In Pharmaceutical Sciences (Pharmacognosy)

A PHARMACOGNOSTICAL STUDY OF 

PHYLLANTHUS ATROPURPUREUS BOJ. HORT. MAURIT. 

FAMILY EUPHORBIACEAE CULTIVATED IN EGYPT 

By 

MAY AHMED MOHAMED EL-SAYED 

A Thesis Submitted in Partial Fulfillment of The Requirements for 

The Degree of Master 

In 

Pharmaceutical Sciences 

(Pharmacognosy) 

Department of Pharmacognosy 

Faculty of Pharmacy 

Zagazig University 

2009




By 


A Thesis Submitted in Partial Fulfillment of The Requirements for 

The Degree of Master 

In 

Pharmaceutical Sciences 

(Pharmacognosy) 

Department of Pharmacognosy 

Faculty of Pharmacy 

Zagazig University 

2009




Submitted By: 


Under The Supervision of: 

(B.Sc. in Pharmaceutical Sciences) 

PROF. DR. TAHA MOUSTAFA SARG 

Professor of Pharmacognosy, Zagazig University. 

PROF. DR. AFAF EL-SAYED ABDEL-GHANI 

Professor of Pharmacognosy, Zagazig University. 

DR. RAWIA ABDEL-HADY ZAYED 

Assistant Professor of Pharmacognosy, Zagazig 

University.

Approval Sheet 




BY 


(B.Sc. in Pharmaceutical Sciences) 

This thesis for M.Sc. degree has been 

Approved by: 

1- Prof. Dr. Taha Moustafa Sarg 

Professor of Pharmacognosy, Faculty of Pharmacy, Zagazig University. 

………………………………………………………………………….. 

2- Prof. Dr. Nabil Ahmed Abd El-Salam 

Professor of Pharmacognosy, Faculty of Pharmacy, Alexandria University. 

……………………………………………………………………………… 

3- Prof. Dr. Ehsan Mahmoud Abd El-Aziz 

Professor and Head of Pharmacognosy Department, Faculty of Pharmacy, 

Zagazig University. 

……………………………………………………………………………. 

Date of examination:7-11-2009

ﻢﻴﺣﺮﻟﺍ ﻦﲪﺮﻟﺍ ﺍ ﻢﺴﺑ 

ﻚﻧﺎﺤﺒﺳ ﺍﻮﻟﺎﻗ 

ﺎﻨـﺘﻤﻠﻋ ﺎﻣ ﻻﺇ ﺎﻨﻟ ﻢﻠﻋ ﻻ 

" ﻢﻴﻜﳊﺍ ﻢﻴﻠﻌﻟﺍ ﺖﻧﺃ ﻚﻧﺇ 

ﻡﻴﻅﻌﻟﺍ ﷲﺍ ﻕﺩﺼ 

" 

32 

ﺔﻴﻵﺍ 

- 

ﺓﺭﻘﺒﻟﺍ ﺓﺭﻭﺴ" 

"

This effort is dedicated to my 

Parents, 

Beloved husband& 

Sweet son, Ra'ed…….

CONTENTS 

List of Figures……………………………….………………….……….. 

List of Tables...…………………………….…………………………….. 

List of Schemes……………………………………….……….………… 

List of Abbreviations…………………………………………………….. 

Introduction………………………………….…………………………... 

Aim of the Present Work…………………………………….………….. 

Materials, Reagents and Apparatus……………………………………… 

PART І 

Macro- and Micromorphology of Phyllanthus atropurpureus 

Boj. Hort. Maurit. 

Chapter 1: Macromorphological study of Phyllanthus atropurpureus 

Boj. Hort . Maurit 

The Leaf…………………………………………….……….……….… 

The stem…………………………………………………………...…… 

The Inflorescence……..………………………………………………... 

The subterranean organs………………….……….…………………… 

Chapter 2: Micromorphological study of Phyllanthus atropurpureus 

Boj. Hort. Maurit 

The Leaf……………………………………………….……….………. 

The petiole……..……………………………………………………….. 

The Powdered Leaf…………………………………………….………. 

The Stem……………………………………………….……….…….... 

The Powdered Stem ……………………………………….……….….. 

The Flower……………………………………………………………… 

The Pedicel ………………………………………….……….………… 

The Calyx …………………………………………….……….……...... 

The Androecium ………………………………….……….…………… 

The Gynaecium …………………………………….………………….. 

The Powdered Flower……………………………………………........... 

The Root & the Rhizome………………………………………………. 

The powdered Root…………………………………………………….. 

I 

V 

VII 

VIII 

1 

39 

40 

45 

46 

46 

48 

55 

64 

68 

70 

79 

80 

80 

84 

89 

93 

98 

100 

107

PART Π 

Phytochemical Investigation Of Phyllanthus atropurpureus 


Chapter 1: Preliminary phytochemical investigation of Phyllanthus 

atropurpureus Boj. Hort. Maurit……………………………. 

Chapter 2: Successive extraction of the powdered Phyllanthus 

atropurpureus Boj. Hort. Maurit. with selective organic 

solvents and examination of the respective extracts………... 

Chapter 3: Extraction of the powdered Phyllanthus atropurpureus Boj. 

Hort. Maurit. and fractionation of the extract……………… 

Chapter 4: Investigation of light petroleum soluble fraction and 

analysis of the fatty acid constituents of Phyllanthus 

atropurpureus Boj. Hort. Maurit……………………………… 

Chapter 5: Isolation and characterization of three materials from light 

petroleum soluble fraction of Phyllanthus atropurpureus 

Boj. Hort. Maurit……………………………………..……… 

Chapter 6: Isolation and characterization of six materials from ethyl 

acetate soluble fraction of Phyllanthus atropurpureus Boj. 

Hort. Maurit…………………………………………………. 

PART ΠІ 

Biological Activities of Phyllanthus atropurpureus Boj. Hort. 

Maurit. 

І- Antihepatotoxic Activity…....………………………….……………. 

Π- Anticancer activity…..………………………….………………....... 

ІΠ - Antimicrobial Activity …..………………………….……............. 

Summary……………………….…………………………… 

References………………………….………………………… 

Arabic summary……………………………………………. 

108 

110 

113 

116 

122 

138 

195 

208 

213 

215 

219

ﷲ ﺭﻜﺸﻟﺍ ﻭ ﺩﻤﺤﻟﺍ 

ACKNOWLEDGEMENT 

First of all, my deepest gratitude to ALLAH, the source of all gifts and 

blessings. 

I would like to express my deep feeling of gratitude and sincere 

appreciation to Prof. Dr. Taha M. Sarg, Professor of Pharmacognosy, Faculty of 

Pharmacy, Zagazig University, for his kind supervision, valuable scientific 

guidance, directions, comments and revision. I am wholeheartedly appreciating 

his effort to complete this work. 

I am very grateful with sincere appreciation to Prof. Dr. Afaf El-Sayed 

Abdel-Ghani, Professor of Pharmacognosy, Zagazig University for her kind 

direct supervision, guidance, direction, comment, continuous help, direct 

encouragement and persistent help during practical work as well as continuous 

unlimited help during this work. 

I am greatly indebted and grateful with the deep thanks to Dr. Rawia 

Abdel-Hady Zayed, Assistant Professor of Pharmacognosy, Faculty of 

Pharmacy, Zagazig University for her kind supervision, directions and 

comments. 

I would like to express my deep feelings of gratitude to Prof. Dr. Ahmed 

Fahmy, Professor of Pharmacology, and Shimaa El-Shazly, Assistant Lecturer 

of Pharmacology, Faculty of Pharmacy, Zagazig University for carrying out the 

pharmacological screening. 

I would like to express my deep feelings of gratitude to Prof. Dr. Fathy 

Serry, Professor of Microbiology, and Ahmed Olwan, Assistant Lecturer of 

Microbiology, Faculty of Pharmacy, Zagazig University for carrying out the 

antimicrobial activity. 

My thanks also due to Dr. Dalia Hamdan, Lecturer of Pharmacognosy, 

Faculty of Pharmacy, Zagazig University for her kind help in carrying out the 

spectral analysis of some of the isolated compounds. 

I wish to express my cordial thanks to the staff members and my 

colleagues in Department of Pharmacognosy, Faculty of Pharmacy, Zagazig 

University, for their positive support, help and encouragement and to all who 

contributed by one way or another to the realization of this work. 

Finally, this work is dedicated to My Parents, My Precious Husband, My 

Son and My Family who helped and supported me throughout all the hard and 

exhausting times till this work comes out to light. 

MAY AHMED MOHAMED AHMED

Figure 

Fig. 1: 

Fig. 2: 

Fig. 3: 

Fig. 4: 

Fig. 5: 

Fig. 6: 

Fig. 7: 

Fig. 8: 

Fig. 9: 

Fig. 10: 

Fig. 11: 

Fig. 12: 

Fig. 13: 

Fig. 14: 

Fig. 15: 

Fig. 16: 

Fig. 17: 

Fig. 18: 

Fig. 19: 

Fig. 20: 

Fig. 21: 

Fig. 22: 

Fig. 23: 

Fig. 24: 

Fig. 25: 

LIST OF FIGURES 

I 


A Photograph of aerial part of Phyllanthus atropurpureus 

Boj. Hort. Maurit…………………………………………. 

A Photograph of female flowers of Phyllanthus 

atropurpureus Boj. Hort. Maurit…………………………. 

A Photograph of the root and rhizome of Phyllanthus 

atropurpureus Boj. Hort. Maurit…………………………… 

Sketch of Phyllanthus atropurpureus Boj. Hort Maurit…… 

Sketch of the flower……………………………………….. 

The leaf, diagrammatic and detailed transverse sections…. 

The epidermal cells and some elements of the leaf………. 

The petiole, diagrammatic and detailed transverse sections. 

The old stem, diagrammatic and detailed transeverse 

section………………………………………………………. 

The young stem……………………………………………. 

The Pedicel, diagrammatic and detailed transeverse section.. 

The calyx, diagrammatic and detailed transeverse section… 

The androecium……………………………………………. 

The gynoecium…………………………………………….. 

The old root………………………………………………… 

The root and rhizome……………………………………….. 

GLC of the fatty acids methyl esters of Phyllanthus 

atropurpureus Boj. Hort. Maurit……………………… 

GC spectrum of the unsaponifiable matter of Phyllanthus 

atropurpureus Boj. Hort. Maurit ……………………….. 

IR spectrum of material "1" of Phyllanthus atropurpureus 

Boj. Hort. Maurit ………………………………………..… 

Mass spectrum of material "1" of Phyllanthus 

atropurpureus Boj. Hort. Maurit …………………………... 

IR spectrum of material "2 "of Phyllanthus atropurpureus 

Boj. Hort. Maurit ………………………………………..…. 


atropurpureus Boj. Hort. Maurit ………………………..… 


Boj. Hort. Maurit ……………………………………..……. 



1 

H-NMR spectrum of material "3" of Phyllanthus 


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Figure 

Fig. 26: 

Fig. 27: 

Fig. 28: 

Fig. 29: 

Fig. 30: 

Fig. 31: 

Fig. 32: 

Fig. 33: 

Fig. 34: 

Fig. 35: 

Fig. 36: 

Fig. 37: 

Fig. 38: 

Fig. 39: 

Fig. 40: 

Fig. 41: 

Fig. 42: 

Fig. 43: 

Fig. 44: 

Fig. 45: 

Fig. 46: 

13 

C-NMR spectrum of material "3" of Phyllanthus 

atropurpureus Boj. Hort. Maurit………………………….. 


Boj. Hort. Maurit ………………………………………. 


atropurpureus Boj. Hort. Maurit 

…………………………………………. 

1 


atropurpureus Boj. Hort. Maurit ……………………….... 

13 


atropurpureus Boj. Hort. Maurit ……………………..….. 

DEPT 135 spectrum of material "4" of Phyllanthus 

atropurpureus Boj. Hort. Maurit ….................................. 


Boj. Hort. Maurit ……………………………………….… 

UV spectrum of material "5" of Phyllanthus atropurpureus 

Boj. Hort. Maurit ……………………………………..…. 


atropurpureus Boj. Hort. Maurit …………………........... 

1 


atropurpureus Boj. Hort. Maurit……………………..….. 


Boj. Hort. Maurit ………………………………………… 


Boj. Hort. Maurit ………………………………………… 

EI-MS spectrum of material "6" of Phyllanthus 

atropurpureus Boj. Hort. Maurit …………………….…… 

FAB-MS spectrum of material "6" of Phyllanthus 

atropurpureus Boj. Hort. Maurit …………………….….. 

1 


atropurpureus Boj. Hort. Maurit ………………………….. 

13 


atropurpureus Boj. Hort. Maurit ……………..……….… 

1 1 

H- H COSY spectrum of material "6" of Phyllanthus 

atropurpureus Boj. Hort. Maurit………………………… 

DEPT 135 spectrum of material "6" of Phyllanthus 

atropurpureus Boj. Hort. Maurit.…………………………. 

APT spectrum of material "6" of Phyllanthus 

atropurpureus Boj. Hort. Maurit………………………….. 

gHSQCAD spectrum of material "6" of Phyllanthus 


gHMBC spectrum of material "6" of Phyllanthus 

II 

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Figure 

Fig. 49: 

Fig. 50: 

Fig. 51: 

Fig. 52: 

Fig. 53: 

Fig. 54: 

Fig. 55: 

Fig. 56: 

Fig. 57: 

Fig. 58: 

Fig. 59: 

Fig. 60: 

Fig. 61 

Fig. 62: 

Fig. 63: 

Fig. 64: 

Fig. 65: 

Fig. 

66&67: 

Fig. 

68&69 

III 




Boj. Hort. Maurit………………………………………….. 


Boj. Hort. Maurit.…………………………………………. 


atropurpureus Boj. Hort. Maurit.………………………… 

1 




Boj. Hort. Maurit.…………… …………………………..... 


Boj. Hort. Maurit.………………………………………. 



1 




Boj. Hort. Maurit………………………………………….. 


Boj. Hort. Maurit.…………….…………………………… 

EI-MS spectrum of material "9" of Phyllanthus 


FAB-MS spectrum of material "9" of Phyllanthus 

atropurpureus Boj. Hort. Maurit………………….………. 

1 



13 


atropurpureus Boj. Hort. Maurit……………………..…… 

1 1 

H- H Cosy spectrum of material "9" of Phyllanthus 


gHSQCAD correlation spectrum of material "9" of 

Phyllanthus atropurpureus Boj. Hort. Maurit……………… 

gHMBCAD correlation spectrum of material "9" of 

Phyllanthus atropurpureus Boj. Hort. Maurit………..….. 

Effect of oral treatment with silymarin, ethanolic extract of 

aerial parts and total ethanolic extract of roots for 30 days 

on ALT and AST levels in adult male cirrhotic rats……… 

Effect of oral treatment with silymarin, total ethanolic 

extract of aerial parts and total ethanolic extract of roots for 

30 days on total protein and albumin levels in adult male 

Page 

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Figure 

Fig. 

70& 71 

Fig. 72 

cirrhotic rats………………………………………………. 

Effect of oral treatment with silymarin, total ethanolic 

extract of aerial parts and total ethanolic extract of roots for 

30 days on malondialdehyde and glutathione levels in adult 

male cirrhotic rats…………………………………………... 

Anti-tumor activity of Phyllanthus atropurpureus Boj. Hort. 

Maurit. against hepatic cell line ………………………….. 

IV 

Page 

204 

212

Table 

Table 1 

Table 2 

Table 3 

Table 4 

Table 5 

Table 6 

Table 7 

Table 8 

Table 9 

Table 10 

Table 11 

Table 12 

Table 13 

Table 14 

Table 15 

Table 16 

Table 17 

Table 18 

Table 19 

Table 20 

Table 21 

Table 22 

Table 23 

Table 24 

Table 25 

Table 26 

LIST OF TABLES 

V 


Some of the oil plants of family Euphorbiaceae.…….…… 

Some dyes present in family Euphorbiaceae ……….……. 

The sterols and terpenes isolated from genus Phyllanthus … 

Alkaloids of genus Phyllanthus…………………………... 

Lignans present in genus Phyllanthus……………………... 

Hydrolyzable (pyrogallol) tannins of genus Phyllanthus... 

Condensed tannins of genus Phyllanthus……………..…. 

Flavonoids of genus Phyllanthus……………..………….. 

Oxygenated compounds present in genus Phyllanthus…. 

Acids isolated from genus Phyllanthus………………..… 

Microscopical numerical values of the leaves of 

Phyllanthus atropurpureus Boj. Hort. Maurit……….……. 

Phytochemical Screening of Powdered Phyllanthus 


Physical, Chromatographic and Chemical Characters of 

Successive Extractives of Aerial Parts …………………… 

Physical, Chromatographic and Chemical Characters of 

Successive Extractives of Subterranean Parts…………….. 

The Results of Extraction and Fractionation of Air-Dried 

Powdered Plant Organs………………………………… 

TLC Investigation of Light Petroleum Soluble Fraction…. 

Results of GLC analysis of fatty acid methyl esters from 

the lipid fraction of Phyllanthus atropurpureus Boj. Hort. 

Maurit………………………………………………………. 

Results of GC analysis of the unsaponifiable matter of 

Phyllanthus atropurpureus Boj. Hort. Maurit……………. 

Column Chromatographic Fractionation of light petroleum 

soluble fraction……………………………………………. 

TLC Investigation of Ethyl Acetate Fractions using solvent 

system (9)…………………………………………………. 

TLC Investigation of Leaf Ethyl Acetate Fraction with 

different visualizing agents………………………………. 

Column Chromatography of Leaf Ethyl Acetate Fraction 

UV Spectral data of material "5" of Phyllanthus 

atropurpureus Boj. Hort. Maurit ………………………… 

1 H-NMR spectral data of material "5" of Phyllanthus 

atropurpureus Boj. Hort. Maurit ……………………….. 

1 13 

HNMR and CNMR Spectral Data of Material "6" of 

Phyllanthus atropurpureus Boj. Hort. Maurit ………….. 

1 

HNMR Spectral Data of Material "7" of Phyllanthus 

Page 

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21 

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31 

59 

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141 

151 

152 

158 

169


Table 

Table 27 

Table 28 

Table 29 

Table 30 

Table 31 

Table 32 

Table 33 

Table 34 

Table 35 

Table 36 


Comparison between robustaside A and material "7" data. 

TLC Investigation of stem and Root Ethyl Acetate Fraction 

with different visualizing agents…………………………. 

Column Chromatography of Ethyl Acetate Fraction of Root 

and Stem………………………………………………….. 


atropurpureus Boj. Hort. Maurit ………………………. 

1 

H-NMR spectral data of material "8" of Phyllanthus 

atropurpureus Boj. Hort. Maurit …………………………. 


atropurpureus Boj. Hort. Maurit ………………………... 

1 

H-NMR (CD3OD, 500 MHz) and 13 C-NMR (CD3OD, 125 

MHz) spectral data of material "9" of Phyllanthus 

atropurpureus Boj. Hort. Maurit ……………………….... 

Drugs and chemicals used in antihepatotoxic experiment… 

Effect of total extract of aerial parts and roots of 

Phyllanthus atropurpureus taken orally for 30 days on liver 

enzymes, plasma protein and antioxidant parameters in sub 

acute male cirrhotic rats………………………………… 

Antimicrobial activities of different fractions of P. 


VI 

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Scheme 

Scheme 1: 

Scheme 2: 

Scheme 3: 

Scheme 4: 

Scheme 5: 

Scheme 6: 

Scheme 7: 

Scheme 8: 

Scheme 9: 

LIST OF SCHEMES 

VII 

LIST OF SCHEME 

Extraction and Fractionation of Air-Dried Powdered 

Plant Organs of Phyllanthus atropurpureus Boj. Hort. 

Maurit…………………………………………………. 

Mass fragmentation pattern of ß-sitosterol…………..… 

Mass fragmentation pattern of material "3" of 

Phyllanthus atropurpureus Boj. Hort. Maurit……….. 




Phyllanthus atropurpureus Boj. Hort. Maurit………..… 









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194

LIST OF ABBREVIATIONS 

AIDS 

ALT 

ANOVA 

APT 

AST 

ATCC 

BCG 

CCl4 

CDCl3 

COSY 

DEPT 

DMEM 

DMSO 

DNA 

EDTA 

EI-MS 

ELISA 

EtOAc 

eV 

FAB-MS 

FBS 

GSH 

gHMBCAD 

gHSQCAD 

HepG2 

HETCOR 

Hz 

IC50 

I.P. 

IR 

MS 

m/z 

nm 

LIST OF ABBREVIATIONS 

:Acquired immunodeficiency syndrome 

:Alanine aminotransferase 

:Analysis of variance 

:Attached Proton Test 

:Aspartate aminotransferase 

:American Type Culture Collection 

:Bromocresol green 

:Carbon tetrachloride 

:Deuterated chloroform 

:Correlation Spectroscopy 

:Destortionless Enhancement by Polarization Transfere 

:Dulbeco’s Modified Eagle’s Medium 

:Dimethyl sulphoxide 

:Deoxyribonucleic acid 

:Ethylene diamine tetra-acetic acid 

:Electron Impact Mass Spectrum 

:Enzyme Linked Immuno-Sorbent Analysis 

:Ethyl acetate 

:Electron volt 

:Fast atomic bombardment Mass Spectrum 

:Fetal bovine serum 

:Reduced glutathione 

:Heteronuclear Multiple Bond Correlation Spectroscopy 

:Heteronuclear Single Quantum Correlation Spectroscopy 

:Hepatocellular carcinoma cell line 

:Heteronuclear Correlation Spectroscopy 

: Hertz 

:half maximal inhibitory concentration 

:Intraperitoneal 

:Infrared 

:Mass Spectrum 

:Mass-to-charge ratio 

:Nanometers or 10 -9 meters 

VIII

NMR 

r.p.m. 

RPMI-1640 

ROS 

S.E.M 

SRB 

TCA 

TMS 

Tris 

:Nuclear Magnetic Resonance 

:Rotation per minute 

:Roswell Park Memorial Institute medium 

:Reactive Oxygen Species 

:Standard Error of Mean 

: Sulphorhodamine-B 

: Trichloroacetic acid 

:Tetra methyl silane 

:Hydroxyl methyl amino methane 

IX 

LIST OF ABBREVIATIONS

Introduction 

- 1 - 

INTRODUCTION 

Family Euphorbiaceae (Spurge) represented all over the world by 

almost 326 genera and about 7750 species distributed mainly in both 

tropical and temperate regions except arctic regions. Over 60 genera 

and about 350 species have been reported from India (1) . Flora of 

Egypt comprises 7 genera and 52 species in this family (2) . 

Taxonomy 

Plants of family Euphorbiaceae are mostly trees or shrubs, rarely 

herbaceous or climber. Some are xerophytic or cactus-like or 

phylloclades; some are marshy; usually the plants contain milky sap, 

latex or acrid juice (1, 3-5) . 

The stems are herbaceous or woody; become cactus-like in 

several species of Euphorbia or phylloclades (4) . 

The leaves are usually simple, alternate, stipulate; often reduced 

or deciduous, sometimes palmetly lobed or deeply so. Leaves are 

rarely opposite or whorled . The stipules are present in the form of 

hairs, glands or thorns and the venation is pinnate or palmate (1, 6, 7) . 

The inflorescence is usually complex and varied from a raceme, 

a spike, a dichasium or even the flowers are solitary axillary but in 

majority of the cases, the inflorescence is a cyathium which appears 

like a single flower. Each cyathium contains terminally a single naked 

female flower, usually represented by a tricarpellary gynoecium. The 

female flower is surrounded by a cup-like involucre formed by 4 or 5 

connate sepaloid bracts; stamens in a scropioid manner; each stamen 

represented a naked male flower. On the rim of the cup-like involucres

- 2 - 

INTRODUCTION 

are present nectar-secreting glands, these glands are oval or crescent- 

shaped and often brightly coloured (1, 5) . 

The flowers are usually bracteate, bracteolate, actinomorphic, 

hypogynous, rarely perigynous and generally unisexual. The plant 

may be monoecious or dioecious (3, 6, 7) . 

Flower structure of Euphorbiaceae varies greatly from genus to 

genus. The most highly specialized type is that of Euphorbia, which 

has a single pistillate flower aggregated with a number of staminate 

flowers forming cyathium and enclosed in a cup-like structure with 

glands on its margins (3) . 

• The perianth is consisting of both calyx and corolla or only 

of corolla or sometimes both are absent; valvate or imbricate; calyx 

are composed of 3-6 free, sometimes united sepals; corolla of 0 to 6 

free or united petals but commonly 5 and usually none petals are 

present (1, 3, 6, 7) . 

• The androecium is of 1 to 100 or more but usually as many 

or twice as many as petals, free or monadelphous stamens forming a 

staminal column by the fusion of the filaments; anther bilocular, rarely 

3 to 4 locular; longitudinal or transversely dehiscent. The stamens 

have a jointed stalk, and the part below the joint is interpreted as a 

pedicel, with the filament above. Sometimes staminodes are present in 

pistillate flower. Pollen grains often tricolaporate or polyporate (1, 5, 7) . 

• The gynoecium is usually tricarpellary, rarely bicarpellary 

or pentacarpellary; syncarpus; superior, rarely semi-inferior; trilocular 

ovary with one or two anatropous ovule in each locule; of axial

- 3 - 

INTRODUCTION 

placentation. Styles are 3, each entire, bifid or bifurcating apically into 

2 feathery stigmas. A nectariferous disc is present at the base of the 

ovary. In staminate flowers; the gynoecium is sometimes present as a 

pistillode (1, 3, 6, 7) . 

The fruit is commonly capsule or schizocarpic, rarely 

indehiscent, berry or drupe (1, 5-7) . 

The seeds are sometimes arillate or showing a caruncle and 

usually with oily endosperm and straight to curved embryo (1, 6, 7) . 

General floral formula (1) : 

(A) Male flower: 

Br, ⊕, ♂, P3+3 or (3-5) or 0, A1-∞ or (3-∞ ) , G 0 or pistillode 

(B) Female flower: 

Br, ⊕, ♀, P 3+3 or (3-5) or (5), A0 or staminodes, G (3). 

The most fundamental division in Euphorbiaceae (8) is based on 

ovule number with a grouping of two biovulate subfamilies which 

lack latex and laticifers and three uniovulate ones. 

• Biovulate subfamilies: 

1. Phyllanthoideae. 

2. Oldfieldioideae. 

• Uniovulate subfamilies: 

1. Acalyphoideae. 

2. Crotonoideae. 

3. Euphorbioideae.

Genus Phyllanthus 

- 4 - 

INTRODUCTION 

Genus Phyllanthus is a widespread tropical genus and has been 

much employed in traditional medicine. It comprises 550 species. This 

genus name come from the Greek name Phyllan-thus which means 

leaf-flower; the flower of some species apparently born on the leaves 

(9, 10) . Plant of the genus Phyllanthus (10) are shrubs, trees or herbs. 

v The leaves are alternate and entire. It is arranged in opposite 

rows along the smaller branches as to give them the 

appearance of pinnate leaves. 

v The flowers are apetalous, in lateral panicles or solitary. 

