Phyt oc he m ic a l a nd Biologic a l
St udie s of Ce rt a in Pla nt s Be longing
t o Fa m ily Ac a nt ha c e a e
A Thesis Presented By
Marwa Abdel-Aziz Ali Fayed
B. Pharm. Sciences, Assiut University, Assiut
M. Pharm. Sci. (Pharmacognosy), Assiut University, Assiut
Submitted for the Partial Fulfillment of the requirements for the
Degree of Doctor of Philosophy in Pharmaceutical Sciences
(Pharmacognosy)
Under Supervision of
Prof. Dr. Mohamed A. El -Shanawany
Professor of Pharmacognosy,
Faculty of Pharmacy,
Assiut University
Prof. Dr. Hanaa M. Sayed
Professor of Pharmacognosy,
Faculty of Pharmacy,
Assiut University
Dr.Sabri n Ragab Mohamed
Associate Professor of Pharmacognosy,
Faculty of Pharmacy, Assiut University
Pharmacognosy Department
Faculty of Pharmacy
Assiut University
Assiut, Egypt
(2013)
ACKNOWLEDGEMENT
To the almighty God Allah who have granted me all these graces to
fulfill this work and who supported and blessed me by his power and his
mercy in all my life.
I am greatly indebted to Prof. Dr. Mohamed A. El-Shanawany,
Professor of Pharmacognosy, Faculty of Pharmacy, Assiut University,
for his kind supervision, kind help, valuable information, continuous
support, suggesting the subject of research and encouragement during this
work.
I would like to express my deep hearty appreciation to Prof. Dr.
Hanaa M. Sayed, Professor of Pharmacognosy, Faculty of Pharmacy,
Assiut University, for her great help, sincere supervision, continuous
guidance and valuable support during preparation and presentation of this
work.
I am profoundly thankful to Dr.Sabrin Ragab Mohamed,
Associate Professor of Pharmacognosy, Faculty of Pharmacy, Assiut
University, for her admirable help, kind supervision and ultimate support.
I wish to express my sincere hearty thanks and gratitude for
Prof.Dr.Abdel-Aziz A. Fayed, Professor of Taxonomy, Faculty of
Science, Assiut University, for identification and providing the plants.
I am thankful to Prof. Dr. Hanaa M. Sayed, Prof.Dr.Azza
A.Khalifa and Prof.Dr.Zedan Z. Ibraheim, Heads of Pharmacognosy
Department, Faculty of Pharmacy, Assiut University, for the facilities,
great help and encouragement during this work.
I am really thankful to Prof.Dr.Samir A.Ross, Professor of
Natural Products Chemistry, School of Pharmacy, University of
Mississipi (USA), for facilities to carry out the spectral analysis and
biological studies.
I am thankful to all of Prof.Dr.Mohamed S. Kamel, Professor of
Pharmacognosy and head of Pharmacognosy Department, Faculty of
Pharmacy, El-Menya University, Dr.Wael M.Abdel-Megied and
Dr.Refaat B.Hamed, Lecturers of Pharmacognosy, Faculty of Pharmacy,
Assiut University, for carrying the spectral analysis.
I am thankful to All Staff members of the Pharmacognosy
Department, Faculty of Pharmacy Assiut University for the facilities,
great help and encouragement throughout this work.
I am thankful to the head of the Pharmacology Department, Faculty
of Medicine, Assiut University for the facilities for carrying
the
biological studies.
Many thanks to the technical staff of Pharmacognosy Department
and everybody who contributed in bringing this work to completion.
I am greatly thankful to my parents, my brother and sisters for their
great help, encouragement, unfailing love, spiritual and constant prayers.
Finally, I am indebted to my husband, he tolerated and scarified
much and supported me to finish this work. Really, I am very grateful for
his unfailing help and great support during this arduous work, his
excellent help and continuous encouragement. So, I highly appreciate his
efforts and pray to Allah to bless my family all the time.
Marwa A.A.Fayed
i
CONTENTS
Title
Page
INTRODUCTION ................................................................................1
TAXONOMY .......................................................................................2
REVIEW OF LITERATURE ..............................................................9
I- Chemical Review of Family Acanthaceae ..................................9
II- Biological Review of Some Plants of
Family Acanthaceae ............................................................... 52
AIM OF WORK ................................................................................ 54
MATERIALS, APPARATUS AND TECHNIQUES ........................... 55
PART I
Phytochemical Study of Anisotes trisulcus
(Forssk.) Nees. Aerial Parts
Chapter I: Preliminary Phytochemical Screening of the Air Dried
Powdred Anisotes trisulcus (Forssk.)Nees Aerial Parts................. 66
Chapter II: Extraction, Fractionation and Isolation of the Main
Constituents from Anisotes trisulcus (Forssk.)Nees Aerial Parts ..... 68
1- Extraction and Fractionation of Anisotes trisulcus
(Forssk.) Nees. Aerial Parts ...................................................... 68
2- Investigation of the Lipoidal Content of the n-Hexane
Fraction of Anisotes trisulcus (Forssk.) Nees. Aerial Parts ........ 70
3- Isolation of the Main Constituents of the n-Hexane
Fraction of Anisotes trisulcus (Forssk.) Nees. Aerial Parts ........ 78
4- Isolation of the Main Constituents of the Chloroform
Fraction of Anisotes trisulcus (Forssk.) Nees. Aerial Parts ........ 80
ii
Contents
Title
Page
5- Isolation of the Main Constituents of the Ethyl Acetate
Fraction of Anisotes trisulcus (Forssk.) Nees. Aerial Parts ........ 82
6- Isolation of the Main Constituents of the n-Butanol
Fraction of Anisotes trisulcus (Forssk.) Nees. Aerial Parts ........ 84
Chapter III: Identification of the Isolated Compounds from
Anisotes trisulcus (Forssk.) Nees. Aerial Parts ................ 86
− Identification of Compound AT-1............................................... 86
− Identification of Compound AT-2 ............................................... 87
− Identification of Compound AT-3 ............................................... 88
− Identification of Compound AT-4 ............................................... 90
− Identification of Compound AT-5 ............................................... 97
− Identification of Compound AT-6 ............................................. 102
− Identification of Compound AT-7 ............................................. 107
− Identification of Compound AT-8 ............................................. 110
− Identification of Compound AT-9 ............................................. 114
− Identification of Compound AT-10 ........................................... 119
− Identification of Compound AT-11 ........................................... 128
− Identification of Compound AT-12 ........................................... 132
− Identification of Compound AT-13 ........................................... 135
− Identification of Compound AT-14 ........................................... 139
− Identification of Compound AT-15 ........................................... 144
− Identification of Compound AT-16 ........................................... 150
− Identification of Compound AT-17 ........................................... 155
− Identification of Compound AT-18 ........................................... 160
− Identification of Compound AT-19 ........................................... 164
− Identification of Compound AT-20 ........................................... 168
iii
Contents
Title
Page
PART II
Phytochemical Study of Blepharis ciliaris (L.)
B.L. Burtt. Aerial Parts
Chapter I: Preliminary Phytochemical Screening of the Powdered
Blepharis ciliaris (L.) B.L.Burtt aerial parts .................... 172
Chapter II: Extraction, Fractionation and Isolation of the Main
Constituents from Blepharis ciliaris (L.) B.L.Burtt aerial
parts……………………………………………………174
1- Extraction and Fractionation of Blepharis ciliaris (L.)
B.L.Burtt. Aerial Parts ............................................................ 174
2- Investigation of the Lipoidal Content of the n-Hexane
Fraction of Blepharis ciliaris (L.) B.L.Burtt. Aerial Parts ........ 176
3- Isolation of the Main Constituents of the n-Hexane
Fraction of Blepharis ciliaris (L.) B.L.Burtt. Aerial Parts ....... 184
4- Isolation of the Main Constituents of the Chloroform
Fraction of Blepharis ciliaris (L.) B.L.Burtt. Aerial Parts ........ 186
5- Isolation of the Main Constituents of the Ethyl Acetate
Fraction of Blepharis ciliaris (L.) B.L.Burtt. Aerial Parts ....... 188
Chapter III: Identification of the Isolated Compounds from
Blepharis ciliaris (L.) B.L.Burtt. Aerial Parts ............... 190
− Identification of Compound BC-1............................................. 190
− Identification of Compound BC-2............................................. 191
− Identification of Compound BC-3............................................. 192
− Identification of Compound BC-4............................................. 198
− Identification of Compound BC-5............................................. 205
iv
Contents
Title
Page
− Identification of Compound BC-6............................................. 208
− Identification of Compound BC-7............................................. 211
− Identification of Compound BC-8............................................. 214
− Identification of Compound BC-9............................................. 216
− Identification of Compound BC-10 ........................................... 217
− Identification of Compound BC-11 ........................................... 220
− Identification of Compound BC-12 ........................................... 224
− Identification of Compound BC-13 ........................................... 230
− Identification of Compound BC-14 ........................................... 236
− Identification of Compound BC-15 ........................................... 243
− Identification of Compound BC-16 ........................................... 249
PART III
Biological Studies of Anisotes trisuclus (Forssk.) Nees.
and Blepharis ciliaris (L.)B.L.Burtt. Aerial Parts
1- Antioxidant Activity ...................................................................... 255
2- Acute Toxicity Study..................................................................... 259
3- Brine-Shrimp Assay (Cytotoxic Activity) ...................................... 260
4- Anti-inflammatory Activity............................................................ 262
5- Anti-malarial Activity .................................................................... 265
6- Anti-hyperglycaemic Activity ........................................................ 267
GENERAL SUMMARY AND CONCLUSION ............................... 270
REFERENCES ................................................................................. 288
ARABIC SUMMARY ............................................................................
v
Contents
LIST OF TABLES
No.
Page
1
List of some alkaloids isolated from family Acanthaceae ................... 9
2
List of some flavonoids isolated from family Acanthaceae ............... 17
3
List of some iridoids isolated from family Acanthaceae ................... 25
4
List of some lignans isolated from family Acanthaceae .................... 31
5
List of some sterols, di- and triterpeens isolated from
family Acanthaceae .......................................................................... 39
6
List of some phenolic, phenyl ethanoid and phenyl propanoid
glycosides isolated from family Acanthaceae ................................... 44
7
List of some megastigmans isolated from family Acanthaceae ......... 48
8
List of some simple aliphatic compounds isolated from
family Acanthaceae .......................................................................... 49
9
List of some quaternary ammonium compounds isolated
from family Acanthaceae ................................................................. 51
10
List of some miscellaneous compounds isolated from
family Acanthaceae .......................................................................... 51
11
Some biological activities carried on different plants of
family Acanthaceae .......................................................................... 52
12
Conditions for Gas/Liquid chromatographic analysis ....................... 64
13
Results of the preliminary phytochemical screening of the
dried aerial parts of Anisotes trisulcus (Forssk.) Nees. ..................... 66
14
Results of GC-MS analysis of fatty acids methyl esters of
the n-hexane of Anisotes trisulcus (Forssk.) Nees. aerial parts ......... 71
15
Results of GC-MS analysis of unsaponifiable matter
of the n-hexane fraction of Anisotes trisulcus (Forssk.) Nees.
aerial parts ....................................................................................... 74
16
13
C-, DEPT 13C- and 1H-NMR data of compound AT-4
(C5D5N-d5, 400 & 100 MHz) ............................................................ 91
17
The UV spectral data of compound AT-5 with methanol and
different ionizing and complexing reagents ...................................... 97
18
13
C- and 1H-NMR data of compound AT-5
(DMSO-d6, 400 & 100 MHz) ........................................................... 98
vi
Contents
No.
Page
19
The UV spectral data of compound AT-6 with methanol and
20
13
21
22
1
23
different ionizing and complexing reagents .................................... 102
C- and 1H-NMR data of compound AT-6
(DMSO-d6, 400 & 100 MHz) ......................................................... 103
H-NMR data of compound AT-7 (DMSO-d6, 400 MHz) .............. 107
C-, DEPT 13C- and 1H-NMR data of compound AT-8
13
(DMSO-d6, 400 & 100 MHz) ......................................................... 110
13
C- and 1H-NMR data of compound AT-9
(DMSO-d6, 400 & 100 MHz) ......................................................... 115
24
13
25
13
26
C- & DEPT 13C-NMR, HMBC, 1H-NMR and 1H-1H COSY
data of compound AT-10 (CDCl3, 400 & 100 MHz) ...................... 119
C-, DEPT 13C- and 1H-NMR data of compound AT-11
(DMSO-d6, 400 & 100 MHz) ......................................................... 128
13
C- and 1H-NMR data of compound AT-12
(DMSO-d6, 400 & 100 MHz) ......................................................... 132
H-NMR data of compound AT-13 (C5D5N-d5, 400 MHz) ............ 135
27
1
28
13
29
C- and DEPT 13C-NMR data of compound AT-13
(C5D5N-d5, 100 MHz) .................................................................... 136
13
C- & DEPT 13C-NMR, HMBC and 1H-NMR data of
30
compound AT-14 (DMSO-d6, 400 & 100 MHz) ............................ 140
13
C- & DEPT 13C-NMR, HSQC, 1H-NMR and 1H-1H COSY
31
data of compound AT-15 (DMSO-d6, 400 & 100 MHz) ................ 145
13
C-, DEPT 13C- and 1H-NMR data of compound AT-16
32
(DMSO-d6, 400 & 100 MHz) ......................................................... 151
13
C-, DEPT 13C- and 1H-NMR data of compound AT-17
(DMSO-d6, 400 & 100 MHz) ......................................................... 156
33
13
C- and 1H-NMR data of compound AT-18
(DMSO-d6, 400 & 100 MHz) ......................................................... 160
34
13
35
C- & DEPT 13C-NMR, HMQC and 1H-NMR data of
compound AT-19 (DMSO-d6 + TFA, 400 & 100 MHz) ................. 164
13
C- & DEPT 13C-NMR, HMQC and 1H-NMR data of
36
compound AT-20 (DMSO-d6 + TFA) (400 & 100 MHz) ............... 168
Results of the preliminary phytochemical screening of the
dried aerial parts of Blepharis ciliaris (L.) B.L.Burtt...................... 172
vii
Contents
No.
37
Page
Results of GC-MS analysis of fatty acids methyl esters of
the n-hexane Blepharis ciliaris (L.) B.L.Burtt. aerial parts ............. 177
38
Results of GC-MS analysis of unsaponifiable matter
of the n-hexane fraction of Blepharis ciliaris (L.)B.L.Burtt.
aerial parts ..................................................................................... 180
39
13
C- and 1H-NMR data of compound BC-3
(CDCl3, 400 MHz) ........................................................................ 193
40
13
C-, DEPT 13C- and 1H-NMR data of compound BC-4
(C5D5N-d5, 400 & 100 MHz) ......................................................... 199
41
13
C- and 1H-NMR data of compound BC-5
(DMSO-d6, 400 & 100 MHz) ......................................................... 205
42
13
C- and 1H-NMR data of compound BC-6
(DMSO-d6, 400 & 100 MHz) ......................................................... 208
43
13
C- and 1H-NMR data of compound BC-7
(DMSO-d6, 400 & 100 MHz) ......................................................... 211
44
The UV spectral data of compound BC-8 with different
45
1
46
The UV spectral data of compound BC-11 with different
47
1
48
The UV spectral data of compound BC-12 with different
49
13
ionizing and complexing reagents .................................................. 214
H-NMR data of compound BC-10 (DMSO-d6, 400 MHz) ............ 217
ionizing and complexing reagents .................................................. 220
H-NMR data of compound BC-11 (DMSO-d6, 400 Hz) ............... 221
ionizing and complexing reagents .................................................. 224
C- and 1H-NMR data of compound BC-12
(DMSO-d6, 400 &100 MHz) .......................................................... 225
50
The UV spectral data of compound BC-13 with different
51
13
ionizing and complexing reagents .................................................. 230
C- and 1H-NMR data of compound BC-13
(DMSO-d6, 400 & 100 MHz) ......................................................... 231
52
The UV spectral data of compound BC-14 with different
53
13
ionizing and complexing reagents .................................................. 236
C-, DEPT 13C- and 1H-NMR data of compound BC-14
(DMSO-d6, 400 & 100 MHz) ......................................................... 237
Contents
No.
54
55
56
57
58
59
60
61
62
63
64
65
66
67
viii
Page
The UV spectral data of compound BC-15 with different
ionizing and complexing reagents .................................................. 243
13
C-, DEPT 13C- and 1H-NMR data of compound BC-15
(DMSO-d6, 400 &100 MHz) .......................................................... 244
13
C-, DEPT 13C- and 1H-NMR data of compound BC-16
(DMSO-d6, 400 & 100 MHz) ......................................................... 250
Results of the antioxidant activity of the different extracts
of the aerial parts of Anisotes trisulcus (Forssk.) Nees.
and Blepharis ciliaris (L.) B.L.Burtt. and some of
the isolated compounds .................................................................. 257
Results of the toxic activity of the different extracts of the
dried aerial parts of Anisotes trisulcus (Forrsk.) Nees.................... 261
Results of the toxic activity of the different extracts of the
dried aerial parts of Blepharis ciliaris (L.)B.L.Burtt....................... 261
Results of the anti-inflammatory effect of the different
extracts of the aerial parts of Anisotes trisulcus
(Forssk.) Nees. ............................................................................... 264
Results of the anti-inflammatory effect of the different
extracts of the aerial parts of Blepharis ciliaris (L.) B.Burtt. .......... 264
In-vitro anti-malarial screening for the different extracts
of Anisotes trisulcus (Forssk.) Nees ............................................... 266
In-vitro anti-malarial screening for the different extracts
of Blepharis ciliaris L.B.Burtt ....................................................... 266
Effect of the different extracts of the dried aerial parts
of Anisotes trisulcus (Forssk.) Nees. on the Blood
Glucose Levels (BGLs) when they fed concurrently
with the glucose load ..................................................................... 268
Effect of the different extracts of the dried aerial parts of
Blepharis ciliaris (L.)B.L.Burtt. on the Blood Glucose Levels
(BGLs) when they fed concurrently with the glucose load ............. 268
List of the isolated compounds from Anisotes trisulcus
(Forssk.) Nees.aerial parts .............................................................. 274
List of the isolated compounds from Blepharis ciliaris (L.)
B.L.Burtt.aerial parts...................................................................... 280
ix
Contents
LIST OF SCHEMES
No.
1
Page
Extraction and fractionation of the total methanolic extract
of Anisotes trisulcus (Forssk.) Nees. aerial parts .............................. 69
2
Isolation of the main constituents from the n-hexane fraction
of Anisotes trisulcus (Forssk.) Nees. aerial parts .............................. 79
3
Isolation of the main constituents from the chloroformic
fraction of Anisotes trisulcus (Forssk.) Nees. aerial parts ................. 81
4
Isolation of the main constituents from the ethyl acetate
fraction of Anisotes trisulcus (Forssk.) Nees. aerial parts ................. 83
5
Isolation of the main constituents from the n-butanol fraction
of Anisotes trisulcus (Forssk.) Nees. aerial parts .............................. 85
6
Extraction and fractionation of the total methanolic extract
of Blepharis ciliaris (L.)B.L.Burtt. aerial parts............................... 175
7
Isolation of the main constituents of the n-hexane fraction
of Blepharis ciliaris (L.) B.L.Burtt.aerial parts............................... 185
8
Isolation of the main constituents of the chloroform fraction
of Blepharis ciliaris (L.) B.L.Burtt.aerial parts............................... 187
9
Isolation of the main constituents of the ethyl acetate
fraction of Blepharis ciliaris (L.)B.L.Burtt. aerial parts.................. 189
x
Contents
LIST OF FIGURES
No.
Page
1
Photo of Anisotes trisulcus (Forssk.) Nees. ........................................ 5
2
Photo of Blepharis ciliaris (L.) B.L.Burtt........................................... 6
3
Chromatogram of GC-MS analysis of FAMEs of the n-hexane
fraction of Anisotes trisulcus (Forssk.) Nees. aerial parts ................. 73
4
Chromatogram of GC-MS analysis of hydrocarbons of the
n-hexane fraction of Anisotes trisulcus (Forssk.) Nees.
aerial parts ....................................................................................... 76
5
Chromatogram of GC-MS analysis of steroids of the n-hexane
fraction of Anisotes trisulcus (Forssk.) Nees. aerial parts…............. 77
6
IR spectrum of compound AT-1 (KBr) ............................................ 86
7
IR spectrum of compound AT-2 (KBr) ............................................ 88
8
IR spectrum of compound AT-3 (KBr) ............................................ 89
9
+FAB-MS spectrum of compound AT-4 .......................................... 94
10
EI-MS spectrum of FAME of compound AT-4 ................................ 94
11
EI-MS spectrum of LCB of compound AT-4 ................................... 95
12
1
13
13
14
DEPT13C-NMR spectrum of compound AT-4 (C5D5N-d6, 100 MHz) ....... 96
15
EI-MS spectrum of compound AT-5 .............................................. 100
16
1
17
13
18
EI-MS spectrum of compound AT-6 .............................................. 103
19
1
20
13
21
EI-MS spectrum of compound AT-7 .............................................. 109
22
1
23
EI-MS spectrum of compound AT-8 .............................................. 112
24
1
25
13
26
DEPT 13C-NMR of compound AT-8 (DMSO-d6, 100 MHz) .......... 113
27
EI-MS spectrum of compound AT-9 .............................................. 117
H-NMR spectrum of compound AT-4 (C5D5N-d6, 400 MHz) ......... 95
C-NMR spectrum of compound AT-4 (C5D5N-d6, 100 MHz)........ 96
H-NMR spectrum of compound AT-5 (DMSO-d6, 400 MHz) ...... 101
C-NMR spectrum of compound AT-5 (DMSO-d6, 100 MHz) ..... 101
H-NMR spectrum of compound AT-6 (DMSO, 400 MHz) ........... 106
C-NMR spectrum of compound AT-6 (DMSO-d6, 100 MHz) ..... 106
H-NMR spectrum of compound AT-7 (DMSO-d6, 400 MHz) ...... 109
H-NMR spectrum of compound AT-8 (DMSO-d6, 400 MHz) ...... 112
C-NMR of compound AT-8 (DMSO-d6, 100 MHz) .................... 113
xi
Contents
No.
Page
28
1
29
13
30
EI-MS spectrum of compound AT-10 ............................................ 123
31
1
32
Expanded 1H-NMR spectrum of compound AT-10
H-NMR spectrum of compound AT-9 (DMSO-d6, 400 MHz) ...... 118
C-NMR spectrum of compound AT-9 (DMSO-d6, 100 MHz) ..... 118
H-NMR spectrum of compound AT-10 (CDCl3, 400 MHz) .......... 123
(CDCl3, 400 MHz) ......................................................................... 124
33
13
34
1
35
HSQC spectrum of compound AT-10 ............................................ 126
36
HMBC spectrum of compound AT-10 ........................................... 127
37
EI-MS spectrum of compound AT-11 ............................................ 130
38
1
39
13
C-NMR spectrum of compound AT-10 (CDCl3, 100 MHz)......... 125
H-1H COSY spectrum of compound AT-10 (CDCl3, 400 MHz) ... 126
H-NMR spectrum of compound AT-11 (DMSO-d6, 400 MHz) .... 130
C-NMR spectrum of compound AT-11
(DMSO-d6, 100 MHz).................................................................... 131
40
DEPT 13C-NMR spectrum of compound AT-11
(DMSO-d6, 100 MHz).................................................................... 131
41
EI-MS spectrum of compound AT-12 ............................................ 134
42
1
43
13
44
1
45
13
46
DEPT 13C-NMR spectrum of compound AT-13
H-NMR spectrum of compound AT-12 (DMSO-d6, 400 MHz) .... 134
C-NMR spectrum of compound AT-12 (DMSO-d6, 100 MHz) ... 134
H-NMR spectrum of compound AT-13 (C5D5N-d5, 400 MHz) ..... 137
C-NMR spectrum of compound AT-13 (C5D5N-d5, 100 MHz) .... 138
(C5D5N-d5, 100 MHz) .................................................................... 138
47
EI-MS spectrum of compound AT-14 ............................................ 142
48
1
49
13
50
DEPT 13C-NMR spectrum of compound AT-14
H-NMR spectrum of compound AT-14 (DMSO-d6, 400 MHz) .... 142
C-NMR spectrum of compound AT-14 (DMSO-d6, 100 MHz) ... 143
(DMSO-d6, 100 MHz).................................................................... 143
51
HMBC spectrum of compound AT-14 (DMSO-d6, 700 MHz) ....... 143
52
EI-MS spectrum of compound AT-15 ............................................ 147
53
1
54
13
H-NMR spectrum of compound AT-15 (DMSO-d6, 400 MHz) .... 147
C-NMR spectrum of compound AT-15 (DMSO-d6, 100 MHz) ... 148
xii
Contents
No.
55
Page
DEPT 13C-NMR spectrum of compound AT-15
(DMSO-d6, 100 MHz).................................................................... 148
56
1
H-1H COSY spectrum of compound AT-15
(DMSO-d6, 700 MHz).................................................................... 149
57
HSQC spectrum of compound AT-15 (DMSO-d6, 700 MHz) ........ 149
58
EI-MS spectrum of compound AT-16 ............................................ 153
59
1
60
13
61
DEPT 13C-NMR spectrum of compound AT-16
H-NMR spectrum of compound AT-16 (DMSO-d6, 400 MHz) .... 153
C-NMR spectrum of compound AT-16 (DMSO-d6, 100 MHz) ... 154
(DMSO-d6, 100 MHz).................................................................... 154
62
EI-MS spectrum of compound AT-17 ............................................ 158
63
1
64
13
65
DEPT 13C-NMR spectrum of compound AT-17
H-NMR spectrum of compound AT-17 (DMSO-d6, 400 MHz) .... 158
C-NMR spectrum of compound AT-17 (DMSO-d6, 100 MHz) ... 159
(DMSO-d6, 100 MHz).................................................................... 159
66
+ MALDI-TOF-MS spectrum of compound AT-18 ....................... 162
67
1
68
13
H-NMR spectrum of compound AT-18 (DMSO-d6, 400 MHz) .... 162
C-NMR spectrum of compound AT-18 (DMSO-d6, 100 MHz) ... 163
69
+MALDI-TOF-MS spectrum of compound AT-19 ........................ 166
70
1
71
13
H-NMR spectrum of compound AT-19
(DMSO-d6 + TFA, 400 MHz) ........................................................ 166
C-NMR spectrum of compound AT-19
(DMSO-d6 + TFA, 100 MHz) ........................................................ 167
72
DEPT 13C-NMR spectrum of compound AT-19
(DMSO-d6 + TFA, 100 MHz) ........................................................ 167
73
HMQC spectrum of compound AT-19
74
1
75
13
(DMSO-d6 + TFA, 100 MHz) ........................................................ 167
H-NMR spectrum of compound AT-20
(DMSO-d6 + TFA, 400 MHz) ........................................................ 170
C-NMR spectrum of compound AT-20
(DMSO-d6 + TFA, 100 MHz) ........................................................ 170
76
DEPT 13C-NMR spectrum of compound AT-20
(DMSO-d6 + TFA, 100 MHz) ........................................................ 171
xiii
Contents
No.
Page
77
HMBC spectrum of compound AT-20 (DMSO-d6 +TFA).............. 171
78
Chromatogram of GC-MS analysis of FAMEs of the
n-hexane of Blepharis ciliaris (L.) B.L.Burtt. aerial parts .............. 179
79
Chromatogram of GC-MS analysis of hydrocarbons of the
n-hexane of Blepharis ciliaris (L.) B.L.Burtt. aerial parts .............. 182
80
Chromatogram of GC-MS analysis of steroids of the
n-hexane of Blepharis ciliaris (L.) B.L.Burtt. aerial parts .............. 183
81
+FAB-MS spectrum of compound BC-3 ........................................ 196
82
EI-MS spectrum of FAME of compound BC-3 .............................. 196
83
1
84
13
85
+FAB-MS spectrum of compound BC-4 ........................................ 202
86
EI-MS spectrum of LCB of compound BC-4 ................................. 202
87
EI-MS spectrum of FAME of compound BC-4 .............................. 203
88
1
89
13
90
DEPT 13C-NMR spectrum of compound BC-4
H-NMR spectrum of compound BC-3 (CDCl3, 400 MHz)............ 197
C-NMR spectrum of compound BC-3 (CDCl3, 100 MHz)........... 197
H-NMR spectrum of compound BC-4 (C5D5N-d6, 400 MHz) ....... 203
C-NMR spectrum of compound BC-4 (C5D5N-d6, 100 MHz)...... 204
(C5D5N-d6, 100 MHz) .................................................................... 204
91
EI-MS spectrum of compound BC-5 .............................................. 207
92
1
93
13
94
EI-MS spectrum of compound BC-6 .............................................. 210
95
1
96
13
97
EI-MS spectrum of compound BC-7 .............................................. 213
98
1
99
13
100
1
H-NMR spectrum of compound BC-10 (DMSO-d6, 400 MHz) .... 219
101
1
H-NMR spectrum of compound BC-11 (DMSO-d6, 400 MHz) .... 223
H-NMR spectrum of compound BC-5 (DMSO-d6, 400 MHz) ...... 207
C-NMR spectrum of compound BC-5 (DMSO-d6, 100 MHz) ..... 207
H-NMR spectrum of compound BC-6 (DMSO-d6, 400 MHz) ...... 210
C-NMR spectrum of compound BC-6 (DMSO-d6, 100 MHz) ..... 210
H-NMR spectrum of compound BC-7 (DMSO-d6, 400 MHz) ...... 213
C-NMR spectrum of compound BC-7 (DMSO-d6, 100 MHz) ..... 213
102
+FAB-MS spectrum of compound BC-12 ...................................... 228
103
1
104
Expanded 1H-NMR spectrum of compound BC-12
H-NMR spectrum of compound BC-12 (DMSO-d6, 400 MHz) .... 228
(DMSO-d6, 400 MHz).................................................................... 229
xiv
Contents
No.
Page
105
13
106
+FAB-MS spectrum of compound BC-13 ...................................... 234
107
1
108
13
109
+FAB-MS spectrum of compound BC-14 ...................................... 240
110
1
111
Expanded 1H-NMR spectrum of compound BC-14
C-NMR spectrum of compound BC-12 (DMSO-d6, 100 MHz) ... 229
H-NMR spectrum of compound BC-13 (DMSO-d6, 400 MHz) .... 235
C-NMR spectrum of compound BC-13 (DMSO-d6, 100 MHz) ... 235
H-NMR spectrum of compound BC-14 (DMSO-d6, 400 MHz) .... 241
(DMSO-d6, 400 MHz).................................................................... 241
112
13
113
DEPT 13C-NMR of compound BC-14 (DMSO-d6, 100 MHz) ........ 242
114
+FAB-MS spectrum of compound BC-15 ...................................... 247
115
1
116
13
117
DEPT 13C-NMR spectrum of compound BC-15
C-NMR spectrum of compound BC-14 (DMSO-d6, 100 MHz) ... 242
H-NMR spectrum of compound BC-15 (DMSO-d6, 400 MHz) .... 247
C-NMR spectrum of compound BC-15 (DMSO-d6, 100 MHz) ... 248
(DMSO-d6, 100 MHz).................................................................... 248
118
+FAB-MS spectrum of compound BC-16 ...................................... 253
119
1
120
13
121
DEPT 13C-NMR spectrum of compound BC-16
H-NMR of compound BC-16 (DMSO-d6, 400 MHz) ................... 253
C-NMR spectrum of compound BC-16 (DMSO-d6, 100 MHz) ... 254
(DMSO-d6, 100 MHz).................................................................... 254
122
Results of the antioxidant activity of the different extracts
of the aerial parts of Anisotes trisulcus (Forssk.) Nees ................... 257
123
Results of the antioxidant activity of the different extracts
of the aerial parts and some of the isolated compounds
of Blepharis ciliaris (L.) B.L.Burtt................................................. 258
xv
LIST OF ABBREVIATIONS
Aqu.
Aqueous
br.s
Broad singlet
BuOH
Butanol
°C
Degrees Celsius
C.C.
Column Chromatography
CHCl3
Chloroform
CDCl3
Deutrated chloroform
COSY
Chemical Shift Correlation Spectroscopy
d
Doublet
dd
Doublet doublet
DEPT
Distortionless Enhancement by Polarization
Transfer
DMSO-d6
Deuterated dimethyl sulfoxide
EI-MS
Electron Impact Mass Spectroscopy
EtOAc
Ethyl acetate
ext.
Extract
FAB
Fast Atomic Bombardment
FAME
Fatty acid methyl ester
FAMEs
Fatty acid methyl esters
Fig.
Figure
fr.
Fraction
GC/MS
Gas Chromatography/Mass Spectrometry
GLC
Gas Liquid Chromatography
glu.
Glucose
gm.
Gram
hex.
Hexane
1
Proton Nuclear Magnetic Resonance
H-NMR
HMBC
Heteronuclear Multiple Bond Connectivity
HSQC
Hetero Nuclear Single Quantum Coherence
xvi
List of Abbreviations
hr.
Hour
Hz.
Hertz
IR
Infra Red
J
Coupling Constant
KBr
Potassium bromide
kg
Kilogram
LCB
Long chain base
m
Multiplet
µ
Micron
M+
Molecular ion peak
MALDI-TOF-
Matrix Assisted Laser Desorption Ionization
MS
Time of Flight Mass Spectrometry
mg
Milligram
m.p.
Melting point
Me
Methyl
MHz
Mega Hertz
min.
Minutes
MeOH
Methanol
N
Normal
NMR
Nuclear Magnetic Resonance
Rf
Retardation factor
Rt
Retension time
rha.
Rhamnose
RP
Reversed phase
s
Singlet
t
Triplet
TLC
Thin Layer Chromatography
TMS
Tetra Methyl Silane
UV
Ultraviolet
v/v
Volume/volume
1
INTRODUCTION
Many medicinal plants have always had an important place in the
therapeutic harmony of mankind. According to the WHO(1), 80% of the
world population relies on medicinal plants for their primary health care
needs. Plants and their extracts continue to provide effective treatment for
different kinds of diseases therefore such plants should be investigated to
understand better about their properties, safety and efficacy.
Family Acanthaceae, a taxon of dicotyledonous flowering plants, is
considered as one of the richest families with medicinal plants(2). It
includes about 346 genera and around 4300 species(3,4,5). The most
common genera are Barleria (230 spp.), Blepharis (129 spp.), Acanthus
(50 spp.), Anisotes (23 spp.)(6), Andrographis (20 spp.) and Adhatoda
(20spp.)(4).
Most are tropical herbs, shrubs, or twining vines; some are
epiphytes. Only a few species are distributed in temperate regions. The
four main centres of distribution are Indonesia, Malaysia, Africa, Brazil
and Central America(4,5). The representatives of the family can be found in
nearly every habitat, including dense or open forests, in scrublands, on wet
fields, valleys, at the sea coast, in marine areas, in swamps and as an
element of mangrove woods. Several plants of family Acanthaceae were
used in traditional herbal medicine, some are grown as ornamentals(3) since
they have large flowers with colourful petals, also they are used as a
source of natural dyes(3). Therefore, the two plants under investigation
were selected to find new pharmaceutical compounds and potential
applications for them.
2
TAXONOMY
The plants under investigation could be classified taxonomically as
follows(7,8):
Kingdom:
Plantae = Plants
Subkingdom:
Tracheobionta = Vascular plants
Division:
Magnoliophyta = flowering plants
Class:
Magnoliopsida = Dicotyledons
Subclass:
Asteridae
Order:
Scrophulariales
Family:
Acanthaceae
Subfamily:
Acanthoideae
Tribe:
Acantheae
Genus:
Anisotes
Blepharis
Species:
trisulcus – Nees.
ciliaris
Botanical name:
Anisotes trisulcus (Forssk.) Nees.
Blepharis ciliaris (L.)B.L.Burtt.
Arabic names:
Amodh, Madh ()ﺍﻟﻤﺽ, Moze ()ﻣﻮوز
Shawk-ul-Dab ()ﺸﻭﻙ ﺍﻟﻀﺏ, and
Modaid (() ﺍﻟﻤﻀﻴﺽ7,9)
Zughaf ()ﺯﻏﺎﻑ, Shuqaf ()ﺸﻐﻑ,
Kohl-el-agouz ()ﻜﺤل ﺍﻟﻌﺠﻭﺯ
Synonyms:
Dianthera trisulca Forssk.,
B.edulis (Nees.)Pers.,
Anisotes velutinus Lindau,
B.persica (Burm.f.) Kuntze
Calasias bracteata Raf.(7,9,10)
Acanthodium
spicatum(2,10,11,12,13)
3
Taxonomy
Plants of this family are characterized morphologically by the
following(2-5,8,10,14): The leaves: are simple, opposite mostly decussate,
rarely whorled, exstipulate, usually entire with lobed or spiny or toothed
margin and without stipules. The stem: the main trunk is erect, branched
mainly herbaceous or woody. The stem breaks with complete or
incomplete fracture, especially in its upper part and fibrous in its lower
one. The inflorescence: either spike, dichasial cymes, in its ultimate
branching tending to be monochasial and frequently condensed in the leaf
axils. Racemose inflorescences also occur and solitary inflorescences are
common. Bracts and bracteoles: are usually present, often coloured. The
bracteoles are frequently large, enclosing the flower. They are usually two,
sometimes more under the individual flowers, either free or connected to
an epicalyx. The flowers: are solitary or aggregated as inflorescence.
They
are
hermaphrodite,
small,
usually
irregular,
zygomorphic,
hypogenous and with nectariferous disk below the ovary. The calyx: is
composed of 5 (rarely 4) free or gamosepalous sepals. The corolla: is
tubular two-lipped or sub-equally five lobed. The lobes are imbricate,
sometimes are nearly regular. The androecium: is composed of 4
epipetalous stamens in two sets, didynamous rarely five. Sometimes a
staminodium is present, hypogenous disk is present but mostly small. The
anthers are two or one celled. They are often with one lobe smaller than
the other or abortive, connective is often long. The pollen grains exhibit a
great variety of patterns. The gynaecium: is bicappillary and syncarpous.
The ovary is superior, bilocular with two to many ovules in each locule.
The ovules are anatropous with an axile placentation. The style is simple,
filiform and linear. The stigma is usually two-lobed, often of unequal size.
The floral formula:
Taxonomy
4
%, , K(4-5), C(4-5), A2 or 4,G(2)
The fruit: is bilocular capsule opening by two valves which are often
elastically, dehiscent. The septum splitting and the seeds are borne on each
half. The seeds: are usually exalbuminous, possess curved projections or
peculiar hook-like outgrowth (retinacula or jaculators) arising from their
stalks. The seeds have hairs on the surface which swell up when wetted
and may be of one use in anchoring the seed to its place of germination.
Genus Anisotes Nees.(6,15,16):
This genus comprises about 23 species, distributed in Tropical
regions and Southern Africa to Madagascar. It is represented by perennial
glabrous or hairy shrubs, the leaves are opposite petiolated rarely sessile.
Flowers are sessile in axillary clusters or in dense spike like cyme, the
calyx is distinct of 5 sepals, the corolla bilobed, red to orange red in
colour, the stamens 2, the ovary 2-loculed and the ovules 2 per locule. The
fruits are capsules, four seeded lacking hygroscopic hairs.
Anisotes trisulcus (Forssk.) Nees.(7,9,15,16,17,18) is a stiffy erect
shrub with a height of 1-3.5 m. It is endogenous to Yemen and widely
distributed in the Southern mountainous regions of the Kingdom of Saudi
Arabia particularly in Wadi Jabal Abu Hassan between Abha and Najran.
The main stem of the plant is erect, cylindrical, monopodially branched
carrying numerous branches showing more or less long internodes. The
young stem is purplish-green in colour, while the old one has rough surface
covered with brownish cork. The branches bear opposite and decussate
leaves at the nodes. The leaves are simple, petiolate and exstipulated. The
lamina is ovate to elliptic in shape with obtuse or sub-acute apex, entire
margin and symmetric base. The venation is pinnately reticulate. The
lamina is hairy and yellowish green in colour. Flowers are shortly
pedunculate, 2-flowered axillary clusters. Bracts are small and linear.
5
Taxonomy
Calyx segments are 5 and linear. Corrolla brick red, 3-5 cm long, divided
nearly to the base into 2 lips, the upper lip is notched, the lower one is
much narrower and weakly 3-lobed. Stamens have 2 anthers which are 2celled, one cell above the other. Fruit capsule, 4 seeded. Seeds are rough.
Fig. (1): Photo of Anisotes trisulcus (Forssk.) Nees.
Genus Blepharis A. Juss.(3,10,17,18):
It is an Afroasiatic genus comprising about 129 species which occur
in arid and semi-arid habitats. It is widespread in Tropical Africa, Arabia,
India extending to South East Asia and China. The genus is represented by
annual or perennial thistle like spiny herbs or shrubs. The leaves are in
false whorls, one pair is smaller than the other. Flowers are blue in spikelike cymes, calyx of 4 glumaceous sepals, corolla one lipped, the lip is 3-5
lobed with four stamens, two anterior filaments broader than the posterior
6
Taxonomy
and bilocular ovary. Fruits are two seeded capsule with discoid seeds
covered with hygroscopic hairs.
Blepharis ciliaris (L.) B.L.Burtt.(3,10,18) is a perennial, greyishpubescent to glabrescent ascending, prickly herb branched from the base,
reaching up to 30 cm in height, distributed in East Tropical Africa, Saudi
Arabia and Egypt. The leaves appearing in whorls of 4-outer pairs in each
whorl often smaller, they are oblong to lanceolate, coriacious lamina with
silvery appearance and long petiole, margins are entire to remotely
spinulose, puberulous above, with spine tipped apex, evanescent.
Inflorescence is a dense, strobilate spike with purplish-blue flowers,
bracteoles are linear-subulate villose. Calyx is scarious, membranous, soft
hairy, 4-lobed, lobes are unequal, upper lobe is the longest. Corolla tube is
short, upper lip is absent, lower lip is flat with 3 obtuse lobes and
puberulous. Fruit is a capsule, ovate to compressed shining brown and
glabrous with two flat seeds.
Fig. (2): Photo of Blepharis ciliaris (L.) B.L.Burtt.
Folk Medicine:
7
Taxonomy
Many plants belonging to this family are of great biological
importance since they are widely used in folk medicine as a remedy for
several ailments as:
Adhatoda vasica Nees. the leaves, flowers and roots extracts are
commonly used in India as an expectorant and bronchodilator,
consequently act as a remedy for cold, cough and asthma, in addition they
are used as antibacterial, anticholinestrerase. Also they are used in wound
healing,
abortifacient,
inflammatory,
hepatoprotective,
antimutagenic,
antioxidant,
cardioprotective,
antiulcer
and
antias
hypoglycaemic(19).
The leaves extracts of Justicia gendarussa Burm.f. are used in
treatment of arthritis(20), anti-inflammatory, strongly active against HIV
type 1 reverse transcriptase and asthma(21).
Andrographis paniculata (Burm.f.) Wall. ex Nees. is useful in
treatment of diarrhea, anti-diabetic, anti-oxidant, anti-leishmanial, antiHIV, anti-malarial, hepatoprotective, anti-bacterial, anti-fungal, anticancer, cold and fever(22).
Justicia pectoralis Jacq. is used as a relaxant and general tonic. Its
preparations can possess anti-inflammatory, anti-fungal, anti-lieshmanial,
relaxant effects and it supports recovery from influenza(21,23).
Barleria cristata L. seeds are used in cases of snake bites while the
juice of the leaves is used for cough(24) while roots are ground into a paste
and taken orally for diarrhea(25).
Taxonomy
8
Ruella asperula Benth. & Hook.f. leaves and roots maceration and
infusion are used against bronchitis, asthma, flu, fever and uterus
inflammation(26).
Hypostes forskaolei R.Br. extract possess an anti-neoplastic
activity(27).
Hypostes purpurea R.Br. is used in Taiwan in folk medicine to
promote blood circulation, an antipyretic, antiphlogistic, liver protective
agent and remove stasis, while they are used in Chinese folk medicine for
the treatment of cough, fever, sore throat, mumps, hepatitis and incised
wounds with hemorrhage(28).
Hygrophilla spinosa T. commonly used in traditional medicine for
the treatment of inflammation, pain, jaundice, rheumatism, arthritis,
anaemia and its leaves extract was investigated to have anti-inflammatory
and anti-pyretic effects(29).
Anisotes trisulcus (Forssk.) Nees. is used as traditional herbal
medicine in the Arabian Peninsula as a treatment for all hepatic conditions
including hepatitis, jaundice, gallstone and other hepatic disorders. It is
also used as anti-diabetic, bronchodilator, hypo-tensive and it has a local
anesthetic effect (15,29). The plant extract is used in several pharmaceutical
forms to limit tobacco consumption and to suppress appetite(15,29,30).
Blepharis ciliaris (L.) B.L.Burtt. seeds (roasted or crushed) were
applied on sores, wounds and boils as an antibacterial. The seeds are also
considered to be attenuant, resolvent, diuretic, aphrodisiac and
expectorant(29) in addition to charcoal from the roots is applied to the eyes
to improve vision, hence the Arabic name "Kohl-el-agouz"(10,31).
9
REVIEW OF LITERATURE
Reviewing
the
available
literature
concerning the
chemical
constituents and biological studies of family Acanthaceae, it was found
that alkaloids, flavonoids, iridoids, phenylpropanoids, lignans, neo-lignans,
sterols and terpenes in addition to some quaternary ammonium compounds
are reported from different plants of this family.
I- Chemical Review:
1- Alkaloids:
Table (1): List of some alkaloids isolated from family Acanthaceae.
No.
Name
Structure
Plant source
R2
Organ
Ref.
N
N
R1
1
Vasicine
(peganine)
2
Vasicinol
3
Vasicoline
R1= OH, R2= H
R1= R2= OH
CH3
N
R =
1
R2= H
CH3
Adhatoda vasica Nees.
Anisotes sessiliflorus
C.B.Cl.
Anisotes trisulcus
(Forssk.) Nees.
Leaves
32,32'
Aerial parts 33
Aerial parts
34,
35
Adhatoda vasica Nees.
Roots, stem
seeds &
leaves
32,
36
Adhatoda vasica Nees.
Leaves
32
10
Review of Literature
Table (1): Continued.
No.
Name
Structure
Plant source
Organ
Ref.
Leaves,
Stem &
roots
Aerial parts
32,
36
Aerial parts
34
Aerial parts
Aerial parts
Aerial parts
32,
33
32
34
Adhatoda vasica Nees.
Leaves
32
Anisotes sessiliflorus
C.B.Cl.
Aerial parts
33
Anisotes sessiliflorus
C.B.Cl.
Aerial parts
33
Adhatoda vasica Nees.
Aerial parts
32
Anisotes sessiliflorus
C.B.Cl.
Aerial parts
33
Adhatoda vasica Nees.
Aerial parts
32
O
R3
N
N
R
R1
R2
4
Vasicinone
R1= R2= R3= H
R= OH
Adhatoda vasica Nees.
Anisotes sessiliflorus
C.B.Cl.
Anisotes trisulcus
(Forssk.) Nees.
5
Anisotine
R1= R2= R3= H,
R=
CO 2-CH3
NH-CH3
6
Hydroxyanisotine
R1= R2= OH, R3= H,
Anisotes sessiliflorus
C.B.Cl.
Adhatoda vasica Nees.
Anisotes trisulcus
(Forssk.) Nees.
33
R=
CO 2-CH3
NH-CH3
7
Sessiflorine
R= R3 = H, R2 = OCH3,
R1=
NH-CH3
8
Anisessine
R1= R2= R3= H,
N-H
CO2Et
R=
9
Aniflorine
R1= OH, R2= OCH3,
R3= H,
CH3
N
R=
CH3
11
Review of Literature
Table (1): Continued.
No.
10
Name
DeoxyAniflorine
Structure
R1= R3= H, R2= OCH3,
CH 3
N
Desmethoxyaniflorine
Organ
Ref.
Anisotes sessiliflorus
C.B.Cl.
Aerial parts
33
Adhatoda vasica Nees.
Aerial parts
32
Adhatoda vasica Nees.
Aerial parts
37
CH3
R=
11
Plant source
R1= OH, R2= R3= H,
CH 3
N
CH3
R=
12
Vasicinolone
R1= R2= H, R3= R= OH
Adhatoda vasica Nees.
Leaves,
Flowers &
roots
38
13
7-Methoxyvasicinone
R1= R2= H, R3= OCH3,
R= OH
Adhatoda vasica Nees.
Leaves
37
14
Asicolinone
R1= R2= R3= H,
Adhatoda vasica Nees.
Aerial parts
32
Adhatoda vasica Nees.
Anisotes sessiliflorus
C.B.Cl.
Leaves
Aerial parts
32
33
Acanthus ilicifolius L.
Aerial parts
39
Acanthus ilicifolius L.
Roots
40
N(CH3)2
R=
15
Vasnetine
R1= R2= R3= H,
HN
O
R=
16
OCH3
Acanthicifoline
N
NH
O
OMe
17
BAO
O
OH
N
12
Review of Literature
Table (1): Continued.
No.
Name
Structure
Plant source
Organ
Ref.
MeO
N
CH3
R
MeO
OMe
18
Hypoestestatin 1
R=H
Hypostes vertcillaris
(Linn. f.) Soland.
Leaves
41
19
Hypoestestatin 2
R = OH
Hypostes vertcillaris
(Linn. f.) Soland.
Leaves
41
(14-hydroxyhypoestatin)
RO
H
N
H3CO
O
20
Ruspolinone
R = CH3
Ruspolia
hypercrateriformis
(Vahl) Milne-Redh.
Seeds
42
21
2-nor
ruspolinone
R=H
Ruspolia
hypercrateriformis
(Vahl) Milne-Redh.
Seeds
42
22
Nor- ruspoline
Ruspolia
hypercrateriformis
(Vahl) Milne-Redh.
Seeds
42
Ruspolia
hypercrateriformis
(Vahl) Milne-Redh.
Roots
43
HO
H
N
MeO
23
NH
Hypercratine
N
H
O
O
HO
OMe
O
13
Review of Literature
Table (1): Continued.
No.
Name
Structure
Plant source
Organ
Ref.
Aerial parts
44
Aerial parts
45
Macrorungia
longistrobus
(C.B.Clarke) Baden
Aerial parts
44
Macrorungia
longistrobus
(C.B.Clarke) Baden
Rungia grandis T.
Anders.
Aerial parts
44
Aerial parts
45
N
N
N
R
24
Macrorine
25
Nor- macrorine
26
Iso-macrorine
R = CH3
Macrorungia
longistrobus
(C.B.Clarke) Baden
Rungia grandis T.
Anders.
R=H
CH3
N
N
N
O
O
N
OR
R1
27
4-Hydroxyacanthamine
R = H, R1 = OH
Acanthus arboreus
Forssk.
Whole plant
46
28
Acanthaminoside
R = gluc., R1= H
Acanthus arboreus
Forssk.
Whole plant
46
29
Longistrobine
Macrorangia
longistrobus
(C.B.Clarke) Baden
Aerial parts
47
O
N
O
N
CH3
N
O
14
Review of Literature
Table (1): Continued.
No.
30
Name
Structure
Plant source
O
Isolongistrobine
Organ
Ref.
Macrorangia
longistrobus
(C.B.Clarke) Baden
Aerial parts
47
Macrorangia
longistrobus
(C.B.Clarke) Baden
Aerial parts
47
Leaves
48
O
Thomandersia
laurifolia T. Anderson
ex Baill.
Leaves
48
O
Thomandersia
laurifolia T. Anderson
ex Baill.
Aerial parts
49
CH3
N
N
O
N
HO
31
O
Dehydro-
CH3
N
isolongistrobine
N
O
N
O
32
33
H
N
Thomandersine
H
N
Isothomandersine
R1
R2
34
DIBOA-Glu.
R= OH,
R1= R2= H
R
N
O
O
O-glu
Acanthus ebracteatus
Vahl.
Acanthus ilicifolius L.
Acanthus volubilis
Wall.
Baphicanthus cusia
(Nees.) Bremek.
Acanthus mollis L.
Roots &
49,50
aerial parts
Aerial parts 51
Roots
52
Seeds
53,54
15
Review of Literature
Table (1): Continued.
No.
35
Name
HIBOA-Glu.
Structure
R= R1= R2= H
Plant source
Organ
Ref.
Acanthus ebracteatus
Vahl.
Acanthus ilicifolius L.
Aerial parts
49
Acanthus volubilis
Wall.
Baphicanthus cusia
(Nees.) Bremek.
36
7-Cl-DIBOAGlu.
R= OH, R1= H, R2= Cl
Acanthus ebracteatus
Vahl.
Acanthus ilicifolius L.
Roots and 49,50
aerial parts
Aerial parts 51
Roots
52
Aerial parts
49
Roots
49
37
5-HydroxyHBOA-glu.
R= R2= H, R1= OH
Acanthus ilicifolius L.
Acanthus volubilis
Wall.
Aerial parts
Aerial parts
49
51
38
DHBOA-glu.
R= R1= H, R2= OH
Acanthus ilicifolius L.
Aerial parts
40
R
N
O
O
39
HBOA
R= H
Acanthus ilicifolius L.
Acanthus mollis L.
Acanthus spinosus L.
Roots
50
Aerial parts 53,54
Aerial parts 54
40
DIBOA
R= OH
Acanthus ilicifolius L.
Roots
50
41
Justiciamide
Justicia
ghiesbreghtiana Lem.
Aerial parts
55
Strobilanthus cusia
Blume
Leaves
56
OH
H3CO
O
H3CO
OH
N
H
COONH3+
42
Tryptanthrine
O
N
N
O
16
Review of Literature
Table (1): Continued.
No.
43
Name
6-hydroxybenzoxazoline
Structure
HO
Plant source
O
O
Organ
Ref.
Acanthus arboreus
Forssk.
Whole plant
46
Macrorungia
longistrobus
C.B.Clarke
Aerial parts
44
Adhatoda vasica Nees.
Roots
Flowers
N
H
44
Macrorungine
H
N
N
O
N
CH3
45
Vasicol
NH2
N
O
OH
32,36
17
Review of Literature
2- Flavonoids:
Table (2): List of some flavonoids isolated from family Acanthaceae.
No.
Name
Structure
Plant source
R5
R1
R2
Organ
Ref.
R6
O
R7
R3
R
R4
R9
R8
O
1
Quercetin
R1=R3=R5=R8=R9=H
R= R2= R4= R6= R7= OH
Adhatoda vasica Nees
Ruellia brittoniana
Leonard.
Whole plant
Leaves &
flowers
57
58
2
Quercetin-3O-β-D-glucopyranoside
R1=R3=R5=R8=R9=H
R = O-β-D-glucose,
R2= R4= R6= R7= OH
Adhatoda vasica Nees
Whole plant
57
Ruellia brittoniana
Leonard.
Leaves &
flowers
58
3
Serpyllin
R=R3=R5=R9=H, R1= R2=
R6= R7=R8= OCH3,
R4= OH
Andrographis
serpyllifolia Vahl.
Aerial parts
59
4
Apigenin
R=R1= R3= R5= R6= R8=
R9= H,
R2= R4= R7= OH
Barleria cristata L.
Ruellia praeterrmissa
Sceinf. ex. Lindau
Barleria cristata Linn.
Flowers
Aerial parts
59
60
Flowers
61
5
Apigenin-7-Oglucoside
R= R1= R3= R5= R6= R8=
R9= H, R4= R7= OH,
R2= O-glucose
Ruellia brittoniana
Flowers &
Leonard.
leaves
Eranthemum nervosum Aerial parts
(Vahl)R.Br.
58
Aerial parts
Flowers &
leaves
63
Aerial parts
Flowers
64
65
Blepharis ciliaris(L.)
B.L.Burtt.
Ruellia brittoniana
62
58
Leonard.
Sanchezia nobilis Hook.
Barleria cristata L.
18
Review of Literature
Table (2): Continued.
No.
6
7
Name
Structure
Plant source
Organ
Apigenin-7-O-
R= R1= R3= R5= R7= R8=
Eranthemum nervosum Aerial parts
neohesperidosi-
R9= H, R2= O-glu.- glu.,
(Vahl.) R.Br.
de
R4=R6= OH
Apigenin-7-O-
R=R1=R3=R5=R6=R8=R9=H,
rutinoside
R2= O-rutinose,
Ref.
62
Barleria prionitis L.
Aerial parts
61,65
Acanthus spinosus L.
Aerial parts
66
Aerial parts
67
58
R4= R7 = OH
8
Apigenin-7-O-
R2= O-galactose, R4= R7=
galactoside
OH, R= R1= R3= R5= R6=
R8= R9= H
9
Apigenin-7-O-
R2= O-glucuronic acid, R4=
Acanthus ebracteatus
glucuronide
R7= OH, R= R1= R3= R5=
Vahl.
R6= R8= R9= H
10
7-O-Acetyl
R2= O-acetyl, R4= R7= OH,
Ruellia brittoniana
Flowers &
apigenin
R= R1= R3= R5= R6= R8=
Leonard
leaves
R2=R4= R6= R7= OH, R=
Ruellia praeterrmissa
Whole plant
60
R1= R3= R5= R8= R9= H
Sceinf. ex. Lindau
Luteolin-7-O-
R2= O-glucose, R4= R6= R7=
Asystasia gangentica
Leaves
67
β-gluco-
OH, R= R1= R3= R5= R8=
(L.) T.Anderson
pyranoside
R9= H
Acanthus spinosus L.
Aerial parts
66
Wightin
R1=R2= R3= OCH3, R4= OH,
Andrographis wightiana
Stem &
67
R= R3= R6= R7= R8= R9= H
Arn. ex. Nees.
roots
Barleria montana L.
