only be 

assigned to 3H-20. This 3H-20 resonance was then used to assign C-10 and C-9 at δC 

39.05 and δC 59.83 respectively because of HMBC correlations to them. 

The tertiary carbon atom, C-13 could be assigned to δC 39.57 because of a HMBC 

correlation to H-16a and H-16b. The methine resonance at δΗ 3.85 could then be assigned 

to H-11 because of a HMBC to C-13. Since this proton resonance is situated downfield, 

it suggests the presence of a hydroxyl group at this position even though the 

corresponding carbon resonance at δC 66.19 is not as deshielded as other C-O carbon 

resonances (with chemical shifts between 72-80). 

The methylene carbon resonances could not be assigned from the spectra available, 

therefore, literature with NMR data of similar compounds was used to assign them 

(Tables 19 and 20) (Ansell, 1989; Kang et al., 2005). 

12 

We have placed the proton at C-3 in the axial/alpha position and the hydroxyl group in 

the beta/equatorial position in accordance with the literature (Ansell, 1989). The 

stereochemistry in Ansell (1989) was determined using coupling constants of the ABX 

13 

8 

17 

14 

15 

16 

Figure 17: Fragment of Ring C 

114

system where coupling constants of 5.3 Hz and 9.7 Hz indicated an axial proton (α) and 

an equatorial hydroxyl group (β). The coupling constants of compound III compared 

well to this with values of 3.84 Hz and 10.84 Hz indicating that the hydroxyl group at 

position 3 in compound III was equatorial (β). This was supported by a NOESY 

correlation between H-3 and H-5 indicating a 1,3-diaxial interaction between the two 

protons. 

The NOESY spectrum was then used to determine the configuration of the hydroxyl 

group at C-11 as well as the methyl group situated at C-13. The methyl group CH3-20ax 

showed a correlation to the proton at H-11 which puts this hydrogen in a beta/axial 

position and the hydroxyl group in the alpha/equatorial position (Figure 18). It is for this 

reason that the compound III has been named as ent-pimara-8(14),15-diene-3β,11α-diol. 

The methyl group, CH3-17 was assigned to the equatorial (alpha) position as it showed 

NOESY correlations with the H-12α proton at δΗ 1.82. The protons at H-12 was 

assigned using values from the literature of similar compound, 3.3 (Meragelman et al., 

2003) (Table 20, page 117) as compound III in the literature does not distinguish 

between the H-12 protons. 

HO 

H 

3 

2 

4 

19 

1 

18 

5 

H ax 

20 ax 

10 

6 

9 

11 

7 

8 

H 

12 

Figure 18: NOESY correlations for compound III 

HO 

H 

H 

14 

15 

13 

16 

17 

115

The proton NMR data for compound III was similar to that in the literature with the 

exception of the resonance for the methylene group at C-6 (Table 19). Ansell (1989) 

reported 2H-6 having a resonance of δΗ 2.37 (ddd, J = 2.3, 3.3, 13.2 Hz) while in 

compound III, the methylene group at C-6 resonated as singlets at δΗ 1.38 and δΗ 1.63 

(Table 19). The 13 C NMR data was not reported in the literature and therefore the 

diacetylated compound was used for comparison (Table 19). Due to insufficient sample, 

compound III could not be acetylated for absolute comparison. 

In comparing our compound to that of literature, we have had to make an assumption that 

the compound in the literature had the hydroxyl groups in the equatorial positions (3β and 

11α) as concluded in the discussion and not by what was depicted in the structure. The 

discussion of the compound in Ansell (1989) did not match the structure (where the 

hydroxyl groups were axial, 3α and 11β). The 13 C NMR data also compared well to 

similar compounds 3.1 to 3.3 (Table 20, pages 118 and 119). 

Table 19: NMR data of compound III (CDCl3) and its acetylated equivalent 


1 CH2 

2 CH2 

3 CH-OH 

Compound III 

(400MHz) 

2.35 

2.02 

1.69 

1.57 

3.26, dd 

(J = 3.84, 10.84 Hz) 

1 H 

Ansell, 1989 

(80MHz) 

3.26, sx 

(J = 5.3, 9.7, Hz) 

Compound III 

(400MHz) 

13 C 

# Ansell, 1989 

(20MHz) 

35.74 36.5 

27.59 24.0 

78.86 80.6 

*4 C 39.11 38.0 

5 CH 1.08 53.99 54.0 

6 CH2 

1.63 

1.38 

2.37, ddd 

(J = 2.3, 3.3, 13.2 Hz) 22.55 22.4 

7 CH2 

1.48 

1.85 

38.38 35.6 

8 C=C 137.46 136.4 

9 CH 1.61 59.83 55.4 

*10 C 39.05 38.7 


116


Compound III 

(400MHz) 

11 CH-OH 3.85, m 

1 H 

Ansell, 1989 

(80MHz) 

3.86, ddd 

(J = 4.7, 7.1, 11.7 Hz) 

Compound III 

(400MHz) 

13 C 

# Ansell, 1989 

(20MHz) 

66.19 69.3 

12 CH2 

1.82 (α) 

1.35 (β) 

45.85 40.7 

*13 C 39.57 38.7 

14 CH= 5.20, s 

5.21, d 

(J = 1.5 Hz) 

127.64 128.4 

15 CH= 

5.72, dd 

(J = 10.57, 17.21 Hz) 

5.74, sx 

(J = 9.5, 18.1 Hz) 

4.87, dd 

146.18 145.4 

16 CH2 

4.89, dd 

(J = 9.28, 17.4 Hz) 

(J = 1.9, 18.1 Hz) 

4.91, dd 

(J = 1.9, 9.5 Hz) 

112.69 112.5 

17 CH3 1.01, s 1.02, s 29.51 29.5 

18 CH3 0.99, s 1.00, s 28.43 28.3 

19 CH3 0.78, s 0.79, s 15.59 15.7 

20 CH3 0.75, s 0.76, s 15.82 16.6 

3 OCOCH3 --- --- --- 170.2 

3 OCOCH3 --- --- --- 21.1 

11 OCOCH3 --- --- --- 170.6 

11 OCOCH3 --- --- --- 21.5 

* these resonances can be used interchangeably 

# The 13 C NMR data corresponds to the acetylated product of compound III 

Position 

1 

2 

3 

Table 20: 1 H NMR data for compound III (CDCl3) compared with two similar 

compounds 

Compound III 

(400MHz) 

Compound 3.1 

(80MHz) 

1 H 

Compound 3.2 

(300MHz) 

Compound 3.3 

(600MHz) 

Ansell, 1989 Kang et al., 2005 Meragelman et al., 2003 

2.35 2.68, s 

1.71, dt 

(J = 13.1, 3.5 Hz) 

2.02 0.98 

1.69 1.61, m 

1.57 1.61, m 

3.26, dd 

(J = 3.84, 10.84 Hz) 

3.31, dd 

(J = 4.5, 9.6 Hz) 

4.0, s 

3.21, dd 

(J = 11.1, 5,2 Hz) 

5 1.08 1.68, m 0.82 


117

Position 

6 

7 

Compound III 

(400MHz) 

9 1.61 

Compound 3.1 

(80MHz) 

1 H 

Compound 3.2 

(300MHz) 

Compound 3.3 

(600MHz) 


1.63 1.73, m 

1.63, qd 

(J = 13.4, 3.2 Hz) 

1.38 1.49 

1.48 2.13, m 1.22 

1.85 2.35, m 

2.02, d 

(J = 5.7 Hz) 

11 3.85 4.0, m 

12 1.82 1.63, m 

15 

16 

5.72, dd 

(J = 10.57, 17.21 

Hz) 

4.89, dd 

(J = 9.28, 17.4 Hz) 

5.74, septet 

(J = 18.4, 8.6 Hz) 

4.90, dd 

(J = 2.1, 18.4 Hz) 

4.96, dd 

(J = 2.2, 8.5 Hz) 

5.83, dd 

(J = 10, 17.8 Hz) 

4.94, d 

(J = 10 Hz) 

5.00, d 

(J = 17.8 Hz) 

1.78, dt 

(J = 13.4, 3.2 Hz) 

0.85 

1.47 (α), qd 

(J = 13.4, 3.1 Hz) 

1.47 (β), m 

2.01, dq 

(J = 13.7, 3.1 Hz) 

5.98, dd 

(J = 17.9, 11.0 Hz) 

5.09, dd 

(J = 11.0, 1.2 Hz) 

5.14, dd 

(J = 17.9, 1.2 Hz) 

17 1.01, s *1.09, s 1.05, s 0.91, s 

18 0.99, s *1.10, s 1.19, s 0.99, s 

19 0.78, s *0.84, s 0.69, s 0.81, s 

20 0.75, s *0.92, s 0.80, s 0.93, s 

* Resonance for each methyl group not specified 

Table 21: 13 C NMR data for compound III (CDCl3) compared with two similar 

compounds 

Compound 3.1 Compound 3.2 Compound 3.3 

Position Compound III (80MHz) (75MHz) 