Often pistillate and staminate flowers are together in axils of 

leaves. 

• The calyx: 

Imbricated and consisting of 4-6 sepals. 

• The androecium: 

Stamens are 3 or 4. 

• The gynoecium: 

Ovary consists of 3 to 4 locules with 2 ovules in each 

locule and each of the 3 styles usually bifid. 

v The fruit is usually berry or capsule.

- 5 - 

INTRODUCTION 

Taxonomical position of Phyllanthus atropurpureus Boj. 

Hort. Maurit. 

Taxonomically, Phyllanthus atropurpureus Boj. Hort. Maurit. is a 

species of Phyllanthus : 

Kingdom: Plant 

Subkingdom: Emprophyta-Siphonogama 

Phyllum: Angiospermeae 

Class: Dicotyledoneae 

Subclass: Archichlamydeae 

Order: Geraniales 

Family: Euphorbiaceae 

Subfamily: Phyllanthoideae 

Tribe: Phyllanthus 

Recent taxonomical study of subfamily Phyllanthoideae 

separates this subfamily giving it a new classification termed family 

Phyllanthaceae on the basis of molecular data and DNA sequence 

(internal transcribed spacer (ITS) regions of the nuclear ribosomal 

DNA and plastide matK gene sequences) (8, 11, 12) .

- 6 - 

INTRODUCTION 

Medicinal and Economical Importance of Family 

Euphorbiaceae (6) 

Family Euphorbiaceae has evidently provided many plants with 

economical and medicinal importance. 

The following are selected examples of pronounced values:- 

[A] Medicinally used plants: 

1. Jatropha gossypifolia leaves are used in eczema, while the roots 

are used in leprosy and snakebites (1) . 

2. Entire plant of Synadenium grantii is used as CNS stimulant (1) . 

3. Croton cascarilla and C. elateria bark are used as tonic (1) . 

4. Euphorbium, a drug obtained from latex of Euphorbia 

resinifera, is used as a purgative (1) . 

5. Twig of P. reticulates and P. muellerianus were used as diuretic 

in South Africa and Nigeria (1) . 

6. Phyllanthus emblica syn. Emblica officinalis (Amla) is used as 

diuretic, laxative, digestive, emetic, anti-inflammatory and 

antiscorbutic agent in treatment of scurvy as their tannin 

contents retards the oxidation of vit.C, in preparing shampoo, 

in making hair dyes, ophthalmic anti-inflammatory agent and 

in the treatment of colic and other abdominal illness due to the 

presence of tannins and vit.C (1, 13- 14) . 

7. P. niruri has positive antihepatotoxic properties (13) . 

8. P. acuminatus root has antitumor properties (13) . 

9. Mallotus philippensis (Kamela tree) fruits are used as cathartic, 

anthelmintic and oral contraceptive (1, 13) . 

10. Ricinus communis (Castor) oil is used as purgative (1) and 

antidote in food poisoning (1, 13) .

- 7 - 

INTRODUCTION 

11. P. urinaria and /or P. niruri are used as diuretic, expectorant, 

emmenagogue, and a remedy for colic and dysentery (14) . 

12. P. acidus is used as emetic, purgative, remedy for smallpox if 

accompanied by a cough (14) . 

13. P. elegans roots are used as antipyretic (14) . 

14. P. reticulates is used in treatment of bleeding gums, smallpox, 

syphilis, asthma, sore throat, dysentery (14) . 

15. Fruits of P. engleri are used as stomachic and in the treatment 

of constipation and cough (15) . 

16. Latex of many genera of Euphorbiaceae is used to treat 

toothache as it placed carefully in the hollow of carious teeth 

for relief (15) . 

Several Euphorbiaceae are poisonous, causing sickness or death 

if ingested, or dermatitis if juice contacts the skin (1) . 

[B] Economically important plants: 

(A) Source of Rubber: 

Euphorbiaceae trees are considered as the main source of latex, 

which is the main commercial source of rubber. 

Hevea brasiliensis and Manihot glaziovii are common and 

important species of latex yielding plants (1, 6) . 

(B) Ornamental plants: 

Euphorbia plucherrima ,Codiaeum variegatum and Phyllanthus 

(Otaheite gooseberry) is the famous ornamental plant (with red , pink 

or white floral leaves) (1, 5, 6) .

(C) Cassava or Manioc: 

- 8 - 

INTRODUCTION 

Tuberous root of Manihot esculents (cassava) are rich in starch 

(Arrowroot) and used for preparation of bread, biscuits and other 

foodstuff (1, 6) . 

(D) Oil plants (1, 6) : 

Table (1): Some of the oil plants of family Euphorbiaceae: 

Name of oil Source Uses of the oil 

Croton oil 

Castor oil 

Seeds of Croton tiglium 

(jamalghota) 

Seeds of Ricinus communis 

(Arandi) 

Jatropha oil Seeds of Jatropha curcas. 

Tung oil 

(E) Dyes (1) : 

Seeds of Aleurites fordii, A. 

moluccana and A. montana 

As powerful purgative 

1- As a vegetable oil. 

2- As purgative (13) . 

3- as lubricant. 

4- In paint, varnish and 

plastic industries. 

1- As purgative. 

2- In skin disease. 

3- In rheumatism. 

4- In manufacture of 

soaps , lubricants, 

candles,….etc 

1- In preparation of 

paints, varnishes, 

linoleum, India ink. 

2- In waterproofing the 

paper, wood,….etc 

Table (2): Some dyes present in plants of family Euphorbiaceae: 

Type of dye source uses 

Mallotus philippinensis 

Kamela dye 

fruits. 

For dyeing wool and silk. 

Blue dye Jatropha curcus bark For dyeing fishing nets. 

Purple dye 

Chrozophora tinctoria bark For dyeing in textile 

industry 

Red dye Kirganelia reticulate roots

(F) Timber plants (1) : 

- 9 - 

INTRODUCTION 

Timber used for packing cases, tea boxes, veneers, plywood, and 

match industry is from: Aporosa dioica, Bischofia javanica, Drypetes 

roxburghii, Gelonium multiflorum, Hemicyclia andamanica, 

Hemicyclia elata, Hura crepitans and Trewia nudiflora.

R= H; 

ß-Sitosterol 

Chemical Review on Genus Phyllanthus 

- 10 - 

INTRODUCTION 

Literature survey on genus Phyllanthus reveals the presence of 

sterols and/ or terpenes, flavonoids, lignans, alkaloids, polyphenolic 

compounds and tannins in addition to acids. 

These different classes can be presented as follows: 

1. Sterols and/or terpenes: 

The most common sterols and terpenes isolated from the genus 

Phyllanthus are represented in the following table: 

Table (3): The sterols and terpenes isolated from the genus 

Phyllanthus. 

Name of Compound Structure Species & ref. 

R= glu.; 

ß-Sitosterol-O- glucoside RO 

R= H; 

Stigmasterol 

R= glu.; 

Stigmasterol-O-glucoside 

Campesterol 

RO 

HO 

oxyphyllus (16) 

(17, 18) 

flexuosus 

muellerianus (19) . 

(20, 21) 

reticulatus 

watsonii (22) 

urinaria 

(23, 24) 

corcovadensis (25) 

(26, 27) 

sellowianus 

oligospermus (28) 

anisolobus (29) 

singmapattiana (30) 

debilis (31) 

niruri (32) 

flexuosus (18) 

reticulatus (21) 

urinaria (23) 

corcovadensis (25) 

(26, 27) 

sellowianus 


corcovodensis (25) 

sellowianus (27)

R= Me; 

Friedelin 

R= COOH; 

Polpunonic acid 

R= α- H; 

Epifriedelanol 

R= β- H; 

Friedelan-3ß-ol 

- 11 - 

INTRODUCTION 


1ß ,22ß-dihydroxyfriedelin 

R1= R2= H, R3= OH; 

22 ß-hydroxyfriedel-1-ene 

R1= OH, R2= Me, R3= H; 

11ß-hydroxy-D:A-friedoolean-1-en-3one 

21α-hydroxyfriedelan-3-one 

21α-hydroxyfriedel-4(23)-en-3-one 

26- nor-D:A-friedoolean-14-en-3ß-ol 

R1= R3= OH, R2= Me; 

Olean-12-en-3ß,15α diol 

R1=OH, R2=CH2OH, R3=H; 

Olean-12-en-3ß, 24-diol 

R1= R3= OH, R2=CH2OH; 

Olean-12-en-3ß, 15α, 24-triol 

O 

O 

O 

O 

O 

HO 

HO 

R 1 

OH 

R 1 

R 2 

R2 

R 

R3 

R 

OH 

R 3 

OH 

OH 



reticulates (20) 

watsonii (22) 

urinaria (23) 

singampattiana (30) 


watsonii (22) 

muellerianus (19) 


muellerianus (19) 



watsonii (22) 

flexuosus 

watsonii (22) 

(17, 18, 33)

- 12 - 

INTRODUCTION 


Oleana-9(11):12-diene-3ß-ol 

R= Me; 

Oleana-11:13(18)-diene-3ß-ol 

R= CH2OH; 

Oleana-11:13(18)-diene-3ß, 

24-diol 

R1= CH3, R2= H; 

α – amyrin 

R1= H, R2= CH3; 

ß - amyrin HO 

δ -amyrin acetate 

Phyllanthol 

Phyllanthone 

R=α- OH; 

Phyllanthosterol 

R=β- OH; 

Pyllanthostigmasterol 

Phyllanthosecosteryl ester 

O 

CO 

HO 

HO 

AcO 

HO 

R 

O 

OH 

(CH2)5 CH 

R 

(CH 2) 8 

R 1 

OH 

CH 

(CH 2) 7 

R 2 

CH 3 


flexuosus 

(19, 33) 


urinaria (24) 


acidus (34) 

polyanthus (35) 

acidus (34) 


sellowianus (36) 


fraternus (37) 

fraternus (37)

Fraternusterol 

- 13 - 

INTRODUCTION 


29-nor-3,4-seco-friedelan-4(23),20(30)diene-3-oic 

acid 

R1= H, R2= OH, R3= Me; 

Lupeol 

R1= H, R2= OAc, R3= Me; 

Lupeol acetate 

R1=H, R2= OH, R3=CH2OH; 

Lup-20(29)-en-3ß, 24-diol 

R1= R2= β OH, R3= Me; 

Lup-20(29)-ene-1ß, 3ß-diol 

R= ═O; 

Lupenone 

R= CH3(CH2)14COO; 

Lupenyl palmitate 

R= CH2OH; 

Betulin 

R= COOH; 

Betulinic acid 

R= H; 

Glochidone 

R= OH; 

Glochidonol 

Glochidiol 

HO 

R2 

O 

HO 

HO 

O 

COOH 

R 1 

R 

OH 

R 3 

O 

R 

fraternus (37) 




watsonii (22) 

urinaria (24) 



watsonii (22) 






watsonii (22) 

sellowianus 

toxodiifolius (39) 

virgatus (40) 

sellwoianus (26) 

(26, 27, 38)

- 14 - 

INTRODUCTION 

Name of Compound 

R=α OAc; 

Structure Species & ref. 

(20 S)-3α-acetoxy-24-methylene 

dammaran-20-ol 

HO 

R= ß OAc; 

(20 S)-3ß -acetoxy-24-methylene 

dammaran-20-ol 

β- daucosterol 

5-hydroxy-6,9-epoxy-guaiane 

R1= R2= OH; 

phyllaemblic acid B 

R1=H, R2= OH; 

phyllaemblic acid C 

R1= H, R2= O-glu.; 

phyllaemblicin D 

R= H; 

Spruceanol 

R= OH; 

Cleistanthol 

Trans phytol 

(2Z,6Z,10Z,14E,18E)-farnesyl farnesol 

HO 

R 

HO 

O 

OH 

OH 

O 

HO 

Bisabolane-type sesquiterpenoids 

R 1 

HOOC 

H 

OH 

OH 

O 

H O 

O HO 

Cleistanthane diterpenes 

R 

HO 

Acyclic diterpenes 

HO 

Acyclic triterpene 

OH 

OH 

R2 


emblica (41) 


emblica (42) 


niruri (43) 

niruri (44)

2. Alkaloids: 

- 15 - 

INTRODUCTION 

Alkaloids are widely distributed through different species of the 

genus Phyllanthus; they are represented in the following table: 

Table (4): Alkaloids of genus Phyllanthus. 

Phyllanthimide 

R= H; 

Securinine 

R= OMe; 

Phyllanthine 

Dihydrosecurinine 

R= α- OH; 

Isobubbialine 

R= β- OH; 

Epibubbialine 

R= H; 

Norsecurinine 


R= OMe; 

4-methoxy-norsecurinine 

R= H; 

14,15-dihydroallo-securinin-15ß-ol 

R= OMe; 

Simplexine 

Viroallosecurinine 

R 

R 

R 

CH 2CH 2 

N 

N 

N 

N 

O 

N 

O 

O 

O 

N 

O 

H O 

N 

H 

O 

O 

O 

R 

O 

O 

O 

O 

O 

OH 

N(CH3)2 


discoideus (46) 

amarus (47) 

simplex (48) 

niruri (49) 


amarus (47) 


amarus (47) 

niruri (49) 


simplex (48) 

discoideus (46)

Niruroidine 

- 16 - 

INTRODUCTION 


3. Lignans: 

H 

N 

H 

O 

O 

H 

H H 

OH 

niruroides (50) 

The literature survey of the genus Phyllanthus reveals the 

presence of large number of lignans that are summarized in the 

following table: 

Table (5): Lignans present in genus Phyllanthus: 

Name of Compound Structure 

A- Diarylbutane type: 

Species & ref. 

R1= R2= OMe, R3= H; 

Phyllanthin 

R1 CH2OMe urinaria (23) 

R1= R2= OH, R3= OMe; 

Demethylenedioxyniranthin 

R= H; 

5-demethoxyniranthin 

R= OMe; 

Niranthin 

R1= H, R2= ═O; 

Hinokinin 

R1= ═O, R2= H; 

Heliobuphthalmin lactone 

(+)-8-(3,4-(methylenedioxy) benzyl)-8'- 

(3',4'-dimethoxy-benzyl) butyrolactone 

R 2 

O 

O 

O 

O 

O 

O 

R 3 

R 

OMe 

CH2OMe 

OMe 

CH 2 OMe 

OMe 

OMe 

O 

OMe 

CH 2OMe 

O 

R 1 

O 

R 2 

O 

O 

OMe 

debilis (31) 

(32, 51- 59) 

niruri 

amarus (60) 

niruri 

urinaria 

virgatus (63) 

(32, 51, 52, 56) 

urinaria 

niruri (56) 

virgatus (63) 

virgatus (63) 

(23, 61, 62) 

(23, 61, 62)

Dextrobursehernin 

- 17 - 

INTRODUCTION 


R1= R2 = R3 = OH, R4= CH2OH; 

Seco-isolariciresinol 

R1= R2 = R3 = OMe, R4= CH2OMe; 

Seco-isolariciresinol trimethyl ether 

Linnanthin 

R= H; 

Hydroxyniranthin 

R= OMe; 

7'- hydroxyl-3',4',5,9,9'-pentamethoxy-3, 

4-methylenedioxy lignan 

Nirphyllin 

Hypophyllanthin 

R1=H, R2=R3= OMe; 

Phyltetralin 

R1=O-glu., R2=OH, R3=H; 

Phyllamyricoside C 

MeO 

MeO 

R 1 

R 2 

MeO 

MeO 

O 

O 

O 

O 

R 

OMe 

OMe 

R 3 

OMe 

OH 

OMe 

O 

R 4 

O 

O 

O 

CH 2OH 

OMe 

CH 2OMe 

CH 2OMe 

OMe 

CH 2 OMe 

CH 2 OMe 

OMe 

CH 2 OMe 

MeO OMe 

OH 

B- Aryltetralin type: 

MeO 

O 

MeO 

MeO 

O 

MeO 

R 1 

R 2 

CH2OMe 

CH 2 OMe 

CH 2OMe 

OMe 

CH 2R 2 

CH 2R 3 

OMe 

urinaria 

(23, 61, 62) 


niruri (64) 

niruri (59) 

niruri (64) 

urinaria (65) 

niruri (66) 

debilis (31) 

(32, 51, 52, 54-58, 67, 

niruri 

68) 

amarus (60) 

virgatus (63) 

(23, 61, 62) 

urinaria 

(32, 51, 52) 

niruri 

urinaria 

virgatus (63) 

myrtifolius (69) 

(23, 61, 62)

- 18 - 

INTRODUCTION 


R1= OMe, R2= Me; 

Nirtetralin 

R1= H, R2= Me; 

Isolintetralin 

R1= H, R2= H; 

2,3-desmethoxy seco-isolintetralin 

R1= H, R2= COMe; 

2,3-desmethoxy seco-isolintetralin 

diacetate 

Neonirtetralin 

R1= H, R2= R3= OMe; 

Lintetralin 

R1= R3= H, R2= OH; 

Phyllamyricin F 

R1= O-β-Glu, R2= OH, R3= H; 

Phyllamyricoside A 

R1= O-β-Glu, R2= R3= OH; 

Phyllamyricoside B 

Urinatetralin 

Seco-4-hydroxylintetralin 

Virgatusin 

O 

O 

O 

O 

MeO 

MeO 

R 1 

OMe 

C- Seco-lignan type: 

O 

O 

MeO 

MeO 

CH 2OR 2 

CH 2OR 2 

OMe 

OMe 

OMe 

O 

R 1 

O 

OH 

D- Tetrahydrofuran type: 

O 

O 

MeO 

O 

O 

CH 2 OMe 

CH 2OMe 

OMe 

O 

CH 2 R 2 

O 

CH2R3 

CH 2 OMe 

CH 2OMe 

CH 2OMe 

CH 2OMe 

O 

OMe 

OMe 

OMe 

urinaria 

niruri 

(23, 32, 61, 62) 

(54, 56, 59) 

virgatus (63) 

niruri (55) 

urinaria 

niruri 

(23, 61, 62) 

( 56, 64) 


urinaria 

niruri (64) 

urinaria 

(23, 61, 62) 

(23, 61, 62)

Urinaligran 

Pinoresinol 

R1= ═O, R2=H; 

Retrojusticidin B 

R1= OH, R2= ═O; 

Piscatorin 

R1= R2= OH; 

Haplomyrtin 

R1 = OH, R2= OMe; 

Diphyllin 

R1 = R2= OMe; 

Justicidin A 

R1 = H, R2 = OMe; 

Justicidin B 

Phyllanthusmin A 

R= COOMe; 

Phyllanthusmin B 

R= OH; 

Phyllanthusmin C 

- 19 - 

INTRODUCTION 


O 

O 

MeO 

E- Furofuran type: 

HO 

OMe 

F- Arylnaphthalene type: 

MeO 

MeO 

MeO 

MeO 

R 2 

MeO 

MeO 

HO 

O 

O 

O 

O 

O 

R 1 

O 

OMe 

O 

O 

O 

O 

OMe 

O 

O 

O 

R 1 

O 

R 2 

O 

O 

O 

O 

O 

O 

OH 

OMe 

H H 

OH 

O O H 

OH 

H 

R 

urinaria 

(23, 61, 62) 



piscatorum (70) 



oligospermus (28)

- 20 - 

INTRODUCTION 


G- Arylnaphthalide lignan type: 

R1= R3= OMe, R2=H; 

Phyllamyricin D 

R1= R2= H, R3= OMe; 

Phyllamyricin E 

R= OH; 

Cleistanthin A 

R= OMe; 

Cleistanthin A methyl ester 

R= OH; 

Cleistanthoside A 

R=OAc; 

Taxodiifoloside 

Phyllnirurin 

Virgatyne 

4. Tannins: 

MeO 

MeO 

MeO 

MeO 

MeO 

MeO 

MeO 

MeO 

O 

R 

O 

O 

H-Neolignan type : 

O 

O 

O 

O 

I- Norlignan type: 

O 

O 

O 

O 

O 

HO 

O 

OMe 

CH 2OH 

OH 

O 

O 

O 

O 

R 

O 

O 

OH 

OH 

O 

OH 

O 


taxodiifolius (39) 

taxodiifolius (39) 

niruri (66) 

virgatus (40) 

Tannins are reported to be widely distributed through different 

species of the genus Phyllanthus; they are presented in the following 

tables: 

MeO 

MeO 

R 1 R2 R 3 

O 

O 

O 

O

- 21 - 

INTRODUCTION 

Table (6): Hydrolyzable (pyrogallol) tannins of genus Phyllanthus. 


1- Gallotannins 

R1= H, R2= COOH; 

Protocatechuic acid 

R1= OH, R2= H; 

Pyrogallol 

R1= OH, R2= COOH; 

Gallic acid 

R1= OH, R2= COOMe; 

Methyl gallate 

R1= OH, R2= COOCH2CH3; 

Gallic acid ethyl ester 

R1= O-glu., R2= COOH; 

Gallic acid 3-O-glucoside 

m-digallic acid 

1,6-digalloylgluco-pyranose 

3,6-di-O-galloylglucose 

HO 

HO 

HO 

CO 

O 

R 1 

OH 

OH 

COOH 

OH 

O 

OH 

O 

HO 

O 

R 2 

OH 

HO 

O 

O 

O 

OH 

OH 

OH 

OH 

O 

C OH 

O OH 

Gallic acid 3-O-(6'-O-galloyl)-ß-D- 

HO 

HO 

glucoside O 

HO 

HO 

HO 

HO 

OH 

CO 

CO 

OH 

O 

C 

HO 

O 

OH 

OH 

HO HO 

OH 

COOH 

O 

OH 

OH 

OH 


sellowianus 

(27, 38) 

virgatus (40) 

(67, 68, 71, 72) 

niruri 

emblica 

tenellus (75) 

amarus (76) 

urinaria (77) 

emblica (78) 

emblica 

virgatus (40) 

amarus (76) 

(41, 73, 74) 

(74, 78) 

emblica (74) 

emblica (74)

Bergenin 

Virganin 

Furosin 

- 22 - 

INTRODUCTION 


Phyllanthusiin A 

Phyllanthusiin B 

Phyllanthusiin C 

HO 

MeO 

HO 

HOOC 

O 

OH 

H 

CH2OH O 

OH 

O 

O 

O 

HO HO 

HO 

O 

HO 

HO 

HO 

HO 

HO 

O 

CO CO 

O 

O 

O 

O 

O O 

CO CO 

OH 

O 

HOOC 

HO 

HO 

CO 

OH 

O 

O 

O 

O 

O 

CO 

CO 

O 

CO 

COOH HO 

OH 

O 

O 

O 

O 

O 

CO 

HO 

HOOC 

CO 

OH 

H 

O 

CO 

O 

O 

O 

O 

O 

O 

CO 

HO 

HO 

O 

O 

CO 

H 

O 

CH 2OH 

O 

H 

OH 

OH 

O 

O OH 

OH 

OH 

OH 

OH 

OH 

O 

C OH 

OH 

OH 

HO OH 

OH 

OH 

OH 

CO 

O 

C OH 

OH 

OH 

HO OH 

OH 

OH 

OH 

CO 

O 

C OH 

OH 

OH 

HO OH 

OH 

OH 

CO 

OH 

O 

C OH 

OH 

OH 

OH 

flexuosus 

virgatus (40) 

(18, 79) 

emblica (74) 

amarus (80) 

virgatus (40) 




flexuosus (79)

- 23 - 

INTRODUCTION 


Phyllanthusiin D 

Phyllanthusiin F 

Phyllanthusiin G 

Amariin 

Geraniinic acid B 

HO 

O 

HO 

CO 

O 

O 

HO 

CO 

O 

O 

O 

CO O 

CO 

H 

HO 

HO 

CH2 CO 

O 

HO 

O 

CH3 

OH 

O 

HO CO 

HO 

HO 

HO 

HO 

HO 

O 

O 

O 

CO 

OH 

OH 

OH 

OH 

HO OH 

OH 

O 

C OH 

OH 

OH 

OH 

OH 

O 

O 

HOH2C 

O 

C OH 

OH 

O 

O 

OH 

CO 

O 

CH 2 

OH 

OH 

O 

O 

HO 

HO 

HO 

O 

HO 

HO 

O 

CO 

OC 

O 

O 

OH 

OH 

OH 

OH 

O CO OH 

OH 

OH 

OH 

O O 

C 

O O 

OH 

O 

O 

O 

H 

CO CO 

CO O 

CO 

HO 

H 

CO 

O 

O 

HOOC 

OH 

O 

O 

OH 

OH 

OH 

OH 

CO 

O 

CO O 

CO 

H 

H 

OH 

O 

OH 

O 

O CO 

HO 

OH 

OH 

OH 

OH 

OH 

OH 

OH 

OH 


urinaria (81) 

urinaria (82) 

amarus (80) 

amarus (80) 

flexuosus (79)

G = OOC 

HO O 

OH 

- 24 - 

OH 

OH 

OH 

OH 

OH 

OH 

INTRODUCTION 


R= OH; 

Pinocembrin-7-O-[4'',6''-(S)-hexahydroxyldiphenoyl]- 

ß-D-glucose 

R= G; 

Pinocembrin-7-O-[3"-O-galloyl- 

4'',6''-(S)-hexahydroxydiphenoyl] 

-ß-D-glucose 

Chebulanin 

R= H; 

Chebulagic acid 

R= OH; 

Amariinic acid 

Neochebulagic acid 

Elaeocarpusin 

HO 

HO 

HO 

HO 

O 

O 

O 

O 

CO 

H 

CO 

H 

HOOC 

HO 

OH 

O OH 

HO 

O 

OH HO OH 

HO 

OH 

OH 

CO 

O 

O 

O 

O 

O 

O 

CO CO 

R 

HOOC 

HO 

OH 

O OH 

HO 

O 

OH HO OH 

HO 

CO CO 

O 

O 

O 

O 

OH 

OH 

CO 

O 

C OH 

OH 

O O HO 

HO OH 

HOOC H 

H 

OH 

H 

HOOC 

O 

HO OH 

O 

HO OH 

HO 

H 

HO 

O 

O 

O 

O 

C O 

O 

C O 

CO 

O 

O OH 

R 

O 

CO 

O 

O 

O 

O CO 

O 

H 

CH 2 

O 

O 

O 

OH 

OH 

OH 

CO 

CO 

O O 

OH 

O 

OH 

OH 

OH 

OH 

OH 

OH 

OH 

OH 

OH 

OH 

tenellus (75) 

emblica (74) 

emblica (74) 

amarus (80) 


emblica (74) 

emblica (74) 

amarus (80)

- 25 - 

INTRODUCTION 


Putranjivain A 

R= OH; 

Ellagic acid 

R=OMe; 

2,7-di-O- methylellagic acid 

Geraniin 

Corilagin 

Isocorilagin 

Phyllanemblinin a 

HO 

OH 

O 

HO 

HO OH 

O 

O 

CO 

O 

HO 

O CO 

O 

O 

O O 

CO CO 

H 

OH 

O 

OH 

2-Ellagitannins 

HO 

HO 

HO 

O 

HO 

R 

CO 

HO HO 

HO 

HO 

HO 

CO 

HO 

OH 

O CH 2 

O 

O 

O 

O 

O 

OO 

O 

CO CO 

H 

HO OH 

O 

CO 

OH 

O OH 

HO OH 

CO 

OH 

OH 

OH 

R 

OH 

OH 

OH 

OH 

CO OH 

OH 

OH HO OH 

CO CO 

O 

O CH 2 

OH 

O 

OH 

OH 

OH 

O 

O CO OH 

O 

OH 

OH OH 

OHHO 

OH 

HO OH O 

CO 

OH 

OH 

OH 

O 

O CO OH 

O 

O O 

CO 

CO 

CO 

HO OH 

HO 

O 

OH 

OH 

OH 

OH 

OH 

OH 

emblica 

(74, 78) 



emblica (41) 

(71, 72) 

niruri 

urinaria (83) 


virgatus (40) 

niruri (72) 

emblica 

tenellus (75) 

(74, 84) 

(76, 80) 

amarus 


urinaria (85) 


virgatus (40) 

(67, 68, 71, 86) 

niruri 

emblica 

tenellus (75) 

amarus (76) 


niruri 

(73, 74) 

(67, 68) 

emblica (84) 

emblica (74)

- 26 - 

INTRODUCTION 


Phyllanemblinin B 

Phyllanemblinin C 

R1= A, R2= R3= OH; 

Phyllanemblinin D 

R1= R3= OH, R2= A; 

Phyllanemblinin E 

R1= R2= OH, R3= A; 

Phyllanemblinin F 

HO 

HO 

HO 

HO 

CO 

O 

O 

HO OH 

HO 

CO 

O 

OH HO 

OH HO 

O 

CO 

HOOC 

HO 

HOOC 

R2 R1 R 3 

HOOC 

HOOC 

O O CO 

CO 

OH 

OH 

OH 

OH 

OH 

CO 

O CO OH 

O 

O 

OH 

H 

O 

CO 

O 

O 

OH 

OH 

OH 

OH 

OH 

O CO 

Table (7): Condensed tannins of genus Phyllanthus: 

A = 

COO 

HO 

H 

H 

O 

O 

OH 

OH 

OH 

OH 

OH 

emblica (74) 

emblica (74) 

emblica (74) 

Name of Compound Structure Species& ref. 