Aerial parts
68
Aerial parts
49
R9= H
11
12
13
14
Luteolin
OH
Acylated
HO
O
luteolin-7-O-
β-D-gluco-
R2=
HO
O
CO
pyranoside
HO
R= R1= R3= R5= R8= R9= H,
R4= R6= R7= OH
15
Luteolin-7-O-
R= R1= R3= R5= R6= R9= H,
Acanthus ebracteatus
β-glucuronide
R2= O-glucuronic acid, R4=
Vahl.
R7= R8= OH
19
Review of Literature
Table (2): Continued.
No.
Name
16
6-Hydroxy-
R= R1= R5= R8= R9= H, R2=
Lepidagathis cristata
luteolin-7-O-
O-apioside, R3= R4= R6=
Willd.
apioside
R7= OH
5,7,2′,3′,4′-
R= R1= R3= R8= R9= H,
Andrographis lineata
Pentamethoxy
R2= R4= R5= R6=R7=
Nees.
flavones
OCH3
5-Hydroxy-
R= R3= R8= R9= H, R1=
Andrographis lineata
7,8,2′,3′,4′-
R2= R5= R6=R7= OCH3,
Nees.
17
18
Structure
Plant source
Organ
Ref.
Leaves
69
Whole plant
70
Whole plant
70
Whole plant
70
Whole plant
70
Whole plant
70
Whole plant
70
Whole plant
71
Whole plant
71
pentamethoxyf R4=OH
lavone
19
20
21
5,2′-Dihydroxy-
R1= R2= OCH3, R4= R5=
Andrographis lineata
7,8-dimethoxy-
OH, R= R3= R6= R7= R8=
Nees.
flavone
R9= H
5,2'-Dihydroxy-
R= R1= R3= R6= R7= R8=
Andrographis lineata
7-methoxy-
R9= H, R2= OCH3, R4=
Nees.
flavones
R5= OH
5,2'-Dihydroxy-
R= R1= R3= R6= R7= R8=
Andrographis lineata
7-methoxy-
R9= H, R2= OCH3, R4=
Nees.
flavone-2'-O-β-
OH, R5= O-glu.
D-glucopyranoside
22
5,2'-Dihydroxy-
R= R1= R3= R6= R7= R8=
Andrographis alata
7-methoxy-
R9= H, R2= OCH3, R4=
(Burm.f.) Wall. ex
flavone-5-O-β-
O-glu., R5= OH
Nees.
5,7,2'-Tri-
R= R1= R3= R6= R7=
Andrographis
methoxy
R8=R9= H, R2= R4= R5=
viscosula Nees.
flavones
OCH3
5,7,2',4',6'-
R= R1= R3= R6= R8= H,
Andrographis
Pentamethoxy
R2= R4= R5= R7= R9=
viscosula Nees.
flavone
OCH3
D-glucopyranoside
23
24
20
Review of Literature
Table (2): Continued.
No.
Name
Structure
Plant source
Organ
Ref.
25
5,2',6'-Tri-
R= R1= R3= R6= R7= R8=
Andrographis
Whole plant
71
hydroxy-7-
H, R2= OCH3, R4= R5=
viscosula Nees.
methoxy-
R9= OH
Whole plant
71
Whole plant
72
Whole plant
71
Whole plant
71
Andrographis
Stems &
67
wightiana Arn. ex
roots
flavone
26
Echiodin
R= R1= R3= R5= R6= R7=
Andrographis
R8= H, R2= OCH3, R4=
viscosula Nees.
OH, R9= O-glu.
Andrographis alata
Nees.
27
Echiodinin
R= R1= R3= R5= R6= R7=
Andrographis
R8= H, R2= OCH3, R4=
viscosula Nees.
R9= OH
Andrographis echioids
(L.) Nees.
Nees.
28
29
30
7-O-Methyl
R= R3= R4= R5= R6= R7=
Andrographis
dihydro
R8= R9= H, R1= R2=
paniculata (Burm.f.)
wogonin
OCH3
Wall. ex Nees.
7-O-Methyl
R= R3= R5= R6= R7= R8=
Andrographis
wogonin -5-
R9= H, R1= R2= OCH3,
paniculata (Burm.f.)
glucoside
R4= O-glu.
Wall. ex Nees.
5-Hydroxy-
R= R3= R5= R7= R8= H,
Andrographis
7,8,2',5'-tetra-
R1= R2= R6= R9= OCH3,
paniculata (Burm.f.)
methoxy
R4= OH
Wall. ex Nees.
5-Hydroxy-
R= R3= R5= R6= R7= H,
Andrographis
7,8,2',3'-tetra-
R1= R2= R8=R9= OCH3
paniculata (Burm.f.)
Whole plant
73
Whole plant
73
Whole plant
73
Whole plant
73
Whole plant
73
flavones
31
Wall. ex Nees.
methoxy
flavones
32
5-Hydroxy-
R= R1= R3= R6= R7= R8=
Andrographis
7,2',6'-tri-
H, R2= R5= R9= OCH3,
paniculata (Burm.f.)
methoxy
R4= OH
Wall. ex Nees.
flavones
21
Review of Literature
Table (2): Continued.
No.
33
Name
Tectochrysin
Structure
Plant source
R= R1= R3= R5=R6= R7=
Andrographis
R8= R9= H, R2= OCH3,
serpyllifolia Wt.Ic
Organ
Ref.
Leaves
74
Whole plant
73
Whole plant
73
62
R4= OH
34
35
36
Skullcap-
R= R3= R5= R6= R7= R8=
Andrographis
flavone I- 2'-
H, R1= R2= R9= OCH3,
paniculata (Burm.f.)
methyl ether
R4= OH
Wall. ex Nees.
Skullcap-
R= R3= R5= R6= R7= R8=
Andrographis
flavone I-2'-
H, R1= R2= R4= OCH3,
paniculata (Burm.f.)
glucoside
R9= O-glu.
Wall. ex Nees.
Kaempferol
R= R2= R4= R7= OH,
Eranthemum nervosum Aerial parts
R1= R3= R5= R6= R8= R9= (Vahl.) R.Br.
H
37
38
39
40
Kaempferol-3-
R= O-glu., R1= R3= R5= Eranthemum nervosum Aerial parts
O-β-D-gluco-
R6= R8= R9= H, R2= R4= (Vahl.) R.Br.
pyranoside
R7= OH
6-Hydroxy
R= R1= R5= R8= R9= H, Lepidagathis cristata
luteolin-7-O-
R2= O-apiose, R3= R4= Willd.
apioside
R6= R7= OH
Scutellarein-7-
R= R1= R5= R6= R8= R9=
O-gluco-
H, R2= O-glu.-rham., R3=
rhamnoside
R4= R7= OH
5,2',6'-Tri-
62
Leaves
69
Barleria prionitis L.
Aerial parts
75
R= R1= R3= R6= R7= R8=
Andrographis alata
Whole plant
76
hydroxy-7-
R9= H, R2= OCH3, R5=
Nees.
methoxyflavone
R9= OH, R4= O-glu.-Ac.
Aerial parts
77
Aerial parts
49
Aerial parts
51
-5-O-β-D-(6"O-octyl)-glucopyranoside
41
Schaftoside
R1= O-arab., R3= O-glu.,
Yeastesia viridiflora
R2= R4= R7= OH, R= R5=
Nees.
R6= R8= R9= H
Acanthus ebracteatus
Vahl.
Acanthus volubilis
Wall.
22
Review of Literature
Table (2): Continued.
No.
42
Name
Structure
Plant source
Iso-
R1= O-glu., R3=O- arab.,
Yeastesia viridiflora
schaftoside
R2= R4= R7= OH,
Nees.
Organ
Ref.
Aerial parts
77
Aerial parts
77
Aerial parts
77
Aerial parts
77
Aerial parts
49
Aerial parts
77
Aerial parts
51
Aerial parts
78
59
R= R5= R6= R8= R9= H
43
44
Isovitexin
R= R1= R5= R6= R8= R9= H,
Yeastesia viridiflora
R2= R4= R7= OH, R3=O-glu.
Nees.
Isovitexin-7-
R= R1= R5= R6= R8= R9=
Yeastesia viridiflora
O-rhamnoside
H, R2= O-rha., R4= R7=
Nees.
OH
45
Vicenin-2
R1= R3= O-glu., R2= R4=
Yeastesia viridiflora
R7= OH, R= R5= R6= R8=
Nees.
R9= H
Acanthus ebracteatus
Vahl.
46
Saponarin
R2= R3= O-glu., R4= R7=
Yeastesia viridiflora
OH, R= R1=R5= R6= R8=
Nees.
R9= H
47
Hispidulin-7-
R2= O-glu.-rha., R3=
Acanthus volubilis
O-rutinoside
OCH3, R4= R7= OH, R=
Wall.
R1= R5= R6= R8= R9= H
48
49
Lespedin
3-Methoxy-
R=R2=O-rh., R7=OH, R1=
Brillantaisia palisatii
R3=R4=R5=R6=R8=R9=H
Lind.
R= OCH3
Eremophilia
Leaves &
alternifolia R.Br.
branches
Andrographis lineata
Whole plant
70
Andrographis neesiana Whole plant
79
flavone
50
2′-Hydroxy2,4′,6′-tri-
H3CO
OCH3
methoxy
OH
chalcone
51
O
OCH3
2',4',6',2,3,4Hexamethoxy
Nees.
OCH3
OCH3
H3CO
OCH3
chalcone
OCH3 O
Wt.Ic
OCH3
23
Review of Literature
Table (2): Continued.
No.
52
Name
Structure
Plant source
OH
Isosalipuroside
HO
OH
Organ
Ref.
Leaves
67
Rungia repens Nees.
Leaves
61
Adhatoda vasica Nees.
Flowers
80
Asystasia gangetica
(L.) T.Anderson
O-glu. O
53
O-glu.
2′,4-Dihydroxychalcone, 4-O-
OH
β-D-glucopyranoside
O
HO
H3CO
O
OR3
OH
O
O
R2O
R1O
54
5,2′,6′-Trihydroxy-7-
O
OR
R= Ac., R1= R2= R3= H
Andrographis alata
Nees.
Whole plant
76
R= R2= H, R1= R3= Ac.
Andrographis alata
Nees.
Whole plant
76
R= R1= R2= R3= H
Andrographis alata
Nees.
Whole plant
76
R= Ac., R1= R2= H,
R3= Ac.
Andrographis alata
Nees.
Whole plant
76
methoxyflavone
-2′-O-β-D(2″O-acetyl)glucopyranoside
55
5,2',6'-Trihydroxy-7methoxyflavone
-2'-O-βD(3'',6''- di-Oacetyl)glucopyranoside
56
5,2',6'-Trihydroxyflavone2'-O-β-glucopyranoside
57
5,2′,6′-Trihydroxy-7methoxyflavone
-2′-O-β-D(2″,6″
-di-O-acetyl)-
24
Review of Literature
No.
Name
glucopyranoside
Structure
Plant source
Organ
Ref.
25
Review of Literature
Table (2): Continued.
No
.
Name
Structure
Plant source
Organ
Ref.
Whole plant
46
Whole plant
81
Whole plant
81
seeds
82
O
O
OH
OH
O
HO
O
O
RO
OH
OH
58
Apigenin 7-O-(6''-
O
R= H
Acanthus arboreus
E-p-coumaroyl-β-
W.B.Turill
D- glucopyranoside)
59
Apigenin 7-O-(3''-
R= CO-CH3
Blepharis ciliaris
acetyl-6''-E-p-
(L.) B.L.Burtt.
coumaroyl-β-Dgluco-pyranoside)
O
60
Naringenin 7-O-
Blepharis ciliaris
O
(3''-acetyl-6''-E-pcoumaroyl- β-D-
HO
H3 C
gluco-pyranoside)
O
OH
OH
O
OH
(L.) B.L.Burtt.
O
O
O
OH
O
O
61
naringenin-7-O-
Blepharis sindica
O
(6''-E-pcoumaroyl-β-D-
HO
HO
OH1''
OH
OH
O
O
T.Anderson
O
glucopyranoside
OH
O
R1
H3CO
O
R2
OR
62
7-O-Methyl-
O
R= R2= R3= H, R1= OCH3
dihydro-wogonin
63
Dihydro-skullcapflavone I
R3
Whole plant
73
Andrographis lineata Nees.
Whole plant
70
Andrographis paniculata
Whole plant
73
Whole plant
70
Andrographis paniculata
(Burm.f.) Wall. ex Nees
R= R3= H, R1= OCH3, R2= OH
(Burm.f.) Wall. ex Nees.
Andrographis lineata Nees.
26
Review of Literature
3- Iridoids:
Table (3): List of some iridoids isolated from family Acanthaceae.
No.
Name
Structure
R1
R
Plant Source
Organ
Ref.
O
R2
O-glu.
1
Hygrophiloside
R= OH, R1= H,
R2= CHO
Hygrophila
diformis Blume.
Whole
plant
83
2
Teuhircoside
R= OH, R1= =O,
R2= CH3
Andrographis
laxiflora Blume.
Whole
plant
84
3
Iso-aucubin
R= OH, R2= H, R2=
CH2OH
Hygrophila
poysperma Anderson
Whole
plant
84
4
Aucubin
R= H, R1= OH,
R2= CH2OH
Hygrophila
triflora Roxburg
Hygrophila
diformis Blume
Aerial
parts
Aerial
parts
67
Thunbergia alata
Bojer ex Sims
Leaves
85
Phaulopsis
imbricate Forssk.
Leaves
84
Asystasia bella
(Harvey) Benth. et
Hook. fil.
Leaves
84
5
Thunaloside
HO
O
HO
6
O-glu.
HO
8(S)-7,8dihydroaucubin
67
O
HO
7
O-glu.
COOCH3
Gardoside methyl
ester
HO
O
O-glu.
27
Review of Literature
Table (3): Continued.
No.
8
Name
Structure
HO
Linarioside
Plant Source
HO
Cl
O
OH
9
7-Chlorodeutziol
O-glu.
CH3
HO
Cl
O
OH
10
O
H3C
11
O-glu.
COO-glu.
Asystasioside B
O
H3C
12
O-glu.
COO-glu.
Asystasioside C
O
H2C
13
O-glu.
COO-glu.
Asystasioside D
O
HO
14
Cl
O
HO
HO
15
Asystasia bella
(Harvey) Benth. et
Hook. fil.
Leaves
86
Asystasia bella
(Harvey) Benth. et
Hook. fil.
Leaves
86
Asystasia bella
(Harvey) Benth. et
Hook. fil.
Leaves
86
Asystasia bella
(Harvey) Benth. et
Hook. fil.
Leaves
86
Asystasia bella
(Harvey) Benth. et
Hook. fil.
Leaves
86
Asystasia bella
(Harvey) Benth. et
Hook. fil.
Leaves
86
Asystasia bella
(Harvey) Benth. et
Hook. fil.
Leaves
86
Asystasia bella
(Harvey) Benth. et
Hook. fil.
Leaves
86
O-glu.
HO
Asystasioside E
Ref.
O-glu.
COO-glu.
Asystasioside A
Organ
O-glu.
OH
Rehmaglutins D
Cl
HO
O
28
Review of Literature
Table (3): Continued.
No.
16
Name
Structure
Plant Source
OH
Rehmaglutins B
OH
Cl
HO
17
Cistachlorin
Cl
HO
Asystasia bella
(Harvey) Benth. et
Hook. fil.
Leaves
86
Asystasia bella
(Harvey) Benth. et
Hook. fil.
Leaves
86
Thunbergia alata
Bojer ex Sims
Thunbergia
mysorensis (Wight)
T.Anderson ex
Bedd
Leaves
84
Leaves
84
Thunbergia alata
Bojer ex Sims
Thunbergia
laurifolia Lindl.
Thunbergia
grandiflora Roxb.
Leaves
85,87
Aerial
parts
Aerial
parts
88
Leaves
84,86
Leaves
85
Leaves
84
O
R1
R
R2
O
R3
Thunbergioside
Ref.
O
O
18
Organ
O-glu
R= R1= R2= R3= OH
19
Alatoside
R= H,R1= OH, R2= H,
R3= OH
20
Grandifloric acid
R= R1= R2= H, R3=
COOH
R1
89
R
O
O
R2
O-glu.
21
Catalpol
R= H, R1= OH, R2=
CH2OH
22
Stilbericoside
R= OH, R1= β-OH, R2=
H
Asystasia bella
Harvey Benth. et
Hook. fil.
Thunbergia
alataBojer ex Sims
Thunbergia
mysorensis (Wight)
T.Anderson ex
Bedd
29
Review of Literature
Table (3): Continued.
No.
Name
23
6-Epistilbericoside
Structure
Plant Source
R= OH, R1= α-OH, R2=
H
Thunbergia alata
Bojer ex Sims
Thunbergia
mysorensis (Wight)
T.Anderson ex
Bedd
Organ
Leaves
84
Leaves
84
84
24
Galiridoside
R= OH, R1= H, R2= CH3 Nelsonia canescens Leaves
(Lam.) Spreng.
25
Anthirrinoside
R= R1= OH, R3= CH3
R2
27
84,90
O
R3
Shanzhiside
methyl ester
Chamaeranthemum Whole
gaudichaudii Nees. plant
COOR
R1
26
Ref.
R4
R= R4= CH3, R1= R3=
OH, R2=H
O-glu.
Barleria prionitis
L.
Barleria lupulina
Lindley
Nelsonia canescens
(Lam.) Spreng.
Barleria cristata L.
Aerial
parts
Aerial
parts
Leaves
84
Whole
plant
65
Barleria lupulina
Lindley
Barleria prionitis
L.
Aerial
parts
Leaves
& stems
92
84,91,
92
84
6-O-Acetyl
shanzhiside methyl
ester
R= R4= CH3, R1= Oacetyl, R2= H, R3= OH
28
Barlerin
R= CH3, R1= OH, R2=
H, R3= O-acetyl, R4=
CH3
Barleria prionitis
L.
Barleria lupulina
Lindley
Leaves
& stems
Aerial
parts
65,84
,
93
84,91
29
Acetyl barlerin
R= CH3, R1= R3= Oacetyl, R2= H, R4= CH3
Barleria prionitis
L.
Barleria lupulina
Lindley
Barleria cristata L.
Leaves
& stems
Aerial
parts
Whole
65,93
65
84,91
,
92
30
Review of Literature
Table
Name
No. (3): Continued.
Structure
Plant Source
Organ
Ref.
plant
65
30
Mussaenosidic
acid
R= R1= R2= H, R3= OH,
R4= CH3
Hygrophila
poysperma
Anderson
Aerial
parts
84
31
Mussaenoside
R= R4= CH3, R1= R2=
H, R3= OH
Asystasia bella
Harvey Benth. et
Hook. fil.
Leaves
84
32
8-Acetyl
mussaenoside
R= R4= CH3, R1= R2=
H, R3= O-acetyl
Barleria lupulina
Lindley
Aerial
parts
94
33
8-Epiloganin
R= R3= CH3, R1= R4=
H, R2= OH
Asystasia bella
Harvey Benth. et
Hook. fil.
Leaves
84,86
34
Ipolamudoside
Barleria lupulina
Lindley
Leaves
92
Asystasia bella
(Harvey) Benth. et
Hook. fil.
Leaves
86
Asystasia bella
(Harvey) Benth. et
Hook. fil.
Leaves
86
Eranthemum
pulchellum Andr.
Aerial
parts
84,
95
Thunbergia
laurifolia Lindl.
Aerial
parts
88
HO
COOCH3
O
O
O
O-glu.
35
HO
Glutinoside
Cl
O
O
HO
O-glu.
36
O
Eutoside
O
OH
O
HO
O-glu.
Cl
37
Eranthemoside
O
HO
O-glu.
HO
38
3'-O-βglucopyranosylstilbericoside
HO
O
HO
O
O-glu.-glu.
31
Review of Literature
Table (3): Continued.
No.
39
Name
Structure
Plant Source
COOCH3
Gardioside methyl
ester
HO
O
H2C
Asystasia bella
(Harvey) Benth. et
Hook. fil.
Organ
Ref.
Leaves
84
O-glu.
O
O
COOR1
O
O
O-glu.
R
O
40
6-(p-Methoxytrans cinnamoyl)
barlerin
R= OCH3, R1= CH3
Barleria lupulina
Lindley
Aerial
parts
94
41
6-p-Coumaroyl
barlerin
R= OH, R1= CH3
Barleria lupulina
Lindley
Aerial
parts
94
42
Saletpangponoside R= O-glu., R1= CH3
A
Barleria lupulina
Lindley
Aerial
parts
94
43
Shanzhigenin
R= OH, R1= H
Shanzhigenin; 6O-(4-hydroxy-Ecinnamoyl),8Ac,1-O-β-Dglucopyranoside
R= OH, R1= H
Aerial
parts
Leaves
94
44
Barleria lupulina
Lindley
Barleria lupulina
Lindl.
94
32
Review of Literature
4- Lignans:
Table (4): List of some lignans isolated from family Acanthaceae.
No.
Name
Structure
Plant source
Organ
Ref.
OCH3
H3CO
O
H3CO
O
H3CO
R
1
Cillinaphthalide A
R= OH
Justicia ciliate Jacq.
Whole
plant
96
2
Cillinaphthalide B
R= OCH3
Justicia ciliate Jacq.
96
Justicia procumbens
L.
Whole
plant
Aerial
parts
Justicia hyssopifolia
L.
Aerial
parts
98
Justicia procumbens
L.
Aerial
parts
99
Acanthus
ebracteatus Vahl.
Acanthua ilicifolius
L.
Acanthus volubilis
Wall.
Aerial
parts
Aerial
parts
Aerial
parts
49
3
O
Justicidone
OCH3
O
O
97
H3CO
O
O
4
Justicidinoside A
O
OCH3
O
H 3CO
O
H 3CO
glu.-O
O
O
5
(+) - Lyoniresinol
3α-O-βglucopyranoside
H3CO
OH
O-glu.
HO
OCH3
H3CO
OCH3
OH
50,100
51
33
Review of Literature
Table (4): Continued.
No.
6
Name
(-) - Lyoniresinol
3α-O-βglucopyranoside
Structure
Plant source
H3CO
OH
O-glu.
HO
OCH3
H3CO
Organ
Ref.
Aerial
parts
Aerial
parts
50
Justicia hayatai Var. Aerial
parts
Justicia procumbens Aerial
L.
parts
101
Justicia hayatai Var. Aerial
parts
Justicia procumbens Aerial
L.
parts
101
Aerial
parts
99,
Acanthus ilicifolius
L.
Acanthus volubilis
Wall.
50
OCH3
OH
R
H3CO
O
H3CO
O
R1
O
O
7
8
9
Justicidin A
Justicidin B
Diphyllin
R= OCH3, R1= H
R= R1= H
R= OH, R1= H
Justicia procumbens
L.
99,
102
99,
102
102,
103
10
Tuberculatin
R= O-apio., R1= H
Justicia procumbens
L.
Aerial
parts
99,
104
11
Procumbenoside
A
R= O-apio.-arab.,
R1= H
Justicia procumbens
L.
Aerial
parts
104
12
Justicidinoside B
R= OCH3,
R1= O-glu.
Justicia procumbens
L.
Aerial
parts
99
13
Justicinoside C
R= H, R1= O-glu.
Justicia procumbens
L.
Aerial
parts
99
34
Review of Literature
Table (4): Continued.
No.
14
Name
Justicidin F
Structure
Plant source
OCH3
O
O
Organ
Ref.
Justicia procumbens
L.
Aerial
parts
104
Justicia procumbens
L.
Aerial
parts
99,
104
O
O
O
15
Justicidin C
(Neojusticin B)
O
OCH3
O
H3CO
O
H3CO
O
O
R1
R
O
O
O
O
R2
O
O
16
Justicidin D
R= OCH3, R1= R2= H
Justicia procumbens
L.
Aerial
parts
99,
102,
103
17
Justicidin E
R= R1= R2= H
Justicia procumbens
L.
Justicia hyssopifolia
L.
Aerial
parts
Leaves
102,
103
98
18
Prostalidin A
R= H, R1= OCH3, R2=
OH
Justicia prostrata
Gamble
Aerial
parts
105,
106
19
Prostalidin B
R= H, R1= R2= OCH3
Justicia prostrata
Gamble
Aerial
parts
106
20
Prostalidin C
R= R1= H, R2= OH
Justicia prostrata
Gamble
Aerial
parts
106
35
Review of Literature
Table (4): Continued.
No.
Name
Structure
Plant source
Organ
Ref.
OR
O
R1O
O
OR1
O
O
21
Orosunol
R= H, R1= CH3
Justicia flava Vahl.
Roots
107
22
8-dimethyl
orosunol
R= CH3, R1= H
Justicia flava Vahl.
Roots
107
O
O
O
O
R
R1
23
Prostalidin D
R= R1= OH
Justicia prostrata
Gamble
Aerial
parts
106
25
Retrochinensin
R= R1= OCH3
Justicia prostrata
Gamble
Aerial
parts
106
OCH3
O
O
O
O
R
OH
26
Justalakonin
R= O-glu.
Justicia purpurea
Lour.
Whole
plant
108
27
4'-De-O-methylO-methylchinensinaphthol
R= OCH3
Justicia procumbens
L.
Aerial
parts
104
36
Review of Literature
Table (4): Continued.
No.
28
Name
7,7'-Dihydrotaiwanin C
Structure
Plant source
O
O
O
Organ
Ref.
Justicia neesii
T.P.Ramamoorthy
Aerial
parts
109
Justicia neesii
T.P.Ramamoorthy
Aerial
parts
109
O
O
O
29
OH
Justirumalin
O
O
O
O
O
O
R
O
O
O
O
O
O
30
Jusneesiinol
R= OH
Justicia neesii
T.P.Ramamoorthy
Aerial
parts
109
31
Jusneesiin
R= H
Justicia neesii
T.P.Ramamoorthy
Aerial
parts
109
32
Juspurpurin
Justicia purpurea
Lour.
Whole
plant
108
O-glu.
O
O
O
O
O
O
37
Review of Literature
Table (4): Continued.
No.
Name
Structure
Plant source
Organ
Ref.
R
O
O
O
R1
O
R2
O
O
33
Justicinol
R= R1= H, R2= OH
Justicia flava Vahl.
Aerial
parts
110
34
Helioxanthin
R= R1= R2= H
Justicia flava Vahl.
Justicia hyssopifolia
L.
Aerial
parts
Leaves
110
98
35
7-Methoxyhelioxanthin
R= OCH3, R1= R2= H
Justicia hyssopifolia
L.
Aerial
parts
98
36
Jusmicranthin
R= R2= H, R1= OH
Justicia neesii
T.P.Ramamoorthy
Aerial
parts
111
Whole
plant
Whole
plant
Whole
plant
108
Whole
plant
108
O
O
O
O
R
O
R1
O
OCH3
37
Justin A
R= H, R1= OH
38
Justin B
R= CH3, R1= OH
39
Dihydroclusin
diacetate
R= R1= OCH3
Justicia procumbens
L.
Justicia procumbens
L.
Justicia procumbens
L.
108
108
O
O
H3CO
H3CO
O
R
R1
O
OCH3
40
Dimethylsecoisolariciresinol diacetate
R= H, R1= OCH3
Justicia procumbens
L.
38
Review of Literature
Table (4): Continued.
No.
Name
41
Justin C
42
Seco-isolariciresinol
dimethyl
ether
Structure
R= OCH3, R1= OH
OH
H3CO
H3CO
HO
Plant source
Organ
Ref.
Justicia
procumbens L.
Whole
plant
108
Justicia
procumbens L.
Whole
plant
108
OCH3
OCH3
OCH3
OH
H3CO
OR
O
H3CO
43
Lariciresinol,
4'-methyl
ester
R= H
Monchema
ciliatum Jacq.
Leaves
112
44
Lariciresinol
dimethyl
ether
R= CH3
Monchema
debile
Leaves
113
45
Eudesmin
Phaylopis
falcisepala
C.B.Cl.
Leaves
114
Ecbolium
linneanum Kurz.
Roots
115
OCH3
OCH3
O
H3CO
O
H3CO
46
OCH3
Ecbolin A
H3CO
O
O
O
OCH3
O
O
O
39
Review of Literature
Table (4): Continued.
No.
47
Name
Structure
HO
Justisolin
O
O
O
O
Plant source
Organ
Ref.
Justicia simplex
D.Don
Whole
plant
116
Justicia simplex
D.Don
Whole
plant
117
Justicia simplex
D.Don
Whole
plant
117
Justicia simplex
D.Don
Whole
plant
117
Justicia simplex
D.Don
Justicia
hyssopifolia L.
Phaulopsis
falcisepala
C.B.Cl.
Whole
plant
Leaves
117
Leaves
114
Justicia simplex
D.Don
Whole
plant
116
O
O
48
O
Simplexolin
O
O
O
O
O
O
O
49
O
Sesamolin
O
O
O
O
O
O
50
O
Sesamin
O
O
O
O
O
51
O
Asarinin
O
O
O
O
O
52
O-glu.
Simplexoside
O
O
O
OCH3
O
98
40
Review of Literature
5- Sterols, di-, triterpenes and other related substances:
Table (5): List of some sterols, di- and triterpenes isolated from family
Acanthaceae.
No.
1
Name
Structure
Plant source
Acanthus spinosus
L.
Acanthus mollis L.
β-sitosterol
Organ
Aerial
parts
Aerial
parts
Whole
plant
Ref.
66
Flowers
& leaves
Whole
plant
Aerial
parts
Aerial
parts
Aerial
parts
58
Ruellia brittoniana
Leonard.
Barleria prionitis
L.
Eranthemum
nervosum (Vahl)
R.Br.
Flowers
& leaves
Whole
plant
Aerial
parts
58
Sanchezia nobilis
Hook.
Barleria prionitis
L.
Barleria cristata L.
Aerial
parts
Whole
plant
Whole
plant
Aerial
parts
64
Andrographis
paniculata
(Burm.f.) Wall. ex
Nees.
Ruellia brittoniana
Leonard.
Sanchezia nobilis
Hook.
Barleria prionitis
L.
Barleria cristata L.
HO
Eranthemum
nervosum (Vahl)
R.Br.
2
H3 C
β-amyrin
CH3
CH3
H
HO
CH3
H3C
H
H3 C
3
CH3
CH3
α-amyrin
CH3
CH3
H
HO
H
H3 C
CH3
CH3
H3 C
Eranthemum
nervosum (Vahl)
R.Br.
118
73
64
65
65
62
65
62
65
65
62
41
Review of Literature
Table (5): Continued.
No.
Name
4
Stigmasterol
Structure
Plant source
HO
5
Stigmasterol
-3- Oglucoside
Organ
Ref.
Acanthus ilicifolius
L.
Acanthus arboreus
W.B.Turill
Sanchezia nobilis
Hook.
Eranthemum
nervosum (Vahl.)
R.BR
Aerial
parts
Whole
plant
119
Barleria prionitis
L.
Barleria cristata L.
Whole
plant
Whole
plant
65
Sanchezia nobilis
Hook.
Aerial
parts
64
Ruellia brittoniana
Leonard.
Eranthemum
nervosum (Vahl)
R.Br.
Flowers
& leaves
Aerial
parts
58
Sanchezia nobilis
Hook.
Eranthemum
nervosum (Vahl)
R.Br.
Ruellia brittoniana
Leonard
Aerial
parts
Aerial
parts
64
Leaves
&
flowers
58
46
64
Aerial
parts
Aerial
parts
62
65
glu-O
6
Cholesterol
HO
7
CH2
Lupeol
H3C
H
CH3
H
HO
H3C
CH3
H
H3C
8
CH3
62
β-Sitosterol3-O-βglucopyranoside
glu.-O
CH3
62
42
Review of Literature
Table (5): Continued.
No.
Name
Structure
Plant source
Organ
Ref.
O
O
OH
HO
RO
9
Andrographolide
R= H
Andrographis
paniculata
(Burm.f.) Wall. ex
Nees.
Roots &
aerial
parts
73
120
10
Andrographoside
R= glu.
Andrographis
paniculata
(Burm.f.) Wall. ex
Nees.
Whole
plant
73
11
14-Deoxy-15isopropylidene11,12-dihydroandrographolide
Andrographis
paniculata
(Burm.f.) Wall. ex
Nees.
Roots &
aerial
parts
73
Andrographis
paniculata
(Burm.f.) Wall. ex
Nees.
Roots &
aerial
parts
73
H3C
CH3
O
O
CH3
CH2
HO
CH2OH
H3C
12
O
14-Deoxy-11,12didehydroandrographolide
O
CH3
HO
H 3C
HO
CH2
43
Review of Literature
Table (5): Continued.
No.
13
Name
Structure
Plant source
O
17,19,20Trihydroxy-5-β8αH, 9βH,10αlabd-13-en-16,15Olactone
O
CH3
Organ
Ref.
Andrographis
lineata Nees.
Whole
plant
70
CH2OH
HO
HOH2C
CH2OH
O
O
R
R1
OR2
14
Neoandrographolide
R= R1= H, R2= glu.
Andrographis
paniculata
(Burm.f.) Wall. ex
Nees.
Roots &
aerial
parts
73
120
15
14-Deoxyandrographolide
R= R2= H, R1= OH
Andrographis
paniculata
(Burm.f.) Wall. ex
Nees.
Whole
plant
73
16
14-Deoxy-11hydroxyandrgrapholide
R= R1= OH, R2= H
Andrographis
paniculata
(Burm.f.) Wall. ex
Nees
Whole
plant
73
17
Phlogantholide-A
Phlogacanthus
thyrsiflorus
(Roxb.) Nees.
Leaves
121
O
O
HO
HO
44
Review of Literature
Table (5): Continued.
No.
18
Name
[α-L-arabinofuranosyl-(1→4)β-D-glucuronopyranosyl (1→3)]3-β-hydroxy-lup20(29)-ene
Structure
Plant source
Organ
Acanthus ilicifolius Roots
L.
Ref.
122
O
O
HO
COOH
O
OH
O
OH
OH
HO
RO
OH
O
OH
O
HO
HO
HO
O
OH
O
HO
HO
OR1
19
Justicoside A
R=H, R1=H
20
Justicoside B
R=H, R1= β-D-glu.
21
Justicoside C
R= CH3, R1= H
22
Justicoside D
R= CH3, R1= β-D-glu.
Justicia betonica
L.
Justicia betonica
L.
Justicia betonica
L.
Justicia betonica
L.
Aerial
parts
Aerial
parts
Aerial
parts
Aerial
parts
123
123
123
123
RO
OH
O
O
HO
HO
O
OH
O
HO
HO
OR 1
23
Justicoside E
R= R1= H
24
Justicoside F
R= H, R1= β-D-glu.
25
Justicoside G
R= CH3, R1= β-D-glu.
Justicia
betonica L.
Justicia
betonica L.
Justicia
betonica L.
Aerial parts
124
Aerial parts
124
Aerial parts
124
45
Review of Literature
6-
Phenolic, phenyl ethanoid and phenyl propanoid
glycosides:
Table (6): List of some phenolics, phenyl ethanoids and phenyl propanoid
glycosides isolated from family Acanthaceae.
No.
1
Name
Structure
Plant Source
O
Caffeic acid
OH
HO
OH
2
O
Ferulic acid
OH
HO
OCH3
3
O
Cinnamic acid
OH
4
p-hydroxy-ciscinnamic acid
H
H
O
Organ
Ref.
Andrographis
paniculata
(Burm.f.) Wall. ex
Nees
Whole
plant
73
Andrographis
paniculata
(Burm.f.) Wall. ex
Nees
Whole
plant
73
Andrographis
paniculata
(Burm.f.) Wall. ex
Nees.
Whole
plant
73
Ruellia brittoniana
Leonard
Leaves
&
flowers
58
Eranthemum
nervosum(Vahl)
R.Br.
Aerial
parts
62
Ruellia brittoniana
Leonard
Leaves
&
flowers
58
Eranthemum
nervosum(Vahl)
R.Br.
Aerial
parts
62
OH
OH
5
Benzoic acid
6
Para-methoxy
benzoic acid
O
OH
COOH
OCH3
7
Di-butyl
phthalate
O
O
O
O
46
Review of Literature
Table (6): Continued.
No.
8
Name
Structure
Plant Source
O
Bis-(2-ethyl
hexyl) phthalate
O
O
Organ
Ref.
Eranthemum
nervosum(Vahl)
R.Br.
Aerial
parts
62
Eranthemum
nervosum (Vahl)
R.Br.
Aerial
parts
62
Acanthus ilicifolius
L.
Aerial
parts
49
Acanthus ilicifolius Aerial
L.
parts
100
Acanthus volubilis
Vahl.
Aerial
parts
51
Acanthus
ebracteatus Vahl.
Thunbergia
laurifolia Lindl.
Aerial
parts
Aerial
parts
49
Acanthus ilicifolius
L.
Aerial
parts
49
Aerial
parts
Aerial
parts
Aerial
parts
Aerial
parts
51
O
9
Syringin
OCH3
.glu-O
OH
H3CO
10
OCH3
Syringic acid βD-glucopyranosyl ester
OH
O
OCH3
O-glu.
11
Cistanoside F
HO
O-glu.-rha.
HO
12
CH3
Canthoside B
H3C
CH3
O-glu.
13
14
Benzyl β-glucopyranosyl(1→2)-β-glucopyranoside
api.
glu.-glu.-O
H3CO
2,6-Dimethoxyp-hydroquinone1-O-β-glucopyranosyl ester
OH
glu-O
88
OCH3
OH
HO
R1O
O
O
OR
OR2
OR3
15
16
Phenylethyl-O-β- R= β-D-glu., R1 =R2 =R3 =H
D-glucopyranosyl(1→2)-β-Dglucopyranoside
Cistanoside E
Acanthus volubilis
Vahl.
Acanthus ilicifolius
L.
Acanthus
ebracteatusVahl.
R= H, R1= α-L-rha., R2= R3= Acanthus ilicifolius
OH
L.
100
49
100
47
Review of Literature
Table (6): Continued.
No.
Name
Structure
O
Plant Source
Ref.
OH
RO
R2
O
O
O
HO
OH
OH
O
H3C
HO
Organ
OR1
O
OH
OH
17
18
Verbascoside
R= R1= R2= H
Leucoscepto- R= CH3, R1= R2= H
side A
19
Martynoside
R= R1= CH3, R2= H
20
β-hydroxyaceteside
R= R1= H, R2= OH
21
22
23
Aerial
parts
Aerial
parts
Aerial
parts
Blepharis edulis
Aerial
(Forssk.) Pers.
parts
Acanthus
Aerial
ebracteatus Vahl. parts
Blepharis edulis
Aerial
(Forssk.) Pers.
parts
Acanthus
Aerial
ebracteatus Vahl. parts
Acanthus
Aerial
ebracteatus Vahl. parts
Acanthus
Aerial
ilicfolius L.
parts
Acanthus
Aerial
ilicfolius L.
parts
Acanthus
Aerial
ilicfolius L.
parts
Acanthus
Aerial
ebracteatus Vahl. parts
Acanthus volubilis Aerial
Vahl.
parts
Blepharis edulis
Aerial
(Forssk.) Pers.
parts
Acanthus
ebracteatus Vahl.
Acanthus
ilicifolius L.
Acanthus volubilis
Vahl.
Campneoside I
Ilicifolioside
A
Isoverbascoside
R= R1= H,R2= OCH3
R= R1= H, R2= OC2H5
O
HO
O
HO
O
HO
O
H3C
HO
OH
OH
O
OH
O
OH
49
49,
100
51
125
49
125
49
49
49
100
100
49
50
125
OH
24
3',4'-Dihydroxy-βphenyl ethyl
caffeate-4'-βO-D-galactopyranosyl(1'''→ 4'')-αO-L-rhamnopyranoside
O
HO
O
OH
O
H3C
OH
OH
O
O
OH
O
OH
OH
OH
OH
Blepharis ciliaris
L.
Aerial
parts
82
48
Review of Literature
Table (6): Continued.
No.
25
26
27
28
29
30
Name
9-O-β-glucopyranosyl
trans
cinnamyl
alcohol
4-O-β-glucopyranosyl3,5-dimethoxy
trans
cinnamyl
alcohol
(Syringin)
β-xylopyranosyl(1→6)-βglucopyranosyl-(1→6)-βgucopyranoside trans
cinnamyl
alcohol
β-glucopyranosyl
benzyl
alcohol
β-apiofuranosyl(1→6)-βglucopyranosyl benzyl
alcohol
4-O-β-glucopyranosyl
dehydrodiconiferyl
alcohol
Structure
HO
O
O
HO
Plant Source
Organ
Ref.
Sanchezia nobilis
Hook.
Aerial
parts
64
Sanchezia nobilis
Hook.
Aerial
parts
64
Sanchezia nobilis
Hook.
Aerial
parts
64
Sanchezia nobilis
Hook.
Aerial
parts
64
Sanchezia nobilis
Hook.
Aerial
parts
64
Sanchezia nobilis
Hook.
Aerial
parts
64
OH
HO
OH
O
H3CO
HO
OH
HO
CH2OH
O
H3CO
HO
O
HO
OH
HO
O
O
HO
OH
HO
O
O
O
HO
OH
HO
OH
O
H2
C
O
HO
OH
HO
O
O
OH
HO
O
HO
H2
C
O
HO
HO
OH
CH2 OH
HOH3C
O
HO
O
HO
HO
OH
O
CH3
CH3
49
Review of Literature
No.
31
Name
Vanillic acid
Structure
O
OH
OCH3
OH
Plant Source
Acanthus
arboreus
W.B.Turill
Organ
Ref.
Whole
plant
46
50
Review of Literature
7- Megastigmane glycosides:
Table (7): List of some megastigmanes isolated from family Acanthaceae.
No.
Name
Structure
Plant source
Organ
Ref.
OR2
RO
OR1
1
Plucheoside B
R= glu., R1= R2= H
Acanthus
ebracteatus Vahl.
Acanthus
ilicifolius L.
Aerial
parts
Aerial
parts
49
49
2
Alangionoside
C
R= R2= H, R1= glu.
Acanthus
ebracteatus Vahl.
Aerial
parts
49
3
Ebracteatoside
A
R= R1= H, R2= glu.(2′-1″)apio
Acanthus
ebracteatus Vahl.
Aerial
parts
49
4
Premnaionoside
C
Acanthus
ebracteatus Vahl.
Aerial
parts
49
O-glu(2'-1'')apio.
O
HO
51
Review of Literature
8- Simple aliphatic compounds:
Table (8): List of some simple aliphatic compounds isolated from family
Acanthaceae.
No.
Name
1
Ebracteatoside
D
2
Structure
Plant source
HO
CH2
O-glu.-xyl.
O
Ilicifolioside C
HO
CH2
Organ
Ref.
Acanthus
Aerial
ebracteatus Vahl. parts
49
Aerial
parts
49
Sanchezia nobilis Aerial
Hook.
parts
64
Sanchezia nobilis Aerial
64
Acanthus
ilicifolius L.
O-glu.-glu.-xyl.
3
Palmitic acid
4
1-octen-3-ol
(Matsutake
alcohol)
5
6
5-aminononane-2,8dione
CH3-(CH2)14-COOH
OH
O
H
O
H3C
CH3
NH2
3-O-β-glucopyranosyl-1octen-3-ol
O
O
HO
Hook.
parts
Ruellia
brittoniana
Leonard
Leaves
&
flowers
58
Sanchezia nobilis Aerial
Hook.
parts
64
H
Sanchezia nobilis Aerial
Hook.
parts
64
H
Sanchezia nobilis Aerial
Hook.
parts
64
H
OH
HO
HO
7
3-O-β-glucopyranosyl(1→6)-βglucopyranosyl
-1- octen-3-ol
O
O
HO
O
HO
OH HO
O
OH
HO
HO
8
3-O-α-arabinopyranosyl(1→6)-βglucopyranosyl
-1- octen-3-ol
O
O
HO
HO
O
HO
OH HO
O
OH
52
Review of Literature
Table (8): Continued.
No.
Name
9
3-O-α-arabinopyranosyl(1→6)-βglucopyranosyl
-(1→6)-βglucopyranosyl
-1-octen-3-ol
Structure
Plant source
O
O
O
O
O
O
HO
OH HO
H
Organ
Ref.
Sanchezia nobilis Aerial
Hook.
parts
64
Acanthus
ebracteatus Vahl.
Acanthus
ilicifolius L.
Aerial
parts
Aerial
parts
49
Acanthus
ebracteatus Vahl.
Acanthus
ilicifolius L.
Aerial
parts
Aerial
parts
49
Acanthus
ebracteatus Vahl.
Acanthus
ilicifolius L.
Aerial
parts
Aerial
parts
51
OH
HO
OH HO
HO
HO
R
OR1
O
O
HO
HO
OH
HO
HO
3
4
5
Ebracteatoside
B
Ebracteatoside
C
Ilicifolioside B
6
n-Hexyl-βglucopyranoside
7
(E)-2-hexenylβ-glucopyranoside
O
O
OH
R= H, R1= xyl.
R= OH, R1= H
R= OH, R1= xyl.
49
49
100
H 3C
O-glu.
Thunbergia
laurifolia Lindl.
Aerial
parts
88
H 3C
O-glu.
Thunbergia
laurifolia Lindl.
Aerial
parts
88
53
Review of Literature
9- Quaternary ammonium compounds:
Table (9): List of some quaternary ammonium compounds isolated from
family Acanthaceae.
No.
1
Name
Structure
Plant source
-
COO
Trigonelline
Organ
Ref.
Acanthus ilicifolius Aerial
L.
parts
100
Eranthemum
pulchellum Andr.
Aerial
parts
95
Eranthemum
pulchellum Andr.
Aerial
parts
95
N+
CH3
2
O
Betaine
N+
O
3
(Me)3-N+-CH2-CH2-OH
Choline
10- Miscellaneous compounds:
Table (10): List of some miscellaneous compounds isolated from family
Acanthaceae.
No.
1
Name
Structure
Plant source
H2N
Adenosine
Acanthus ebracteatus
Vahl.
Acanthus ilicifolius L.
N
N
N
O
N
HO
HO
OH
Acanthus volubilis
Wall.
Organ
Aerial
parts
Aerial
parts
Aerial
parts
Ref.
49
50,
100
51
54
Review of Literature
II- Biological Review:
Table (11): Some biological activities carried on different plants of family
Acanthaceae.
No.
1
2
3
Pharmacological
action
Cardio-vascular
activity
Anti-oxidant activity
Anti-microbial
activity
4
Vasorelaxant effect
5
Anti-platelet activity
6
Anti-angiogenic
activity
Plant source
Plant extract
Ref.
Andrographis paniculata
(Brum.f.) Nees.
Crude water extract
n- butanol fraction
126
Blepharis lineariifolia
Pers.
Dicliptera verticillata
Forrsk.
Dyschoriste perrottetii
Nees.
Hygrophila auriculata
Schumach
Lepidagathis anobrya
Nees.
Nelsonia canescens (Lam)
Spreng
Phaulopsis fascisepala
C.B.Cl
Andrographis echioides
(L.) Nees.
Boerhavia diffusa L.
Monchema ciliatum Jacq.
Andrographis paniculata
(Brum.f.) Nees.
Barleria prionitis L.
Blepharis ciliaris
(L.)B.L.Burtt.
Brillantaisia nitens Lindau
Aqueous-acetone
extract
Aqueous fraction
127
127
Aqueous fraction
127
Crude extract
Ethanolic extract
Ethanolic extract
Methanolic extract
Ethanolic extract
127
Crude extract
128
Aqueous fraction
129
Aqueous fraction
Aqueous fraction
Chloroform extract
129
130
Bark extracts
Total extract
65
63
Methylene
chloride/methanol
extract
Total extract
131
132
Total extract
133
Andrographis paniculata
(Brum.f.) Nees.
Andrographis paniculata
(Brum.f.) Nees.
127
127
55
Review of Literature
Table (11): Continued.
No.
7
Pharmacological
action
Anti-inflammatory
effect
Plant source
8
Immuno-stimulant
activity
Andrographis paniculata
(Brum.f.) Nees.
Hygrophila spinosa
T.Anders
Blepharis lineariifolia
Pers.
Dicliptera verticillata
Forrsk.
Dyschoriste perrottetii
Nees.
Hygrophila auriculata
Schumach
Lepidagathis anobrya
Nees.
Andrographis paniculata
(Brum.f.) Nees.
9
Hepatoprotective
activity
Andrographis paniculata
(Brum.f.) Nees.
10
Choleretic effect
11
Anti-malarial effect
12
Anti-diabetic effect
Andrographis paniculata
(Brum.f.) Nees.
Andrographis paniculata
(Brum.f.) Nees.
Andrographis paniculata
(Brum.f.) Nees.
Asteracantha longifolia
Nees.
Hygrophila auriculata
Schumach.
Strobilanthes crispus
Blume
Hygrophila spinosa
T.Anders
13
Anti-pyretic effect
14
Acanthus ilicifolius L.
15
Anti-mutagenic
effect
Cytotoxic effect
16
Purgative effect
Ruellia praetermissa
Sceinf.ex. Lindau
17
Analgesic effect
Lepidagathis anobrya Nees.
Justicia hyssopifolia Linn.
Plant extract
Ref.
Active component:
andrographolide
Aqueous fraction
134
Aqueous-acetone
extract
Crude extract
127
Ethanolic extract
127
Ethanolic extract
127
Methanolic extract
127
Ethyl-acetate
extract
Active component:
andrographolide
Total extract
Active component:
andrographolide
Active component:
andrographolide
Chloroformic
extract
Ethanolic extract
135
127
127
136
137
138
139
140
141
Aqueous extract
142
Alcoholic extract
Chloroformic
extract
Totat extract
143
144
Active component:
Elenoside
Methanolic extract
145
Methanolic extract
147
146
Review of Literature
56
54
AIM OF WORK
Previous Phytochemical and biological studies of Acanthaceous
plants especially Anisotes trisulcus (Forssk.) Nees. and Blepharis ciliaris
(L.)B.L. Burtt. showed the isolation and identification of diverse secondary
metabolites as well as different biological activities. Thus, it was deemed
important to carry out comprehensive phytochemical and biological
investigations of the selected plants. A work plan for the study was
designed to explore the phytoconstituents and the biological activity for the
two plants under investigation as follows:
1. Phytochemical study of Anisotes trisuclus (Forssk.) Nees. Aerial
parts.
a- Screening of Anisotes trisulcus (Forssk.) Nees. aerial parts for its
different phytoconstituents content.
b- Extraction, fractionation, isolation and identification of the
different phytoconstituents of the aerial parts of Anisotes trisuclus
(Forssk.) Nees. aerial parts.
2. Phytochemical study of Blepharis ciliaris (L.)B.L. Burtt. aerial
parts:
a- Screening of Blepharis ciliaris (L.)B.L. Burtt. aerial parts for the
different phytoconstituents content.
b- Extraction, fractionation, isolation and identification of the
different phytoconstituents of the aerial parts of Blepharis ciliaris
(L.)B.L. Burtt. aerial parts.
3. Biological studies:
a- Biological studies of Anisotes trisuclus (Forssk.) Nees.
b- Biological studies of Blepharis ciliaris (L.)B.L. Burtt.
55
MATERIALS, APPARATUS AND
TECHNIQUES
1- Materials:
1.1. Plant Material:
The plant material used in this work consists of the air-dried aerial
parts of Anisotes trisulcus (Forssk.) Nees.. The plant material was
collected during the flowering season on March, 2005 from AL-Baha: AlAbnaa escarpment (KSA). In addition to the air-dried aerial parts of
Blepharis ciliaris (L.) B.L.Burtt., collected during the flowering season on
May, 2005 from Wadi Al-Ratam Southern Sinaii, Egypt. The two plants
were kindly identified by Prof. Dr.A.A.Fayed, Prof. of Plant Taxonomy,
Faculty of Science, Assiut University, Assiut, Egypt. Voucher samples
were kept in the Herbarium of the Pharmacognosy Department, Faculty of
Pharmacy, Assiut University, Assiut, Egypt.
1.1.1. Materials for Phytochemical Study:
The aerial parts were collected, air dried at room temperature, then
powdered and macerated in 70% methanol. The alcoholic extract of
Anisotes trisulcus (Forssk.) Nees. (2.5 kg) and Blepharis ciliaris (L.)
B.L.Burtt. (3 kg) were concentrated under reduced pressure to get a
viscous residue (300 gm) and (270 gm) respectively which were used for
isolation of their different constituents.
1.2. Authentic Reference Materials:
• Sterols and triterpenes: β-sitosterol, β-sitosterol glucoside, stigmasterol,
α-amyrin, were obtained from the Pharmacognosy Department, Faculty
of Pharmacy, Assiut University, Assiut, Egypt.
56
Materials, Apparatus and Techniques
• Flavonoids and Phenolics were obtained from the Pharmacognosy
Department, Faculty of Pharmacy, Assiut University, Assiut, Egypt.
• Sugars:
glucose
and
rhamnose
were
obtained
from El-Nasr
Pharmaceutical and Chemical Co., Egypt (ADWIC).
1.3. Solvents:
1.3.1. Solvents for Extraction:
• The solvents used in this work include: n-hexane, chloroform, ethyl
acetate, n-butanol, methanol and ethanol were purchased from El-Nasr
Pharmaceutical and Chemical Co.. These solvents were subjected to
purification process as described by Vogel(148) and were used for
chromatographic and analytical purposes as well as for crystallization.
1.3.2. NMR-Solvents:
• The following deuterated solvents have been used in the NMR spectral
analysis: deuterated pyridine, CDCl3 and DMSO-d6.
• Tetramethyl silane (TMS) was used as an internal standard.
1.4. Materials for Chromatographic Study:
1.4.1. Adsorbents:
• Silica
gel
G60
(70-230
mesh,
Prolabo,
India)
for
column
chromatography.
• Precoated silica gel plates G60 F254 (Aluminum sheets, E-Merck,
Germany) for TLC.
• Precoated alumina plates (Aluminum sheets, E-Merck, Germany) for
TLC.
• Precoated reversed phase silica gel plates RP-18 F254 (Aluminum sheets,
E-Merck, Germany) for TLC.
• Reversed phase (RP-18) silica gel (E-Merck, Germany) for column
chromatography.
57
Materials, Apparatus and Techniques
• Sheets of Whatmann paper No.I chromatographic paper (Whatmann,
Ltd., England).
• Sheets of Whatmann paper No.III chromatographic paper (Whatmann,
Ltd., England) for preparative purposes.
1.4.2. Solvent Systems:
• I- n-hexane – ethyl acetate
(9.5:0.5) v/v
• II- n-hexane – ethyl acetate
(9:1) v/v
• III- n-hexane – ethyl acetate
(8:2) v/v
• IV- n-hexane – ethyl acetate
(7:3) v/v
• V- Chloroform – methanol
(9.5:0.5) v/v
• VI- Chloroform – methanol
(9:1) v/v
• VII- Chloroform – methanol
(8:2) v/v
• VIII- Chloroform – methanol
(7:3) v/v
• IX- n-Butanol – acetic acid – water
(4:1:2) v/v
• X- n-butanol – acetic acid – water
(5:4:3) v/v
1.4.3. Spraying Reagents:
• Sulphuric acid(149): is used for general detection 10% v/v concentrated
sulphuric acid in methanol, the chromatograms were allowed to dry for
15 min at room temperature then heated at 110⁰C till characteristic
colours developed and reached their maxima.
• Modified Dragendorff’s reagent(150,151) for detection of alkaloids.
• Aluminum chloride reagent(152): 5% aluminum chloride in methanol,
for detection of flavonoids.
• Ammonium hydroxide vapour(153): for detection of flavonoids.
• Ferric chloride solution(154): 10% solution of Ferric chloride in 0.5 N
HCl.
• Aniline hydrogen phthalate(155): for detection of sugar spots after
spraying the chromatograms they are allowed to dry at room
temperature then heated in hot air oven at 120°C.
Materials, Apparatus and Techniques
58
1.5. Reagents for Phytochemical Screening:
The following reagents were used during the preliminary
phytochemical screening of the plants under investigation which include:
Molish’s reagent(156), Antimony trichloride(157), Baljet’s reagent(150), aniline
hydrogen phthalate(155), Modified Dragendorff’s reagent(150,151), Kedd’s
reagent(150), Wagner’s reagent(158,159), Mayer’s reagent(158), concentrated
ammonia, glacial acetic acid, acetic anhydride, benzidine, hydrogen
peroxide, concentrated sulphuric acid (ADWIC Co., Egypt), Trim-Hill
reagent(155,,160), Fehling and Barfoed’s reagents(150). These reagents were
prepared according to their corresponding references mentioned.
1.6. Reagents for UV Spectral Analysis(161):
• Sodium methoxide (NaOMe): 2.5 g freshly cut metallic sodium were
added cautiously in small portions to 100 ml dry spectroscopic
methanol.
• Aluminum chloride (AlCl3): 5 g fresh anhydrous AlCl3 were added
cautiously to 100 ml spectroscopic methanol.
• Hydrochloric acid (HCl): (50%).
• Sodium acetate (NaOAc)
• Boric acid (H3BO3).
1.7. Materials for Biological Activity:
1.7.1. Preparation of the Sample for Biological Activity:
• Preparation of the different extracts for biological activity:
The air dried powdered aerial parts of both plants (200 gm) were
extracted with methanol by percolation and the percolate was evaporated
under reduced pressure till complete dryness. The concentrated total
methanolic extract was digested in the least amount of distilled water,
59
Materials, Apparatus and Techniques
transferred to a separating funnel and partitioned with successive portions
of n-hexane, chloroform and ethyl acetate. Each extractive was
concentrated under reduced pressure to yield solvent free residue.