(50MHz) 


1 35.74 37.3 52.2 37.8 

2 27.59 27.7 211.0 27.2 

3 78.86 79.2 82.2 79.1 

4 39.11 38.3 45.3 38.9 

5 53.99 54.3 53.3 55.6 

6 22.55 22.3 22.3 17.8 

7 38.38 35.8 35.2 42.0 

8 137.46 137.9 134.6 72.3 

9 59.83 51.3 59.1 56.2 

Continued on next page… 

118

HO 

Compound 3.1 Compound 3.2 Compound 3.3 

Position Compound III (80MHz) (75MHz) 

(50MHz) 


10 39.05 38.6 44.7 37.0 

11 66.19 19.2 65.8 17.4 

12 45.85 35.8 44.0 36.1 

13 39.57 38.6 37.8 36.5 

14 127.64 128.3 129.1 53.4 

15 146.18 147.4 148.3 147.5 

16 112.69 112.8 110.9 112.0 

17 29.51 29.5 26.5 28.3 

18 28.43 28.5 29.3 32.4 

19 15.59 15.7 16.4 15.5 

20 15.82 14.8 16.5 15.5 

1 

3 

4 

H 

6 

5 

7 

16 

17 

12 

15 

20 11 13 

9 14 

10 8 

O 

HO 

H 

HO 

1 

3 

4 6 

5 

18 19 

7 

12 

20 11 13 

9 

10 8 

18 19 

17 

15 

16 

HO 

1 

3 

4 

OH 

6 

5 

7 

18 

15 

12 

20 11 13 

17 

9 

10 8 

19 

3.1 3.2 3.3 

119 

16

3.4.3 Structure elucidation of Compound IV 

HO 

HO 

2 

23 

1 

4 

24 

25 26 

9 

5 

10 

6 7 

8 

12 

29 

27 

18 

14 

OH 

15 

17 

30 

21 

22 

COOH 

Compound IV is a white compound soluble in methanol with a melting point of 255°C 

and an optical rotation of [ ] 20 

α D +32.26° (c 0.0186g/100ml, MeOH). A molecular ion peak 

of m/z 488 was anticipated as this molecular weight corresponded to the molecular 

formula of compound IV, C30H48O5. This peak could not be detected on the mass 

spectrum but a fragment ion peak of m/z 426 and a base peak of m/z 218 were 

documented. The loss of one of the hydroxyl (-OH) group together with the carboxylic 

acid (-COOH) group, accounts for the fragment ion peak of m/z 426. Tertiary alcohols 

and carboxylic acids seldom show the molecular ion peaks in the mass spectrum. The IR 

spectrum showed O-H stretching bands at 3424 cm -1 , C-H stretching vibrations at 2927 

cm -1 and a stretching band at 1687 cm -1 which is suggestive of a carboxylic acid group 

since this group has less double bond character than an aliphatic ketone. The UV 

spectrum showed a maximum absorbance at a wavelength of 211nm (log ε = 2.05). The 

UV spectra of pentacyclic triterpenoids with a single double bond, a carboxylic acid 

group and several hydroxyl groups normally show absorbance values at approximately 

205-210 nm. 

The 1 H NMR spectrum in conjuction with the HSQC and DEPT spectrum showed the 

presence of seven methyl resonances at δΗ 0.78 (s), δΗ 0.86 (s), δΗ 0.92 (d, J = 7.8 Hz), 

δΗ 0.98 (s, 2 x CH3), δΗ 1.19 (s) and δΗ 1.35 (s), one broadened triplet olefinic proton 

120

esonance at δΗ 5.29 and two oxygenated methine resonances at δΗ 3.33 (d, J = 2.28 Hz) 

which overlapped with the solvent peaks and δΗ 3.93, a broad doublet of triplets 

respectively. 

The two characteristic olefinic resonances at δC 127.95 (CH=) and δC 138.66 (C=) 

suggested an urs-12-ene skeleton. However the tertiary methyl group (3H-28) which is 

usually present at C-17 in urs-12-ene triterpenes was replaced by a carboxylic acid group 

evidenced by the carbonyl resonance of the carboxyl group at δC 180.88. The C-17 

carbon resonance overlapped with the resonances of the deuterated methanol solvent 

peak between δ 47 and δ 48 and showed HMBC correlations to a methylene and methine 

resonance at δΗ 2.57 (2H-16) and δΗ 2.49 (H-18), respectively. The proposed resonance 

for quartenary carbon C-17, falls within the range as documented for similar compounds 

4.1, 4.4 and 4.5 (Table 23). 

The two proton resonances at δH 3.93 and δH 3.33 were attributed to H-2ax and H-3eq 

respectively based on a comparison with the C-17 methylated compounds, where H-2 and 

H-3 are axial and equatorial respectively (Lontsi et al., 1992) and where H-2 and H-3 are 

both axially situated (Lontsi et al., 1998). Our compound compared favourably with the 

H-2ax and H-3eq configuration. The corresponding carbon resonances at δC 65.90 (C-2) 

and δC 78.70 (C-3) are deshielded owing to the oxygenation at these carbons. 

In addition to C-2 and C-3, a further deshielded carbon resonance at δC 72.18 suggested 

the presence of another oxygenated group. This carbon resonance was however 

quaternary and was placed at C-19 with a hydroxyl and methyl attached to it. Literature 

supported this assignment as C-19 in the literature with the same substitution pattern 

resonates between δ 71 and δ 74, Table 22 (da Graca Rocha et al., 2007; Fang et al., 

1991; Lontsi et al., 1992; 1998). 

The C-3 carbon resonance showed HMBC correlations to two tertiary methyl resonances, 

3H-23eq (δΗ 0.98) and 3H-24ax (δΗ 0.86) whose corresponding methyl proton resonances 

121

showed HMBC correlations to another methine resonance and one quartenary carbon 

resonance at δC 47.90 and δC 38.07, which were then attributed to C-5 and C-4, 

respectively. 

The quartenary carbon resonance at δC 39.86 (C-8) showed correlations to two tertiary 

methyl resonances at δH 1.35 and δH 0.78 which were assigned as 3H-27 and 3H-26, 

respectively. The 3H-27 methyl resonance was then used to assign the quaternary 

carbon resonance of C-14 and the methylene resonance of C-15 to δ 41.35 and δ 28.21 

due to HMBC correlations. 

The C-19 carbon resonance showed HMBC correlations to two methyl resonances at δH 

1.19 and δH 0.92 (d, J = 7.8 Hz), which were then attributed to 3H-29eq and 3H-30eq 

respectively. The two methyl groups were distinguished as the resonance at δH 0.92 was 

a doublet and had to be situated at C-20, which was assigned to δC 41.73 because of a 

HMBC correlation to 3H-29 and 3H-30. The H-18 methine resonance could also be 

assigned because of a HMBC correlation to C-19. 

In addition to the HMBC correlation to C-8, 3H-26 showed correlations to the carbon 

resonances at δC 32.68 and δC 46.79 which were then assigned to C-7 and C-9 

respectively. The distinction between the two was made as δC 32.68 was a methylene 

carbon resonance and δC 46.79 was a methine carbon resonance. The C-9 carbon 

resonance in turn was used to assign 3H-25, the remaining methyl resonance as C-9 

showed HMBC correlations to it. The 3H-25 methyl resonance then showed a HMBC 

correlation to δC 41.09 which was then attributed to C-1. 

The carboxylic acid carbon resonance at δC 180.88 and the two olefinic carbon 

resonances at δC 127.95 and δC 138.66 showed correlations to H-18 and were assigned to 

C-28 of the carboxylic acid group and C-12 and C-13 of the double bond. The quaternary 

carbon atom, C-17 was assigned to a resonance underneath the solvent peak due to a 

122

HMBC correlation with H-18. The H-18 resonance was also used to assign the 

methylene proton resonance of 2H-16 due to HMBC coupling with this resonance. 

The remaining methylene carbon resonances of C-6, C-11, C-21 and C-22 were assigned 

using literature values (Table 22) (da Graca Rocha et al., 2007; Fang et al., 1991; Lontsi 

et al., 1992; 1998). We think that da Graca Rocha et al. (2007) has incorrectly assigned 

C-15 and C-21 and that these values are in fact switched around. We have based our 

assignment of C-15 on a HMBC correlation with 3H-27. 

Compounds having the ursene skeletal structure posess seven methyl groups with the 

orientation of five methyl groups (3H-23 to 3H-27) being certain as a result of 

biosynthesis and two methyl groups (3H-29 and 3H-30) being either alpha or beta. To 

determine the stereochemistry of these two methyl groups as well as the two oxygenated 

methines (H-2 and H-3), correlations in the NOESY spectrum was used, in conjunction 

with a molecular model. 