R1= OH, R2= H; 

(-)- Epiafzelechin 

R1= β- OH, R2= OH; 

(+)-Catechin 

R1= α- OH, R2= OH; 

(-)-Epicatechin 

R1= α- G, R2= OH; 

(-)-Epicatechin-3- O- gallate 

(±) -Gallocatechin 

HO 

OH 

G = COO 

HO 

OH 

O 

O 

R 1 

OH 

OH 

OH 

OH 

OH 

OH 

R2 

OH 

OH 

emblica 

emblica 

(73, 74) 

(73, 74) 

amarus (76)

- 27 - 

INTRODUCTION 

Name of Compound Structure Species& ref. 

R= OH; 

(-)-Epigallocatechin 

R= G; 

(-)-Epigallocatechin 3-O-gallate 

Epicatechin (4ß→8)epigallocatechin 

3-O- gallate 

R1= H, R2= OH; 

Epicatechin (4ß→8)-gallocatechin 

R1= R2= OH; 

Prodelphinidin B-1 

R1= OH, R2= G; 

Prodelphinidin B-2-3'-O-gallate 

Prodelphinidin A-1 

5. Flavonoids: 

HO 

HO 

HO 

HO 

G = 

HO 

G = 

OH 

OH 

OH 

HO 

COO 

OH 

HO 

HO 

O 

COO 

O 

O 

O 

OH 

O 

O 

OH 

O 

R 

OH 

OH 

OH 

OH 

O 

OH 

OH 

OH 

R1 

OH 

OH 

OH 

OH 

R 2 

OH 

OH 

OH 

OH 

OH 

OH 

OH 

OH 

OH 

OH 

OH OH 

O 

OH 

OH 

O 

OH 

OH 

OH 

OH 

OH 

emblica 

emblica (73) 

emblica 

emblica (73) 

(73, 74) 

(73, 74) 

Flavonoidal compounds are reported in many different species of 

genus Phyllanthus; they are represented in the following table:

Luteolin 

R= H; 

Kaempferol 

R= glu.; 

Astragalin 

Table (8): Flavonoids of genus Phyllanthus: 

- 28 - 

INTRODUCTION 


R= rham-glu.; 

Kaempferol-3-O- rutinoside 

5,7-dihydroxy-4'-methoxy 

flavonol 

R= H; 

Quercetin 

R= rham. ; 

Quercetrin 

R= glu.; 

Isoquercitrin 

R=glu-glu.; 

Quercetin-3-O-ß-D-glucosyl-(1→6)-ß- 

D-glucoside 

R= glu-rham.; 

Quercetin-3-O-ß-D-glucopyranosyl 

(1→4)-α- rhamnopyranoside 

R= rham-glu.; 

Rutin 

Myricitrin 

HO 

HO 

HO 

HO 

HO 

OH 

OH 

OH 

OH 

OH 

O 

O 

O 

O 

O 

O 

O 

O 

O 

O 

O 

OH 

O 

O 

R 

OH 

R 

OH 

OH 

Rhamnose 

OH 

OH 

OCH 3 

OH 

OH 

OH 



virgatus (40) 

( 53) 

niruri 

tenellus (75) 

emblica 

niruri (87) 

(78, 84) 

virgatus (40) 


sellowianus 

virgatus (40) 

tenellus (75) 

amarus (76) 

emblica 

niruri 

(27, 38 ) 

(41, 78, 84) 

(67, 68, 87) 

tenellus (75) 

virgatus (40)

- 29 - 

INTRODUCTION 


R1= R2= R3= R4= H; 

8-(3-methyl-but-2-enyl)-2-phenylchroman-4-one 


R1= R2= R3= H, R4= OH; 

2-(4-hydroxyphenyl)-8-(3-methylbut-2-enyl)-chroman-4-one 

R2= R3=R4= OH, R1= rham-glu.; 

Nirurin 

R1= OH, R2= H; 

Galangin-8-sulfonate 

R1=O-glu., R2= H; 

Galangin-3-O-ß-D-glucoside-8sulfonate 

R1= R2= OH; 

Kaempferol-8-sulfonate 

Niruriflavone 

3,5,7-trihydroxyflavonol-4'-O-α- 

rhamnopyranoside 

Eriodictyl-7- rhamnopyranoside 

R= H; 

Eriodictyol-7-O-glucoside 

R= A; 

(S)-Eriodictyol-7-O-(6"-O-trans-pcoumaroyl)-β-D- 

glucopyranoside 

R= G; 

(S)-Eriodictyol-7-O-6"-O-galloyl)β-D-glucopyranoside 

HO 

R 3 

R 2 

HO 

HO 

HO3S 

OH 

rhamnose 

HO 

HO 

RO 

A = 

O 

O 

O 

OH 

G = 

R1 

SO 3Na 

OH 

OH 

CH 2 

O 

O 

C 

O 

O 

O 

O 

O 

O 

OH 

OH 

COO 

OH 

R 1 

O 

O 

O 

O 

C 

H 2 

OH 

OH 

R 4 

R2 

OMe 

O rhamnose 

OH 

OH 

OH 

OH 

OH 

OH 

niruri 

(87, 88) 

virgatus (40) 

(67, 68) 

niruri 

niruri (89) 

niruri (89) 

emblica (90)

6. Other oxygenated compounds: 

- 30 - 

INTRODUCTION 

The literature survey of genus Phyllanthus reveals the presence 

of large number of oxygenated non-phenolic compounds that are 

summarized in the following table: 

Table (9): Oxygenated compounds present in genus Phyllanthus: 


Descinnamoylphyllanthocindiol 

O 

Niruriside O 

R1= R3= OAc, R2= OH; 

Phyllanthoside 

R1= R2= R3= OH; 

Didesacetylphyllanthoside 

R1= OH, R2= R3= OAc; 

Phyllanthostatin 1 

R1= R2= OAc; 


R1= R2= OH; 


R1= OAc, R2= OH; 


R1= OH, R2= OAc; 


R= OH; 

Didesacetyl-phyllanthostatin 3 

R= OAc; 


O 

O 

OH 

HO 

CH3 O 

CH2 CH 3 

O 

O 

O 

O 

O 

O 

O 

O 

O 

HO 

O 

O 

HO 

O 

O 

OH 

O H2C O 

O 

CH2 OH 

O 

CH 3 

O 

O 

O 

O 

CH O 

3 

O 

R2 R1 O 

HO 

O 

CH2OH 

O 

O 

HO 

R1 O 

HO 

O 

O 

O 

CH O 

3 

R2 R1 O 

HO 

O 

O 

OH 

O 

CH 2OH 

O 

HO 

R O 

HO 

O 

O 

O 

OH 

O 

O 

O 

O 

O 

O 

O 

O 

O 

R 3 

R 2 

OH 

R 

CH 3 

OH 

CH 3 

OH 

CH 3 

OAc 

CH 3 

OH 

acuminatus (91) 

niruri 

( 53) 

acuminatus (91-93) 

acuminatus 

(91, 92) 

acuminatus (91) 

acuminatus (91)

- 31 - 

INTRODUCTION 


R= glu.; 

Phyllaemblicin A 


R= glu-glu.; 

Phyllaemblicin B 

R= glu-glu-glu.; 

Phyllaemblicin C 

7. Acids: 

R 

O 

O 

HO 

O 

OH 

O 

O 

O 

O 

emblica (73) 

Acids have been found to be widely distributed through different 

species of the genus Phyllanthus; they are represented in the following 

table: 

Table (10): Acids isolated from genus Phyllanthus. 

Name of Structure Structure Name of the plant 

Ricinoleic acid 

Linoleic acid 

Cinnamic acid 

Trephthalic acid mono-[2-(4carboxy-phenoxycarbonyl) 

-vinyl ]ester 

(E)-3-(5'-hydroepoxy-2,2'dihydroxy[1,1'-biphenyl]-4yl)-2-propenoic 

acid 

3,4,5-trihydroxybenzoic acid 

(gallic acid) 

2,3,4,5,6pentahydroxybenzoic 

acid 

OH 

O 

OH 

niruri (94) 

HOOC niruri (94) 

O 

HO 

HO 

MeO 

O 

HO 

HO 

HO 

H 

C C H 

O 

O O 

HO 

COOH 

OH 

COOH 

OH 

COOH 

OH 

OH 

OH 

O 

OH 

O 

OH 

emblica (41) 

urinaria (95) 

urinaria (95) 

urinaria (95) 

urinaria (95)

- 32 - 

INTRODUCTION 

Name of Structure Structure Name of the plant 

4-O-galloylquinic acid 

R= COOH; 

Indol-3-carboxylic acid 

R= CHO; 

Indol-3-carboxaldehyde 

HO 

HO COOH 

O 

O 

OH 

OH 

H 

N 

OH 

R 

OH 

amarus (80) 

virgatus 

(40, 63)

- 33 - 

INTRODUCTION 

The Biological Activities of Genus Phyllanthus 

Reviewing the current literature concerning the biological 

activities of genus Phyllanthus, the following findings were 

abstracted: 

1. Antioxidant effect : 

• Extract of Phyllanthus emblica (syn. Emblica officinalis) 

( 84, 96- 

100) , P. niruri (67, 101-103) , P. simplex (104) , P. oxyphyllus (16) , P. 

ussurensis (105) and P. reticulatus (106, 107) show strong antioxidant 

activity as they have direct free radical scavenging role and they 

protect the antioxidant enzyme like superoxide dismutase enzyme 

required for cellular defence (96) . 

2. Antihepatotoxic activity: 

• Phyllanthus amarus (108) have antiviral activity against hepatic 

damage caused by HBV (i.e. cause hepatitis B and C), woodchuck and 

aflatoxin B1 through antioxidant activity which is associated with 

phyllanthin and hypophyllanthin lignans (109) . 

• Phyllanthus emblica (110, 111) , P. maderaspatensis (112, 113) , P. 

fraternus (114, 115) , P. rheedii (116) , P. niruri (101-103, 117- 120) and P. amarus 

(108, 121- 123) show antihepatotoxic activity against different models of 

hepatic rats. This protective activity is quite similar to silymarin (111, 

122) , a standard hepatoprotective herbal drug, or it found to be better 

than silymarin in certain cases (113) , with the possible mechanisms of 

antioxidation (103, 111, 112, 116, 118, 119, 122) or the lipid lowering activity of 

the herb (102) which might be related to the high contents of 

polyphenolic compounds (123) .

3. Anticancer effect: 

- 34 - 

INTRODUCTION 

It was reported that extracts of Phyllanthus amarus (124-127) , 

P. emblica (128, 129) , P. urinaria (65, 126) , P. oligospermus, P. 

acuminatus, P. flexuosus , P. toxodiifolius (26, 39, 91, 130) and P. 

polyphyllus (131) exhibit significant antitumor activity without toxic 

side effects on normal cells against skin tumorigenesis (129) , 

hepatocellular carcinoma (121, 125, 126) , KB (human epidermal 

carcinoma), P-388 (the mouse leukaemia), murine P-388 

lymphocytic leukemia and mammalian cancer cell lines 

(28, 39, 91, 130, 

131) and Ehrlich ascites carcinoma (131) . This activity is attributed to 

many varieties of mechanisms (124, 126, 127) . 

4. Anti-diabetic effect: 

• Extract of Phyllanthus emblica (97, 132 ) , P. amarus ( 133 ) , P. 

sellowianus (134 ) , P. fraternus (135) and P. niruri ( 136) produced a 

significant reduction in blood glucose level in model diabetic rats with 

no harmful side effects and this activity is more effective than that 

observed with glibenclamide or similar to it which was used as a 

reference for the hypoglycemic activity. 

• Phyllanthus emblica and P. niruri used in the management of 

diabetic complication such as cataract and this anticataract effect (71) 

was proved to be due to inhibition of sugar-induced osmotic changes 

and Aldose Reductase enzyme which prevents aggregation and 

insolubilization of lens proteins caused by hyperglycemia (137) .

5. Effect on respiratory system: 

- 35 - 

INTRODUCTION 

• Phyllanthus emblica has antitussive activity which is less 

effective than shown by classical narcotic antitussive drug codeine, 

but more effective than the non-narcotic antitussive agent 

dropropizine. This action may be due to antispasmolytic, antioxidant 

efficiency effects and its effect on mucus secretion in the airways (145) 

also it contains natural vitamin C which is used in treatment of 

pulmonary tuberculosis (139) . 

• Phyllanthus urinaria cause graded and complete relaxation of 

guinea pig trachea pre-contracted by carbachol (140) or by histamine 

(141) . This effect may be used for the antinociceptive effect toward 

neurogenic pain (142) . 

6. Effect on urinary tract: 

Phyllanthus amarus, P. niruri, P. fraternus, P. urinaria, P. 

maderaspatensis and P. sellowianus have diuretic action (143) as it may 

have a direct action on smooth muscle of urinary bladder (144) . 

7. Effect on GIT: 

Phyllanthus amarus (145) and P. emblica (146, 147) have anti- 

diarrhoeal, gastro-intestinal protective effect and anti-ulcer activity. 

They significantly inhibited gastric lesions in different acute gastric 

ulcer models in rats (146, 147) with different mechanisms (146- 149) . 

8. Effect on cardiovascular system (150- 152) : 

• Extracts of Phyllanthus emblica and P. amarus showed a 

cardioprotective activity through reduction of the blood pressure, 

level of serum cholesterol and triglycerides or elevation of HDL

- 36 - 

INTRODUCTION 

cholesterol level which indicates the usefulness of this drug in 

coronary artery diseases. 

• Phyllanthus emblica has a significant antiatherogenic effect 

through inhibition oxidized LDL-induced vascular smooth muscle 

cells proliferation. 

9. Hypolipidemic effect: 

Phyllanthus emblica and P. amarus are effective hypolipidemic 

agents without any reported side effects through antioxidant activity 

(99, 153, 154) and other different mechanisms (102, 154-157) . 

10. Antimutagenic effect: 

Extract of Phyllanthus orbicularis (158- 160) and P. emblica 

(161, 162) 

produced a significant antimutagenic activity (163) against many 

promutagenic agents (159- 164) . 

11. Antinociceptive activity: 

Extract of Phyllanthus amarus, P. orbicularis, P. fraternus, 

P.stipulatus, P. urinaria, P. corcovadensis, P. niruri and P. 

sellowianus given intraperitoneally or orally, produced significant 

antinociceptive action against different models of abdominal 

constrictions or pains (25, 27, 77, 165, 166) . 

12. Anti-inflammatory, antipyretic and immunomodulatory effects 

(148, 167- 169 ) : 

Different extracts of Phyllanthus emblica (170- 172) , P. amarus (148, 

167, 168, 173, 174) and P. polyphyllus (169) have been used widely for its

- 37 - 

INTRODUCTION 

anti-inflammatory, antipyretic and immunomodulatory effects through 

different mechanisms (148, 170-173) . 

13. Effect on enzymes: 

• Phyllanthus maderaspatensis has mild α-amylase inhibitory 

activity which may be used in control of obesity and diabetes (175) . 

• Phyllanthus niruri is glucosyltransferase inhibitor which is 

suitable for use in a food, feed or dentifrice compounds for prevention 

of dental plaque formation and periodontal disease (176) . 

14. Effect on HIV (53, 78, 177, 178) : 

Extracts of Phyllanthus niruri (1, 53, 179) , P. myrtifolius and P. 

emblica (78, 177) show strong inhibitory effect on HIV (virus caused 

human immunodeficiency disease). 

15. Antisnake venom activity (99) : 

Methanolic root extract of Phyllanthus emblica has potent 

antisnake venom activity as it significantly antagonize snake venom- 

induced lethal activity as haemorrhage, coagulant, defibrinogenating 

and inflammatory activities (99, 180) . While P. niruri and P. urinaria 

cause a protection against snake bite (181) . 

16. Effect on brain (182) : 

Phyllanthus amarus reversed successfully the amnesia induced 

by scopolamine and diazepam so it has a therapeutic potential in the 

management of Alzheimer patients as it improved the memory of both 

young and old mice through lowering serum cholesterol level, 

inhibition of acetylcholinesterase enzyme and elevation of 

acetylcholine concentration in brain.

17. Effect on sexual organs (183) : 

- 38 - 

INTRODUCTION 

• Phyllanthus amarus extract was used in traditional medicine in 

India and China as a tool for birth control as it is at a dose of 100 mg/ 

kg showed antifertility (contraceptive) effect in female mice while at 

a dose of 500 mg/kg induce functional sterility as it causes gradual 

inhibition of fertility potential. In the quest for the search for male 

contraceptive, more research light may be focused on P. amarus in 

order to discover its full potential. This means that appropriate dose 

that will not expose the testes to toxic injury should be determined. 

Further searchlight should be shed on the effect of this plant on the 

testosterone.

AIM OF THE PRESENT WORK 

- 39 - 

INTRODUCTION 

The previously mentioned literature survey reveals that many 

plants belonging to genus Phyllanthus were phytochemically studied 

and the occurrence of several active compounds such as lignans, 

flavonoids, tannins, polyphenolic compounds, sterols and terpenes 

were reported. These compounds are characterized by their 

physiological and medicinal values such as antioxidant, anticancer, 

antihepatotoxic, antitussive, antidiabetic, antiatherogenic, antiulcer, 

antimutagenic, antinociceptive, anti-inflammatory, antipyretic, 

diuretic, cardioprotective and hypolipidemic effects. 

As there is almost no report on the chemistry and botanical 

study of Phyllanthus atropurpureus Boj. Hort. Maurit. cultivated in 

Egypt as well as biological screening of this plant. It was deemed of 

interest and importance to carry out a pharmacognostical study on this 

plant. 

The proposed study of this plant presented in this thesis includes: 

1. A macro- and micromorphology of the leaf, stem, root and 

flower to help in the identification of the plant and these organs 

in both entire and powdered forms. 

2. A phytochemical investigation of different extracts of the plant 

and isolation of the major constituents whenever possible which 

may have a potential pharmacological activity. 

3. Physical, chemical and spectral characterization of the isolated 

compounds and hence identifying them if possible. 

4. A biological study of different extracts as well as the major 

compound including antihepatotoxic, antioxidant, anticancer 

activities and microbiological screening to reveal the medicinal 

value of this plant.

I- Materials: 

1. Plant material: 

Materials, Reagents and Apparatus 

- 40 - 

INTRODUCTION 

The plant material used in this work, Phyllanthus 

atropurpureus Boj. Hort. Maurit. family Euphorbiaceae (spurge), was 

collected in the flowering stage on June 2004 from the plant cultivated 

in medicinal plants garden of Faculty of Science, Ain Shams 

University. 

The identification was kindly verified by Dr. Hesham abdel- 

Aal Elshamy, Assistant Professor of medicinal, aromatic and 

ornamental plants, Horticulture Department, Faculty of Agriculture, 

Zagazig University, Egypt. 

A voucher specimen is deposited in Department of 

Pharmacognosy, Faculty of Pharmacy, Zagazig University, Egypt. 

The plant was shade dried and ground by electric mill to 

moderately fine powder and extracted by maceration in ethyl alcohol 

75%. 

The material used for macro- and micromorphological study 

was either fresh or preserved in glycerin-alcohol mixture (1:1). 

2. Solvents and chemicals: 

The solvents used in this work viz.: Light petroleum (60-80 

o C), diethyl ether, chloroform, ethyl acetate, benzene, methanol and 

ethanol (95%) were of the analytical grade for chromatography and 

crystallization. Solvents for 1 H- and 13 C-NMR determination viz.: 

deuterated chloroform (CD3Cl3) and deuterated methanol (CD3OD) 

were of spectroscopic grade for spectral analysis.

- 41 - 

INTRODUCTION 

These solvents and chemicals were prepared according to the 

procedures described by Vogel (184) . 

3. Reagents: 

A. Qualitative test reagents and solutions: 

The reagents and test solutions used in this work viz.: 

diluted acids, diluted alkalies, alcoholic α– naphthol, Fehling's 

solution, ferric chloride, Iodine solution, Mayer's and Wagner's 

reagents were prepared according to E.p (185) . 

B. Spray reagents (186) : 

• Anisaldehyde-sulphuric acid reagent. 

• Ammonium hydroxide. 

• Ferric chloride. 

• Modified Dragendorff's reagent. 

• 50 % aqueous sulphuric acid. 

C. Reagents for U.V. spectroscopic analyses (187) : 

• Sodium methoxide, 2.5% in methanol. 

• Aluminium chloride, 5% in methanol. 

• Hydrochloric acid, 18% aqueous solution. 

• Anhydrous sodium acetate "El Nasr Pharmaceutical 

Chemicals". 

• Anhydrous boric acid "El Nasr Pharmaceutical 

Chemicals". 

4. Materials for chromatography (adsorbents): 

• Silica gel for column (Merck) 60-230 mesh. 

• Silica gel for thin-layer chromatography, 60 GF254

• Precoated TLC sheets, silica gel 60 GF254 (Merck). 

5. Apparatus and equipments: 

1. Chromatographic equipments: 

- 42 - 

INTRODUCTION 

Glass jars for TLC, capillaries for spotting, precoated 

layers for TLC and chromatographic glass column. 

2. BÜCHI rotary evaporator. 

3. Melting point apparatus, Digital, electro-thermal LTD 

(England). 

4. U.V. lamp for TLC visualization U.V.P., GL-58(λ max 254 

and 365 nm). 

5. Circulating hot air oven, W.T-binder 7200, (Germany). 

6. Heraeus Sepatech centrifuge (Labofuge 200) 

7. Shimadzu U.V.-1700 spectrophotometer (Japan) for U.V. 

spectral analysis. 

8. Infrared spectral analysis were recorded in potassium 

bromide disks on a pye Unicam SP 3000 and carried out on 

IR spectrophotometer, Jasko, FT/IR-460 plus. 

9. Mass spectra were carried on: 

• Shimadzu GC-MS-QP 1000 EX mass spectrometer at 

70 e.v. 

• VG- Quattro II waters, Masslynx V40 SP4, 508, Copy 

rights© 2004 Micromass Ltd. 

10. 1 H- and 13 C-NMR spectra were obtained using JEOL and 

Varian MAT 500, 300, 125 and 75 MHz respectively, 

chemical shifts were given in ppm with the TMS as internal 

standard.

- 43 - 

INTRODUCTION 

11. GLC analysis of methyl ester of the total fatty acids were 

carried out on GC V pye Unicam gas chromatograph under 

the following operating conditions: 

• Detector Dual flame ionization detector 

• Temp. of detector 300 o C 

• Recorder Dual channel recorder 

• Temp. of injector 250 o C 

• Column temp. 70 to 190 o C (8 o C/min) 

• Column package Diatomite C (100-120 mesh) 

• Liquid phase 10 % polyethylene glycol adipate (PEGA) 

• Column dimensions 1.5m × 4mm 

• Hydrogen flow rate 33 ml / min. 

• Chart speed 1 cm / min 

6. Authentics: 

Authentic sample of β – sitosterol and stigmasterol mixture and 

quercetin-7-O-glucoside were obtained from Department of 

Pharmacognosy, Faculty of Pharmacy, Zagazig University, Egypt. 

Authentic sample of Ampicillin and Gentamycin as well as 

Nystatin were obtained from Department of Microbiology, Faculty of 

Pharmacy, Zagazig University, Egypt. 

Silymarin for determination of antihepatotoxic activity was 

supplied by Unipharma Pharmaceutical Company, Egypt 

Authentic sample of fatty acids methyl esters were obtained from 

Central Research Laboratory, Faculty of Agriculture, Cairo 

University, Egypt. 

7. Kits: 

Kits for determination of ALT and AST were supplied by 

Plasmatek (Germany) while that of total protein and serum albumin 

supplied by Biocon (Germany).

8. Chromatographic Solvent Systems: 

- 44 - 

INTRODUCTION 

1. Ethyl acetate: Light Petroleum 30: 70 

2. Methanol: Chloroform 2: 98 






8. Methanol: Ethyl acetate 

15:85 

9. Ethyl acetate: Acetic acid: Formic acid: H2O 100: 11: 11: 27 

10. Ethyl acetate: Benzene 14: 86 

11. Light petroleum: Chloroform: Methanol 15: 15: 1 

12. Ethyl acetate: Light Petroleum 15: 10 

13. Benzene: Ethyl acetate: Formic acid: H2O 30: 50: 14: 6 

14. Light petroleum: chloroform: glacial acetic acid 75: 25: 0.5

- 45 - 

PART I 

Phyllanthus atropurpureus Boj. Hort. Maurit. (Fig.1) is a perennial 

monoecious shrub. The plant shows a brownish to purple monopodially 

branched stem and measures 0.5 to 1.5 m in height. It carries simple 

alternate ovate leaves with dark olive green surfaces showing red or 

purple, occasionally white coloration; the young leaves are purplish in 

colour. The plant shows green or purple axillary flowers (Fig. 2& 4A). 

Female flowers are present at the top while male flowers are present 

below. The plant flowers from June to November and is cultivated by 

cutting and sucker divisions. 