1.7.2. Materials for Antioxidant Activity:
• 1,1-Diphenyl-2-Picryl-Hydrazyl
radical
(DPPH),
Sigma-Aldrich
Chemicals Co., Germany.
• Quercitin, Sigma-Aldrich Chemicals Co., Germany.
1.7.3. Materials for Acute Toxicity:
• Male albino rats each (120-150 gm) obtained from the pre-clinical
animal house of the Pharmacology Department, Faculty of Medicine,
Assiut University, Assiut.
• 2% tween-80 obtained from the Pharmaceutical Department, Faculty of
Pharmacy, Assiut University.
1.7.4. Materials for Brine-Shrimp Assay:
• Commercial sea-salt (339 salt/liter).
• Brine shrimp eggs (Artemia salina Leach.), Dohse, Aquaristik GmbH,
Bonn, Germany.
• Magnetic stirrer.
1.7.5. Materials for Anti-inflammatory Activity:
• Male albino rats each (120-150 gm) were bred and housed under
standardized environmental conditions in the pre-clinical animal house,
Pharmacology Department, Faculty of Medicine, Assiut University,
Assiut, Egypt.
• 3% PEG 600 in normal saline.
• Normal saline 0.9% (El-Nasr Pharmaceutical and Chemical Co., Egypt).
Materials, Apparatus and Techniques
60
• Indomethacin as a standard anti-inflammatory drug (El-Nile Co.,
Egypt).
• 1% Cargeenin for inducing oedema (Sigma Chemical Co.)
• Smiec Vernier Caliper (China) for measuring the paw thickness in the
anti-inflammatory activity.
• Disposable syringes.
1.7.6. Materials for Anti-malarial Activity:
• Plasmodium falciparum obtained from the American type culture
collection (Manassas, AV).
• Standard antimalarial drug: Chloroquine.
1.7.7. Materials for Anti-hyperglycaemic Activity:
• Male adult albino mice (20-25 gm) were bred and housed under
standardized environmental conditions in the pre-clinical animal house,
Pharmacology Department, Faculty of Medicine, Assiut University,
Assiut, Egypt.
• Disposable syringes.
• 1% Tween 80 obtained from the Pharmaceutical Department, Faculty of
Pharmacy, Assiut University.
• Glucostar device for measuring blood glucose level (Miles-Sankyo Co.,
Ltd., Tokyo, Japan).
• Blood glucose test strips (Acon Laboratories, San Diego, CA 92121,
USA).
2- Apparatus and Equipments:
• Rectangular glass jars of different sizes.
• Glass plates of different dimensions.
• Glass columns of variable dimensions were employed for column
chromatography.
Materials, Apparatus and Techniques
61
• Vacuum-Liquid chromatography column (VLC).
• Capillary glass tubes.
• Atomizers for spraying the chromatograms.
• Separating funnel for fractionation of different plant extracts.
• Circulating hot air oven (Lotus, Egypt).
• Rotary flash evaporator, Heidolph WB 2000 (Germany).
• Digital balance, Sartorius electrical balance TE 2145 (Hanover,
Germany).
• Ultrasonic cleaner (Cole-Parmer, Chicago, USA).
• Agilent GC/MS (7890A/5975B) spectrometer (USA) used for the
analysis of saponifiable and unsaponifiable contents.
• Electrothermal 9100 Digital Melting Point apparatus (Electrothermal
Engineering Ltd., Essex, England).
• Shimadzu 1601 UV/VIS spectrophotometer.
• Portable UV-lamp (254, 366 nm, VL, 6LC, Marine Lavalee-Cedex,
France).
• Shimadzu Infrared-400 spectrometer (Kyoto, Japan).
• 1H and 13C NMR were run at 400 and 100 MHz respectively using Jeol
Oxford NMR YH-400, Varian-Mercury 400 MHz (Oxford) and Bruker
AVANCE AVIII 700 MHz NMR spectrometer (Oxford).
• EI and FAB-MS were recorded on Jeol the MS route JMS 600H.
• ADP410 Automatic Digital Polarimeter (Bellingham and Stanely Ltd.)
for measuring the opltical rotation.
• Matrix Assisted Laser Desorption Ionization Time of Flight Mass
Spectrometry (MALDI-TOF-MS) was recorded on a Bruker microTOF-mass spectrometer; +TOF MS: 0.239 min. Max. 2.5e6 counts .
3- Techniques:
Materials, Apparatus and Techniques
62
3.1. Chromatographic Techniques:
3.1.1-Techniques for TLC(162,163):
TLC was used to monitor the identity of each of the fractions and
the qualitative purity of the isolated compounds. It was also utilized to
optimize the solvent system that would be applied for column
chromatography.
3.1.2. Vacuum Liquid Chromatography(152):
Vacuum liquid chromatography (VLC) is a normal phase column
using silica gel as a stationary phase. It is a useful method for initial
isolation procedure for large amounts of sample.
3.1.3. Column Chromatographic Technique(152):
Fractions derived from VLC were screened by TLC and subjected
to repeated separation by column chromatography (c.c.) using appropriate
stationary phases and mobile phases.
The wet method was followed in packing the column. The column
was developed and eluted with the suitable solvent in the order of
increasing polarities.
3.1.4. Paper Chromatographic Technique(161):
Sheets of Whatmann paper No.1 were used. A horizontal line
(starting line) was drawn with a pencil across the width of the sheet about
2cm from the lower edge. The solutions of the samples were applied on
the starting line by a micro-pipette and left to dry spontaneously.
The sheets were introduced into the developing chambers in which
the chosen solvent system was placed and left for equilibrium. After
development, the wet paper was carefully removed and dried. The spots
Materials, Apparatus and Techniques
63
were visualized by UV light and/or by spraying with the suitable spray
reagents.
3.2. Preparation of the Fatty Acids(164):
About 5 gm of the n-hexane extract of the aerial parts of both plants
were saponified by refluxing with 0.5 N alc. KOH for three hours on a
boiling water bath. The major part of the alcohol present was distilled off
and the concentrated extract was diluted with distilled water then extracted
with several portions of ether till exhaustion. The alkaline aqueous solution
(soap) that remained after the removal of the unsaponifiable matter was
acidified with sulphuric acid (20%) and the liberated fatty acids were
extracted with ether. The combined ether extract was washed several times
with distilled water till the washings were free from any acidity. The ether
extract was dried over anhydrous sodium sulphate. The solvent was
distilled off under reduced pressure to give a viscous residue of the free
fatty acids, which have yellowish brown colour. A part of the residue was
subjected to methylation.
3.3. Preparation of the Fatty Acids Methyl Esters(164):
The residue obtained after solvent evaporation was subjected to
methylation by mixing with anhydrous K2CO3 (2 gm) and (CH3)2SO4 (5ml)
in dry acetone and refluxed for 4 hrs followed by filtration. The filterate
was concentrated to remove acetone, diluted with water and extracted with
ethyl acetate. The ethyl acetate layer was washed with water, dried over
anhydrous sodium sulphate and then concentrated to yield an oily residue
kept for further investigation.
3.4. Gas/Liquid Chromatography Technique(155):
64
Materials, Apparatus and Techniques
The following conditions were used in GLC for analysis of methyl
esters of fatty acids and silylated un-saponifiable matter.
Table (12): Conditions for Gas/Liquid chromatographic analysis.
Condition
Column
Program
Injector
temp.
Detector
temp.
Fatty acid methyl ester
3% OV-17 on 80% CWHP, 6',
1/8'' x 0.085'', S.S
Silylated unsaponifiable matter
4% OV-101 + 6% OV-210 on
80% CWHP, 200 cm x 6.35 mm x
2mm, S.S
160°C for 2 min. then increase
100°C for 2 min. then increase by
by rate 15°C/min. till 300°C
rate 10°C and isothermal for 25
and isothermal for 15 min.
min.
250°C
150°C
320°C with FID
250°C with FID
where: OV-17 = methyl phenyl silicone type of stationary phase; OV-101, OV210 = methyl silicone type of stationary phase; CWHP = carbowax HP; FID =
Dual flame ionization detector.
3.5. Acid Hydrolysis(161):
A solution of the isolated glycoside (5 mg in 10 ml MeOH) was
treated with 3% H2SO4 (1.5 ml) and heated at 100°C for 1hr. The aglycone
was extracted with EtOAc, concentrated under reduced pressure and
identified by co-TLC with an authentic sample using system VI. The
sugars in the aqueous layer were identified by paper chromatography with
authentic materials using system IX & X.
3.6. Alkaline Hydrolysis(165):
Materials, Apparatus and Techniques
65
Certain amount of the pure compound was dissolved in 10%
ethanolic potassium hydroxide (10 ml) and left overnight at room
temperature. The reaction mixture was neutralized with 10% acetic acid
and extracted with ether (10 ml x 4). The combined ethereal fractions were
dried over anhydrous sodium sulphate then prepare the fatty acid methyl
ester (P.63, 64).
3.7. Methanolysis for ceramides(166, 167):
About 20 mg of the compound were continuously stirred with 0.9 N
HCl in 82% aqueous methanol at 80°C for 18 hrs. The reaction mixture
was extracted with n-hexane (3x10 ml) and the combined organic layer
was dried over anhydrous Na2SO4. Evaporation of the hexane under
reduced pressure to yield the fatty acid methyl ester (FAME).
The aqueous methanol layer was neutralized with conc. NH4OH and
extracted with ether (3x10 ml). The ether layer was dried over anhydrous
Na2SO4, filtered and then concentrated to yield the long chain base (LCB).
Powdered Aerial Parts
(2.5 Kg)
Maceration with methanol
(300gm)
-solventfractionation
n-hex. fr.
CHCl3 fr.
EtOAc fr.
n-BuOH.fr.
Aqu.fr.
(50 gm)
(35 gm)
(22 gm)
(8 gm)
(145 gm)
Scheme (1): Extraction and fractionation of the total methanolic extract of Anisotes trisulcus (Forssk.) Nees. aerial parts.
n- hexane fraction
(45 gm)
VLC, gradient elution using n-hexane- ethyl acetate
n-hexane
(10 gm)
n-hex.: EtOAc (9:1)
(8.5 gm)
n-hex.: EtOAc (8:2)
(5.5 gm)
ATH-I
ATH-II
ATH-III
- silica gel c.c
- n-hex/EtOAc
(gradient elution)
n-hex.: EtOAc (9.5:0.5)
(1 gm)
AT-1
(50 mg)
n-hex.: EtOAc (9:1)
(3 gm)
n-hex.: EtOAc (7:3)
(6.5 gm)
ATH-IV
n-hex.: EtOAc (6:4)
(5.5gm)
EtOAc
(5.5 gm)
ATH-V
ATH-VI
- silica gel c.c
- n-hex/EtOAc (gradient elution)
AT-4 (50 mg)
AT-2 (100 mg)
AT-3 (30 mg)
Scheme (2): Isolation of the main constituents from the n-hexane fraction of Anisotes trisulcus (Forssk.) Nees. aerial parts.
Chloroform Fraction
(30 gm)
VLC, gradient elution using n-hexane: ethyl acetate
n-hexane
(2.5 gm)
n-hex.: EtOAc (9:1)
(8 gm)
n-hex.: EtOAc (8:2)
(8 gm)
ATC-II
ATC-III
ATC-I
- silica gel c.c
- n-hex/EtOAc
(gradient elution)
AT-5 (20 mg)
AT-6 (40 mg)
n-hex.: EtOAc (7:3)
(6.5 gm)
ATC-IV
- silica gel c.c
- n-hex/EtOAc
(gradient elution)
AT-7 (50 mg)
EtOAc
(3.5 gm)
AT-8 (40 mg)
ATC-V
- silica gel c.c
- n-hex/EtOAc
(gradient elution)
AT-9 (20 mg)
AT-10 (20 mg)
Scheme (3): Isolation of the main constituents from the chloroformic fraction of Anisotes trisulcus (Forssk.) Nees. aerial parts.
EtOAc fraction
(18 gm)
VLC, gradient elution using CHCl3/MeOH
Chloroform
(1.5 gm)
CHCl3: MeOH (9.5:0.5)
(4.5 gm)
ATE-I
AT-11
(50 mg)
CHCl3:MeOH (9:1)
(5.5 gm)
ATE-II
AT-12
(100 mg)
CHCl3:MeOH (8:2)
(3.5 gm)
CHCl3:MeOH (7:3)
(1.5 gm)
ATE-IV
ATE-V
ATE-III
-silica gel c.c.
-CHCl3/MeOH
-silica gel c.c.
-CHCl3/MeOH
-silica gel c.c.
- CHCl3/MeOH
-silica gel c.c.
- CHCl3/MeOH
(gradient elution)
(gradient elution)
(gradient elution)
(gradient elution)
AT-13
AT-14
AT-15
AT-16
AT-17
(300 mg)
(30 mg)
(20 mg)
(10 mg)
(12 mg)
Scheme (4): Isolation of the main constituents from the ethyl acetate fraction of Anisotes trisulcus (Forssk.) Nees. aerial parts.
n-Butanol Fraction
(8 gm)
-silica gel c.c
-CHCl3: MeOH (gradient elution)
CHCl3: MeOH (9.5:0.5)
(2.5 gm)
CHCl3: MeOH (9:1)
(2.5 gm)
ATB-I
ATB-II
-aluminium oxide c.c
-CHCl3/MeOH (gradient elution)
AT-18 (10 mg)
CHCl3: MeOH (8:2)
(1.2 gm)
ATB-III
CHCl3: MeOH (7:3)
(1 gm)
ATB-IV
-alumina c.c
-CHCl3/MeOH (gradient elution)
AT-19 (30 mg)
AT-20 (15 mg)
Scheme (5): Isolation of the main constituents from the n-butanol fraction of Anisotes trisulcus (Forssk.) Nees. aerial parts.
Powdered aerial parts
(3 Kg)
Maceration with methanol
(270 gm)
-solvent fractionation
n-hex. fr.
CHCl3 fr.
EtOAc fr.
n-BuOH fr.
(55 gm)
(37 gm)
(45 gm)
(12 gm)
(kept for further study)
Aqu.fr.
(105 gm)
(kept for further study)
Scheme (6): Extraction and fractionation of the total methanolic extract of Blepharis ciliaris (L.)B.L.Burtt. aerial parts.
n- hexane fraction
(45 gm)
VLC, gradient elution using n-hexane- ethyl acetate
n-hexane
(6.5 gm)
BCH-I
n-hex.: EtOAc (9:1)
(9.5 gm)
n-hex.: EtOAc (8:2)
(5.5 gm)
n-hex.: EtOAc (7:3 )
(7.5 gm)
n-hex.: EtOAc (6:4 )
(5.5 gm)
BCH-III
BCH-IV
BCH-V
BCH-II
- silica gel c.c
- n-hex/EtOAc
(gradient elution)
n-hex.: EtOAc (9.5:0.5)
(2.5 gm)
- silica gel c.c
-n-hex./EtOAc
(gradient elution)
n-hex.: EtOAc (9:1)
(3 gm)
EtOAc
(6 gm)
BCH-VI
- silica gel c.c
- n-hex/EtOAc
(gradient elution)
n-hex.: EtOAc (8:2)
(1 gm)
BC-4
(100 mg)
- silica gel c.c
-n-hex./EtOAc
(gradient elution)
BC-1
BC-2
BC-3
(80 mg)
(10 mg)
(80 mg)
Scheme (7): Isolation of the main constituents of the n-hexane fraction of Blepharis ciliaris (L.) B.L.Burtt.aerial parts.
Chloroform Fraction
(30 gm)
VLC, gradient elution using CHCl3: MeOH
CHCl3
CHCl3: MeOH (9:1)
CHCl3: MeOH (8:2)
CHCl3: MeOH (7:3)
(2 gm)
(14 gm)
(3 gm)
BCC-I
BCC-II
BCC-III
(2 gm)
BCC-IV
-silica gel c.c.
- CHCl3:MeOH
(gradient elution)
CHCl3: MeOH (9.5:0.5)
( 3 gm)
-silica gel c.c.
- CHCl3:MeOH
(gradient elution)
MeOH
(4 gm)
BCC-V
-silica gel c.c.
- CHCl3:MeOH
(gradient elution)
CHCl3: MeOH (9:1)
(6 gm)
-silica gel c.c.
- CHCl3:MeOH
(gradient elution)
BC-11
(30 mg)
BC-5
BC-6
BC-7
BC-8
BC-9
(30 mg)
(20 mg)
(30 mg)
(15 mg)
(50 mg)
BC-10
(50 mg)
Scheme (8): Isolation of the main constituents of the chloroform fraction of Blepharis ciliaris (L.) B.L.Burtt.aerial parts.
EtOAc fraction
(40 gm)
VLC, gradient elution using CHCl3:MeOH
Chloroform
(3.5 gm)
BCE-I
CHCl3: MeOH (9:1)
(12.5 gm)
BCE-II
CHCl3: MeOH (8:2)
(8.5 gm)
BCE-III
-silica gel c.c.
- CHCl3:MeOH
(gradient elution)
CHCl3:MeOH (9.5:0.5)
(3 gm)
-silica gel c.c.
- CHCl3:MeOH
(gradient elution)
BC-12
(30 mg)
CHCl3: MeOH (7:3)
(7 gm)
BCE-IV
-silica gel c.c.
- CHCl3:MeOH
(gradient elution)
BC-14 (105 mg)
BC-15 (120 mg)
CHCl3:MeOH (9:1)
(4 gm)
MeOH
(4 gm)
BCE-V
-RP-18 c.c.
-H2O/MeOH
(gradient elution)
BC-16 (20 mg)
-silica gel c.c
- CHCl3:MeOH
(gradient elution)
BC-13
(50 mg)
Scheme (9): Isolation of the main constituents of the ethyl acetate fraction of Blepharis ciliaris (L.)B.L.Burtt. aerial parts.
66
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
CHAPTER I
Preliminary Phytochemical Screening of the Air
Dried Powdered Anisotes trisulcus (Forssk.) Nees.
Aerial Parts
The air-dried powdered aerial parts of Anisotes trisulcus (Forssk.)
Nees. were subjected to preliminary phytochemical screening for their
different constituents. The tests and results are summarized in the
following table:
Table (13): Results of the preliminary phytochemical screening of the
dried aerial parts of Anisotes trisulcus (Forssk.) Nees.
Test
No.
Results
1
Microsublimation test(168)
—
2
Steam distillation(153)
—
3
Carbohydrates and/or glycosides(156)
+
4
Cardenolides(157)
—
5
Unsaturated sterols and/or triterpenes(169,170)
+
6
Tannins(159,171)
+
7
Flavonoids(172)
+
8
Saponins(173)
—
9
Oxidase enzyme(174)
—
10
Lactones and/or esters(153)
+
11
Alkaloids and/or basic nitrogenous substances(171)
+
12
Cyanogenic glycosides(150)
—
13
Anthraquinones(168)
—
14
Coumarins(175)
—
15
Iridoids(155, 176)
—
+ present
— absent
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
67
Conclusion:
The following results could be concluded from Table (13):
The dried aerial parts of Anisotes trisulcus (Forssk.) Nees. contains
carbohydrates and/or glycosides, unsaturated sterols and/or triterpenes,
tannins, flavonoids, lactones and/or esters in addition to alkaloids and/or
basic nitrogenous substances.
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
68
CHAPTER II
Extraction, Fractionation and Isolation of the
Main Constituents of Anisotes trisulcus (Forssk.)
Nees. Aerial Parts
1- Extraction and Fractionation of Anisotes trisulcus
(Forssk.) Nees. Aerial Parts:
The air-dried powdered aerial parts (2.5 kg) of Anisotes trisulcus
(Forssk.) Nees. were extracted by maceration and percolation with (70%)
methanol till complete exhaustion [four times each 10 L, overnight]. The
combined methanolic extracts were concentrated under reduced pressure
till constant weight to give a dark brown syrupy residue (300 gm).
The methanolic extract (300 gm) was digested in the least amount of
distilled water subjected to successive solvent fractionation using a
separating funnel with n-hexane, chloroform, ethyl-acetate and n-butanol.
The n-hexane, chloroform, ethyl-acetate and n-butanol extracts were
concentrated separately under reduced pressure to give 50, 35, 22 and 8
gm respectively and 145 gm of the aqueous extract was left after
extraction with n-butanol which was kept for further study.
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
69
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
70
2- Investigation of the Lipoidal Content of the n-Hexane
Fraction of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Reviewing the current literature, no information could be traced
concerning the lipoidal matter of Anisotes trisulcus (Forssk.) Nees. aerial
parts although preliminary phytochemical screening revealed the presence
of sterols and/or triterpenes (P.66,67) screening.
Accordingly, investigation of the unsaponifiable matter and fatty
acid methyl esters of the n-hexane extract were carried to identify sterols
and/or triterpenes as well as fatty acid content using GC-MS analysis is
undertaken in this chapter.
1- Investigation of the Fatty Acids Composition:
5 gm of the n-hexane fraction of the aerial parts of Anisotes
trisulcus (Forssk.) Nees. was saponified as mentioned before (P.63) the
fatty acids fraction converted to its methyl esters according to the
previously mentioned method (P.63,64), then analysed by GC-MS. The
results are listed in Table (14) and shown in Fig. (3).
71
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Table (14): Results of GC-MS analysis of fatty acids methyl esters of the nhexane of Anisotes trisulcus (Forssk.) Nees. aerial parts.
Peak
Rt
Relative
Mol.
No. of
[M+]
No.
(min.)
%
formula
unsaturation
m/z
1
15.702
0.088
C10H20O2
1
172
Nonanoic acid methyl ester
2
17.19
0.104
C11H22O2
1
186
Decanoic acid methyl ester
3
20.775
0.252
C13H26O2
1
214
Dodecanoic acid methyl ester
4
21.289
1.039
C11H20O4
2
216
Nonanedioic acid dimethyl ester
5
23.2
0.118
C12H22O4
2
230
Decanedioic acid dimethyl ester
6
23.423
0.136
C16H32O2
1
256
3,7,11-trimethyl-Dodecanoic acid
7
24.413
3.104
C15H30O2
1
242
Tetradecanoic acid methyl ester
8
24.752
0.164
C13H24O4
2
244
Undecanedioic acid dimethyl ester
9
25.07
0.324
C17H34O2
1
270
10
25.774
0.447
C16H32O2
1
256
11
26.272
0.408
C18H36O2
1
284
12
26.934
0.109
C17H32O2
2
268
11-Hexadecanoic acid methyl ester
13
27.024
14.971
C17H34O2
1
270
Hexadecenoic acid methyl ester
14
27.58
0.426
C17H34O2
2
268
2-Hexadecenoic acid methyl ester
15
27.654
0.762
C20H40O2
1
312
16
27.844
0.233
C12H20O3
3
212
Methyl 11-oxo-9-undecenoate
17
27.977
0.433
C5H9O2Br
4
179
Methyl 2-bromo-isobutyrate
18
28.125
1.731
C23H43O3
3
381
Heptadecanoic acid methyl ester
19
28.374
0.181
C17H34O3
1
286
20
28.792
0.366
C13H24O3
2
228
Methyl 4-oxododecanoate
21
29.089
5.410
C19H36O2
2
296
9-Octadecenoic acid methyl ester
22
29.470
5.887
C19H38O2
1
298
Octadecanoic acid methyl ester
23
29.719
1.407
C19H34O2
3
294
24
29.719
1.85
C25H38O2
7
370
25
29.798
0.305
C19H36O3
2
312
Component
4,8,12-trimethyl-Tridecanoic acid
methyl ester
Pentadecanoic acid methyl ester
5,9,13-trimethyl-Tetradecanoic acid
methyl ester
2,6,10,14-tetramethyl-Pentadecanoic
acid methyl ester
2-hydroxy-Hexadecanoic acid methyl
ester
7,10-Octadecadienoic acid methyl
ester
9,12- Octadecadienoic acid methyl
ester
3-octyl-Oxiraneoctanoic acid methyl
ester
72
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Table (14): Continued.
Peak
Rt
Relative
Mol.
No. of un-
No.
(min.)
%
formula
saturation
26
30.503
3.279
C19H34O2
4
294
27
30.879
0.418
C20H38O2
2
310
28
31.147
0.324
C20H40O2
1
312
29
31.668
0.897
C18H34O4
2
314
30
31.768
1.392
C19H32O2
4
292
31
33.558
0.802
C17H32O2
2
268
32
34.739
0.416
C20H40O2
1
312
33
35.02
0.158
C9H16O4
2
188
34
35.274
0.228
C22H44O2
1
340
Heneicosanoic acid methyl ester
35
35.644
0.168
C20H38O4
2
342
Octadecanedioic acid dimethyl ester
36
35.904
0.120
C19H38O3
1
314
37
36.243
0.563
C21H38O2
3
322
38
36.566
0.28
C20H40O2
2
312
39
37.122
0.335
C13H26O2
1
214
40
37.424
1.111
C21H42O2
1
326
Eicosanoic acid methyl ester
41
37.424
1.111
C24H48O2
1
368
Tricosanoic acid methyl ester
42
37.964
0.847
C15H30O2
1
242
43
38.287
3.102
C25H50O2
1
382
Tetracosanoic acid methyl ester
44
39.229
0.519
C26H52O2
1
396
Pentacosanoic acid methyl ester
45
40.331
2.029
C27H54O2
1
410
Hexacosanoic acid methyl ester
46
43.211
1.713
C29H58O2
1
438
Octacosanoic acid methyl ester
[M+] m/z
Component
9,11-Octadecadienoic acid methyl
ester
2-octyl-Cyclopropaneoctanoic acid
methyl ester
15-ethyl-Heptadecanoic acid methyl
ester
Hexadecanedioic acid dimethyl ester
6,9,12-Octadecatrienoic acid methyl
ester
9-Hexadecenoic acid methyl ester
2-methyl-Octadecanoic acid methyl
ester
3-Propylglutaric acid monomethyl
ester
9-hydroxy-Octadecanoic acid methyl
ester
11,13-Eicosadienoic acid methyl ester
3-pentyl-Oxiraneundecanoic acid
methyl ester
2,4,6-trimethyl-Nonanoic acid methyl
ester
2-methyl-Tetradecanoic acid methyl
ester
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
73
Fig. (3): GC-MS analysis chromatogram of FAMEs of the n-hexane
fraction of Anisotes trisulcus (Forssk.) Nees. aerial parts.
Conclusion:
GC-MS analysis of fatty acids methyl esters of the n-hexane
revealed the presence of 46 fatty acids which have been identified through
their GC-MS data (Table 14, Fig.3). The major fatty acids identified were
hexadecanoic acid methyl ester (14.971%), octadecanoic acid methyl ester
(5.887%), (Z)-9-octadecenoic acid methyl ester (5.410%), 9,11octadecadienoic acid methyl ester (3.279%), tetradecanoic acid methyl
ester (3.104%) and tetracosanoic acid methyl ester (3.102%).
2- Investigation of the Unsaponifiable Matter:
The unsaponifiable matter was analysed by GC-MS. The results are
listed in Table (15) and shown in Figs. (4 & 5).
74
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Table (15): Results of GC-MS analysis of unsaponifiable matter of the nhexane fraction of Anisotes trisulcus (Forssk.) Nees. aerial
parts.
Peak
Rt
Relative
No. of
No. of
[M+]
No.
(min.)
%
carbons
unsaturation
m/z
1
12.519
0.418
0
142
3-Ethyl-2,5-dimethylhexane
2
13.054
0.059
0
100
3-Methylhexane
3
13.245
0.583
1
98
3-Methyl-1-hexene
4
13.441
2.044
—
98
Ethylcyclopentane
5
13.504
1.299
C10H22
C7H16
C7H14
C7H14
C7H14
1
98
2-Methyl-3-hexene
6
13.573
1.536
C10H20
1
140
1-Methyl-2-(3-methylpentyl)cyclopropane
7
13.79
0.581
1
98
2,4-Dimethyl-2-pentene
8
13.838
0.551
1
98
4,4-Dimethyl-2-pentene
9
13.891
0.936
1
98
2,3-Dimethyl-1-pentene
10
14.013
1.247
1
98
5-Methyl-1-hexene
11
14.15
2.487
1
98
3-Ethyl-1-pentene
12
14.246
1.351
1
98
1,2-Dimethylcyclopentane
13
14.304
0.439
C7H14
C7H14
C7H14
C7H14
C7H14
C7H14
C7H14
1
98
1,1-Dimethylcyclopentane
14
14.426
0.546
C9H18
1
126
1,2,3-Trimethylcyclohexane
15
14.473
0.765
C11H24
0
156
2,2,6-Trimethyloctane
16
14.521
0.120
C12H24
1
168
6-Methyl-4-undecene
17
14.722
0.577
1
182
4,5-Dimethyl-2-undecene
18
14.865
0.224
C13H26
C12H24
1
168
8-Methyl-1-undecene
19
14.908
0.335
C8H16
1
112
1-butyl-2-methylcyclopropane
20
15.077
0.829
C8H16
1
112
3,3-Dimethyl-1-hexene
21
15.141
1.030
C8H16
1
112
1-Ethyl-3-methylcyclopentane
22
15.225
1.177
C7H14
1
98
4,4-Dimethyl-1-pentene
23
15.273
1.179
C8H16
1
112
4-Methyleneheptane
24
15.315
0.491
C8H16
1
112
Cyclooctane
25
15.395
0.798
C8H16
1
112
4-Octene
26
15.49
0.215
1
252
5-Octadecene
27
15.538
0.713
1
126
1,1,2-Trimethylcyclohexane
28
15.575
0.734
C18H36
C9H18
C10H20
1
140
5-Methyl-4-nonene
29
15.623
0.613
C8H16
1
112
trimethylcyclopentane
30
15.776
0.498
C8H16
1
112
1-Ethyl-1-methylcyclopentane
31
15.813
0.854
C8H16
1
112
1,4-Dimethylcyclohexane
32
15.855
0.119
C8H16
1
112
Propylcyclopentane
Component
75
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Table (15): Continued.
Peak
Rt
Relative
Mol.
No. of un-
[M+]
No.
(min.)
%
formula
saturation
m/z
33
15.882
0.139
C8H16
1
112
Methylcycloheptane
34
16.004
0.117
C17H34
1
238
1-Heptadecene
35
16.353
0.112
C9H18
1
126
3,3,5-Trimethyl-1-hexene
36
16.523
0.155
C9H18
1
126
2,4,4-Trimethyl-1-hexene
37
17.812
0.36
C28H48O4
5
448
ergost-25-ene-3,5,6,12-tetrol
38
18.315
0.55
C29H46O
7
410
4,22-Stigmastadiene-3-one
39
18.538
0.52
C28H42O2
8
410
epoxy-methylcholesta-4,6-diene-3one
40
18.728
0.22
C29H50O
5
414
4,4-Dimethyl-(5-alpha) cholestan3-one
41
18.76
0.28
C27H46O
5
386
16,22-Epoxycholestane
42
21.0
0.51
C28H46O
6
398
5-Ethenyl-5-beta-A-norcholestan3-one
43
22.432
0.089
2
138
2,6-Dimethyl-2,6-octadiene
44
23.004
0.126
C10H18
C11H7NO
12
169
1-Isocyanatonaphthalene
45
23.047
0.130
0
168
Cyclododecane
46
24.241
0.59
C12H24
C27H46O
5
386
cholest-5-en-3-ol
47
24.41
0.26
4
388
Cholestan-3-ol
48
24.535
0.118
C27H48O
C7H28
1
196
7-Tetradecene
49
24.815
0.753
8
182
Phenanthrene
50
25.938
0.142
C13H26
C11H18O3
3
240
Heptadecane
51
26.001
1.614
C8H18
0
114
3,3-Dimethylhexane
52
26.075
0.070
C10H20
1
140
Ethylpropylcyclopentane
53
26.221
3.35
C28H48O
5
400
ergost-5-en-3-ol
54
26.478
0.046
C14H28
0
196
Cyclotetradecane
55
26.841
3.536
C16H32
1
224
5-Cyclohexyldecane
56
26.843
0.088
C15H32
0
212
2,6,11-Trimethyldodecane
57
27.458
0.27
C14H18
6
186
1,2,3,4,5,6,7,8octahydrophenanthrene
58
28.307
10.77
5
414
Ethylcholest-5-en-3-beta-ol
59
28.44
0.91
C29H50O
C29H52O
4
416
23-Ethylcholestanol
60
28.877
0.148
1
252
1-Octadecene
61
29.827
0.882
C18H36
C29H50O
6
412
4,4-Dimethyl-cholest-7-en-3-one
62
30.219
0.81
C31H52O
6
440
3-Methoxy-3-beta-olean-12-ene
63
31.021
10.11
C29H50O
5
414
β-sitosterol
Component
76
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Table (15): Continued.
Peak
Rt
Relative
Mol.
No. of
[M+]
No.
(min.)
%
formula
unsaturation
m/z
64
31.601
0.56
C31H52O
6
440
24-Methylene-3-beta-9,19cyclolanostan-3-ol
65
31.853
7.23
C29H48O
6
412
Stigmasterol
66
32.438
0.57
C29H46O
7
410
Stigmasta-4,6,22-trien-3-alpha-ol
67
34.566
0.44
6
412
3-Acetylcholestene
68
34.797
0.483
0
196
69
35.639
0.071
2
432
37.932
0.237
C12H2
2
2
166
39.547
0.259
C15H28
2
208
70
71
C14H2
8
C31H6
0
Component
1,2,4,5-Tetraethylcyclohexane
1-(1-Decylundecyl)decahydronaphthalene
3,4,5,6-Tetramethyl-2,5-octadiene
Decahydro-1,6-dimethyl-4-(1methylethyl)naphthalene
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
77
Fig. (4): GC-MS analysis chromatogram of unsapnifiable matter of the
n-hexane fraction of Anisotes trisulcus (Forssk.) Nees. aerial
parts.
Fig. (5): Chromatogram of GC-MS analysis of unsaponifiable matter of
the n-hexane fraction of Anisotes trisulcus (Forssk.) Nees. aerial
parts
Conclusion:
GC-MS analysis of the unsaponifiable matter of the n-hexane
fraction of the aerial parts of Anisotes trisulcus (Forssk.) Nees. (Table 15,
Figs.4&5) revealed the presence of 53 hydrocarbons representing
(34.018%) and 18 sterols representing (38.922%).
This study is the first report for the lipid investigation of Anisotes
trisulcus (Forssk.) Nees.
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
78
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
79
3- Isolation of the Main Constituents of the n-Hexane
Fraction of Anisotes trisulcus (Forssk.) Nees. Aerial Parts:
A part of the n-hexane soluble fraction (45 gm) was subjected to
vacuum liquid chromatography (VLC) using n-hexane-ethyl acetate
gradient. Fractions of each polarity were collected together and
concentrated under reduced pressure to give six sub-fractions labeled
(ATH-I−ATH-VI).
Sub-fraction ATH-II (8.5 gm) was re-chromatographed on silica gel
column chromatography (350 gm) and eluted with n-hexane followed by nhexane : ethyl acetate gradient, fractions 100 ml each were collected and
monitored by TLC. The fractions eluted with n- hexane : ethyl acetate
(9.5:0.5) afforded compound AT-1 (50 mg) and fractions eluted with nhexane : ethyl acetate (9:1) afforded compounds AT-2 (100 mg) and AT-3
(30 mg).
Sub-fraction ATH-V (5.5 gm) was re-chromatographed on silica gel
column chromatography (220 gm) and eluted with n-hexane : ethyl acetate
gradient. Fractions (100 ml each) were collected and monitored by TLC.
Fractions eluted with n-hexane : ethyl acetate (7:3) afforded compound
AT-4 (50 mg).
The other fractions were subjected to colomn chromatography and
TLC monitored but nothing significant was isolated.
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
80
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
81
4- Isolation of the Main Constituents of the Chloroform
Fraction of Anisotes trisulcus (Forssk.) Nees. Aerial Parts:
A part of the chloroform soluble fraction (30 gm) was subjected to
vacuum liquid chromatography (VLC) using n-hexane : ethyl acetate
gradient elution. Fractions soluble in each polarity were collected together
and concentrated under reduced pressure to give 5 sub-fractions labeled
ATC-I−ATC-V.
Sub-fraction ATC-II (8 gm) was re-chromatographed on silica gel
column chromatography (320 gm) and eluted with n-hexane and followed
n-hexane : ethyl acetate gradient. Fractions (100 ml each) were collected
and monitored by TLC and similar fractions were collected and grouped
together. The fractions eluted with n-hexane : ethyl acetate (9.5:0.5)
afforded compounds AT-5 (20 mg) and AT-6 (40 mg).
Sub-fraction ATC-III (8 gm) was re-chromatographed on silica gel
column chromatography (320 gm) and eluted with n-hexane : ethyl acetate
gradient. Fractions (100 ml each) were collected, grouped and similar
fractions were collected together. The fractions eluted with n-hexane :
ethyl acetae (9:1) yielded compounds AT-7 (50 mg) and AT-8 (40 mg).
Sub-fraction ATC-IV (6.5 gm) were re-chromatographed on silica
gel column chromatography (250 gm) and eluted with n-hexane : ethyl
acetate gradient. Fractions (100 ml each) were collected and monitored by
TLC. Similar fractions were grouped together where those eluted with nhexane : ethyl acetate (8:2) yielded compounds AT-9 (20 mg) and AT-10
(20 mg).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
82
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
83
5- Isolation of the Main Constituents of the Ethyl Acetate
Fraction of Anisotes trisulcus (Forssk.) Nees. Aerial Parts:
A part of the ethyl acetate soluble fraction (18 gm) was subjected to
vacuum liquid chromatography (VLC) with chloroform : methanol gradient
elution. Fractions soluble in each polarity were collected together and
concentrated under reduced pressure to give 5 sub-fractions labeled ATEI−ATE-V.
Sub-fraction ATE-II (4.5 gm) was re-chromatographed on silica gel
column chromatography (180 gm) and eluted with chloroform : methanol
gradient. Fractions (100 ml each) were collected and monitored by TLC.
Similar fractions were collected and grouped together. Fractions eluted
with chloroform : methanol (9.5:0.5) afforded compounds: AT-11 (50 mg),
AT-12 (100 mg) and AT-13 (300 mg).
Sub-fraction ATE-III (5.5 gm) was re-chromatographed on silica gel
column chromatography (220 gm) and eluted with chloroform : methanol
gradient. Fractions (100 ml each) were collected and grouped together.
Fractions eluted with chloroform : methanol (9:1) yielded compound AT14 (30 mg) and compound AT-15 (20 mg).
Sub-fraction ATE-IV (3.5 gm) was re-chromatographed on silica gel
column chromatography (140 gm) and eluted with chloroform : methanol
gradient. Similar fractions eluted with chloroform : methanol (8:2) afforded
compound AT-16 (10 mg).
Sub-fraction ATE-V (1.5 gm) was re-chromatographed on silica gel
column chromatography (60 gm) and eluted with chloroform : methanol
gradient. Similar fractions eluted with chloroform : methanol (7:3) yielded
compound AT-17 (12 mg).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
84
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
85
6- Isolation of the Main Constituents of the n-Butanol
Fraction of Anisotes trisulcus (Forssk.) Nees. Aerial Parts:
A part of the n-butanol soluble fraction (8 gm) was subjected to
silica gel column chromatography (320 gm) and eluted with chloroform :
methanol gradient elution. Fractions (100 ml each) were collected and
monitored with TLC. Similar fractions were grouped together and
concentrated under reduced pressure and labeled ATB-I−ATB-IV.
Sub-fraction ATB-I (2.5 gm) was re-chromatographed on aluminium
oxide column chromatography (80 gm) and eluted with chloroform :
methanol gradient elution. Fractions eluted with chloroform : methanol
(9.5:0.5) afforded compound AT-18 (5 mg).
Sub-fraction ATB-II (2.5 gm) was re-chromatographed on
aluminium oxide column chromatography and eluted with chloroform :
methanol gradient. Fractions eluted with chloroform : methanol (9:1)
yielded compound AT-19 (30 mg) and AT-20 (15 mg).
The other fractions were subjected to colomn chromatography and
TLC monitored but nothing significant was isolated.
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
86
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
86
CHAPTER III
Identification of the Isolated Compounds from
Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Identification of Compound AT-1
Physical Properties:
Compound AT-1 (50 mg) was obtained as colourles needles from
acetone, m.p. 184-186°C. It is soluble in n-hexane, chloroform and
insoluble in methanol. It responded to Salkowski’s and LiebermannBurchard’s tests(169,170), suggesting it's steroidal and/or triterpenoidal
nature. It showed a single spot which attained a reddish brown colour
after spraying with 10 % v/v H2SO4 in methanol and heating at 110°C for
10 min. using precoated silica gel plates. It has Rf value of 0.38 upon
using systems I.
Spectroscopic Analysis:
IR (KBr) of compound AT-1 (Fig. 6) showed the following
absorption bands at υ cm-1: 3380 for OH stretching, 2920 for aliphatic CH stretching, 1639 for C=C stretching, 1451, 1362, 1066 and 1016 for
geminal dimethyl bending.
HO
Fig. (6): IR spectrum of compound AT-1 (KBr).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
87
Conclusion:
From the above mentioned physical, chemical and IR spectral data
in addition to co-chromatography with an authentic sample of α-amyrin
which showed the same Rf values, colour reaction and mixed melting
point, compound AT-1 is identified as α-amyrin. To our knowledge this
represents the first report for its identification from Anisotes trisulcus
(Forssk.) Nees..
Identification of Compound AT-2
Physical Properties:
Compound AT-2 (100 mg) was obtained as white fine needles
from n-hexane, m.p. 134-136°C. It is soluble in n-hexane, chloroform and
sparingly soluble in methanol. It responded to Salkowski’s and
Liebermann-Burchard’s tests(169,170) suggesting its steroidal and/or
triterpenoidal nature. It showed a single spot which attained a violet
colour after spraying with 10 % v/v H2SO4 in methanol and heating at
110°C for 10 min. using precoated silica gel plates. It showed Rf value
of 0.64 upon using system III.
Spectroscopic Analysis:
The IR (KBr) spectrum of compound AT-2 (Fig. 7) showed the
following absorption bands at υ cm-1: 3385 for OH stretching, 2920 for
aliphatic C-H stretching, 1639 for C=C stretching, 1452, 1362, 1068 and
1017 for geminal dimethyl bending.
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
88
OH
Fig. (7): IR spectrum of compound AT-2 (KBr).
Conclusion:
From the above mentioned physical, chemical and IR spectral data
in addition to co-chromatography with an authentic sample of β-sitosterol
which showed the same Rf values, colour reaction and mixed melting
point, compound AT-2 is identified as β-sitosterol and confirmed from
the GC-MS of the lipoidal content of the n-hexane fraction (P.73). To our
knowledge it is the first report for its identification from Anisotes
trisulcus (Forssk.) Nees..
Identification of Compound AT-3
Physical Properties:
Compound AT-3 was obtained as white crystalline needles (30 mg,
CHCl3), m.p. 158-160°C. It is soluble in n-hexane, chloroform and
insoluble in methanol. It responded to Salkowski’s & LiebermannBurchard’s tests(169,170) indicating its steroidal and/or triterpenoidal nature.
It showed a single spot which attained a violet colour after spraying with
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
89
10 % v/v H2SO4 in methanol and heating at 110°C for 10 min. using
precoated silica gel plates. It has Rf value of 0.66 upon using system III.
Spectroscopic Analysis:
The IR (KBr) of compound AT-3 (Fig. 8) showed the following
absorption bands at υ cm-1: 3385 for OH stretching, 2920 for aliphatic CH stretching, 1639 for C=C stretching, 1452, 1362, 1068 and 1017 for
geminal dimethyl bending.
HO
Fig. (8): IR spectrum of compound AT-3 (KBr).
Conclusion:
From the above mentioned physical, chemical and IR spectral data
in addition to co-chromatography with an authentic sample of
stigmasterol which showed the same Rf values, colour reaction and mixed
melting point, compound AT-3 is identified as Stigmasterol and
confirmed from the GC-MS of the lipoidal content of the n-hexane
fraction (P.73). To our knowledge this represents the first report for its
identification from Anisotes trisulcus (Forssk.) Nees..
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
90
Identification of Compound AT-4
Physical Properties:
Compound AT-4 was obtained as white powder (50 mg, n-hexane),
[α] 25
D
+12.5 (co.1, CHCl3). It is soluble in n-hexane, chloroform and
insoluble in ethyl-acetate, methanol and ethanol. It showed a single spot
with Rf value of 0.56 using system IV which gave orange colour after
spraying then heating for 10 min. with 10 % v/v H2SO4 in methanol using
precoated silica gel plates.
Spectroscopic Analysis:
1- IR (KBr) spectrum showed the following absorption bands at υ cm-1 :
3580-3230 (OH or NH stretching), 1626 (amide carbonyl), 1554 (NH
bending).
2- (a) FAB-MS (+ve ion mode) of compound AT-4 (rel.int.%) (Fig. 9):
m/z 683 [M+H]+ (35%), 340 (20%), 169 (30%), 120 (100%) and 57
(35%).
After methanolysis:
(b) EI-MS of the FAME (rel.int.%) (Fig. 10): m/z 398 [M]+ (15%),
380 [M-H2O]+ (5%), 367 [M-CH3O]+ (12%), 350 [M-CH3-OH]+
(5%), 337 [M-CH3COO]+ (10%), 228 (100%), 95 (70%), 69 (75%)
and 55 (85%).
(c) EI-MS of the LCB (rel.int. %) (Fig. 11): m/z 317 [M]+ (15%), 299
[M-H2O]+ (3%), 281 [M-2H2O]+(5%) and 263 [M-3H2O]+(10%).
3- NMR: 13C-, DEPT 13C- and 1H-NMR spectral data are listed in Table
(16) and illustrated in Figs. (12-14).
91
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Table (16):
13
C-, DEPT
13
C- and 1H-NMR data of compound AT-4
(C5D5N-d5, 400 & 100 MHz).
13
No.
C-NMR
DEPT
1
62.16
CH2
2
3
4
53.09
76.91
73.12
CH
CH
CH
5
34.25
CH2
(CH2)n
CH3 x 2
1'
2'
3'
CH3CH2CH2
CH3CH2
-NH
1-OH
3&4-OH
2'-OH
29.63 – 32.16
14.29
175.80
72.56
35.78
32.16
22.96
—
—
—
—
CH2
CH3
—
CH
CH2
CH2
CH2
—
—
—
—
1
H-NMR (m, J in Hz)
a 4.51 (1H, dd, J = 11.2, 4.4)
b 4.42 (1H, dd, J = 11.2, 4.4)
5.09 (1H, m)
4.35 (1H, m)
4.29 (1H, m)
A 2.23 (1H, m)
b 1.93 (1H, m)
1.27−1.32 (m)
0.88 (6H, d, J = 6.7)
—
4.62 (1H, m)
2.23 (m)
—
—
8.55 (1H, d, J = 9)
6.12 (br.s)
6.59 (2H, br.s)
7.53 (br.s)
Discussion:
Inspection of the IR spectrum of compound AT-4 revealed the
presence of absorption bands at υ cm-13580-3230 for bonded OH or N-H
stretching, 1626 for amide carbonyl and 1554 for N-H bending in
addition
to
CH2
functionalities
suggesting
a
sphingolipid
structure(166,167,177).
The 1H-NMR spectrum (Table 16, Fig. 12) showed characteristic
signals for an amide proton at δH 8.55 (d, J = 9 Hz), resonances for four
hydroxyl groups at δH 7.53, 6.59 (for two protons) and 6.12 ppm all
appearing as broad singlets, a signal at δH 5.09 (m, H-2) for a methine
bonded to a nitrogen, signals at δH 4.51 (dd, J = 11.2, 4.4 Hz, H-
92
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
1a) and 4.42 (dd, J = 11.2, 4.4 Hz, H-1b) for a hydroxymethylene, as well
as signals at δH 4.62 (m, H-2'), 4.35 (m, H-3) and 4.29 (m, H-4)
corresponding to three oxymethines. In addition to two terminal methyls
at δH 0.88 (d, J = 6.7 Hz, 3H-18 & 3H-24) and several methylene
hydrogens at δH 1.32-1.27 (m) corresponding to two saturated straight
aliphatic chains were also observed (178).
The 13C- and DEPT 13C-NMR spectrum of compound AT-4 (Table
16, Figs. 13 & 14) exhibited one downfield signal at δc 175.80 (C-1')
corresponding to an amide carbonyl. Signals for a nitrogenated methine at
53.09 (C-2), an oxymethylene at δc 62.16 (C-1) and three oxymethines at
δc 76.91 (C-3), 73.12 (C-4) and 72.56 (C-2'). In addition to several carbon
signals in the range of δc 32.16-29.63 related to methylene groups and a
carbon signal at δc 14.29 corresponding to two terminal methyls were also
deduced from
13
C-NMR spectra and all are in agreement with those of
similar compounds of the sphingolipid(178,179).
The mass spectral studies (Figs. 9-11) showed positive FAB-MS at
m/z 683 [M+1]+ (35%) calculated for C42H85NO5. Methanolysis(166, 167) of
compound AT-4 as mentioned (P.65) resulting in n-hexane fraction
containing fatty acid methyl ester (FAME) and ethyl acetate fraction
containing a long chain base (LCB).
AT-4
1N HCl
aq. MeOH
reflux H CO
3
H2N
O
(CH2)20
OH
CH3
+
OH
2
3
1
(CH2)12
4
CH3
5
OH
OH
Methanolysis of compound AT-4.
The fatty acid methyl ester (FAME) was crystallized from
25
methanol to give an amorphous whitish powder (9 mg) [α] D -27° (co.l,
CHCl3) its 1H-NMR spectrum in CDCl3 revealed the presence of a
93
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
terminal methyl at δH 0.80 (t, J = 6.4 Hz), a wide signal at δH 1.18 for
(CH2)n a singlet at δH 3.72 for COOCH3 and an oxomethine at δH 4.33 (m,
H-2'). The EI-MS of FAME showed molecular ion peak at m/z 398 [M]+
(15%) calculated for C25H50O3 and characteristic mass fragments at m/z
367 [M - CH3O]+ and 337 [M - CH3COO]+ followed by sequential loss of
CH2 units. It was identified as 2-hydroxytetracosanoic acid methyl ester.
The mass fragment at m/z 380 [M-H2O]+ provided another evidence for
the hydroxylated fatty acid residue.
While the (LCB) was crystallized from methanol to give whitish
25
residue (10 mg) [α] D +18.5° (co.l, CHCl3) its 1H-NMR spectrum in
CDCl3 showed a terminal methyl at δH 0.85 (t, J = 6.4 Hz), a wide signal
at δH 1.22 for (CH2)n, an amide NH at δH 8.9 (d, J = 6.8 Hz) in addition to
protons for one oxymethylene and two oxymethines at δH 4.61 (m, H-2),
4.33 (m, H-3) and 3.98 (m, H-4). The EI-MS of LCB showed molecular
ion peak at m/z 317 [M]+ (15%) calculated for the molecular formula
C18H39NO3, 299 [M-H2O]+ (17%), 281 [M-2H2O]+ ( 5%), 263 [M-3H2O]+
(10%) confirmed by the presence of three hydroxyl functions in the
(LCB) moiety, followed by sequential loss of CH2 units. It was identified
as 2-amino-octadecan-1,3,4-triol.
Reading the stereochemistry, the formulated absolute configuration
of compound AT-4 was based on the carbon chemical shifts at δC 62.16
(C-1), 53.09 (C-2), 76.91 (C-3), 175.80 (C-1'), 72.56 (C-2'), which
appeared to be fairly close to those previously reported for (2S,3S, 4R,
2'R) sphingolipid moieties(177).
By comparison of the obtained data with those of analogous
compounds(166,167,177-179),
sphingolipid.
Conclusion:
compound
AT-4
could
be
identified
as
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
94
On the basis of the above mentioned spectroscopic data and
comparison with those previously reported(180), the structure of compound
AT-4 is established as (2S, 3S, 4R)-2[(2'R)-2'-hydroxytetracosanoyl
amino] octadecane 1,3,4-triol. To our knowledge this represents the first
report for the identification of sphingolipids from family Acanthaceae.
O
1'
2'
3'
(CH2)20
CH3
OH
HN
OH
2
3
1
(CH2 )12
4
CH3
5
OH
OH
(2S, 3S, 4R)-2[(2'R)-2'-hydroxytetracosanoyl amino] octadecane 1,3,4-triol.
Fig. (9): +FAB-MS spectrum of compound AT-4.
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Fig. (10): EI-MS spectrum of FAME of compound AT-4.
Fig. (11): EI-MS spectrum of LCB of compound AT-4.
95
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
96
Fig. (12): 1H-NMR spectrum of compound AT-4 (C5D5N-d6, 400 MHz).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
97
Fig. (13): 13C-NMR spectrum of compound AT-4 (C5D5N-d6, 100 MHz).
Fig. (14): DEPT 13C-NMR spectrum of compound AT-4 (C5D5N-d6, 100 MHz).
97
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Identification of Compound AT-5
Physical Properties:
Compound AT-5 was isolated as yellow fine needles (20 mg,
MeOH) with m.p. 236-238°C. It is soluble in chloroform, methanol,
ethanol and insoluble in n-hexane. It showed a single spot which gave a
yellow florescence under UV light and a yellow colour with 5% AlCl3
using
precoated
silica
gel plates.
It
gave
positive
tests
for
flavonoids(155,172). It has Rf value of 0.66 using system II.
Spectroscopic Analysis:
1- UV: The UV spectral analysis of compound AT-5 in methanol and
with different ionizing and complexing reagents were determined and
the data are listed in Table (17).
Table (17): The UV spectral data of compound AT-5 in methanol and
with different ionizing and complexing reagents.
λmax (nm)
Band
MeOH
+NaOMe
+ AlCl3
+AlCl3 /HCl
+ NaOAc
+NaOAc/H3BO3
λmax
λmax
Δλmax
λmax
Δλmax
λmax
Δλmax
λmax
Δλmax
λmax
Δλmax
I
375
390
+15
415
+40
395
+20
395
+20
385
+10
II
270
285
+15
279
+9
275
+5
282
+12
278
+8
2- EI-MS (Fig. 15) (rel.int.%): 436 [M]+ (4%), 331 [M-benzoyl]+ (50%),
316 (12%), 300 [M+-(benzoyl + OCH3)] (15%), 181 (15%), 172
(100%), 139 (24%), 134 (57%).
3- NMR: 13C- and 1H-NMR spectral analysis are listed in Table (18) and
illustrated in Figs. (16 & 17).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Table (18):
98
13
C- and 1H-NMR data of compound AT-5 (DMSO-d6, 400
& 100 MHz).
No.
1
2
3
4
5
6
7
8
9
10
1'
2'
3'
4'
5'
6'
7-OH
5-OCH3
1''
2''
3''
4''
5''
6''
7''
13
C-NMR
—
143.47
135.26
174.30
152.94
93.44
157.82
150.60
156.21
108.81
122.05
115.27
141.45
139.84
117.72
122.84
—
55.46
131.59
127.74
127.46
128.58
127.46
127.74
167.42
1
H-NMR (m, J in Hz)
—
—
—
—
—
6.07 (1H, s)
—
—
—
—
—
7.66 (1H, br.s)
—
—
7.36 (1H, d, J = 8)
8.06 (1H, d, J = 8)
11.03 (1H, s)
3.94 (3H, s)
—
7.33-7.27(m)
7.17 (t, J = 7.6 Hz)
7.33-7.27 (m)
—
Discussion:
The UV spectral data of compound AT-5 in methanol (Table 17)
showed two absorption bands at λmax 270 and 375 nm for a flavonol(161).
The UV spectral data with different ionizing and complexing reagents
revealed the following:
1- The absence of a shift in band I with NaOMe indicating that C-4'
hydroxyl may be absent or blocked.
2- A bathochromic shift with NaOAc in band II (Δλmax +12 nm) revealed
the presence of a free hydroxyl group at C-7.
3- A bathochromic shift with AlCl3 in band I (Δλmax +40 nm) indicating
the presence of a free hydroxyl group at C-3, which decreased upon
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
99
adding HCl (Δλmax +20) indicating the presence of an orthodihydroxy group in ring A.
4- The bathochromic shift with NaOAc/H3BO3 mixture in band I (Δλmax
+ 10 nm) confirmed the ortho-dihydroxy groups in ring A.
The mass and 13C-NMR of compound AT-5 suggested its
flavonoidal nature with an extra aromatic ring with the molecular ion
peak at m/z 436 equivalent to the molecular formula C23H16O9.
The 1H-NMR spectrum (Table 18, Fig. 16) displayed several
aromatic protons; four of them were attributed to the flavonoidal skeleton
and other protons for a mono-substituted benzene moiety. Three protons
at δH 8.07 (d, J = 8 Hz), 7.66 (br.s) and 7.36 (d, J = 8 Hz) for a
trisubstituted benzene of ring B(161). In addition to an upfield singlet
signal at δH 6.10 for one hydrogenated carbon in ring A(161), a singlet at δH
3.94 attributed to a methoxy group and a singlet for a hydroxyl group at
δH 11.03 which confirm the presence of a free 7-OH group(181). The triplet
signal at δH 7.17 (J = 7.6 Hz) was assigned for C-4" proton of a monosubstituted benzene and a multiplet at δH 7.27-7.33 for the other benzoyl
protons.
From the UV spectral studies with different ionizing and
complexing reagents (Table 17), the methoxy group could be attached to
C-5 which was confirmed by the absence of 5-OH at δH 12.5 in the 1HNMR and comparison with similar compounds(181).