The H-2 proton resonance at δΗ 3.93 showed correlations to the H-3, 3H-24ax (δΗ 0.86) 

and the overlapping methyl group resonances of 3H-23eq and 3H-25ax at δΗ 0.98. We 

have assumed that H-2 correlates to the resonance of 3H-25ax since this 3H-25ax is closer 

in proximity to H-2. Thus, H-2, based on this correlation would be axial and beta and 

since there is a NOESY correlation between H-2 and H-3, H-3 must also be beta and 

equatorial. All these NOESY correlations were also observed as possible on a molecular 

model. 

Assuming that all the rings in the molecule are in the more stable chair conformation, H- 

18 would be in the axial/alpha position. A NOESY correlation between H-18ax and 3H- 

27 confirms the methyl group (CH3-27) to be axial/alpha and a further correlation 

between H-18ax and 3H-29 puts the methyl group (CH3-29) in the alpha/equatorial 

position as well (Figure 19). 

123

H 

HO 

3 

OH 

H 

2 

4 

24 eq 

1 

23 

5 

ax 

Hax 25 

10 

6 

9 

11 

7 

12 

26 

8 

14 

27 

13 

15 

29 

18 

H 

OH 

19 

16 

20 

COOH 

H 

17 

Figure 19: NOESY correlations for compound IV 

Based on the analytical data reported for compound IV as well as the comparison of this 

data with literature, compound IV was identified as 2α,3α,19α-trihydroxyurs-12-en-28- 

oic acid (Table 22). A slight difference with regard to chemical shift has been obeserved 

between compound IV and the reference compound (Table 22). 

Table 22: NMR data of compound IV (CD3OD) compared with a reference compound 


Compound IV 

(600MHz) 

1 H 

*da Graca Rocha et 

al., 2007 b 

(200MHz) 

Compound 

IV 

(600MHz) 

30 

22 

13 C 

21 

da Graca Rocha et al., 

2007 b 

(200MHz) 

1 CH2 

1.28 

1.56 

41.09 

37.2 

2 CH 3.93, dt 

4.10, m 65.90 64.6 

3 CH 

(J = 

3.33, 

3.72, 

d 

3.18 

(J = 2.28 Hz) 

3.90, d 78.70 77.9 

4 C 38.07 41.6 

5 CH 1.26 47.90 46.5 

6 CH2 

1.39 

1.45 

17.93 17.7 

7 CH2 

1.31 

1.58 

32.68 32.6 

8 C 39.86 41.1 

9 CH 1.85 46.79 47.6 

10 C 37.99 38.1 

11 CH2 2.00 23.41 25.1 

12 CH= 

5.29, t 

(J = 3.72 Hz) 

a 5.20, s 127.95 126.9 

13 C=C 138.66 138.6 


124


Compound IV 

(600MHz) 

1 H 

*da Graca Rocha et 

al., 2007 b 

(200MHz) 

Compound 

IV 

(600MHz) 

13 C 

da Graca Rocha et al., 

2007 b 

(200MHz) 

14 C 41.35 40.7 

15 CH2 

1.01 

1.79 

28.21 25.9 

16 CH2 

1.51 

2.57 

25.25 23.1 

17 C 47-48 46.8 

18 CH 2.49 53.66 53.1 

19 C 72.18 71.6 

20 CH 1.35 41.73 41.4 

21 CH2 

1.23 

1.73 

25.96 29.0 

22 CH2 

1.61 

1.71 

37.65 38.0 

23 CH3 0.98 0.69, s 27.89 28.9 

24 CH3 0.86 1.30, s 21.09 21.8 

25 CH3 0.98 0.89, s 16.15 16.3 

26 CH3 0.78 0.95, s 15.51 16.1 

27 CH3 1.35 1.22, s 23.55 24.1 

28 COOH 180.88 178.9 

29 CH3 1.19 1.06, s 25.68 26.4 

30 CH3 0.92, d 

(J = 7.8 Hz) 

* coupling constants not documented 

0.90, d 15.25 16.6 

a 

Reported as resonance for H-5 by De Graca Rocha et al. (2007) but suspect that this resonance belongs to 

H-12 

b DMSO 

The 1 H and 13 C NMR data of three previously isolated compounds (da Graca Rocha et 

al., 2007; Lontsi et al., 1998; Fang et al., 1991) were compared with that of compound 

IV (Tables 23 and 24) as they closely resemble euscaphic acid, the differences being the 

orientation of the hydroxyl group at C-3 and C-11 as well as the substituent at the C-17 

position. Once again it should be noted that reference compounds 4.1 to 4.3 were 

analysed in dimethylsulphoxide and chloroform and at different frequencies for 1 H and 

13 C NMR analysis (Tables 23 and 22). 

125

HO 

HO 

Position 

2 

23 

4 

10 

24 

25 26 

9 

6 

8 

7 

12 

29 

18 

14 

27 

OH 

30 

20 

21 

22 

COOH 

16 

HO 

R 

12 

25 26 

9 

1 

10 8 

7 

4 

6 

24 23 

4.1 R 

4.2 β-OH 

4.3 α-OH 

29 

18 

14 

27 

OH 

17 

30 

20 

21 

22 

COOCH 3 

Table 23: Comparison of 13 C NMR data of compound IV (CD3OD) with two similar 

compounds 

Compound IV 

(600MHz) 

4.1 4.2 4.3 

b da Graca 

Rocha et al., 

2007 

(200MHz) 

a Fang et al., 

1991 

(75MHz) 

a Lontsi et al., 

1992 

(75MHz) 


1991 

(75MHz) 

28 


1998 

(75MHz) 

1 41.09 39.1 47.7 41.7 41.4 46.34 

2 65.9 67.1 68.4 66.5 77.2 68.47 

3 78.70 82.3 83.2 78.9 78.6 83.25 

4 38.07 41.4 39.1 38.2 39.9 39.14 

5 47.95 53.1 55.0 48.0 53.0 55.05 

6 17.93 18.1 18.3 18.1 17.9 18.28 

7 32.68 32.6 32.4 32.5 32.3 32.44 

8 39.86 42.4 39.7 40.1 41.0 39.72 

9 46.79 46.7 47.7 46.9 47.7 46.95 

10 37.99 37.6 37.9 38.3 37.3 37.94 

11 23.41 25.2 23.5 23.6 23.5 23.54 

12 127.95 126.7 128.5 129.0 128.8 128.59 

13 138.66 138.9 138.0 138.1 138.0 137.99 

14 41.35 40.7 41.0 41.2 41.0 40.99 

15 28.21 30.4 25.8 28.1 28.0 27.99 

16 25.25 25.1 25.2 25.4 25.3 25.23 

17 47-48 47.0 46.3 47.9 47.8 47.68 

18 53.66 54.8 53.0 53.2 51.5 53.02 

19 72.18 71.7 72.8 73.1 72.9 72.83 

20 41.73 41.4 41.0 41.1 46.7 40.99 

21 25.96 28.1 28.0 26.0 25.3 25.83 

22 37.65 39.1 37.2 37.4 37.3 37.24 


126

Position 

Compound IV 

(600MHz) 

4.1 4.2 4.3 

b 

da Graca 

Rocha et al., 

2007 

(200MHz) 


1991 

(75MHz) 


1992 

(75MHz) 


1991 

(75MHz) 

a Lontsi et al., 1998 

(75MHz) 

23 27.89 28.8 

28.5 

28.5 

28.5 

28.55 

24 21.09 17.1 16.7 21.8 21.8 16.72 

25 15.51 16.6 16.4 16.2 16.1 16.36 

26 16.15 23.3 16.7 16.6 17.9 16.42 

27 23.55 24.0 224.3 24.7 24.6 24.37 

28 180.88 179.0 178.2 178.2 178.4 178.26 

29 25.68 26.4 28.0 27.4 27.2 27.18 

30 15.25 16.5 15.9 16.1 16.5 15.97 

COOCH3 -- -- 51.4 51.6 66.2 51.46 

a CDCl3 

b DMSO 

Table 24: Comparison of 1 H NMR data of compound IV (600MHz, CD3OD) with three 

reference compounds 

Position Compound IV 

1 

2 

3 

1.28 

1.56 

3.93, dt 

(J = 3.72, 3.18 Hz) 

3.33, d 

(J = 2.28 Hz) 

4.1 4.2 4.3 

b # da Graca Rocha et al., 

2007 

(200MHz) 

4.10, m 

3.91, s 


(300MHz) 

1.21, m 

1.62, m 

3.97, ddd 

(J = 11.8, 4.4, 2.8 Hz) 

3.40, d 

(J = 2.8 Hz) 


(300MHz) 

0.92, m 

1.96, m 

3.36, ddd 

(J = 14.7, 9.5, 4.7 Hz) 

2.98, d 

(J = 9.5 Hz) 

5 1.26 0.81, m 

6 

1.39 

1.45 

1.32, m 

1.52, m 

7 

1.31 

1.58 

1.26, m 

1.50, m 

9 1.85 1.70, m 1.65, m 

11 2.00 1.96, m 1.98, m 

12 

15 

16 

5.29, t 

(J = 3.72 Hz) 

*5.19, s 

5.33, dd 

(J = 3.6, 3.5 Hz) 