The leaf: 

The leaves (Fig. 1, 2, 4A& 4B) are simple, shortly petiolate, 

stipulate, with obtuse apex showing epiculus, entire margin and 

symmetric base. The leaves have greenish smooth glabrous surfaces, 

mottled with whitish, pale pink or purple coloration, and reticulate 

pinnate venation. The midrib and the big veins are prominent on the 

lower surface only. The leaf measures 1.5 to 5.5 cm in length and 1 to 4 

cm in breadth. 

Two stipules are present, each on both sides of the leaf base. They 

are triangular in shape with entire margin, acute apex, smooth green 

surfaces and measure 1.6 mm in length and 0.4 mm in breadth. 

The petiole of the leaf (Fig. 4B) is short cylindrical, solid with dark 

green to purple smooth surface and measures 0.8 to 1.3 mm in length 

and 0.5 to 0.8 mm in diameter. 

The leaves have faint characteristic odour and a slightly bitter taste.

The stem: 

- 46 - 

PART I 

The old stem is rigid, solid, monopodially branched and cylindrical 

with brownish rough surface. The internodes measure 2.5 to 3.5 cm in 

length and 1.5 to 3.5 mm in diameter 

The young stem and small branches (Fig. 1& 4A) have dark purple 

smooth glabrous surface. The stem is flexible when fresh, hard and 

rigid when dry, broken with fibrous fracture exposing greenish- white 

interior. The stem has slight odour and slightly bitter taste. 

The inflorescence: 

The flowers occur either single or in small inflorescences. The 

inflorescences are axillary fascicles of unisexual flowers, being 

pistillate at the top of branches, and staminate below. 

• The pedicel: 

The pedicel of the flower (Fig. 2, 5B& 5D) is short cylindrical, 

solid with purple smooth surface and measures 2.5 to 5 mm in length 

and 0.5 to 1 mm in diameter. 

• The flower: 

The flowers (Fig. 2, 5B& 5D) are small, green or purple in colour. 

They are unisexual and the plant is monoecious. They are shortly 

pedicelate and have no petals. 

The male flowers (Fig. 5B) are small measuring 4 to 7 mm in 

length and 0.5 to 2 mm in diameter at the widest part while female 

flowers measure 3 to 5 mm in length and 2 to 7 mm in diameter. The 

female flower (Fig. 2& 5D) shows a calyx with 6, occasionally 5, 

spreaded lobes while those of male flower are unspreaded. 

The flowers have slight characteristic odour and bitter taste.

• The calyx: 

- 47 - 

PART I 

The calyx (Fig. 5D) is persistent cupuliform gamosepalous, 

consists of 6, occasionally 5 united sepals with free apical lobes. The 

apical lobe has entire margin and rounded apex showing fine epiculus. 

The calyx of female flower is bell-shaped with six apical spreaded 

lobes, green or purple smooth surfaces and shows pinnately- reticulate 

venation; the main veins are six, occasionally 5, with small lateral ones. 

The apical lobes of the female flowers measure 0.2 to 0.8 mm in 

length and 2.5 to 3.5 mm in breadth while the tubular part measures 3 to 

4.2 mm in length and 0.5 to 2 mm in diameter at its widest part. 

The calyx of male flower is funnel-shaped with six small 

unspreaded lobes which measure 1 to 1.4 mm in length and 0.1 to 0.4 

mm in diameter. 

• The androecium: 

The androecium is present in the male flower only (Fig. 5C) and 

consists of six monodelphous stamens with 6 free anthers arranged in 

one whorl. 

The filaments are united into solid, cylindrical, whitish staminal 

column measuring 1 to 1.2 mm in length and 0.1 to 0.3 mm in diameter. 

The anther (Fig. 5C) is more or less oblong, pale yellow in colour, 

dorsifixed and bilobed, each lobe shows one pollen sac. It measures 

0.55 to 0.65 mm in length and 0.13 to 0.18 mm in diameter.

• The Gynaecium: 

- 48 - 

PART I 

The gynaecium of the female flower (Fig. 5E) is tricarpellary, 

syncarpous and consists of superior ovoid trilocular ovary, gynobasic 

style and trifid ligulate stigma. 

The ovary is ovoid, three-sided and trilocular; each locule contains 

two axile placented ovules. It has light green smooth glabrous surface 

and measures 0.85 to 1 mm in length and 0.45 to 0.8 mm in diameter. 

The style is solid cylindrical, gynobasic with light green smooth 

surface. It is branched into three separated parts just above the ovary; 

each is traversed longitudinally by one small vascular strand. It 

measures 0.3 to 0.5 mm in length and 0.15 to 0.23 mm in diameter. 

The stigma is trifid; each lobe is ligulate obtriangular, with entire 

margin, pale yellowish surfaces and slightly emarginated papillosed 

apex. Each lobe measures 0.3 to 0.45 mm in length and 0.34 to 0.64 

mm in breadth at its widest part. 

The subterranean organs: 

The plant has well developed highly branched underground part. It 

comprises a vertical rhizome gives on its lower end three to four roots. 

The rhizome (Fig. 3) is vertical showing two to three internodes, dark 

reddish-brown rough, longitudinally wrinkled outer surface. It breaks 

with fibrous fracture showing reddish-brown narrow outer bark, wide 

yellowish-white wood and a wide pith in the center. The rhizome 

measures 1.5 to 3 cm in length and 1 to 1.5 cm in diameter.

- 49 - 

PART I 

The root (Fig. 3) is cylindrical, teret and gives few wiry secondary 

roots and numerous rootlets. The main root measures 10 to 25 cm in 

length and 0.2 to 0.4 cm in diameter while the secondary root measures 

10 to 15 cm in length and 1 to 1.5 mm in diameter. The root and its 

branches have rough, reddish-brown outer surface and breaks with a 

fibrous fracture exposing a reddish bark and a yellowish wood. 

The rhizome and root have a characteristic bitter taste and a 

characteristic dusty odour.

Fig. 1: A photograph of aerial parts of 

Phyllanthus atropurpureus Boj. Hort. 

Maurit. (x 0.25) 

- 50 - 

PART I 

Fig. 3: A photograph of the root and rhizome of Phyllanthus 

atropurpureus Boj. Hort. Maurit. (x 0.5) 

Fig. 2: A photograph of female 

flowers of Phyllanthus atropurpureus 

Boj. Hort. Maurit. (x 1.92)

Fig. (4): Sketch of Phyllanthus atropurpureus Boj. Hort Maurit. 

A. A flowering branch 

B. A leaf. 

C. A stipule. 

- 51 - 

(x 0.58) 

(x 3.80) 

(x 36.6) 

F.fl., female flower; m. midrib; m.fl., male flower; pet., petiole; st., 

stem. 

PART I

B 

A 

m 

. 

pet. 

- 52 - 

st. 

f.fl 

. 

m.fl 

. 

PART I 

C

Fig. (5): The flower. 

A. Two lobes of dissected calyx. (x 30.6) 

B. Male flower. (x 22.75) 

C. Androecium. (x 45) 

D. Female flower. (x 20.1) 

E. Gynaecium (x 26.7) 

An., anther; cal., calyx; ov., ovary; ped., pedicel; sep., sepal; stg., 

stigma; stm.col., staminal column; sty., style. 

- 53 - 

PART I

D 

B 

ped. 

A 

stg. 

sep. 

sty. 

ov. 

cal. 

ped. 

- 54 - 

an. 

stm.col. 

E 

C 

PART I

THE LEAF 

- 55 - 

PART I 

A transverse section of the leaf (Fig. 6A) shows a dorsiventral, 

heterogenous mesophyll with one row of palisade below the upper 

epidermis and is interrupted by collenchyma in the midrib region. 

The midrib is more prominent on the lower surface and shows a 

parenchymatous cortex with peripheral collenchyma and is transversed 

longitudinally by a crescent-shaped vascular tissue with a 

collenchymatous pericycle above and below the vascular bundle. 

The Upper Epidermis of The Lamina: 

The upper epidermis (Fig. 6B& 7A) is formed of polygonal 

isodiametric cells with thin straight anticlinal walls and is covered with 

thin smooth cuticle. They measure 16 to 35 µ in length, 16 to 43 µ in 

breadth and 10 to 16 µ in height. 

Many cells have short conical-shaped papillae measuring 17 to 21µ 

in height. 

Many cells contain mucilage and transparent secretions which 

appear as transparent dots. 

The Upper Epidermis of The Midrib: 

The upper epidermal cells (Fig.6C& 7C) are polygonal axially 

elongated cells containing mucilage. They have thin straight anticlinal 

walls and are covered with moderately thick smooth cuticle and 

measure 33 to 62 µ in length, 10 to 21µ in breadth and 14 to 16 µ in 

height.

The Lower Epidermis of The Lamina: 

- 56 - 

PART I 

The lower epidermal cells (Fig. 6B& 7B) are isodiametric, 

polygonal, with thin slightly wavy anticlinal walls and covered with 

thin smooth cuticle; numerous cells show short conical-shaped papillae 

and most of them contain mucilage. They measure 17to 41 µ in length, 

10 to 41 µ in breadth and 8 to 12 µ in height. 

The Lower Epidermis Over The Midrib: 

The lower epidermis (Fig. 6C&7D) is formed of polygonal axially 

elongated cells containing mucilage, with moderately thick beaded 

anticlinal walls and covered with smooth cuticle. They measure 27 to 

58 µ in length, 10 to 23 µ in breadth and 10 to 14µ in height. 

Stomata: 

The stomata (Fig.7B) are numerous and present on the lower 

surface only, being absent on neural regions. They are of paracytic type; 

each is surrounded by two cells, oval in shape and in the same level of 

the epidermal cells measuring 19 to 35 µ in length and 10 to 19 µ in 

breadth. 

Trichomes: 

Both glandular and covering trichomes are completely absent. 

Mesophyll: 

The mesophyll (Fig. 6A &6B) is dorsiventral with an upper 

palisade discontinuous in the midrib region and a wide spongy tissue. 

The palisade (Fig. 6B) consists of one row of cylindrical columnar 

and radially elongated cells with straight anticlinal walls. The cells 

measure 47 to 70 µ in length and 8 to 16 µ in diameter.

- 57 - 

PART I 

The spongy tissue (Fig. 6B) consists of few rows of more or less 

polygonal parenchymatous cells with thin walls and wide intercellular 

spaces. They measure 29 to 35 µ in length and 19 to 45 µ in breadth. 

The mesophyll cells contain scattered cluster crystals of calcium 

oxalate measuring 16 to 21µ in diameter. In addition, the mesophyll 

contains few irregular lignified idioblast measuring 14 to 31µ in length 

and 16 to 21 µ in breadth. 

The Midrib: 

The Cortical Tissue: 

The cortical tissue of the midrib (Fig. 6A& 6C) is 

parenchymatous with two collenchymatous hypodermal bands, one row 

below each epidermis. The collenchymatous cells are more or less 

rounded with thick cellulosic walls, and measure 8 to 17 µ in diameter. 

The parenchyma is formed of more or less rounded, isodiametric cells 

with thin cellulosic walls and small intercellular spaces, and measure 8 

to 33 µ in diameter. Scattered cluster crystals of calcium oxalate 

measuring 10 to 29 µ in diameter and very few prisms of calcium 

oxalate measuring 6 to 19 µ in diameter are present in the cortical 

parenchyma. 

The endodermis is not differentiated. 

The Pericycle: 

The pericycle (Fig. 6C) is collenchymatous present below and 

above the vascular bundle. Below the vascular stele the arc is 3 to 6 

cells wide; the cells are isodiametric with moderately thick cellulosic 

walls and measure 6 to 16 µ in diameter. Above the vascular stele the 

band is more or less hemispherical formed of 10 to 12 cell wide and 2

- 58 - 

PART I 

to 4 cells high; the cells have moderately thin cellulosic walls and 

measure 8 to 16 µ in diameter. 

The Vascular Tissue: 

The vascular tissue (Fig. 6C) consists of upper radiated xylem 

with a phloem band underneath. 

The xylem (Fig. 6C & 7G) is formed of polygonal cellulosic 

wood parenchyma in addition to lignified spiral and annular vessels 

measuring 12 to 21µ in diameter. 

The phloem (Fig. 6C) is formed of polygonal moderately thin- 

walled cellulosic elements. Many phloem parenchyma contain starch 

granules which are more or less rounded with indistinct hilum and no 

striations measuring 2 to 4 µ in diameter, the cells measure 6 to 12 µ in 

diameter. 

The medullary rays (Fig.6C) are uni- to biseriate, the cells are 

rectangular, radially elongated in xylem region and polygonal 

isodiametric in the phloem region; they have moderately thin cellulosic 

walls and measure 8 to 17 µ in length and 6 to 12 µ in breadth. 

The Microscopical Numerical Values of The Leaf: 

The numerical values of the leaf as a mean of four determinations 

are presented in table (10).

- 59 - 

PART I 

Table (10): Microscopical numerical values of the leaves of 

Phyllanthus atropurpureus Boj. Hort. Maurit. 

1-Stomatal Index 

Lower Epidermis 

2-Vein-Islet Number 

The numerical value Recorded value 

3-Veinlet-Termination Number 

4-Palisade Ratio 

The Upper Epidermis of Stipule: 

THE STIPULE 

64- 86 

3.63 

4.86 

3.0- 3.8 

The upper epidermis (Fig.7E) is formed of papillosed cells which 

are polygonal axially elongated with straight anticlinal walls and 

covered with thin smooth cuticle, the outer periclinal walls of the cells 

are prolonged into cylindrical or conical-shaped papillae with rounded 

apices; they measure 29 to 49 µ in length and 12 to 19 µ in breadth. 

The Lower Epidermis of Stipule: 

The lower epidermis (Fig.7F) is formed of polygonal isodiametric 

cells with thin straight anticlinal walls and is covered with smooth 

cuticle showing paracytic stomata measuring 8 to 12 µ in length and 4 

to 6 µ in breadth. 

The cells measure 10 to 19 µ in length and 10 to 25 µ in breadth.

Fig. (6): The Leaf. 

- 60 - 

PART I 

A. Diagrammatic transverse section of the leaf. (x 91.7) 

B. Detailed transverse section of the lamina. (x 430) 

C. Detailed transverse section of the midrib (x 364) 

C.cr., cluster crystal of calcium oxalate; col., collenchyma ; col.p., 

collenchymatous pericycle; l.ep., lower epidermis; lig.i., lignified 

idioblast; m.r., medullary ray; pa., papilla; pal., palisade; ph., 

phloem; pr.cr., prismatic crystal of calcium oxalate; sp.t., spongy 

tissue; u.ep., upper epidermis; v., vessels; xy., xylem.

A 

B 

pr.cr. 

- 61 - 

u.ep. 

col. 

pal. 

lig.i. 

xy. 

c.cr. 

ph. 

l.ep. 

col.p. 

col. 

v. 

m.r. 

ph. 

u.ep. 

pal. 

lig.i. 

col. 

sp.t. 

l.ep. 

l.ep. 

pa. 

PART I 

C

Fig. (7): The epidermal cells and some elements of the Leaf. 

A. Upper epidermal cells of the lamina. (x 253) 

B. Lower epidermal cells of the lamina. (x 284) 

C. Upper neural epidermal cells. (x 270) 

D. Lower neural epidermal cells. (x 253) 

E. Upper epidermal cells of the stipule. (x 344) 

F. Lower epidermal cells of the stipule. (x 505) 

- 62 - 

PART I 

G. Some elements of the leaf. (pr.cr. x 344, lig.i. x 

332, c.cr.& v.x 317) 

C.cr., cluster crystal of calcium oxalate; lig.i., lignified idioblast; 

pa., papilla; pr.cr., prismatic crystal of calcium oxalate; sec., 

secretion; v., vessel.

A 

E 

C 

F 

- 63 - 

pa. 

sec. 

Pr.cr. 

c.cr. 

G 

D 

B 

PART I 

lig.i. 

v.

THE PETIOLE 

- 64 - 

PART I 

A transverse section of the petiole (Fig. 8A& 8B) is semicircular in 

outline. It consists of epidermis surrounding a wide parenchymatous 

cortex showing a complete layer of sub-epidermal collenchyma. The 

vascular system consists of a large crescent -shaped vascular tissue, 

consists of a radiated xylem with phloem underneath, and a 

collenchymatous pericycle. 

The Upper Epidermis: 

The cells of the upper epidermis (Fig. 8B& 8C) are axially 

elongated cells with straight thick beaded anticlinal walls and covered 

with thick smooth cuticle. They contain mucilage and measure 29 to 58 

µ in length, 19 to 29µ in breadth and 10 to 17 µ in height. 

The Lower Epidermis: 

The lower epidermal cells (Fig. 8B& 8D) are polygonal, 

isodiametric with thin straight anticlinal walls and covered with thin 

smooth cuticle. The cells measure 12 to 21µ in length, 6 to 17µ in 


Stomata: 

The stomata are absent on both upper and lower epidermis. 

Trichomes: 

Both glandular and covering trichomes are completely absent on 

the epidermis of the petiole. 

The Cortical Tissue: 

The cortical tissue (Fig. 8A& 8B) is parenchymatous with a 

continuous layer of 2 to 4 rows of collenchyma below the epidermis. 

The collenchymatous cells are oval or polyhedral with moderately thick 

cellulosic walls and measure 10 to 35 µ in diameter.

- 65 - 

PART I 

The rest of the cortex is parenchymatous consisting of 8 to10 rows 

of rounded, somewhat elongated cells with small intercellular spaces. 

They measure 14 to 39µ in diameter. 

Scattered cluster crystals of calcium oxalate measuring 6 to 19 µ in 

diameter, and starch granules measuring 2 to 4µ in diameter are found 

in the cells of the cortical tissue. 

The endodermis is indistinct. 


The pericycle is collenchymatous formed of 4 to 6 rows of 

polygonal cells with thick cellulosic walls and measure 4 to 16 µ in 

length and 6 to 17 µ in width. 


The vascular tissue (Fig. 8A& 8B) is formed of an upper 

radiating xylem and lower cellulosic phloem. 

The xylem (Fig. 8B & 8E) is formed of polygonal cellulosic 

wood parenchyma and lignified spiral and annular vessels, measuring 6 

to 26 µ in diameter. 

The phloem (Fig. 8B) is formed of polygonal, moderately thin- 

walled cellulosic elements. 

The medullary rays are uni- to biseriate; the cells are rectangular, 

radially elongated in xylem region and polygonal isodiametric in the 

phloem region; they have moderately thin cellulosic walls and measure 

16 to 23 µ in length and 6 to 14 µ in breadth.

Fig. (8): The petiole. 

- 66 - 

PART I 

A. Diagrammatic transverse section of the petiole. (x 87) 

B. Detailed transverse section of the petiole. (x 273) 

C. Upper epidermal cells of the petiole. (x 333) 

D. Lower epidermal cells of the petiole. (x 469) 

E. Some elements of the petiole. (x 319) 

C.col., cortical collenchyma; c.cr.; cluster crystal of calcium oxalate; 

c.par., cortical parenchyma; col.p., collenchymatous pericycle; ep., 

epidermis; m.r., medullary rays; ph., phloem; pr.cr., prismatic 

crystal of calcium oxalate; v., vessels; xy., xylem.

C 

c.cr. 

A 

E 

D 

pr.cr. 

v. 

ep. 

c.col 

xy. 

c.par. 

ph. 

col. 

p. 

v. 

m.r. 

ph. 

col. 

p. 

c.par. 

c.cr. 

c.col. 

- 67 - 

B 

PART I

THE POWDERED LEAF 

- 68 - 

PART I 

The powdered leaf is yellowish green in colour with slight 

characteristic odour and slightly bitter taste. It is characterized 

microscopically by the following features: 

1. Fragments of the upper epidermis of the lamina; the cells are 

polygonal with thin straight anticlinal walls and thin smooth 

cuticle showing transparent secretion which appear as 

transparent dots and many cells have short conical-shaped 

papillae. 

2. Fragments of the lower epidermis of the lamina; the cells are 

polygonal with thin slightly wavy anticlinal walls and thin 

smooth cuticle showing paracytic stomata and many cells have 

short conical-shaped papillae. 

3. Fragments of the upper epidermis of the midrib; the cells are 

axially elongated with thin straight anticlinal walls and covered 

with thin smooth cuticle showing no stomata. 

4. Fragments of the lower epidermis of the midrib; the cells are 

axially elongated with thick beaded anticlinal walls and covered 

with thin smooth cuticle showing no stomata. 

5. Fragments of the upper epidermis of the petiole; the cells are 

axially elongated with thick straight beaded anticlinal walls and 

covered with thick smooth cuticle showing no stomata. 

6. Fragments of the lower epidermis of the petiole; the cells are 

polygonal isodiametric with thin straight anticlinal walls and 

covered with thin smooth cuticle showing no stomata. 

7. Fragments of cortical collenchyma and parenchyma.

- 69 - 

PART I 

8. Fragments of the lamina showing dorsiventral structure with one 

layer of palisade and numerous cluster crystals of calcium 

oxalate. 

9. Fragments of lignified spiral vessels. 

10. Numerous cluster crystals of calcium oxalate and very few 

prisms of calcium oxalate. 

11. Fragments of the lamina showing lignified irregular idioblasts 

with very thick walls. 

12. Fragments of the upper epidermis of the stipule; the cells are 

papillosed, polygonal axially elongated with straight anticlinal walls 

and covered with thin smooth cuticle showing no stomata. 

13. Fragments of the lower epidermis of the stipule; the cells are 

polygonal isodiametric with thin straight anticlinal walls and 

covered with thin smooth cuticle showing paracytic stomata.

THE STEM 

- 70 - 

PART I 

A transverse section of the young stem (Fig. 10 A) is more or less 

circular in outline and show an outer epidermis followed by a 

collenchymatous hypodermis and parenchymatous cortex. The 

pericycle is collenchymatous. The vascular tissue is formed of a 

complete ring of 16 to 20 vascular bundles each with an outer phloem 

and inner xylem surrounding wide parenchymatous pith. 

The transverse section of the old stem (Fig. 9A) is circular in 

outline. It shows an outer cork arising below the epidermal layer, then 

the parenchymatous cortex occupying one-tenth of the diameter. The 

pericycle is parenchymatous and showing batches of non-lignified 

pericyclic fibres. The vascular tissue forms a complete ring of an outer 

phloem and inner xylem (which occupy two-third of the diameter) and 

is transversed by numerous medullary rays. The pith is narrow and 

parenchymatous. 

The Cork: 

The cork (Fig. 9B1& 10D) consists of 6 to 7 rows of radially 

arranged polygonal to rectangular cells with moderately thick lignified 

walls and brown contents. They measure 37 to 41 µ in length, 31 to 35 

µ in breadth and 9 to 11 µ in height. 

The Phellogen: 

The phellogen arises in the subepidermal layer and is 

indistinguished.

The Epidermis: 

- 71 - 

PART I 

The epidermal cells of the young stem (Fig. 10B& 10C) are 

polygonal, slightly axially elongated with straight anticlinal walls and 

covered with thin smooth cuticle. 

They measure 27 to 31µ in length, 14 to 18 µ in breadth and 5 to 7 

µ in height. 

Stomata: 

The epidermis of the young stem (Fig. 10C) shows very few 

stomata of paracytic type, measuring 17 to 21 µ in length and 19 to 23 µ 

in breadth. 

Trichomes: 

Both glandular and covering trichomes are completely absent in 

the young stem. 

The Cortex: 

The cortex (Fig. 9B1) is formed of 5 to 7 rows of more or less 

rounded parenchymatous cells with narrow intercellular spaces; they 

measure 19 to 33 µ in length and 17 to 58µ in breadth. The cortex show 

tangentially elongated lacunae which measure 21 to 28µ in length and 

21 to 30µ in breadth. 

The endodermis is formed of tangentially elongated rectangular 

parenchymatous cells, which measure 18 to 20 µ in length and 66 to 

70µ in breadth. 

In young stem, the cortex (Fig. 10B) is formed of one row of more 

or less rounded collenchymatous hypodermal cells with moderately 

thick walls measuring 15 to 20 µ in diameter followed by 6 to 8 rows of 

rounded parenchymatous cells with narrow intercellular spaces. They

- 72 - 

PART I 

measure 17 to 24 µ in length and 20 to 29 µ in breadth. The endodermis 

is formed of tangentially elongated rectangular parenchymatous cells, 

which measure 14 to 20 µ in length and 25 to 33 µ in breadth. 

Numerous cluster crystals of calcium oxalate are scattered in the 

cortical cells. They measure 10 to 16 µ in diameter. 


The pericycle of old stems (Fig. 9B1) is formed of an interrupted 

ring formed of groups of non-lignified pericyclic fibres, separated by 

tangentially elongated rectangular parenchymatous cells measuring 15 

to 17µ in length and 37 to 41 µ in breadth. Each group of pericyclic 

fibres consists of 3 to 5 rows of non-lignified fibres. Fibres (Fig.10D) 

are spindle-shaped with smooth thick non-lignified walls, moderately 

narrow lumens and blunt apices; they measure 385 to 395 µ in length 

and 18 to 21µ in diameter. The pericyclic parenchyma is formed of 

polygonal or rounded cells with thin cellulosic walls and measure 10 to 

25 µ in length, 14 to 39 µ in breadth. 

Between the pericyclic fibres, there are some tangentially 

elongated lacunas that measure 14 to 18 µ in length and 37 to 41 µ in 

breadth. 

In the young stem (Fig.10B), the pericycle is formed of an 

interrupted ring of 3 to 5 rows of collenchymatous cells. The cells are 

polygonal with thin cellulosic walls, and measure 6 to 14 µ in length, 4 

to 16 µ in breadth.

Fig. (9): The old stem. 

- 73 - 

PART I 

A. Diagrammatic transverse section of the old stem. (x 31.3) 

B1 & B2. Detailed transverse section of the old stem. (x 307) 

C.par., cortical parenchyma; ck., cork; end., endodermis; lac., lacuna; 

m.r., medullary rays; p., pith; p.f., pericyclic fibre; p.par., pericyclic 

parenchyma; ph., phloem; pt.par., pitted parenchyma; v., vessel; w.f., 

wood fibre; w.p., wood parenchyma; xy., xylem.

B1 

- 74 - 

ck. 

p.f. 

ph. 

xy. 

m.r. 

c.par. 

p. 

end. 

p.f. 

p.par. 

m.r. 

ph. 

p. 

v. 

w.f. 

pt.par. 

w.par. 

B2 

PART I 

A

Fig. (10): The young stem. 

- 75 - 

PART I 

A. Diagrammatic transverse section of the young stem. (x 61) 

B. Detailed transverse section of the young stem. (x 345) 

C. Epidermal cells of the young stem. (x 364) 

D. Some elements of the old stem. (ck. x 239, p.f.& w.f. x 148, 

c.cr.x 369, w.par., m.r.& 

v. x 322.5and pt.par. x 284) 

C.cr., cluster crystal of calcium oxalate; c.par., cortical parenchyma; 

ck., cork; col., collenchyma; col.p., collenchymatous pericycle; end., 

endodermis; ep., epidermis; m.r., medullary ray; p., pith; p.f., 

pericyclic fibre; ph., phloem; pt.par., pitted parenchyma; v., vessels; 

w.f., wood fibre; w.par., wood parenchyma; xy., xylem.

ck. 

w.par. 