The mass spectrum (Fig. 15) showed [M]+ at m/z 436 and other
mass fragments at m/z 331 (50%) [M-benzoyl]+ confirmed the presence
of benzoyl substitution in the flavonoidal skeleton(182,183). The appearance
of mass fragment at m/z 134 (57%) agree with Retro Diels Alder
fragmentation pattern indicating ring B to contain two hydroxyl groups
while the mass fragment at m/z 181 (15%) indicating the presence of two
hydroxyls and a methoxy group in ring A.
Depending on the 1H-NMR data the proton signal at δH 6.07 could
be attributed to C-6 which was confirmed by δC 93.4 ppm upon
comparison with similar data for a monohydrogenated ring A(182).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
100
The 13C-NMR spectrum (Table 18, Fig. 17) exhibited 23 signals
attributed to 23 carbons, fifteen for the flavonoidal skeleton in full
agreement with similar compounds(184) and seven for the benzoyl moiety
appear at δC 127.74 (C-2'', 6"), 127.46 (C-3", 5"), 128.58 (C-4") and
131.59 (C-1") with an ester carbonyl which appear at δC 167.42.
Depending upon the UV spectral data with NaOMe the benzoyl moiety
could be attached to C-4'.
Conclusion:
By Comparison of the obtained spectral data with those previously
reported for similar compounds(181-184), compound AT-5 is identified as
3,7,8,3'-tetrahydroxy 5-methoxy–4'–benzoyl flavone. To our
knowledge this represents the first report for its identification from a
natural source (new compound)(185).
OH
O
OH
HO
O
O
OH
OCH3 O
3,7,8,3'-Tetrahydroxy 5-methoxy−4'−benzoyl flavone.
Fig. (15): EI-MS spectrum of compound AT-5.
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
101
Fig. (16): 1H-NMR spectrum of compound AT-5 (DMSO-d6, 400 MHz).
Fig. (17): 13C-NMR spectrum of compound AT-5 (DMSO-d6, 100 MHz).
102
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Identification of Compound AT-6
Physical Properties:
Compound AT-6 was isolated as fine yellow needles (40 mg,
MeOH) with m.p. 245-247°C. It is soluble in chloroform, methanol and
ethanol while it is insoluble in n-hexane. It showed a single spot which
gave a yellow florescence under UV light and a yellow colour with 5%
AlCl3 using precoated silica gel plates. It gave positive tests for
flavonoids(155,172). It has Rf value of 0.66 in system III.
Spectroscopic Analysis:
1- UV: The UV spectral analysis of compound AT-6 in methanol and
with different ionizing and complexing reagents were determined and
the data are listed in Table (19).
Table (19): The UV spectral data of compound AT-6 with methanol and
different ionizing and complexing reagents.
λmax (nm)
Band
MeOH
+NaOMe
+ AlCl3
+AlCl3 /HCl
+ NaOAc
+NaOAc/H3BO3
λmax
λmax
Δλmax
λmax
Δλmax
λmax
Δλmax
λmax
Δλmax
λmax
Δλmax
I
365
415
+50
395
+30
385
+20
395
+30
400
+35
II
265
280
+15
280
+15
270
+15
280
+15
275
+10
2- EI-MS (rel.int %) Fig. (18): m/z 332 [M]+ (20%), 300 [M-OCH3]+
(20%), 181 (17%), 172 (100%), 139 (22%) and 134 (50%).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
103
Fig. (18): EI-MS spectrum of compound AT-6.
3- NMR: 13C- and 1H-NMR spectral analysis are listed in Table (20) and
illustrated in Figs. (19 & 20).
Table (20):
13
C- and 1H-NMR data of compound AT-6 (DMSO-d6, 400
& 100 MHz).
No.
1
2
3
4
5
6
7
8
9
10
1'
2'
3'
4'
5'
6'
7-OH
5-OCH3
13
C-NMR
—
144.23
135.44
175.91
152.56
139.18
158.0
96.89
152.56
112.1
126.54
114.71
140.78
141.76
117.18
121.74
—
55.46
1
H-NMR (m, J in Hz)
—
—
—
—
—
—
—
6.61 (1H, s)
—
—
—
7.68 (1H, br.s)
—
—
7.29 (1H, d, J = 7.6)
8.05 (1H, d, J =7.6)
10.81 (1H, s)
3.91 (3H, s)
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
104
Discussion:
The UV spectral data of compound AT-6 in methanol (Table 19)
showed absorption bands at λmax 265 and 365 nm for a flavonol(161). The
UV spectral data with different ionizing and complexing reagents
revealed the following:
1- A bathochromic shift in band I with NaOMe (Δλmax+50 nm) without
decrease in intensity indicating the presence of a free hydroxyl group
at C-4'.
2- A bathochromic shift with NaOAc in band II (Δλmax +15 nm)
indicating the presence of a free hydroxyl group at C-7.
3- A bathochromic shift with AlCl3 in band I (Δλmax +30 nm) indicating
the presence of a free hydroxyl group at C-3 which decreased upon
adding HCl (Δλmax+25 nm) due to the presence of ortho dihydroxy
groups in ring B and/or A.
4- The bathochromic shift with NaOAc/H3BO3 mixture in band I
(Δλmax+35 nm) confirmed the ortho dihydroxy groups in ring B
and A.
The mass and
13
C-NMR of compound AT-6 confirmed its
flavonoidal nature with molecular ion peak at m/z 332 equivalent for the
molecular formula C16H12O8.
The 1H-NMR (Table 20, Fig. 19) showed four signals in the
aromatic region, a singlet at δH 6.61 indicating the presence of only one
hydrogenated carbon in the ring A. The other three appear at δH 7.29 (d, J
= 7.6 Hz, H-5'), 7.68 (br.s, H-2') and 8.05 (d, J = 7.6 Hz, H-6') for a trisubstituted benzene of the ring B as those of compound AT-5 (P.97). In
addition to a three proton singlet at δH 3.91 attributed to a methoxy group
105
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
and another singlet at δH 10.81 (s) attributed to the hydroxyl group on C7(182) which was deduced from the UV spectral shifts (NaOAc).
The mass spectrum (Fig. 18) showed [M]+ at m/z 332 (20%) and
other mass fragments at m/z 300 (20%) [M-OCH3]+ and at m/z 134 (50%)
which complies with Retro Diels-Alder cleavage indicating ring B to
contain two hydroxyl groups which was deduced from UV data and 1HNMR spectral shifts. Further EI-MS data showed another ion at m/z
181(17%) indicating the presence of two hydroxyls and a methoxy group
in ring A, the two hydroxy were present as ortho-dihydroxy based on the
UV shifts and the 1H-NMR signal at 6.61 which was attributed to H-8(161).
From the UV spectral studies with different ionizing and
complexing reagents (Table 19) the methoxy group could be attached to
C-5 which was confirmed by the absence of 5-OH signal at δH 12.5 in the
1
H-NMR and comparison with similar compounds(182,186).
The
13
C-NMR spectrum (Table 20, Fig. 20) displayed fifteen
signals assigned for sixteen carbons of the flavonoidal skeleton. The
upfield signal at δC 96.9 was attributed to C-8 of 6,7-dihydroxyflavone
depending on comparison with similar data(184). In addition to a signal at
δC 55.46 attributed to the methoxy group attached to C-5.
Conclusion:
By Comparison of the obtained spectral data with those previously
reported for such compounds(182,184,186), compound AT-6 is identified as
3,6,7,3',4'-pentahydroxy
-5-methoxy
flavone
(5-methoxy
quercetagetin) (allopateuletin)(187). To our knowledge this represents the
first report for its identification from family Acanthaceae.
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
106
OH
OH
HO
O
HO
OH
OCH3 O
3,6,7,3',4'-Pentahydroxy -5-methoxy flavone
(5-methoxy quercetagetin)
Fig. (19): 1H-NMR spectrum of compound AT-6 (DMSO, 400 MHz).
Fig. (20): 13C-NMR spectrum of compound AT-6 (DMSO-d6, 100 MHz).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
107
Identification of Compound AT-7
Physical Properties:
Compound AT-7 was isolated as colourless needles (50 mg,
MeOH) with m.p. 188-190°C. It is soluble in chloroform and methanol,
insoluble in n-hexane. It gave orange colour with Dragendorrf’s
reagent(155) using precoated silica gel plates. It has Rf value of 0.63 in
system IV.
Spectroscopic Analysis:
1- UV (MeOH) λmax: 280 and 295 nm.
2- IR (KBr) υ cm-1: 3240 (OH), 1685 (C-N), 1625 (C=N), 1610, 1570
and 1500 (aromaticity).
3- EI-MS m/z (rel.int. %) Fig. (21): 188 [M]+ (25%), 187 [M-H]+ (5%),
170 [M-OH]+ (4%), 130 [M-C3H6O]+ (18%), 69 (25%) and 58 (13%).
4- NMR: 1H-NMR spectral data are listed in Table (21) and illustrated
in Fig. (22).
Table (21): 1H-NMR data of compound AT-7 (DMSO-d6, 400 MHz).
No.
1
2
3
3a
4
4a
5,6,7,8
8a
9
3-OH
1
H-NMR (m, J in Hz)
a 3.48 (1H, m)
b 3.83 (1H, m)
a 2.46 (1H, m)
b 1.92 (1H, m)
4.11 (1H, br.s)
—
—
—
7.23-7.51 (4H, m)
—
4.89 (2H, dd, J = 15,15)
6.02 (1H, d, J =5.0)
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
108
Discussion:
Compound AT-7 gave positive results for alkaloids with Mayer's
and Dragendorff's reagents(155), its 1H-NMR spectrum suggested the
presence of pyrrolo-quinazoline nucleus(188). Also the UV (MeOH)
spectrum exhibited absorption maxima at λmax: 280 and 295 nm indicating
the presence of a quinazoline chromophore in the molecule(188).
Inspection of the 1H-NMR spectrum (Table 21, Fig. 22) revealed
four sets of multiplets at δH 1.92 (1H, m), 2.46 (1H, m), 3.83 (1H, m),
3.48 (1H, m) attributed to methylene protons at C-2 and C-1 respectively
and a singnal at δH 4.11 (1H, br.s) attributed to C-3 methine, which
represent the pyrrole protons in addition to an aliphatic hydroxyl group at
δH 6.02 (1H, d, J = 5.0 Hz) which is confirmed by an absorption band at
3240 cm-1 in the IR spectrum and a mass fragment at m/z 170 (4%) [MOH]+. The 1H-NMR spectrum also displayed four aromatic protons as
multiplet at δH 7.23-7.51 assigned for an ortho-disubstituted benzene ring
in addition to two proton doublets at δC 4.89 assigned to C-9 methylenic
protons(188).
The mass spectrum (Fig. 21) showed molecular ion peak at m/z
188 indicating the molecular formula C11H12N2O. The obtained spectral
data was in full agreement with those previously reported for the alkaloid
peganine(32).
Conclusion:
From the above mentioned physical and spectral data, compound
AT-7 is identified as Vasicine (Peganine) which was previously isolated
from Adhatoda vasica L.(32) and Anisotes trisulcus (Forssk.) Nees.(34,35).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
8
7
8a
9
109
1
N
2
6
4a
5
N
3a
3
4
OH
Vasicine (Peganine)
Fig. (21): EI-MS spectrum of compound AT-7.
Fig. (22): 1H-NMR spectrum of compound AT-7 (DMSO-d6, 400 MHz).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
110
Identification of Compound AT-8
Physical Properties:
Compound AT-8 was isolated as colourless needles (40 mg, MeOH)
with m.p. 175-177°C. It is soluble in chloroform and methanol, insoluble
in n-hexane. It gave an orange colour with Dragendorrf’s reagent(155)
using precoated silica gel plates. It has Rf value of 0.58 in system IV.
Spectroscopic Analysis:
1- UV (MeOH) λmax: 265, 295 and 309 nm.
2- IR (KBr) υ cm-1: 3150 (OH), 1665 (-O=C-N-), 1630 (C=N-), 1610
and 1485 (aromaticity).
3- EI-MS m/z (rel.int. %) Fig. (23): 202 [M]+ (5%), 185 [M-OH]+ (3%),
146 (3%), 135 (100%), 105 (77%), 69 (10%) and 58 (13%).
4- NMR: 13C-, DEPT 13C- and 1H-NMR spectral data are listed in Table
(22) and illustrated in Figs. (24-26).
Table (22):
13
C-, DEPT
13
C- and 1H-NMR data of compound AT-8
(DMSO-d6, 400 & 100 MHz).
No.
13
C-NMR
DEPT
1
43.00
CH2
2
30.0
CH2
3
3a
4
4a
5
6
7
8
8a
9
3-OH
71.59
158.03
—
142.74
124.18
109.16
109.16
129.17
122.03
160.19
—
CH
C
—
C
CH
CH
CH
CH
C
C
—
1
H-NMR (m, J in Hz)
a 3.84 (1H, m)
b 4.04 (1H, m)
a 2.46 (1H, m)
b 1.92 (1H, m)
4.86 (1H, m)
—
—
—
7.38-7.51 (3H, m)
7.53 (1H, d, J = 6.7)
—
—
5.99 (1H, d, J = 4.88)
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
111
Discussion:
Compound AT-8 gave positive results for alkaloids with Mayer's
and Dragendorff's reagents(155), its 1H-NMR spectrum suggested the
presence of pyrrolo-quinazoline nucleus(188). Also the UV (MeOH)
spectrum exhibited absorption maxima at υ cm-1: 265, 295 and 309 nm
indicating the presence of a quinazoline chromophore in the molecule(32).
Inspection of the 1H-NMR spectrum (Table 22, Fig. 24) revealed
great similarity to that of compound AT-7 showing four sets of multiplets
at δH 1.92 (1H, m), 2.46 (1H, m), 3.84 (1H, m), 4.04 (1H, m) attributed to
methylene protons at C-2 and C-1 respectively and a signal at δH 4.86
(1H, m) attributed to C-3 methine which represent the pyrrole protons in
addition to an aliphatic hydroxyl group at δH 5.99 (1H, d, J = 4.88 Hz)
which was confirmed by an absorption band at 3150 cm-1 in the IR
spectrum and a mass fragment at m/z 185 [M-OH]+. The 1H-NMR
spectrum also displayed three aromatic protons as multiplet at δH 7.387.51 (H-5,6,7) and a doublet at δH 7.53 (H-8) assigned for an orthodisubstituted benzene ring in addition to the absence of the methylenic
protons attached to C-9
(188)
which suggested the presence of a carbonyl
group.
The 13C- and DEPT 13C-NMR (Table 22, Figs. 25 & 26) exhibited
the presence of 10 signals assigned for the presence of 11 carbon atoms, 2
methylenes, 5 methines and 4 quaternary carbons one of them appears
downfield at δC 160.19 which was attributed to the presence of a carbonyl
group (C-9) which was absent in compound AT-7.
The mass spectrum (Fig. 23) showed molecular ion peak at m/z
202 indicating the molecular formula C11H10N2O2. The obtained spectral
data was in full agreement with those previously reported for the alkaloid
vasicinone(33,34,35).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
112
Conclusion:
From the above mentioned physical and spectral data, compound
AT-8 is identified as Vasicinone which was previously isolated from
Adhatoda vasica L.(32) and Anisotes trisulcus (Forssk.) Nees.(34,35).
O
8
8a
7
9
1
N
2
6
5
4a N 3a
4
3
OH
Vasicinone
Fig. (23): EI-MS spectrum of compound AT-8.
Fig. (24): 1H-NMR spectrum of compound AT-8 (DMSO-d6, 400 MHz).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
113
Fig. (25): 13C-NMR of compound AT-8 (DMSO-d6, 100 MHz).
Fig. (26): DEPT 13C-NMR of compound AT-8 (DMSO-d6, 100 MHz).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
114
Identification of Compound AT-9
Physical Properties:
Compound AT-9 was obtained as orange needles (20 mg,
chloroform), m.p. 189-190°C, soluble in chloroform, ethyl-acetate and
insoluble in n-hexane and methanol. It showed a single spot which gave
an orange colour with Dragendorff's reagent(155) using precoated silica gel
plates. It has Rf value of 0.76 using system V.
Spectroscopic Analysis:
1- UV (CHCl3) showed λmax : 225, 298, 310 and 359 nm.
2- IR (KBr) showed the following characteristic bands υ cm-1: 3340 (NH), 1740 (C=O), 1625 (C=N), 1610, 1584 and 1500 (aromaticity).
3- EI-MS m/z (rel.int.%) (Fig. 27): 349 [M]+ (30%), 348 [M-H]+ (10%),
317 [M-CH3OH]+ (10%), 316 [M-H-CH3OH]+ (30%), 290 [MCO2CH3]+ (25%) and 186 [M-C9H10NO2]+ (5%), 166 (20%) and 98
(32%).
4- NMR: 13C- and 1H-NMR spectral data are listed in Table (23) and
illustrated in Figs. (28 & 29).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Table (23):
115
13
C- and 1H-NMR data of compound AT-9 (DMSO-d6, 400
& 100 MHz).
No.
1
2
3
3a
4
4a
5
6
7
8
8a
9
10
11
12
13
14
15
16
17
18
NH
13
C-NMR
45.74
29.10
49.93
140.17
—
144.92
127.33
132.60
128.16
128.53
122.30
159.86
109.05
131.40
152.61
152.31
113.43
134.87
169.21
51.86
29.36
—
1
H-NMR (m, J in Hz)
3.30, 3.42 (2H, m) under solvent
2.31, 2.66 (2H, m)
4.8 (1H, s)
—
—
—
7.33-7.42 (3H, m)
8.10 (1H, d, J = 8.8)
—
—
—
8.0 (1H, d, J =4.8)
—
—
6.84 (1H, d, J = 8.8)
7.30 (1H, s)
—
3.86 (3H, s)
2.92 (3H, d, J = 4.8)
9.01 (1H, s)
Discussion:
Compound AT-9 gave positive results with Mayer's, Wagner's and
Dragendorff's reagents which suggested its alkaloidal nature(155).
The UV spectrum was very similar to that of 4-quinazoline
alkaloids(188). The IR spectrum indicated the presence of an –NH group at
υ 3340 cm-1 and two carbonyl functions assigned to an amide and an
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
116
aromatic carbo-methoxy group at υ 1625 and 1740 cm-1 respectively. The
13
EI-MS and
C-NMR spectrum suggested the molecular formula
C20H19N3O3.
Inspection of the 1H-NMR spectrum (Table 23, Fig. 28) revealed
the presence of pyrrolo-quinazoline system(188) with great similarity to the
desoxyvasicinone(34), the appearance of seven aromatic protons indicated
the presence of a second aromatic ring. Four aromatic protons of ring A
appears as a downfield doublet at δH 8.10 (J= 8.8 Hz) was characteristic
for C-8 proton(34) situated peri to the amide carbonyl in a 4-quinazoline
moiety(34), the remaining protons appeared as multiplets at δH 7.33-7.42.
The substitution pattern of the extra phenyl group (Table 23) (ring B) was
deduced from the presence of an exceptionally high field position at δH
6.84 (d, J = 8.8 Hz, H-14), this was due to the shielding effect of an
orthoamino group(34).
The presence of a carbo-methoxy group was represented by a
singlet signal at δH 3.86 (3H, s, H-17) and a carbon signal at δC 51.86 (C17) and δC 169.21 (C-16) which was further confirmed by mass fragment
at m/z 317 [M-CH3OH]+ and m/z 316 [M-H-CH3OH]+. The proton at δH
8.00 (d, J = 4.8 Hz, H-11) must be situated next to the deshielding carbomethoxy group.
The
13
C-NMR spectrum (Table 23, Fig. 29) displayed 20 carbons
including one methoxy, one methyl, two methylenes, eight methines and
eight quaternary. The two methyl carbon signals at δC 29.36 and 51.86
were assigned to CH3-NH at (δH 2.92, d, J = 4.8 Hz) and OCH3 at δH
(3.86, s).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
117
The remaining of the recorded carbon signals were in full
agreement with those previously reported for the alkaloid Anisotine
previously isolated from Anisotes sessiliflorus C.B.Cl(33) and Anisotes
trisulcus (Forssk.) Nees.(34).
Conclusion:
By comparison of the obtained data with those reported
previously(33,34), compound AT-9 is identified as Anisotine which was
previously isolated from Anisotes trisulcus (Forssk.) Nees.(34).
O
8
8a
7
A
6
5
9
4a
1
N
C
D
2
3a
N
3
4
10
11
15
B
14
12
13
18
16 17
COOCH3
NHCH3
Anisotine
Fig. (27): EI-MS spectrum of compound AT-9.
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
118
Fig. (28): 1H-NMR spectrum of compound AT-9 (DMSO-d6, 400 MHz).
Fig. (29): 13C-NMR spectrum of compound AT-9 (DMSO-d6, 100 MHz).
119
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Identification of Compound AT-10
Physical Properties:
Compound AT-10 was obtained as colourless needles (20 mg,
methanol), m.p. 204-205°C, soluble in n-hexane, chloroform, ethylacetate and insoluble in methanol. It showed a single spot which gave an
orange colour with Dragendorff's reagent(155) using precoated silica gel
plates. It has Rf value of 0.49 using system V.
Spectroscopic Analysis:
1- UV (CHCl3) showed λmax: 315, 274 and 208 nm.
2- IR (KBr) showed the following characteristic bands at υ cm-1: 3480
(OH), 3340 (N-H), 1740 (C=O), 1630 (C=N), 1610, 1584 and 1500
(aromaticity).
3- EI-MS m/z (rel.int. %) Fig. (30): 351 [M]+ (20%), 350 [M-H]+ (12%),
334 [M-OH]+ (70%), 335 (100%), 319 [M-CH3OH]+ (10%), 302
(17%), 274 (77%), 200 [M-150]+ (25%) and 183 (37%).
4- NMR: 13C- & DEPT
13
C-NMR, HMBC, 1H-NMR and 1H-1H COSY
spectral data are listed in Table (24) and illustrated in Figs. (31-36).
Table (24):
13
C- & DEPT 13C-NMR, HMBC, 1H-NMR and 1H-1H COSY
data of compound AT-10 (CDCl3, 400 & 100 MHz).
13
C-NMR
HMBC
1
43.75
2,3,9
2
29.95
1, 3, 3a
3
3a
4
4a
56.42
127.80
—
149.30
1, 2, 3a, 10
—
—
—
No.
1
H-NMR (m, J in Hz)
a 4.05 (1H, m)
b 4.39 (1H, m)
a 2.92 (1H, m)
b 2.14 (1H, m)
5.02 (1H, m)
—
—
—
1
H -1H COSY
1b, 2a, 2b
1a, 2a, 2b
1a, 2b, 3
1a, 1b, 2a, 3
2a, 2b
—
—
—
120
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Table (24): Continued.
No.
5
6
7
8
8a
9
10
11
12
13
14
15
16
17
-NH
5-OH
13
C-NMR
HMBC
158.08
134.19
126.82
126.36
121.07
161.08
111.53
149.72
131.82
116.25
134.60
112.11
169.15
51.96
—
—
—
7, 4a
8, 8a
4a, 8a
—
—
—
—
11, 14, 16
15
11, 12
13
—
16
—
4a, 6
1
H-NMR (m, J in Hz)
—
7.48 (1H, dd, J = 8, 4)
7.72 (1H, t, J = 8)
7.78 (1H,dd J = 8, 4)
—
—
—
—
7.95 (1H, dd, J = 8, 4)
6.71 (1H, dt, J = 8, 4)
7.41 (1H, dt, J = 8, 4)
6.90 (1H, br.d.)
—
3.85 (1H, s)
8.46 (1H,d, J = 4)
8.30 (1H,br.s)
1
H -1H COSY
—
7
6, 8
6, 7
—
—
—
—
13
12, 14
13, 15
14
—
—
3
6
Discussion:
Compound AT-10 gave positive results with Mayer's, Wagner's
and Dragendorff's reagents which suggested its alkaloidal nature(155).
The UV spectrum exhibited absorption bands at λmax: 315, 274 and
208 nm suggesting the presence of a quinazoline chromophore in the
molecule(188).
Preliminary inspection of 1H- & 13C-NMR and mass spectral data
established the presence of a pyrrolo-quinazoline system(188) with an extra
aromatic ring, which is proved by the presence of an extra 7 aromatic
protons at δH (6.71- 8.31 ppm.).
The IR spectrum indicated the presence of a hydroxyl group (3480
cm-1), NH group (3340 cm-1), two carbonyl functions assigned to an
amide (1630 cm-1) and an aromatic ester carbonyl (1740 cm-1) in addition
to an aromatic skeleton (1610, 1584 & 1500 cm-1). The EI-MS and
NMR suggested the molecular formula C19H17N3O4.
13
C-
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
121
The 1H-NMR spectrum in addition to 1H-1H COSY correlations
(Table 24, Fig. 31, 32 &34) revealed the presence of pyrrolo-quinazoline
system with great similarity to that of AT-9 (P.114) signals for a trisubstituted benzene (ring A) [δH 7.48 (dd, J = 8.0, 4.0 Hz, H-6), 7.72 (t, J
= 8.0 Hz, H-7), and 7.78 (dd, J = 8.0, 4.0 Hz, H-8)], in addition to an
ortho-disubstituted phenyl ring (ring B) [δH 7.95 (dd, J = 8.0, 4.0 Hz, H12), 7.41 (dt, J = 8.0, 4.0 Hz, H-14), 6.90 (br.d, H-15), and 6.71 (dt, J =
8.0, 4.0 Hz, H-13)]. The upfield shift of H-3 and C-3 compared with the
corresponding chemical shifts of anisotine (P.114) are indicative of the
iminosubstituted at C-3 of the quinazoline ring.
The
13
C-NMR spectrum (Table 24, Fig. 33) displayed resonances
for 19 carbons, including one methoxy and the other carbons are in full
agreement with those previously reported for related compounds(32).
The HMBC experiments (Table 24, Fig. 36) displayed a 3JCH
coupling between H-6 and C-4a and H-8 with C-8a which could place
ring A at C-4a and C-8a of ring C. Also, 1H-NMR and COSY spectra
(Table 24, Figs. 31, 32 & 34) showed signals for two coupled methylene
groups at δH 4.39 (m, H-1b), 4.05 (m, H-1a) and 2.92 (m, H-2a), 2.14 (m,
H-2b), which further coupled with one methine proton at δH 5.02 (m, H3) to give the structural fragment -CH2-CH2-CH- and confirmed by the
observed HMBC correlations (Fig. 36)(32). Its attachment to ring C was
established by the observed HMBC and HSQC correlations of H-1 with
C-9 and H-2 with C-3a as have been found in the other quinazoline
alkaloids of the plant contain deoxy vasicinone skeleton(32), also
supported by the mass spectrum which exhibited a fragment ion peak at
m/z 183(32). The presence of a carbomethoxy group was supported by the
singlet signal at δH 3.85 (3H, s, H-17), along with the carbon signals at δC
51.96 (C-17) and δC 169.15 (C-16). Its situation at C-11 in ring B was
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
122
proved by the HMBC correlations of H-12 and H-17 with C-16 and
further confirmed by the mass fragment peak at m/z 319 [M-CH3OH]+
due to loss of methanol (carbo-methoxy group was substituted ortho to
the amino substituent of the aromatic ring(32). In addition, the signals at δH
8.30 (br.s) and 8.46 (d, J = 4.0 Hz) were attributed to 5-OH and 3-NH
groups, respectively. The OH group located at C-5 in ring A based on the
carbon signal at δC 158.08 (C-5) and further confirmed by the HMBC
correlations of 5-OH with C-6 and C-4a, and a mass fragment at m/z 334
[M-OH]+ and an IR band at υ 3480 cm-1. The presence of the NH group at
C-3 in ring D was supported by the signals at δH 5.02/δC 56.3(H-3/C-3)
and confirmed by the COSY cross peak of NH with H-3 to give 5hydroxy-3-amino deoxy-vasicinone moiety. Finally, full assignment of
compound AT-10 was achieved by extensive interpretation of 1D and
2D-NMR spectroscopic data.
Conclusion:
The obtained spectral data of compound AT-10 was in good
agreement with those previously reported for vasnetine(32) except for the
presence of a hydroxyl group proved to be attached to C-5. Thus the
compound is identified as 5-hydroxyvasnetine. To our knowledge it
represents the first report for its identification from natural source (new
compound) (185).
O
8
9
7
1
2
N
6
5
4a
N
3a
3
4
O
NH
OH
10
15
16
17
C-OCH3
11
12
14
13
5-hydroxyvasnetine
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
123
Fig. (30): EI-MS spectrum of compound AT-10.
Fig. (31): 1H-NMR spectrum of compound AT-10 (CDCl3, 400 MHz).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
124
O
8
9
7
1
N
6
5
2
3
N
4
O
NH
OH
10
15
16
17
C-OCH3
11
12
14
13
Fig. (32): Expanded 1H-NMR spectrum of compound AT-10 (CDCl3,
400 MHz).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
125
Fig. (33): 13C-NMR spectrum of compound AT-10 (CDCl3, 100 MHz).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
126
Fig. (34): 1H-1H COSY spectrum of compound AT-10 (CDCl3, 400 MHz).
Fig. (35): HSQC spectrum of compound AT-10.
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Fig. (36): HMBC spectrum of compound AT-10.
127
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
128
Identification of Compound AT-11
Physical Properties:
Compound AT-11 was obtained as colourless to beige fine crystals
(50 mg, MeOH), m.p. 180-181°C, soluble in chloroform, methanol and
insoluble in n-hexane. It showed a single spot which gave a brown colour
with 10 % v/v H2SO4 using precoated silica gel plates. It has an Rf value
of 0.78 using system VI.
Spectroscopic Analysis:
1- IR (KBr) showed the following characteristic bands at υ cm-1: 2900
(C-H), 1671 (C=O), 1615, 1593 and 1501 (aromaticity).
2- EI-MS m/z (rel.int. %) (Fig. 37): 182 [M]+ (18%), 165 [M-OH]+
(17%), 167 [M-CH3]+ (100%), 151 [M-OCH3]+ (20%) and 120 [M(OCH3)2]+ (5%).
3- NMR: 13C-, DEPT 13C- and 1H-NMR spectral data are listed in Table
(25) and illustrated in Figs. (38-40).
Table (25):
13
C-, DEPT
13
C- and 1H-NMR data of compound AT-11
(DMSO-d6, 400 & 100 MHz).
No.
1
2
3
4
5
6
(O-CH3)2
-CO
13
C-NMR
121.95
112.78
147.12
150.92
114.92
123.34
55.51
167.20
DEPT
C
CH
C
C
CH
CH
(CH3)2
C
1
H-NMR (m, J in Hz)
—
6.49 (1H, s)
—
—
6.83 (1H, d, J = 8.6)
7.43 (1H, dd, J = 7.6, 2.0)
3.80 (6H, s)
—
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
129
Discussion:
The mass and
13
C-NMR spectral data of compound AT-11
suggested the molecular formula C9H10O4.
The
1
H-NMR spectrum (Table 25, Fig. 38) exhibited three
aromatic protons indicating a trisubstituted benzene ring with ABX
system at δH 6.83 (d, J = 8.6 Hz, H-5), 6.49 (s, H-2) and 7.43 (dd, J = 7.6,
2.0 Hz, H-6) while the singlet at δH 3.80 (6H) suggested the presence of
two methoxy groups.
The
13
C-NMR including DEPT spectra (Table 25, Figs. 39 & 40)
displayed nine carbon signals including four quaternary, three methine
and two methoxy groups. The downfield signal at δC 167.20 was
displayed for carboxylic carbonyl and the upfield one at δC 55.51
confirmed the methoxyl function.
Conclusion:
By comparing the obtained data with those previously reported for
similar compounds(189,190) compound AT-11 is identified as 3,4dimethoxybenzoic acid (Veratric acid), which was previously isolated
from Tabebuia aurea (Silva Manso) Benth. & Hook.f. ex. S.Moore,
family Bignoniaceae(189). To our knowledge this represents the first report
for its identification from family Acanthaceae.
O
7 C-OH
6
1
2
3
5
4
OCH3
OCH3
Veratric acid
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
130
Fig. (37): EI-MS spectrum of compound AT-11.
Fig. (38): 1H-NMR spectrum of compound AT-11 (DMSO-d6, 400 MHz).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Fig. (39):
131
13
C-NMR spectrum of compound AT-11 (DMSO-d6,
100 MHz).
Fig. (40): DEPT
13
C-NMR spectrum of compound AT-11 (DMSO-d6,
100 MHz).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
132
Identification of Compound AT-12
Physical Properties:
Compound AT-12 was isolated as white amorphous powder (100
mg, MeOH). It is soluble in chloroform, ethyl acetate, methanol and
insoluble in n-hexane. It showed a single spot which gave a brown colour
after spraying with 10 % v/v H2SO4 and heating for 10 min., also it gave a
green colour with FeCl3 (T.S) using precoated silica gel plates(155). It has
Rf value of 0.66 using system VII.
Spectroscopic Analysis:
1- IR (KBr) υ cm-1 showed the following bands: 3485 (OH), 1680
(C=O), and 1500-1000 (aromaticity).
2- EI-MS m/z (rel.int. %) Fig. (41): 168 [M]+ (10%), 167 [M-H]+
(100%), 153 [M-CH3]+ (70%), 151 [M-OH]+ (20%), 124 [M-COO]+
(27%) and 107 [M-(CH3 + COO)]+.
3- NMR: 13C- and 1H-NMR data are listed in Table (26) and illustrated
in Figs. (42 & 43).
Table (26):
13
C- and 1H-NMR data of compound AT-12 (DMSO-d6, 400
& 100 MHz).
No.
1
2
3
4
5
6
7
3-OCH3
4-OH
13
C-NMR
121.72
112.69
147.21
151.04
115.01
123.47
167.28
55.53
—
1
H-NMR (m, J in Hz)
—
7.38 (1H, br.s)
—
—
6.82 (1H, d, J = 8.4)
7.42 (1H, dd, J = 8.4, 2)
—
3.78 (3H, s)
9.86 (1H, br.s)
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
133
Discussion:
The mass and
13
C-NMR spectral data of compound AT-12
suggested the presence of an aromatic acid(191) with the molecular ion
peak at m/z 168 for the molecular formula C8H8O4.
The 1H-NMR spectrum (Table 26, Fig. 42) showed three signals in
the aromatic region at δH 7.38 (br.s, H-2), 7.42 (dd, J = 8.4, 2 Hz, H-6)
and 6.82 (d, J = 8.4 Hz, H-5) indicated the presence of a tri-substituted
benzene ring. A singlet for three protons at δH 3.78 (3H) suggested the
presence a methoxy group which is confirmed by δC at 55.53 and another
singlet at δH 9.86 (1H, br.s) attributed to a hydroxyl group which is
confirmed by an absorption band in the IR spectrum at υ 3485 cm-1.
The
13
C-NMR spectrum (Table 26, Fig. 43) revealed a downfield
signal at δC 167.28 attributed to a carboxy carbonyl in addition to other
six aromatic carbons at δC 151.04 (C-4), 147.21 (C-3), 123.47 (C-6),
121.72 (C-1), 112.69 (C-2) and 115.01 (C-5) which are in good
agreement with those for similar compounds(192,193). The mass spectral
data confirmed the suggested structure by the appearance of peaks at m/z
153 [M-CH3]+ (70%), 151 [M-OH]+ (20%), 124 [M-COO]+ (27%).
Conclusion:
Comparing the obtained data with those previously reported in the
literature for similar compounds(192, 193), compound AT-12 is concluded to
be 3-methoxy,4-hydroxy benzoic acid (Vanillic acid). To our
knowledge it represents the first report for its identification from the
genus Anisotes.
O
7 OH
C
1
6
2
3
5
4
OCH3
OH
Vanillic acid
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
134
Fig. (41): EI-MS spectrum of compound AT-12.
Fig. (42): 1H-NMR spectrum of compound AT-12 (DMSO-d6, 400 MHz).
Fig. (43): 13C-NMR spectrum of compound AT-12 (DMSO-d6, 100 MHz).
135
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Identification of Compound AT-13
Physical Properties:
Compound AT-3 was isolated as a white amorphous powder (300
mg, MeOH) It is soluble in hot methanol, ethanol, insoluble in
chloroform and n-hexane. It showed a single spot which attained a violet
colour after spraying with 10% v/v H2SO4 and heating for 10 min. at
110°C using precoated silica gel plates. It gave a positive LiebermannBurchard's test(169,170) positive Molish's test(156) suggesting its steroidal
glycosidic nature. It has Rf value of 0.75 using system VII.
Spectroscopic Analysis:
NMR: 1H-, 13C- and DEPT 13C-NMR data are listed in Tables (27
& 28) and illustrated in Figs. (44-46).
Table (27): 1H-NMR data of compound AT-13 (C5D5N-d5, 400 MHz).
1
H-NMR (m, J in Hz)
No.
H-3
3.98 (1H, m)
H-1'
H-6
5.37 (1H, br.s)
No.
1
H-NMR (m, J in Hz)
5.08 (1H, d, J = 7.2 Hz)
Other sugar
4.06-5.10 (m)
protons
CH3-18
0.68 (3H, s)
CH3-19
0.95 (3H, s)
Other-CH3
0.84-0.99
136
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
13
C- and DEPT 13C-NMR data of compound AT-13 (C5D5N-
Table (28):
d5, 100 MHz).
13
13
No.
CNMR
1
37.96
CH2
2
30.74
3
13
13
CDEPT No.
NMR
CNMR
DEPT
No.
CNMR
DEPT
11
21.76
CH2
21
19.91
CH3
2'
78.55
CH
CH2
12
39.82
CH2
22
34.79
CH2
3'
79.00
CH
79.11
CH
13
42.96
C
23
26.83
CH2
4'
72.26
CH
4
40.42
CH2
14
57.30
CH2
24
46.51
CH
5'
75.83
CH
5
141.36
C
15
24.99
CH2
25
29.93
CH
6'
63.31
CH2
6
122.42
CH
16
29.03
CH2
26
19.49
CH3
7
32.66
CH2
17
56.71
CH
27
20.47
CH3
8
32.53
CH
18
12.46
CH3
28
24.99
CH2
9
50.81
CH
19
19.69
CH3
29
12.64
CH3
10
37.41
C
20
36.88
CH
1'
103.06
CH
DEPT No.
Discussion:
The 1H-NMR spectrum of compound AT-13 (Table 27, Fig. 44)
displayed signals for sitosteryl glycoside(194) by the appearance of six
methyl group signals at δH 0.68-0.99 including two tertiary methyl groups
at δH 0.68 (3H, s, CH3-18) and δC 0.95 (3H, s, CH3-19), a methine proton
at δH 3.98 (1H, m, H-3), an olefinic proton at δH 5.37 (1H, br.s, H-6),
sugar protons at δH 4.06-5.10 and an anomeric proton at δH 5.08 (1H, d, J
= 7.2 Hz) indicating its β-configuration.
The 13C- and DEPT 13C-NMR data (Table 28, Figs. 45 & 46) was
in full agreement with those previously reported for sitosteryl
glucoside(194) with characteristic signals at δC 141.4, 122.4 for Δ5-6 and
anomeric carbon at δC 103.1 for C-1' of glucose in addition to other sugar
carbons at δC 78.55 (C-2'), 79.00 (C-3'), 72.26 (C-4'), 75.83 (C-5') and
63.31 (C-6').
137
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Acid hydrolysis of compound AT-13 was carried out as mentioned
before (P.64), the hydrolysates were confirmed to be β-sitosterol by direct
comparison with authentic samples (co-TLC) and glucose by paper
chromatography using system IX.
Conclusion:
Comparing the obtained spectral data with those reported for
similar compounds(194) and by direct comparison with an authentic sample
of β-sitosterol glucoside (co-TLC), compound AT-13 is identified as βsitosterol-3-O-β-D-glucopyranoside. To our knowledge this represents
the first report for its identification from the genus Anisotes.
29
28
21
22
20
23
18
24
26
25
12
19
1
OH
2
6´
O
HO
HO
OH
O
9
10
5
4
17
27
16
8
3
1´
13
11
14
15
7
6
β-sitosterol-3-O-β-D-glucopyranoside
H-1
Fig. (44): 1H-NMR spectrum of compound AT-13 (C5D5N-d5, 400 MHz).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
138
Fig. (45): 13C-NMR spectrum of compound AT-13 (C5D5N-d5, 100 MHz).
Fig. (46): DEPT
13
C-NMR spectrum of compound AT-13 (C5D5N-d5,
100 MHz).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
139
Identification of Compound AT-14
Physical Properties:
Compound AT-14 was isolated as colourless needles (30 mg,
MeOH), m.p. 259°C, soluble in methanol, insoluble in n-hexane and
chloroform. It showed a single spot which gave an orange colour with
Dragendorff's reagent(155) using precoated silica gel plates. It has Rf 0.81
using system VII.
Spectroscopic Analysis:
1- UV (MeOH) showed absorbance at λmax: 294 and 205 nm.
2- IR (KBr) showed the following characteristic bands at υ cm-1: 3395
(OH), 1678 (C=N), 1590 and 1498 (aromaticity).
3- EI-MS (rel.int. %) Fig. (47): m/z 204 [M]+ (30%), 188 (25%), 187
[M-OH]+ (14%), 170 [M-2OH]+ (17%), 146 (3%), 130 (17%) and 58
(17%).
4- NMR:
13
C- & DEPT
13
C-NMR, HMBC and 1H-NMR spectral data
are listed in Table (29) and illustrated in Figs. (48-51).
140
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Table (29):
13
C- & DEPT
13
C-NMR, HMBC and 1H-NMR data of
compound AT-14 (DMSO-d6, 400 & 100 MHz).
No.
13
C-NMR
1
52.07
2
31.17
3
3a
4
4a
5
6
7
8
8a
72.48
163.58
DEPT
HMBC
CH2
1, 2, 3
CH2
1, 2, 3
124.77
120.52
117.52
158.10
115.20
120.22
CH
C
—
C
CH
CH
C
CH
C
2, 3a
—
—
—
4a, 5, 6, 7
4a, 5, 6, 8
—
7, 9
—
9
47.84
CH2
3a, 6, 8, 8a, 9
3-OH
7-OH
—
—
—
—
2, 3, 3a
7
—
1
H-NMR (m, J in Hz)
a 4.01 (1H, m)
b 4.13 (1H, m)
a 2.47 (1H, m)
b 2.43 1H, m)
5.51 (1H, dd, J = 7.3)
—
—
—
7.51 (1H, d, J = 8.6)
7.21 (1H, dd, J = 8.6, 2.3)
—
7.08 (1H, br.s)
—
a 5.24 (1H, d, J = 15.5)
b 5.12 (1H, d, J = 15.5)
6.94 (1H, d, J = 7.7)
10.31 (1H, s)
Discussion:
Compound
AT-14
gave
positive
Dragendorff's
tests
for
alkaloids(155), its mass and 13C-NMR spectral data suggested the presence
of pyrrolo-quinazoline system(188) with a molecular formula C11H12N2O2.
The 1H-NMR spectrum (Table 29, Fig. 48) revealed four sets of
multiplets at δH 2.43, 2.47, 4.01 and 4.13 ppm characteristic for the
pyrrolo-quinazoline type alkaloids(188) which was further confirmed by
the appearance of a mass fragment at m/z 146 [M-58]+. In addition to an
aliphatic hydroxyl at δH 6.94 ppm (1H, d, J = 7.7 Hz) and an aromatic one
at δH 10.31 (s). The appearance of an absorption band at υ 3395 cm-1 in
the IR spectrum and a mass fragment at m/z 187 [M-OH]+ and m/z 170
[M-2OH]+ proved the presence of the hydroxyl groups.
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
141
The 1H-NMR spectrum displayed 3 aromatic protons at δH 7.08
(1H, br.s), 7.51 (1H, d, J = 8.6 Hz) and 7.21 (1H, dd, J = 8.6, 2.3 Hz)
assigned for a tri-substituted benzene ring.
The 13C- and DEPT 13C-NMR spectrum (Table 29, Figs. 49 & 50)
displayed resonances for 11 carbons. The DEPT spectrum showed 3
methylenes, 4 methines and 4 quaternary carbons. The 3 methylenes
showed signals at δH 31.17, 47.84, 52.07 assigned for C-2, C-9 and C-1
respectively. The 4 methines showed signals at δH 72.48, 115.20, 117.52
and 120.52 assigned for C-3, C-8, C-6 and C-5, in addition to 4
quaternary carbons resonating at δH 120.22, 124.77, 158.10 and 163.58
attributed to C-8a, C-4a, C-7 and C-3a respectively.
The aliphatic hydroxyl group in ring C was located at C-3 based on
the carbon signal at δC 72.4 (C-3)(37,188), and further confirmed by the
HMBC correlations (Fig. 51) of C3-OH with C-2 and C-3a, while the
aromatic hydroxyl group was located at C-7 based on the carbon signal at
δC 158.10 (C-7) and further confirmed by the HMBC correlations with
C-7.
Conclusion:
By comparison of the obtained data with those reported
previously(32,37,188), compound AT-14 is identified as 7-hydroxyvasicine
(vasicinol) previously isolated from Linaria vulgaris Mill., family:
Scrophulariaceae(195). It is the first report for carrying out 2D-NMR
correlations to confirm the structure. To our knowledge this represents
the first report for its identification from the genus Anisotes.
8
HO
7
6
8a
4a
5
9
1
N
N
3a
4
7-hydroxyvasicine
2
3
OH
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
142
Fig. (47): EI-MS spectrum of compound AT-14.
Fig. (48): 1H-NMR spectrum of compound AT-14 (DMSO-d6, 400 MHz).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
143
Fig. (49): 13C-NMR spectrum of compound AT-14 (DMSO-d6, 100 MHz).
Fig. (50): DEPT 13C-NMR spectrum of compound AT-14 (DMSO-d6, 100 MHz).
Fig. (51): HMBC spectrum of compound AT-14 (DMSO-d6, 700 MHz).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
144
Identification of Compound AT-15
Physical Properties:
Compound AT-15 was isolated as colourless needles (20 mg,
MeOH), m.p. 280°C, soluble in methanol, insoluble in hexane and
chloroform. It showed a single spot which gave an orange colour with
Dragendorff's reagent(155) using precoated silica gel plates. It has Rf value
of 0.88 using system VII.
Spectroscopic Analysis:
1- UV (MeOH) λmax: 326, 274 and 225 nm.
2- IR (KBr) showed the following characteristic bands at υ cm-1: 3395
(OH), 1670 (C=N), 1615 (C=O), 1600 and 1485 (aromaticity).
3- EI-MS m/z (rel.int. %) Fig. (52): 218 [M]+ (14%), 200 [M-18]+ (3%),
190 (7%), 184 [M-2OH]+ (5%), 160 [M-C3H6O]+ (3%), 135 (100%),
and 58 (15%).
4- NMR:
13
C- & DEPT
13
C-NMR, HSQC, 1H-NMR and 1H-1H COSY
spectral data are listed in Table (30) and illustrated in Figs. (53-57).
145
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Table (30):
13
C- & DEPT 13C-NMR, HSQC, 1H-NMR and 1H-1H COSY
data of compound AT-15 (DMSO-d6, 400 & 100 MHz).
No.
13
C-NMR
DEPT
HSQC
1
H-NMR (m, J in Hz)
1
42.92
CH2
1
2
29.69
CH2
2
3
3a
4
4a
5
6
7
8
8a
9
3-OH
7-OH
71.09
157.46
—
121.55
128.57
123.54
155.85
108.69
142.23
159.56
—
—
CH
C
—
C
CH
CH
C
CH
C
C
—
—
3
—
—
—
5
6
—
8
—
—
3
7
a 3.91 (1H, m)
b 4.06 (1H, m)
a 2.50 (1H, m)
b 2.01 (1H,m)
4.95 (1H, m)
—
—
—
7.58 (1H, d, J = 8.6)
7.30 (1H, dd, J = 8.3, 3.1)
—
7.46 (1H, d, J = 3.1)
—
—
5.98 (1H, d, J = 6.04)
10.01 (1H, s)
AT-15
gave
positive
1
H-1H COSY
1, 2
1, 2, 3
2 (a&b)
—
—
—
6, 8
5, 8
—
5, 6
—
—
3
—
Discussion:
Compound
Dragendorff's
tests
for
alkaloids(155), its mass and 13C-NMR spectral data suggested the presence
of pyrrolo-quinazoline system(188) with a molecular formula C11H10N2O3.
The 1H-NMR spectrum in addition to the 1H-1H COSY correlations
(Table 30, Figs. 53& 56) revealed four sets of multiplets at δH 2.50, 2.01,
3.91 and 4.06 characteristic for geminally coupled pyrrole protons at C2
and C1, protons characteristic for the pyrrolo-quinazoline type
alkaloids(188) which was further confirmed by the appearance of a mass
fragment at m/z 160 [M-58]+. In addition to an aliphatic hydroxyl at δ
5.96 (d, J = 6.04 Hz) and an aromatic one at δH 10.01 (s). The appearance
of an absorption band at υ 3395 cm-1 in the IR spectrum and a mass
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
146
fragment at m/z 200 [M-18]+ and m/z 184 [M-2OH]+ confirmed the
presence of the hydroxyl functional group.
The 1H-NMR spectrum displayed three aromatic protons at δH 7.58
(d, J = 8.6 Hz), 7.30 (dd, J = 8.4, 3.1 Hz) and 7.46 (d, J = 3.1 Hz)
assigned for a tri-substituted benzene ring.
The
13
C-NMR spectrum (Table 30, Fig. 54) displayed resonances
for 11 carbons. The DEPT spectra (Table 30, Fig. 55) showed 2
methylenes, 4 methines and 5 quaternary carbons. The 2 methylenes
showed signals at δC 29.69 and 42.92 assigned for C-2 and C-1
respectively. The 4 methines showed signals at δC 71.09, 128.57, 123.54
and 108.69 assigned to C-3, C-5, C-6 and C-8 respectively. In addition to
5 quaternary carbon atoms resonating at δC 159.56, 157.46, 155.85,
121.55 and 142.23 assigned to C-9, C-3a, C-7, C-4a and C-8a
respectively.
The aliphatic hydroxyl group in ring C could be located at C-3
based on the carbon signal at δC 71.0 (C-3)(37,
188, 195)
and further
confirmed by the HMBC correlations (Table 30, Fig. 57) of C3-OH with
C-3, while the aromatic hydroxyl was located at C-7 based on the carbon
signal at δC 155.85 (C-7) and further confirmed by the HMBC
correlations with C-7.
Conclusion:
Based on the previous mentioned data and by comparison with
those previously published(32,37,188,195), compound AT-15 is identified as
7-Hydroxyvasicinone (Vasicinolone) which was previously isolated
from Adhatoda vasica Nees.(32,37,188) but it is the first report for carrying
2D-NMR correlations to confirm the hydroxyl substitution. To our
knowledge this represents the first report for its identification from the
genus Anisotes.
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
O
HO
8
7
8a
9
1
N
6
4a
5
N
4
3a
2
3
OH
7-Hydroxyvasicinone
Fig. (52): EI-MS spectrum of compound AT-15.
Fig. (53):
1
H-NMR spectrum of compound AT-15 (DMSO-d6,
400 MHz).
147
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Fig. (54):
148
13
C-NMR spectrum of compound AT-15 (DMSO-d6,
100 MHz).
Fig. (55): DEPT
13
C-NMR spectrum of compound AT-15 (DMSO-d6,
100 MHz).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Fig. (56):
1
149
H-1H COSY spectrum of compound AT-15 (DMSO-d6,
700 MHz).
Fig. (57): HMBC spectrum of compound AT-15 (DMSO-d6, 700 MHz).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
150
Identification of Compound AT-16
Physical Properties:
Compound AT-16 was isolated as brown fine needles (10mg,
MeOH) with m.p. 98-100°C. It is soluble in methanol, insoluble in nhexane and chloroform. It gave an orange colour with Dragendorff's
reagent(155) using precoated silica gel plates. It has Rf value of 0.67 using
system VII.
Spectroscopic Analysis:
1- IR (KBr) showed the following absorption bands at υ cm-1: 3320
(OH), 1690 (C-N), 1630 (C=N), 1610, 1550 and 1500 (aromaticity).
2- EI-MS m/z (rel.int. %) (Fig. 58): 233 [M]+ (3%), 217 [M-OH]+ (4%),
197 [M-2H2O]+ (10%), 173 (90%), 164 (100%), 138 (25%), 120
(40%), 86 (10%) and 72 (5%).
3- NMR: 13C-, DEPT 13C- and 1H-NMR spectral data are listed in Table
(31) and illustrated in Figs. (59-61).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Table (31):
13
C-, DEPT
151
13
C- and 1H-NMR data of compound AT-16
(DMSO-d6, 400 & 100 MHz).
No.
1
2
3
4
4a
5
5a
6
7
8
9
10
11
11a
8-NH2
6-OH
2-OH
13
C-NMR
113.24
156.12
115.57
118.52
122.70
—
161.59
70.58
29.36
48.45
50.09
—
45.86
118.26
—
—
—
DEPT
CH
C
CH
CH
C
—
C
CH
CH2
CH
CH2
—
CH2
C
—
—
—
1
H-NMR (m, J in Hz)
6.58 (1H, d, J = 2.3)
—
6.70 (1H, dd, J = 8.6, 2.3)
7.02 (1H, d, J = 8.6)
—
—
—
5.01 (1H, q, J =7.9)
1.94 ( 2H, m)
4.05 (1H, m)
3.49, 3.62 (2H, m)
—
4.75, 4.63 (2H, each d, J = 15.8)
—
11.99 (2H, br.s)
6.41 (1H, d, J = 7.5)
9.77 (1H, s)
Discussion:
Compound AT-16 gave positive results for alkaloids with Mayer's
and Dragendorff's reagents(155), its mass and
13
C-NMR spectral data
suggested the presence of a tricyclic quinazoline system with a molecular
formula C12H15N3O2.
The 1H-NMR spectrum (Table 31, Fig. 59) exhibited similar
pattern to that of 7-hydroxyvasicine (P.144) representing the tricyclic
quinazoline alkaloids with an extra methine at δH 4.05 (m) which could
be attributed to ring C and an amino substitution, which was confirmed
by a downfield signal at δH 11.99 (2H, br.s).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
152
The 1H-NMR spectrum also exhibited two hydroxyl functions at δH
6.41 and 9.77, which were confirmed by the appearance of an absorption
band in the IR at υ 3320 cm-1 and mass fragments at m/z 217 [M-OH]+
and 197 [M-2H2O]+. The attachment of the hydroxyl functions to C-2 and
C-6 was confirmed from comparing the chemical shifts of δC at 156.12,
70.58 with those of C-7 and C-3 of 7-hydroxyvasicine (P.139). In
addition to three aromatic protons at δH 6.58 (1H, d, J = 2.3 Hz), 6.70
(1H, dd, J = 8.6, 2.3 Hz) and 7.02 (1H, d, J = 8.6 Hz) representing a tri
substituted benzene ring.
The
13
C-NMR spectrum (Table 31, Fig. 60) displayed twelve
signals (7-hydroxyvasicine showed eleven carbons only). The DEPT
experiment (Table 31, Fig. 61) showed three methylenes, five methines
and four quaternary carbons. The amino group could be attached to C-8
depending on comparison of the chemical shift (δH and δC) at positions 6,
7 and 9 with the corresponding 1, 2 and 3 in 7-hydroxyvasicine.
Conclusion:
Based on the previous spectroscopic data and by comparison with
that reported in the literature(196,197), compound AT-16 is identified as 8amino-7,8,9,11-tetrahydro-6H-pyrido[2,1-b]quinazoline-2,6-diol.
To
our knowledge this represents the first report for its identification from
natural source (new compound) (185).
1
HO
2
3
11a
4a
4
11
9
N
5
5a
NH2
8
N
7
6
OH
8-Amino-7, 8, 9, 11-tetrahydro-6H-pyrido [2,1-b]quinazoline-2,6-diol
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
153
Fig. (58): EI-MS spectrum of compound AT-16.
Fig. (59): 1H-NMR spectrum of compound AT-16 (DMSO-d6, 400 MHz).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Fig. (60):
154
13
C-NMR spectrum of compound AT-16 (DMSO-d6,
100 MHz).
Fig. (61): DEPT
13
C-NMR spectrum of compound AT-16 (DMSO-d6,
100 MHz).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
155
Identification of Compound AT-17
Physical Properties:
Compound AT-17 was isolated as brown needles (12 mg, MeOH)
with m.p. 120-122°C. It is soluble in methanol, insoluble in n-hexane and
chloroform. It gave an orange colour with Dragendorff's reagent(155) using
precoated silica gel plates. It has Rf value of 0.56 using system VIII.
Spectroscopic Analysis:
1- IR (KBr) showed the following absorption bands at υ cm-1: 3350
(OH), 1690 (C-N), 1630 (C=N), 1615 (C=O), 1620, 1560 and 1500
(aromaticity).
2- EI-MS m/z (rel.int. %) (Fig. 62): 247 [M]+ (50%), 230 [M-OH]+
(5%), 213 [M-2OH]+ (8%), 173 (92%), 164 (100%), 138 (25%), 120
(42%) and 86 (10%).
3- NMR: 13C-, DEPT 13C- and 1H-NMR spectral data are listed in Table
(32) and illustrated in Figs. (63-65).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Table (32):
13
C-, DEPT
156
13
C- and 1H-NMR data of compound AT-17
(DMSO-d6, 400 & 100 MHz).
No.