5.33, dd 

(J = 3.6, 3.6 Hz) 

1.01 1.00, m 

1.79 1.60, m 

1.51 1.56, m 

2.57 

2.49, ddd 

(J = 13.7, 11.5, 4.7 Hz) 

2.49, ddd 

(J = 14.5, 12.6, 4.8 Hz) 

18 2.49 2.57, s 2.57, s 


127

Position Compound IV 

4.1 4.2 4.3 

b # da Graca Rocha et al., 

2007 

(200MHz) 


(300MHz) 


(300MHz) 

20 1.35 

1.38, m 

21 

1.23 

1.73 

1.25, m 

1.66, m 

22 

1.61 

1.71 

1.52, m 

1.72, m 

23 0.98 0.99, s 1.01, s 

24 0.86 0.84, s 0.80, s 

25 0.98 0.93, s 0.95, s 

26 0.78 0.64, s 0.65, s 

27 1.35 1.23, s 1.23, s 

29 1.19 1.18, s 1.19, s 

30 

0.92, d 

0.92, d 

0.92, d 

28- 

OCH3 

(J = 7.8 Hz) 

(J = 6.6 Hz) 

3.57, s 

(J = 6.7 Hz) 

3.58, s 

NB 1 H NMR data for compounds 4.2 and 4.3 were not documented by Fang et al. (1991) 

* Reported as resonance for H-5 by da Graca Rocha et al. (2007) but suspect that this resonance belongs to 

H-12 

# coupling constants not documented 

a CDCl3 

b DMSO 

3.4.4 Structure elucidation of Compounds V and VI 

HO 

3 

19 

5 

10 

6 

7 

21 

18 

14 

20 

22 

29 

16 

23 

28 

27 

25 

26 

HO 

3 

25 26 

24 23 

(V) (VI) 

Compound V is a white compound soluble in both dichloromethane and methanol having 

a molecular formula of C29H42O. It has a melting point of 168°C and an optical rotation 

10 

30 

20 

29 

27 

18 

19 

17 

22 

28 

128

of [α] 20 D -40.8° (c 0.12g/100ml, CDCl3). The IR spectrum showed O-H stretching bands 

at 3305 cm -1 , C-H stretching vibrations at 2932 cm -1 and alkene stretching bands at 1641 

cm -1 . 

The 1 H NMR spectrum showed the characteristic pattern of the ubiquitous stigmasterol 

with the three olefinic proton resonances at δH 4.99 (dd, J = 8.64, 15.2 Hz, H-22), δH 5.13 

(dd, J = 8.56, 15.2 Hz, H-23) and δH 5.32 (bd, J = 5.04 Hz, H-6) and the deshielded 

methine resonance at δΗ 3.49 (m, H-3), owing to the hydroxy group at this position. On 

closer examination of the methyl group region of the 1 H NMR spectrum and in 

comparison with the literature (Langlois, 2000), the methyl groups numbered as 18, 19, 

21, 26, 27 and 29 were assigned to δH 0.65, 0.67, 0.99 (d, J = 8.12 Hz), 0.89 (d, J = 7.18 

Hz), 0.80 (d, J = 6.24 Hz) and 0.82 (t, J = 6.28 Hz), respectively. 

The 13 C NMR spectrum indicated the presence of characteristic olefinic resonances at δC 

121.71 (C-6), δC 129.29 (C-23), δC 138.31 (C-22) and δC 140.76 (C-5). The deshielded 

oxygenated methine carbon resonance of C-3 occurred at δC 71.81. Compound V was 

positivley identified as stigmasterol by comparison with the 1 H and 13 C NMR spectra of 

an authentic sample in the laboratory. The NMR data also compared well with that in 

literature (Langlois, 2000; Kongduang et al., 2008) 

Compound VI is a white compound soluble in both dichloromethane and methanol 

having a molecular formula of C30H50O. It has a melting point of 214°C and an optical 

rotation of [α] 20 D +25.3° (c 1.2g/100ml, CDCl3). The IR spectrum showed O-H 

stretching bands at 3332 cm -1 , C-H stretching vibrations at 2941 cm -1 and alkene 

stretching bands at 1637 cm -1 . 

The 1 H NMR spectrum showed characteristic resonances for the well known pentacyclic 

triterpenoid, lupeol with the characteristic olefinic proton resonances at δH 4.57 (brs, H- 

29α) and δH 4.69 (brs, H-29β), one deshielded methine group resonance at δΗ 3.19 (dd, J = 

5.04, 11.17 Hz, H-3), the H-19 triplet of doublets at δΗ 2.38 (J = 5.88, 11.03 Hz), the 

129

multiplet at δH 1.91 (H-21) and a very shielded doublet at δH 0.68 (J = 9.12 Hz, H-5) 

(Makatini, 2007). The methyl groups identified as 3H-23 (δH 0.97), 3H-24 (δH 0.76), 3H- 

25 (δH 0.83), 3H-26 (δH 1.03), 3H-27 (δH 0.94), 3H-28 (δH 0.79) and a methyl group 

bonded to an olefinic carbon at δH 1.68 (3H-30). 

The 13 C NMR spectrum indicated the presence of thirty carbons with characteristic 

olefinic resonances at δC 150.98 (C-20) and δC 109.33 (C-29). The C-3 and C-5 carbon 

resonance could also be singled out at δC 79.01 and δC 55.30, respectively, Compound VI 

was identical to an authentic sample of lupeol with regard to physical appearance, 

melting point, optical rotation and IR spectroscopy. A comparison of the NMR data of 

compound VI with that documented by Burns et al. (2000) for lupeol, also proved that 

the two compounds were identical. 

Table 25: 1 H and 13 C NMR data of stigmasterol (V) compared with reference data 


Position 

1 H 

Compound V Langlois, 2000 Compound V 

13 C 

Kongduang et al., 2008 

(125MHz) 

1 37.26 37.21 

2 31.66 231.62 

3 3.49, m 3.50, m 71.81 71.80 

4 42.31 42.18 

5 140.76 140.72 

6 5.32, bd 5.35, brs 121.71 121.71 

7 31.87 31.87 

8 31.91 31.87 

9 50.15 50.08 

10 36.51 36.48 

11 21.08 21.07 

12 39.68 39.64 

13 42.32 42.29 

14 56.87 56.83 

15 24.36 24.34 

16 28.91 28.92 

17 55.96 55.90 

18 0.65, s 0.66, s 12.04 12.03 

0.99, d 

21 

(J = 8.12 Hz) 


1.00, d 

(J = 6.65 Hz) 

21.21 21.05 

130

1 H 

Position 

Compound V Langlois, 2000 Compound V Kongduang et al., 2008 

19 0.67, s 0.70, s 19.39 19.38 

20 40.48 40.50 

22 

23 

4.99, dd 

(J = 8.56, 15.12 Hz) 

5.13, dd 

(J = 8.64, 15.16 Hz) 

5.00, dd 

(J = 7.60, 15.38 Hz) 

5.15, dd 

(J = 11.53, 15.38 Hz) 

13 C 

138.31 138.32 

129.29 129.22 

24 2.25, m 2.21, m 51.24 51.21 

25 32.42 31.87 

26 

27 

0.89, d 

(J = 7.18 Hz) 

0.80, d 

(J = 6.24 Hz) 

0.90, d 

(J = 6.53 Hz) 

0.78, d 

(J = 6.35 Hz) 

20.20 21.20 

18.98 18.95 

28 25.40 25.40 

29 

0.82, t 

(J = 6.28 Hz) 

0.80, t 

(J = 6.5 Hz) 

12.24 12.25 

NB. Langlois (2000) did not report the 13 C NMR data for stigmasterol 

Kongduang et al. (2008) did not report the 1 H NMR data for stigmasterol 

Table 26: 1 H and 13 C NMR data of lupeol (VI) compared with reference data (CDCl3, 

400MHz) 

1 H 

Position 

Compound VI 

Burns et al., 2000 

(500MHz) 

Compound VI 


(500MHz) 

1 

0.90 

1.67 

38.71 38.72 

2 

1.60 

1.56 

27.42 27.42 

3 

3.19, dd 

(J = 5.04, 11.17 Hz) 

3.19 79.01 79.02 

-OH 1.26 

4 38.86 38.87 

5 

0.68, d 

(J = 9.12 Hz) 

55.30 55.31 

6 

1.51 

1.39 

18.32 18.33 

7 1.39 34.29 34.29 

8 40.84 40.84 

9 1.27 50.44 50.35 

10 37.17 37.18 

11 

1.41 

1.23 

20.93 20.94 

12 

1.07 

1.67 

25.15 25.16 

13 1.66 38.06 38.07 


13 C 

131

1 H 

Position 

Compound VI 


(500MHz) 

Compound VI 


(500MHz) 

14 42.84 42.85 

15 

1.00 

1.68 

27.45 27.46 

16 

1.37 

1.47 

35.59 35.60 

17 43.00 43.01 

18 1.36 47.99 48.32 

19 

2.38, dt 

(J = 5.88, 11.03 Hz) 