D 

m.r. 

A 

C 

p.f. 

pt.par. 

c.cr. 

w.f. 

v. 

p. 

- 76 - 

ep. 

col. 

c.cr. 

p. 

c.par. 

end. 

col.p. 

ph. 

xy. 

m.r. 

v. 

PART I 

B


- 77 - 

PART I 

The vascular tissue (Fig. 9A & 9B 1, 2) is formed of a complete 

ring of an outer phloem and inner xylem and transversed by medullary 

rays. 

The Phloem: 

The phloem (Fig. 9B1) consists of wide band of soft tissue 

composed of large moderately thick walled polygonal cellulosic 

elements. The phloem contains scattered elongated lacuna containing 

tannin, measuring 7 to 9 µ in length and 10 to 13 µ in diameter. 

The Xylem: 

The xylem (Fig. 9B1, 2& 10D) is wholly lignified and mainly 

consists of numerous wood fibres with scattered wood parenchyma and 

vessels. Vessels are spiral and annular, they measure 19 to 23 µ in 

diameter. 

The wood fibres (Fig. 10D) are spindle-shaped with thick or thin 

lignified walls, narrow or wide lumens and acute or blunt apices. They 

measure 19 to 22 µ in length and 10 to 13 µ in diameter. 

The wood parenchyma (Fig. 10D) are diffused and consist of 

polygonal, usually axially elongated cells with slightly lignified, thick 

pitted walls, showing simple pits and measuring 27 to 31 µ in length 

and 14 to 18 µ in diameter. 

The Medullary Rays: 

The medullary rays (Fig. 9A, 9B&10D) are mostly uniseriate rarely 

biseriate. The cells are rectangular radially elongated with lignified 

thick pitted walls in the xylem and rectangular parenchymatous with 

cellulosic walls in phloem. The cells measure 33 to 37 µ in length and 8 

to 12 µ in breadth.

- 78 - 

PART I 

In young stem (Fig. 10A & 10 B), the vascular tissue is formed of 

a complete ring of 16 to 20 vascular bundles, each with an outer phloem 

of cellulosic elements and inner xylem showing lignified spiral and 

annular vessels and cellulosic wood parenchyma. The vessels measure 

10 to 19 µ in diameter. 

The Pith: 

The pith (Fig. 9A& 9B2) is narrow, composed of somewhat 

rounded cells. At the periphery, the cells are rounded, small in size and 

with thin cellulosic walls. Becoming larger in size, with slightly thick 

pitted walls in the centre (i.e. pitted parenchyma), cluster crystals of 

calcium oxalate and starch granules are present in the parenchyma of 

pith. The cluster crystal measure 21 to 25 µ in diameter. The starch 

granules are simple, without hilum or striations and measuring 2 to 4 µ 

in diameter. 

In young stem (Fig. 10A& 10B), the pith is formed of more or less 

rounded thin-walled small cellulosic cells at premedullary zone 

measuring 10 to 14 µ in diameter, followed by more larger rounded 

cells with wide intercellular spaces. The cells measure 43 to 47 µ in 

diameter. 

Cluster crystals of calcium oxalate measuring 19 to 27 µ in diameter 

and rounded or oval-shaped simple starch granules measuring 6 to 8 µ 

in diameter are present in the parenchyma of pith.

THE POWDERED STEM 

- 79 - 

PART I 

The powdered stem is greenish-brown in color with slight 

characteristic odour and faint characteristic taste. It is characterized 

microscopically by the following fragments: 

1. Fragments of polygonal to rectangular cork cells with moderately 

thick lignified straight walls and brown contents. 

2. Fragments of epidermis consisting of polygonal elongated cells 

with straight anticlinal walls and covered with thin smooth 

cuticle, and shows few stomata of paracytic type. 

3. Numerous cluster crystals of calcium oxalate free or in 

parenchymatous cells. 

4. Fragments of non-lignified pericyclic fibres, being spindle- 

shaped with narrow lumens and straight, thick walls and blunt or 

pointed apices. 

5. Fragments of lignified vessels, having spiral and annular 

thickenings. 

6. Fragments of lignified wood parenchyma with simple pits. 

7. Fragments of wood fibres with thin or thick lignified pitted walls, 

wide or narrow lumens and blunt apices. 

8. Fragments of parenchymatous cortical cells, sometimes 

containing cluster crystal of calcium oxalate. 

9. Numerous simple starch granules which are more or less rounded 

with indistinct hilum.

THE FLOWER 

1-THE PEDICEL 

- 80 - 

PART I 

A transverse section of the flower stalk (Fig. 11A& 11B) is almost 

circular in outline. It shows an outer epidermis surrounding a wide 

cortex formed of an outer collenchymatous hypodermis and inner 

parenchyma. The vascular tissue is formed of a complete ring of 8 to 12 

separated collateral vascular bundles surrounding narrow 

parenchymatous pith. The pericycle is formed of collenchyma abutting 

each vascular bundle. 

The Epidermis: 

The epidermal cells of the pedicel (Fig. 11B& 11C) are polygonal 

and slightly axially elongated with straight anticlinal walls and covered 

with thin smooth cuticle. They measure 21 to 48 µ in length, 10 to 17 µ 

in breadth and 8 to 12 µ in height. 

Stomata and trichomes are completely absent on the surface of the 

pedicel. 

The Cortex: 

The cortex (Fig. 11B) is formed of a single layer of 

collenchymatous hypodermis and 5 to 7 rows of parenchymatous cells. 

The hypodermal cells are large polyhedral collenchymatous with thin 

walls and measure 12 to 17 µ in diameter. The rest of the cortex is 

formed of rounded, irregular parenchymatous cells with thin cellulosic 

walls and small intercellular spaces; they measure 16 to 27 µ in 

diameter. Scattered cluster crystals of calcium oxalate measuring 14 to 

17 µ in diameter are present in the cortical parenchyma. 

The endodermis is undifferentiated.


- 81 - 

PART I 

The pericycle (Fig. 11B) is formed of small groups of 1 to 3 rows 

of polygonal collenchymatous cells present abutting the vascular 

bundle. The pericyclic collenchyma have moderately thick walls and 

measure 6 to 14 µ in diameter. 


The stele (Fig. 11A& 11B) is formed of a ring of 8 to 12 vascular 

bundles separated by narrow medullary rays; each composed of an outer 

phloem and inner xylem. 

The Phloem: 

The phloem (Fig.11B) consists of polygonal moderately thin- 

walled cellulosic elements. 

The Xylem: 

The xylem (Fig. 11B & 11E) is formed of lignified spiral and 

annular vessels measuring 4 to 10 µ in diameter and cellulosic wood 

parenchyma. 

The Medullary Rays: 

The primary medullary rays (Fig. 11A& 11B) are multiseriate, the 

cells are rectangular, radially elongated and cellulosic. The secondary 

medullary rays are uni- to biseriate, radially elongated in xylem region 

and polygonal isodiametric in the phloem region. They have moderately 

thin cellulosic walls and measure 6 to 14 µ in length and 6 to 10 µ in 

breadth. 

The Pith: 

The pith (Fig. 11A& 11B) is narrow and composed of somewhat 

rounded parenchymatous cells with thin cellulosic walls and small 

intercellular spaces. The cells measure 16 to 23 µ in diameter.

Fig (11): The Pedicel: 

- 82 - 

PART I 

A. Diagrammatic transverse section of the pedicel. (x 96.5) 

B. Detailed transverse section of the pedicel. (x 722.5) 

C. Epidermal cells of the pedicel. (x 500.6) 

D. Cluster crystals of calcium oxalate. (x 322.5) 

E. Xylem vessels. (x 451.5) 

C., cortex; c.cr, cluster crystals of calcium oxalate; c.par., cortical 

parenchyma; col., collenchyma; ep.,epidermis; m.r., medullary 

rays; p., pith; p.col., pericyclic collenchyma; ph., phloem; v., vessel; 

w.p., wood parenchyma; xy., xylem.

C 

A 

D 

E 

ep. 

col. 

c. 

xy. 

p. 

c.cr. 

c.par. 

p.col. 

ph. 

w.p. 

m. 

r. v. 

p. 

- 83 - 

B 

PART I

2-THE CALYX 

- 84 - 

PART I 

A transverse section of the sepal (Fig. 12A& 12B) shows an outer 

and inner epidermis enclosing a parenchymatous mesophyll with one 

row of a hypodermal layer formed of rectangular collenchymatous cells 

below outer and inner epidermis. It is traversed longitudinally by few 

vascular bundles and contains red contents that are localized mainly in 

outer epidermis and the hypodermal layer. 

The Outer Epidermis: 

The outer epidermis of the sepal consists of polygonal cells with 

straight occasionally beaded anticlinal walls and smooth cuticle. 

At the lobe and at the tube (Fig. 12C& 12D); the cells have 

straight occasionally beaded anticlinal walls, smooth cuticle and show 

paracytic stomata. They measure 8 to 25 µ in length, 6 to 33 µ in 


Some cells show papilla, which measure 6 to 10 µ in height. 

Over the vein (Fig. 12E), the cells are axially elongated with 

straight occasionally beaded anticlinal walls and smooth cuticle. The 

cells measure 25 to 56 µ in length and 8 to 17 µ in breadth. 

The Inner Epidermis: 

The inner epidermis of the sepal consists of polygonal cells with 

straight occasionally beaded anticlinal walls and smooth cuticle. 

It shows no stomata, papillosed cells or trichomes. 

At the lobe (Fig. 12F); the cells are small isodiametric with very 

thin straight anticlinal walls and smooth cuticle. They measure 10 to 25 

µ in length and 12 to 25 µ in breadth.

- 85 - 

PART I 

The epidermal cells show cluster crystals of calcium oxalate 

measuring 12 to17 µ in diameter and other cell contents which are not 

affected by strong alkalis or acids. 

Of the tubular part (Fig. 12G); the cells are axially elongated 

with thick straight anticlinal walls and smooth cuticle. They measure 23 

to 39 µ in length and 14 to 23 µ in breadth. 

Over the veins of the lobe (Fig. 12 H), the cells are axially 

elongated with thick straight occasionally beaded anticlinal walls and 

smooth cuticle. They measure 27 to 45 µ in length and 10 to 16 µ in 

breadth. 

It shows cluster crystals of calcium oxalate measuring 8 to 12 µ in 

diameter and other cell contents which are not affected by strong alkalis 

or acids. 

Over the veins at the tubular part (Fig. 12I), the cells are 

axially elongated with thick straight occasionally beaded anticlinal 

walls and smooth cuticle. They measure 19 to 35 µ in length and 10 to 

17 µ in breadth. 

It shows cluster crystals of calcium oxalate measuring 10 to 14 µ 

in diameter and other cell contents which are not affected by strong 

alkalis or acids. 

Stomata: 

Stomata are present only on the outer epidermis (Fig.12C& 12D), 

they are of paracytic type and measure 12 to 16 µ in diameter.

Trichomes: 

Trichomes are completely absent. 

The Mesophyll: 

- 86 - 

PART I 

The mesophyll (Fig.12B) consists of one row of a hypodermal 

layer formed of rectangular collenchymatous cells below outer and 

inner epidermis; the cells measure 12 to 23 µ in length and 14 to 27 µ in 

breadth. The rest of the mesophyll is formed of almost rounded 

parenchymatous cells with small intercellular spaces. They measure 16 

to 33 µ in diameter. Many of the parenchymatous cells of the mesophyll 

contain cluster crystals of calcium oxalate which measure 8 to 16 µ in 

diameter. 


The pericycle is parenchymatous formed of moderately thick- 

walled cells. They measure 8 to 12 µ in length and 10 to 16µ in breadth. 

The vascular bundles are fifteen to eighteen, each consists of an 

upper xylem showing lignified spiral vessels measuring 6 to 10 µ in 

diameter and a lower phloem formed of moderately thick-walled 

cellulosic elements.

Fig. (12): The calyx. 

- 87 - 

PART I 

A. Diagrammatic transverse section of the calyx. (x 105) 

B. Detailed transverse section of the calyx. (x 352) 

C. Outer epidermis of calyx- lobe. (x 352) 

D. Outer epidermis of calyx - tube. (x 307) 

E. Outer epidermis of calyx over the vein. (x 322.5) 

F. Inner epidermis of calyx - lobe. (x 288.5) 

G. Inner epidermis of calyx - tube. (x 405) 

H. Inner epidermis of calyx - lobe over the vein. (x 288.5) 

I. Inner epidermis of calyx - tube over the vein. (x 322.5) 

C.cr, cluster crystal of calcium oxalate; col.hy., collenchymatous 

hypodermis; i.ep., inner epidermis; m., mesophyll; o.ep., outer 

epidermis; pap., papilla; ph., phloem; v.b., vascular bundle; xy., 

xylem.

C 

F 

A 

pap. 

G 

i.ep 

. 

col.hy. 

c.cr. 

m. 

D 

xy. 

ph. 

v.b. 

col.hy. 

o.ep. 

- 88 - 

H 

B 

E 

PART I 

I

3-THE ANDROECIUM 

- 89 - 

PART I 

The androecium (Fig.13A) consists of six monodelphous stamens 

with 6 free anthers arranged in one whorl. 

1- Anther: 

A transverse section of the anther (Fig.13A) consists of two lobes 

separated by the connective; each lobe consists of one pollen sac 

containing pollen grains. 

The anther wall (Fig.13B) is thin and formed of an outer epidermis 

followed by a single row of fibrous layer and the remaining of tapetum. 

Epidermis: 

The epidermal cells (Fig. 13B) of the anther-lobes are polygonal 

with straight anticlinal walls and covered with thin smooth cuticle. 

They measure 10 to 14µ in length, 17 to 23 µ in width and 6 to 10 µ in 

height. The epidermis of the anther is devoid of stomata and trichomes. 

Fibrous layer: 

The fibrous layer of the anther (Fig.13B) is formed of one row of 

polygonal axially elongated cells with straight anticlinal walls showing 

lignified bar-like thickening appear beaded in surface view (Fig.13E). 

They measure 10 to 17µ in length, 31 to 41µ in breadth and 35 to 39µ 

in height. 

Tapetum: 

Tapetum (Fig.13B) is formed of collapsed cells.

Pollen grains: 

- 90 - 

PART I 

The pollen grains (Fig.13 F) are yellow in colour, spherical with 

three germ pores, three germinal furrows and finely granular exine. 

They measure 19 to 23 µ diameter. 

2-Staminal column (Filaments): 

A transverse section of the staminal column (Fig.13 C) is nearly 

rounded in outline and formed of an outer epidermis surrounding a 

ground tissue formed of one or two rows of moderately thick-walled 

rectangular collenchymatous cells followed by 7 to 9 rows of thin- 

walled rounded parenchymatous cells. The hypodermal cells measure 

12 to 18 µ in length and 10 to 16 µ in breadth while the 

parenchymatous cells measure 10 to 19µ in diameter. It is traversed 

longitudinally by three small vascular bundles. The vascular strand is 

formed of few delicate spiral vessels measuring 4 to 6µ in diameter and 

cellulosic elements of the phloem. 

Few cluster crystals of calcium oxalate measuring 8 to 16 µ in 

diameter and many reddish contents are present in the cells of the 

ground tissue. 

Epidermis: 

The epidermal cells of staminal column at the apex (Fig.13D& 

13G) are polygonal isodiametric to rectangular axially elongated cells 

with straight thin anticlinal walls and covered with thin smooth cuticle. 

They measure 16 to 25µ in length, 12 to 20 µ in breadth and 8 to 12 µ 

in height. 

At the base, (Fig.13D& 13H) the epidermal cells are rectangular 

axially elongated and measure 35 to 48µ in length, 8 to 19µ in breadth 

and 8 to 14µ in height.

Fig. (13): The Androecium: 

- 91 - 

PART I 

A. Diagrammatic transverse section of androecium in anthers 

region. (x 117) 

B. Detailed transverse section of anther wall. 

C. Diagrammatic transverse section of the staminal column 

(x 495.5) 

below the anthers. 

D. Detailed transverse section of the staminal column below 

(x 143.5) 

the anthers. (x 660) 

E. Fibrous layer in surface view. (x 279) 

F. Pollen grains. (x 436.6) 

G. Epidermal cells of the staminal column at apex. (x 383) 

H. Epidermal cells of the staminal column at base. (x 279) 

C., connective; c.cr., cluster crystals of calcium oxalate; col.hy., 

collenchymatous hypodermis; c.par., cortical parenchyma; ep., 

epidermis; f.l., fibrous layer; g.t., ground tissue; ph., phloem; tap., 

tapetum; v., vessels; v.st., vascular strand.

E 

A 

C 

G 

F 

ep. 

ep. 

f.l. 

c. 

tap 

. 

ph. 

v. 

v.st. 

col.hy. 

v.st. 

g.t 

. 

c .par. 

c.cr. 

H 

- 92 - 

B 

D 

PART I

1- The ovary: 

4-THE GYNAECIUM 

- 93 - 

PART I 

A transverse section of the tricarpellary ovary (Fig.14A& 14B) is 

almost rounded or three-sided in outline. It is formed of an outer and 

inner epidermis enclosing a parenchymatous mesophyll in between. 

The mesophyll (Fig. 14B) shows one row of collenchymatous cells 

under outer epidermis; measuring 10 to 14 µ in height. The rest of the 

mesophyll is formed of rounded, moderately thin-walled parenchyma 

with small intercellular spaces and traversed by three main vascular 

strands. Each vascular strand is composed of a xylem showing few 

spiral vessels and a phloem formed of thin-walled cellulosic elements. 

Many cluster crystals of calcium oxalate are scattered in the 

mesophyll cells, measuring 8 to 12 µ in diameter. 

Epidermis: 

The outer epidermal cells of the ovary at the apex (Fig. 14B& 14C) 

are polygonal axially elongated cells with thin straight occasionally 

beaded anticlinal walls and covered with thin smooth cuticle. They 

measure 27 to 45µ in length, 14 to 19 µ in breadth and 6 to 10 µ in 

height. 

At the base, the epidermal cells of the ovary (Fig.14D) are polygonal 

isodiametric to axially elongated cells with thin straight anticlinal walls 

and thin smooth cuticle. They measure 23 to 35 µ in length, 10 to 23 µ 

in breadth and 4 to 8 µ in height. 

Red contents are present in outer epidermal cells. 

Stomata and trichomes are completely absent.

2- The style: 

- 94 - 

PART I 

A transverse section of the style (Fig.14E& 14F) is nearly 

lenticular in outline with a notched on the middle of the inner side and 

formed of an epidermis surrounding a parenchymatous ground tissue 

traversed longitudinally by one vascular strand. 

Epidermis: 

The epidermis of the style (Fig.14G) is formed of polygonal 

isodiametric cells with straight anticlinal moderately thick walls and 

covered with thin smooth cuticle. They measure 10 to 17 µ in length, 10 

to 19 µ in breadth. The epidermal cells of the style over the vein 

(Fig.14G) measure 10 to 21µ in length, 12 to 15µ in breadth. 

Some epidermal cells contain transparent secretions which appear as 

transparent dots. Stomata and trichomes are completely absent. 

The outer parenchymatous cells of the ground tissue (Fig. 14F) 

are polygonal cells with thin cellulosic walls, narrow intercellular 

spaces and contain few calcium oxalate cluster crystals which measure 

12 to 14µ in diameter. Near the vascular strand, the cells are polygonal 

isodiametric to rectangular, axially elongated cells with thin walls and 

narrow intercellular spaces. Near the inner side (below the vascular 

bundle) the cells are polygonal isodiametric and small with thin 

cellulosic walls, narrow intercellular spaces and many cells have red 

contents. 

The vascular strand shows few lignified spiral vessels of the xylem 

and thin walled cellulosic elements of the phloem.

3- The stigma: 

- 95 - 

PART I 

The epidermis of the stigma (Fig.14H) is composed of papillosed 

cells which are polygonal axially elongated with straight anticlinal 

walls and covered with smooth cuticle, the outer periclinal walls of the 

cells are prolonged into cylindrical or conical-shaped papillae with 

rounded apices; they measure 10 to 17µ in length, 8 to 14µ in breadth. 

Stomata and trichomes are completely absent.

Fig. (14): The gynaecium: 

A. Diagrammatic transverse section of the ovary. (x 81) 

B. Detailed transverse section of ovary wall. (x 421) 

- 96 - 

PART I 

C. Outer epidermis of the ovary wall at the apex. (x 303.5) 

D. Outer epidermis of the ovary wall at the base. (x 283) 

E. Diagrammatic transverse section of the style. (x 81) 

F. Detailed transverse section of the style. (x 536) 

G. Epidermis of the style. (x 317.5) 

H. Epidermis of the stigma. (x 368.5) 

C.cr, cluster crystal of calcium oxalate; col., collenchyma; ep., 

epidermis; g.t., ground tissue; i.ep., inner epidermis; m., mesophyll; 

o.ep, outer epidermis; ph., phloem; v.st., vascular strand; xy., 

xylem.

C 

G 

E 

A 

D 

o.ep. 

col. 

m 

c.cr . 

. Ph. 

xy. 

i.ep. 

ep. 

c.cr 

v.st. 

ph. 

xy. 

H 

ep. 

g.t. 

g.t 

- 97 - 

B 

F 

PART I

THE POWDERED FLOWER 

- 98 - 

PART I 

The powdered flower is reddish-brown in colour with slight odour 

and slightly bitter taste. It is characterized microscopically by the 

following features: 

1. Numerous cluster crystals of calcium oxalate free or in 

parenchymatous cells. 

2. Fragments of the epidermal cells of the pedicel which are 

polygonal and slightly axially elongated with straight anticlinal 

walls, occasional paracytic stomata and covered with thin smooth 

cuticle. 

3. Fragments of the epidermal cells of sepals formed of polygonal 

cells with straight occasionally beaded anticlinal walls and smooth 

cuticle. 

4. Fragments of the epidermal cells of the anther which are polygonal 

with straight anticlinal walls and covered with thin smooth cuticle. 

5. Fragments of the fibrous layer of the anther, the cells are 

polygonal, axially elongated having lignified beaded anticlinal 

walls. 

6. Fragments of the epidermis of the staminal column formed of 

polygonal isodiametric to rectangular axially elongated cells with 

straight thin anticlinal walls and covered with thin smooth cuticle. 

7. Numerous spherical yellow pollen grains with three germ pores, 

three germinal furrows and finely granular exine. 

8. Fragments of the epidermal cells of the ovary formed of 

polygonal, axially elongated or isodiametric cells with straight 

occasionally beaded anticlinal walls and covered with thin smooth 

cuticle.

- 99 - 

PART I 

9. Fragments of the epidermal cells of the style formed of 

polygonal, isodiametric cells with straight anticlinal moderately 

thick walls and covered with thin smooth cuticle. 

10. Fragments of the epidermal cells of the apical part of the stigma 

lobes formed of papillosed cells which are polygonal axially 

elongated with straight anticlinal walls and covered with smooth 

cuticle. 

11. Fragments of lignified spiral vessels.

THE ROOT 

- 100 - 

PART I 

The transverse section of the root (Fig. 15A& 15B) is almost 

circular in outline. It is formed of an outer brownish cork followed by a 

wide parenchymatous phelloderm, surrounding a cylinder of vascular 

tissue comprises a narrow outer phloem and a wide inner xylem with 

cambium in between and a central diarch primary xylem. 

The transverse section of the rhizome (Fig.16B) is almost circular 

showing an outer brown cork, somewhat wide phelloderm, interrupted 

bands of sclerides in the pericycle and a wide cylinder of vascular 

strand traversed longitudinally by the medullary rays and surrounding a 

central narrow parenchymatous pith. 

The Cork: 

The cork (Fig. 15B1& 16 C) consists an outer tangentially 

elongated cells arranged in 2 to 3 rows of lignified, polygonal, 

moderately thick-walled cells, followed by a band of 3 to 4 rows of 

polygonal, moderately thick-walled, suberized cells. The cells are 

polygonal, tangentially elongated and arranged in radial rows. They 

measure 21 to 48µ in length, 16 to 39µ in breadth and 8 to 16µ in 

height. 

The Phelloderm: 

The Phelloderm (Fig. 15B1) is wide and parenchymatous, formed of 

5 to 6 rows of thin-walled polygonal cellulosic cells with narrow 

intercellular spaces; few mucilage cavities are present. The cells 

measure 16 to 39µ in length and 39 to 77µ in breadth. Many of these 

cells contain brownish granular contents which give bluish black

- 101 - 

PART I 

colour with ferric chloride (T.S) and a yellow colour with caustic alkali. 

Numerous cluster crystals of calcium oxalate measuring 14 to 25 µ in 

diameter are present in some of these parenchymatous cells. 

More or less rounded or polyhedral starch granules with indistinct 

hilum and no striations measuring 2 to 4µ in diameter are present in the 

phelloderm cells. 


The vascular tissue (Fig. 15A& 15B1, 2) is formed of a cylinder 

consists of an outer narrow phloem and a wide inner xylem with 

cambium in between and transversed by uni- or biseriate medullary 

rays. 

The phloem (Fig. 15B1) consists of polygonal, thin-walled 

cellulosic elements. Many cells of phloem parenchyma show brownish 

granular contents which give bluish black colour with ferric chloride 

(T.S) and a yellow colour with caustic alkali. Few starch granules are 

present which is more or less rounded or polyhedral with indistinct 

hilum and no striations measuring 2 to 4 µ in diameter. The phloem 

shows no fibres. 

The cambium (Fig. 15B1) consists of 3 to 4 rows of thin-walled 

tangentially elongated rectangular meristematic cells. 

The xylem (Fig. 15B1) is wide, lignified and consists mainly of 

wood fibres, xylem vessels with few tracheides and wood parenchyma. 

It is transversed radially by moderately thick-walled cellulosic 

medullary rays. In the center of the young root (Fig. 16A) there is a 

diarch primary xylem formed of few spiral vessels.

- 102 - 

PART I 

• Wood fibres (Fig. 16C) are spindle-shaped with moderately thick 

lignified walls with slit-like pits, wide lumen and acute apices. They 

measure 291 to 330µ in length and 8 to 29µ in diameter. 

• Xylem vessels (Fig. 16C) are diffused and occur either isolated 

or in radial rows of 2 to 6 vessels. They have pitted lignified walls with 

oval or rounded bordered pits and measure 12 to 48µ in diameter. 

• Few tracheides (Fig.16C) are present showing blunt apices and 

moderately thick lignified walls showing rounded bordered pits. They 

measure 126 to 136 µ in length and 10 to 23 µ in breadth. 

• The wood parenchyma (Fig. 15B1, 15B2 & 16C) is either 

metatracheal or vasocentric and formed of polygonal, axially elongated 

cells. They have moderately thick lignified walls showing simple pits 

and measure 39 to 64 µ in length and 17 to 31µ in breadth. Most of 

these cells contain brownish granular contents, which give bluish black 

colour with ferric chloride (T.S). They also contain starch granules 

which are more or less rounded with indistinct hilum and no striations 

and measure 2 to 4 µ in diameter. 