1
2
3
4
4a
5
5a
6
7
8
9
10
11
11a
8-NH2
6-OH
3-OH
13
C-NMR
109.39
122.24
156.55
129.28
124.26
—
158.16
71.79
30.38
49.26
43.62
—
160.29
142.99
—
—
—
DEPT
CH
C
CH
CH
C
—
C
CH
CH2
CH
CH2
—
C
C
—
—
—
1
H-NMR (m, J in Hz)
7.55 (1H, d, J = 8.7)
7.25 (1H,dd, J = 8.7, 2.7)
—
7.42 (1H, dd, J = 2.7)
—
—
—
4.93 (1H, d, J = 6.2)
1.98 ( 2H, m)
4.10 (1H, m)
3.89 (2H, m)
—
—
—
11.99 (2H, br.s)
5.95 (1H, d, J = 5.6)
10.0 (1H, s)
Discussion:
Compound AT-17 gave positive results for alkaloids with Mayer's
and Dragendorff's reagents(155), its mass and
13
C-NMR suggested the
presence of a tricyclic quinazoline system with a molecular formula
C12H13N3O3.
The 1H-NMR spectrum (Table 32, Fig. 63) revealed a great
similarity to that of compound AT-16 (P.150), showing a downfield
signal at δH 11.99 (2H, br.s) suggesting an amino substitution, two
hydroxyl functions at δH 10.0 and 5.95 which were confirmed by the
appearance of an absorption band in the IR spectrum at υ 3350 cm-1 and
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
157
mass fragments at m/z 230 [M-OH]+ and 213 [M-2OH]+. It also exhibited
three aromatic protons at δH 7.25 (1H, dd, J = 8.7, 2.7 Hz), 7.42 (1H, dd,
J = 2.7 Hz) and 7.55 (1H, d, J = 8.7 Hz) representing a trisubstituted
benzene.
The 13C- and DEPT 13C-NMR (Table 32, Figs. 64 & 65) displayed
twelve signals while the 7-hydroxyvasicinone showed only eleven
carbons. The DEPT experiments showed 2 methylenes, 5 methines and 5
quaternary carbons with a downfield quaternary carbon at δC 160.29 (C11)`. The amino group could be attached to C-8 depending on comparison
of the chemical shift (δH and δC) at positions 6,7 and 9 with the
corresponding 1, 2 and 3 in 7-hydroxyvasicinone.
The absence of the two doublets at δH 4.75, 4.63 (d, J = 15.8 Hz)
attributed for C-11 methylene in AT-16 (P.150) and appearance of carbon
signal at δC 160.29 suggested the oxo form of AT-17.
Conclusion:
Based on the previous spectroscopic data and by comparison with
that reported in the literature(196,197), compound AT-17 is identified as 8amino-3,6-dihydroxy-7,8,9-trihydro-6H-pyrido[2,1-b]quinazoline-11one. To our knowledge this represents the first report for its identification
from natural source ( new compound)(185).
O
1
11a
2
9
11
N
3
HO
4
4a
N
5
5a
NH2
8
7
6
OH
8-Amino-3,6-dihydroxy-7,8,9-trihydro-6H-pyrido[2,1-b]quinazoline-11-one
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Fig. (62): EI-MS spectrum of compound AT-17.
Fig. (63):
1
H-NMR spectrum of compound AT-17 (DMSO-d6,
400 MHz).
158
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Fig. (64):
159
13
C-NMR spectrum of compound AT-17 (DMSO-d6,
100 MHz).
Fig. (65): DEPT
13
C-NMR spectrum of compound AT-17 (DMSO-d6,
100 MHz).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
160
Identification of Compound AT-18
Physical Properties:
Compound AT-18 was obtained as colourless needles (10 mg,
MeOH), m.p. 290-291°C, soluble in hot methanol, sparingly soluble in
cold methanol and insoluble in n-hexane, chloroform and ethyl acetate. It
showed a single spot which gave a brick red colour with Dragendorff's
reagent(155) using precoated alumina plates. It has Rf value of 0.51 using
system V.
Spectroscopic Analysis:
1- Positive ion mode MALDI-TOF-MS: Fig. (66), m/z (rel.int.%): 169
[M+H]+ (78%), 171 [M+2]+ (25%), 126 (17%), 104 (100%) and 102
(49%).
2- NMR:
13
C- and 1H-NMR spectral data are listed in Table (33) and
illustrated in Figs. (67 & 68).
Table (33):
13
C- and 1H-NMR data of compound AT-18 (DMSO-d6, 400
& 100 MHz).
No.
13
C-NMR
1
H-NMR (m, J in Hz)
1
67.41
3.81 (2H, dist.m*)
2
—
—
3
67.38
3.38 (2H, m)
4-N(CH3)2
53.60
3.10 (6H, s)
2-N(CH3)2
55.55
3.32 (6H, s)
1-OH
—
5.42 (1H, br.s)
*dist.m = distorted multiplet
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
161
Discussion:
Compound AT-18 gave brick red colour with Dragendorff's reagent
which suggested it to be a quaternary ammonium compound(155).
The positive ion mode MALDI-TOF-MS spectrum (Fig. 66)
exhibited a molecular ion peak at m/z 169 [M+H]+ and 171 [M+2]+ in a
ratio of 3:1 indicating a chloride salt(191). The mass and 13C-NMR spectra
suggested a molecular formula C6H17N2OCl.
The 1H-NMR (Table 33, Fig. 67) showed the presence of two
singlets at δH 3.32 and 3.10 assigned for methyls attached to nitrogen
which was confirmed by two signals in the 13C-NMR (Table 33, Fig. 68)
at δC 55.55 and 53.60 consistent with methyl ammonium groups(192) in
addition to two multiplets each for two protons at δH 3.81 and 3.38
assigned for two methylenes attached to nitrogen (CH2N) which are
observed at δC 67.41 and 67.38 in the 13C-NMR spectrum while the broad
singlet δH 5.42 which was attributed to the terminal hydroxyl group.
Conclusion:
Based on the previous spectroscopic data and by comparison with
that reported in the literature for quaternary methyl ammonium
compounds(198, 199), compound AT-18 is identified as (dimethylamino)-N(hydroxymethyl)-N,N-dimethylmethanaminium chloride. According to
the available literature quaternary ammonium compounds were traced in
Acanthus montanus (Nees.) T.Andres and Crossandra nilitoca Oliver
(subfamily: Acanthoideae)(90). To our knowledge this represents the first
report for identification of such compound from a natural source (new
compound)(185).
The presence of such compounds in plants have been connected
with salt stress conditions(198) and some mangrove plants have been found
to accumulate
compounds(90).
large amounts of quaternary
methyl ammonium
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
4
N
2
3
N
1
162
OHCl
(dimethylamino)-N-(hydroxymethyl)-N,N-dimethylmethanaminium chloride
Fig. (66): + MALDI-TOF-MS spectrum of compound AT-18
Fig. (67):
1
H-NMR spectrum of compound AT-18 (DMSO-d6,
400 MHz).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Fig. (68):
13
163
C-NMR spectrum of compound AT-18 (DMSO-d6,
100 MHz).
164
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Identification of Compound AT-19
Physical Properties:
Compound AT-19 was obtained as colourless needles (30 mg,
MeOH), m.p. 295-296°C, soluble in hot methanol, sparingly soluble in
cold methanol and insoluble in n-hexane, chloroform and ethyl acetate. It
showed a single spot which gave a brick red colour with Dragendorff's
reagent(155) using precoated alumina plates. It has Rf value of 0.78 using
system VI.
Spectroscopic Analysis:
1- IR (KBr) showed the following characteristic bands at υ cm-1: 34753290 (OH or NH stretching), 1615 (C=O) and 1545 (NH bending).
2- Positive ion mode MALDI-TOF-MS spectrum m/z (rel.int. %) (Fig.
69): 169 [M+H]+ (70%), 171 [M+2]+ ( 23%),126 (33%), 118 (95%)
and 102 (57%).
3- NMR: 13C- & DEPT 13C-NMR, HMQC and 1H-NMR spectral data are
listed in Table (34) and illustrated in Figs. (70-73).
Table (34):
13
C- & DEPT
13
C-NMR, HMQC and 1H-NMR data of
compound AT-19 (DMSO-d6 + TFA, 400 & 100 MHz).
No.
13
C-NMR
DEPT
HMQC
1
H-NMR (m, J in Hz)
1
166.55
—
—
—
2-NH
—
—
—
8.07 (1H, s)
3
62.90
CH2
3
4.24 (2H, br.s)
N(CH3)3
53.14
CH3
4
3.17 (9H, br.s)
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
165
Discussion:
Compound AT-19 gave brick red colour with Dragendorff's
reagents which suggested being a quaternary ammonium compound(155).
The positive ion mode MALDI-TOF-MS spectrum (Fig. 69)
exhibited a molecular ion peak at m/z 168 with [M+H]+ and [M+2]+ in a
ratio of 3:1 indicating a chloride salt(191). The mass and 13C-NMR spectra
suggested a molecular formula C5H13N2O2-Cl.
The 1H-NMR (Table 34, Fig. 70) exhibited three singlets, one at δH
4.24 assigned for two protons of a methylene group attached to nitrogen,
the other at δH 3.17 assigned for 9 protons representing three methyl
groups attached to nitrogen and the third at δH 8.07 which was attributed to
NH function and confirmed by IR absorption band at υ 3475 cm-1.
The 13C-NMR (Table 34, Fig. 71) revealed three signals at δC 53.14,
62.90 and 166.55 with the signal at δC 53.14 about three times as intense as
that at δC 62.9. The chemical shift of the signal at δC 53.14 was consistent
with that of a trimethyl ammonium group(199) while that at δC 62.9 was
consistent with a CH2-N group(84,199) which was confirmed from the DEPT
spectra (Table 34, Fig. 72) while the signal at δC 166.55 was assigned to a
free carboxylate group and confirmed by IR absorption band at υ
1615 cm-1.
The HMQC correlations (Table 34, Fig. 73) confirmed the
suggested structure where C-3 showed correlation with H-3 and the Nmethyls in the 1H-NMR showed correlations with their carbons in the
13
C-NMR.
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
166
Conclusion:
Based on the previous spectroscopic data and by comparison with
that reported in the literature(84, 199), compound AT-19 is identified as N[(carboxyamino)methyl]–N,N–dimethyl ethanaminium chloride. To
our Knowledge this represents the first report for identification of such
compound from a natural source (new compound) (185).
4
Cl
N
3
H
N
2
1
COOH
N-[(carboxyamino)methyl]-N,N-dimethylethanaminium chloride
Fig. (69): +MALDI-TOF-MS spectrum of compound AT-19.
Fig. (70):
1
H-NMR spectrum of compound AT-19 (DMSO-d6 + TFA,
400 MHz).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Fig. (71):
167
13
C-NMR spectrum of compound AT-19 (DMSO-d6 + TFA,
100 MHz).
Fig. (72): DEPT 13C-NMR spectrum of compound AT-19 (DMSO-d6 +
TFA, 100 MHz).
Fig. (73): HMQC spectrum of compound AT-19 (DMSO-d6 + TFA, 100
MHz).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
168
Identification of Compound AT-20
Physical Properties:
Compound AT-20 was obtained as colourless needles (15 mg,
MeOH), soluble in hot methanol, sparingly soluble in cold methanol and
insoluble in n-hexane, chloroform and ethyl acetate. It showed a single
spot which gave a brick red colour with Dragendorff's reagent(155) using
precoated alumina plates. It has Rf value of 0.78 using system VII.
Spectroscopic Analysis:
1- IR (KBr) showed the following characteristic bands υ cm-1: 3440 (OH)
and 1615 (C=O).
2- NMR: 13C- & DEPT 13C-NMR, HMQC and 1H-NMR spectral data are
listed in Table (35) and illustrated in Figs. (74-77).
Table (35):
13
C- & DEPT
13
C-NMR, HMQC and 1H-NMR data of
compound AT-20 (DMSO-d6 + TFA) (400 & 100 MHz).
No.
1
2-NH
3
4
1'
2'
3'
13
C-NMR
167.67
—
62.90
53.14
67.60
55.48
53.48
DEPT
—
—
CH2
CH3
CH2
CH2
CH3
HMQC
—
—
3
4
2'
1'
3'
1
H-NMR (m, J in Hz)
—
8.10 (1H, s)
4.15 (2H, m)
3.14 (9H, br.s)
3.77 (2H, m)
3.31 (2H, m)
3.02 (9H, br.s)
Discussion:
Inspection of the 1H-NMR spectrum of compound AT-20 (Table 35,
Fig. 74) revealed the presence of two broad singlets at δH 3.02 & 3.14 each
attributed to nine protons representing two sets each of three methyl
groups attached to a quaternary nitrogen(199). In addition to three multiplets
169
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
attributed to three methylene groups at δH 3.31, 3.77 & 4.15. The signals at
δH 3.14, 4.15 and 8.10 were in full agreement with those of compound
AT-19 (P.194) in addition to three signals attributed to another quaternary
ammonium compound but in little ratio.
The broad singlet at δH 3.02 assigned for three methyls and the two
methylene singlets at δH 3.31 and 3.77 were in accordance with those
previously reported for choline(90).
The
13
C-NMR spectrum (Table 35, Fig. 75) revealed signals at δC
53.14, 62.90 and 167.67 in complete accordance with those of compound
AT-19 (P.194) in addition to further carbon signals at δC 53.48, 55.48 and
67.60 assigned for choline(90) which was confirmed from the HMQC
correlations.
This mixture could be identified as N-[(carboxyamino)methyl]–
N,N–dimethyl ethanamine and choline base in a ratio of 5:1 according to
the signal intensity.
Conclusion:
Based on the previous spectroscopic data and by comparison with
that reported in the literature(90,199), compound AT-20 is identified as a
mixture of N-[(carboxyamino)methyl]–N,N–dimethyl ethanamine and
Choline base.
4N
3
H
N
2
1
HO
COO+
N-[(carboxyamino)methyl]N,N-dimethylethanamine
2
1
N
Choline base
3
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Fig. (74):
1
170
H-NMR spectrum of compound AT-20 (DMSO-d6 + TFA,
400 MHz).
Fig. (75):
13
C-NMR spectrum of compound AT-20 (DMSO-d6 + TFA,
100 MHz).
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees. Aerial Parts
Fig. (76): DEPT
171
13
C-NMR spectrum of compound AT-20 (DMSO-d6 +
TFA, 100 MHz).
Fig. (77): HMBC spectrum of compound AT-20 (DMSO-d6 +TFA).
172
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
CHAPTER I
Preliminary Phytochemical Screening of the
Powdered Blepharis ciliaris (L.) B.L.Burtt. Aerial
Parts
The air-dried powdered aerial parts of Blepharis ciliaris (L.)
B.L.Burtt. were subjected to preliminary phytochemical screening for their
different constituents. The tests and results are summarized in the
following table:
Table (36): Results of the preliminary phytochemical screening of the
dried aerial parts of Blepharis ciliaris (L.) B.L.Burtt.
Test
No.
Results
1
Microsublimation test(168)
—
2
Steam distillation(153)
—
3
Carbohydrates and/or glycosides(156)
+
4
Cardenolides(157)
—
5
Unsaturated sterols and/or triterpenes(169,170)
+
6
Tannins(159,171)
+
7
Flavonoids(172)
+
8
Saponins(173)
—
9
Oxidase enzyme(174)
—
10
Lactones and/or esters(153)
+
11
Alkaloids and/or basic nitrogenous substances(171)
—
12
Cyanogenic glycosides(150)
—
13
Anthraquinones(168)
—
14
Coumarins(175)
—
15
Iridoids(155, 176)
—
+ present
— absent
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
173
Conclusion:
The following results could be concluded from Table (36):
The dried aerial parts of Blepharis ciliaris (L.)B.L.Burtt. contain
carbohydrates and/or glycosides, unsaturated sterols and/or triterpenes,
tannins, flavonoids in addition to lactones and/or esters.
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
174
CHAPTER II
Extraction, Fractionation and Isolation of the
main Constituents of Blepharis ciliaris (L.)
B.L.Burtt. Aerial Parts
1- Extraction and Fractionation of Blepharis ciliaris (L.)
B.L.Burtt. Aerial Parts:
The air-dried powdered aerial parts (3 kg) of Blepharis ciliaris (L.)
B.L.Burtt. were extracted by maceration and percolation with (70%)
methanol till complete exhaustion [four times each 10 L., overnight]. The
combined methanolic extracts were concentrated under reduced pressure
till constant weight to give a dark green syrupy residue (270 gm).
The methanolic extract (270 gm) was digested in the least amount of
distilled water subjected to successive solvent fractionation using a
separating funnel with n-hexane, chloroform, ethyl-acetate, n-butanol. The
n-hexane, chloroform, ethyl-acetate, n-butanol extracts were concentrated
separately under reduced pressure to give 55, 37, 45 and 12 gm
respectively and 105 gm of the aqueous extract was left after extraction
with n-butanol which was kept for further study.
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
175
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
176
2- Investigation of the Lipoidal Content of the n-Hexane
Fraction of Blepharis ciliaris (L.)B.L.Burtt. Aerial Parts
Reviewing the current literature, no information could be traced
concerning the lipoidal matter of Blepharis ciliaris (L.)B.L.Burtt. aerial
parts although preliminary phytochemical screening revealed the presence
of sterols and/or triterpenes (P.172).
Accordingly, investigation of the unsaponifiable matter and fatty
acid methyl esters of the n-hexane extract were carried to identify sterols
and/or triterpenes as well as fatty acid content using GC-MS analysis is
undertaken in this chapter.
1- Investigation of the Fatty Acids Composition:
5 gm of the n-hexane fraction of the aerial parts of Blepharis ciliaris
(L.)B.L.Burtt. was saponified as mentioned before (P.63). The fatty acids
fraction is converted to its methyl esters according to the previously
mentioned method (P.63&64), then analysed by GC-MS. The results are
listed in Table (37) and shown in Fig. (78).
177
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
Table (37): Results of GC-MS analysis of fatty acids methyl esters of the
n-hexane Blepharis ciliaris (L.) B.L.Burtt. aerial parts.
Peak
No.
Rt
(min.)
Relativ
e%
Mol.
formula
No. of unsaturation
[M+]
m/z
1
2
3
4
14.024
15.702
17.19
20.77
0.053
0.218
0.111
0.504
C9H18O2
C10H20O2
C11H22O2
C13H26O2
1
1
1
1
158
172
186
214
5
21.283
0.755
C11H20O4
2
216
6
22.226
0.08
C16H30O2
2
254
7
23.2
0.064
C12H22O4
2
230
8
23.417
0.069
C16H32O2
1
256
9
24.397
2.418
C15H30O2
1
242
10
24.752
0.058
C15H28O4
1
272
11
25.064
0.29
C11H22O2
—
186
12
25.387
0.547
C16H32O2
1
256
13
14
25.562
25.769
0.086
0.695
C13H24O2
C16H32O2
2
1
212
256
15
26.266
0.435
C18H36O2
1
284
16
17
18
26.547
26.934
27.034
0.258
0.585
29.77
C14H28O2
C17H34O2
1
2
1
228
268
270
19
27.654
0.347
C12H24O2
—
200
20
27.781
1.002
C18H36O2
1
284
21
27.971
0.262
C11H20O2
2
184
22
28.114
1.884
C18H36O2
1
284
C17H32O2
Component
Methyl octanoate
Nonanoic acid methyl ester
Methyl decanoate
Methyl dodecanoate
Nonadecanoic acid methyl
ester
2,4,6-Trimethyl-11dodecenoic acid methyl ester
Decanedioic acid dimethyl
ester
Methyl-3,7,11-trimethyldodecanoate
Methyl tetradecanoate
1,15-Pentadecanedioic acid
methyl ester
3-Methyldecanoic acid methyl
ester
Methyl-12methyltetradecanoate
Methyl-9-dodecenoate
Methyl n-pentadecanoate
Methyl-5,9,13-trimethyltetradecanoate
Methyl tridecanoate
Methyl-11-hexadecenoate
Methyl hexadecanoate
2,2- Dimethyl Decanoic acid
methyl ester
Methyl-14methylhexadecanoate
Methyl-2-methyl-2-nonenoate
Heptadecanoic acid methyl
ester
178
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
Table (37): Continued.
Peak
No.
Rt
(min.)
Relative
%
Mol.
formula
No. of unsaturation
[M+]
m/z
23
28.586
0.56
C13H26O2
1
214
24
28.835
0.11
C18H34O2
2
282
25
28.919
1.033
C19H38O2
2
298
26
28.978
1.574
C19H34O2
3
294
27
28
29
29.073
29.152
29.475
3.607
1.32
14.589
C19H36O2
C19H36O2
C19H38O2
2
2
1
296
296
298
30
29.708
0.7
C19H34O2
2
294
31
30.063
0.177
C19H36O2
2
296
32
30.508
1.806
C20H40O2
1
312
33
30.598
0.409
C20H40O2
1
312
34
30.863
0.555
C5H10O2
1
102
35
32.292
1.15
C19H32O2
4
292
36
33.346
3.519
C21H42O2
1
326
37
34.728
0.175
C17H34O2
1
270
38
35.258
0.713
C22H44O2
1
340
39
36.486
3.938
C23H45O2
1
354
40
36.672
0.5
C17H32O2
1
268
41
37.111
0.344
C20H40O2
1
312
42
37.418
1.874
C24H48O2
1
368
43
38.276
2.91
C25H50O2
1
382
44
45
40.315
43.18
0.602
0.331
C27H54O2
C29H58O2
1
1
410
438
Component
Methyl-2,4,6trimethylnonanoate
Oleic acid methyl ester
Methyl-16-methylheptadecanoate
8,11-Octadecadienoic acid
methyl ester
Methyl-8-octadecenoate
Methyl-9-octadecenoate
Methyl octadecanoate
9,12-Octadecadienoic acid
methyl ester
Methyl-7-octadecenoate
Methyl-2,6,10,14tetramethylpenta-decanoate
Methyl-17-methyloctadecanoate
Methyl butanoate
Methyl-6,9,12-octadecatrienoate
Methyl eicosanoate
Methyl-2,4-dimethyltetradecanoate
Methyl heneicosanoate
Docosanoic acid methyl
ester
9-Hexadecenoic acid
methyl ester
Methyl-2-methyloctadecanoate
Methyl tricosanoate
Tetracosanoic acid methyl
ester
Methyl hexacosanoate
Methyl octacosanoate
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
179
Fig. (78): GC-MS analysis chromatogram of FAMEs of the n-hexane of
Blepharis ciliaris (L.) B.L.Burtt. aerial parts.
Conclusion:
GC-MS analysis of fatty acids methyl esters of the n-hexane fraction
of the aerial parts of Blepharis ciliaris (L.) B.L.Burtt. (Table 37) revealed
the presence of 45 fatty acids which have been identified through their
mass spectra (Fig.78). The major fatty acids identified were methyl
hexadecanoate (29.77%), methyl octadecanoate (14.589%), docosanoic
acid methyl ester (3.938%), methyl-(8E)-8-octadecenoate (3.607%) and
methyl eicosanoate (3.519%).
2- Investigation of the Unsaponifiable Matter:
The unsaponifiable matter was analysed by GC-MS. The results are
listed in Table (38) and shown in Figs. (79 & 80).
180
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
Table (38): Results of GC-MS analysis of unsaponifiable matter of the nhexane fraction of Blepharis ciliaris (L.)B.L.Burtt. aerial
parts.
Peak
Rt
Relativ
Mol.
No. of un-
[M+]
No.
(min.)
e%
formula
saturation
m/z
1
2
3
4
5
5.769
13.039
14.156
14.882
15.538
0.066
0.12
0.04
0.07
1.898
C19H32
C9H18
C19H40
C7H14
C4H8
4
1
—
1
—
260
126
268
98
56
6
15.829
0.13
C7H9D5
6
103
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
16.47
18.361
19.69
22.189
22.321
22.435
23.047
23.693
24.016
24.177
24.244
24.53
24.805
25.038
25.435
25.459
25.515
25.859
25.991
26.11
26.256
26.316
26.473
26.674
26.677
26.817
0.057
0.137
0.134
0.228
0.175
0.023
0.45
0.219
0.124
0.278
1.44
0.411
0.112
0.068
0.41
0.637
0.168
0.063
5.223
2.579
0.077
0.47
0.27
0.3
4.618
0.147
C12H24
C14H30
C16H32
C16H32
C16H34
C29H50O
C9H18
C15H30
C17H36
C27H46O
C18H20
C14H28
C13H22
C18H38
C18H38
1
—
1
1
—
5
1
1
—
5
9
1
3
—
—
5
—
4
—
5
1
4
1
—
1
4
168
198
224
224
226
414
126
210
240
386
236
196
178
254
254
414
282
260
128
400
210
402
196
268
350
260
C29H50O
C20H42
C19H32
C9H20
C28H48O
C15H30
C28H50O
C14H28
C19H40
C29H48O
C19H32
Table (38): Continued.
Component
(1-Pentyloctyl)-benzene
2,6-Dimethyl-1-heptene
2,6,10,14-Tetramethylpentadecane
Cycloheptane
Cyclobutane
1,1-D2-2-(D3-methyl)-4-methyl-1pentene
Cyclododecane
Tetradecane
Cyclohexadecane
1-Hexadecene
Hexadecane
Stigmast-5-en-3-beta-ol
2,trans-3-Trimethylcyclo-hexane
1-Pentadecene
Heptadecane
Cholest-5-en-3-ol
1,1,3-Trimethyl-3-phenylindan
3-Tetradecene
1-Isopropyladamantane
3-Methylheptadecane
Octadecane
29-Nornalost-8-en-3-beta-ol
2,6,10,14-Tetramethylhexadecane
(1-butynonyl)-benzene
2,4-Dimethylheptane
Methylcholesterol
Cyclopentadecane
Ergostanol
Cyclotetradecane
Nonadecane
(4-Octyldodecyl)-cyclopentane
1-Methyldodecylbenzene
181
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
Peak
Rt
Relative
Mol.
No. of un-
[M+]
No.
(min.)
%
formula
saturation
m/z
33
26.872
0.805
C29H50O
5
414
34
27.018
0.112
C12H6D6
6
162
35
36
37
38
39
40
41
27.217
27.389
27.497
27.844
28.096
28.302
28.906
0.226
0.076
1.084
0.153
9.372
1.615
2.046
C29H50O
C24H50
C28H46O
C18H27
C29H50O
C29H52O
C30H50O
5
—
6
6
5
4
6
414
338
398
242
414
416
426
42
28.935
0.145
C19H40
—
236
43
29.046
0.459
C21H44
—
296
44
29.764
0.572
C29H48O
6
412
45
46
47
48
49
50
51
52
53
54
55
56
29.928
30.214
30.383
30.508
31.021
31.543
31.774
31.839
32.348
32.425
32.653
0.188
5.963
1.293
1.23
8.11
1.757
5.556
0.474
1.489
4.19
7.36
C29H48O
C30H50O
C30H50O2
C19H38
C29H50O
C31H52O
C25H50
C29H46O
C29H48O
C20H40
C22H46
6
6
6
1
—
6
6
7
6
1
1
412
426
442
266
414
440
412
410
412
280
310
33.402
0.702
C29H48O
6
412
57
33.91
0.457
C29H48O
6
412
58
33.979
0.687
C29H48O
6
412
59
34.545
0.487
C29H48O
6
412
60
61
62
34.723
34.802
36.084
1.28
0.987
4.6
C24H48
C24H50
C18H36
1
9
1
336
338
252
Table (38): Continued.
Component
Ethyl-cholest-22-en-3-alpha-ol
1,2,3,6,7,8-D6-4,5-dimethylnaphthalene
14-Methylergost-8-en-3-ol
2-Methyltricosane
Dehydrocholesterol-1-ether
1,4-Dicyclohexylbenzene
Ethylcholest-5-en-3-beta-ol
23-Ethylcholestan-3-beta-ol
Urs-12-en-3-beta-ol
6,6'-Dimethylidene-2,2'bi(tricycle[3.3.0.0(3,7)]octylidene)]
Heneicosane
24,26-Dimethylcholesta-5,26-dien3-beta-ol
Stigmasta-5,22-dien-3-beta-ol
Urs-20(30)-en-3-ol
Ergost-8(14)-en-3-yl acetate
1-Nonadecene
β-sitosterol
24-Methylenecycloartanol
Stigmasterol
Stigmasta-4,6,22-trien-3-alpha-ol
Stigmasta-7,22-dien-3-ol
Cycloeicosane
Docosane
4,4-Dimethylcholesta-7,14-dien-3ol
Conicasterol
24,26-Dimethylergosta-5,24-dien3-beta-ol
24,27-Dimethylcholesta-5,26-dien3-beta-ol
Cyclotetracosane
Tetracosane
1-Octadecene
182
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
Peak
Rt
Relativ
Mol.
No. of un-
[M+]
No.
(min.)
e%
formula
saturation
m/z
63
64
65
66
67
68
69
70
71
72
73
74
75
76
36.163
37.148
37.884
37.945
37.99
38.747
38.853
38.896
39.521
39.833
40.956
41.12
42.391
42.735
4.417
0.362
0.608
1.265
5.26
0.19
0.265
0.38
1.86
1.161
0.117
0.61
2.527
0.363
C25H52
C26H54
C23H48
C29H6O
C21H36
C23H46
C30H62
C28H58
C18H38
C13H28
C20H43
C19H40
C35H70
—
—
—
5
—
4
1
—
—
1
—
—
—
1
352
366
324
402
408
288
322
422
394
254
184
282
268
490
Pentacosane
Hexacosane
Tricosane
16,22-Epoxycholestan-3-ol
Nonacosane
14B-Pregnane
9-Tricosene
Triacontane
Octacosane
4-Methylheptadecane
Tridecane
Eicosane
2,6,10-Trimethylhexadecane
17-Pentatriacontene
77
78
44.403
46.304
0.24
0.928
C27H56
C31H64
1
—
380
436
8-Hexyl-8-pentylhexadecane
11-Decylhenicosane
C27H46O2
Component
Fig. (79): GC-MS analysis chromatogram of unsaponifiable matter of
the n-hexane of Blepharis ciliaris (L.) B.L.Burtt. aerial parts.
183
(4-Octyldodecyl)cyclopentane
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
Fig. (80): GC-MS analysis chromatogram of unsaponifiable matter of the
n-hexane of Blepharis ciliaris (L.) B.L.Burtt. aerial parts.
Conclusion:
GC-MS analysis of the unsaponifiable matter of the n-hexane
fraction of the aerial parts of Blepharis ciliaris (L.)B.L.Burtt. (Table 38,
Figs.79 & 80) revealed the presence of 53 hydrocarbon representing
(57.27%) and 24 sterol representing (40.025%).
This study is considered as the first report for the lipid investigation
of Blepharis ciliaris (L.) B.L.Burtt..
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
184
3- Isolation of the Main Constituents of the n-Hexane
Fraction of Blepharis ciliaris (L.) B.L.Burtt. Aerial Parts:
A part of the n-hexane soluble fraction (45 gm) was subjected to
vacuum liquid chromatography (VLC) using n-hexane-ethyl acetate
gradient. Fractions of each polarity were collected together and
concentrated under reduced pressure to give six sub-fractions labeled
(BCH-I−BCH-VI).
Sub-fraction BCH-II (9.5 gm) was re-chromatographed on silica gel
column chromatography (360 gm) and eluted with n-hexane followed by nhexane : ethyl acetate gradient. Fractions 100 ml each were collected and
monitored with TLC then similar fractions were grouped together. The
fractions eluted with n- hexane : ethyl acetate (9.5:0.5) afforded
compounds BC-1 (80 mg) and BC-2 (10 mg) while fractions eluted with
n- hexane : ethyl acetate (9:1) afforded compound BC-3 (80 mg).
Sub-fraction BCH-V (5.5 gm) was re-chromatographed on silica gel
column chromatography (200 gm) and eluted with n-hexane : ethyl acetate
gradient. Fractions (100 ml each) were collected and monitored by TLC.
Similar fractions were collected and grouped together where fractions
eluted with n-hexane : ethyl acetate (6:4) afforded compound BC-4 (100
mg).
The other sub-fractions were subjected to further isolation and
purification of their constituents but nothing significant was isolated.
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
185
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
186
4- Isolation of the Main Constituents of the Chloroform
Fraction of Blepharis ciliaris (L.) B.L.Burtt. Aerial Parts:
A part of the chloroform soluble fraction (30 gm) was subjected to
vacuum liquid chromatography (VLC) using chloroform : methanol
gradient elution. Fractions soluble in each polarity were collected together
and concentrated under reduced pressure to give 5 sub-fractions labeled
BCC-I−BCC-V.
Sub-fraction BCC-II (14 gm) was re-chromatographed on silica gel
column chromatography (560 gm) and eluted with chloroform, followed by
chloroform : methanol gradient. Fractions (100 ml each) were collected
and monitored by TLC and similar fractions were collected and grouped
together. The fractions eluted with chloroform : methanol (9.5:0.5)
afforded compounds BC-5 (30 mg) and fractions eluted with polarity (9:1)
after repeated purification steps through small columns afforded 5
compounds which are: BC-6 (20 mg), BC-7 (30 mg), BC-8 (15 mg), BC-9
(50 mg) and BC-10 (50 mg).
Sub-fraction BCC-III (3 gm) was re-chromatographed on silica gel
column chromatography (120 gm) and eluted with chloroform : methanol
gradient. Fractions (50 ml each) were collected, grouped and similar
fractions were collected together. The fractions eluted with chloroform :
methanol (9:1) yielded compound BC-11 (30 mg).
The other sub-fractions were subjected to further isolation and
purification of their constituents but nothing significant was isolated.
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
187
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
188
5- Isolation of the Main Constituents of the Ethyl Acetate
Fraction of Blepharis ciliaris (L.) B.L.Burtt. Aerial Parts:
A part of the ethyl acetate soluble fraction (40 gm) was subjected to
vacuum liquid chromatography (VLC) using chloroform : methanol
gradient elution. Fractions soluble in each polarity were collected together
and concentrated under reduced pressure to give 5 sub-fractions labeled
BCE-I−BCE-V.
Sub-fraction BCE-II (12.5 gm) was re-chromatographed on silica
gel column chromatography (500 gm) and eluted with chloroform :
methanol gradient. Fractions (100ml each) were collected and monitored
by TLC. Similar fractions were collected and grouped together. Fractions
eluted with chloroform : methanol (9.5:0.5) afforded compound BC-12 (30
mg) and those eluted with polarity (9:1) yielded compound BC-13 (50
mg).
Sub-fraction BCE-III (8.5 gm) was re-chromatographed on silica gel
column chromatography (350 gm) and eluted with chloroform : methanol
gradient. Fractions (100 ml each) were collected, grouped and similar
fractions were collected together. The fractions eluted with chloroform :
methanol (9:1) yielded compound BC-14 (105 mg) and BC-15 (120 mg).
Sub-fraction BCE-IV (7 gm) was re-chromatographed on reversed
phase column chromatography (5 gm) and eluted with water : methanol
gradient. Fractions (15 ml each) were collected and monitored by TLC.
Similar fractions were collected and grouped together. Fractions eluted
with 30% methanol yielded compound BC-16 (20 mg).
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
189
190
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
CHAPTER III
Identification of the Isolated Compounds from
Blepharis ciliaris (L.) B.L.Burtt. Aerial Parts
Identification of Compound BC-1
Physical Properties:
Compound BC-1 (50 mg) was obtained as white fine needles from
n-hexane, m.p. 134-136°C. It is soluble in n-hexane, chloroform and
sparingly soluble in methanol. It responded to Salkowski’s and
test(169,170)
Liebermann-Burchard’s
suggesting
its
steroidal
or
triterpenoidal nature. It showed a single spot which attained a violet
colour after spraying with 10% v/v H2SO4 and heating at 110°C for 10
min. using precoated silica gel plates. It showed Rf value of 0.65
upon using system III.
Conclusion:
From the above mentioned physical, chemical data in addition to
co-chromatography with an authentic sample of β-sitosterol which
showed the same Rf values, colour reaction and mixed melting point.
Compound BC-1 is identified as β-sitosterol and confirmed from the GCMS of lipoidal content of the n-hexane fraction (P.176).To our knowledge
this represents the first report for its identification from Blepharis ciliaris
(L.) B.L.Burtt..
29
28
21
20
22
23
18
12
19
1
2
11
25
17
9
8
10
4
5
26
27
16
3
HO
13
24
14
15
7
6
β-sitosterol
191
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
Identification of Compound BC-2
Physical Properties:
Compound BC-2 was obtained as white crystalline needles (30 mg,
CHCl3), m.p. 158-160°C. It is soluble in n-hexane, chloroform and
insoluble in methanol. It responded to Salkowski’s & LiebermannBurchard’s tests(169,170) indicating its steroidal or triterpenoidal nature. It
showed a single spot which attained a violet colour after spraying with
10% v/v H2SO4 and heating at 110°C for 10 min. using precoated silica
gel plates. It has Rf value of 0.67 upon using system III.
Conclusion:
From the above mentioned physical, chemical data in addition to
co-chromatography with an authentic sample of Stigmasterol which
showed the same Rf values, colour reaction and mixed melting point,
compound BC-2 is identified as Stigmasterol and confirmed from the
GC-MS of the lipoidal content of the n-hexane fraction (P.176). To our
knowledge this represents the first report for its identification Blepharis
ciliaris (L.) B.L.Burtt..
29
28
21
18
12
19
11
1
2
8
3
HO
17
15
7
5
4
14
23
24
26
25
27
16
9
10
13
22
20
6
Stigmasterol
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
192
Identification of Compound BC-3
Physical Properties:
Compound BC-3 was isolated as a white amorphous powder (80
mg, n-hexane). It is soluble in chloroform and n-hexane and insoluble in
ethyl-acetate and methanol. It showed a single spot which attained a
violet colour after spraying with 10% v/v H2SO4 and heating for 10 min.
at 110°C using precoated silica gel plates. It gave a positive LiebermannBurchard's test(169,170) suggesting its steroidal nature. It has Rf value of
0.75 using system I.
Spectroscopic Analysis:
1- IR (KBr) showed the following absorption bands at υ cm-1: 2920 (CH stretching), 1720 (ester carbonyl), 1632 (C=C) stretching and 724
(long aliphatic chain).
2- a) +FAB-MS of compound BC-3 (Fig. 81): 763 [M+1]+ (3%), 439
[M-fatty acid]+ (13%), 413 (80%), 351 (60%), 349 (27%), 301 (58%)
and 274 (40%).
b) EI-MS of fatty acid methyl ester at m/z (Fig. 82): 382 [M]+ (15%),
367 [M-15]+ (11%) and 351 [M-OCH3]+ (15%).
3- NMR: 13C- and 1H-NMR data are listed in Table (39) and illustrated in
Figs. (83 & 84).
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
Table (39):
13
1
C- and
H-NMR
data of compound BC-3 (CDCl3,
400 MHz).
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
1'
13
C-NMR
36.95
31.87
73.61
39.57
139.66
122.56
34.71
31.92
51.20
36.56
20.97
38.10
42.25
56.7
22.98
28.23
56.61
11.96
19.82
40.54
21.12
129.18
138.34
49.93
21.20
31.92
14.14
28.23
14.14
173.37
2'
39.57
3'
4'-23'
24'
25.05
29.02-29.69
12.01
193
1
H-NMR (m, J in Hz)
—
—
3.98 (1H, t, J = 6.4)
—
—
5.30 (1H, d, J = 5.2 Hz)
—
—
—
—
—
—
—
—
—
—
—
0.77 (3H, s)
0.94 ( 3H, s)
—
1.21 (3H, s)
4.94 (1H, dd, J = 15.2, 8.8)
5.08 (1H, dd, J = 15.2, 8.8)
—
—
0.85 (3H, d, J = 6.8)
0.85 (3H, d, J = 6.8)
—
0.82 (3H, m)
—
a 2.22 (1H, d, J = 8.0)
b 2.18 (1H, d, J = 8.0)
1.62 (2H, m)
1.14-1.21(m)
0.60 (3H, t, J = 6.8)
194
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
Discussion:
The IR spectrum of compound BC-3 showed characteristic
absorption bands indicating the presence of an ester group (1720 cm-1),
unsaturation (1632 cm-1) and long aliphatic chain (724 cm-1).
The 1H-NMR spectrum (Table 39, Fig. 83) exhibited signals for
sitosteryl derivative with two double bond protons at δH 4.94 (1H, dd, J =
15.2, 8.8 Hz), 5.08 (1H, dd, J = 15.2, 8.8 Hz) and 5.30 (1H, d, J = 5.2 Hz)
respectively, multiple aliphatic protons at δH 1.14-1.21 suggesting the
presence of long chain aliphatic moiety.
The 1H-NMR spectrum (Table 39, Fig. 83) also displayed two
tertiary, three secondary and one primary methyl groups characteristic for
sitosteryl unit(165, 194). Two one proton doublets at δH 2.22 and 2.18 were
attributed to C-2' methylene protons adjacent to an ester group(200,201). A
triplet at δH 0.60 (3H, t, J = 6 Hz) was due to terminal primary methyl
protons.
The
13
C-NMR spectrum (Table 39, Fig. 84) displayed 53 signals,
29 of them were attributed to sitosteryl moiety with vinylic carbons at δC
139.66 (C-5), 122.56 (C-6), 129.18 (C-22) and 138.34 (C-23). A carbonyl
carbon at δC 73.61 (C-3), in addition to other carbon signals in full
agreement with those previously reported for stigmasterol(165,
194)
. The
signal at δC 173.37 was assigned to an ester carbonyl which was
confirmed by IR absorption band at υ 1720 cm-1, while the carbon signal
at δC 12.01 was attributed to a terminal methyl group of a straight chain
fatty acid ester(200, 201).
195
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
The positive FAB-MS of compound BC-3 showed molecular ion
peak at m/z 763 [M+1]+ and other characteristic fragments at m/z 439,
413, 351 and 274 indicated the presence of stigmasterol units.
Upon alkaline hydrolysis compound BC-3 (P.65) yielded
stigmasterol which was identified by co-chromatography with authentic
sample in addition to a fatty acid. The prepared fatty acid methyl ester
(P.64) was identified by mass spectroscopy which gave a molecular ion
peak at m/z 382, 351 [M-OCH3]+ and a series of fragment ions with the
difference of m/z 28 or 14 indicated a methyl tetracosanoate moiety.
Conclusion:
From the above mentioned spectral data and by comparison with
the previously reported data of similar compounds(200-202), compound BC3 is identified as Stigmasterol tetracosanoate. To our knowledge it is
the first time for its identification from natural source (new
compound)(185).
29
28
21
22
20
23
18
24
26
25
12
19
1
8
3
1'
O
C
27
9
10
15
7
5
4
17
16
14
2
O
13
11
6
23'
(CH2)22' CH
3
Stigmasterol tetracosanoate
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
196
Fig. (81): +FAB-MS spectrum of compound BC-3.
Fig. (82): EI-MS spectrum of fatty acid methyl ester of compound BC-3.
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
197
Fig. (83): 1H-NMR spectrum of compound BC-3 (CDCl3, 400 MHz).
Fig. (84): 13C-NMR spectrum of compound BC-3 (CDCl3, 100 MHz).
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
198
Identification of Compound BC-4
Physical Properties:
Compound BC-4 was obtained as white powder (100 mg, nhexane). It is soluble in n-hexane, chloroform and insoluble in ethylacetate and methanol. It showed a single spot which gave an orange
colour after spraying with 10 % v/v H2SO4 then heating for 10 min. using
precoated silica gel plates. It has Rf value of 0.76 using system IV.
Spectroscopic Analysis:
1- IR (KBr) spectrum showed the following absorption bands at υ cm-1 :
3470-3230 (OH or NH stretching), 1626 (amide carbonyl), 1554 (NH
bending).
2- (a) + FAB-MS of compound BC-4 (rel.int.%) (Fig. 85): m/z 629
[M+1]+ (10%), 583 (12%), 327 (6%) and 312 (15%).
After methanolysis:
(b) EI-MS of the LCB (rel.int. %) (Fig. 86): m/z 317 [M]+ (25%), 299
[M-H2O]+ (27%), 281 [M-2H2O]+ (10%) and 263 [M-3H2O]+ (5%).
(c) EI-MS of the FAME (rel.int. %) (Fig. 87): m/z 342 [M+] (27%),
325 [M-OH]+ (4%), 311 [M-OCH3]+ (15%), 283 [M-CH3COO]+
(5%), 253 (2%) and 225 (5%).
3- NMR: 13C-, DEPT 13C- and 1H-NMR spectral data are listed in Table
(40) and illustrated in Figs. (88-90).
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
Table (40):
13
C-, DEPT
199
13
C- and 1H-NMR data of compound BC-4
(C5D5N-d5, 400 & 100 MHz).
No.
13
C-NMR
DEPT
1
61.94
CH2
2
3
4
52.89
76.67
72.94
CH
CH
CH
5
34.05
CH2
6
25.78
7
26.63
(CH2)n
29.58 – 30.30
CH3 x 2
14.26
1'
175.23
2'
72.43
3'
35.67
CH3CH2CH2
32.10
CH3CH2
22.92
-NH
—
1-OH
—
3&4-OH
—
2'-OH
—
C
C
CH2
CH3
—
CH
CH2
CH2
CH2
—
—
—
—
1
H-NMR (m, J in Hz)
a 4.54 (1H, dd, J = 4.8,10.8)
b 4.45 (1H, dd, J = 4.8,10.8)
5.14 (1H, m)
4.40 (1H, m)
4.31 (1H, m)
a 2.05 (1H, m)
b 1.94 (1H, m)
—
—
1.23 1.41 (m)
0.84 (6H, t, J = 6)
—
4.63 (1H, m)
2.24 (1H, m)
—
—
8.62 (1H, d, J = 9.2)
6.31 (1H, d, J = 6.4)
6.81 (2H, br.s)
7.73 (d, J = 4.8)
Discussion:
Inspection of the IR spectrum of compound BC-4 revealed the
presence of absorption bands atυ cm-1: 3470-3230 for a bonded OH or NH stretching, 1626 for an amide carbonyl and 1554 for N-H bending in
addition to CH2 functionalities suggesting a sphingolipid structure(177).
The 1H-NMR spectrum (Table 40, Fig. 88) showed characteristic
signals for an amide proton at δH 8.62 (d, J = 9.2 Hz), resonances for four
hydroxyl groups: a doublet at δH 7.73 (J = 4.8 Hz), a singlet at δH 6.81
200
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
(integrating for two hydrogens) and δH 6.31 which appears as a doublet (J
= 6.4 Hz) attributed to 2'-OH, 3&4-OH and 1-OH respectively(166, 167,177).
Also a signal at δH 5.14 (m, H-2) for a methine bonded to a nitrogen(178),
signals at δH 4.54 (dd, J = 10.8, 4.8 Hz, H-1a) and δH 4.45 (dd, J = 10.8,
4.8 Hz, H-1b) for a hydroxymethylene, as well as signals at δH 4.31 (m,
H-4), 4.40 (m, H-3) and 4.63 (m, H-2') corresponding to three
oxymethines. In addition to two terminal methyls at δH 0.84 (t, J = 6 Hz)
and several methylene hydrogens at δH 1.23-1.41 (m) corresponding to
two saturated straight aliphatic chains were also observed.
The
13
C-NMR spectrum of compound BC-4 (Table 40, Fig. 89)
exhibited one downfield signal at δC 175.23 (C-1') corresponding to an
amide carbonyl. Signals for a nitrogenated methine at δC 52.89 (C-2), an
oxymethylene at δC 61.94 (C-1) and three oxymethines at δC 76.67 (C-3),
72.94 (C-4) and 72.43 (C-2'). In addition to several carbon signals in the
range of δC 30.30 − 29.58 related to methylene groups and a carbon signal
at δC 14.26 corresponding to two terminal methyls were also deduced
from 13C-NMR spectra and in agreement with similar compounds(177-179 )
of the sphingolipid.
The mass spectral studies (Figs. 85-87) showed positive ion FABMS
at
m/z
629
[M+1]+
(10%)
calculated
for
C38H77NO5..
Methanolysis(166,167) of compound BC-4 as mentioned (P.65) resulting in
n-hexane fraction containing fatty acid methyl ester (FAME) and ethyl
acetate fraction containing a long chain base (LCB).
BC-4
1N HCl
aq. MeOH
reflux
H2N
O
OH
2
(CH2)16
H3CO
OH
CH3
+
3
1
(CH2)12
4
5
OH
Methanolysis of compound BC-4
OH
CH3
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
201
The fatty acid methyl ester (FAME) was crystallized from
methanol to give an amorphous whitish powder (8 mg).The EI-MS of
(FAME) showed molecular ion peak at m/z 342 [M]+ (14%) calculated
for the molecular formula C21H42O3and other fragments at m/z325 [MOH]+ indicating the presence of hydroxylated fatty acid moiety. In
addition to other mass fragments at m/z 311 [M+-CH3O]+(5%), 281 [MCH3COO]+ (5%) followed by sequential loss of CH2 units. It was
identified as 2'-hydroxyeicosanoic acid methyl ester.
While the (LCB) was crystallized from methanol to give whitish
residue (7mg). the EI-MS of the (LCB) showed molecular ion peak at m/z
317 [M]+ (25%) calculated for the molecular formula C18H39NO3 and
other mass fragments at m/z 299 [M-2H2O]+, 281 [M-2H2O]+ and 263
[M-3H2O]+ confirmed the three hydroxyl functional groups in the (LCB).
In addition to sequential loss of CH2 units, the base was identified as 2aminooctadecane-1,2,3-triol.
Reading the stereochemistry, the formulated absolute configuration
of compound BC-4 was based on the carbon chemical shifts at δC 61.94
(C-1), 52.89 (C-2), 76.67 (C-3), 175.23 (C-1'), 72.43 (C-2'), which
appeared to be fairly close to those previously reported for (2S,3S, 4R,
2'R) sphingosine moieties(177).
By comparison of the obtained data with those of analogous
compounds(177-179), compound BC-4 could be identified as sphingolipid.
Conclusion:
On the basis of the above mentioned spectroscopic data and
comparison those previously reported(203), the structure of compound BC4 is established as (2S, 3S, 4R)-2[(2'R)-2'-(hydroxyeicosanoyl amino)
octadecane-1,3,4-triol. To our knowledge this represents the first report
for the identification of sphingolipids from family Acanthaceae.
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
202
O
1'
2'
3'
(CH2)16
CH3
OH
HN
OH
2
1
3
4
(CH2)12
CH3
5
OH
OH
(2S, 3S, 4R)-2[(2'R)-2'-(hydroxyeicosanoyl amino) octadecane-1,3,4-triol
Fig. (85): +FAB-MS spectrum of compound BC-4.
Fig. (86): EI-MS spectrum of LCB.
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
203
Fig. (87): EI-MS spectrum of FAME.
Fig. (88): 1H-NMR spectrum of compound BC-4 (C5D5N-d6,400 MHz).
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
204
Fig. (89): 13C-NMR spectrum of compound BC-4 (C5D5N-d6, 100 MHz).
Fig. (90): DEPT
13
C-NMR spectrum of compound BC-4 (C5D5N-d6,
100 MHz).
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
205
Identification of Compound BC-5
Physical Properties:
Compound BC-5 was isolated as colourless needles (25 mg,
MeOH) with m.p. 59-62°C. It is soluble in chloroform, ethyl acetate and
methanol while insoluble in n-hexane. It showed a single spot which gave
a brown colour with 10% v/v H2SO4 and heating for 10 min. also gave a
green colour with FeCl3 (T.S) (155) using precoated silica gel plates. It has
an Rf value of 0.67 using system VI.
Spectroscopic Analysis:
1- IR (KBr) showed the following absorption bands at υ cm-1: 3420
(OH), 1670 (C=O) and 1510-1100 (aromaticity).
2- EI-MS m/z (rel.int. %) Fig. (91): 196 [M]+ (40%), 168 (100%), 165
[M-(OCH3)]+ (3%), 153 [M-43]+ (40%) and 134 [M-(2OCH3)]+ (3%).
3- NMR: 13C- and 1H-NMR data are listed in Table (41) and illustrated
in Figs. (92 & 93).
Table (41):
13
C- and 1H-NMR data of compound BC-5 (DMSO-d6, 400
& 100 MHz).
No.
1
2
3
4
5
6
7
8-OCH3
3&4-OCH3
13
C-NMR
121.79
112.66
147.21
151.04
115.01
123.45
167.33
51.41
55.51, 55.87
1
H-NMR (m, J in Hz)
—
7.40 (1H, br.s)
—
—
6.82 (1H, d, J = 8)
7.41 (1H, d, J = 8)
—
3.57 (3H, s)
3.79 (6H, s)
Discussion:
The mass and
13
C-NMR spectral data of compound BC-5
suggested the presence of an aromatic acid derivative(191) with a
molecular ion peak at m/z 196 for the molecular formula C10H12O4.
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
The
206
1
H-NMR spectrum (Table 41, Fig. 92) exhibited three
aromatic signals at δH 7.40 (br.s), 6.82 (d, J = 8 Hz) and 7.41 (d, J = 8
Hz) attributed to H-2, H-5 and H-6 respectively indicated the presence of
a tri-substituted benzene ring. A singlet signal representing three protons
of a methoxy group at δH 3.57 which is located on C-8 in addition to
another singlet representing six protons appear at δH 3.79 attributed to
two methoxy groups located on C-3 and C-4 respectively.
The 13C-NMR spectrum (Table 41, Fig. 92) displayed 10 signals, a
downfield signal at δC 167.3 attributed to an ester carbonyl in addition to
a methoxy group signal at δC 51.41 assignable to ester function and two
methoxy groups attached to the benzene ring appear at δC 55.51 and
55.87. The spectrum also exhibited carbon signals with chemical shift in
accordance with similar compounds(189, 190, 204).
EI-MS spectral data of compound BC-5 (Fig. 91) revealed a
molecular ion peak at m/z 196 and other significant peaks at m/z 165
[M+-OCH3] and 134 [M+-(2OCH3)] confirming the presence of benzoic
acid derivative with C3 and C4 methoxy substitution.
Conclusion:
Comparing the obtained data with those reported for similar
compounds(189,
190, 204)
, compound BC-5 is identified as 3,4-dimethoxy
benzoic acid methyl ester (methyl veratrate). To our knowledge this
represents the first report for its identification from family Acanthaceae.
7
8
COOCH3
1
2
6
5
3
4
OCH3
OCH3
Methyl veratrate
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
207
Fig. (91): EI-MS spectrum of compound BC-5.
Fig. (92): 1H-NMR spectrum of compound BC-5 (DMSO-d6, 400 MHz).
Fig. (93): 13C-NMR spectrum of compound BC-5 (DMSO-d6, 100 MHz).
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
208
Identification of Compound BC-6
Physical Properties:
Compound BC-6 was obtained as pale yellow needles (20mg,
MeOH), m.p. 65-67°C. It is soluble in chloroform, ethyl acetate,
methanol and insoluble in n-hexane. It showed a single spot which gave
brown colour with 10% v/v H2SO4 and heating at 110°C for 10 min. also
it gave a green colour with FeCl3 (T.S)(155) using precoated silica gel
plates. It has an Rf value of 0.52 using system VI.
Spectroscopic Analysis:
1- IR (KBr) cm-1 showed the following absorption bands atυ cm-1: 3440
(OH), 1675 (C=O), and 1520-1100 (aromaticity).
2- EI-MS m/z (rel.int. %) Fig. (94): 182 [M]+ (17%), 168 (100%), 167
[M-CH3]+ (5%), 165 (2%), 151 [M-OCH3]+ (13%) and 138 [MCOO]+ (3%).
3- NMR: 13C- and 1H-NMR data are listed in Table (42) and illustrated
in Figs. (95 & 96).
Table (42):
13
C- and 1H-NMR data of compound BC-6 (DMSO-d6, 400
& 100 MHz).
No.
1
2
3
4
5
6
7
3-OCH3
8-OCH3
4-OH
13
C-NMR
121.79
112.62
147.0
150.93
114.94
123.38
167.30
55.46
51.31
—
1
H-NMR (m, J in Hz)
—
7.40 (1H, br.s)
—
—
6.81 (1H, d, J = 8.8)
7.41 (1H, d, J = 6.8)
—
3.78 (3H, s)
3.56 (3H,s)
9.86 (1H, br.s)
Discussion:
The mass and
13
C-NMR spectral data of compound BC-6
suggested the presence of an aromatic acid derivative(191) with a
molecular formula C9H10O4. It showed a molecular ion peak at m/z 182.
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
209
The 1H-NMR spectrum (Table 42, Fig. 95) revealed three signals in
the aromatic region for a tri-substituted benzene ring at δH 7.40 (br.s),
7.41 (d, J = 6.8 Hz) and 6.81 (d, J = 8.8 Hz) attributed to H-2, H-6 and H5 respectively. Two signals each for three protons at δH 3.78 and 3.56
indicating the presence of two methoxy groups and a singlet at δH 9.86
(1H, br.s) attributed to a hydroxyl group attached to C-4 which is
confirmed by an absorption band in the IR spectrum at υ 3440 cm-1.
The 13C-NMR spectrum (Table 42, Fig. 96) displayed nine signals
with a downfield one at δC 167.3 attributed to an ester carbonyl in
addition to two methoxy signals at δC 51.31 for a methoxy ester and 55.46
for a methoxy attached to aromatic system. The other six aromatic
carbons at δC 150.93, 147.0, 123.47, 121.79, 114.94 and 112.62 which are
in good agreement with those for similar compounds (190,193, 204).
The mass spectral data (Fig. 94) confirmed the suggested structure
by the appearance of fragment peaks at m/z 167 [M-CH3]+ and 151[MOCH3]+.