2.38 48.31 48.00 

20 150.98 150.98 

21 

1.91, m 

1.32 

1.92 

29.85 29.86 

22 

1.19 

1.38 

40.01 40.02 

23 0.97, s 0.97 27.99 28.00 

24 0.76, s 0.76 15.38 15.38 

25 0.83, s 0.83 16.12 16.13 

26 1.03, s 1.03 15.98 15.99 

27 0.94, s 0.95 14.55 14.56 

28 0.79, s 0.79 18.01 18.02 

29 

4.57, brs 

4.69, brs 

4.56 

4.69 

109.33 109.32 

30 1.68, s 1.68 19.31 19.32 

13 C 

132

Physical data for compounds I-VI 

Compound I 

Chemical name: 7β-acetoxy-6β,12-dihydroxy-8,12-abietadiene-11,14-dione 

Common name: 7β-acetoxy-6β-hydroxyroyleanone 

Molecular formula: C22H30O6 

Physical description: yellow crystals 

Melting point: 197 o C (228 o C, Mehrotra et al., 1989) 

Optical rotation:[ ] 20 

α D +29 o (c 0.0052g/100ml, DCM) (lit. value = +23 o (CHCl3; Cl), 

Mehrotra et al., 1989) 

Infrared spectrum ν KBr 

max cm-1 : 3377 (O-H stretching bands), 1734 (carbonyl stretching 

bands), 1375 (C-H stretching vibrations), 1641 (alkene stretching bands) 

Ultraviolet spectrum (λ max): 275 nm (log ε = 1.61) 

GC-MS (70eV, direct inlet), relative intensity %: m/z 390 (1.46) [M] + , m/z 372 (4.85) [M 

– H2O] + , m/z 357 (0.97) [M - H2O - Me] + , m/z 71 (100) 

1 H NMR and 13 C NMR data (CDCl3, 400MHz): Refer to tables 15 and 16 on pages 109 

and 110 

Compound II: 

Chemical name: 6β,7β,12-trihydroxy-8,12-abietadiene-11,14-dione 

Common name: 6β,7β-dihydroxyroyleanone 


Physical description: yellow residue 

Melting point: 125 o C (203-205 o C, Hensch et al., 1975) 

Optical rotation: [ ] 20 

α D -47 o (c 0.0063g/100ml, DCM) (lit. value = -77 o , Hensch et al., 

1975) 


vibrations), 1638 (alkene stretching bands) 

max cm-1 : 3369 (O-H stretching bands), 1376 (C-H bending 

133

Ultraviolet spectrum (λ max): 192nm (log ε = 2.73) 

LC-MS (70eV, direct inlet): [M] + at 347 (negative ion mode) 

1 H and 13 C NMR data (CDCl3, 400MHz): Refer to tables 17 and 18 on pages 111 and 112 

Compound III: 

Chemical name: ent-pimara-8(14),15-dien-3α,11β-diol 

Physical description: white residue 


Melting point: 89 o C (125-130 o C, Ansell, 1989) 


α D -79° (c 0.0082g/100ml, DCM) 


vibrations) 

max cm-1 : 3396 (O-H stretching bands), 2930 (C-H stretching 


GC-MS (70eV, direct inlet), relative intensity %: m/z 304 (1.94) [M] + , m/z 135 (100), m/z 

286 (10.68) [M – H2O] + , m/z 268 (5.34) [M – 2Me] + 

1 H NMR and 13 C NMR data (CDCl3, 400MHz): Refer to table 19 on page 116 

Compound IV: 

Chemical name: 2α,3α,19α-trihydroxyurs-12-en-28-oic acid 

Common name: euscaphic acid, jacarandic acid 



Melting point: 255 o C 


α D +32.26° (c 0.0186g/100ml, MeOH) 


1687 (C=O) 

max cm-1 : 3424 (O-H stretching bands), 2927 (C-H stretching bands), 


134

GC-MS (70eV, direct inlet), relative intensity %: m/z 426 (8.56) [M - COOH - OH] + , m/z 

218 (100) 

1 H NMR and 13 C NMR data (CD3OD, 600MHz): Refer to table 22 on page 124 

Compound V: 

Chemical name: 5,22-Stigmastadien-3β-ol 

Common name: Stigmasterol 

Molecular formula: C29H42O 


Melting point: 168 o C (165 o C, Jamal et al., 2009) 


α D -40.8° (c 0.12g/100ml, CDCl3) 


max cm-1 : 3305 cm -1 (O-H stretching bands), 2932 cm -1 (C-H 

stretching vibrations), 1641 cm -1 (alkene stretching bands) 

1 H NMR and 13 C NMR data: Refer to table 25 on page 130 

Compound VI: 

Chemical name: lup-20(29)-en-3β-ol 

Common name: Lupeol 

Molecular formula: C30H50O 

Physical description: white residue/powder 

Melting point: 214 o C (213 o C, Fotie et al., 2006; 214-215 o C, Saeed et al., 2003) 


α D +25.3° (c 1.2g/100ml, DCM) (lit. value = +25.7° (c 0.70, CHCl3), 

Fotie et al., 2006) 


max cm-1 : 3332 cm -1 (O-H stretching bands), 2941 cm -1 (C-H 

stretching vibrations), 1637 cm -1 (alkene stretching bands) 

1 H NMR and 13 C NMR data: Refer to table 26 on pages 131 

135

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Wellsow, J., Grayer, R.J., Veitch, N.C., Kokubun, T., Lelli, R., Kite, G.C. and 

Simmonds, M.S.J. (2006) Insect-antifeedant and antibacterial activity of diterpenoids 

from species of Plectranthus, Phytochemistry, 67, 1818-1825 

140

Chapter 4 Antibacterial and anticancer activity of the 

compounds isolated 

The compounds isolated were tested for their antibacterial and anticancer activity in 

collaboration with the CSIR as these biological screens were available at the time. The 

bacterial strains used were the Gram-negative Pseudomonas aeruginosa American Type 

Culture Collection 25922 (ATCC25922) and the Gram-positive Enterococcus faecalis 

(ATCC29212) whilst breast (Michigan Cancer Foundation – Seventh sample (MCF-7)), 

renal (TK-10) and melanoma (UACC-62) cell lines were assayed for anticancer purposes. 

There is no record of antibacterial, antifungal or anticancer testing being done on 

compounds I to IV. However, compounds 33 (6β,7α-dihydroxyroyleanone) and 36 (7α- 

acetoxy-6β-hydroxyroyleanone) which are 7α-isomers of compounds I and II 

respectively, have proven to posess antibacterial activity against six strains of methicillin 

resistant Staphylococcus aureus (MRSA), two strains of vancomycin-resistant 

Enterococcus faecalis (VRE), Bacillus subtilis, Staphylococcus aureus, Vibrio cholera 

and Xanthomonas campestris (Laing et al., 2006; Teixera et al., 1997; Gaspar-Marques et 

al., 2006), antifungal activity against Rhizoctonia solani, Sclerotinia sclerotiorum, 

Pythium ultimum and an unknown Candida species (Laing et al., 2006; Teixera et al., 

1997; Gaspar-Marques et al., 2006), anticancer activity against human lymphocytes 

induced by phytohaemagglutinin (PHA) and expression of CD69 by T- and B- mouse 

lymphocytes, antitumor activity against human cancer cell ines MCF-7, NCI-H460, SF- 

268, TK-10 and UACC-62 (Cerqueira et al., 2004; Gaspar-Marques et al., 2002) and 

antimalarial activity against Plasmodium falciparum (van Zyl et al., 2008) (Tables 12 and 

14). 

Compound V has been isolated twice before from the Plectranthus species (Simoes, 

2010; Yao, 2002) but this is the first time that it has been isolated from P. hadiensis. 

This is the first report of lupeol (VI) being isolated from the Plectranthus species. 

141

Since the 7α-isomers of compounds I and II (36 and 33, respectively) exhibited the 

above mentioned activity, it was decided to determine the antibacterial activity and 

anticancer activity of these two compounds. The bacterial strains tested were that of E. 

faecalis and P. aeruginosa with the hope of these two compounds exhibiting greater 

activity than that reported in literature where 33 and 36 (having MIC values of 

62.50µg/mL and 31.25µg/mL and 15.63µg/mL and 31.25µg/mL against two VRE strains, 

respectively) displayed stronger activity against VRE than vancomycin (having a 

minimum MIC value of 125µg/mL) and where 36 displayed more potent activity against 

MRSA (with MIC values averaging 7.8µg/mL) than oxacillin (having a MIC value of 

15.63µg/mL) (Gaspar-Marques et al., 2006). The 7-acetoxy function also proved 

superior in activity to the 7-hydroxy function when tested for anticancer activity against 

MCF-7, NCI-H460, SF-268, TK-10 and UACC-62 cell lines (Gaspar-Marques et al., 

2002). The purpose of the anticancer testing performed in this study was to see how the 

isomers of 36 and 33, I and II respectively, faired in the anticancer assays. 