The medullary rays (Fig. 15B1& 16C) are usually uni- or 

biseriate and formed of radially elongated rectangular cellulosic cells 

with moderately thick walls in the xylem region and of subrectangular 

parenchymatous cells with cellulosic walls in the phloem region; few 

cells show brownish granular contents which give bluish black colour 

with ferric chloride (T.S) and a yellow colour with caustic alkali. The 

cells measure 12 to 29 µ in length and 21 to 33 µ in breadth in the 

phloem region and 17 to 31 µ in length and 16 to 25 µ in breadth in the 

xylem region.

Fig. (15): The old root: 

- 103 - 

PART I 

A. Diagrammatic transverse section of the old root. (x 30.5) 

B1 &B2. Detailed transverse section of the old root. (x 307) 

B.gr., brown granules; c.cr., cluster crystals of calcium oxalate; 

cb., cambium; lig.ck., lignified cork; m.r., medullary rays; pd., 

phelloderm; ph., phloem; sub.ck., suberized cork; w.f., wood 

fibre; w.p., wood parenchyma; v., vessels.

.gr. 

A 

B2 

lig.ck. 

sub.ck. 

Pd. 

Ph. 

cb. 

c.cr. 

m.r. 

pd. 

b.gr. 

w.p. 

v. 

cb. 

w.f. 

v. 

m.r. 

w.p. 

- 104 - 

ph. 

b.gr. 

B1 

PART I

Fig. (16): The root and rhizome: 

- 105 - 

PART I 

A. Diagrammatic transverse section of the young root. (x 85) 

B. Diagrammatic transverse section of the rhizome. (x 26) 

C. Some elements of the root and rhizome. (ck.x 315, c.cr.x 561, 

w.f., tra. & v. x 383 and 

scl., w.p. & m.r. x 360) 

C.cr., cluster crystals of calcium oxalate; cb., cambium; ck., cork; 

end., endodermis; m.c.; mucilage cavity; m.r., medullary rays; p.l., 

piliferous layer; pd., phelloderm; ph., phloem; pr., pericycle; scl., 

scleride; tra., tracheid; w.f., wood fibres; w.p., wood parenchyma; v., 

vessels; xy., xylem.

ck. 

w.f. 

A 

scl. 

tra. 

p.l. 

pr. 

end. 

ph. 

xy. 

m.c 

v. 

v. 

- 106 - 

ck. 

c.cr. 

scl. 

ph. 

cb. 

m.r. 

w.p. 

v. 

p. 

C 

w.p. 

c.cr. 

PART I 

B 

B 

m.r.

THE POWDERED ROOT AND RHIZOME 

- 107 - 

PART I 

The powdered root and rhizome (Fig. 16C) is reddish-brown in 

colour with faint characteristic dusty odour and a bitter taste. It is 

characterized microscopically by the following fragments: 

1. Fragments of polygonal thick-walled lignified or suberized cork 

cells. 

2. Lignified sclerides isolated or in groups with moderately wide 

lumen and thin lignified walls. 

3. Fragments of lignified xylem vessels with pitted moderately 

thick walls showing rounded or oval bordered pits. 

4. Fragments of lignified wood fibres .each with moderately thick, 

lignified walls with slit-like pits, wide lumen and acute apices. 

5. Fragments of wood parenchyma. The cells have thick, lignified 

pitted walls and may contain brownish contents or starch grains. 

6. Fragments of the phelloderm parenchymatous cells containing 

starch granules, cluster crystals of calcium oxalate and/or 

brownish granular contents. 

7. Cluster crystals of calcium oxalate, scattered or in the 

phelloderm cells. 

8. More or less rounded starch granules with indistinct hilum and 

no striations measuring 2 to 4 µ in diameter in the phelloderm 

cells.

PART II 

The air-dried powdered plant material of Phyllanthus 

atropurpureus Boj. Hort. Maurit. collected in flowering stage was 

subjected to the following experiment and the result were recorded in 

table (12). 

Table (12): Phytochemical Screening of Powdered Phyllanthus 

atropurpureus Boj. Hort. Maurit. 

Test & ref. Result Conclusion 

1-Steam distillation (185) No oily residue was observed There is no volatile oil. 

2-For carbohydrate and/ or 

glycoside (188) : 

• Molisch's test. 

• Fehling test. 

3-For flavonoids (187) : 

• NaOH (T. S) 

• AlCl3 (0.1M) 

• Amyl alcohol. 

4-For isoflavones (187) 

(HNO3) Durham's test. 

5-For tannins (189) : 

• FeCl3 (T. S). 

6-For Saponin (190) : 

• Froth test. 

• Haemolysis of (RBCS) 

7-For alkaloids and/ or basic 

nitrogenous substances (188) 

(Mayer's & Wagner tests) 

8-For mucilage (188) 

(Rhuthenium red). 

9- For Sterols and/ or 

triterpenes. 

• Libermann's test (191) . 

• Salkowski's test (192) . 

It gave violet ring at the junction. 

Reduction of Fehling's solution 

Yellow colour. 

Yellow colour. 

Faint yellow colour. 

Deep red colour. 

Bluish black colour. 

No Froth. 

No Haemolysis of (RBCS) 

No colour or precipitate 

Red colour was produced 

Intense reddish brown colour was 

produced changing to dark green. 

Reddish brown colour was produced 

at the junction of two layers 

- 108 - 

The presence of 

carbohydrate and/ or 

glycosides 


flavonoids 


isoflavonoides 

The presence of pyrogallol 

tannins 

The absence of saponins. 

Absence of alkaloid or 

basic nitrogenous 

compound. 

The presence of mucilage. 

The presence of sterols 

and/ or triterpenes. 

The presence of sterols 

and/ or triterpenes

CONCLUSION 

- 109 - 

PART II 

From the previous preliminary chemical tests, it could be 

suggested that Phyllanthus atropurpureus Boj. Hort. Maurit. contains 

the following constituents: 

• Carbohydrates and/ or glycosides 

• Sterols and/ or triterpenes. 

• Flavonoids and isoflavonoids. 

• Tannins. 

• Mucilage. 

Determination of the moisture content and ash contents: 

A. Moisture Content (185) : 

The air dried powdered plant material was used for this 

determination according to the method described in the Egyptian 

Pharmacopoeia, the mean of three determinations for aerial parts of 

the plant was 15% , while that of root was 10%. 

B. Total Ash and Acid Insoluble Ash (185) : 

The mean of three determinations for the total ash and acid 

insoluble ash were found to be 8.5% and 1.1 % respectively for the 

aerial part of the plant, while that for the root of the plant, it found to 

be 6.81% and 1.3% respectively.

PART II 

Successive Extraction of Powdered 


and Examination of the Respective Extracts. 

Successive Extraction: 

The air dried powdered aerial part (50 g) and root (35 g) of 

Phyllanthus atropurpureus Boj. Hort. Maurit. plant materials were 

successively extracted till exhaustion in a hot continuous extraction 

apparatus (soxhlet) with the following solvents: 

1. Light petroleum (b.r. 60-80 o C). 

2. Diethyl ether. 

3. Chloroform. 

4. Ethyl alcohol (95%). 

The powder, after each extraction, was freed from the solvent 

before the next extraction. The extract was filtered, the solvent was 

distilled off from each extract under reduced pressure (45 o C) and the 

respective residue was dried at 100 o C to constant weight then 

examined. The average of three determinations for each extraction 

was concluded and the percentage of weight, physical properties, TLC 

screening and chemical properties of the extracts are summarized in 

table (13 & 14). 

- 110 -

Table (13): Physical, Chromatographic and Chemical Characters of Successive Extractives of Aerial Parts. 

Items Light Petroleum Extract Diethyl Ether Extract Chloroform Extract Ethyl Alcohol Extract 

I- Percentage 0.3 % 0.11 % 0.07 % 13.27 % 

II- Physical 

Characters 

III- TLC 

Screening 

Dark green residue with greasy 

touch, characteristic odour and 

no characteristic taste, soluble in 

chloroform and ether but 

slightly soluble in alcohol. 

Rf (11)* 

0.97 (m) blue 

0.88 (m) blue 

0.87 (m) violet 

0.83 (m) brown 

0.79 (j) violet 

0.61 (j) purple 

0.48 (m) blue 





Greenish black residue, faint 

odour and slightly bitter taste, 

soluble in chloroform, ether, 

ethyl acetate and to less extent 

in alcohol. 

Rf (4)* 

0.97 (j) green 

0.92 (m) yellow 


0.84 (m) pink 

0.46 (m) purple 


0.33 (m) blue 


0.20 (j) greenish yellow 

0.13 (m) pink 



Dark greenish brown residue 

with faint odour and slightly 

bitter taste, soluble in 

chloroform, ethyl acetate and 

insoluble in light petroleum. 

Rf (12)* 


0.92 (m) green 

0.91 (j) yellow 

0.86 (m) greenish yellow 

0.84 (j) pink 

0.66 (j) pink 

0.53 (j) dark violet 

0.47 (j) pink 

0.46 (m) pink 

0.34 (m) pink 


0.32 (j) brown 





Dark brown residue with 

characteristic odour and slightly 

bitter taste, soluble in methanol 

and insoluble in benzene and 

light petroleum. 

Rf (9)** 



0.92 (j) brownish orange 


0.89 (j) brown 



0.84 (j) brownish orange 

0.74 (j) yellowish brown 




0.35 (m) yellowish brown 


IV- Chemical Tests 

1- For sterols and/ 

or triterpenes 

+ 

+ - - 

2- For alkaloids - - - - 

3- For flavonoids - - + + 

4- For glycoside - - - + 

-System (4) Methanol: Chloroform (1: 9) -System (9) Ethyl acetate: Acetic acid: Formic acid: H2O (100: 11: 11: 27) 

-System (11) Light petroleum: Chloroform: Methanol (15: 15: 1) -System (12) Ethyl acetate: Light Petroleum (1.5: 1) 

* Anisaldehyde Sulphuric Acid ** 50 % Aqueous Sulphuric Acid m = minor; j = major

Table (14): Physical, Chromatographic and Chemical Characters of Successive Extractives of Subterranean Parts. 

Items Light Petroleum Extract Diethyl Ether Extract Chloroform Extract Ethyl Alcohol Extract 

I- Percentage 0.18% 0.02% 0.01% 4.86% 

II- Physical 

Characters 

III- TLC 

Screening 

IV- Chemical 

Tests 

1- For sterols 

and/ or 

Yellowish brown residue with 

greasy touch, characteristic 

odour and no characteristic 

taste, soluble in chloroform 

and ether but slightly soluble 

in alcohol. 

Rf (11)* 

0.97 (m) blue 

0.88 (m) blue 





0.48 (m) blue 





Dark brown residue, faint 


soluble in chloroform, ether, 

ethyl acetate and to less 

extent in alcohol. 

Rf (4)* 

0.84 (m) pink 

0.70 (m) pink 


0.55 (m) blue 


0.46 (j) pink 

0.43 (m) pink 




0.20 (j) greenish yellow 

Dark brown residue with faint 


soluble in chloroform, ethyl 

acetate and insoluble in light 

petroleum. 

Rf (12)* 






Dark brown residue with 

characteristic odour and 

slightly bitter taste, soluble in 

methanol and insoluble in 

benzene and light petroleum. 

Rf (9)** 



0.92 (m) brownish orange 



0.84 (m) brownish orange 






+ 

+ - - 

2- triterpenes For alkaloids - - - - 

3- For flavonoids - - + + 

4- For glycoside - - - + 

System (4) Methanol: Chloroform (1: 9) -System (9) Ethyl acetate: Acetic acid: Formic acid: H2O (100: 11: 11: 27) 

-System (11) Light petroleum: Chloroform: Methanol (15: 15: 1) -System (12) Ethyl acetate: Light Petroleum (1.5: 1) 

* Anisaldehyde Sulphuric Acid ** 50 % Aqueous Sulphuric Acid m = minor; j = major

- 113 - 

PART II 

The air-dried powdered leaves of Phyllanthus atropurpureus Boj. 

Hort. Maurit. (575g) was extracted by cold maceration with 75% 

ethanol (4 L.) till complete exhaustion. The combined extract was 

evaporated under reduced pressure at 50 o C to give 65.6 g of greenish 

brown residue. 

To the concentrated ethanolic leaves extract about 500 ml of 

MeOH: H2O mixture (9:1) was added to dissolve it then extracted with 

light petroleum (b.r 60-80 o C) till exhaustion (4 x 250 ml). The 

combined light petroleum fractions were washed with distilled water, 

dried over anhydrous sodium sulphate and then distilled off under 

reduced pressure at 45 o C to afford 0.56 g of light petroleum soluble 

fraction. 

The aqueous layer was then extracted with chloroform (4 x 

250ml). The combined fractions were washed with distilled water, 

dried over anhydrous sodium sulphate and then distilled off under 

reduced pressure at 45 o C to afford 0.5 g of chloroform soluble 

fraction. 

Finally, the remainder aqueous layer was extracted with ethyl 

acetate (6 x 250). The combined ethyl acetate fractions were then 

washed with distilled water, dried over anhydrous sodium sulphate 

and then the solvent was distilled off under reduced pressure at 50 o C 

to afford 6.5 g of ethyl acetate soluble fraction. The fractionation 

process was summarized in Scheme (1).

PART II 

The air-dried powdered stem and subterranean organs of 

Phyllanthus atropurpureus Boj. Hort. Maurit. were extracted with 

75% ethanol and the extracts were fractionated in the same manner as 

that of leaf. The weights in grams are presented in table (15) 

The results of extraction and fractionation carried out on the 

air-dried powdered leaves, stems and roots of Phyllanthus 

atropurpureus Boj. Hort. Maurit. are summarized in table (15). 

Table (15): The Results of Extraction and Fractionation of Air-Dried 

Powdered Plant Organs. 

Organ (g) 

Total Ethanolic 

Extract 

% 

Petroleum 

Ether 

Fraction % 

- 114 - 

Chloroform 

Fraction 

% 

Ethyl Acetate 

Fraction 

% 

Leaves 575 11.41 0.10 0.10 1.13 

Stems 1270 5.91 0.11 0.07 0.22 

Roots 241 10.86 0.05 0.03 0.52

Air – Dried Powdered organs 

Total Ethanolic Extract of 

- 115 - 

PART II 

Scheme (1): Extraction and Fractionation of Air-Dried Powdered 

Plant Organs of Phyllanthus atropurpureus Boj. Hort. 

Maurit. 

1- 75% Ethanol 

2- Concentration 

1- Methanol: Water (4:1) (500ml) 

2- Light petroleum 

Aqueous phase Light Petroleum Soluble 

Chloroform 

Fraction of 

Aqueous phase 

Ethyl Acetate 

Aqueous phase 

Chloroform Soluble 

Fraction of 

Leaves 

(0.5g) 

Leaves 

(0.56g) 

Ethyl Acetate Soluble Fraction of 

Leaves 

(6.5 g) 

Stems 

(0.91g) 

Stems 

(2.75g) 

Roots 

(0.072g) 

Roots 

(1.25g) 

leaves (65.6 g) 

stem (75 g) 

root (26.17 g) 

Stems 

(1.33g) 

Roots 

(0.31g)

PART II 

Chromatographic Investigation of Leaf, Stem and Root 

Light Petroleum Soluble Fraction: 

A. TLC Screening of Leaf, Stem and Root Light Petroleum 

Soluble Fraction. 

Thin layer chromatographic screening of the light petroleum 

soluble fraction of leaf, stem and root was carried out using silica gel 

GF245 chromatoplates and solvent system (1, 10 and 11). The 

developed plates were visualized by anisaldehyde-sulfuric acid spray 

reagent. The chromatoplates revealed that light petroleum extract of 

leaf, stem and root showed the same spots. These fractions were 

collected together and designated as the light petroleum extract of 


The chromatoplates of the light petroleum extract of Phyllanthus 

atropurpureus Boj. Hort. Maurit. revealed the presence of three major 

and eight minor spots. Results of TLC investigation are illustrated in 

table (16). 

Table (16): TLC Investigation of Light Petroleum Soluble Fraction. 

Spot n 

Rf value using system 

o 

1 10 11 

The Isolated 

Materials 

1 0.98 (blue) 0.93 (blue) 0.97 (blue) -- 

2 0.95 (blue) 0.82 (blue) 0.88 (blue) -- 

3 0.89 (violet) 0.77 (violet) 0.87 (violet) -- 

4 0.85 (brown) 0.69 (brown) 0.83 (brown) -- 

5 0.83* (violet) 0.67* (violet) 0.79* (violet) Material 1 

6 0.78*(purple) 0.47* (purple) 0.61* (purple) Material 2 

7 0.56 (blue) 0.33 (blue) 0.48 (blue) -- 

8 0.52*(violet) 0.30* (violet) 0.45* (violet) Material 3 


10 0.35 (brown) 0.18 ( brown) 0.30 ( brown) -- 


*major spot 

- 116 -

I. Saponification of Light Petroleum Soluble Fraction (193, 194) : 

- 117 - 

PART II 

About 1 g of light petroleum soluble fraction was refluxed with 

16 ml alcoholic potassium hydroxide (10%) for 5 hours, and then 

diluted with distilled water .The alcohol was distilled off and the 

obtained residue was diluted with water (10 ml) and extracted with 

ether (5 × 50 ml). The combined ethereal extract was washed several 

times with water (to remove any alkalinity), dried over anhydrous 

sodium sulphate and evaporated to give 0.57 g of the unsaponifiable 

matter (USM). 

The alkaline aqueous layer remained after extraction of the 

unsaponifiable matter with the ether was acidified with concentrated 

hydrochloric acid and extracted with successive portions of ether (5 × 

50 ml). The combined ethereal extract was washed several times with 

water, dried over anhydrous sodium sulphate and distilled off to afford 

0.43 g of fatty acid contents. 

II. Preparation of Fatty Acid Methyl Esters (195, 196) : 

About 0.2 g of fatty acids residue was dissolved in 15 ml. of 

methanol / sulphuric acid (5% v/v) and left at room temperature (25 o 

C) overnight, then refluxed for four hours. Methanol was distilled off 

under reduced pressure and 15 ml of brine solution was added to the 

residue. The solution was extracted with successive portions of 

mixture of (1: 1) light petroleum: ether (3 ×50 ml). The combined 

ethereal extract was washed several times with distilled water, dried 

over anhydrous sodium sulphate, and evaporated to afford about 0.21 

g of fatty acids methyl esters.

PART II 

III. GLC Analysis of Fatty Acid Methyl Esters: 

Gas liquid chromatography analysis of fatty acids methyl esters 

was carried out (adapting conditions mentioned in page 43) against 

references of methyl esters of many fatty acids including caprylic, 

capric, lauric, myristic , palmitic, palmiteolic, margaric, stearic, oleic 

and linoleic. 

Identification of fatty acids methyl esters (Fig. 17) was carried out 

by comparison of the retention times of the fatty acid methyl esters 

with that of the authentic samples. The quantitative estimation was 

carried out by the peak area measurements and the results were 

recorded in table (17). 

Table (17): Results of GLC analysis of fatty acid methyl esters from 

the light petroleum fraction of Phyllanthus atropurpureus Boj. Hort. 

Maurit. 

Retention 

Time 

Conc. % 

No .of Carbon: 

Double Bond 

- 118 - 

Systematic Name Trivial 

Name 

16.517 0.200 8 : 0 Octanoic Caprylic 

17.417 0.098 10 : 0 Decanoic Capric 

19.617 1.322 12 : 0 Dodecanoic Lauric 

22.767 2.909 14 : 0 Tetradecanoic Myristic 

25.083 0.647 unidentified -------------- ---------- 

26.083 59.318 16 : 0 Hexadecanoic Palmitic 

28.800 1.464 16 : 1 Cis-9hexadecanoic 

Palmitoleic 

31.983 0.367 17 : 0 Heptadecanoic Margaric 

39.467 5.397 18 : 0 Octadecanoic Stearic 

41.417 14.221 18 : 1 Cis-9octadecanoic 

Oleic 

46.267 11.600 18 : 2 9,12- 

Octadecadienoic Linoleic

Results and conclusion: 

- 119 - 

PART II 

From the results shown in table (17) it could be concluded that: 

• Eleven fatty acid methyl esters were observed in Phyllanthus 

atropurpureus Boj. Hort. Maurit. plant; constituting about 97.5435% 

of its fatty acids content. 

• Ten fatty acids were identified and constitute 96.897%. 

• Seven fatty acids (Caprylic, Capric, Layric, Myristic, Palmitic, 

Margaric, and Stearic) represent the saturated fatty acids which 

comprise about 69.611% of the total amount of the analyzed fatty acid 

of the plant. 

• The monounsaturated fatty acid (Palmiteolic& oleic) 

represents 15.686% of the total fatty acid contents. 

• The diunsaturated fatty acid (Linoleic) represents 11.6 % of 

the total fatty acid contents. 

• Palmitic (59.32%), Oleic (14.22%), Linoleic (11.6%) are the 

major fatty acids. 

• Palmitic acid has an antioxidant activity while Linoleic acid is 

required for the biosynthesis of prostaglandin hormones that 

responsible of the anti-inflammatory and analgesic activities ( 197, 198) . 

IV. GC Analysis of The Unsaponifiable Matter 

Gas chromatography analysis of unsaponifiable matter was 

carried out against references following conditions mentioned in page 

43. Identification of the unsaponifiable matter (Fig.18) was carried out 

by comparison of the retention times with that of the authentic samples. 

The quantitative estimation was carried out by the peak area 

measurements and the results were recorded in table (18).

PART II 

Table (18): Results of GC analysis of the unsaponifiable matter 

of Phyllanthus atropurpureus Boj. Hort. Maurit. 

Retention time 

Area 

% 

No. of Carbon 

26.962 1.455 19 

Results and conclusion: 

From the results shown in table (18) it could be concluded that: 

• Twelve compounds were detected in unsaponifiable matter of 

Phyllanthus atropurpureus Boj. Hort. Maurit. plant; constituting about 

77.569 % of its contents. 

• β -sitosterol and stigmasterol were identified; constituting 

about 3.061% of unsaponifiable matter of Phyllanthus atropurpureus 


28.882 56.117 20 

29.522 1.325 21 

33.437 1.074 22 

33.995 8.913 23 

35.118 0.596 24 

37.967 1.288 25 

39.058 2.689 26 

40. 260 0.699 27 

43.573 0.352 28 

44.728 1.310 29 (β- sitosterol) 

45.023 1.751 29 (Stigmasterol) 

- 120 -


- 121 - 

PART II 

Fig. 17: GLC of the fatty acids methyl esters of Phyllanthus atropurpureus 



Fig. 18: GC spectrum of the unsaponifiable matter of Phyllanthus atropurpureus 

Boj. Hort. Maurit.

PART II 

B- Column Chromatography of Light Petroleum Soluble Fraction: 

About 2 g of the light petroleum soluble fraction of leaf, stem and 

root was dissolved in the least amount of chloroform, adsorbed on 5g 

silica gel and the solvent was completely evaporated at low temperature. 

The resulted dry powder was placed on the top of silica gel column (3×85 

cm, 100 g silica). The column (A) packed by wet method using benzene. 

The elution of the column (A) was carried out starting with benzene then 

the polarity was increased gradually using chloroform then methanol. The 

eluate was collected in fractions each of 250 ml, concentrated under 

reduced pressure, monitored by TLC using solvent systems (100% 

chloroform, system 4) and similar fractions were combined. Column 

chromatographic fractionation was illustrated in table (19). 

Table (19): Column Chromatographic Fractionation of Light petroleum 

Soluble Fraction (A). 

Eluent % 

Composition 

Fr. 

No. 

122 

Fr. 

wt. 

( g ) 

Rf value (solvent system 

11 ) 

Isolated 

material 

Benzene 

Benzene: chloroform 

100 % 

95: 5 

1 & 2 

3 & 4 

0.34 Undifferentiated spots 

-- 

-- 

Benzene: chloroform 90: 10 5-7 0.12 

0.97, 0.88 + 

Undifferentiated spots 

-- 

Benzene: chloroform 75: 25 8 & 9 0.10 0.88, 0.87, 0.83 -- 

Benzene: chloroform 50: 50 10- 13 0.15 0.87, 0.83, 0.79 * material 1 

Benzene: chloroform 25: 75 14- 21 0.71 0.79 *, 0.61*, 0.48, 0.45 

material 

1, 2 

Chloroform 100 % 22 & 23 0.12 0.45* material 3 

Chloroform: methanol 99: 1 24 & 25 0.11 0.45, 0.37, 0.30 -- 

Chloroform: methanol 98: 2 26 & 27 0.10 

0.30, 0.17 + 


-- 

Chloroform: methanol 95:5 28 -- 

Methanol 100% 29 & 30 

0.25 Undifferentiated spots 

-- 

*Rf Corresponding to the isolated compound.

• Isolation of materials (1, 2): 

123 

PART II 

TLC investigation of fractions (14-21) using solvent system (11) 

revealed the presence of two major spots with Rf value 0.79 (violet) and 

0.61 (purple) respectively. 

Fractions (14-21) (0.708 g) were rechromatographed over silica gel 

column (B) (1×50 cm, 20 g) packed in light petroleum (60-80). The 

elution of the column was started with light petroleum then the polarity 

increased gradually with chloroform and methanol , fractions 100 ml each 

were collected , concentrated under reduced pressure , monitored by TLC 

using solvent system 10 and 11 and similar fractions were pooled. 

a- Isolation of material (1): 

TLC screening of fractions in previous subcolumn (B) eluted by 50 

% chloroform in light petroleum indicated the presence of one major 

violet spot with Rf values of 0.67 and 0.79 (System 10 and 11 

respectively with anisaldehyde-sulphuric acid spray reagent). 

Crystallization from chloroform - methanol afforded 80 mg of white 

powder with m.p. 62 - 63 o C. This substance was designated as material 1. 

This material was previously separated (70 mg) from fractions (10-13) of 

the light petroleum column (A). 

b- Isolation of material (2): 

TLC screening of fractions in previous subcolumn (B) eluted by 60 

% chloroform in light petroleum revealed the presence of one major 

purple spot with Rf values of 0.47 and 0.61 (solvent systems 10 and 11 

respectively with anisaldehyde-sulphuric acid spray reagent). 

Crystallization from chloroform - methanol afforded 350 mg of colourless 

needles crystals with m.p. 144-146 o C. This substance was designated as 

material 2.

PART II 

• Isolation of material (3): 

TLC screening of fractions (22& 23) of column (A) indicated the 

presence of one major violet spot with Rf values of 0.52 and 0.45 (using 

solvent systems 1 and 11 respectively with anisaldehyde-sulphuric acid 

spray reagent). Crystallization from chloroform - methanol afforded 75 

mg of white powder with m.p.30- 33 o C. This substance was designated as 

material 3. 

Characterization of material 1 

Material "1" (150 mg) occurs in the form of white powder; with m.p. 

62-63 o C and Rf value 0.67 and 0.79 (System 10 and 11). It is soluble in 

light petroleum, benzene, chloroform and insoluble in methanol, acetone. 

It gives negative Libermann's and Salkowiski's tests for sterols and / 

or triterpenes (191, 192) . 