Conclusion:
Comparing the obtained spectral data with those previously
reported in the literature for similar compounds(193,204), compound BC-6 is
concluded to be 3-methoxy,4-hydroxy benzoic acid methyl ester
(methyl vanillate). To our knowledge this represents the first report for
its identification from the genus Blepharis.
O
8
7C
OCH3
1
6
2
5
3
4
OCH3
OH
Methyl vanillate
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
210
Fig. (94): EI-MS spectrum of compound BC-6.
Fig. (95): 1H-NMR spectrum of compound BC-6 (DMSO-d6, 400 MHz).
Fig. (96): 13C-NMR spectrum of compound BC-6 (DMSO-d6, 100 MHz).
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
211
Identification of Compound BC-7
Physical Properties:
Compound BC-7 was obtained as colourless needles (30mg,
MeOH), m.p. 196-198°C. It is soluble in chloroform, methanol and
insoluble in hexane. It showed a single spot which gave a brown colour
with 10 % v/v H2SO4 and heating at 110°C for 10 min. also it gave a
green colour with FeCl3 (T.S) (155) using precoated silica gel plates. It has
an Rf value of 0.88 using system VI.
Spectroscopic Analysis:
1- IR (KBr) showed the following absorption bands at υ cm-1: 3400
(OH), 1615 (C=O), 1600 and 1550 (aromaticity).
2- EI-MS m/z (rel.int. %) Fig. (97): m/z 154 [M]+ (100%), 153 [M- H]+
(42%), 137 [M-17]+ (30%), 120 [M-(2OH)]+ (15%) and 109 [MCOO]+ (30%).
3- NMR: 13C- and 1H-NMR data are listed in Table (43) and illustrated
in Figs. (98 & 99).
Table (43):
13
C- and 1H-NMR data of compound BC-7 (DMSO-d6, 400
& 100 MHz).
No.
1
2
3
4
5
6
7
3,4-OH
13
C-NMR
121.82
115.26
144.97
149.92
116.83
122.84
168.04
—
1
H-NMR (multiplicity, J in Hz)
—
7.30 (1H,d, J =1.2)
—
—
6.73 ( 1H, d, J = 8.4)
7.24 ( 1H, dd, J = 8.4, 1.2)
—
10.46 (2H, s)
Discussion:
The mass and
13
C-NMR spectral data of compound BC-7
suggested the presence of a phenolic acid(191) with the molecular formula
C7H6O4.
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
212
The 1H-NMR spectrum (Table 43, Fig. 98) exhibited the behavior
of a tri-substituted benzene ring due to the presence of 3 signals in the
aromatic region at δH 7.30 (d, J = 1.2 Hz), 7.24 (dd, J = 8.4, 1.2 Hz) and
6.73 (d, J = 8.4 Hz) attributed to H-2, H-6 and H-5 respectively(204, 205).
The
13
C-NMR (Table 43, Fig. 99) revealed the presence of seven
signals assigned for 7 carbon atoms, one of them at δC 168.04 attributed to
a carboxy carbonyl (C-7), in addition to two downfield oxygenated
carbons at δC 149.92 (C-4) and 144.97 (C-3) which was further confirmed
by an absorption band at υ 3400 cm-1 in the IR spectrum indicating the
presence of aromatic hydroxyls. In addition to four carbon signals at δC
121.82 (C-1), 116.83 (C-5), 115.26 (C-2) and 122.84 (C-6), they were in
full agreement with those previously reported for 3,4-dihydroxybenzoic
acid(204, 205).
EI-MS spectral data of compound BC-7 (Fig. 97) revealed a
molecular ion peak at m/z 154 and other significant peaks at m/z 137 [MOH]+, 120 [M-(2OH)]+ and m/z 109 [M-COOH]+ confirming the
presence of benzoic acid with C3 and C4 hydroxyl substitution.
Conclusion:
Comparing the obtained spectral data with those previously
reported in the literature for similar compounds(204,205), compound BC-7 is
concluded to be 3,4-dihydroxy benzoic acid (Protocatechuic acid). To
our knowledge this represents the first report for its identification from
the genus Blepharis.
O
OH
OH
OH
Protocatechuic acid
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
213
Fig. (97): EI-MS spectrum of compound BC-7.
Fig. (98): 1H-NMR spectrum of compound BC-7 (DMSO-d6, 400 MHz).
Fig. (99): 13C-NMR spectrum of compound BC-7 (DMSO-d6, 100 MHz).
214
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
Identification of Compound BC-8
Physical Properties:
Compound BC-8 was obtained as yellow amorphous powder (15
mg, MeOH). It is soluble in methanol, sparingly soluble in chloroform
and insoluble in n-hexane. It showed a single spot which gave deep
purple fluorescence under UV light and a yellow colour after spraying
then heating at 110°C for 10 min. with 10% v/v H2SO4 and 5% AlCl3
using precoated silica gel plates. It also gave positive tests for
flavonoids(155,172). It has Rf value of 0.55 using system VI.
Spectroscopic Analysis:
The UV spectral data of compound BC-8 in methanol showed
characteristic absorption bands for flavones λmax 270 and 335 nm(161). The
UV spectral analysis with different ionizing and complexing reagents
were determined and the data are listed below in Table (44).
Table (44): The UV spectral data of compound BC-8 with different
ionizing and complexing reagents.
λmax
Band MeOH
+ NaOMe
λmax
λmax
I
335
383
II
270
276
Δλ
+4
8
+6
+AlCl3
+AlCl3/HCl
+NaOAc
+NaOAc/H3BO3
λmax
Δλ
λmax
Δλ
λmax
Δλ
λmax
Δλ
390
+55
380
+45
363
+28
337
+2
280
+10
280
+10
280
+10
273
+3
Discussion:
The UV spectral data with different ionizing and complexing
reagents (Table 44) showed the following:
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
215
1- A bathochromic shift with NaOMe in band I (Δλmax +48 nm)
indicating the presence of a free hydroxyl group at C-4'.
2- A bathochromic shift with AlCl3 in band I (Δλmax +55 nm) indicating
the presence of a free hydroxyl group at C-5.
3- A bathochromic shift with NaOAc in band II (Δλmax +10 nm)
indicating the presence of a free hydroxyl group at C-7.
4- Lacking of ortho-dihydroxy group in ring B since the complex
formed with AlCl3 was acid stable and this was confirmed by the
absence of a bathochromic shift with NaOAc/H3BO3 mixture.
Conclusion:
From the above mentioned physical, chemical, UV spectral data in
addition to co-chromatography with authentic sample of apigenin and
comparison with reported literature(161,206), compound BC-8 is identified
as Apigenin which was previously isolated from Blepharis sindica(82)
Stocks ex T. Anders.and to our knowledge this represents the first time
for its identification from Blepharis ciliaris (L.)B.L.Burtt..
OH
HO
O
OH
O
Apigenin
216
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
Identification of Compound BC-9
Physical Properties:
Compound BC-9 was isolated as a white amorphous powder
(50mg, MeOH) It is soluble in hot methanol, ethanol, sparingly soluble in
chloroform and insoluble in n-hexane. It showed a single spot which
attained a violet colour after spraying with 10% v/v H2SO4 and heating
for 10 min. at 110°C using precoated silica gel plates. It gave a positive
Liebermann-Burchard's test
(169,170)
and positive Molish's test(156)
suggesting its steroidal glycosidic nature. It has Rf value of 0.75 using
system VI.
Discussion:
By direct comparison with an authentic sample of β-sitosterol
glucoside (co-TLC) and carrying acid hydrolysis as mentioned before
(P.64) the hydrolysate was confirmed to be β-sitosterol by direct
comparison with authentic samples (co-TLC) and glucose using paper
chromatography and system IX.
Conclusion:
Compound BC-9 is identified by direct authentication as βsitosterol-3-O-β-D-glucopyranoside. To our knowledge this represents
the first report for its identification from Blepharis ciliaris (L.)B.L.Burtt.
29
28
21
22
20
23
18
24
26
25
12
19
1
OH
6´
2
O
HO
HO
OH
O
27
9
10
8
14
15
7
5
4
17
16
3
1´
13
11
6
β-sitosterol-3-O-β-D-glucopyranoside
217
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
Identification of Compound BC-10
Physical Properties:
Compound BC-10 was isolated as a white amorphous powder (50
mg, MeOH). It is soluble in hot methanol, sparingly soluble in
chloroform and insoluble in n-hexane. It showed a single spot which
attained a violet colour after spraying with 10 % v/v H2SO4 and heating
for 10 min. at 110°C using precoated silica gel plates. It gave a positive
Liebermann-Burchard's
test(169,170)
and
positive
Molish's
test(156)
suggesting its steroidal glycosidic nature. It has Rf value of 0.33 using
system VI.
Spectroscopic Analysis:
NMR: 1H-NMR data are listed in Table (45) and illustrated in Fig. (100).
Table (45): 1H-NMR data of compound BC-10 (DMSO-d6, 400 MHz).
No.
H-6
H-22
H-23
H-3
18-CH3
21-CH3
26-CH3
29-CH3
19-CH3
27-CH3
CH2 and CH protons
H-1'
H-2'−H-6'
1
H-NMR (m, J in Hz)
5.31 (1H, br.s)
5.00 (1H, dd, J = 15.2, 8.4)
5.13 (1H, dd, J = 15.2, 8.4 )
3.63 (m)
0.63 (s)
1.0 (s)
0.88 (3H, d, J = 8.8)
0.82 (t, J = 7.4)
0.94 (s)
0.80 (3H, d, J = 6.8)
1.00-2.88
4.20 (1H, d, J = 8.0)
3.00-4.21
other sugar protons
Discussion:
The 1H-NMR spectrum of compound BC-10 (Table 45, Fig. 100)
exhibited signals for sitosteryl glycoside(194) with two unsaturation
centers, one at δH 5.31 (1H, br.s) for Δ5-6 and the others at δH 5.00 (1H,
218
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
dd, J = 15.2, 8.4 Hz) and δH 5.13 (1H, dd, J = 15.2, 8.4 Hz) for Δ22-23, in
addition to six methyl group signals in full agreement with sitosteryl
moiety(194,207). The signal at δH 3.63 (1H, m) was assigned as H-3 and the
doublet signal at δH 4.20 (1H, d, J = 8 Hz) was assigned as an anomeric
sugar proton with β-configuration in addition to other sugar protons at δH
3.00-4.21 assigned for glucose moiety(194).
Acid hydrolysis of compound BC-10 was carried out as mentioned
before (P.64), it yielded an aglycone and a sugar moiety. The aglycone
was identified as stigmasterol by co-chromatography with authentic
sample and the sugar was identified as glucose by paper chromatography
with authentic reference of glucose using paper chromatography and
system IX.
The
1
H-NMR spectral data of compound BC-10 and acid
hydrolysis
suggested
the
presence
Stigmasterol-3-O-β-D-
of
glucopyranose.
Conclusion:
From the above mentioned spectral data as well as comparison with
the published data(207,208), compound BC-10 is identified as Stigmasterol3-O-β-D-glucopyranoside. To our knowledge this represents the first
report for its identification from Blepharis ciliaris (L.) B.L.Burtt..
29
28
21
22
20
23
18
24
26
25
12
19
1
OH
6´
2
O
HO
HO
OH
O
27
9
10
8
14
15
7
5
4
17
16
3
1´
13
11
6
Stigmasterol-3-O-β-D-glucopyranoside
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
219
Fig. (100): 1H-NMR spectrum of compound BC-10 (DMSO-d6, 400 MHz).
220
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
Identification of Compound BC-11
Physical Properties:
Compound BC-11 was isolated as yellow amorphous powder (30
mg, MeOH). It is soluble in methanol, sparingly soluble in ethyl-acetate
and insoluble in chloroform and n-hexane. It showed a single spot which
gave a dark brown colour under UV light and a yellow colour after
spraying then heating at at 110°C for 10 min. with 10% v/v H2SO4 and
5% AlCl3 using precoated silica gel plates. It also gave positive tests for
flavonoids(155,172) and positive Molish's test(156) suggesting its glycosidic
nature. It has an Rf value of 0.35 using system VII.
Spectroscopic Analysis:
1- The UV spectral data in methanol of compound BF-AG showed
characteristic absorption bands of a flavone at λmax 268 and 330
nm(161). The UV spectral analysis with different ionizing and
complexing reagents were determined and the data are listed below in
Table (46).
Table (46): The UV spectral data of compound BC-11 with different
ionizing and complexing reagents.
λmax
Band
MeOH
+ NaOMe
+AlCl3
+AlCl3/HCl
+NaOAc
+NaOAc/H3BO3
λmax
λmax
Δλ
λmax
Δλ
λmax
Δλ
λmax
Δλ
λmax
Δλ
I
330
382
+52
372
+42
372
+42
330
—
330
—
II
268
273
+5
276
+8
274
+6
268
—
268
—
2- NMR: 1H-NMR data are listed in Table (47) and illustrated in Fig.
(101).
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
221
Table (47): 1H-NMR data of compound BC-11 (DMSO-d6, 400 Hz).
No.
3
6
8
2', 6'
3', 5'
1'
Sugar protons
5-OH
1
H-NMR (m, J in Hz)
6.86 (1H, s)
6.43 (1H, d, J = 2)
6.82 (1H, d, J = 2)
7.94 (2H, d, J = 8.8)
6.92 (2H, d, J = 8.8)
5.05 (1H, d, J = 7.2)
5.43-3.47
12.96 (1H, s)
Discussion:
Compound BC-11 gave positive colour reactions for flavonoids
and/or glycosides, indicating its flavonoidal glycosidic nature(172).
The UV spectral data in MeOH (Table 46) showed two absorption
bands at λmax 330 nm (band I) and 268 nm (band II) characterized for
flavones(161). The UV spectral data with different ionizing and
complexing reagents showed the following:
1- A bathochromic shift with NaOMe in band I (Δλmax +52 nm)
indicating the presence of a free hydroxyl group at C-4'.
2- There was no shift in band II on the addition of NaOAc which
revealed that C-7 lacks a hydroxyl group or blocked.
3- A bathochromic shift with AlCl3 in band I (Δλmax +42) revealed the
presence of a free hydroxyl group at C-5.
4- Lacking of ortho-dihydroxy group in ring B since the complex
formed with AlCl3 was acid stable and this was confirmed by the
absence of bathochromic shift in band I upon the addition of
NaOAc/H3BO3 mixture.
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
222
The 1H-NMR spectrum (Table 47, Fig. 101) exhibited a flavone
skeleton(161). It showed a pair of doublets attributed to para disubstituted
benzene ring at δH 7.94 (d, J = 8.8 Hz) and 6.92 (d, J = 8.8 Hz) assigned
for H-2', 6' and H-3',5' in ring B respectively(161,206). Also two doublets at
δH 6.43 (1H, d, J = 2) and δH 6.82 (1H, d, J = 2) attributed to meta
coupled protons at H-8 and H-6 respectively in ring A(206,209). In addition
to a singlet at δH 6.86 (s) assigned for H-3(206,209) and a downfield singlet
at δH 12.96 for hydroxyl at C-5. The obtained 1H-NMR data of compound
BC-11 was in full agreement with those previously reported for
apigenin(206,210) while the doublet at δH 5.05 (d, J = 7.2 Hz) was assigned
for β-anomeric proton suggesting the presence of one β-D-glucosyl
moiety(206) and the other sugar protons were detected as a multiplet at δH
5.43-3.47 the position of attachment of glucose unit to apigenin was
determined on the basis of UV spectral data to be at C-7.
Acid hydrolysis of compound BC-11 as mentioned before (P.64)
yielded an aglycone moiety and one sugar unit. The aglycone was
identified as apigenin (by co-chromatography with authentic sample of
apigenin) while the sugar unit was identified as glucose (by cochromatography with authentic sample using system IX.
Conclusion:
From the above mentioned physical, chemical, UV and 1H-NMR
spectral data in addition to acid hydrolysis, compound BC-11 is identified
as apigenin-7-O-β-D-glucpyranoside which was previously isolated
from Blepharis ciliaris (L.)B.L.Burtt.(81).
OH
glu-O
O
OH
O
Apigenin -7-O- β-D-glucopyranoside
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
Fig. (101):
1
223
H-NMR spectrum of compound BC-11 (DMSO-d6,
400 MHz).
224
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
Identification of Compound BC-12
Physical Properties:
Compound BC-12 was isolated as pale yellow needles (30 mg,
MeOH) with m.p. 265-267°C. It is soluble in hot methanol, sparingly
soluble in cold methanol and insoluble in n-hexane and chloroform. It
showed a single spot which gave a deep purple colour under UV light and
a yellow colour after spraying then heating at 110°C for 10 min. with
10% v/v H2SO4 and 5% AlCl3 using precoated silica gel plates. It also
gave positive tests for flavonoids(155,172) in addition to positive Molish's
test suggesting its glycosidic nature(156). It has an Rf value of 0.69 using
system V.
Spectroscopic Analysis:
1- The UV spectral data in methanol of compound BC-12 showed
characteristic absorption bands of a flavone at λmax 268 and 316
nm(161). The UV spectral analysis with different ionizing and
complexing reagents were determined and the data are listed below in
Table (48).
Table (48): The UV spectral data of compound BC-12 with different
ionizing and complexing reagents.
λmax
Band
MeOH
λmax
I
316
368
300
II
268
272
+ NaOMe
λmax Δλ
+AlCl3
λmax Δλ
375
+52 325 +59
319
298
+4
+12
276
+AlCl3/HCl
λmax
Δλ
375
325 +59
318
298
+12
276
+NaOAc
λmax Δλ
+NaOAc/H3BO3
λmax
Δλ
375
318
—
382
318
—
268
—
268
—
2- + FAB-MS m/z (rel.int. %) (Fig. 102): 579 [M+H]+ (25%), 417 [M-(pcoumaroyl unit) +H]+ (17%), 301 (100%) and 271 [M-(p-coumaroyl
unit + hexose unit) + H]+ (12%).
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
225
3- NMR: 13C- and 1H-NMR data are listed in Table (49) and illustrated
in Figs. (103-105).
Table (49):
13
C- and 1H-NMR data of compound BC-12 (DMSO-d6, 400
&100 MHz).
No.
2
3
4
5
6
7
8
9
10
1'
2', 6'
3', 5'
4'
1''
2''
3''
4''
5''
6''
1'''
2''', 6'''
3''', 5'''
4'''
7'''
8'''
9'''
5-OH
4'-OH
4'''-OH
13
C-NMR
165.61
105.09
181.88
162.58
99.56
163.94
94.59
158.86
103.05
121.01
128.52
115.96
159.88
100.23
73.02
76.28
70.07
73.69
63.65
124.85
130.10
115.62
161.11
144.92
113.66
166.53
—
—
—
1
H-NMR (m, J in Hz)
—
6.83 (1H, s)
—
—
6.45 (d, J = 2.0)
—
6.80 (d, J = 2.0)
—
—
—
7.93 (2H, d, J = 8.4)
6.90 (2H, d, J = 8.4)
—
5.15 (1H, d, J = 7.2)
5.75-3.29 (m)
—
7.35 (2H, d, J = 8.8)
6.64 (2H, d, J = 8.4)
—
7.47 (1H, d, J = 15.6)
6.33 (1H, d, J = 15.6)
—
12.97 (s)
9.84 (s)
9.01 (s)
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
226
Discussion:
The UV spectral data of compound BC-12 with different ionizing
and complexing reagents (Table 48) revealed the following:
1- A bathochromic shift in band I with NaOMe (λmax +52) indicating the
presence of C-4' hydroxyl group.
2- Lacking of a free hydroxyl group at C-7 due to the absence of a
bathochromic shift in band II upon the addition of NaOAc.
3- A bathochromic shift in band I with AlCl3 (λmax +59) indicating the
presence of a free hydroxyl group at C-5.
4- Absence of ortho-dihydroxy group in ring B since the complex
formed with AlCl3 was acid stable and this was confirmed by the
absence of bathochromic shift in band I upon the addition of
NaOAc/H3BO3.
The 1H-NMR spectrum (Table 49, Figs. 103 & 104) and UV
spectral data suggested a flavone skeleton(161) with free 4'-OH, 5-OH and
substituted at C-7, which is deduced from the meta-coupled protons at δH
6.80, 6.45 (each d, J = 2.0 Hz) assignable to H-6 and H-8 of ring A. Also
two doublets at δH 7.93, 6.90 (each d, J = 8.4 Hz) assignable to a para
substituted benzene of ring B and singlet signal at δH 6.83 characteristic
for H-3 of flavones(161). The proton and carbon signals of the flavonoid
skeleton were in full agreement with those previously reported for
apigenin(161).
The 13C-NMR spectrum (Table 49, Fig. 105) displayed 26 signals,
thirteen of them were assignable for apigenin(184), seven for p-coumaroyl
moiety with an ester carbonyl at δC 166.53 in addition to sugar carbons.
227
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
The 1H- and
13
C-NMR spectrum (Table 49, Figs. 103-105) also
displayed characteristic pattern of trans p-coumaroyl moiety. In addition
to β-D-glucopyranoside moiety with δH1'' at 5.15 (d, J = 7.2 Hz), the
position of glycosilation at C-7 was deduced from the UV spectral data.
The position of the linkage of the p-coumaroyl moiety to C-6'' of glucose
was deduced by comparing the chemical shifts of δH and δC with similar
previously published data(46,209).
The positive ion FAB-MS (Fig. 102) showed [M+H]+ at m/z 579
with three significant fragment peaks at m/z 417 [M-(p - coumaroyl unit)
+H]+ confirming the p-coumaroyl unit and at m/z 271 [M-(p-coumaroyl
unit + hexose unit) + H]+ confirming that BC-12 is apigenin p-coumaroyl
glucoside.
Upon acid hydrolysis of BC-12 (P.64) apigenin was confirmed by
co-TLC alongside with authentic samples and the sugar was identified as
glucose by paper chromatography using system IX.
Conclusion:
On the basis of the previous mentioned spectral data and
comparison with literature(46,81,209), compound BC-12 is identified as
apigenin-7-O-(6''-E-p-coumaroyl-β-D-glucopyranoside).
To
our
knowledge this represents the first report for its identification from
Blepharis ciliaris (L.) B.L.Burtt..
O
7'''
9'''
2'''
1'''
O
4''
HO
HO
3'''
8'''
4'''
6'''
6''
O
2''
OH1''
3'
OH
5'''
8
O
9
7
4'
2'
1'
O
OH
5'
2
6'
3
6
10
4
5
OH
O
Apigenin-7-O-(6''-E-p-coumaroyl-β-D-glucopyranoside)
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
228
Fig. (102): +FAB-MS spectrum of compound BC-12.
Fig. (103):
1
H-NMR spectrum of compound BC-12 (DMSO-d6,
400 MHz).
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
229
Fig. (104): Expanded 1H-NMR spectrum of compound BC-12
(DMSO-d6, 400 MHz).
C-4
Fig. (105):
13
C-NMR spectrum of compound BC-12 (DMSO-d6, 100
MHz).
230
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
Identification of Compound BC-13
Physical Properties:
Compound BC-13 was isolated as yellow powder (50 mg, MeOH),
soluble in methanol, ethanol and insoluble in chloroform and n-hexane. It
showed a single spot which gave a deep purple colour under UV light
changed to dull brown with ammonium hydroxide and a yellow to orange
colour after spraying then heating at 110°C for 10 min. with 10% v/v
H2SO4 using pre-coated silica gel plates. It also gave positive tests for
flavonoids(155,172), in addition to positive Molish's test suggesting its
glycosidic nature(156). It has Rf value of 0.75 using system VI.
Spectroscopic Analysis:
1- The UV spectral data of compound BC-13 in methanol (Table 50)
showed characteristic absorption bands of an iso-flavone at λmax 270
and 301sh nm(161). The UV spectral analyses with different ionizing
and complexing reagents were determined and the data are listed
below in Table (50).
Table (50): The UV spectral data of compound BC-13 with different
ionizing and complexing reagents.
λmax
Band MeOH
+ NaOMe
λmax
λmax
I
301sh
338
II
270
275
+AlCl3
+AlCl3/HCl
+NaOAc
+NaOAc/H3BO3
λmax
Δλ
λmax
Δλ
λmax
Δλ
λmax
Δλ
+37
356
+55
352
+51
302
+1
302
+1
+5
282
+12
280
+10
272
+2
272
+2
2- + FAB-MS m/z (rel.int. %) (Fig. 106): 595 [M+H]+ (15%), 433 [M(caffeoyl unit) + H]+ (3%), 271 [M-(caffeoyl unit + hexose unit) +
H]+ (8%), 229 (100%), 165 (63%) and 94 (73%).
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
3- NMR:
231
13
C- and 1H-NMR spectral data are listed in Table (51) and
illustrated in Figs. (107 & 108).
Table (51):
13
C- and 1H-NMR data of compound BC-13 (DMSO-d6, 400
& 100 MHz).
No.
2
3
4
5
6
7
8
9
10
1'
2', 6'
3', 5'
4'
1''
2''
3''
4''
5''
6''
1'''
2'''
3'''
4'''
5'''
6'''
7'''
8'''
9'''
5-OH
13
C-NMR
156.87
120.34
181.88
162.15
99.44
164.40
94.62
161.13
105.32
120.34
130.05
115.77
160.21
102.77
73.03
73.79
69.86
76.28
63.44
124.55
115.98
145.07
145.02
116.16
120.34
145.02
113.43
166.63
—
1
H-NMR (m, J in Hz)
8.43 (1H, br.s)
—
—
—
6.45 (1H, br.s)
—
6.55 (1H, br.s)
—
—
—
7.33 (2H, d, J = 8.8)
6.67 (2H, d, J = 8.8)
—
5.15 (1H, d, J = 8.0)
3.14-5.03 (m)
—
6.81 (1H, d, J = 3.2)
—
—
7.91 (1H, d, J = 8.0)
6.93, 1H (dd, J = 8.0, 3.2)
7.47 (1H, d, J = 15.6)
6.30 (1H, d, J = 15.6)
—
12.92 (1H, br.s)
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
232
Discussion:
The UV spectral data of compound BC-13 (Table 50) with
different ionizing and complexing reagents revealed the following:
1- A bathochromic shift in band I with NaOMe (λmax +37) indicating the
presence of a free hydroxyl at C-4'.
2- Lacking of a free hydroxyl group at C-7 due to the absence of a
bathochromic shift in band II upon the addition of NaOAc.
3- A bathochromic shift in band I with AlCl3 (λmax +55 nm) indicating
the presence of a free hydroxyl group at C-5.
4- Absence of ortho-dihydroxy group in ring B since the complex
formed with AlCl3 was acid stable and this was confirmed by the
absence of bathochromic shift in band I upon the addition of
NaOAc/H3BO3.
The 1H-NMR spectrum (Table 51, Fig. 107) showed two singlet
signals at δH 12.92 and 8.43 were assigned to 5-chelated hydroxyl group
and H-2 of an isoflavone(161). The proton signals at δH 7.33 (d, J = 8.8 Hz)
and 6.67 (d, J = 8.8 Hz) indicated the presence of a para-disubstituted
benzene moiety (ring B) they were attributed to H-2', 6' and 3', 5'
respectively as well as other protons resonated at δH 6.55 and 6.45 (each
1H, br.s) were assigned to H-8 and H-6 respectively(211,212). The 1H-NMR
spectrum also displayed an ABX system for a 1,3,4- trisubstituted
aromatic ring at δH [6.81 (1H, d, J = 3.2 Hz), 7.91 (1H, d, J = 8.0 Hz),
6.93 (1H, dd, J = 8.0, 3.2 Hz)] and two trans-olefinic protons at δH [7.47
(1H, d, J = 15.6 Hz), 6.30 (1H, d, J = 15.6 Hz)], suggesting the presence
of a caffeoyl moiety(213-215). Furthermore, anomeric proton for sugar
moiety at δH 5.15 (1H, d, J = 8.0 Hz) indicated β-glucopyranose unit(209).
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
The
233
13
C-NMR (Table 51, Fig. 108) displayed 28 signals, 13 for
flavonoid moiety with an downfield signal at δC 181.88 assigned for C-4
which confirmed the presence of iso-flavone nucleus(216) and the other
carbons of flavonoidal moiety agree with those previously reported for
genisetin(216). In addition to six signals for glucose moiety with an
anomeric carbon at δC 102.77. Also it displayed eight carbons for the
caffeoyl moiety(213-215) and an ester carbonyl at δC 166.63. The positive
ion FAB-MS spectrum (Fig. 108) showed [M+H]+ at m/z 595 with two
significant fragment peaks at m/z 433 [M-(caffeoyl unit) + H]+
confirming the presence of caffeoyl ester and at m/z 271 [M-(caffeoyl
unit+ hexose unit) + H]+ indicating that BC-13 is a flavonoidal caffeoyl
glucoside.
Upon acid hydrolysis of compound BC-13 (P.64) genistein and
caffeic acid were confirmed by co-TLC alongside with authentic samples,
and β-D-glucose was authenticated using paper chromatography and
system IX.
On the basis of the above results, it was assumed that BC-13
contained caffeoyl glucose linked at the 7-hydroxyl group of genistein
which was confirmed by UV spectral data with NaOAc. The significant
downfield shift of C-6'' of glucose (δC 63.44) indicated the attachment of
caffeoyl moiety at C-6'' of glucose(81).
Conclusion:
On the basis of the previous mentioned data, compound BC-13 is
identified
as
Genistein-7-O-(6''-O-E-caffeoyl)-β-D-glucopyranoside
which could be considered as a new natural product (new compound)(185).
234
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
According to the available literature it is the first report for identification
of an isoflavone from family Acanthaceae.
O
7```
9```
O
3``` OH
8```
4```
6```
4`` 6``
HO
HO
2```
1```
O
2``
OH 1``
5```
OH
8
O
7
9 O
2
3
6
5
OH
10
1`
2`
3`
4
O
4`
6`
5`
OH
Genistein-7-O-(6''-O-E-caffeoyl)-β-D-glucopyranoside
Fig. (106): +FAB-MS spectrum of compound BC-13.
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
235
Fig. (107): 1H-NMR spectrum of compound BC-13 (DMSO-d6, 400 MHz).
Fig. (108): 13C-NMR spectrum of compound BC-13 (DMSO-d6, 100 MHz).
236
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
Identification of Compound BC-14
Physical Properties:
Compound BC-14 was isolated as yellow powder (105 mg,
MeOH). It is soluble in hot methanol, sparingly soluble in cold methanol
and insoluble in n-hexane and chloroform. It showed a single spot which
gave a deep purple colour under UV light and a yellow to orange colour
after spraying then heating at 110°C for 10 min. with 10% v/v H2SO4
using pre-coated silica gel plates. It also gave positive tests for
flavonoids(155,172), in addition to positive Molish's test suggesting its
glycosidic nature(156). It has Rf value of 0.81 using system VII.
Spectroscopic Analysis:
1- The UV spectral data in methanol of compound BC-14 (Table 52)
showed characteristic absorption bands of a flavanone(161) at λmax 287
and 316 nm. The UV spectral analysis with different ionizing and
complexing reagents were determined and the data are listed below in
Table (52).
Table (52): The UV spectral data of compound BC-14 with different
ionizing and complexing reagents.
λmax
Band
MeOH
λmax
I
316
II
287
+ NaOMe
+AlCl3
λmax
Δλ
λmax
Δλ
382sh
357
+41
+66
329sh
308sh
285
305
—
+18
242sh
284sh
+AlCl3/HCl
λmax
372sh
325sh
305
284sh
Δλ
+56
+18
+NaOAc
+NaOAc/H3BO3
λmax
Δλ
362sh
+46
320sh
λmax
Δλ
314
—
—
283
—
287
2- + FAB-MS m/z (rel.int. %) (Fig. 109): 623 [M+H]+ (15%), 580 [M(CH3CO) + H]+ (30%), 415 [M-(CH3CO + p-coumaroyl unit)+H]+
(5%), 273 [M-(CH3CO+ p-coumaroyl unit + hexose unit) + H)]+
(25%), 229 (100%), 165 (60%) and 94 (78%).
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
237
3- NMR: 13C-, DEPT 13C- and 1H-NMR data are listed in Table (53) and
illustrated in Figs. (110-113).
Table (53):
13
C-, DEPT
13
C- and 1H-NMR data of compound BC-14
(DMSO-d6, 400 & 100 MHz).
No.
2
13
C-NMR
78.68
DEPT
CH
3
42.0
CH2
4
5
6
7
8
9
10
1'
2',6'
3',5'
4'
1''
2''
3''
4''
5''
197.43
163.02
96.38
164.73
95.48
157.79
103.26
128.57
128.48
115.21
162.99
98.75
70.83
77.05
67.67
73.45
C
C
CH
C
CH
C
C
C
CH
CH
C
CH
CH
CH
CH
CH
6''
62.82
CH2
1'''
2''',6'''
3''',5'''
4'''
7'''
8'''
9'''
5-OH
4'-OH
2"-OH
4"-OH
4'''-OH
3''-COCH3
3''-COCH3
125.05
130.29
115.80
159.90
145.07
113.86
166.40
—
—
—
—
—
21.14
169.75
C
CH
CH
C
CH
CH
C
—
—
—
—
—
C
C
1
H-NMR (m, J in Hz)
5.45 (1H, dd, J = 12.8, 3.2)
3 a 3.44 under solvent
3b 2.70 (1H, dd, J = 16.8, 3.2)
—
—
6.15 (1H, d, J = 2.4)
—
6.19 (1H, d, J = 2.4)
—
—
—
7.27 (2H, d, J = 8.4)
6.77 (2H, d, J = 8.4)
—
5.23 (1H, d, J = 8.0)
3.46 (1H, m)
4.91 (1H, t, J = 9.2)
3.45 (1H, m)
3.91 (1H, t, J = 6.4)
a 4.37 (1H,d, J = 10.8)
b 4.15 (1H, dd, J = 12.4, 6.4)
—
7.50 (2H, d, J = 8.4)
6.75 (2H, d, J = 8.4)
—
7.51 (1H, d, J = 16.0)
6.36 (1H, d, J = 16.0)
—
12.08 (1H, s)
10.05 (1H, s)
5.71 (1H,d, J = 6)
5.59 (1H, d, J = 6)
9.62 (1H, s)
2.04 (3H, s)
—
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
238
Discussion:
The UV spectral data of compound BC-14 (Table 52) with
different ionizing and complexing reagents revealed the following:
1- A bathochromic shift in band I with NaOMe (λmax +41) indicating the
presence of C-4' hydroxyl group.
2- Lacking of a free hydroxyl group at C-7 due to the absence of a
bathochromic shift in band II upon the addition of NaOAc.
3- A bathochromic shift in band I with AlCl3 (λmax +66) indicating the
presence of a free hydroxyl group at C-5.
4- Absence of ortho-dihydroxy group in ring B since the complex
formed with AlCl3 was acid stable and this was confirmed by the
absence of bathochromic shift in band I upon the addition of
NaOAc/H3BO3.
The 1H- and 13C-NMR spectra of BC-14 (Table 53, Figs. 110-113)
revealed a flavanone skeleton(161) with two meta coupled protons at δH
6.15, 6.19 (d, J = 2.4 Hz) assignable to H-6 and H-8 of ring A. Also two
doublet signals attributed to para di-substituted benzene at δH 7.27, 6.77
(d, J = 8.4 Hz) for ring B(161), which was further confirmed from UV
spectral shift in band I with NaOMe.
The compound was further confirmed to be a flavanone by the
appearance of diagnostic signals at δH 5.45, 3.44 and 2.70 attributed to C2 and C-3 protons of a flavanone skeleton
(161)
and carbon signals at δC
78.68 and 42.0(181). The proton signal at δH 12.08 belonging to 5hydroxylation shown unsubstituted 5-position. Also a singlet assignable
to a methyl group at δH 2.04 and δC 21.14 was observed. The 1H-NMR
spectrum also displayed further aromatic protons at δH 6.77, 7.50 as (d, J
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
239
= 8.4 Hz) for para disubstituted benzene and at δH 6.36, 7.51 (d, J = 16.0
Hz), δC 113.86 and 145.07 respectively which were assignable to trans pcoumaroyl moiety(81). In addition to a doublet at δH 5.25 (J = 8.0 Hz) for
H-1 β-linked glucose unit, the remaining sugar protons are observed at δH
3.46-5.71 which were further confirmed by carbon signals at δC 98.7562.82. The glucose moiety was linked to C-7 of the flavanone, which was
easily deduced from the UV spectral shifts after addition of NaOAc(161).
The point of attachment of p-coumaroyl moiety to glucose was deduced
to be at H-6" depending on comparison of the chemical shifts of the
corresponding protons and carbons with those previously reported for
similar compounds(81).
The
13
C- and DEPT
13
C-NMR spectrum (Table 53, Figs. 112 &
113) displayed 28 signals, thirteen of them were in full agreement with
those previously reported for naringenin(181), seven carbon signals with
the same chemical shifts for p-trans coumaroyl moiety(81) with an ester
carbonyl at δC 166.40. The carbonyl signals at δC 21.14, 169.75 were in
accordance with acetyl substitution in the glucose unit(46). The linkage of
the acetyl group to glucose was deduced to be at H-3'' depending on the
chemical shift of H-3'' δH 4.91 (t, J = 9.2 Hz) and δC 77.05 in comparison
with literature(81).
The positive ion FAB-MS (Fig. 109) showed [M+H]+ at m/z 623
with three significant fragment peaks at m/z 580 [M-(CH3CO+H]+
confirming the presence of an acetate group, at m/z 415 [M-(CH3CO + pcoumaroyl unit)+H]+ confirming the p-coumaroyl unit and at m/z 273
[M-(CH3CO + p-coumaroyl unit + hexose unit) + H]+ indicating that BC14 is a flavonoidal p-coumaroyl glucoside.
240
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
Conclusion:
On the basis of the previous mentioned spectral data and
comparison with literature(46,81), compound BC-14 is identified as
naringenin-7-O-(3''-acetyl-6''-E-p-coumaroyl-β-D-glucopyranoside
which was previously isolated from Blepharis ciliaris (L.) B.L.Burtt.(81).
O
7'''
9'''
2'''
1'''
O
4''
O
2''
3''
3'
4'''
6'''
6''
HO
RO
3'''
8'''
8
O
9
7
OH 1''
1'
O
OH
5'
2
6'
6
R = COCH3
4'
2'
OH
5'''
5
OH
3
10
4
O
Naringenin-7-O-(3''-acetyl-6''-E-p-coumaroyl-β-D-glucopyranoside
Fig. (109): +FAB-MS spectrum of compound BC-14.
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
Fig. (110):
1
241
H-NMR spectrum of compound BC-14 (DMSO-d6,
400 MHz).
Fig. (111): Expanded
1
H-NMR spectrum of compound BC-14
(DMSO-d6, 400 MHz).
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
242
Fig. (112): 13C-NMR spectrum of compound BC-14 (DMSO-d6, 100 MHz).
Fig. (113): DEPT 13C-NMR of compound BC-14 (DMSO-d6, 100 MHz).
243
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
Identification of Compound BC-15
Physical Properties:
Compound BC-15 was isolated as yellow powder (120 mg,
MeOH). It is soluble in hot methanol, sparingly soluble in cold methanol
and insoluble in n-hexane and chloroform. It showed a single spot which
gave a deep purple colour under UV light and a yellow to orange colour
after spraying then heating at 110°C for 10 min. with 10% v/v H2SO4
using pre-coated silica gel plates. It also gave positive tests for
flavonoids(155,172), in addition to positive Molish's test suggesting its
glycosidic nature(156). It has Rf value of 0.79 using system VII.
Spectroscopic Analysis:
1- The UV spectral data in methanol of compound BC-15 showed
characteristic absorption bands of a flavanone at λmax 287 and 312
nm(161). The UV spectral analysis with different ionizing and
complexing reagents were determined and the data are listed below in
Table (54).
Table (54): The UV spectral data of compound BC-15 with different
ionizing and complexing reagents.
λmax
Band
MeOH
λmax
I
312
II
285
+ NaOMe
λmax
309sh
358
240sh
283
Δλ
+46
+4
+AlCl3
λmax
327sh
380sh
282sh
307
Δλ
+AlCl3/HCl
λmax
327sh
+68
379sh
282sh
+22
+NaOAc
+NaOAc/H3BO3
Δλ
λmax
Δλ
λmax
Δλ
—
360sh
+48
312
—
—
318sh
285
—
285
—
2- + FAB-MS (Fig. 114) m/z (rel.int.%): 581 [M+H]+ (15%), 433 [M(p-coumaroyl unit)+H]+ (25%), 273 [M-(p-coumaroyl unit + hexose
unit) + H)]+ (17%), 163 (52%) and 147 (17%).
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
244
3- NMR: 13C-, DEPT 13C- and 1H-NMR data are listed in Table (55) and
illustrated in Figs. (115-117).
Table (55):
13
C-, DEPT
13
C- and 1H-NMR data of compound BC-15
(DMSO-d6, 400 &100 MHz).
No.
2
13
C-NMR
78.58
DEPT
CH
3
41.98
CH2
4
5
6
7
8
9
10
1'
2', 6'
3', 5'
4'
1''
2''
3''
4''
5''
6''
1'''
2''', 6'''
3''', 5'''
4'''
7'''
8'''
9'''
5-OH
4'-OH
4'''-OH
197.23
163.05
96.30
165.06
95.51
157.79
103.28
128.62
128.40
115.17
162.63
99.18
72.92
76.11
69.76
73.79
63.29
125.08
130.26
115.77
159.86
144.91
113.91
166.44
—
—
—
C
C
CH
C
CH
C
C
C
CH
CH
C
CH
CH
CH
CH
CH
CH2
C
CH
CH
C
CH
CH
C
—
—
—
1
H-NMR (m, J in Hz)
5.47 (1H, dd, J = 16.2, 4.2)
3 a: 3.33 (1H, dd, J= 16.8, 2.7)
3 b: 2.69 (1H, br.d, J = 16.8)
—
—
6.13 (1H, br.s)
—
6.17 (1H, br.s)
—
—
—
7.27 (2H, br.d, J = 7.8)
6.76 (2H, d, J = 7.8)
—
5.06(1H, d, J = 7.6)
5.48-3.33 (m)
—
7.49 (2H, d, J = 7.8)
6.77 (2H, d, J = 7.8)
—
7.51 (1H, d, J = 16.4)
6.35 (1H, d, J = 16.4)
—
12.08 (1H, s)
10.06 (s)
9.62 (s)
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
245
Discussion:
The UV spectral data of compound BC-15 with different ionizing
and complexing reagents (Table 54) revealed the following:
1- A bathochromic shift in band I with NaOMe (λmax +46) indicating the
presence of 4'- hydroxyl groups.
2- Lacking of a free hydroxyl group at C-7 due to the absence of a
bathochromic shift in band II upon the addition of NaOAc.
3- A bathochromic shift in band I with AlCl3 (λmax +68) indicating the
presence of a free hydroxyl group at C-5.
4- Absence of ortho-dihydroxy group in ring B since the complex
formed with AlCl3 was acid stable and this was confirmed by the
absence of bathochromic shift in band I upon the addition of
NaOAc/H3BO3.
The 1H- and
13
C-NMR spectrum of BC-15 (Table 55, Figs. 115-
117) revealed a flavanone skeleton with β-D glucopyranose unit and a pcoumaroyl moiety showing many similarities with those corresponding
spectrums of compound BC-14 (P.236). However the change in the
chemical shift of δ H-3", δ C-2", C-3" and C-4" of the β-D glucopyranose
unit indicated the absence of the acetyl moiety at C-3" which was further
confirmed by the absence of the corresponding signals at δH 2.04 and δC
21.14 and 169.75 of BC-14.
The positive ion FAB-MS of BC-15 (Fig. 114) m/z 581 in
comparison with the positive ion FAB-MS of BC-14 at m/z 623 also
proved the absence of the acetyl group and the appearance of the mass
fragment at m/z 273, 163 and 147 for the naringenin, glucose and p-
246
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
coumaroyl fragments confirmed that compound BC-15 is completely
similar to BC-14 with the absence of the C-3" acetyl group.
Conclusion:
On the basis of the previous mentioned spectral data, and
comparison with those for BC-14 (P.236), compound BC-15 is identified
as
naringenin-7-O-(6''-E-p-coumaroyl-β-D-glucopyranoside
which
was previously isolated from Blepharis sindica T.Anderson(82) seeds.To
our knowledge this represents the first report for its identification from
Blepharis ciliaris (L.) B.L.Burtt..
O
7'''
9'''
O
3''
2'''
3'''
8'''
O
2''
OH1''
3'
4'''
6'''
4'' 6''
HO
HO
1'''
5'''
8
O
2'
OH
9 O
7
1'
2
4' OH
5'
6'
6
5
10
OH
3
4
O
Naringenin-7-O-(6''-E-p-coumaroyl-β-D-glucopyranoside
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
247
Fig. (114): +FAB-MS spectrum of compound BC-15.
Fig. (115):
1
H-NMR spectrum of compound BC-15 (DMSO-d6,
400 MHz).
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
Fig. (116):
248
13
C-NMR spectrum of compound BC-15 (DMSO-d6,
100 MHz).
Fig. (117): DEPT
13
C-NMR spectrum of compound BC-15
(DMSO-d6, 100 MHz).
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
249
Identification of Compound BC-16
Physical Properties:
Compound BC-16 was isolated as a dark red residue (20 mg,
MeOH). It is soluble in methanol, insoluble in chloroform and n-hexane.
It showed a single spot which gave a red colour which changes to brown
after spraying with 10% v/v H2SO4 and heating for 10 min. at 110°C
using precoated silica gel plates. It has Rf value of 0.65 using system VIII.
Spectroscopic Analysis:
1- UV (MeOH) λmax: 229, 260, 290 and 330 nm.
2- IR (KBr) showed the following absorption bands at υ cm-1: 3400
(OH), 1705 (α, β-unsaturated ester, 1600, 1510 and 1480
(aromaticity).
3- + FAB-MS m/z (rel.int.%) Fig. (118): 625 [M+H]+ (20%), 461 [M(caffeoyl)+H]+ (5%), 305 [M-(caffeoyl unit + hexose unit)+H]+ (2%)
and 163 [caffeoyl]+ (10%).
4- NMR: 13C-, DEPT 13C- and 1H-NMR data are listed in Table (56) and
illustrated in Figs. (119-121).
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
Table (56):
13
C-, DEPT
13
C- and 1H-NMR data of compound BC-16
(DMSO-d6, 400 & 100 MHz).
No.
13
C-NMR
250
DEPT
1
H-NMR (m, J in Hz)
1
129.18
C
—
2
116.36
CH
6.60 (1H, br.s)
3
145.04
C
—
4
143.59
C
—
5
115.52
CH
6.61 (1H, d, J = 8)
6
119.62
CH
6.47 (1H, dd, J = 8, 2.0)
7
35.06
CH2
2.67 (2H, t, J = 7.6)
8
70.25
CH2
α 4.15 (1H, dd, J = 16.4, 6.8)
β 3.86 (1H, dd, J = 16.4, 6.8)
1'
102.32
CH
4.33 (1H, d, J = 8.4)
2'
74.55
CH
3'
79.14
CH
4'
68.79
CH
5'
74.55
CH
6'
62.78
CH2
1''
101.29
CH
2''
70.43
CH
3''
70.58
CH
4''
71.70
CH
5''
68.79
CH
6''
18.21
CH3
0.93 (3H, d, J = 5.6)
1'''
125.56
C
—
2'''
114.73
CH
3'''
145.61
C
7.04 (1H, br.s)
—
4'''
148.53
C
—
5'''
115.83
CH
6.74 (1H, d, J = 7.2)
6'''
121.51
CH
6.96 (1H, d, J = 7.2)
7'''
145.61
CH
7.44 (1H, d, J = 16.4)
8'''
113.61
CH
6.19 (1H, d, J = 16.4)
9'''
165.77
C
—
-OH
—
—
8.73 (1H, br.s)
(3.44 - 3.70, m)
4.69 (1H, t, J = 7.6)
(3.44 - 3.70, m)
5.01 (1H, br.s)
(3.09-3.27, m)
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
251
Discussion:
The mass and
13
C-NMR spectral data of compound BC-16 gave
molecular ion peak at m/z 625 calculated for the molecular formula
C29H36O15.
The 1H-NMR,
13
C- and DEPT
13
C-NMR spectra (Table 56, Figs.
119-121) revealed the presence of aromatic protons of two ABC systems
at δH 6.43-7.04 and δC 114.7-148.5, trans olefinic protons at δH 6.19, 7.44
(d, J = 16.4 Hz), δC 145.04, 113.61 attached to one of the trisubstituted
benzene rings along with an ester carbonyl at δC 165.77 confirmed by IR
band at υ 1705 cm-1 indicating the presence of trans caffeoyl
moiety(217,218). In addition to two methylene groups typical of phenethyl
alcohol(217) at δH 2.67 (2H, t, J = 7.6 Hz), 3.86 (1H, dd, J = 16.4, 6.8 Hz),
4.15 (1H, dd, J = 16.4, 6.8 Hz), δC 35.06 and 70.25 respectively. While
the sugar protons revealed the presence of β-glucopyranosyl and αrhamnopyranosyl moieties with chemical shift values at δH 4.33 (1H, d, J
= 8.4 Hz), δC 102.32 and δH 5.01 (1H, br.s), δC 101.29 in addition to and
secondry methyl at δH 0.93 (3H, d, J = 5.6 Hz), δC 18.21 indicating its
diglucosidic nature.
The attachment of rhamnose unit to C-3'' of glucose was indicated
from the downfield shift of this carbon at δC 79.14 and the location of the
caffeoyl moiety at C-4'' of glucose was established by comparison with
the reported data of acteoside(219--221) while the linkage of glucose unit at
the methylene group (C-8) of the phenethyl alcohol moiety was proved
from the chemical shift values of this carbon at δC 70.25(219--221).
The positive ion FAB-MS of compound BC-16 (Fig. 118) showed
a molecular ion peak at m/z 625 [M+H]+ and other characteristic
252
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
fragments at m/z 461 [M-(caffeoyl)+H]+, 305 [M-(caffeoyl unit + hexose
unit)+H]+ and 163 [caffeoyl]+ which confirmed the glucose caffeoyl
nature of the compound.
Conclusion:
From the above mentioned spectral data and by comparison with
similar compounds in literature(218-221), compound BC-16 is identified as
3,4-dihydroxy-phenethyl
alcohol
8-O-[(4'-O-caffeoyl)-α-L-
rhamnopyranosyl-(1''→3')]-β-D-glucopyranoside (acteoside). To our
knowledge this represents the first report for its identification from the
genus Blepharis.
5'''
HO
6'''
4'''
1'''
HO 3'''
2'''
8'''
7'''
9'''
OH
O 6`
4`
6``
4`` H3C
HO
HO
O
5`
2`
O
3`
O
O
2``
1`
OH
O
8
1``
3``
7
OH
1
2
6
3
5
4
OH
Acteoside
OH
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
Fig. (118): +FAB-MS spectrum of compound BC-16.
Fig. (119): 1H-NMR of compound BC-16 (DMSO-d6, 400 MHz).
253
Phytochemical Study of Blepharis ciliaris (L.) B.L. Burtt. Aerial Parts
Fig. (120):
254
13
C-NMR spectrum of compound BC-16 (DMSO-d6,
100 MHz).
Fig. (121): DEPT
13
C-NMR spectrum of compound BC-16
(DMSO-d6, 100 MHz).
255
Biological Studies of the Different Extracts of the
Aerial Parts of Anisotes trisulcus (Forssk.) Nees.
and Blepharis ciliaris (L.) B.L.Burtt.
1- Antioxidant Activity:
Recent developments in biomedical science have shown that free
radicals are involved in many diseases. They attack the unsaturated fatty
acids in the biomembrane resulting in membrane lipid per-oxidation, which
is strongly connected to aging, carcinogenesis and atherosclerosis. Free
radicals also attack the DNA and cause mutation leading to cancer.
Therefore, it is important to look for new radical scavengers.
Antioxidant compounds play an important role as a health protecting
factor. Antioxidant compounds like phenolic acids, polyphenols,
phenylpropanoids and flavonoids scavenge free radicals and thus inhibit
the oxidative mechanisms that lead to degenerative diseases.
Various antioxidant activity methods have been used to monitor and
compare the antioxidant activity of different extracts and compounds. One
of which is the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical method. It is
a rapid, simple and inexpensive method to measure the antioxidant
activity.
Antioxidant activity was determined by the DPPH method(222). The
stable organic radical DPPH has been widely used in the assessment of
antioxidant activity studies of single compounds, plant extracts and foods.
Because of its odd electron, DPPH• gives a strong absorption band at 517
nm in visible spectrometry (deep violet colour).
The method is based on the reduction of the alcoholic DPPH•
solutions at 517 nm in the presence of a hydrogen donating antioxidant
Biological Studies of Anisotes trisuclus (Forssk.) Nees.& Blepharis ciliaris (L.)B.L.Burtt. Aerial Parts
256
(AH) due to the formation of the non radical form DPPH-H by the
reaction:
DPPH• + AH
DPPH-H + A•
As this electron becomes paired off in the presence of a free radical
scavenger, the absorption vanishes and the decrease in the DPPH
absorption at 517 nm was measured 10 min. later. The actual decrease in
absorption induced by the test compound was calculated by subtracting
that of the control. The method is rapid, a sample analysis takes 15 min. in
total and little manpower, no expensive reagents or sophisticated
instruments are required.
The antioxidant activity was quantified by the decrease in
absorption of each of the isolated compounds or soluble fraction in
118x10-5 % DPPH solution (final concentration of the sample in the
cuvette was 20 µM for pure compounds while 0.25, 0.5 and 1mg/ml for
soluble fractions) monitored at 517 nm using a spectrophotometer. The
absorbance of DPPH in ethanol (with or without compounds) was
measured after 2 min. the antioxidant capacity was measured in relation to
quercetin (as a reference antioxidant) set as 100% antioxidant activity.
Determinations were performed in triplicates. The antioxidant activity was
calculated using the following equation:
A 0 of the blank − A 0 with compound
A 0 of the blank
X 100
% Antioxidant activity =
where ; A° = Absorbance
The obtained results are listed in Table (57) and illustrated in Figs
(122 & 123).
Biological Studies of Anisotes trisuclus (Forssk.) Nees.& Blepharis ciliaris (L.)B.L.Burtt. Aerial Parts
257
Table (57): Results of the antioxidant activity of the different extracts of
the aerial parts of Anisotes trisulcus (Forssk.) Nees. and
Blepharis ciliaris (L.) B.L.Burtt. and some of the isolated
compounds.
% Antioxidant activity
Anisotes trisulcus
Blepharis ciliaris (L.) B.L.Burtt.
(Forrsk.) Nees.
Extractives
Conc.
DPPH (blank)
Quercetin (reference)
Total meth.ext.
n-Hexane ext.
Chloroformic ext.
Ethyl acetate ext.
Apigenin (BC-8)
Apigenin -7-O- β-Dglucopyranoside (BC-11)
Genistein-7-O-(6''-OE-caffeoyl)-β-Dglucopyranoside (BC-12)
Naringenin-7-O-(6''-Ep-coumaroyl-β-Dglucopyranoside (BC-13)
0.25 mg
0.5 mg
1 mg
0.25 mg
0.5 mg
1 mg
20 µM
—
100
51
19
35
55
—
—
100
67
22
36
63
—
—
100
75
28
38
68
—
—
100
75
19
18
79
—
—
100
85
15
30
83
—
—
100
89
17
35
86
—
—
—
—
—
—
—
30
—
—
—
—
—
—
28
—
—
—
—
—
—
25
—
—
—
—
—
—
23
Antioxidant activity
120
100
80
60
0.25 mg/ml
40
0.5 mg/ml
1 mg/ml
20
0
Quercitin
Tolal
meth.ex.
n-Hexane
ex.
Extracts
Chloroform
Ethyl
ex.
acetate ex.
Fig. (122): Results of the antioxidant activity of the different extracts of
the aerial parts of Anisotes trisulcus (Forssk.) Nees.
258
120
100
80
60
0.5 mg/ml
1 mg/ml
...
co
um
oy
pE-
-E
'' -
O
-(6
N
ar
in
ge
ni
n-
7-
O
7-
20 µM/ml
O
-( 6
''-
-g
-D
nei
af
fe
py
ra
lu
co
Ap
is
t
en
G
n.
..
ni
ig
e
e
-β
-O
-7
in
en
ig
Ap
-c
.
ex
.
ce
t
la
Et
hy
ro
f
lo
Ch
at
or
m
e
an
ex
H
n-
ex
ex
.
ex
h.
et
lm
To
la
Q
...
0
n
0.25 mg/ml
.
20
rc
itin
40
ue
Antioxidant activity
Biological Studies of Anisotes trisuclus (Forssk.) Nees.& Blepharis ciliaris (L.)B.L.Burtt. Aerial Parts
Extracts/Compound
Fig. (123): Results of the antioxidant activity of the different extracts of
the aerial parts and some of the isolated compounds of
Blepharis ciliaris (L.) B.L.Burtt.
Conclusion:
The obtained results (Table 57, Fig. 122) proved that the total
methanolic and the ethyl acetate extracts of Anisotes trisulcus (Forssk.)
Nees. showed the highest antioxidant activity 75% and 68% at 1 mg
concentration while Blepharis ciliaris (L.) B.L.Burtt. also exhibited high
antioxidant activity 89% and 86% at 1 mg concentration respectively for
the same extracts. The obtained results could be attributed to the presence
of phenolic compounds (flavonoids, phenylpropanoids and phenolic acids).