Even though, compound III has been isolated by Ansell (1989) there hasn’t been any 

report of this compound showing biological activity. The only report of an ent-pimarene 

displaying antibacterial activity was by Porto et al. (2009) where ent-8(14),15- 

pimaradien-3β-ol was found to be active against E. faecalis having an an MIC value of 

20µg/mL. It is still unknown as to whether ent-pimarene compounds possess anticancer 

activity as they have not been tested against any cell lines to date. 

There were three reports of anticancer testing of compound IV, however none of these 

studies tested for activity against the TK-10 (renal) and UACC-62 (melanoma) cell lines. 

Cheng et al. (2010) tested compound IV for activity against four human cancer cell lines 

(MCF-7, tested in this work, NCI-H460, HT-29 and CEM) (Cheng et al., 2010). The 

results indicated that this compound was inactive as an anticancer agent based on cancer 

cell proliferation (Cheng et al., 2010). Studies by Ogura et al. (1977) and Mujovo (2010) 

proved that compound IV does possess anticancer activity by testing positive for activity 

against P-388 lymphocytic leukaemia cell lines (Ogura et al., 1977) and exhibiting strong 

142

2 

19 

1 

4 

18 

cytotoxic activity against monkey kidney Vero cell lines, having IC50 values ranging 

between 4.52µg/mL and 19.21µg/mL (Mujovo, 2010). 

4.1 Antibacterial testing 

Compounds I to IV (7β-acetoxy-6β-hydroxyroyleanone (I), 7β,6β-dihydroxyroyleanone 

(II), ent-pimara-8(14),15-diene-3β,11α-diol (III) and 2α,3α,19α-trihydroxyurs-12-en-28- 

oic acid (IV)) were sent to the Council for Science and Industrial Research (CSIR) in 

Pretoria, South Africa to be assessed for their antibacterial activity. The bacterial strains 

used were the Gram-negative Pseudomonas aeruginosa (ATCC25922) and the Gram- 

positive Enterococcus faecalis (ATCC29212). 

O 

20 

9 

10 

5 

11 

6 7 

OH 

OH 

12 

14 

8 

R 

R 

13 

16 

(I) OC(O)CH3 

(II) OH 

15 

O 

17 

4.1.1 Experimental 

HO 

2 

3 

1 

HO 

20 11 

9 

4 5 6 7 

10 8 

18 19 

12 

The samples were dissolved in acetone to a known concentration (1.0 mg/mL) prior to 

being tested, with the exception of compound IV which was prepared to a concentration 

of 0.8 mg/mL due to there being insufficient sample. The antibacterial assays followed 

the format of the serial microdilution assay (Eloff, 1998). In brief, two-fold serial 

dilutions of the samples were carried out and made up to 100µL in 96-well microtitre 

plates. Bacteria (100µL of an overnight culture) was then added to each well before 

143 

13 

17 

14 

15 

16 

HO 

HO 

1 

4 

25 26 

9 

5 

10 

6 7 

8 

23 24 

(III) (IV) 

2 

12 

29 

27 

18 

14 

OH 

15 

17 

30 

21 

22 

COOH

eing incubated for 24 hours at 37°C. A volume of 40µL of 0.2mg/mL 

Iodonitrotetrazolium chloride (INT, Sigma) was added to each well as indicator of 

bacterial growth. INT, a colourless tetrazolium salt, is converted to a red-coloured 

formazan product by actively dividing cells. The minimum inhibitory concentration 

(MIC) was visually read as the lowest concentration of sample that inhibited microbial 

growth, as indicated by a visible reduction in the red colour of the INT formazan. In each 

assay, a negative solvent control and a positive control of the antibiotic was used. 

Gentamicin (Sigma) was used as the antibacterial control and the samples were tested in 

triplicate. 

4.1.2 Results and discussion 

The results of the Minimum Inhibitory Concentration (MIC) determinations of the four 

compounds against Enterococcus faecalis and Pseudomonas aeruginosa are given in 

Table 27. MIC values for the samples and the reference antibiotic, gentamicin are 

reported in µg/mL. 

Table 27: Antibacterial activity of compounds I to IV 

Compound 

Average MIC (µg/mL) 

24 hours 

E. faecalis P. aeruginosa 

I 62.50 62.50 

II 31.30 7.80 

III 125.00 15.60 

IV 200.00 200.00 

Gentamicin 0.39 0.78 

The antibacterial activity of compounds I to IV were evaluated relative to gentamicin. 

The lower the MIC value, the better the antibacterial activity of the compound. Since the 

MIC value for gentamicin is below 1µg/mL for both the bacterial strains tested, 

compounds with MIC values below 10µg/mL were noted to be potent antibacterial agents 

while compounds having MIC values between 10µg/mL and 100µg/mL were considered 

to be good antibacterial agents. Compounds exhibiting MIC values between 100µg/mL 

144

and 125µg/mL were categorised as being moderately active. MIC values above 

125µg/mL suggested poor activity. 

Of the four compounds tested for activity against E. faecalis and P. aeruginosa, 

compound IV was the only compound which showed poor activity against both 

microorganisms, having MIC values of 200µg/mL. This compound however was 

structurally different to the other three, being a tetracyclic triterpenoid whilst the other 

three compounds were abietane diterpenoids. In the assay against E. faecalis, compound 

III exhibited moderate activity having a MIC value of 125µg/mL. Compounds II and III 

showed good antibacterial activity with royleanone II, having a hydroxy group at 

position 7, having the highest activity at 31.30µg/mL. 

With regard to P. aeruginosa the highest activity (regarded as potent) was shown by 

compound II, the royleanone with the hydroxy group at position 7. In comparison to it’s 

acetylated counterpart, compound I, it was eight times more active. The pimarane, ent- 

pimara-8(14),15-diene-3β,11α-diol (III), was also very active with a MIC value of 

15.60µg/mL. 

In general, the abietanes showed good to potent activity against both strains being more 

active against P. aeruginosa than E. faecalis, with the highest activity being shown by 

compound II (7β,6β-dihydroxyroyleanone). The acetyl group at position 7 of compound 

I seemed to lower the activity in both bacterial strains in relation to the unacetylated 

compound II. 

In comparison to the antibiotic gentamicin, compounds I-III are less potent, however 

they may have less side effects and be less toxic than standard antimicrobials used in the 

drug industry and are worth being investigated for development into a new range of 

antibiotics. 

Compounds I (7β-acetoxy-6β-hydroxyroyleanone) and II (7β,6β-dihydroxyroyleanone) 

exhibited MIC values of 62.50 µg/mL and 31.30 µg/mL against E. faecalis, respectively. 

145

This indicated that the dihydroxy compound had better activity than its monoacetylated 

counterpart. This is different from that in the literature where the dihydroxy isomer (33) 

showed worse activity with MIC values of 62.50 and 31.25µg/mL than the isomer of the 

acetylated compound (36) with MIC values of 15.63 and 31.25µg/mL against two strains 

of vancomycin-resistant E. faecalis. Even though our compounds displayed weaker 

activity than the antibiotic, gentamicin, they do exhibit stronger activity than that reported 

for vancomycin in Gaspar-Marques et al. (2006). 

4.2 Anti-cancer screening 

An in-house test method which was developed by the CSIR was employed for the 

evaluation of the anticancer activity of compounds I to IV. This test method was 

implemented by the CSIR in 1999 and is known as the three cell prescreening method 

(Fouche et al., 2006; 2008). Breast (MCF-7), renal (TK-10) and melanoma (UACC-62) 

cell lines were three human cell lines chosen for anticancer testing due to their high 

sensitivity to detect anticancer activity (Fouche et al., 2008). Compounds need to 

demonstrate growth inhibitory activity in this pre-screen panel before proceeding to 

advanced testing in the full 60-cell-line screen. 

Melanoma refers to cancer of the skin and occurs when a UV photon strikes a 

chromophore in a skin cell. A chromophore is the part of a molecule which gives it 

colour. When the chomophore is struck by the UV photon, a singlet oxygen ( 1 O2) or 

hydroxyl (•OH) free radical is produced, which then travels about the body until it finds a 

home in a melanocyte, where DNA is mutated by means of oxidation. The damaged 

melanocyte then becomes a malignant tumour, which is apparent by a change in skin 

colour, usually to a darker shade. Genetic predisposition, excessive, unprotected 

exposure to the sun as well as the use of tanning or sun beds are some of the factors 

which lead to the formation of melanomas. 

Breast cancer is a form of cancer which originates from breast tissue, commonly found in 

the inner lining of milk ducts or lobules, the ducts responsible for the supply of milk. 

146

Breast cancer can develop in both males and females and is detectable by means of self- 

examination of the breast area. The presence of lumps or masses in this area is usually an 

indication of cancerous matter, however a mammogram which is performed at most 

medical centres, must be done to confirm the presence of such matter. Prognosis and 

survival rate varies greatly depending on cancer type and staging. Treatment includes 

surgery, drugs (hormonal therapy and chemotherapy) and radiation. 