Spectral Analysis: 

IR Spectral Analysis: 

The IR spectrum of material "1"(Fig.19) shows the following 

absorption frequencies: 

Vmax (KBr) cm -1 : 3500-3300, 2920-2850, 1705, 1466, 1296-1040, 940, 

723, 687 and 549. 

Mass Spectral Analysis: 

mass fragments: 

The mass spectrum of material "1" (Fig.20) shows the following 

m/z (relative intensity %): 284 (M + , 1.5), 265 (2.1), 264 (2.2), 256 (M + , 

22.9), 239 (3.3), 213 (10.6), 199 (4.2 ), 185 (8.5), 171(8.5), 169 (0.5), 157 

(8.4), 143 (4.3), 129 (22), 115 (9.2),101 (7.1), 97 (20.5), 83(30), 73 

(81.8), 71 (37.3), 69 (46.5), 60 (97.2), 57 (75.9) and 55 (100). 

124

Discussion and conclusion 

125 

PART II 

The IR spectrum of material "1" (Fig.19) indicated the presence of 

aliphatic stretching peaks at 3500-3300 cm -1 for OH group, 2925-2845 

cm -1 for aliphatic -CH, 1705 cm -1 for C = O of the carboxylic group, 1466 

(CH2- bending) and 1296- 1040 (C - C stretching) (199) . 

The mass spectrum showed two distinct parent ions peaks at m/z 

284 (1.5%) and at m/z 256 (22.9%) together with other significant 

fragments at m/z 129, 73, 55. 

There are ions representing fragmentations between most methylene 

groups as mass spectrum showed the pattern of long chain fatty acid by 

successive loss of 14 (CH2) mass units. An ion at m/z = 239 [M + -17] 

presumably reflects a loss of OH from carboxyl group. 

Comparison of mass spectrum with that of the standard stearic and 

palmitic acid individually, elevates the following data: 

Molecular ion at m/z 284 together with other fragments at m/z 255, 

241, 227, 213, 199, 185, 171, 157, 143, 129, 115,101, 87, 73, 60, 55 

indicating the presence of stearic acid and a molecular ion peak at m/z 

256 together with other fragments at m/z 239, 227, 213, 199, 185, 171, 

157, 143, 129, 115, 101, 87, 73, 60, and 55 indicating the presence of 

palmitic acid. 

From the previously mentioned data, material 1 was proved to be a 

mixture of palmitic acid and stearic acid. 

CH3-(CH2)14- COOH CH3-(CH2)16- COOH 

Palmitic acid Stearic acid

PART II 

Fig. (19): IR spectrum of material "1" of Phyllanthus atropurpureus 


Fig. (20): Mass spectrum of material "1" of Phyllanthus atropurpureus 


126


127 

PART II 

Material "2" (350 mg) occurs as white needle-shaped crystals 

(chloroform/ methanol) with m.p. 144-146 o C and Rf values of 0.47 and 

0.61 (solvent systems 10 and 11 respectively). It is soluble in chloroform, 

benzene and insoluble in methanol and acetone. It gives positive 

Libermann's and salkowski's (191, 192, 200) tests for sterols and/ or 

triterpenes. It also gives negative tests for reducing properties and the 

glycosidic nature of the compound (188) . 

Spectral analysis: 


The IR spectrum of material "2" (Fig.21) shows the following 


Vmax (KBr) cm -1 : 3416-3213, 2940-2870, 1461, 1376, 1185, 1053, 959, 

837, 805 and 593. 



The mass spectrum of material "2" (Fig.22) shows the following 

m/z (% relative abundance): 414 (M + , 11.4) for C29H50O, 412 (M + , 2.4) 

for C29H48O, 399 (4.7), 397 (2.2), 396 (5.4), 381 (6), 329 (5), 303 (6.5), 

289 (6.1), 273 (4.9), 271(2.6), 255 (9.7), 231 (6.9), 229 (3.6), 213 (13.2), 

211 (2.9), 173 (10.3), 145 (22.5), 133(17.9), 109 (18.8), 107 (31.8), 105 

(34.9), 95 (40.3), 83 (19), 81 (41.5), 79 (31.7), 69 (47.1), 67 (41.9), 57 

(68.2) and 55 (100). 

Preparation of Acetyl Derivative of material "2" (201, 202) : 

Mass spectrum of material "2" shows 414 (M + ) and 412 (M + ) which 

indicates the presence of two compounds. Acetylation was made to

PART II 

confirm this probability. About 20 mg of material "2" was dissolved in 15 

ml acetic anhydride containing 1 g fused sodium acetate. The mixture 

was refluxed for 30 minutes on water bath and poured into ice-cooled 

water (50 ml) followed by stirring, where white precipitate was formed. It 

was filtered and washed several times with cold water till completely free 

from acidity. On repeated crystallization (chloroform/ methanol) about 28 

mg of colourless needle-shaped crystals with m.p. 126-128 o C were 

obtained. 

Preparation of Reactive Layer (silver nitrate) and TLC Examination 

of Acetates (202) : 

Silica gel GF was slurred with 10% aqueous silver nitrate solution; 

wedge shape plate was prepared and activated in hot oven for 90 minutes 

at 110 o C. Spotting was made on narrow part of the plate (solvent system 

14) (197) . The dried chromatogram was examined under light UV lamp, to 

show two fluorescent spots with Rf values 0.22 and 0.30. 

Discussion and Conclusion 

The physical properties and colour reactions of material "2" 

suggested a steroidal or triterpenoidal skeleton (191, 192, 200) . 

IR spectrum of material "2" showed broad absorption band at 3416- 

3213 cm -1 for bonded –OH group together with C-O stretching at 1053 

cm -1 besides the presence of bands at 2940-2870 cm -1 for C-H aliphatic 

and another absorption peak at 959 cm -1 indicating the trans disubstituted 

side chain. 

The mass spectrum (Fig. 22) showed two distinct parent ions at m/z 

414 (11.4%) and at m/z 412 (2.4%) together with other significant 

128

129 

PART II 

fragments at m/z 396, 381, 303, 271 and 255, these data are indicative for 

steroidal compound (203) . 

Direct comparison of mass spectrum with that of the standard 

β-sitosterol and stigmasterol individually, elevates the following data: 

molecular ion at m/z 414 together with other fragments at m/z 399, 396, 

273, 231, 229 and 213 indicating the presence of β-sitosterol, and a 

molecular ion peak at m/z 412 with other fragments at m/z 397, 396, 271, 

255, 229, 213 and 211 are characteristic for stigmasterol. 

However, it showed two parent ion peaks at m/z 414 and m/z 412 

corresponding to β-sitosterol and stigmasterol respectively. The relative 

abundance of parent ion at m/z 414 (11.4%) and m/z 412 (2.4%) 

indicated that β-sitosterol is the major compound. 

The suggested fragmentation pattern is shown in scheme (2). 

From the previously mentioned data and direct comparison (MS, IR, 

m.p. and coTLC of acetyl derivatives on silver nitrate plate with authentic 

samples), material "2" was proved to be a mixture of β-sitosterol and 

stigmasterol. 

HO HO 

β-sitosterol 

stigmasterol

PART II 




Boj Hort. Maurit. 

130

+ 

H 

HO 

m/z 273 

m/z 303 

+ 

-R 

HO 

HO 

m/z 213 

-CH3 

Scheme (2): Mass fragmentation pattern of β-sitosterol (major one) 

+ 

m/z 231 

m/z 414 

+ 

m/z 381 

- H 2O m/z 396 

-R 

+ 

m/z 255

PART II 


Material "3" (75 mg) occurs in the form of white residue; with 

m.p. 30 - 33 o C and Rf values of 0.52 and 0.45 (System 1 and 11 

respectively). It is soluble in light petroleum, benzene, chloroform and 

insoluble in methanol, acetone. It gave negative Libermann's and 

Salkowiski's tests for sterols and / or triterpenes (191, 192, 200) . 



The IR spectrum of material "3" (Fig. 23) shows the following 


Vmax (KBr) cm -1 : 3500- 3350, 2920-2850, 1707, 1460 and 597. 


The mass spectrum of material "3" (Fig. 24) shows the following 


m/z (% relative intensity): 254 (1.6), 225 (2.4), 156 (1.4), 139 (3.5), 

126 (3.5), 125 (4.1), 124 (1.6), 111 (3.5), 97 (10.3), 83 (23.6), 69 

(29.6) and 55 (100). 

NMR Spectral Analysis: 

The 1 H-NMR spectral analysis of material "3" (CD3OD, 500 

MHz) (Fig. 25) showed the following signals: 5.25 (2H, m), 2.17 (2H, 

t, J= 6Hz), 1.96 (2H, m), 1.5 (2H, m) and 0.81(3H, t, J= 6Hz) ppm. 

The 13 C-NMR spectral analysis of material "3" (CD3OD, 125 

MHz) (Fig. 26) revealed the presence of several signals which are 

- 132 -

- 133 - 

PART II 

177.79 (C-1), 130.89 (C-10), 129.05 (C-9), 14.43 (C-18), 35.01, 

33.07, 32.67, 30.76, 30.48, 30.43, 30.25, 28.11, 26.11, 23.73, 23.63 

ppm. 

Discussion and conclusion: 

The IR spectrum of material "3" (Fig. 23) indicated the presence 

of hydroxyl group at 3500-3350, aliphatic stretching peaks at 2920- 

2850 (of methyl and methylene groups), 1707 (C = O stretching 

vibration of the carboxylic group) and 1460 (CH2- bending). 

The 1 HNMR spectrum of material "3" (Fig. 25) showed one 

multiplet at δ 5.25 ppm integrated for two protons each assigned to 

olefinic protons H-9 and H-10. Two proton triplet at δ 2.17 ppm (J= 6) 

is attributed to C-2 methylene protons adjacent to carboxylic group. 

Two multiplets at δ1.96 and 1.50, both intergrated for two protons 

each accounted C-8 and C-11 methylene adjacent to the olefinic 

linkage, respectively. A three-proton triplet at δ 0.81 ppm (J= 6) for 

terminal primary CH3 protons. The remaining methylene proton 

resonated as a broad signal at δ 1.25 ppm. 

H 3C 

0.81 ppm 

1.5 ppm 

10 

5.25ppm 

H 

5.25 ppm 

1.96ppm 2.17 ppm 

The 13 C-NMR spectrum of material "3" (Fig. 26) revealed 

important signals for carboxylic carbonyl (δ 177.79), unsaturated 

carbons at δ 130.89 (C- 10) and 129.05 (C-9) and methyl carbon at δ 

14.43 (C- 18). The remaining methylene carbon resonated between 

H 

9 

O 

1 

C 

OH

PART II 

δ 23.63 - 35.01 ppm. The absence of any signals between δ 129.05- 

35.01 ppm ruled out the presence of a hydroxyl group in the molecule. 

14.43 

H 3C 

The mass fragmentation pattern of material 3 is represented in 

scheme (3).The prominent ion fragments generated at m/z 156, 126 

(C9-C10 fission) + , 111 (156 -COOH) + , 124 (139 –Me) + suggested the 

location of double bond at C9 -C10 

Molecular ions at m/z 139, 125, 111, 97, 83, 69 and 55 are 

corresponding to the sequential loss of 14 mass units (-CH2) which 

revealed the presence of a C18 straight chain acid with an olefinic 

unsaturation at C-9. 

On the basis of spectral data analysis, chemical reactions and 

available literature (204- 206) , the structure has been characterized as 

oleic acid. 

H 3C 

23.63 

33.07 

30.43- 30.48 

26.11 

130.89 

H 

H 

129.0532.76 

28.11 

Oleic acid 

- 134 - 

30.43 

30.76 

30.25 

23.73 

O 

C 

35.01 177.79 

OH 

COOH

- 135 - 

PART II 




Boj. Hort. Maurit.

PART II 

Fig. (25): 1 H-NMR spectrum of material "3" of Phyllanthus atropurpureus 


Fig. (26): 

- 136 - 

13 C-NMR spectrum of material "3" of Phyllanthus atropurpureus 

Boj. Hort. Maurit.

m/z 254 

- CO 

m/z 124 (1.6%) 

H 3C 

H 3C + 

m/z 139 (3.5%) 

- CH3 

M + 282 

C18H34O2 

H 3C + 

m/z 126 

(3.5%) 

m/z 97 (10.3%) 

m/z 83 (23.6%) 

- 137 - 

- CH3 

m/z 111 (3.5%) 

- CH2 

- CH2 

- CH2 

m/z 69 (29.6%) 

-CH2 

m/z 55 (100%) 

PART II 

Scheme (3): Mass fragmentation pattern of material "3" of Phyllanthus 


+ 

COOH 

+. 

COOH 

m/z 156 (1.4%) 

- COO 

m/z 112 (1.6%) 

- H 

m/z 111 (3.5%)

Spot n o 

PART II 

Chromatographic Investigation of Ethyl Acetate Soluble 

Fractions: 

TLC Screening of Ethyl acetate Fractions of Leaf, Stem and Root. 

Thin layer chromatographic screening of the ethyl acetate 

fractions of leaf, stem and root were carried out using silica gel GF245 

chromatoplates and solvent systems (6, 9 and 13). The developed 

plates were visualized by UV lamp λmax 365nm and spray reagents 

(ammonia and 50 % aqueous sulfuric acid). The chromatoplates 

revealed that the ethyl acetate fractions of stem and root are similar 

and in the same time are different from leaf ethyl acetate fraction. 

Results of TLC investigation are illustrated in table (20). 

Table (20): TLC Investigation of Ethyl Acetate Fractions using 

solvent system (9). 

Rf value using 

solvent system 

(9) 

+ present 

─ absent 

+++ major spot 

Leaf ethyl 

acetate 

fraction 

Stem & root 

ethyl acetate 

fractions 

- 138 - 

Visualizing agent 

UV lamp (365 

NH4OH 

nm) 

1 0.99 + + blue fluorescence yellow violet 

2 0.97 + + violet yellow violet 

3 0.95 + ─ violet yellow yellow 

4 0.92 +++ + blue fluorescence yellow 

50% 

H2SO4 

blue fluorescence yellow 

brownish 

orange 

violet 

violet yellow brown 

5 0.90 + ─ 

6 0.89 +++ ─ 

7 0.88 ─ + violet. yellow violet 

8 0.87 + ─ violet yellow yellow 

9 0.86 + +++ blue fluorescence yellow yellow 

10 0.84 +++ + 

blue 

fluorescence 

yellow 

11 0.74 +++ ─ violet yellow 

brownish 

orange 

yellowish 

brown 

12 0.60 ─ +++ purple yellow yellow 

13 0.48 + + blue fluorescence yellow yellow 

14 0.30 + + blue fluorescence yellow yellow 

15 0.15 + + blue fluorescence Yellow 

yellowish 

brown

- 139 - 

PART II 

The chromatoplates of ethyl acetate soluble fraction of leaf 

revealed the presence of four major and nine minor spots. Results of 

TLC investigation are illustrated in tables (20& 21). While that of 

stem and root ethyl acetate fractions revealed the presence of two 

major and eight minor spots. Results of TLC investigation are 

illustrated in tables (20 & 28). 

Column Chromatography of Leaf Ethyl Acetate Fraction: 

A. TLC Screening of Leaf Ethyl Acetate Fraction. 

Thin layer chromatographic investigation of leaf ethyl acetate 

soluble fraction was carried out using silica gel GF245 chromatoplates 

using solvent systems (6, 9, 13), UV lamp λmax 365nm and spray 

reagents (ammonia and 50 % aqueous sulfuric acid) for visualization. 

The chromatographic study revealed the presence of four major and 

nine minor spots. The Rf values and behaviors with different reagents 

are illustrated in table (21).

PART II 

Table (21): TLC Investigation of Leaf Ethyl Acetate Soluble 

Fraction with different visualizing agents and different solvent 

systems. 

Spot 

n 

Rf value Visualizing agent 

*major spot 

o 6 9 13 

UV lamp 

(365 nm) NH4OH 50 % H2SO4 

Isolated 

materials 

1 0.97 0.99 0.98 

Blue 

fluorescence 

Yellow Violet --- 

2 0.82 0.97 0.96 Violet Yellow Violet --- 

3 0.80 0.95 0.93 Violet Yellow Yellow --- 

4 0.75* 0.92* 0.91* Blue 

fluorescence 

Yellow Brownish orange Material 4 

5 0.69 0.90 0.87 Blue 

fluorescence 


6 0.59* 0.89* 0.82* Violet Yellow Brown Material 5 

7 0.51 0.87 0.79 Violet. Yellow Yellow --- 

8 0.49 0.86 0.78 Blue 

fluorescence 

Yellow Yellow --- 

9 0.39* 0.84* 0.76* Blue 

fluorescence 

Yellow Brownish orange Material 6 

10 0.33* 0.74* 0.65* Violet Yellow Yellowish brown Material 7 

11 0.29 0.48 0.43 Blue 

fluorescence 


12 0.20 0.3 0.24 Blue 

fluorescence 


13 0.09 0.15 0.11 Blue 

fluorescence 

Yellow Yellowish brown --- 

B. Column Chromatography of Leaf Ethyl Acetate Soluble 

Fraction. 

6.5 g of the leaf ethyl acetate soluble fraction was dissolved in 

the least amount of methanol and adsorbed on 15 g of silica gel for 

column and transferred into a silica column (A) (2.5 × 150 cm, 250 g) 

packed with benzene. Elution was carried out starting with benzene 

and the polarity gradually increased with chloroform and methanol. 

Fractions (250 ml each) were collected, concentrated and examined by 

TLC using solvent system (9) and the similar fractions were pooled. 

- 140 -

- 141 - 

PART II 

Description of the column chromatographic process is illustrated in 

table (22). 

Table (22): Column Chromatography of Leaf Ethyl Acetate Soluble 

Fraction. 

Eluent 

% 

Composition 

Fr. 

No. 

Fr. wt. 

(g) 

Rf value (solvent system 9 ) 

Isolated 

Material 

Benzene 100% 1-5 -- 

Benzene: 

chloroform 

Benzene: 

chloroform 

Benzene: 

chloroform 

75:25 6-15 Undifferentiated spots 

-- 

1.25 

50:50 16-33 -- 

25:75 34- 49 

Chloroform 100% 50- 68 0.58 

Chloroform: 

methanol 

Chloroform: 

methanol 

Chloroform: 

methanol 

Chloroform: 

methanol 

Chloroform: 

methanol 

Chloroform: 

methanol 

Chloroform: 

methanol 

Chloroform: 

methanol 

Chloroform: 

methanol 

0.99, 0.97, 0.95 + undifferentiated 

spots 

99.5: 0.5 69- 73 0.55 0.95, 0.92*, 0.90 

-- 

-- 

Material 

4 

99: 1 74- 80 0.27 0.92, 0.90 -- 

97: 3 81-96 0.34 undifferentiated spots -- 

96: 4 97-99 0.10 0.89* + undifferentiated spots 

Material 

5 

94: 6 100-113 0.16 0.87, 0.86 --- 

92: 8 114-122 1.04 0.86, 0.84* 

84: 16 123-125 0.15 0.84, 0.74* 

Material 

6 

Material 

7 

68: 32 126-135 0.35 0.74, 0.48 , 0.3, 015 --- 

50: 50 136-147 0.40 

0.48, 0.3, 0.15+ undifferentiated 

spots 

Methanol 100% 148 1.31 Undifferentiated spots --- 

*Rf Corresponding to the isolated compound. 

--

PART II 


TLC examination of fractions 69-73 (0.55 g) revealed the 

presence of one major brownish orange spot with Rf value 0.92 

(solvent system 9 and 50 % aqueous sulphuric acid visualizing 

reagent). Crystallization from methanol afforded 250 mg of reddish 

brown needles, m.p. 141-143 o C. This was designated as material 4. 


Fractions 97-99 (0.1 gm) were examined by TLC to reveal the 

presence of one major brown spot with Rf value 0.89 alongside with 

other undifferentiated spots (solvent system 9 and 50 % aqueous 

sulphuric acid visualizing reagent). 

Fractions 97-99 were rechromatographed over silica gel column 

(B) (1x 50 cm, 15 g) and eluted with chloroform and the polarity 

increased gradually with methanol. Fractions (25 ml each) were 

collected, concentrated and examined by TLC using solvent system 

(9) and 50 % aqueous sulphuric acid visualizing reagent. 

TLC examination of fractions of this subcolumn eluted with 4% 

methanol in chloroform indicated the presence of one major brown 

spot with Rf value 0.89 (solvent system 9 and 50 % aqueous sulphuric 

acid visualizing reagent). Crystallization from methanol afforded 30 

mg of yellowish white residue; m.p.179 o C. This was designated as 

material 5. 


TLC investigation of fractions 114-122 (1.04 g) revealed the 

presence of one major brownish orange spot with Rf values of 0.84 

- 142 -

- 143 - 

PART II 

and 0.66 (solvent systems 9 and 100% ethyl acetate respectively 

using 50% aqueous sulphuric acid visualizing reagent). 

Fractions 114-122 was then rechromatographed over a silica gel 

column (C) (1 x 50 cm, 20 g) and eluted by gradient elution technique 

using benzene and ethyl acetate. 50ml fractions were collected, 

monitored by TLC and similar fractions were pooled. 

The pooled fractions eluted from benzene: ethyl acetate (1:1) 

revealed the presence of one major brownish orange spot with Rf 

value 0.84. Crystallization from methanol afforded 800 mg of buff 

fine powder; m.p. 144-146 o C. This was designated as material 6. 


The fractions 123-125 (0.15 g) when examined using solvent 

system (9) and 50 % aqueous sulphuric acid as visualizing reagent 

revealed the presence of one major yellowish brown spot with Rf 

value 0.74 and one minor brownish orange spot Rf value 0.84. 

Crystallization from methanol afforded faint yellow crystals (50 mg); 

m.p. 150-152 o C. This was designated as material 7.

PART II 


Material "4" (250 mg) occurs as reddish brown needles 

(methanol), with m.p. 141-143 o C. It is freely soluble in methanol and 

ethyl acetate, slightly soluble in chloroform and insoluble in benzene 

and light petroleum. It shows Rf value of 0.75 & 0.92 (solvent system 

6 & 9 respectively using 50 % aqueous sulphuric acid as visualizing 

reagent). Material 4 gave yellow colour with ammonia, brownish 

orange with 50% aqueous sulphuric acid reagent and blue 

fluorescence under UV light. 



The IR spectrum of material "4" (Fig. 27) showed the following 


Vmax (KBr) cm -1 : 3621, 3500-3100, 3030, 2929-2800, 1649, 1517, 

1465, 1246, 1194, 1095, 1023, 833 and 760. 

UV Spectral Analysis: 

The UV spectroscopic analysis of material "4" showed 

absorption peak at λ max (methanol) 292 nm. 

Mass Spectral Analysis: (Fig. 28): 

EI-MS: m/z (%): 266 (M + , 4.7), 265 (7.47), 248 (0.23), 140 

(2.47), 125 (3.3), 124 (1.06), 112 (6.79),109 (10.19), 108 (4.51), 107 

(20.84), 99 (12.88), 98 (13.08), 95 (39.08), 84 (20.84), 79 ( 41.92), 67 

( 52.58), 66 ( 4.14), 60 (100). 

1 H-NMR and 13 C-NMR Spectral analysis 

The 1 H-NMR spectrum of material "4" (Fig. 29) (CDCl3/ 

CD3OD, 300 MHz), showed the presence of the following signals: 

- 144 -

• A broad singlet at δ 6.615 - 6.651 ppm. 

• A singlet signal at δ 3.338 ppm. 

- 145 - 

PART II 

The 13 C-NMR of material "4" (Fig. 30) (CDCl3/ CD3OD, 75 

MHz), showed signals at δ (115.29- 117.30) and (149.87- 150.76) 

ppm. 

DEPT 135 of material "4" (Fig. 31) showed signals at δ 115.29- 

117.30 ppm. 


The physical characters, colour reaction and UV absorption of 

material "4" suggest the presence of phenolic skeleton (207) . 

The IR spectrum of material "4" showed an absorption 

frequency at 3621 cm -1 attributed to unbonded -OH, broad absorption 

band at 3500-3100 cm -1 for several –OH groups and 1649, 1517, 1465 

cm -1 for aromaticity in addition to peaks at 1246, 1194, 1095 and 1023 

cm -1 for C-O stretching (ether linkage). 

EI-MS exhibited a molecular ion peak at m/z 266 (M + ) which is 

compatible with the molecular formula C12H10O7. The suggested mass 

fragmentation of this material illustrated in scheme (4). 

The 1 H-NMR spectrum of material "4" showed a broad singlet at 

δ 6.615- 6.651 ppm for aromatic protons and singlet signal at δ 3.338 

ppm for –OH groups. 

13 C-NMR spectral data and DEPT 135 experiments showed that 

material 4 contains only two types of carbons: 

=C-H at δ (115.29- 117.30) ppm and C-O at δ (149.87- 150.76) ppm.

PART II 

From the previously mentioned data and the available literature 

(208) , it is assumed that this material is di (3, 4, 5- trihydroxy phenyl) 

ether. 

For our knowledge, this is the first report about the isolation of 

this compound from nature. Also, this is the first detection of this class 

of natural product (diphenyl ether) in terrestrial plant while many 

marine brown algae (209) contain the phlorotannins which are 

phloroglucinol units exclusively linked by aryl ether bonds and can be 

grouped into several structure subclasses according to the structural 

elements they contain (210) . 

HO 

HO 

HO 

O 

- 146 - 

OH 

OH 

OH 

Di (3, 4, 5- trihydroxy phenyl) ether.

- 147 - 

PART II 




Boj. Hort. Maurit.

PART II 



- 148 -

- 149 - 

PART II 

Fig. (30): 13 C-NMR spectrum of material "4" of Phyllanthus atropurpureus 


Fig. (31): DEPT 135 spectrum of material "4" of Phyllanthus atropurpureus 

Boj. Hort. Maurit.

HO 

O 

- H 

HO 

HO 

HO 

HO 

- CO 

PART II 

HO 

HO 

H 

H 

H 

H 

m/z 98 (13.08 %) 

HO 

- OH 

HO 

HO 

m/z 248 ( 0.23 %) 

m/z 266 


- 150 - 

+ 

- OH 

m/z 109 (10.19 %) 

- H 

O 

m/z 108 (4.51 %) 

- H 

m/z 107 (20.84 %) 

+ . 

OH 

O O 

O H 

m/z 140 (2.47 %) 

- CO 

m/z 112 (6.79 %) 

- CO 

OH 

O H 

m/z 84 (20.84 %) 

- OH 

m/z 67 (52.58 %) 


OH 

OH 

OH


- 151 - 

PART II 

Material "5" (30 mg) occurs as yellowish white residue 

(methanol), with m.p. 179 o C. It is freely soluble in methanol and ethyl 

acetate, slightly soluble in chloroform, insoluble in benzene and light 

petroleum. It shows Rf value of 0.89 & 0.59 (System 9 & 6 

respectively). Material "5" gave blue colour with FeCl3, red colour 

with nitric acid and yellow colour with alkali (T.S) and aluminum 

chloride (T.S). 