While
the
glucopyranoside,
and
pure
compounds
apigenin,
apigenin-7-O-β-D-
genistein-7-O-(6''-O-E-caffeoyl)-β-D-glucopyranoside
naringenin-7-O-(6''-E-p-coumaroyl-β-D-glucopyranoside
showed
moderate activity, which was in agreement with the conclusion that the
hydroxylation pattern of the flavonoidal compounds is highly effective
against free radicals(223).
Biological Studies of Anisotes trisuclus (Forssk.) Nees.& Blepharis ciliaris (L.)B.L.Burtt. Aerial Parts
259
2- Acute Toxicity Study(224):
a- Animals:
Male albino rats each (120-150 gm), were bred and housed under
standardized environmental conditions. The animals were fed with
standard diet and free access to water. They were kept at 24-28°C
temperature, 60-70% relative humidity, 12 hrs day and night cycle for one
week to acclimatize to the environmental conditions.
b- Method:
The previously prepared total methanolic extract (P.58) of both
plants: Anisotes trisulcus (Forssk.) Nees.and Blepharis ciliaris (L.)
B.L.Burtt. was dissolved in 2% tween-80 and administrered orally at
increasing doses (1, 2, 4 and 8 gm/kg). The animals were remained for 48
hrs under observation for any signs and symptoms of toxicity(224).
c- Results and Conclusion:
The symptoms of toxicity were characterized by irritability,
writhing, hypothermia, loss of motor coordination, sedation and deep
sleep, followed by death. The total methanolic extract of Anisotes trisulcus
(Forssk.) Nees. didn't show any signs of toxicity and mortality up to
5gm/kg dose while Blepharis ciliaris (L.) B.L.Burtt. didn't show any signs
of toxicity and mortality up to 4 gm/kg.
Biological Studies of Anisotes trisuclus (Forssk.) Nees.& Blepharis ciliaris (L.)B.L.Burtt. Aerial Parts
260
3- Brine-Shrimp Assay (Cytotoxic Activity) (225,226):
This technique is an in vivo lethality test involving the whole body
of a tiny crustacean, the brine shrimp (Artemia salina Leach.). This test
takes into account the basic premise that pharmacology is simply
toxicology at a lower dose, and that toxic substances might indeed elicit, at
a lower non-toxic dose, interesting pharmacological effects. The procedure
determines LC50 values in µg/ml of active fractions in the brine medium.
a- Sample Preparation:
The test samples previously prepared (P.58) were dissolved in
DMSO and the appropriate amount was transferred to a 10 ml sample vial
and then serially diluted in artificial sea water to the desired concentration.
Bioassay was done on 0.5 mg of the different fractions of the total
alcoholic extract and the different extracts of the aerial parts of both
plants: Anisotes trisulcus (Forssk.) Nees. and Blepharis ciliaris
(L.)B.L.Burtt. were performed in triplicates. The samples were then dried
and reconstituted with 20 µl DMSO. Control vials containing the same
amount of DMSO were also prepared.
b- Technique:
As much as 33 gm salt mixture (Biomarine, Erkrath, Germany) was
dissolved in 1000 ml distilled water, thoroughly mixed with the help of
magnetic stirrer. A small amount of the dried brine shrimp eggs (Dohse,
Bonn, Germany) were hatched in a small tank filled with 1000 ml artificial
sea water. After 48 hours, the eggs would hatch.
Twenty nauplii were taken from the hatching tank, and transferred
into each test vial (test substance + artificial sea water + DMSO). After
the transfer of 20 nauplii, artificial sea water was then added to make the
Biological Studies of Anisotes trisuclus (Forssk.) Nees.& Blepharis ciliaris (L.)B.L.Burtt. Aerial Parts
261
final volume 5ml.The experimental vials were left overnight, under
illumination, in normal room temperature. After 24 and 48 hours, survivors
and dead brine shrimp were counted under microscope and recorded. The
more the death of brine shrimp the more toxic is the test substance. The
test substance resulting in 50% death of the brine shrimp was considered
bioactive. Determination was performed and the toxic activity was
calculated using the following equation:
% Mortality =
Control — test
Control
x 100
The obtained results are listed in Tables (58) and (59).
Table (58): Results of the toxic activity of the different extracts of the
dried aerial parts of Anisotes trisulcus (Forrsk.) Nees.
Extract
Meth.ex.
n-Hexane ex.
Chloroform ex.
Ethyl-acetate ex.
Control
A
After 24 hrs
B
C
% Mortality
A
After 48 hrs
B
C
% Mortality
5
16
7
11
20
8
13
9
13
20
3
10
1
7
19
7
10
0
5
19
10
20
8
16
20
61
18
60
33
0
8
15
1
7
19
70
41
96
68
1
-A, B and C: Experiments in triplicates.
Table (59): Results of the toxic activity of the different extracts of the
dried aerial parts of Blepharis ciliaris (L.)B.L.Burtt.
Extract
Meth. ex.
n-Hexane ex.
Chloroform ex.
Ethyl-acetate ex.
Control
A
After 24 hrs
B
C
% Mortality
A
After 48 hrs
B
C
% Mortality
20
20
10
20
20
20
20
18
16
20
15
15
9
10
19
14
19
16
14
19
19
17
10
19
20
-A, B and C: Experiments in triplicates.
Conclusion:
2
5
36
8
0
17
10
8
18
19
23
26
45
30
1
Biological Studies of Anisotes trisuclus (Forssk.) Nees.& Blepharis ciliaris (L.)B.L.Burtt. Aerial Parts
262
The obtained results showed that the total methanolic extract and
the chloroformic extract of the dried aerial parts of Anisotes trisulcus
(Forrsk.) Nees. had a strong activity in the brine shrimp bioassay 61% and
60% after 24 hrs while the methanolic, chloroformic and ethyl acetate
showed 70%, 96% and 68% after 48 hrs respectively which may be
attributed to their alkaloidal, steroidal and/or triterpenoidal content while
on the other hand the n-hexane and ethyl acetate extracts had mild activity.
While the different extracts of the dried aerial parts of Blepharis
ciliaris (L.) B.L.Burtt. showed poor to mild activity after 24 and 48 hrs
respectively.
4- Anti-Inflammatory Activity(227,228):
Carageenin-induced rat hind paw oedema model described by
Winter et al.(229-230). This method of testing anti-inflammatory activity
depends on measuring the inhibition of oedema produced acutely by
injection of an irritant (phlogistic agent) into the tissues of the plantar
surface of the hind paw of the rat.
In this test, sixty albino rats were divided into six groups of six
animals each. The first group was kept as negative control, injected
intraperitoneally by 2% tween 80 in normal saline (mixture solvent) while
the other group was injected by indomethacin (reference group) at a dose
of 8 mg/kg and kept as a reference for both studied plants. The other
groups were intraperitoneally injected with the different extracts of the
aerial parts both plants: Anisotes trisulcus (Forssk.) Nees. and Blepharis
ciliaris (L.) B.L.Burtt. at a dose of 400 mg/kg body weight. One hour later
Biological Studies of Anisotes trisuclus (Forssk.) Nees.& Blepharis ciliaris (L.)B.L.Burtt. Aerial Parts
263
foot paw oedema was induced by injecting 0.1 ml of 1% carrageenin
subcutaneously into the sub-planter portion of the right hind paw of each
rat while the left one was injected by an equal volume of saline solution.
The difference between the thickness of the paws was taken as a measure
of paw oedema. The paws thicknesses were measured in mm using
Vernier Caliper.
The anti-inflammatory efficacy of the tested extracts was estimated
by comparing the magnitude of paw swelling in the pretreated animals
with those induced in control animals receiving the vehicle only. The
measurements were carried out immediately after injection of the
phlogistic agent (0 hr.) and at 1, 2, 3, 4 and 5 hrs after the injection of
carrageenin.
The % protection of oedema was calculated according to the
formula:
% inhibition of inflammation = 100 X (1- Pt /PC)
where Pt is the increase in paw thickness of the treated and PC is that of
the control.
The results of the measurements of percentage of anti-inflammatory
activity are listed in Tables (60 & 61).
Biological Studies of Anisotes trisuclus (Forssk.) Nees.& Blepharis ciliaris (L.)B.L.Burtt. Aerial Parts
264
Table (60): Results of the anti-inflammatory effect of the different
extracts of the aerial parts of Anisotes trisulcus (Forssk.)
Nees..
Dose
(mg/kg)
Group
Control
Indomethacin
n-hexane
Cholroform
Ethyl Acetate
Total methanolic
extract
% inhibition of inflammation after time (hrs)
1 hr
2 hrs
3 hrs
4 hrs
5 hrs
—
8
400
400
400
100
34.43
28.16
20.67
56.93
100
61.75
56.84
49.28
87.53
100
84.66
79.19
51.84
90.67
100
91.82
90.41
52.56
90.84
100
93.17
90.49
52.30
91.18
400
37.83
65.71
81.47
87.55
92.10
Table (61): Results of the anti-inflammatory effect of the different
extracts of the aerial parts of Blepharis ciliaris (L.)
B.L.Burtt..
Group
Control
Indomethacin
n-hexane
Cholroform
Ethyl Acetate
Total methanolic
extract
Dose
(mg/kg)
% inhibition of inflammation after time (hrs)
1 hr
2 hrs
3 hrs
4 hrs
5 hrs
—
8
400
400
400
100
34.43
29.92
22.64
55.18
100
61.75
54.90
44.83
86.03
100
84.66
86.07
54.14
90.17
100
91.82
89.90
59.16
90.30
100
93.17
90.10
59.98
91.20
400
34.03
67.90
83.14
85.75
92.81
Conclusion:
From the previous data listed in Table (60 & 61), it was observed
that
all the tested extracts showed anti-inflammatory activity in
carageenin-induced rat hind paw oedema model when the animals were
pretreated with the different extracts of the aerial parts of both plants
(Anisotes trisulcus (Forssk.) Nees. and Blepharis ciliaris (L.) B.L.Burtt.)
where the total methanol, n-hexane and ethyl acetate extracts showed
significant anti-inflammatory effect, where marked reduction of the paw
Biological Studies of Anisotes trisuclus (Forssk.) Nees.& Blepharis ciliaris (L.)B.L.Burtt. Aerial Parts
265
edema was observed due to their high content of sterols and their
corresponding glucosides, flavonoids and phenolic compounds.
5- Anti-Malarial Assay:
a- Preparation of the Tested Samples and the Parasite Used:
The different extracts of Anisotes trisulcus (Forssk.) Nees.and
Blepharis ciliaris (L.) B.Burtt. were tested for in vitro anti malarial
activity against the chloroquine sensitive (D6, Sierraleon) Plasmodium
falciparum; using the parasite lactate dehydrogenase (pLDH) assay using
Malstat reagent(231,232).
The previously prepared different extracts (P.58) of Anisotes
trisulcus (Forssk.) Nees.and Blepharis ciliaris (L.) B.L.Burtt. were
dissolved in DMSO at concentrations of 50 mg/ml sonicated for 10 min.
and then diluted in malaria culture medium to prepare 2mg/ml solution.
The appropriate dilutions were added to the cultures of P.falciparum set
up in clear flat bottomed 96 well micro-liter plates.
b- Technique:
The parasites were maintained in continuous culture on human
erythrocytes in RPMI-1640 medium. The plates were placed into a
humidified chamber and flushed with a gas mixture of (90% N2, 5% CO2
and 5% O2). Medium RBCs controls were also set up in each plate.The
cultures were incubated at 37°C for 48 hrs. Growth of the parasite in each
well was determined spectrophotometrically at 650 nm by measuring the
activity of the pLDH in control and drug treated cultures using a
microplate reader. The standard anti-malarial agent, chloroquine was used
as the positive control while DMSO was used as the negative one. The
Biological Studies of Anisotes trisuclus (Forssk.) Nees.& Blepharis ciliaris (L.)B.L.Burtt. Aerial Parts
266
anti-malarial activity of the tested extracts was expressed as a percentage
of inhibition of the parasite growth (Tables 62 & 63).
Table (62): In-vitro anti-malarial screening for the different extracts of
Anisotes trisulcus (Forssk.) Nees.
Tested Extracts
Total methanolic extract
n-Hexane extract
Chloroformic extract
Ethyl acetate extract
Chloroquine
% inhibition
(P. falciparum, D6 clone)
37
35
35
20
100
Conclusion:
The methanolic, hexane and chloroform extracts showed mild antimalarial activity against the tested P.falciparum (D6 clone) relative to
chloroquine.
Table (63): In-vitro anti-malarial screening for the different extracts of
Blepharis ciliaris (L.)B.L.Burtt.
Tested Extracts
Total methanolic extract
n-Hexane extract
Chloroformic extract
Ethyl acetate extract
Chloroquine
% inhibition
(P. falciparum, D6 clone)
28
20
19
31
100
Conclusion:
The ethyl acetae and the total methanolic extract showed mild to
weak anti-malarial activity against the tested P.falciparum (D6 clone)
relative to chloroquine.
Biological Studies of Anisotes trisuclus (Forssk.) Nees.& Blepharis ciliaris (L.)B.L.Burtt. Aerial Parts
267
6- Anti-Hyperglycaemic Activity(233):
Technique:
1- Overnight fasting adult male albino mice ( 20-25 gm each), (18 hrs)
received different doses of glucose (from 1 to 5 gm/kg) orally using a
stomach tube and grouped in groups six mice each, blood samples
were collected at 15, 30, 45, 60, 90, 120 and 180 minutes. The dose at
which the animals became hyperglycaemic was determined to be 2.25
gm/kg by trial and error. Increasing the glucose load above this dose
didn't change the glucose tolerance curve.
2- Specific weight of the different dried extracts (4 gm) were dissolved in
distilled water by the aid of 1% tween 80 and the volume was
completed to 100 ml with distilled water.
Effect on Blood Glucose Level when the Extracts were Fed
Simultaneously with Glucose Load:
Overnight fasting mice (18 hrs) received the different extracts
solutions (400 mg/Kg) of the investigated plants with glucose load (2.25
gm/kg) orally using a stomach tube at 0 min., blood samples were
withdrawn from the cavernous sinus at 0, 30, 60, 90, 120 and 180 minutes
and the blood glucose level in mg/dl was determined using GlucostarGlucostix device. The results are listed in Tables (64 & 65).
Biological Studies of Anisotes trisuclus (Forssk.) Nees.& Blepharis ciliaris (L.)B.L.Burtt. Aerial Parts
268
Table (64): Effect of the different extracts of the dried aerial parts of
Anisotes trisulcus (Forssk.) Nees. on the Blood Glucose Levels
(BGLs) when they fed concurrently with the glucose load.
Group
No.
Group I
Group II
Group III
Group IV
Group V
Zero
M ± S.E
30 min.
M ± S.E
60 min.
M ± S.E
90 min.
M ± S.E
120 min.
M ± S.E
180 min
M ± S.E
85.00
± 3.00
98.11
± 6.99
84.59
± 1.98
106.12
± 5.77
89.23
± 5.19
215.00
±10.00
121.89
± 5.91
200.47
± 9.33
203.38 ±
6.56***
125.38
± 8.45
224.50
± 4.50
135.99
± 2.36***
218.21
± 7.34
91.15
± 5.67***
141.28
± 4.32
194.50
± 6.50
98.61
± 5.86***
194.19
± 1.29
105.00
± 6.93***
129.29
± 4.31
163.00
± 1.00
95.98
± 4.48***
161.17
± 2.49
70.15
± 5.67***
85.65
± 9.46
120.00
± 5.00
78.99
± 6.76**
115.95
± 5.66
65.02
± 1.53***
75.18
± 7.56
Table (65): Effect of the different extracts of the dried aerial parts of
Blepharis ciliaris (L.)B.L.Burtt. on the Blood Glucose Levels
(BGLs) when they fed concurrently with the glucose load.
Group
No.
Group I
Group II
Group III
Group IV
Group V
Zero
M ± S.E
30 min.
M ± S.E
60 min.
M ± S.E
90 min.
M ± S.E
120 min.
M ± S.E
180 min
M ± S.E
85.00
± 3.00
106.12
± 5.77
84.99
± 1.98
114.99
± 4.68
88.23
± 9.39
215.00
± 10.00
203.38
± 6.56
205.87
± 9.33
202.87
± 10.33
205.38
± 7.65
224.50
± 4.50
231.15
± 5.67
219.21
± 7.14
229.21
± 9.94
241.28
± 7.32
194.50
± 6.50
185.00
± 6.93
184.69
± 1.49
194.69
± 2.79
191.29
± 9.31
163.00
± 1.00
170.15
± 4.67
158.17
± 2.79
169.17
± 4.99
159.65
± 5.46
120.00
± 5.00
115.02
± 1.53
119.95
± 6.16
109.97
± 5.26
115.18
± 7.56
Group I: Received glucose load only (2.25 gm/kg).
Group II: Received glucose load and the n-hexane extract 400 mg/kg.
Group III: Received glucose load and the chloroform extract 400 mg/kg.
Group IV: Received glucose load and the ethyl-acetate extract 400 mg/kg.
Group V: Received glucose load and the total methanolic extract 400 mg/kg.
Data are expressed as mean ± S.E, n = 6
*P < 0.05, **P < 0.05, *** P < 0.001 (using student t-test).
Biological Studies of Anisotes trisuclus (Forssk.) Nees.& Blepharis ciliaris (L.)B.L.Burtt. Aerial Parts
269
Conclusion:
From the previous results listed in Table (64 & 65), it was observed
that the total methanolic extract of the aerial parts of Anisotes trisulcus
(Forssk.) Nees. and its n-hexane and ethyl acetate extracts when given
concurrently with the glucose load dose at a dose of 400mg/Kg a
significant hypoglycaemic effect was obtained after 1 hr and this continued
for 2 hrs while the chloroform extract showed the least activity.
The obtained results support the use of powdered Anisotes trisulcus
(Forssk.) Nees. as an anti-diabetic in folk medicine.
Concerning the methanolic extract of the dried aerial parts of
Blepharis ciliaris (L.) B.L.Burtt. and its fractions when given concurrently
with glucose load showed a non-significant anti-hypergycaemic activity.
270
GENERAL SUMMARY AND CONCLUSION
Phytochemical and Biological Studies of Some
Plants Belonging to Family Acanthaceae
Family Acanthaceae is considered as one of the richest families with
medicinal plants. It includes about 346 genera and around 4300 species.
The plants under investigation are Anisotes trisulcus (Forssk.) Nees.
& Blepharis ciliaris (L.) B.L.Burtt. both belonging to family Acanthacaea.
Anisotes trisulcus (Forssk.) Nees.: A stiffy erect shrub with a
height of 1-3.5 m. It is endogenous to Yemen and widely distributed in the
Southern mountainous regions of the kingdom of Saudi Arabia particularly
in the Wadi Jabal Abu Hassan between Abha and Najran. The main stem
of the plant is erect, cylindrical, monopodially branched carrying numerous
branches showing more or less long internodes. The young stem is
purplish-green in colour, while the old one has rough surface covered with
brownish cork. The branches bear opposite and decussate leaves at the
nodes. Flowers are shortly pedunculate, 2-flowered axillary clusters. Fruit
capsule, 4 seeded. Seeds are rough.
Blepharis ciliaris (L.) B.L.Burtt.: A perennial, greyish-pubescent
to glabrescent ascending, prickly herb branched from the base, reaching up
to 30 cm in height, distributed in East Tropical Africa, Saudi Arabia and
Egypt. The leaves appearing in whorls of 4-outer pairs in each whorl often
smaller, they are oblong to lanceolate, coriacious lamina with silvery
appearance and long petiole, margins are entire to remotely spinulose,
puberulous above, with spine tipped apex. Inflorescence is a dense,
strobilate spike with purplish-blue flowers, bracteoles are linear-subulate
General Summary and Conclusion
271
villose. Fruit is a capsule, ovate to compressed shining brown and glabrous
with two flat seeds.
Anisotes trisulcus (Forssk.) Nees. is used as traditional herbal
medicine in the Arabian Peninsula as a treatment for all hepatic conditions
including hepatitis, jaundice, gallstone and other hepatic disorders. It is
also used as anti-diabetic, bronchodilator, hypo-tensive and it has a local
anesthetic effect. The plant extract is used in several pharmaceutical forms
to limit tobacco consumption and to suppress appetite.
Blepharis ciliaris (L.) B.L.Burtt. seeds (roasted or crushed) were
applied on sores, wounds and boils as an antibacterial. The seeds are also
considered to be attenuant, resolvent, diuretic, aphrodisiac and expectorant
in addition to charcoal from the roots is applied to the eyes to improve
vision, hence the Arabic name "Kohl-el-agouz".
Previous Phytochemical and biological studies of Acanthaceous
plants especially Anisotes trisulcus (Forssk.) Nees. and Blepharis ciliaris
(L.)B.L. Burtt. showed the isolation and identification of diverse secondary
metabolites as well as different biological activities. Thus, it was deemed
important to carry out comprehensive phytochemical and biological
investigations of the selected plants.
The present work includes the following three parts:
General Summary and Conclusion
272
PART I: Phytochemical Study of Anisotes trisulcus (Forssk.) Nees.
Aerial Parts.
Chapter I:
Preliminary phytochemical screening of the air dried
powdered aerial parts of Anisotes trisulcus (Forssk.) Nees.
Chapter II: Extraction, fractionation and isolation of the constituents
from Anisotes trisulcus (Forssk.) Nees.
Chapter III: Identification of the isolated compounds from Anisotes
trisulcus (Forssk.) Nees. aerial parts.
PART II: Phytochemical Study of Blepharis ciliaris (L.) B.L.Burtt.
Aerial Parts.
Chapter I:
Preliminary phytochemical screening of the powdered
Blepharis ciliaris (L.) B.L.Burtt.aerial parts.
Chapter II: Extraction, fractionation and isolation of the constituents
from Blepharis ciliaris (L.) B.L.Burtt.aerial parts.
Chapter III: Identification of the isolated compounds from Blepharis
ciliaris (L.) B.L.Burtt. aerial parts.
PART III: Biological Studies of Anisotes trisuclus (Forssk.) Nees. and
Blepharis ciliaris (L.)B.L.Burtt. Aerial Parts.
1- Antioxidant Activity.
2- Acute Toxicity Study.
3- Brine-Shrimp Assay (Cytotoxic Activity).
4- Anti-inflammatory Activity.
5- Anti-malarial Activity.
6- Anti-hyperglycaemic Activity.
273
General Summary and Conclusion
PART I
Phytochemical Study of Anisotes trisulcus (Forssk.)
Nees. Aerial Parts
Chapter I
Preliminary Phytochemical Screening of the Air Dried
Powdered Aerial Parts of Anisotes trisulcus (Forssk.) Nees.
The dried aerial parts of Anisotes trisulcus (Forssk.) Nees. contains
volatile substances, carbohydrates and/or glycosides, unsaturated sterols
and/or triterpenes, tannins, flavonoids, lactones and/or esters in addition to
alkaloids and/or basic nitrogenous substances.
Chapter II
Extraction, Fractionation and Isolation of the Constituents
from Anisotes trisulcus (Forssk.) Nees. Aerial Parts
The air-dried powdered aerial parts (2.5 kg) of Anisotes trisulcus
(Forssk.) Nees.were extracted by maceration and percolation with (70%)
methanol till complete exhaustion [four times each 10 L, overnight]. The
combined methanolic extracts were concentrated under reduced pressure
till constant weight to give a dark green syrupy residue (300 gm).
The methanolic extract (300 gm) was digested in the least amount of
distilled water subjected to successive solvent fractionation using a
separating funnel with n-hexane, chloroform, ethyl-acetate, n-butanol.
Each extractive was concentrated separately under reduced pressure to
give 45, 35, 18 and 8 gm respectively.
The lipoidal content of the n-hexane fraction was investigated.
274
General Summary and Conclusion
The obtained different extractives were subjected to different
chromatographic techniques for separations of their constituents where 20
compounds were isolated.
Chapter III
Identification of the Isolated Compounds from Anisotes
trisulcus (Forssk.) Nees. Aerial Parts
Structure elucidation of the isolated compounds was deduced on the
basis of spectroscopic methods: (UV, IR, 1H-NMR,1H-1H COSY,
NMR, DEPT
13
C-
13
C-NMR, HSQC, HMQC, EI-,+FAB- and MALDI-TOF-
MS) in addition to comparison of the physical, chemical and
chromatographic characters of these compounds with the available
authentic samples. A list of the identified compounds listed in Table (66).
Table (66): List of the isolated compounds from Anisotes trisulcus
(Forssk.) Nees.aerial parts.
Sign
Name and Structure
Remarks
First report from
the plant
AT-1
HO
α-Amyrin
First report from
the plant
AT-2
HO
β-Sitosterol
275
General Summary and Conclusion
First report from
the plant
AT-3
HO
Stigmasterol
Table (66): Continued.
Sign
Name and Structure
Remarks
O
CH3
(CH2)20
OH
HN
AT-4
OH
2
1
3
(CH2)12 CH3
4
First report from
the family
Acanthaceae
5
OH
OH
(2S, 3S, 4R)-2[(2'R)-2'-Hydroxytetracosanoyl amino]
octadecane 1,3,4-triol
OH
O
OH
O
HO
O
New Compound
AT-5
OH
OCH3 O
3,7,8,3'-Tetrahydroxy 5-methoxy-4'benzoyl flavone
OH
OH
HO
O
AT-6
HO
OH
OCH3 O
3,6,7,3',4'-Pentahydroxy -5-methoxy flavone
First report from
the family
Acanthaceae
276
General Summary and Conclusion
N
AT-7
N
OH
Vasicine (Peganine)
Previously
isolated from the
plant
277
General Summary and Conclusion
Table (66): Continued.
Sign
Name and Structure
Remarks
O
N
Previously
isolated from the
plant
AT-8
4
OH
Vasicinone
O
N
N
Previously
isolated from the
plant
AT-9
COOCH3
NHCH3
Anisotine
O
N
N
AT-10
NH
OH
O
New compound
C-OCH3
5-Hydroxyvasnetine
O
C-OH
AT-11
OCH3
OCH3
Veratric acid
First report from
the family
Acanthaceae
278
General Summary and Conclusion
Table (66): Continued.
Sign
Name and Structure
O
Remarks
OH
C
First report from
the genus
Anisotes
AT-12
OCH3
OH
3-Methoxy,4-hydroxy benzoic acid (Vanillic acid)
29
28
21
22
20
23
18
19
AT-13
1
HO OH
6´
1´
OH
O
8
10
4
27
16
3
HO
17
9
2
O
13
26
25
12
11
24
5
14
15
First report from
the genus
Anisotes
7
6
β-Sitosterol-3-O-β-D-glucopyranoside
HO
N
AT-14
N
OH
First report from
the genus
Anisotes
7-Hydroxyvasicine (Vasicinol)
O
HO
First report from
the genus
Anisotes
N
AT-15
N
OH
7-Hydroxyvasicinone (Vasicinolone)
HO
NH2
N
AT-16
N
New Compound
OH
8-Amino-7,8,9,11-tetrahydro-6H-pyrido[2,1-b]
quinazoline-2,6-diol
279
General Summary and Conclusion
Table (66): Continued.
Sign
Name and Structure
Remarks
O
NH2
N
AT-17
HO
New compound
N
OH
8-Amino-3,6-dihydroxy-7,8,9-trihydro-6Hpyrido[2,1-b]quinazoline-11-one
4
2
N
N
3
OH Cl
1
New compound
AT-18
(Dimethylamino)-N-(hydroxymethyl)-N,Ndimethylmethanaminium chloride
4
AT-19
N
H
N
3
Cl
1
COOH
2
New compound
N-[(carboxyamino)methyl]–N,N–dimethyl
ethanaminium chloride
2
HO
+
1
N
3
Choline
AT-20
4
Cl
N
3
1
2
COOH
N-[(carboxyamino)methyl]–N,N–dimethyl
ethanaminium chloride
First report from
the plant
280
General Summary and Conclusion
PART II
Phytochemical Study of Blepharis ciliaris (L.)
B.L.Burtt. Aerial Parts
Chapter I
Preliminary Phytochemical Screening of the Powdered
Blepharis ciliaris (L.) B.L.Burtt.aerial parts
The dried aerial parts of Blepharis ciliaris (L.)B.L.Burtt. contain
volatile substances, carbohydrates and/or glycosides, unsaturated sterols
and/or triterpenes, tannins, flavonoids in addition to lactones and/or esters.
Chapter II
Extraction, Fractionation and Isolation of the Constituents
from Blepharis ciliaris (L.) B.L.Burtt.aerial parts
The air-dried powdered aerial parts (3 kg) of Blepharis ciliaris (L.)
B.L.Burtt.were extracted by maceration and percolation with methanol till
complete exhaustion [four times each 10 ml, overnight]. The combined
methanolic extracts were concentrated under reduced pressure till constant
weight to give a dark green syrupy residue (270 gm).
The methanolic extract (270 gm) was digested in the least amount of
distilled water subjected to successive solvent fractionation using a
separating funnel with n-hexane, chloroform, ethyl-acetate, n-butanol.
Each extractive was concentrated separately under reduced pressure to
give 50, 37, 45 and 12 gm respectively.
The lipoidal content of the n-hexane fraction was investigated.
The obtained different extractive were subjected to different
chromatographic techniques for separations of their constituents where 16
compounds were isolated.
281
General Summary and Conclusion
Chapter III
Identification of the Isolated Compounds from Blepharis
ciliaris (L.) B.L.Burtt. Aerial Parts
Structure elucidation of the isolated compounds was deduced on the
basis of spectroscopic methods: (UV, IR, 1H-NMR,13C-NMR, DEPT 13CNMR, EI- and +FAB- MS) in addition to comparison of the physical,
chemical and chromatographic characters of these compounds with
available authentic samples. A list of the identified compounds are listed in
Table (67).
Table (67): List of the isolated compounds from Blepharis ciliaris (L.)
B.L.Burtt.aerial parts.
Sign
Name and Structure
Remarks
28
12
19
BC-1
11
1
2
8
3
HO
4
21
20
18
23
24
26
25
13 17
27
First report from
the plant
16
9
10
22
29
14
15
7
5
6
β-Sitosterol
29
28
12
19
BC-2
1
2
11
10
4
13 17
16
9
8
3
HO
22
21
23
20
18
5
14
15
7
6
Stigmasterol
24
26
25
27
First report from
the plant
282
General Summary and Conclusion
Table (67): Continued.
Sign
Name and Structure
Remarks
29
28
22
21
20
23
18
1
BC-3
14
10
1'
O
C
New compound
15
8
3
7
5
O
27
16
9
2
17
13
11
26
25
12
19
24
4
6
(CH2)22
CH3
Stigmasterol tetracosanoate
O
1'
(CH2)16
CH3
OH
OH
HN
BC-4
3'
2'
2
(CH2)12 CH3
3 4
1
5
OH
OH
First report from
the family
Acanthaceae
(2S, 3S, 4R)-2[(2'R)-2'-(Hydroxyeicosanoyl amino)
octadecane-1,3,4-triol
7
8
COOCH 3
1
2
6
BC-5
5
3
OCH 3
4
First report from
the family
Acanthaceae
OCH 3
Methyl veratrate
O
8
7
C
OCH3
1
6
2
5
3
BC-6
4
OCH3
OH
Methyl vanillate
First report from
the genus
Blepharis
283
General Summary and Conclusion
Table (67): Continued.
Sign
Name and Structure
O
Remarks
OH
First report from
the genus
Blepharis
BC-7
OH
OH
Protocatechuic acid
OH
HO
O
First report from
the plant
BC-8
O
OH
Apigenin
29
28
21
22
20
24
23
18
26
25
12
19
BC-9
6´
2
O
14
10
8
4
OH
First report from
the plant
15
7
5
O
27
9
3
1´
HO
17
16
1
HO OH
13
11
6
β-Sitosterol-3-O-β-D-glucopyranoside
29
28
21
22
20
23
18
24
26
25
12
19
BC-10
1
HO OH
6´
2
O
14
10
4
OH
27
9
8
15
First report from
the plant
7
5
O
17
16
3
1´
HO
13
11
6
Stigmasterol-3-O-β-D-glucopyranose
OH
O
glu-O
BC-11
OH
O
Apigenin -7-O- β-D-glucopyranoside
Previously
isolated from the
plant
284
General Summary and Conclusion
Table (67): Continued.
Sign
Name and Structure
Remarks
O
7'''
9'''
8'''
O
3'''
O
HO
HO
8
1'
9 O 2
7
OH 1''
4' OH
2'
OH
5'''
O
2''
3'
4'''
6'''
4'' 6''
BC12
2'''
1'''
5'
6'
6
5
3
10
First report
from the
plant
4
O
OH
Apigenin-7-O-(6''-E-p-coumaroyl-β-D-glucopyranoside)
O
7```
9```
BC13
3``` OH
8```
O
HO
HO
4```
6```
6``
4``
2```
1```
O
2``
OH
5```
8
O
OH 1``
9 O
3
6
10
5
1`
2`
3`
4
O
OH
New
compound
2
7
4`
6`
OH
5`
Genistein-7-O-(6''-O-E-caffeoyl)-β-Dglucopyranoside
O
7'''
9'''
2'''
1'''
O
4''
BC14
3''
O
2''
3'
4'''
6'''
6''
HO
RO
3'''
8'''
OH
5'''
8
O
9
7
OH 1''
O
1'
2
5'
6'
6
R = COCH3
4'
2'
5
OH
3
10
4
O
Naringenin-7-O-(3''-acetyl-6''-E-p-coumaroyl-β-Dglucopyranoside
OH
Previously
isolated from
the plant
285
General Summary and Conclusion
Table (67): Continued.
Sign
Name and Structure
Remarks
O
7'''
9'''
3'''
8'''
O
HO
HO
3''
O
2''
3'
4'''
6'''
4'' 6''
BC15
2'''
1'''
5'''
1'' O
OH
2'
8
1'
9 O
7
OH
2
4' OH
5'
6'
First report
from the
plant
3
6
5
10
4
OH O
Naringenin-7-O-(6''-E-p-coumaroyl-β-D-glucopyranoside
5'''
HO
6'''
4'''
8'''
1'''
HO 3'''
O
2'''
7'''
9'''
O OH
6`
6``
2`
4`` H3C
BC16
HO
HO
O
3`
2``
1``
3``
O
5`
4`
O
1`
Previously
isolated from
the plant
OH
O
OH
8
7
1
6
2
5
3
4
OH
Acteoside
OH
286
General Summary and Conclusion
PART III
Biological Studies of Anisotes trisuclus (Forssk.) Nees.
and Blepharis ciliaris (L.)B.L.Burtt. Aerial Parts
1- Antioxidant Activity:
The total methanolic and the ethyl acetate extracts of Anisotes
trisulcus (Forssk.) Nees. showed the highest antioxidant activity 75% and
68% at 1 mg concentration while Blepharis ciliaris (L.) B.L.Burtt. also
exhibited high antioxidant activity 89% and 86% at 1 mg concentration
respectively for the same extracts. The obtained results could be attributed
to the presence of phenolic compounds (flavonoids, phenylpropanoids and
phenolic acids).
While
the
glucopyranoside,
and
pure
compounds
apigenin,
apigenin-7-O-β-D-
genistein-7-O-(6''-O-E-caffeoyl)-β-D-glucopyranoside
naringenin-7-O-(6''-E-p-coumaroyl-β-D-glucopyranoside
showed
moderate activity, which was in agreement with the conclusion that the
hydroxylation pattern of the flavonoidal compounds is highly effective
against free radicals.
2- Acute Toxicity Study:
The total methanolic extract of Anisotes trisulcus (Forssk.) Nees.
didn't show any signs of toxicity and mortality up to 5 gm/kg while that of
Blepharis ciliaris (L.) B.L.Burtt. didn't show any signs of toxicity and
mortality up to 4 gm/kg.
3- Brine-Shrimp Assay (Cytotoxic Activity):
The activity of the total methanolic extract and the chloroformic
extract of the dried aerial parts of Anisotes trisulcus (Forrsk.) Nees. was
General Summary and Conclusion
287
strong in the brine shrimp bioassay 61% and 60% after 24 hrs while the
methanolic, chloroformic and ethyl acetate showed 70%, 96% and 68%
after 48 hrs respectively which may be attributed to their alkaloidal,
steroidal and/or triterpenoidal content while on the other hand the nhexane extract had mild activity.
While the different extracts of the dried aerial parts of Blepharis
ciliaris (L.) B.L.Burtt. showed poor to mild activity after 24 and 48 hrs
respectively.
4- Anti-Inflammatory Activity:
It was observed that all the tested extracts showed antiinflammatory activity in carageenin-induced rat hind paw oedema model
when the animals were pretreated with the different extracts of the aerial
parts of both plants (Anisotes trisulcus (Forssk.) Nees. and Blepharis
ciliaris (L.) B.L.Burtt.) where the total methanol, n-hexane and ethyl
acetate extracts showed significant anti-inflammatory effect, where marked
reduction of the paw edema was observed due to their high content of
sterols and their corresponding glucosides, flavonoids and phenolic
compounds.
5- Anti-Malarial Activity:
The methanolic, n-hexane and chloroform extracts of Anisotes
trisulcus (Forssk.) Nees. dried aerial parts showed mild anti-malarial
activity against the tested Plasmodium falciparum (D6 clone) relative to
chloroquine.
While the ethyl acetae and the total methanolic extract of Blepharis
ciliaris (L.) B.L.Burtt. dried aerial parts showed mild to weak anti-malarial
activity against the tested P.falciparum (D6 clone) relative to chloroquine.
General Summary and Conclusion
288
6- Anti-Hyperglycaemic Activity:
It was observed that the total methanolic extract of the aerial parts
of Anisotes trisulcus (Forssk.) Nees. and its n-hexane and ethyl acetate
extracts when given concurrently with the glucose load at a dose of 400
mg/Kg a significant hypoglycaemic effect was obtained after 1 hr and this
continued for 2 hrs while the chloroform and the n-butanol extracts
showed the least activity.
The obtained results support the use of powdered Anisotes trisulcus
(Forssk.) Nees. as an anti-diabetic in folk medicine.
Concerning the methanolic extract of the dried aerial parts of
Blepharis ciliaris (L.) B.L.Burtt. and its fractions when given concurrently
with glucose load showed a non-significant anti-hyperglycaemic activity.
288
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١
ﺍﻟﻤﻠﺨﺹ ﺍﻟﻌـﺭﺒﻰ
ﺩﺭﺍﺴﺔ ﻓﻴﺘﻭﻜﻴﻤﻴﺎﺌﻴﺔ ﻭﺒﻴﻭﻟﻭﺠﻴﻪ ﻟﺒﻌﺽ ﻨﺒﺎﺘﺎﺕ
ﺍﻟﻌﺎﺌﻠﻪ ﺍﻷﻜﺎﻨﺜـﺎﺜـﻴﺔ
ﺘﻌﺘﺒﺭ ﺍﻟﻌﺎﺌﻠﺔ ﺍﻻﻜﺎﻨﺜﺎﺜﻴﺔ ﻤﻥ ﺍﻟﻌﺎﺌﻼﺕ ﺍﻟﻐﻨﻴﻪ ﺒﺎﻟﻨﺒﺎﺘﺎﺕ ﺍﻟﻁﺒﻴﻪ ﻭﻫﻰ ﺘﻀﻡ ٣٤٦ﺠﻨﺱ
ﻭﺤﻭﺍﻟﻰ ٤٣٠٠ﻨﻭﻉ .ﻤﻥ ﺍﻻﺠﻨﺎﺱ ﺍﻟﻭﺍﺴﻌﺔ ﺍﻻﻨﺘﺸﺎﺭ ﺍﻟﺒﺎﺭﻟﻴﺭﻴﺎ ) ٢٣٠ﻨﻭﻉ( ،ﺍﻟﺒﻠﻴﻔﺎﺭﺱ
) ١٢٩ﻨﻭﻉ( ،ﺍﻷﻜﺎﻨﺜﺱ ) ٥٠ﻨﻭﻉ( ،ﺍﻷﻨﻴﺯﻭﺘﺱ ) ٢٣ﻨﻭﻉ( ،ﺍﻷﻨﺩﻭﺠﺭﺍﻓﺱ ) ٢٠ﻨﻭﻉ(
ﻭﺍﻷﺩﻫﺎﺘﻭﺩﺍ ) ٢٠ﻨﻭﻉ(.
ﺍﻟﻨﺒﺎﺘﺎﺕ ﺍﻟﺘﻰ ﺘﺨﻀﻊ ﻟﻠﺩﺭﺍﺴﻪ ﻫﻤﺎ ﺍﻷﻨﻴﺯﻭﺘﺱ ﺘﺭﺍﻴﺴﺎﻟﻜﺱ )ﻓﻭﺭﺴﻙ( ﻨﻴﺱ .
ﻭﺍﻟﺒﻠﻴﻔﺎﺭﺱ ﺴﻴﻠﻴﺎﺭﻴﺱ )ل (.ﺏ .ل .ﺒﻭﺭﺕ .ﺍﻟﺘﺎﺒﻌﺎﻥ ﻟﻠﻌﺎﺌﻠﻪ ﺍﻷﻜﺎﻨﺜﺎﺜﻴﺔ .
ﻴﻌﺘﺒﺭ ﺍﻷﻨﻴﺯﻭﺘﺱ ﺘﺭﺍﻴﺴﺎﻟﻜﺱ )ﻓﻭﺭﺴﻙ( ﻨﻴﺱ .ﺸﺠﻴﺭﺓ ﻗﺎﺌﻤﺔ ﻴﺘﺭﺍﻭﺡ ﺍﺭﺘﻔﺎﻋﻬﺎ ﻤﺎ
ﺒﻴﻥ ٣. ٥- ١ﻤﺘﺭ .ﻭﻴﻨﺘﺸﺭ ﻫﺫﺍ ﺍﻟﻨﺒﺎﺕ ﻓﻰ ﺍﻟﻴﻤﻥ ﻜﻤﺎ ﻴﻨﻤﻭ ﺸﻴﻁﺎﻨﻴﺎ ﻓﻰ ﻤﻨﻁﻘﺔ ﺍﻟﺠﺒﺎل ﺍﻟﺠﻨﻭﺒﻴﺔ
ﺒﺎﻟﻤﻤﻠﻜﺔ ﺍﻟﻌﺭﺒﻴﺔ ﺍﻟﺴﻌﻭﺩﻴﺔ ﻭﺨﺼﻭﺼﺎ ﻓﻰ ﻤﻨﻁﻘﺔ ﻭﺍﺩﻯ ﺠﺒل ﺃﺒﻭ ﺍﻟﺤﺴﻥ ﺒﻴﻥ ﺃﺒﻬﺎ ﻭﻨﺠﺭﺍﻥ .
ﺍﻟﺴﺎﻕ ﺍﻟﺭﺌﻴﺴﻴﺔ ﻟﻠﻨﺒﺎﺕ ﺃﺴﻁﻭﺍﻨﻴﺔ ﺼﺎﺩﻗﺔ ﺍﻟﻤﺤﻭﺭ ﻭﺘﻔﺭﻋﺎﺘﻬﺎ ﻋﺩﻴﺩﺓ ﻭﺍﻟﻌﻘﺩ ﻁﻭﻴﻠﺔ ﻨﺴﺒﻴﺎ .
ﺍﻟﺴﻴﻘﺎﻥ ﺍﻟﺤﺩﻴﺜﺔ ﺨﻀﺭﺍﺀ ﺍﻟﻰ ﺃﺭﺠﻭﺍﻨﻴﻪ ﺍﻟﻠﻭﻥ ﺘﺘﺤﻭل ﺘﺩﺭﻴﺠﻴﺎ ﻟﺘﺼﺒﺢ ﺫﺍﺕ ﺴﻁﺢ ﺨﺸﻥ ﻤﻐﻁﻰ
ﺒﻘﻠﻑ ﺒﻨﻰ ﺍﻟﻠﻭﻥ .ﺘﺤﻤل ﺍﻟﺴﻴﻘﺎﻥ ﺍﻷﻭﺭﺍﻕ ﺒﻜﺜﺎﻓﻪ ﻋﻨﺩ ﺍﻟﻌﻘﺩ ﻭﺘﻜﻭﻥ ﺍﻷﻭﺭﺍﻕ ﻓﻰ ﺃﺯﻭﺍﺝ
ﻤﺘﺼﺎﻟﺒﻪ .ﻭﻫﻰ ﺃﻭﺭﺍﻕ ﺒﺴﻴﻁﻪ ،ﻤﻌﻨﻘﻪ ﻭﻋﺩﻴﻤﺔ ﺍﻷﺫﻴﻨﺎﺕ .ﺍﻷﺯﻫﺎﺭ ﺘﻨﻤﻭ ﻓﺭﺍﺩﻯ ﻭﺘﻜﻭﻥ ﻤﻌﻨﻘﻪ
ﻭﺘﻨﻤﻭ ﻓﻰ ﺜﻨﺎﺌﻴﺎﺕ ﺍﺒﻁﻴﻪ .ﺍﻷﺴﺩﻴﻪ ﺜﻨﺎﺌﻴﻪ ﺍﻟﻤﺘﻭﻙ ﻭﻟﻜل ﻤﺘﻙ ﻏﺭﻓﺘﺎﻥ ﺘﻨﻤﻭﻜل ﻭﺍﺤﺩﻩ ﻤﻨﻬﻤﺎ
ﻓﻭﻕ ﺍﻷﺨﺭﻯ .ﺍﻟﺜﻤﺭﺓ ﻋﻠﺒﻪ ﺘﺤﺘﻭﻯ ﻋﻠﻰ ﺃﺭﺒﻊ ﺒﺫﻭﺭ ،ﺍﻟﺒﺫﺭﻩ ﺨﺸﻨﺔ .
ﻴﻌﺘﺒﺭ ﺍﻟﺒﻠﻴﻔﺎﺭﺱ ﺴﻴﻠﻴﺎﺭﻴﺱ )ل (.ﺏ .ل .ﺒﻭﺭﺕ .ﻋﺸﺏ ﻤﻌﻤﺭ ﺸﺒﻪ ﻗﺎﺌﻡ ﻏﺯﻴﺭ ﺍﻷﺸﻭﺍﻙ
ﻴﻐﻁﻴﻪ ﺯﻏﺏ ﺭﻤﺎﺩﻯ ﺘﻘل ﻜﺜﺎﻓﺘﻪ ﺘﺩﺭﻴﺠﻴﺎ ﻤﻊ ﺍﻟﻭﻗﺕ .ﻭﺍﻟﻨﺒﺎﺕ ﻋﺩﻴﺩ ﺍﻟﺘﻔﺭﻉ ﻋﻨﺩ ﺍﻟﻘﺎﻋﺩﻩ ﻭﻴﺼل
ﺇﺭﺘﻔﺎﻉ ﺍﻷﻓﺭﻉ ﺍﻟﻰ ٣٠ﺴﻡ .ﻭﻴﻨﺘﺸﺭ ﻫﺫﺍ ﺍﻟﻨﻭﻉ ﻤﻥ ﺍﻟﻨﺒﺎﺘﺎﺕ ﻓﻰ ﺃﻓﺭﻴﻘﻴﺎ ﺍﻹﺴﺘﻭﺍﺌﻴﺔ ﻭ ﺍﻟﺴﻌﻭﺩﻴﺔ
ﻭﻤﺼﺭ .ﺘﻨﻤﻭ ﺍﻷﻭﺭﺍﻕ ﻓﻰ ﻤﺤﻴﻁﺎﺕ ﻭﻴﺤﺘﻭﻯ ﻜل ﻤﺤﻴﻁ ﻋﻠﻰ ٤ﺃﺯﻭﺍﺝ .
ﻭﻫﻰ ﺃﻭﺭﺍﻕ ﻤﺴﺘﻁﻴﻠﺔ ﺇﻟﻰ ﺭﻤﺤﻴﻪ ﺍﻟﺸﻜل ﺫﺍﺕ ﺃﻋﻨﺎﻕ ﻁﻭﻴﻠﻪ ﻭﺍﻟﻨﺼل ﺠﻠﺩﻯ ﻟﻪ ﺒﺭﻴﻕ
ﻓﻀﻰ .ﺘﻭﺠﺩ ﺍﻟﻨﻭﺭﺍﺕ ﻤﺘﻜﺎﺜﻔﻪ ﻋﻠﻰ ﺸﻜل ﺴﻨﺎﺒل ﻤﺨﺭﻭﻁﻴﻪ ﻭﺘﺤﻤل ﺃﺯﻫﺎﺭ ﺯﺭﻗﺎﺀ -ﺃﺭﺠﻭﺍﻨﻴﺔ
ﺍﻟﻠﻭﻥ .ﺍﻟﻘﻨﻴﺒﺎﺕ ﺸﺭﻴﻁﻴﺔ -ﻀﻴﻘﺔ ﺯﻏﺒﻴﺔ .ﺍﻟﺜﻤﺭﺓ ﻋﻠﺒﻪ .
٢
ﺍﻟﻤﻠﺨﺹ ﺍﻟﻌﺭﺒﻰ
ﻴﺴﺘﺨﺩﻡ ﺍﻷﻨﻴﺯﻭﺘﺱ ﺘﺭﺍﻴﺴﺎﻟﻜﺱ )ﻓﻭﺭﺴﻙ( ﻨﻴﺱ .ﻓﻰ ﺍﻟﻁﺏ ﺍﻟﺸﻌﺒﻰ ﻓﻰ ﺸﺒﻪ ﺍﻟﺠﺯﻴﺭﺓ
ﺍﻟﻌﺭﺒﻴﺔ ﺤﻴﺙ ﻴﺴﺘﺨﺩﻡ ﻟﻌﻼﺝ ﺠﻤﻴﻊ ﺍﻷﻤﺭﺍﺽ ﺍﻟﻜﺒﺩﻴﺔ ﻭﻤﻨﻬﺎ ﺍﻹﻟﺘﻬﺎﺏ ﺍﻟﻜﺒﺩﻯ ﻭﺍﻟﺼﻔﺭﺍ
ﻭﺍﻟﺤﺼﻭﺍﺕ ﺍﻟﻤﺭﺍﺭﻴﻪ ﻭﺒﻌﺽ ﺍﻷﻤﺭﺍﺽ ﺍﻷﺨﺭﻯ .ﻭﻴﺴﺘﺨﺩﻡ ﺃﻴﻀﺎ ﻓﻰ ﻋﻼﺝ ﺍﻟﺴﻜﺭ ﻭﻜﻤﻭﺴﻊ
ﻟﻠﺸﻌﺏ ﺍﻟﻬﻭﺍﺌﻴﺔ ﻭﺨﺎﻓﺽ ﻟﻠﻀﻐﻁ ﻭﻜﻤﺨﺩﺭ ﻤﻭﻀﻌﻰ .ﻜﻤﺎ ﺘﺴﺘﺨﺩﻡ ﺨﻼﺼﺔ ﺍﻟﻨﺒﺎﺕ ﻓﻰ ﺃﺸﻜﺎل
ﺼﻴﺩﻻﻨﻴﺔ ﻤﺨﺘﻠﻔﺔ ﻟﻴﺤﺩ ﻤﻥ ﺍﺴﺘﻬﻼﻙ ﺍﻟﻨﻴﻜﻭﺘﻴﻥ ﻭﻟﺘﺜﺒﻴﻁ ﺍﻟﺸﻬﻴﺔ .
ﺘﺴﺘﺨﺩﻡ ﺒﺫﻭﺭ ﺍﻟﺒﻠﻴﻔﺎﺭﺱ ﺴﻴﻠﻴﺎﺭﻴﺱ )ل (.ﺏ .ل .ﺒﻭﺭﺕ ) .ﻤﻁﺤﻭﻨﺔ ﺃﻭ ﻤﺤﻤﺼﺔ( ﻓﻰ
ﻋﻼﺝ ﺍﻟﻘﺭﺡ ﻭ ﺍﻟﺠﺭﻭﺡ ﻭﺍﻟﺤﺭﻭﻕ ﺍﻟﺠﻠﺩﻴﺔ ﻜﻤﻀﺎﺩ ﻟﻠﺒﻜﺘﻴﺭﻴﺎ .ﻜﻤﺎ ﺘﻌﺘﺒﺭ ﺍﻟﺒﺫﻭﺭ ﻤﺩﺭﻩ
ﻟﻠﺒﻭل،ﻭﻜﻤﻘﻭﻯ ﻋﺎﻡ ﻭﻁﺎﺭﺩﻩ ﻟﻠﺒﻠﻐﻡ .ﻜﻤﺎ ﻴﺴﺘﺨﺩﻡ ﺍﻟﻔﺤﻡ ﻤﻥ ﺍﻟﺠﺫﻭﺭ ﻟﺘﺤﺴﻴﻥ ﺍﻟﻨﻅﺭ ﻭﺒﻨﺎﺀﺍ ﻋﻠﻰ
ﺫﻟﻙ ﺴﻤﻰ ﻫﺫﺍ ﺍﻟﻨﺒﺎﺕ ﺒﻜﺤل ﺍﻟﻌﺠﻭﺯ .
ﺃﺩﺕ ﺍﻟﺩﺭﺍﺴﺎﺕ ﺍﻟﻔﻴﺘﻭﻜﻴﻤﻴﺎﺌﻴﺔ ﻭﺍﻟﺒﻴﻭﻟﻭﺠﻴﺔ ﻟﻠﻨﺒﺎﺘﺎﺕ ﺍﻟﺘﺎﺒﻌﻪ ﻟﻠﻌﺎﺌﻠﻪ ﺍﻷﻜﺎﻨﺜﺎﺴﻴﺔ
ﻭﺨﺼﻭﺼﺎ ﺍﻷﻨﻴﺯﻭﺘﺱ ﺘﺭﺍﻴﺴﺎﻟﻜﺱ )ﻓﻭﺭﺴﻙ( ﻨﻴﺱ .ﺇﻟﻰ ﻓﺼل ﻭﺍﻟﺘﻌﺭﻑ ﻋﻠﻰ ﻜﺜﻴﺭ ﻤﻥ ﺍﻟﻤﻭﺍﺩ
ﺍﻟﻔﻌﺎﻟﺔ ﺍﻟﺜﺎﻨﻭﻴﺔ ﻭﺍﻟﺘﺄﺜﻴﺭﺍﺕ ﺍﻟﺒﻴﻭﻟﻭﺠﻴﻪ ﺍﻟﻤﺨﺘﻠﻔﺔ ،ﻭﻟﺫﻟﻙ ﺃﺼﺒﺢ ﻤﻥ ﺍﻟﻀﺭﻭﺭﻯ ﺇﺠﺭﺍﺀ ﻫﺫﻩ
ﺍﻟﺩﺭﺍﺴﻪ ﻟﻤﻌﺭﻓﺔ ﺍﻟﺨﻭﺍﺹ ﺍﻟﻔﻴﺘﻭﻜﻴﻤﻴﺎﺌﻴﺔ ﻭﺍﻟﺒﻴﻭﻟﻭﺠﻴﺔ ﻟﻬﺫﻴﻥ ﺍﻟﻨﺒﺎﺘﻴﻥ .
ﻭﺘﻨﻘﺴﻡ ﺘﻠﻙ ﺍﻟﺩﺭﺍﺴﺔ ﺇﻟﻰ ﺜﻼﺜﺔ ﺃﺠﺯﺍﺀ ﺃﺴﺎﺴﻴﺔ ﻋﻠﻰ ﺍﻟﻨﺤﻭ ﺍﻟﺘﺎﻟﻰ :
ﺍﻟﺠﺯﺀ ﺍﻷﻭل :ﺩﺭﺍﺴﺔ ﻓﻴﺘﻭﻜﻴﻤﻴﺎﺌﻴﺔ ﻟﻸﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﺔ ﻟﻨﺒﺎﺕ ﺍﻷﻨﻴﺯﻭﺘﺱ ﺘﺭﺍﻴﺴﺎﻟﻜﺱ
)ﻓﻭﺭﺴﻙ( ﻨﻴﺱ .
ﺍﻟﻔﺼل ﺍﻷﻭل :ﺍﻟﺘﻌﺭﻑ ﺍﻟﻤﺒﺩﺌﻰ ﻋﻠﻰ ﺍﻟﻤﻭﺍﺩ ﺍﻟﻔﻌﺎﻟﺔ ﻟﻸﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﺔ ﻟﻨﺒﺎﺕ ﺍﻷﻨﻴﺯﻭﺘﺱ
ﺘﺭﺍﻴﺴﺎﻟﻜﺱ )ﻓﻭﺭﺴﻙ( ﻨﻴﺱ .
ﺍﻟﻔﺼل ﺍﻟﺜﺎﻨﻰ :ﺇﺴﺘﺨﻼﺹ ﻭﺘﺠﺯﺌﺔ ﻭﻓﺼل ﺍﻟﻤﻭﺍﺩ ﺍﻟﻔﻌﺎﻟﺔ ﻤﻥ ﺍﻷﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﻪ ﻟﻨﺒﺎﺕ
ﺍﻷﻨﻴﺯﻭﺘﺱ ﺘﺭﺍﻴﺴﺎﻟﻜﺱ )ﻓﻭﺭﺴﻙ( ﻨﻴﺱ .
ﺍﻟﻔﺼل ﺍﻟﺜﺎﻟﺙ :ﺍﻟﺘﻌﺭﻑ ﻋﻠﻰ ﺍﻟﻤﺭﻜﺒﺎﺕ ﺍﻟﻤﻔﺼﻭﻟﺔ ﻤﻥ ﺍﻷﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﺔ ﻟﻨﺒﺎﺕ ﺍﻷﻨﻴﺯﻭﺘﺱ
ﺘﺭﺍﻴﺴﺎﻟﻜﺱ )ﻓﻭﺭﺴﻙ( ﻨﻴﺱ .