Renal cancer otherwise known as renal cell carcinoma refers to cancer of the kidney. 

Since the kidney is responsible for the filtration of blood and removal of waste products 

from the body, renal cell carcinoma affects the lining of the small tubes within the kidney 

which in turn affects the filtering system. Unfortunately renal cancer is not detected as 

easily as breast cancer is. Chronic fatigue, hypertension, fever, presence of blood in the 

urine, pain in the side or lower back as well as a mass or lump in the abdomen are a few 

of the signs and symptoms associated with this cancer. Chemotherapy, radiation therapy 

and surgery are some of the treatment options available to patients diagnosed with this 

particular cancer. 

4.2.1 Experimental 

In the three cell prescreening method, the three cell lines were grown in Roswell Park 

Memorial Institute 1640 (RPMI 1640) medium containing 5% fetal bovine serum and 

2µM L-glutamine. The cells were then inoculated into 96-well microtiter plates with 

densities ranging between 5,000 and 40,000 cells per well. A volume of 100µL of the 

medium was introduced into the microtiter plates and subsequently incubated at 37 o C in a 

5:95 (carbon dioxide:air) atmosphere with 100% relative humidity for 24 hours. 

Compounds I-IV were dissolved in dimethyl suphoxide (DMSO) and then added to the 

cells at concentrations ranging between 0.001µg/ml and 100µg/ml. These cells were then 

incubated for a further 48 hours at 37 o C in a humidified atmosphere, followed by the 

fixing of the cells in situ with trichloroacetic acid (TCA) and staining with 100µL 

sulforhodamine B (SRB) solution. Unbound dye was removed by washing with 1% 

147

acetic acid and air drying the plates. Bound stain was solubilized with 10µM trizma base 

and the optical density was read on an automated plate reader at a wavelength of 540nm. 

The percentage growth of human tumor cells was determined spectrophotometrically by 

measuring the difference in optical density of the control (C) at the start (T0) and end of 

drug exposure (T). If T≥T0 either no effect is experienced or inhibition occurs. Inhibition 

occurs if T

surprising since this compound, a pentacyclic triterepenoid, is structurally different to the 

other three abietane diterpenoids. 

The criteria followed by the CSIR states that compounds with TGI > 50µg/mL are 

regarded as being inactive, TGI ranging between 15µg/mL and 50µg/mL are weakly 

active whilst TGI values between 6.25µg/mL and 15µg/mL are indicative of compounds 

which are moderately active. Any compound having a TGI value of less that 6.25µg/mL 

for at least two of the three cell lines, is considered to be potent (Fouche et al., 2008). 

According to these criteria, compounds I to III are weakly active towards the melanoma 

cell line (44.71 µg/mL (I), 42.18µg/mL (II) and 44.64µg/mL (III)) while the TGI values 

obtained for the breast (51.20µg/mL, 52.72µg/mL and 81.22µg/mL, respectively) and 

renal (49.10µg/mL, 55.84µg/mL and 53.99µg/mL, respectively) cell lines were only 

slightly above 50µg/mL suggesting that these compounds could also be regarded as 

weakly active anticancer agents against these cell lines (Table 28). Since I to III are 

regarded as weakly active, slight chemical modifications to the structure may enhance it’s 

anticancer activity. The chemical modifications could not be carried out in this work 

since the samples isolated were insufficient for chemical derivatisation. 

The standard used in the three cell pre-screening method was Etoposide having TGI 

values of >100µg/mL, 36.20µg/mL and 27.00µg/mL for breast (MCF-7), melanoma 

(UACC-62) and renal (TK-10) human cell lines, respectively. In comparison to the 

standard, compounds I to III were proven to be less active with regard to the renal and 

melanoma cell lines. Compounds II and III displayed TGI values of 55.84µg/mL and 

53.99ug/mL respectively for the renal cell line. These values are twice that of etoposide 

having a TGI value of 27.00µg/mL against the same cell line. Compounds I to III were 

found to be more active against the breast cell line than the control with compounds I and 

II having TGI values measuring half at most of that of etoposide with values of 

51.20µg/mL and 52.72µg/mL, respectively. 

Compounds I to III inhibited the growth of the melanoma cell line (UACC-62) more than 

the other two cell lines having GI50 values ranging between 12.05µg/mL and 13.53µg/mL 

149

whereas GI50 values for the renal and breast cell lines ranged between 21.02µg/mL and 

26.50µg/mL, and 20.57µg/mL and 39.08µg/mL, repectively. These GI50 values indicate 

weak activity as considered by the NCI’s criteria (Fouche et al., 2008). 

Net Growth (%) 

150 

100 

50 

0 

-50 

-100 

-150 

Compound 1 

0.01 0.10 1.00 10.00 100.00 

Concentration (µg/ml) 

Cell line 1 (TK-10) Cell line 2 (UACC) Cell line 3 (MCF) 

Figure 20: Dose response curve for 7β-acetoxy-6β-hydroxyroyleanone (I) against TK-10, 



150 

100 

50 

0 

-50 

-100 

-150 

Compound 2 

0.01 0.10 1.00 10.00 100.00 


Cell Line 1 (TK-10) Cell Line 2 (UACC) Cell Line 3 (MCF) 

Figure 21: Dose response curve for 7β,6β-dihydroxyroyleanone (II) against TK-10, UACC-62 and 

MCF-7 cell lines 

150


150 

100 

50 

0 

-50 

-100 

-150 

Compound 3 

0.01 0.10 1.00 10.00 100.00 


Cell line 1 (TK-10) Cell line 2 (UACC) Cell line 3 (MCF) 

Figure 22: Dose response curve for ent-pimara-8(14),15-diene-3β,11α-diol (III) against TK- 

10, UACC-62 and MCF-7 cell lines 

Compound IV, the only triterpenoid tested was only capable of inhibiting the growth 

(GI50) of the melanoma and breast cancer cell lines at concentrations which classified this 

compound as inactive and showed no potential killing activity (LC50 or LC100) even at 

100µg/mL. 


150 

100 

50 

0 

-50 

-100 

-150 

Compound 4 

0.01 0.10 1.00 10.00 100.00 


Cell Line 1 (TK-10) Cell Line 2 (UACC) Cell Line 3 (MCF) 

Figure 23: Dose response curve for 2α,3α,19α-trihydroxyurs-12-en-28-oic acid (IV) against TK-10, 


151

Table 28: Growth inhibition values for compounds I-IV against TK-10, UACC-62 

and MCF-7 cell lines 

Compound 

I 

II 

III 

IV 

Doseresponse 

parameters 

Line 1 

(TK-10) 

renal 

Line 2 

(UACC-62) 

melanoma 

Line 3 

(MCF-7) 

breast 

GI50 21.02 13.42 20.57 

TGI 49.10 44.71 51.20 

LC50 77.18 76.00 81.84 

LC100 N/A N/A N/A 

GI50 24.06 12.05 23.63 

TGI 55.84 42.18 52.72 

LC50 87.63 72.32 81.82 


GI50 26.50 13.53 39.08 

TGI 53.99 44.64 81.22 

LC50 81.48 75.74 N/A 


GI50 N/A 48.67 92.57 

TGI N/A N/A N/A 



Etoposide TGI 27.00 36.20 >100 

NB. N/A denotes inactivity 

The GI50 values for compounds I and II are compared to that of their isomers (Gaspar- 

Marques et al., 2002) in Table 29. In our results, the GI50 values of both I and II are 

similar. However, this is not the case with the GI50 values for the isomers, compounds 33 

and 36 reported in the literature. Compound 36 displayed significantly better activity 

than any of the other three compounds against the three cell lines used. Our two 

compounds I and II however showed better activity than the dihydroxy isomer (33). 

Table 29: GI50 values (µM) of compounds I and II and their 7α-isomers 

Compound 

MCF-7 

Cell line 

TK-10 UACC-62 

I 34.41 53.90 52.74 

36 6.4 7.4 4.5 

II 34.63 69.14 67.90 

33 48.3 107.6 77.9 

Doxorubicin 0.055 0.570 0.094 

152

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155

Chapter 5 Conclusion 

The aim of this research project was to isolate and identify the compounds extracted from 

Plectranthus hadiensis and to compare the phytochemistry with previous studies as well 

as to test the compounds isolated for antibacterial and anticancer proprties. 

According to literature (Abdel-Mogib et al., 2002), abietane diterpenes are the most 

abundantly occurring compounds in the genus Plectranthus with there being two 

instances where ten triterpenes (oleanolic acid, ursolic acid, betulin, β-sitosterol, 

plectranthoic acid, plectranthoic acid A, plectranthoic acid B, acetylplectranthoic acid, 

plectranthadiol and hexacosanol) have been isolated (Misra et al., 1971; Razdan et al., 

1982). 