ﺍﻟﺠﺯﺀ
ﺍﻟﺜﺎﻨﻰ :ﺩﺭﺍﺴﺔ ﻓﻴﺘﻭﻜﻴﻤﻴﺎﺌﻴﺔ ﻟﻸﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﺔ ﻟﻨﺒﺎﺕ ﺍﻟﺒﻠﻴﻔﺎﺭﺱ ﺴﻴﻠﻴﺎﺭﻴﺱ )ل (.
ﺏ .ل .ﺒﻭﺭﺕ ..
ﺍﻟﻔﺼل ﺍﻷﻭل :ﺍﻟﺘﻌﺭﻑ ﺍﻟﻤﺒﺩﺌﻰ ﻋﻠﻰ ﺍﻟﻤﻭﺍﺩ ﺍﻟﻔﻌﺎﻟﺔ ﻟﻸﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﺔ ﻟﻨﺒﺎﺕ ﺍﻟﺒﻠﻴﻔﺎﺭﺱ
ﺴﻴﻠﻴﺎﺭﻴﺱ )ل (.ﺏ .ل .ﺒﻭﺭﺕ .
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ﺍﻟﻤﻠﺨﺹ ﺍﻟﻌﺭﺒﻰ
ﺍﻟﻔﺼل ﺍﻟﺜﺎﻨﻰ :ﺇﺴﺘﺨﻼﺹ ﻭﺘﺠﺯﺌﺔ ﻭﻓﺼل ﺍﻟﻤﻭﺍﺩ ﺍﻟﻔﻌﺎﻟﺔ ﻤﻥ ﺍﻷﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﻪ ﻟﻨﺒﺎﺕ
ﺍﻟﺒﻠﻴﻔﺎﺭﺱ ﺴﻴﻠﻴﺎﺭﻴﺱ )ل (.ﺏ .ل .ﺒﻭﺭﺕ .
ﺍﻟﻔﺼل ﺍﻟﺜﺎﻟﺙ :ﺍﻟﺘﻌﺭﻑ ﻋﻠﻰ ﺍﻟﻤﺭﻜﺒﺎﺕ ﺍﻟﻤﻔﺼﻭﻟﺔ ﻤﻥ ﺍﻷﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﺔ ﻟﻨﺒﺎﺕ ﺍﻟﺒﻠﻴﻔﺎﺭﺱ
ﺴﻴﻠﻴﺎﺭﻴﺱ )ل (.ﺏ .ل .ﺒﻭﺭﺕ .
ﺍﻟﺠﺯﺀ
ﺍﻟﺜﺎﻟﺙ :ﺩﺭﺍﺴﺔ ﺒﻴﻭﻟﻭﺠﻴﺔ ﻟﻸﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﺔ ﻟﻨﺒﺎﺕ ﺍﻷﻨﻴﺯﻭﺘﺱ ﺘﺭﺍﻴﺴﺎﻟﻜﺱ )ﻓﻭﺭﺴﻙ(
ﻨﻴﺱ .ﻭﺍﻟﺒﻠﻴﻔﺎﺭﺱ ﺴﻴﻠﻴﺎﺭﻴﺱ )ل (.ﺏ .ل .ﺒﻭﺭﺕ .ﻭﻫﻰ ﻜﺎﻟﺘﺎﻟﻰ :
- ١ﺍﻟﻨﺸﺎﻁ ﻜﻤﻀﺎﺩ ﻟﻸﻜﺴﺩﻩ
- ٢ﺩﺭﺍﺴﺔ ﺍﻟﺴﻤﻴﺔ ﻟﻠﻨﺒﺎﺘﻴﻥ
- ٣ﺍﻟﻤﻌﺎﻴﺭﺓ ﻹﺭﺒﻴﺎﻥ ﺍﻟﻤﺎﺀ ﺍﻟﻤﺎﻟﺢ
- ٤ﺍﻟﻨﺸﺎﻁ ﺍﻟﻤﻀﺎﺩ ﻟﻺﻟﺘﻬﺎﺏ
- ٥ﺍﻟﻨﺸﺎﻁ ﺍﻟﻤﻀﺎﺩ ﻟﻠﻤﻼﺭﻴﺎ
- ٦ﺍﻟﻨﺸﺎﻁ ﺍﻟﻤﻀﺎﺩ ﻹﺭﺘﻔﺎﻉ ﺍﻟﺴﻜﺭﺒﺎﻟﺩﻡ
ﺩﺭﺍﺳﺔ ﻓﻴﺘﻮﻛﻴﻤﻴﺎﺋﻴﺔ ﻟﻸﺟﺰﺍﺀ ﺍﻟﻌﻠﻮﻳﺔ ﻟﻨﺒﺎﺕ
ﺍﻷﻧﻴﺰﻭﺗﺲ ﺗﺮﺍﻳﺴﺎﻟﻜﺲ )ﻓﻮﺭﺳﻚ ( ﻧﻴﺲ.
ﺍﻟﺘﻌﺭﻑ ﺍﻟﻤﺒﺩﺌﻰ ﻋﻠﻰ ﺍﻟﻤﻭﺍﺩ ﺍﻟﻔﻌﺎﻟﺔ ﻟﻸﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﺔ ﻟﻨﺒﺎﺕ
ﺍﻷﻨﻴﺯﻭﺘﺱ ﺘﺭﺍﻴﺴﺎﻟﻜﺱ )ﻓﻭﺭﺴﻙ( ﻨﻴﺱ .
ﺍﻷﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﺔ ﺍﻟﺠﺎﻓﺔ ﻟﻨﺒﺎﺕ ﺍﻷﻨﻴﺯﻭﺘﺱ ﺘﺭﺍﻴﺴﺎﻟﻜﺱ )ﻓﻭﺭﺴﻙ( ﻨﻴﺱ .ﺘﺤﺘﻭﻯ ﻋﻠﻰ
ﻤﻭﺍﺩ ﻁﻴﺎﺭﺓ ،ﻨﺸﻭﻴﺎﺕ ﻭ /ﺃﻭ ﺠﻠﻴﻜﻭﺯﻴﺩﺍﺕ ،ﺴﺘﻴﺭﻭﻻﺕ ﻏﻴﺭ ﻤﺸﺒﻌﺔ ﻭ /ﺃﻭ ﺘﺭﺒﻴﻨﺎﺕ ﺜﻼﺜﻴﺔ ،ﻭ
ﻓﻼﻓﻭﻨﻴﺩﺍﺕ ،ﻻﻜﺘﻭﻨﺎﺕ ﻭ /ﺃﻭ ﺍﺴﺘﺭﺍﺕ ﺒﺎﻻﻀﺎﻓﺔ ﺍﻟﻰ ﺃﻟﻜﺎﻟﻭﻴﺩﺍﺕ ﻭ /ﺃﻭ ﻤﻭﺍﺩ ﻗﻠﻭﻴﺔ ﻨﻴﺘﺭﻭﺠﻴﻨﻴﺔ .
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ﺍﻟﻤﻠﺨﺹ ﺍﻟﻌﺭﺒﻰ
ﺇﺴﺘﺨﻼﺹ ﻭﺘﺠﺯﺌﺔ ﻭﻓﺼل ﺍﻟﻤﻭﺍﺩ ﺍﻟﻔﻌﺎﻟﺔ ﻤﻥ ﺍﻷﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﻪ
ﻟﻨﺒﺎﺕ ﺍﻷﻨﻴﺯﻭﺘﺱ ﺘﺭﺍﻴﺴﺎﻟﻜﺱ )ﻓﻭﺭﺴﻙ( ﻨﻴﺱ .
ﺘﻡ ﺇﺴﺘﺨﻼﺹ ﺍﻷﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﺔ ﺍﻟﺠﺎﻓﺔ ﺍﻟﻤﻁﺤﻭﻨﺔ ﺤﻭﺍﻟﻰ ٢. ٥ﻜﺠﻡ ﻤﻥ ﻨﺒﺎﺕ
ﺍﻷﻨﻴﺯﻭﺘﺱ ﺘﺭﺍﻴﺴﺎﻟﻜﺱ )ﻓﻭﺭﺴﻙ( ﻨﻴﺱ .ﺒﻭﺍﺴﻁﺔ ﺍﻟﻤﻴﺜﺎﻨﻭل ﺜﻡ ﺭﻜﺯﺕ ﺤﺘﻰ ﺃﻋﻁﺕ ﻤﺭﻜﺯﺍ
ﺨﺎﻟﻴﺎ ﺘﻤﺎﻤﺎ ﻤﻥ ﺍﻟﻤﺫﻴﺏ ﻭﺯﻨﻪ ٣٠٠ﺠﻡ ﻭﺘﻡ ﺘﺠﺯﺌﺘﻪ ﺘﻌﺎﻗﺒﻴﺎ ﺒﻭﺍﺴﻁﺔ ﺍﻟﻬﻜﺴﺎﻥ ﺜﻡ ﺍﻟﻜﻠﻭﺭﻭﻓﻭﺭﻡ
ﺜﻡ ﺨﻼﺕ ﺍﻹﻴﺜﻴل ﻭ ﺃﺨﻴﺭﺍ ﺍﻟﻜﺤﻭل ﺍﻟﺒﻴﻭﺘﻴﻠﻰ .ﺜﻡ ﺭﻜﺯﺕ ﻜل ﺨﻼﺼﺔ ﻋﻠﻰ ﺤﺩﻩ ﻭﺘﻡ ﺍﻟﺤﺼﻭل
ﻋﻠﻰ ٤٥ﺠﻡ ﻤﻥ ﻤﺴﺘﺨﻠﺹ ﺍﻟﻬﻜﺴﺎﻥ ﻭ ٣٥ﺠﻡ ﻤﻥ ﻤﺴﺘﺨﻠﺹ ﺍﻟﻜﻠﻭﺭﻭﻓﻭﺭﻡ ﻭ ١٨ﺠﻡ ﻤﻥ
ﻤﺴﺘﺨﻠﺹ ﺨﻼﺕ ﺍﻹﻴﺜﻴل ﻭ ٨ﺠﻡ ﻤﻥ ﻤﺴﺘﺨﻠﺹ ﺍﻟﻜﺤﻭل ﺍﻟﺒﻴﻭﺘﻴﻠﻰ ،ﻭ ١٥٠ﺠﻡ ﻤﻥ ﺍﻟﺨﻼﺼﺔ
ﺍﻟﻤﺎﺌﻴﻪ .
ﻭﻗﺩ ﺘﻡ ﻓﺼل ﻤﺤﺘﻭﻴﺎﺕ ﻜل ﺨﻼﺼﺔ ﻋﻠﻰ ﺤﺩﻩ ﺒﺎﺴﺘﺨﺩﺍﻡ ﻜل ﺍﻟﻭﺴﺎﺌل ﺍﻟﻜﺭﻭﻤﺎﺘﻭﺠﺭﺍﻓﻴﺔ
ﺍﻟﻤﺨﺘﻠﻔﻪ ﺤﻴﺙ ﺘﻡ ﻓﺼل ٢٠ﻤﺭﻜﺏ ﻤﻨﻬﺎ .
ﺍﻟﺘﻌﺭﻑ ﻋﻠﻰ ﺍﻟﻤﺭﻜﺒﺎﺕ ﺍﻟﻤﻔﺼﻭﻟﺔ ﻤﻥ ﺍﻷﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﺔ ﻟﻨﺒﺎﺕ
ﺍﻷﻨﻴﺯﻭﺘﺱ ﺘﺭﺍﻴﺴﺎﻟﻜﺱ )ﻓﻭﺭﺴﻙ( ﻨﻴﺱ .
ﺗﻢ اﻟﺘﻌﺮف ﻋﻠﻰ اﻟﺘﺮﻛﯿﺐ اﻟﺪﻗﯿﻖ ﻟﻠﻤﺮﻛﺒﺎت اﻟﻤﻔﺼﻮﻟﺔ ﺑﺈﺳﺘﺨﺪام ﻃﺮق اﻟﺘﺤﻠﯿﻞ اﻟﻄﯿﻔﯿﺔ
اﻟﻤﺨﺘﻠﻔﮫ ودراﺳﺔ ﺧﻮاﺻﮭﺎ اﻟﻄﺒﯿﻌﯿﺔ واﻟﻜﯿﻤﯿﺎﺋﯿﺔ ﺑﺎﻹﺿﺎﻓﺔ إﻟﻰ ﻣﻘﺎرﻧﺔ ﺗﻠﻚ اﻟﻨﺘﺎﺋﺞ ﺑﺎﻟﻤﺮاﺟﻊ اﻟﻤﺨﺘﻠﻔﺔ
أو ﺑﻤﺜﯿﻼﺗﮭﺎ ﻣﻦ اﻟﻌﯿﻨﺎت اﻟﻘﯿﺎﺳﯿﺔ وھﻰ ﻋﻠﻰ اﻟﻨﺤﻮ اﻟﺘﺎﻟﻰ:
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ﺃﻟﻔﺎ -ﺃﻤﻴﺭﻴﻥ ﻴﻔﺼل ﻷﻭل ﻤﺭﺓ ﻤﻥ ﺍﻟﻨﺒﺎﺕ
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ﺒﻴﺘﺎ -ﺴﺎﻴﺘﻭﺴﺘﻴﺭﻭل ﻴﻔﺼل ﻷﻭل ﻤﺭﺓ ﻤﻥ ﺍﻟﻨﺒﺎﺕ
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ﺴﺘﻴﺠﻤﺎﺴﺘﻴﺭﻭل ﻴﻔﺼل ﻷﻭل ﻤﺭﺓ ﻤﻥ ﺍﻟﻨﺒﺎﺕ
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)- ‘٢ﻫﻴﺩﺭﻭﻜﺴﻰ ﺘﻴﺘﺭﺍﻜﻭﺯﺍﻨﻭﻴل -ﺃﻤﻴﻨﻭ( -ﺃﻭﻜﺘﺎﺩﻴﻜﺎﻥ - ١،٣،٤-ﺜﻼﺜﻰ ﺍﻟﻜﺤﻭل ﻴﻔﺼل
ﻷﻭل ﻤﺭﺓ ﻤﻥ ﺍﻟﻌﺎﺌﻠﺔ ﺍﻷﻜﺎﻨﺜﺎﺜﻴﺔ
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- ‘٣،٧،٨،٣ﺭﺒﺎﻋﻰ ﻫﻴﺩﺭﻭﻜﺴﻰ - ٥-ﻤﻴﺜﻭﻜﺴﻰ ﻓﻼﻓﻭﻥ ﻤﺭﻜﺏ ﺠﺩﻴﺩ
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- ‘٤،‘٣،٦،٧،٣ﺨﻤﺎﺴﻰ ﻫﻴﺩﺭﻭﻜﺴﻰ - ٥-ﻤﻴﺜﻭﻜﺴﻰ ﻓﻼﻓﻭﻥ ﻴﻔﺼل ﻷﻭل ﻤﺭﺓ ﻤﻥ
ﺍﻟﻌﺎﺌﻠﺔ ﺍﻷﻜﺎﻨﺜﺎﺜﻴﺔ
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ﻓﺎﺯﻴﺴﻴﻥ )ﺒﻴﺠﺎﻨﻴﻥ( ﺴﺒﻕ ﻓﺼﻠﻪ ﻤﻥ ﺍﻟﻨﺒﺎﺕ
٥
ﺍﻟﻤﻠﺨﺹ ﺍﻟﻌﺭﺒﻰ
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ﻓﺎﺯﻴﺴﻴﻨﻭﻥ ﺴﺒﻕ ﻓﺼﻠﻪ ﻤﻥ ﺍﻟﻨﺒﺎﺕ
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ﺃﻨﻴﺯﻭﺘﻴﻥ ﺴﺒﻕ ﻓﺼﻠﻪ ﻤﻥ ﺍﻟﻨﺒﺎﺕ
- ٥ - ١٠ﻫﻴﺭﻭﻜﺴﻰ ﻓﺎﺯﻨﺘﻴﻥ ﻤﺭﻜﺏ ﺠﺩﻴﺩ
- ١١ﺤﻤﺽ ﻓﻴﺭﺍﺘﺭﻙ ﻴﻔﺼل ﻷﻭل ﻤﺭﺓ ﻤﻥ ﺍﻟﻌﺎﺌﻠﺔ ﺍﻷﻜﺎﻨﺜﺎﺜﻴﺔ
- ٣ - ١٢ﻤﻴﺜﻭﻜﺴﻰ - ٤-ﻫﻴﺩﺭﻭﻜﺴﻰ ﺤﻤﺽ ﺍﻟﺒﻨﺯﻭﻴﻙ )ﺤﻤﺽ ﺍﻟﻔﺎﻨﻴﻠﻴﻙ( ﻴﻔﺼل ﻷﻭل ﻤﺭﺓ
ﻤﻥ ﺠﻨﺱ ﺍﻷﻨﻴﺯﻭﺘﺱ
- ١٣ﺒﻴﺘﺎ -ﺴﺎﻴﺘﻭﺴﺘﻴﺭﻭل - ٣-ﺒﻴﺘﺎ -ﺠﻠﻭﻜﻭﺒﻴﺭﺍﻨﻭﺯﻴﺩ ﻴﻔﺼل ﻷﻭل ﻤﺭﺓ ﻤﻥ ﺠﻨﺱ ﺍﻷﻨﻴﺯﻭﺘﺱ
- ٧ - ١٤ﻫﻴﺩﺭﻭﻜﺴﻰ ﻓﺎﺯﻴﺴﻴﻥ ﻴﻔﺼل ﻷﻭل ﻤﺭﺓ ﻤﻥ ﺠﻨﺱ ﺍﻷﻨﻴﺯﻭﺘﺱ
- ٧ - ١٥ﻫﻴﺩﺭﻭﻜﺴﻰ ﻓﺎﺯﻴﺴﻴﻨﻭﻥ ﻴﻔﺼل ﻷﻭل ﻤﺭﺓ ﻤﻥ ﺠﻨﺱ ﺍﻷﻨﻴﺯﻭﺘﺱ
- ٨ - ١٦ﺃﻤﻴﻨﻭ - ٧،٨،٩،١١-ﺭﺒﺎﻋﻰ ﻫﻴﺩﺭﻭ - ٦-ﻫـ -ﺒﻴﺭﻴﺩﻭ ]- ٢،١ﺏ [ ﻜﻴﻨﺎﺯﻭﻟﻴﻥ - ٢،٦-
ﺩﻴﻭل ﻤﺭﻜﺏ ﺠﺩﻴﺩ
- ٨ - ١٧ﺃﻤﻴﻨﻭ - ٣،٦-ﺜﻨﺎﺌﻰ ﻫﻴﺩﺭﻭﻜﺴﻰ - ٧،٨،٩-ﺜﻼﺜﻰ ﻫﻴﺩﺭﻭ - ٦-ﻫـ -ﺒﻴﺭﻴﺩﻭ ] - ٢،١
ﺏ[ ﻜﻴﻨﺎﺯﻭﻟﻴﻥ - ١١-ﺃﻭﻥ ﻤﺭﻜﺏ ﺠﺩﻴﺩ
- ١٨ﺜﻨﺎﺌﻰ ﻤﻴﺜﻴل -ﻥ -ﻫﻴﺩﺭﻭﻜﺴﻰ ﻤﻴﺜﻴل -ﻥ -ﻥ -ﺜﻨﺎﺌﻰ ﻤﻴﺜﻴل ﻤﻴﺜﺄﻤﻴﻨﻴﻡ ﻜﻠﻭﺭﺍﻴﺩ ﻤﺭﻜﺏ ﺠﺩﻴﺩ
- ١٩ﻥ -ﻜﺎﺭﺒﻭﻜﺴﻰ ﺃﻤﻴﻨﻭ -ﻤﻴﺜﻴل -ﻥ -ﻥ -ﺜﻨﺎﺌﻰ ﻤﻴﺜﻴل ﺍﻴﺜﺎﻥ ﺃﻤﻴﻨﻴﻭﻡ ﻜﻠﻭﺭﺍﻴﺩ ﻤﺭﻜﺏ
ﺠﺩﻴﺩ
- ٢٠ﻤﺨﻠﻭﻁ ﻤﻥ ﻥ -ﻜﺎﺭﺒﻭﻜﺴﻰ ﺃﻤﻴﻨﻭ -ﻤﻴﺜﻴل -ﻥ -ﻥ -ﺜﻨﺎﺌﻰ ﻤﻴﺜﻴل ﺍﻴﺜﺎﻥ ﺃﻤﻴﻨﻴﻭﻡ ﻜﻠﻭﺭﺍﻴﺩ
ﻭﻗﺎﻋﺩﺓ ﺍﻟﻜﻭﻟﻴﻥ .
ﺩﺭﺍﺳﺔ ﻓﻴﺘﻮﻛﻴﻤﻴﺎﺋﻴﺔ ﻟﻸﺟﺰﺍﺀ ﺍﻟﻌﻠﻮﻳﺔ ﻟﻨﺒﺎﺕ
ﺍﻟﺒﻠﻴﻔﺎﺭﺱ ﺳﻴﻠﻴﺎﺭﻳﺲ )ﻝ (.ﺏ.ﻝ.ﺑﻮﺭﺕ..
ﺍﻟﺘﻌﺭﻑ ﺍﻟﻤﺒﺩﺌﻰ ﻋﻠﻰ ﺍﻟﻤﻭﺍﺩ ﺍﻟﻔﻌﺎﻟﺔ ﻟﻸﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﺔ ﻟﻨﺒﺎﺕ
ﺍﻟﺒﻠﻴﻔﺎﺭﺱ ﺴﻴﻠﻴﺎﺭﻴﺱ )ل (.ﺏ .ل .ﺒﻭﺭﺕ .
ﺍﻷﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﺔ ﺍﻟﺠﺎﻓﺔ ﻟﻨﺒﺎﺕ ﺍﻟﺒﻠﻴﻔﺎﺭﺱ ﺴﻴﻠﻴﺎﺭﻴﺱ )ل (.ﺏ .ل .ﺒﻭﺭﺕ .ﺘﺤﺘﻭﻯ ﻋﻠﻰ
ﻤﻭﺍﺩ ﻁﻴﺎﺭﺓ ،ﻨﺸﻭﻴﺎﺕ ﻭ /ﺃﻭ ﺠﻠﻴﻜﻭﺯﻴﺩﺍﺕ ،ﺴﺘﻴﺭﻭﻻﺕ ﻏﻴﺭ ﻤﺸﺒﻌﺔ ﻭ /ﺃﻭ ﺘﺭﺒﻴﻨﺎﺕ ﺜﻼﺜﻴﺔ،
ﺘﺎﻨﻴﻨﺎﺕ ،ﻓﻼﻓﻭﻨﻭﻴﺩﺍﺕ ﺒﺎﻻﻀﺎﻓﻪ ﺍﻟﻰ ﻻﻜﺘﻭﻨﺎﺕ ﻭ /ﺃﻭ ﺇﺴﺘﺭﺍﺕ .
٦
ﺍﻟﻤﻠﺨﺹ ﺍﻟﻌﺭﺒﻰ
ﺇﺴﺘﺨﻼﺹ ﻭﺘﺠﺯﺌﺔ ﻭﻓﺼل ﺍﻟﻤﻭﺍﺩ ﺍﻟﻔﻌﺎﻟﺔ ﻤﻥ ﺍﻷﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﻪ ﻟﻨﺒﺎﺕ
ﺍﻟﺒﻠﻴﻔﺎﺭﺱ ﺴﻴﻠﻴﺎﺭﻴﺱ )ل (.ﺏ .ل .ﺒﻭﺭﺕ .
ﺘﻡ ﺍﺴﺘﺨﻼﺹ ﺍﻷﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﺔ ﺍﻟﺠﺎﻓﺔ ﺍﻟﻤﻁﺤﻭﻨﺔ ﺤﻭﺍﻟﻰ ٣ﻜﺠﻡ ﻤﻥ ﻨﺒﺎﺕ ﺍﻷﻨﻴﺯﻭﺘﺱ
ﺘﺭﺍﻴﺴﺎﻟﻜﺱ )ﻓﻭﺭﺴﻙ( ﻨﻴﺱ .ﺒﻭﺍﺴﻁﺔ ﺍﻟﻤﻴﺜﺎﻨﻭل ﺜﻡ ﺭﻜﺯﺕ ﺤﺘﻰ ﺍﻋﻁﺕ ﻤﺭﻜﺯﺍ ﺨﺎﻟﻴﺎ ﺘﻤﺎﻤﺎ ﻤﻥ
ﺍﻟﻤﺫﻴﺏ ﻭﺯﻨﻪ ٢٧٠ﺠﻡ ﻭﺘﻡ ﺘﺠﺯﺌﺘﻪ ﺘﻌﺎﻗﺒﻴﺎ ﺒﻭﺍﺴﻁﺔ ﺍﻟﻬﻜﺴﺎﻥ ﺜﻡ ﺍﻟﻜﻠﻭﺭﻭﻓﻭﺭﻡ ﺜﻡ ﺨﻼﺕ
ﺍﻻﻴﺜﻴل ﻭ ﺍﺨﻴﺭﺍ ﺍﻟﻜﺤﻭل ﺍﻟﺒﻴﻭﺘﻴﻠﻰ .ﺜﻡ ﺭﻜﺯﺕ ﻜل ﺨﻼﺼﺔ ﻋﻠﻰ ﺤﺩﻩ ﻭﺘﻡ ﺍﻟﺤﺼﻭل ﻋﻠﻰ ٥٠
ﺠﻡ ﻤﻥ ﻤﺴﺘﺨﻠﺹ ﺍﻟﻬﻜﺴﺎﻥ ﻭ ٣٧ﺠﻡ ﻤﻥ ﻤﺴﺘﺨﻠﺹ ﺍﻟﻜﻠﻭﺭﻭﻓﻭﺭﻡ ﻭ ٤٥ﺠﻡ ﻤﻥ ﻤﺴﺘﺨﻠﺹ
ﺨﻼﺕ ﺍﻻﻴﺜﻴل ﻭ ١٢ﺠﻡ ﻤﻥ ﻤﺴﺘﺨﻠﺹ ﺍﻟﻜﺤﻭل ﺍﻟﺒﻴﻭﺘﻴﻠﻰ ،ﻭ ١١٠ﺠﻡ ﻤﻥ ﺍﻟﺨﻼﺼﺔ ﺍﻟﻤﺎﺌﻴﻪ .
ﻭﻗﺩ ﺘﻡ ﻓﺼل ﻤﺤﺘﻭﻴﺎﺕ ﻜل ﺨﻼﺼﺔ ﻋﻠﻰ ﺤﺩﻩ ﺒﺎﺴﺘﺨﺩﺍﻡ ﻜل ﺍﻟﻭﺴﺎﺌل ﺍﻟﻜﺭﻭﻤﺎﺘﻭﺠﺭﺍﻓﻴﺔ
ﺍﻟﻤﺨﺘﻠﻔﻪ ﺤﻴﺙ ﺘﻡ ﻓﺼل ١٦ﻤﺭﻜﺏ ﻤﻨﻬﺎ .
ﺍﻟﺘﻌﺭﻑ ﻋﻠﻰ ﺍﻟﻤﺭﻜﺒﺎﺕ ﺍﻟﻤﻔﺼﻭﻟﺔ ﻤﻥ ﺍﻷﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﺔ ﻟﻨﺒﺎﺕ
ﺍﻟﺒﻠﻴﻔﺎﺭﺱ ﺴﻴﻠﻴﺎﺭﻴﺱ )ل (.ﺏ .ل .ﺒﻭﺭﺕ .
ﺘﻡ ﺍﻟﺘﻌﺭﻑ ﻋﻠﻰ ﺍﻟﺘﺭﻜﻴﺏ ﺍﻟﺩﻗﻴﻕ ﻟﻠﻤﺭﻜﺒﺎﺕ ﺍﻟﻤﻔﺼﻭﻟﺔ ﺒﺎﺴﺘﺨﺩﺍﻡ ﻁﺭﻕ ﺍﻟﺘﺤﻠﻴل ﺍﻟﻁﻴﻔﻴﺔ
ﺍﻟﻤﺨﺘﻠﻔﻪ ﻭﺩﺭﺍﺴﺔ ﺨﻭﺍﺼﻬﺎ ﺍﻟﻁﺒﻴﻌﻴﺔ ﻭﺍﻟﻜﻴﻤﻴﺎﺌﻴﺔ ﺒﺎﻻﻀﺎﻓﺔ ﺍﻟﻰ ﻤﻘﺎﺭﻨﺔ ﺘﻠﻙ ﺍﻟﻨﺘﺎﺌﺞ ﺒﺎﻟﻤﺭﺍﺠﻊ
ﺍﻟﻤﺨﺘﻠﻔﺔ ﺃﻭ ﺒﻤﺜﻴﻼﺘﻬﺎ ﻤﻥ ﺍﻟﻌﻴﻨﺎﺕ ﺍﻟﻘﻴﺎﺴﻴﺔ ﻭﻫﻰ ﻋﻠﻰ ﺍﻟﻨﺤﻭ ﺍﻟﺘﺎﻟﻰ :
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ﺒﻴﺘﺎ -ﺴﺎﻴﺘﻭﺴﺘﻴﺭﻭل ﻴﻔﺼل ﻷﻭل ﻤﺭﺓ ﻤﻥ ﺍﻟﻨﺒﺎﺕ .
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ﺴﺘﻴﺠﻤﺎﺴﺘﻴﺭﻭل ﻴﻔﺼل ﻷﻭل ﻤﺭﺓ ﻤﻥ ﺍﻟﻨﺒﺎﺕ .
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ﺴﺘﻴﺠﻤﺎﺴﺘﻴﺭﻭل ﺘﻴﺘﺭﺍﻜﻭﺯﺍﻨﻭﺍﺕ ﻤﺭﻜﺏ ﺠﺩﻴﺩ .
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- ’٢ﻫﻴﺩﺭﻭﻜﺴﻰ ﺍﻴﻜﻭﺯﺍﻨﻭﻴل ﺍﻤﻴﻨﻭﺍﻭﻜﺘﺎﺩﻴﻜﺎﻥ - ١،٣،٤-ﺜﻼﺜﻰ ﺍﻟﻜﺤﻭل ﻴﻔﺼل ﻷﻭل
ﻤﺭﺓ ﻤﻥ ﺍﻟﻌﺎﺌﻠﺔ ﺍﻷﻜﻨﺜﺎﺜﻴﻪ .
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ﻓﻴﺭﺍﺘﺭﺍﺕ ﺍﻟﻤﻴﺜﻴل ﻴﻔﺼل ﻷﻭل ﻤﺭﺓ ﻤﻥ ﺍﻟﻌﺎﺌﻠﺔ ﺍﻷﻜﻨﺜﺎﺜﻴﻪ .
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ﺍﻟﻤﻠﺨﺹ ﺍﻟﻌﺭﺒﻰ
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ﻓﺎﻨﻴﻼﺕ ﺍﻟﻤﻴﺜﻴل ﻴﻔﺼل ﻷﻭل ﻤﺭﺓ ﻤﻥ ﺠﻨﺱ ﺍﻟﺒﻠﻴﻔﺎﺭﻴﺱ .
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ﺤﻤﺽ ﺍﻟﺒﺭﺘﻭﻜﺎﺘﺸﻭﻙ ﻴﻔﺼل ﻷﻭل ﻤﺭﺓ ﻤﻥ ﺠﻨﺱ ﺍﻟﺒﻠﻴﻔﺎﺭﻴﺱ .
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ﺃﺒﻴﺠﻨﻴﻥ ﻴﻔﺼل ﻷﻭل ﻤﺭﺓ ﻤﻥ ﺍﻟﻨﺒﺎﺕ .
-٩
ﺒﻴﺘﺎﺴﺎﻴﺘﻭﺴﺘﻴﺭﻭل - ٣-ﺃ -ﺠﻠﻭﻜﻭﺒﻴﺭﺍﻨﻭﺯﻴﺩ ﻴﻔﺼل ﻷﻭل ﻤﺭﺓ ﻤﻥ ﺍﻟﻨﺒﺎﺕ .
- ١٠ﺴﺘﻴﺠﻤﺎﺴﺘﻴﺭﻭل - ٣-ﺃ -ﺠﻠﻭﻜﻭﺒﻴﺭﺍﻨﻭﺯﻴﺩ ﻴﻔﺼل ﻷﻭل ﻤﺭﺓ ﻤﻥ ﺍﻟﻨﺒﺎﺕ .
- ١١ﺃﺒﻴﺠﻨﻴﻥ - ٧-ﺃ -ﺒﻴﺘﺎ -ﺠﻠﻭﻜﻭﺒﻴﺭﺍﻨﻭﺯﻴﺩ ﺴﺒﻕ ﻓﺼﻠﻪ ﻤﻥ ﺍﻟﻨﺒﺎﺕ .
- ١٢ﺃﺒﻴﺠﻨﻴﻥ - ٧-ﺃ - "٦) -ﺒﺎﺭﺍ -ﻜﻭﻤﺎﺭﻭﻴل( -ﺒﻴﺘﺎ -ﺠﻠﻭﻜﻭﺒﻴﺭﺍﻨﻭﺯﻴﺩ ﻴﻔﺼل ﻷﻭل ﻤﺭﺓ ﻤﻥ
ﺍﻟﻨﺒﺎﺕ .
- ١٣ﺠﻨﺴﺘﻴﻥ - ٧-ﺃ - "٦)-ﺃ -ﺒﺎﺭﺍ -ﻜﺎﻓﻴﻭﻴل( -ﺒﻴﺘﺎ -ﺠﻠﻭﻜﻭﺒﻴﺭﺍﻨﻭﺯﻴﺩ ﻤﺭﻜﺏ ﺠﺩﻴﺩ .
- ١٤ﻨﺎﺭﻨﺠﻨﻴﻥ - ٧-ﺃ - "٣)-ﺃﺴﻴﺘﺎﺕ - "٦-ﺒﺎﺭﺍ -ﻜﻭﻤﺎﺭﻭﻴل( -ﺒﻴﺘﺎ -ﺠﻠﻭﻜﻭﺒﻴﺭﺍﻨﻭﺯﻴﺩ
ﺴﺒﻕ
ﻓﺼﻠﻪ ﻤﻥ ﺍﻟﻨﺒﺎﺕ .
- ١٥ﻨﺎﺭﻨﺠﻨﻴﻥ - ٧-ﺃ - "٦)-ﺃ -ﺒﺎﺭﺍ -ﻜﺎﻓﻴﻭﻴل( -ﺒﻴﺘﺎ -ﺠﻠﻭﻜﻭﺒﻴﺭﺍﻨﻭﺯﻴﺩ ﻴﻔﺼل ﻷﻭل ﻤﺭﺓ ﻤﻥ
ﺍﻟﻨﺒﺎﺕ
- ١٦ﺃﻜﺘﻴﻭﺯﻴﺩ ﻴﻔﺼل ﻷﻭل ﻤﺭﺓ ﻤﻥ ﺍﻟﻌﺎﺌﻠﺔ ﺍﻷﻜﺎﻨﺜﺎﺜﻴﺔ .
ﺩﺭﺍﺳﺔ ﺑﻴﻮﻟﻮﺟﻴﺔ ﻟﻸﺟﺰﺍﺀ ﺍﻟﻌﻠﻮﻳﺔ ﻟﻨﺒﺎﺕ ﺍﻷﻧﻴﺰﻭﺗﺲ ﺗﺮﺍﻳﺴﺎﻟﻜﺲ
)ﻓﻮﺭﺳﻚ ( ﻧﻴﺲ .ﻭﺍﻟﺒﻠﻴﻔﺎﺭﺱ ﺳﻴﻠﻴﺎﺭﻳﺲ )ﻝ (.ﺏ.ﻝ.ﺑﻮﺭﺕ.
- ١ﺍﻟﻨﺸﺎﻁ ﻜﻤﻀﺎﺩ ﻟﻸﻜﺴﺩﻩ:
ﺍﻟﺨﻼﺼﺔ ﺍﻟﻜﻠﻴﺔ ﻟﻸﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﺔ ﺍﻟﺠﺎﻓﺔ ﻟﻨﺒﺎﺕ ﺍﻷﻨﻴﺯﻭﺘﺱ ﺘﺭﺍﻴﺴﺎﻟﻜﺱ )ﻓﻭﺭﺴﻙ(
ﻨﻴﺱ .ﻭﺨﻼﺼﺔ ﺨﻼﺕ ﺍﻻﻴﺜﻴل ﻜﺎﻥ ﻟﻬﻤﺎ ﺃﻋﻠﻰ ﻨﺸﺎﻁ ﻜﻤﻀﺎﺩﻴﻥ ﻟﻸﻜﺴﺩﻩ %٧٥ﻭ %٦٨
ﺒﺘﺭﻜﻴﺯ ١ﻤﺠﻡ /ﻤل ﺒﻴﻨﻤﺎ ﻨﺒﺎﺕ ﺍﻟﺒﻠﻴﻔﺎﺭﺱ ﺴﻴﻠﻴﺎﺭﻴﺱ )ل (.ﺏ .ل .ﺒﻭﺭﺕ .ﻜﺎﻥ ﻟﻪ ﻨﺸﺎﻁ ﻋﺎﻟﻰ
ﺃﻴﻀﺎ ﺒﻨﺴﺒﺔ % ٨٩ﻭ % ٨٦ﺒﺘﺭﻜﻴﺯ ١ﻤﺠﻡ /ﻤل ﻟﻨﻔﺱ ﺍﻟﺨﻼﺼﺎﺕ .ﻫﺫﻩ ﺍﻟﻨﺘﺎﺌﺞ ﻤﻥ ﺍﻟﻤﻤﻜﻥ
ﺃﻥ ﺘﻜﻭﻥ ﻨﺘﻴﺠﺔ ﻭﺠﻭﺩ ﻤﻭﺍﺩ ﻓﻴﻨﻭﻟﻴﺔ ﻤﺜل ﺍﻟﻔﻼﻓﻭﻨﻭﻴﺩﺍﺕ ﻭ ﺍﻟﻔﻴﻨﺎﻴل ﺒﺭﻭﺒﺎﻨﻭﻴﺩ ﻭ ﺍﻷﺤﻤﺎﺽ
ﺍﻟﻔﻴﻨﻭﻟﻴﺔ .
ﺍﻟﻤﻠﺨﺹ ﺍﻟﻌﺭﺒﻰ
٨
ﺒﻴﻨﻤﺎ ﺍﻟﻤﺭﻜﺒﺎﺕ ﺍﻟﻤﻔﺼﻭﻟﺔ ﻓﻰ ﺼﻭﺭﺓ ﻨﻅﻴﻔﺔ ﻤﺜل ﺍﻷﺒﻴﺠﻨﻴﻥ ﻭﺍﻷﺒﻴﺠﻨﻴﻥ - ٧-ﺃ -ﺒﻴﺘﺎ -
ﺠﻠﻭﻜﻭﺒﻴﺭﺍﻨﻭﺯﻴﺩ ﻭ ﺠﻨﺴﺘﻴﻥ - ٧-ﺃ - "٦)-ﺃ -ﻜﺎﻓﻴﻭﻴل( -ﺒﻴﺘﺎ -ﺠﻠﻭﻜﻭﺒﻴﺭﺍﻨﻭﺯﻴﺩ ﻭﻨﺎﺭﻨﺠﻨﻴﻥ - ٧-ﺃ -
)- "٦ﺃ -ﻜﺎﻓﻴﻭﻴل( -ﺒﻴﺘﺎ -ﺠﻠﻭﻜﻭﺒﻴﺭﺍﻨﻭﺯﻴﺩ ﺃﻋﻁﻭﺍ ﻨﺘﺎﺌﺞ ﻤﺘﻭﺴﻁﺔ ﻭﺍﻟﺫﻯ ﻴﺜﺒﺕ ﺃﻥ ﻨﺸﺎﻁ
ﺍﻟﻔﻼﻓﻭﻨﻭﻴﺩﺯ ﻜﻤﻀﺎﺩﺍﺕ ﻟﻸﻜﺴﺩﻩ ﻴﻌﺘﻤﺩ ﻋﻠﻰ ﺃﻤﺎﻜﻥ ﻤﺠﻤﻭﻋﺎﺕ ﺍﻟﻬﻴﺩﺭﻭﻜﺴﻴل .
- ٢ﺩﺭﺍﺴﺔ ﺍﻟﺴﻤﻴﺔ ﻟﻠﻨﺒﺎﺘﻴﻥ:
ﺨﻼﺼﺔ ﺍﻟﻤﻴﺜﺎﻨﻭل ﺍﻟﻜﻠﻴﺔ ﻟﻸﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﻪ ﺍﻟﺠﺎﻓﺔ ﻟﻨﺒﺎﺕ ﺍﻷﻨﻴﺯﻭﺘﺱ ﺘﺭﺍﻴﺴﺎﻟﻜﺱ
)ﻓﻭﺭﺴﻙ( ﻨﻴﺱ .ﻟﻡ ﺘﻅﻬﺭ ﺃﻯ ﺃﻋﺭﺍﺽ ﻟﻠﺴﻤﻴﺔ ﻭﺍﻟﻤﻭﺕ ﻋﻠﻰ ﺤﻴﻭﺍﻨﺎﺕ ﺍﻟﺘﺠﺎﺭﺏ ﺤﺘﻰ ﻭﺼﻠﺕ
ﺍﻟﺠﺭﻋﺔ ﺇﻟﻰ ﺘﺭﻜﻴﺯ ٥ﺠﻡ /ﻜﺠﻡ ﺒﻴﻨﻤﺎ ﺨﻼﺼﺔ ﻨﺒﺎﺕ ﺍﻟﺒﻠﻴﻔﺎﺭﺱ ﺴﻴﻠﻴﺎﺭﻴﺱ )ل (.ﺏ .ل .ﺒﻭﺭﺕ .
ﻟﻡ ﺘﻅﻬﺭ ﻫﺫﻩ ﺍﻷﻋﺭﺍﺽ ﺤﺘﻰ ﺘﺭﻜﻴﺯ ٤ﺠﻡ /ﻜﺠﻡ .
- ٣ﺍﻟﻤﻌﺎﻴﺭﺓ ﻹﺭﺒﻴﺎﻥ ﺍﻟﻤﺎﺀ ﺍﻟﻤﺎﻟﺢ:
ﺃﻅﻬﺭﺕ ﺍﻟﻨﺘﺎﺌﺞ ﺃﻥ ﺍﻟﺨﻼﺼﻪ ﺍﻟﻜﻠﻴﻪ ﻟﻸﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﺔ ﺍﻟﺠﺎﻓﻪ ﻟﻨﺒﺎﺕ ﺍﻷﻨﻴﺯﻭﺘﺱ
ﺘﺭﺍﻴﺴﺎﻟﻜﺱ )ﻓﻭﺭﺴﻙ( ﻨﻴﺱ .ﺒﺎﻹﻀﺎﻓﻪ ﺇﻟﻰ ﺨﻼﺼﺔ ﺍﻟﻜﻠﻭﺭﻭﻓﻭﺭﻡ ﻜﺎﻥ ﻟﻬﻤﺎ ﺘﺄﺜﻴﺭ ﻗﻭﻯ ﻜﻘﺎﺘل
ﻟﺼﻐﺎﺭ ﺍﻹﺭﺒﻴﺎﻥ ﺒﻨﺴﺒﺔ %٦١ﻭ %٦٠ﺨﻼل ٢٤ﺴﺎﻋﻪ ،ﺒﻴﻨﻤﺎ ﺃﻅﻬﺭﺕ ﺍﻟﺨﻼﺼﺔ ﺍﻟﻜﻠﻴﻪ
ﻭﺨﻼﺼﺔ ﺍﻟﻜﻠﻭﺭﻭﻓﻭﺭﻡ ﻭﺨﻼﺼﺔ ﺨﻼﺕ ﺍﻻﺴﻴﺘﺎﺕ ﺘﺄﺜﻴﺭ ﺒﻨﺴﺒﺔ %٧٠ﻭ %٩٦ﻭ %٦٨ﻋﻠﻰ
ﺍﻟﺘﻭﺍﻟﻰ ﺒﻌﺩ ٤٨ﺴﺎﻋﺔ .ﻭﻗﺩ ﻴﻜﻭﻥ ﺫﻟﻙ ﻹﺤﺘﻭﺍﺌﻬﻡ ﻋﻠﻰ ﻨﺴﺒﺔ ﻋﺎﻟﻴﺔ ﻤﻥ
ﺍﻷﻟﻜﺎﻟﻭﻴﺩﺯﻭﺍﻟﺴﺘﻴﺭﻭﻻﺕ ﻭ /ﺃﻭ ﺍﻟﺘﺭﺒﻴﻨﺎﺕ ﺍﻟﺜﻼﺜﻴﺔ .ﺒﻴﻨﻤﺎ ﺃﻅﻬﺭﺕ ﺨﻼﺼﺔ ﺍﻟﻬﻜﺴﺎﻥ ﻨﺸﺎﻁ
ﻤﺘﻭﺴﻁ .
ﺃﻅﻬﺭﺕ ﺍﻟﺨﻼﺼﺎﺕ ﺍﻟﻤﺨﺘﻠﻔﻪ ﻟﻸﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﺔ ﺍﻟﺠﺎﻓﺔ ﻟﻨﺒﺎﺕ ﺍﻟﺒﻠﻴﻔﺎﺭﺱ ﺴﻴﻠﻴﺎﺭﻴﺱ )ل (.
ﺏ .ل .ﺒﻭﺭﺕ .ﻨﺸﺎﻁ ﻀﻌﻴﻑ ﺇﻟﻰ ﻤﺘﻭﺴﻁ ﻜﻘﺎﺘل ﻟﺼﻐﺎﺭ ﺍﻹﺭﺒﻴﺎﻥ ﺨﻼل ٢٤ﺍﻟﻰ ٤٨ﺴﺎﻋﻪ
ﻋﻠﻰ ﺍﻟﺘﻭﺍﻟﻰ .
- ٤ﺍﻟﻨﺸﺎﻁ ﺍﻟﻤﻀﺎﺩ ﻟﻺﻟﺘﻬﺎﺏ:
ﺍﻟﻤﻼﺤﻅ ﻤﻥ ﺇﺨﺘﺒﺎﺭ ﻜل ﺍﻟﺨﻼﺼﺎﺕ ﻅﻬﻭﺭ ﻨﺸﺎﻁ ﻤﻀﺎﺩ ﻟﻺﻟﺘﻬﺎﺏ ﻋﻨﺩﻤﺎ ﺘﻡ ﻤﻌﺎﻟﺠﺔ
ﺍﻟﺤﻴﻭﺍﻨﺎﺕ ﺒﺎﻟﺨﻼﺼﺎﺕ ﺍﻟﻤﺨﺘﻠﻔﺔ ﻟﻸﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﺔ ﻟﻠﻨﺒﺎﺘﻴﻥ }ﺍﻷﻨﻴﺯﻭﺘﺱ ﺘﺭﺍﻴﺴﺎﻟﻜﺱ )ﻓﻭﺭﺴﻙ(
ﻨﻴﺱ .ﻭﺍﻟﺒﻠﻴﻔﺎﺭﺱ ﺴﻴﻠﻴﺎﺭﻴﺱ )ل (.ﺏ .ل .ﺒﻭﺭﺕ {.ﺤﻴﺙ ﺃﻅﻬﺭﺕ ﺍﻟﺨﻼﺼﺔ ﺍﻟﻜﻠﻴﺔ ﻟﻠﻨﺒﺎﺘﻴﻥ
ﺍﻟﻤﻠﺨﺹ ﺍﻟﻌﺭﺒﻰ
٩
ﻭﺨﻼﺼﺘﻰ ﺍﻟﻬﻜﺴﺎﻥ ﻭﺨﻼﺕ ﺍﻷﺴﻴﺘﺎﺕ ﻨﺸﺎﻁﺎ ﻤﻠﺤﻭﻅﺎ ﻭﺫﻟﻙ ﻹﺤﺘﻭﺍﺌﻬﻡ ﻋﻠﻰ ﻨﺴﺒﺔ ﻋﺎﻟﻴﺔ ﻤﻥ
ﺍﻟﺴﺘﻴﺭﻭﻻﺕ ﻭﺠﻠﻴﻜﻭﺯﻴﺩﺍﺘﻬﺎ ﻭ ﺍﻟﻔﻼﻓﻭﻨﻴﺩﺍﺕ ﺒﺎﻹﻀﺎﻓﻪ ﺍﻟﻰ ﺍﻷﺤﻤﺎﺽ ﺍﻟﻔﻴﻨﻭﻟﻴﺔ .
ﺍﻟﻤﻠﺨﺹ ﺍﻟﻌﺭﺒﻰ
١٠
- ٥ﺍﻟﻨﺸﺎﻁ ﺍﻟﻤﻀﺎﺩ ﻟﻠﻤﻼﺭﻴﺎ:
ﺍﻟﺨﻼﺼﺔ ﺍﻟﻜﻠﻴﺔ ﻭﺨﻼﺼﺎﺕ ﺍﻟﻬﻜﺴﺎﻥ ﻭﺍﻟﻜﻠﻭﺭﻭﻓﻭﺭﻡ ﻟﻸﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﺔ ﻟﻨﺒﺎﺕ
ﺍﻷﻨﻴﺯﻭﺘﺱ ﺘﺭﺍﻴﺴﺎﻟﻜﺱ )ﻓﻭﺭﺴﻙ( ﻨﻴﺱ .ﺃﻅﻬﺭﺕ ﻨﺸﺎﻁ ﻤﺘﻭﺴﻁ ﻤﻀﺎﺩ ﻟﻠﺒﻼﺯﻤﻭﺩﻴﻭﻡ ﻓﺎﻟﺴﻴﺒﺎﺭﻡ
ﺒﺎﻟﻨﺴﺒﻪ ﻟﻠﻜﻠﻭﺭﻭﻜﻴﻥ .ﺒﻴﻨﻤﺎ ﺃﻅﻬﺭﺕ ﺨﻼﺼﺎﺕ ﺍﻷﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﺔ ﻟﻨﺒﺎﺕ ﺍﻟﺒﻠﻴﻔﺎﺭﺱ ﺴﻴﻠﻴﺎﺭﻴﺱ
)ل (.ﺏ .ل .ﺒﻭﺭﺕ .ﺍﻟﻜﻠﻴﻪ ﻭﺨﻼﺕ ﺍﻷﺴﻴﺘﺎﺕ ﻨﺸﺎﻁ ﻤﺘﻭﺴﻁ ﺇﻟﻰ ﻀﻌﻴﻑ ﺒﺎﻟﻨﺴﺒﺔ ﺍﻟﻰ
ﺍﻟﻜﻠﻭﺭﻭﻜﻴﻥ .
- ٦ﺍﻟﻨﺸﺎﻁ ﺍﻟﻤﻀﺎﺩ ﻹﺭﺘﻔﺎﻉ ﺍﻟﺴﻜﺭ ﺒﺎﻟﺩﻡ:
ﺍﻟﻤﻼﺤﻅ ﺃﻥ ﺍﻟﺨﻼﺼﺔ ﺍﻟﻜﻠﻴﺔ ﻟﻸﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﺔ ﻟﻨﺒﺎﺕ ﺍﻷﻨﻴﺯﻭﺘﺱ ﺘﺭﺍﻴﺴﺎﻟﻜﺱ )ﻓﻭﺭﺴﻙ(
ﻨﻴﺱ .ﺒﺎﻹﻀﺎﻓﻪ ﺇﻟﻰ ﺨﻼﺼﺔ ﺍﻟﻬﻜﺴﺎﻥ ﻭ ﺨﻼﺕ ﺍﻷﺴﻴﺘﺎﺕ ﻋﻨﺩﻤﺎ ﺘﻡ ﺇﻋﻁﺎﺌﻬﻡ ﻟﺤﻴﻭﺍﻨﺎﺕ
ﺍﻟﺘﺠﺎﺭﺏ ﻤﺼﺤﻭﺒﺎ ﺒﺠﺭﻋﺔ ﻋﺎﻟﻴﺔ ﻤﻥ ﺍﻟﺠﻠﻭﻜﻭﺯ ﺒﺠﺭﻋﺔ ٤٠٠ﻤﺠﻡ /ﻜﺠﻡ ﺤﺩﺙ ﺇﻨﺨﻔﺎﺽ
ﻤﻠﺤﻭﻅ ﻓﻰ ﻤﺴﺘﻭﻯ ﺍﻟﺴﻜﺭ ﺒﺎﻟﺩﻡ ﺒﻌﺩ ﻤﺭﻭﺭ ﺴﺎﻋﻪ ﻭﺇﺴﺘﻤﺭ ﻫﺫﺍ ﺍﻟﺘﺄﺜﻴﺭ ﻟﻤﺩﺓ ﺴﺎﻋﺘﻴﻥ ﺒﻴﻨﻤﺎ
ﺨﻼﺼﺘﻰ ﺍﻟﻜﻠﻭﺭﻭﻓﻭﺭﻡ ﻭﺍﻟﻜﺤﻭل ﺍﻟﺒﻴﻭﺘﻴﻠﻰ ﺃﻅﻬﺭﻭﺍ ﺃﻗل ﻨﺸﺎﻁ .ﻫﺫﻩ ﺍﻟﻨﺘﻴﺠﻪ ﺘﺘﻤﺎﺸﻰ ﻤﻊ
ﺇﺴﺘﺨﺩﺍﻡ ﻨﺒﺎﺕ ﺍﻷﻨﻴﺯﻭﺘﺱ ﺘﺭﺍﻴﺴﺎﻟﻜﺱ )ﻓﻭﺭﺴﻙ( ﻨﻴﺱ .ﻓﻰ ﺍﻟﻁﺏ ﺍﻟﺸﻌﺒﻰ ﻜﻌﻼﺝ ﻹﺭﺘﻔﺎﻉ
ﻤﺴﺘﻭﻯ ﺍﻟﺴﻜﺭ ﻓﻰ ﺍﻟﺩﻡ .
ﺒﺎﻟﻨﺴﺒﻪ ﻟﻨﺒﺎﺕ ﺍﻟﺒﻠﻴﻔﺎﺭﺱ ﺴﻴﻠﻴﺎﺭﻴﺱ )ل (.ﺏ .ل .ﺒﻭﺭﺕ .ﻜﺎﻨﺕ ﺍﻟﺨﻼﺼﺔ ﺍﻟﻜﻠﻴﻪ
ﻟﻸﺠﺯﺍﺀ ﺍﻟﻌﻠﻭﻴﻪ ﻟﻠﻨﺒﺎﺕ ﺒﺎﻹﻀﺎﻓﻪ ﺇﻟﻰ ﺒﺎﻗﻰ ﺍﻟﺨﻼﺼﺎﺕ ﻟﻡ ﻴﻅﻬﺭﻭﺍ ﺃﻯ ﺘﺄﺜﻴﺭ ﻤﻠﺤﻭﻅ ﻋﻠﻰ
ﺇﻨﺨﻔﺎﺽ ﺍﻟﺴﻜﺭ ﻓﻰ ﺍﻟﺩﻡ .
ﺒﻜﺎﻟﻭﺭﻴﻭﺱ ﺍﻟﻌﻠﻭﻡ ﺍﻟﺼﻴﺩﻟﻴﺔ -ﺠﺎﻤﻌﺔ ﺃﺴﻴﻭﻁ
-ﺠﺎﻤﻌﺔ ﺃﺴﻴﻭﻁ
ﻤﺎﺠﺴﺘﻴﺭ ﻓﻲ ﺍﻟﻌﻠﻭﻡ ﺍﻟﺼﻴﺩﻟﻴﺔ )ﻋﻘﺎﻗﻴﺭ(
ﻜﺠﺯﺀ ﻤﻥ ﻤﺘﻁﻠﺒﺎﺕ ﺍﻟﺤﺼﻭل ﻋﻠﻲ ﺩﺭﺠﺔ ﺩﻜﺘﻭﺭﺍﻩ ﺍﻟﻔﻠﺴﻔﺔ ﻓﻲ ﺍﻟﻌﻠﻭﻡ ﺍﻟﺼﻴﺩﻟﻴﺔ )ﻋﻘﺎﻗﻴﺭ(
ﺃﺴﺘﺎﺫ ﺍﻟﻌﻘﺎﻗﻴﺭ
ﻜﻠﻴﺔ ﺍﻟﺼﻴﺩﻟﺔ -ﺠﺎﻤﻌﺔ ﺃﺴﻴﻭﻁ
ﺃﺴﺘﺎﺫ ﺍﻟﻌﻘﺎﻗﻴﺭ
ﻜﻠﻴﺔ ﺍﻟﺼﻴﺩﻟﺔ -ﺠﺎﻤﻌﺔ ﺃﺴﻴﻭﻁ
ﺃﺴﺘﺎﺫ ﺍﻟﻌﻘﺎﻗﻴﺭ ﺍﻟﻤﺴﺎﻋﺩ
ﻜﻠﻴﺔ ﺍﻟﺼﻴﺩﻟﺔ -ﺠﺎﻤﻌﺔ ﺃﺴﻴﻭﻁ
ﻗﺴﻡ ﺍﻟﻌﻘـﺎﻗﻴــــــﺭ
ﻜﻠﻴﺔ ﺍﻟﺼﻴﺩﻟﺔ -ﺠﺎﻤﻌﺔ ﺃﺴﻴﻭﻁ
ﺠﻤﻬﻭﺭﻴﺔ ﻤﺼﺭ ﺍﻟﻌﺭﺒﻴﺔ
)١٤٣٤ﻫـ ٢٠١٣ -ﻡ(
Hanaa Sayed
Marwa A . A . Fayed