Compounds I and II have been isolated only once before (Mehrotra et al., 1989) from 

Coleus zeylanicus which is a synonym for Plectranthus hadiensis. This is the first report 

of compounds III to VI being isolated from P. hadiensis. Lupeol has never been isolated 

in the Plectranthus genus, however an isopimarane (7α,18-dihydroxy-isopimara- 

8(14),15-diene) was reported in Plectranthus diversus (Rasikari, 2007) and stigmasterol 

has been isolated twice before, once from Coleus and once from Plectranthus. 

Three of the six compounds isolated from P. hadiensis, (7β-acetoxy-6β- 

hydroxyroyleanone (I), 7β,6β-dihydroxyroyleanone (II) and ent-pimara-8(14),15-diene- 

3β,11α-diol (III)) were abietanes while the other three were common pentacyclic 

triterpenes known as euscaphic acid (IV), stigmasterol (V) and lupeol (VI). These three 

triterpenoids add to the ten previously isolated from the Plectranthus species. Even 

though ethyl acetate and methanol were used as extraction solvents, nothing of interest 

was isolated from these solvents. The relatively non-polar hexane extract yielded five of 

the six compounds with euscaphic acid IV being isolated from the dichloromethane 

extract. The isolation of triterpenoids in P. hadiensis is not surprising as most plant 

species contain these common sterols. 

156

The isolation of a pimarane in Plectranthus was an interesting find as pimarane-type 

diterpenes are abundant in the Lamiaceae family (to which Plectranthus belongs) being 

found predominantly in the Salvia and Nepeta genera and to a lesser extent in the 

Rabdosia, Callicarpa, Premna, Amaracus, Orthosipho and Satureja genera (Alvarenga et 

al., 2001). The finding of a pimarane from Plectranthus may now provide a biochemical 

link between Plectranthus and these genera. Pimarane-type diterpenes have also been 

isolated from the Erythroxylum genus (Ansell, 1989) which suggests a link between the 

Lamiaceae family to which Plectranthus is a member of and the Erythroxylaceae to 

which Erythroxylum belongs. 

Compounds I and II showed good activity against Enterococcus faecalis and 

Pseudomonas aeruginosa with MICs for compound I being 62.5µg/mL against both 

bacterial strains and that of compound II being 31.30µg/mL and 7.8µg/mL for E. faecalis 

and P. aeruginosa, respectively. This result is unexpected as royleanones having a highly 

oxidised substituent at C-7 (such as the acetyl group in compound I) are usually more 

active than those bearing a hydroxyl group at positions C-6 and C-7 (Teixeira et al., 

1997; Gaspar-Marques et al., 2006). The pimarane, ent-pimara-8(14),15-diene-3β,11α- 

diol (III), although inactive against E. faecalis was very active against P. aeruginosa. 

Based on criteria set by the CSIR where the total growth inhibition (TGI) value is used to 

evaluate the anticancer activity of compounds, compounds I to III are considered to be 

weakly active against breast (MCF-7), renal (TK-10) and melanoma (UACC-62) cell 

lines. However when compared to the positive control, etoposide, compounds I to III 

exhibit better antitumor activity against the breast cell line than the control. Compound 

IV was inactive against all three cell lines. 

Because compounds I to IV showed weak activity in the three cell prescreen against the 

breast, renal and melanoma cell lines, there was no need to to pursue further anticancer 

testing i.e. the 60-cell-line screen. 

157

References 

Abdel-Mogib, M., Albar, H.A. and Batterjee, S.M. (2002) Chemistry of the genus 

Plectranthus, Molecules, 7, 271-301 

Alvarenga, S.A.V., Gastmans, J.P., do Vale Rodrigues, G., Moreno, P.R.H. and de Paulo 

Emerenciano, V. (2001) A computer-assisted approach for chemotaxonomic studies – 

diterpenes in Lamiaceae, Phytochemistry, 56, 583-595 


Natal, South Africa, PhD Thesis, pp. 37-43, 247-248, 250-251 




Mehrota, R., Vishwakarma, R. A. and Thakur, R. S. (1989) Abietane diterpenoids from 


Misra, P.S., Misra, G., Nigam, S.K., Mitra, C.R. (1971) Constituents of Plectranthus 

rugosus, Llyodia, 34, 265-266 




Razdan T.K., Kachroo, V., Harkar, S., Koul, G.L. and Dhar, K.L. (1982) Plectranthoic 

acid, acetyl plectranthoic acid and plectranthadiol – Three new triterpenoids from 

Plectranthus rugosus, Phytochemistry, 21, 409-412 




158

Appendix 

Spectra 

159

Table of Contents 

Page 

Compound I, 7β-acetoxy-6β-hydroxyroyleanone 161 

Compound II, 7β,6β-dihydroxyroyleanone 175 

Compound III, ent-pimara-8(14),15-diene-3β,11α-diol 189 

Compound IV, euscaphic acid 202 

Compound V, stigmasterol 217 

Compound VI, lupeol 223 

160

Compound I, 7β-acetoxy-6β-acetoxyroyleanone 

2 

19 

1 

4 

O 

20 

18 

9 

10 

5 

11 

6 7 

OH 

OH 

12 

14 

8 

13 

16 

15 

O 

OCCH 3 

Spectrum 1a: 1 H NMR spectrum 162 

Spectrum 1b: 13 C NMR spectrum 163 

Spectrum 1c: Expanded 13 C NMR spectrum 164 

Spectrum 1d: Expanded DEPT spectrum 165 

Spectrum1e: HSQC spectrum 166 

Spectrum 1f: Expanded HSQC spectrum 167 

Spectrum 1g: HMBC spectrum 168 

Spectrum 1h: Expanded HMBC spectrum 169 

Spectrum 1i: COSY spectrum 170 

Spectrum 1j: NOESY spectrum 171 

Spectrum 1k: IR spectrum 172 

Spectrum 1l: UV spectrum 173 

Spectrum 1m: GC-MS spectrum 174 

O 

17 

Page 

161

Compound II, 6β,7β-dihydroxyroyleanone 

2 

19 

1 

4 

O 

20 

18 

9 

10 

5 

11 

6 7 

OH 




Spectrum 2d: Expanded DEPT spectrum 179 

Spectrum 2e: HSQC spectrum 180 

Spectrum 2f: Expanded HSQC spectrum 181 

Spectrum 2g: HMBC spectrum 182 

Spectrum 2h: Expanded HMBC spectrum 183 

Spectrum 2i: COSY spectrum 184 

Spectrum 2j: NOESY spectrum 185 

Spectrum 2k: IR spectrum 186 

Spectrum 2l: UV spectrum 187 

Spectrum 2m: LC-MS spectrum 188 

OH 

12 

14 

8 

13 

OH 

16 

15 

O 

17 

Page 

175

Compound III, ent-pimara-8(14),15-diene-3β,11α-diol 

HO 

2 

3 

1 

HO 

20 11 

9 

4 5 6 7 

10 8 

18 19 



Spectrum 3c: Expanded DEPT spectrum 192 

Spectrum 3d: HSQC spectrum 193 

Spectrum 3e: Expanded HSQC spectrum 194 

Spectrum 3f: HMBC spectrum 195 

Spectrum 3g: Expanded HMBC spectrum 196 

Spectrum 3h: COSY spectrum 197 

Spectrum 3i: NOESY spectrum 198 

Spectrum 3j: IR spectrum 199 

Spectrum 3k: UV spectrum 200 

Spectrum 3l: GC-MS spectrum 201 

12 

13 

17 

14 

15 

16 

Page 

189

Compound IV, euscaphic acid 

HO 

HO 

2 

23 

1 

4 

24 

25 26 

9 

5 

10 

6 7 

8 

12 

29 

27 

18 

14 

OH 

15 

17 

30 

21 

22 

COOH 




Spectrum 4d: DEPT spectrum 206 

Spectrum 4e: Expanded DEPT spectrum 207 

Spectrum 4f: HSQC spectrum 208 

Spectrum 4g: Expanded HSQC spectrum 209 

Spectrum 4h: HMBC spectrum 210 

Spectrum 4i: Expanded HMBC spectrum 211 

Spectrum 4j: COSY spectrum 212 

Spectrum 4k: NOESY spectrum 213 

Spectrum 4l: IR spectrum 214 

Spectrum 4m: UV spectrum 215 

Spectrum 4n: GC-MS spectrum 216 

Page 

202

HO 

Compound V, stigmasterol 

3 

19 

5 

10 

6 

7 

21 


Spectrum 5b: Expanded 1 H NMR spectrum 219 

Spectrum 5c: 13 C NMR spectrum 220 

Spectrum 5d: Expanded 13 C NMR spectrum 221 

Spectrum 5e: IR spectrum 222 

18 

14 

20 

22 

29 

16 

23 

28 

27 

25 

26 

Page 

217

HO 

Compound VI, lupeol 

3 

24 

25 26 

23 

10 






30 

20 

29 

27 

18 

19 

17 

22 

28 

Page 

223

HO 

Compound VI, lupeol 

3 

24 

25 26 

23 

10 






30 

20 

29 

27 

18 

19 

17 

22 

28 

Page 

223

HO 

218

219

HO 

220

221

222

HO 

224

225

HO 

226

227

228

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