Vmax (KBr) cm -1 : 3500-3250, 2930-2850, 1733, 1620 and 1458. 


The data obtained by UV spectral analysis of material "5" (Fig. 

33) using different shifting reagents are summarized in table (23). 

Table (23): UV Spectral data of material "5" 

Shift reagent 

λ max nm 

Band II Band I 

MeOH 252 292( sh.) 

MeOH + NaOMe 274 300 

MeOH + AlCl3 272 312 (sh.) 

MeOH + AlCl3 + HCl 260 298 

MeOH + NaOAc 250 292 (sh.) 

MeOH + NaOAc + Boric acid 264 296 


The mass spectrum of material "5" (Fig. 34) showed the 

following fragments:

PART II 

m/z (% relative abundance):118 (0.1), 111 (7.7), 110 (100), 92 (10.2), 

64( 80.4) and 63 (31.3) 

1 H-NMR Spectral analysis: 

The 1 H-NMR spectrum of material "5" (Fig. 35) using CD3OD 

(500 MHz), showed the presence of the following signals which are 

listed in table (24). 

Table (24): 1 H-NMR spectral data of material "5". 

Position of proton δ (chemical shift) 

2 7.416,1H, S 

7 7.416,1H, S 

2', 6' 7.40, 2H, d, J= 8.5 Hz 

3', 5' 6.74 , 2H, d, J= 8.5 Hz 


Material "5" gave yellow colour with alkali (T.S) and AlCl3 in 

addition to blue colour with ferric chloride indicating phenolic 

character (188) . It gave red colour with nitric acid indicating isoflavone 

nature of the material (211) . 

The IR spectrum showed absorption peaks at 3500-3250 cm -1 

indicating the presence of hydroxyl group; peak at 1733 cm -1 for γ 

pyrone ring and peaks at 1620 and 1458 cm -1 for aromaticity. 

The UV data showed one major band at λmax 252 nm (band II) 

and a shoulder at 292 nm (band I) in methanol suggesting an 

isoflavone material (187) . Addition of NaOMe showed a bathochromic 

shift (+8 and +22 nm) in band I and II respectively indicating the 

presence of free hydroxyl groups at ring A. AlCl3 produced a 

- 152 -

- 153 - 

PART II 

bathochromic shift (+20 nm) in both band I and II indicating the 

presence of ortho-dihydroxy groups in ring A and/ or free 5-OH, this 

will confirmed upon addition of HCl, where the absorption of AlCl3 

was reduced but not return back to methanol spectrum values. NaOAc 

caused no bathochromic shift in band II indicating the absence of free 

hydroxyl group at C-7. No bathochromic shift in band I upon the 

addition of NaOAc/ H3BO3. 

The mass fragmentation pattern of material "5" (scheme 5) 

showed parent ion (M + ) at m/z 286 which is in a good accordance with 

a molecular formula C15H10O6 and fragments at m/z 121, 118 

confirmed the presence of one hydroxyl group at ring B. 

The 1 H-NMR spectrum of material "5" showed singlet signal at 

7.416 p.pm which was assigned to H-2 and H-7 ( 187) , two signals at 

δ 7.40 and 6.74 p.p.m (each 2H, d, J=8.5) which were assigned to H- 

2', 6' and H- 3', 5' respectively. 

From the previous spectral analysis; IR, UV, MS and 1 H-NMR 

confirmed that the material "5" is 5, 6, 8, 4'-tetrahydroxy isoflavone. It 

has the following structural formula: 

HO 

For our knowledge this is the first report of isolation of this 

compound from family Euphorbiaceae and it may be a new 

compound. 

OH 

OH 

O 

O 

OH 

5, 6, 8, 4'-tetrahydroxy isoflavone

PART II 

Fig. (32): IR spectrum of material"5" of Phyllanthus atropurpureus 


─ MeOH 

----MeOH+NaOMe 

Fig. (33): UV spectrum of material "5" of Phyllanthus atropurpureus 


- 154 - 

─ MeOH+NaOAc 

---- MeOH+NaOAc+H3BO3 

─ MeOH+ AlCl3 

---- MeOH+AlCl3+ HCl

- 155 - 

PART II 




Boj. Hort. Maurit.

PART II 

HO 

m/z 258 

HO 

OH 

OH 

O 

OH 

O 

- CO 

HO 

OH 

OH 

OH 

O 

O 

M + 286 

HO C O m/z 168 

OH 

m/z 139 

OH 

- CO 

- H 

-CO 

O 

- H 

m/z 110 (100%) 

m/z 111 (7.7%) 

- 156 - 

OH 

O 

HC 

C 

C 

m/z 121 

OH 

OH 

m/z 118 (0.1%) 


atropurpureus Boj. Hort. Maurit.

Characterization of material "6" 

- 157 - 

PART II 

Material "6" (800 mg) occurs as buff fine powder (Chloroform- 

methanol) m.p 144-146 o C; Rf value of 0.84 & 0.66 (100% ethyl 

acetate & system 9 respectively sprayed with 50% aqueous sulphuric 

acid).It is soluble in methanol and ethyl acetate, slightly soluble in 

chloroform, and insoluble in benzene. Material "6" gives yellow 

colour with alkali (T.S) and aluminum chloride (T.S). 



The UV spectroscopic analysis of material "6" (Fig. 36) showed 

absorptions at λ max (methanol) 224.5, 299.5, 313.5 nm and λ max 

(sodium methoxide) at 361 nm. 




Vmax (KBr) cm -1 : 3500-3250, 2920, 1690, 1605, 1512 and 1448. 

The Mass Spectral Analysis: 

The EI-MS spectroscopic analysis of material "6" (Fig. 38) 

showed the following data: 

m/z (% relative abundance): 418 (M + , 8.76), 313 (9.49), 312 (27), 309 

(56.2), 283 (16.06), 219 (8.03), 164(10.22), 147 (100), 119 (5 ), 109 

(5) and 81 (75 ). 

37.96%). 

FAB-MS (positive ion mode) (Fig. 39) showed m/z 419 (M + ,

PART II 


The 1 HNMR and 13 CNMR spectral analysis of material "6" 

(CD3OD, 300 MHz) (Fig. 40) and (CD3OD, 75 MHz) (Fig. 41) 

respectively revealed the presence of several signals which are 

summarized in table (25). 

Table (25): 1 HNMR and 13 CNMR Spectral Data of Material "6": 

H-N o 

δ – value 

(ppm) 

Integration 

- 158 - 

J (Hz) 

δ – value 

(ppm) 

C-1 152.3 s 

C-N o 

H-2 6.94 2H d (9) C-2 119.64 d 

H-3 6.65 2H d (9) C-3 116.65 d 

C-4 153.9 s 

H-5 6.65 2H d (9) C-5 116.65 d 

H-6 6.94 2H d (9) C-6 119.64 d 

H-1' 4.73 1H d (6.9) C-1' 103.67 d 

H-2' 3.44 1H t (6.9) C-2' 75.51 d 

H-3' 3.66 1H t (6.9) C-3' 77.93 d 

H-4' 3.35 1H t (6.9) C-4' 71.95 d 

H-5' 3.66 1H dd (6.9, 2.5) C-5' 74.96 d 

H-6'a 4.34 1H dd (12, 6.9) C-6' 64.74 t 

H-6'b 4.52 1H dd (12, 2.5) 

C-1" 127.37 s 

H -2" 7.45 2H d (8.7) C -2" 131.28 d 

H -3" 6.81 2H d (8.7) C -3" 116.94 d 

C-4" 161.35 s 

H -5" 6.81 2H d (8.7) C-5" 116.94 d 

H -6" 7.45 2H d (8.7) C -6" 131.28 d 

H -7" 7.63 1H d (15.9 ) C -7" 146.88 d 

H -8" 6.34 1H d (15.9) C -8" 114.95 d 

C-9" 169.06 s 

DISCUSSION AND CONCLUSION 

The UV spectrum showed λ max (methanol) at 299.5 and 313.5 

nm which implied the presence of a conjugated chromophore and 

sodium methoxide which caused 48 nm bathochromic shifts in UV 

spectrum indicating the presence of free OH group. 

The IR spectrum showed a hydroxyl stretching band at 3500- 

3250 cm -1 . Also it showed an absorption band at 1690 cm -1 for ester

- 159 - 

PART II 

carbonyl besides another bands at 1605, 1512 and 1448 cm -1 for 

aromaticity. 

The 1 HNMR spectrum of material "6" (Fig. 40& table 25) 

showed signals for two sets of aromatic protons (δ 6.81 and 7.45 ppm, 

J= 8.7 Hz) and (δ 6.65 and 6.94 ppm, J= 8.7 Hz), trans - olefinic 

protons (J= 15.9) at δ 7.63 and 6.34 ppm, and sugar protons (anomeric 

proton at δ 4.73 ppm). 

The 13 C-NMR spectrum of material "6" (Fig. 41& table 25) 

revealed ester carbonyl at δ 169.06 ppm and six carbon signals arising 

from a monosaccharide moiety whose anomeric carbon signal is at 

103.67 ppm indicating the presence of a glucose moiety in the 

molecule (42, 212, 213) . 

The mass fragmentation pattern of material "6" is a suggestive 

one and shown in scheme (6). The parent ion peak at m/z 418 

corresponding to the molecular formula (C21H22O9), the fragments at 

m/z 309 [M- 109] + and 147 support the presence of a cinnamoyl 

group .Finally it was considered to be a trans-cinnamoyl group 

esterified with a hydroxyl group of the glucose moiety and the 

fragment at m/z 164 and 147 implied the presence of p-coumaroyl 

moiety (212, 214) . 

Extensive NMR analysis: 1 H- 1 H COSY (Fig. 42), DEPT 135 

(Fig. 43), APT (Fig. 44), gHSQCAD (Fig. 45), and gHMBC (Fig. 46) 

spectral data of material "6" allowed a complete assignment of protons 

attached to their respective carbons. 

• Only one CH2 group at 64.74 ppm (C-6') and five quaternary 

carbon at 169.06, 161.35, 153.9, 152.3, 127.37 ppm (C-9", C-4",

PART II 

C-4, C-1, and C-1" respectively) present in the material and this is 

confirmed by DEPT 135 and APT. 

• The chemical shift (δ C 152.3) of one of the aromatic carbons 

indicated that it was attached to an oxygen atom, and the chemical 

shift of the sugar anomeric proton [δ H 4.73 (d, J= 6.9 Hz)] and 

carbon [δ C 103.67] signals indicated that the sugar moiety was 

not attached to the carboxyl group but to this phenolic oxygen 

through a glycosidic linkage. 

• The gHMBC correlation achieved between the anomeric proton 

at δ H 4.73 (H-1') and δ C 152.3 (C-1) support a C-1 location for 

the sugar unit in the glucosidic linkage. 

• The existence of cinnamoyl group in the compound, with an 

ester linkage at C-9", was established by gHMBCAD 

correlations: 

H-6' to C- 9"; H-2" and H-6" to C- 7" and C-4"; and H-3" and H- 

5" to C-2", C-6" and C-4" 

Finally the data confirmed that material "6" is 6'-(4"-hydroxy- 

cinnamoyl) arbutin which is known as robustaside A (215) by 

comparing its UV, 1 H-NMR and 13 C-NMR spectra with the reported 

data. This is the first report about isolation of robustaside A from 

genus Phyllanthus and the second from the nature (215) . 

HO 

4 

HO 

4" 

1 

HO 

O 

1" 

- 160 - 

1' 

7" 

O 

O 

8" 9" 

O 

OH 

OH 

6'-(4"-hydroxy- cinnamoyl) arbutin 

6'

- 161 - 

MeOH+ NaOMe 

MeOH 

PART II 




Boj. Hort. Maurit.

PART II 

Fig. (38): EI-MS spectrum of material "6" of Phyllanthus atropurpureus 


Fig. (39): FAB-Mass spectrum of material "6" of Phyllanthus atropurpureus 


- 162 -

- 163 - 

PART II 




Boj. Hort. Maurit.

PART II 

Fig. (42): 1 H - 1 H COSY spectrum of material "6" of Phyllanthus atropurpureus 


Fig. (43): DEPT 135 spectrum of material "6" of Phyllanthus atropurpureus 


- 164 -

- 165 - 

PART II 

Fig. (44): APT spectrum of material "6" of Phyllanthus atropurpureus 

Boj. Hort. Maurit.

PART II 

Fig. (45): gHSQCAD spectrum of material "6" of Phyllanthus atropurpureus 


Fig. (46): gHMBC spectrum of material "6" of Phyllanthus atropurpureus 


- 166 -

+ 

HO 

HO 

HO 

O 

HO 

O 

O 

m/z 309 ( 56.2 %) 

m/z 164 (10.22%) 

-OH 

m/z 147 (100%) 

4 

HO 

OH 

O 

OH 

-CO 

4" 

OH 

1 

HO 

O 

1" 

1' 

7" 

- 167 - 

O 

O 

8" 9" 

O 

m/z 418 (8.76 %) 

OH 

m/z 119 (5 %) 

OH 

6' 

+ 

HO 

+ . 

O 

m/z 109 (5%) 

- CO 

m/z 81 (75 %) 

PART II 



PART II 


Material "7" (50 mg) occurs as pale yellow crystals (Chloroform- 

methanol) m.p 150-152 o C; Rf value of 0.52 (100% ethyl acetate). It 

is soluble in methanol and ethyl acetate, slightly soluble in 

chloroform, and insoluble in benzene. Material "7" gives yellow 

colour with alkali (T.S) and aluminum chloride (T.S). 



The UV spectroscopic analysis of material "7" (Fig. 49) 

showed absorptions at λ max (methanol) 288, 324 nm and λ max 

(sodium methoxide) at 374 nm. 




Vmax (KBr) cm -1 : 3600-3100, 2925, 1715, 1606, 1511 and 1430. 


The EI-MS spectroscopic date of material "7" (Fig. 51) showed 

the following data: 

m/z (% relative abundance): 255 (M + -179, 100%), 135 (8.1), 118 (7), 

117 (3.7), 110 (13.3), 109 (9.6), 101(8.5) and 100 (10). 


The 1 HNMR spectral analysis of material "7" (CD3OD, 500 

MHz) (Fig. 52) revealed the presence of several signals which are 

summarized in table (26). 

- 168 -

Table (26): 1 HNMR Spectral Data of Material "7": 

δ – value 

(ppm) 

Integration J (Hz) 

H-2 6.94 2H d (9) 

H-3 6.64 2H d (9) 

H-5 6.64 2H d (9) 

H-6 6.94 2H d (9) 

H-1' 4.72 1H d (7.6) 

H-2', 3', 4' 3.35-3.5 3H m 

H-5' 3.65 1H dd (6.9, 2.5 ) 

H-6'a 4.35 1H dd (12, 6.9) 

H-6'b 4.52 1H dd (12, 2.5) 

H -2" 6.93 1H d (2.3) 

H -5" 7.06 1H d (9) 

H -6" 6.81 1H dd (9, 2.3 ) 

H -7" 7.57 1H d (16) 

H -8" 6.29 1H d (16) 

H-N o 

DISCUSSION AND CONCLUSION 

- 169 - 

PART II 

The UV spectrum showed λ max (methanol) at 288 and 324 nm 

which implied the presence of a conjugated chromophore and sodium 

methoxide which caused 50 nm bathochromic shifts in UVspectrum 

indicating the presence of free OH group. 

The IR spectrum showed a hydroxyl stretching band at 3600- 

3100 cm -1 .Also it showed an absorption band at 1715 cm -1 for ester 

carbonyl besides another bands at 1606, 1511 and 1430 cm -1 for 

aromaticity. 

The mass fragmentation pattern of material "7" is a suggestive 

one and shown in scheme (7). 

The 1 HNMR spectrum of material "7" (Fig. 52& table 26) 

showed signals at:

PART II 

• δ 6.64 (2H, d, J= 9Hz) and 6.94 (2H, d, J= 9Hz) for H-2, 

H-6 and H- 3, H-5 aromatic protons respectively. 

• δ 6.81 (1H, dd, J= 9, 2.3), 6.93 (1H, d, J=2.3) and 7.06 

(1H d, J= 9) for H- 6", H- 2" and H- 5" aromatic protons 

respectively. 

• δ 7.57 (1H, d, J=16) and 6.29 (1H, d, J=16) for trans- 

olefinic protons H-7" and H-8" respectively. 

• δ 4.72 for anomeric proton of glucose. 

Comparing data of material 7 with robustaside A was illustrated 

in table (27). 

Table (27): Comparison between robustaside A and material "7" data. 

UV 

Mass 

1 HNMR 

Comparison Robustaside A Material 7 

m.p. 144-146 o C 150-152 o C 

Rf value* 0.66 0.52 

buff fine powder faint yellow crystals 

MeOH 299.5, 313.5 nm 288, 324 nm 

NaOMe 361 nm 374 nm 

IR 

3500-3250(OH), 2920, 1690(C=O), 

1605, 1512 and 1448 (aromaticity) 

cm -1 

- 170 - 

3600-3100(OH), 2925, 1715(C=O), 

1606, 1511 and 1430 (aromaticity) 

cm -1 

M+ 418 434 

base 

peak 

147 255 

H-2 6.94 (2H, d, J= 9) 6.94 (2H, d, J= 9) 

H-3 6.65 (2H, d, J= 9) 6.64 (2H, d, J= 9) 

H-5 6.65 (2H, d, J= 9) 6.64 (2H, d, J= 9) 

H-6 6.94 (2H, d, J= 9) 6.94 (2H, d, J= 9) 

H-1' 4.73 (1H, d, J= 6.9) 4.72 (1H, d, J= 7.6) 

H-2' 3.44 (1H, t, J= 6.9) 

H-3' 3.66 (1H, t, J= 6.9) 

3.35-3.5 (3H, m) 

H-4' 3.35 (1H, t, J= 6.9) 

H-5' 3.66 (1H, dd J= 6.9, 2.5) 3.65 (1H, dd, J= 6.9, 2.5) 

H-6'a 4.34 (1H, dd J=12, 6.9) 4.35 (1H, dd, J= 12, 6.9) 

H-6'b 4.52 (1H, dd J=12, 2.5) 4.52 (1H, dd J= 12, 2.5) 

H -2" 7.45 (2H, d, J= 8.7) 6.93 (1H, d, J= 2.3) 

H -3" 6.81 (2H, d, J= 8.7) ---

- 171 - 

PART II 

Comparison Robustaside A Material 7 

H -5" 6.81 (2H, d, J= 8.7) 7.06 (1H, d, J= 9) 

H -6" 7.45 (2H, d, J= 8.7) 6.81 (1H, dd, J= 9, 2.3) 

H -7" 7.63 (1H, d, J= 15.9) 7.57 (1H, d, J= 16) 

H -8" 6.34 (1H, d, J= 15.9) 6.29 (1H, d, J= 16) 

*solvent system (100% ethyl acetate); 

Finally the data confirmed that material 7 is 6'- (3", 4"-dihydroxy 

cinnamoyl) arbutin or it can be named as 3"-hydroxy robustaside A. 

For our knowledge this is the first report about the isolation of this 

compound from the nature. 

HO 

4 

HO 

HO 

3" 

1 

HO 

O 

1" 

1' 

O 

8" 9" 

7'' 

3"-hydroxy robustaside A 

O 

OH 

O 

OH 

6'

PART II 

─ MeOH 






- 172 -

- 173 - 

PART II 




Boj. Hort. Maurit.

PART II 

HO 

HO 

HO 

HO 

m/z 179 

- COO 

O 

O 

m/z 135 (8.1 %) 

- OH 

m/z 118 (7%) 

- OH 

m/z 101 (8.5%) 

HO 

+ 

HO 

HO 



+ 

- 174 - 

HO 

O 

O 

O 

O 

OH 

m/z 434(M + ) 

HO 

OH 

HO 

O 

+ . 

O 

OH 

+CH 2 

(M + -179) 

m/z 255 (100 %) 

HO OH 

m/z 110 (13.3%) 

+ 

HO 

- H 

O 

m/z 109 (9.6%) 

OH

- 175 - 

PART II 

Column Chromatography of Stem and Root Ethyl 

Acetate Soluble Fraction: 

A. TLC Screening of Collected Ethyl Acetate Soluble Fraction 

of Root and Stem. 

Thin layer chromatographic investigation of root and stem ethyl 

acetate soluble fraction was carried out using silica gel GF245 

chromatoplates, solvent systems (6, 9& 13), UV lamp 365nm and 

spray reagents (ammonia and 50 % aqueous sulfuric acid) for 

visualization. The chromatographic study revealed the presence of 

two major and eight minor spots. The Rf values and behaviors with 

different reagents are illustrated in table (28). 

Table (28): TLC Investigation of Collected Ethyl Acetate Soluble 

Fraction of Stem and Root with different visualizing agents. 

Spot 

n 

Rf value Visualizing agent 

o 6 9 13 

UV lamp 

NH4OH 50 % H2SO4 

(365 nm) 

Isolated 

materials 

1 0.79 0.99 0.98 

Blue 

fluorescence 



3 0.69 0.92 0.87 

Blue 

fluorescence 

Yellow 

Brownish 

orange 

--- 


5 0.49* 0.86* 0.78* Blue 

fluorescence 

Yellow Yellow material 8 

6 0.39 0.84 0.76 

Blue 

fluorescence 

Yellow 

Brownish 

orange 

material 6 

7 0.31* 0.60* 0.54* Purple Yellow Yellow material 9 

8 0.29 0.48 0.43 

Blue 

fluorescence 


9 0.20 0.30 0.24 

Blue 

fluorescence 


10 0.09 0.15 0.11 

Blue 

fluorescence 

Yellow 

Yellowish 

brown 

--- 

* major spot

PART II 

B. Column Chromatography of Collected Root and Stem Ethyl 

Acetate Soluble Fraction. 

About 4 g of the collected ethyl acetate soluble fraction of root 

and stem was dissolved in the least amount of methanol and adsorbed 

on 7 g of silica gel for column and transferred onto a silica column ( 2 

× 100 cm, 100 g) packed with benzene. Elution was carried out 

starting with benzene and the polarity gradually increased with 

chloroform and methanol. Fractions (250 ml. each) were collected, 

concentrated 

and examined by TLC using solvent system (9) and 50 % aqueous 

sulphuric acid as visualizing reagent and similar fractions were 

pooled. Description of the column chromatographic process is 

illustrated in table (29). 

Table (29): Column Chromatography of Collected Ethyl Acetate 

soluble Fraction of Root and Stem. 

Eluent 

% 

Compositio 

n 

Fr. 

N o . 

- 176 - 

Fr. 

Wt. 

(g) 

Rf value (solvent 

system 9 ) 

Isolated 

materials 

Benzene 100% 1-5 0.150 Undifferentiated spots --- 

Benzene: chloroform 75 :25 6-13 0.230 0.99 --- 

Benzene: chloroform 60:40 14-17 0.063 0.97 --- 

Benzene: chloroform 30:70 18-27 0.180 0.97, 0.92 --- 

Chloroform 100% 28-33 0.180 

0.92 + 


--- 

Chloroform: methanol 0.5: 99.5 34-36 0.190 Undifferentiated spots --- 

Chloroform: methanol 99: 1 37-42 0.330 Undifferentiated spots --- 

Chloroform: methanol 97: 3 43-69 0.850 Undifferentiated spots --- 

Chloroform: methanol 95: 5 70-78 0.550 0.88 --- 

Chloroform: methanol 92: 8 79-82 0.150 0.86*, 0.84 

material 6 + 

material 8 

Chloroform: methanol 84: 16 83-88 0.170 0.84 , 0.6* material 9 

Chloroform: methanol 68: 32 89&90 0.410 0.48 , 0.30 , 0.15 --- 

Methanol 100% 91-93 0.547 Undifferentiated spots --- 

*Rf Corresponding to the isolated material

• Isolation of material "8": 

- 177 - 

PART II 

Investigation of fractions (79-82) using TLC, solvent system (6 

&9) and 50 % aqueous sulphuric acid as visualizing reagent revealed 

the presence of one major yellow spot with Rf value of 0.49 & 0.86 

and one minor spot with Rf value of 0.39 & 0.84 respectively. To 

obtain pure material from this 

fraction, crystallization from ethyl acetate and MeOH mixture was 

done to give 50 mg of yellow crystals with m.p. 250- 251 o C, this 

compound was designated as material "8". The minor spot give buff 

sandy crystals with m.p. 144-146 o C and this was previously isolated 

and designated as material "6". 

• Isolation of material "9": 

Investigation of fractions (83-88) using TLC, solvent system (6 

and 9) and 50 % aqueous sulphuric acid as visualizing reagent 

revealed the presence of one major yellow spot with Rf value of 0.31 

and 0.6 respectively. Upon crystallization several times from MeOH- 

ethyl acetate mixture gave 43 mg of yellow crystals with m.p. 254-255 

o C, this compound was designated as material "9".

PART II 


Material "8" was obtained as yellow crystals (MeOH / ethyl 

acetate), m.p. 250-251 o C and Rf of 0.49 (solvent system 6). It is 

soluble in ethyl acetate, methanol, insoluble in benzene and 

chloroform. Material "8" developed a yellow colour with ammonia, 

AlCl3 and showed negative Molisch's and Fehling's tests. 





Table (30): UV spectral data of material "8". 

Shift reagent 

Band II 

λ max nm 

Band I 

MeOH 278 302 

MeOH + NaOMe 290 368 

MeOH + AlCl3 282 314, 344 

MeOH + AlCl3 + HCl 282 314, 344 

MeOH + NaOAc 276, 310(sh.) 342 

MeOH + NaOAc + H3BO3 274, 310(sh.) 348 




Vmax (KBr) cm -1 : 3500-3100, 2924, 2853, 1648, 1453, 1386 and 1181. 




- 178 -

- 179 - 

PART II 

m/z (% relative abundance): 329 (M + -1, 7.8), 280 (9.8), 212 (9.8), 

155 (25.5), 154 (5.9), 121 (29.4), 120 (29.4), 118 (11.8), 93 (78.4), 92 

(9.8) and 89 (100). 

1 H-NMR Spectral Analysis: 

The 1 H-NMR spectrum of material "8" (Fig. 56), using CD3OD 

(500 MHz) is represented in table (31) 

Table (31): 1 H-NMR spectral data of material "8". 

H-No. δ (ppm) integration J (Hz) 

H- 3 6.70 1H S 

H- 2', 6' 7.99 2H d, J= 8.75 

H- 3', 5' 7.10 2H d, J= 8.75 

O-CH3 at C-6 3.89 3H S 

O-CH3 at C-8 3.92 3H S 


The IR spectrum of material "8" showed a hydroxyl stretching 

band at 3500-3100 cm -1 . Also it showed an absorption band at 2924 

and 2853 cm -1 for aliphatic C-H. It showed an absorption band at 1648 

cm -1 for γ-pyrone ring beside another three bands at 1453, 1386 and 

1181 cm -1 for -CH2 bending, -CH3 bending and C-C respectively. 

Material "8" was recognized as a flavone from its UV absorption 

maxima at 302 nm (band I) and 278 nm (band II). A bathochromic 

shift (+66 nm) in band I was observed upon addition of NaOMe 

indicated a free hydroxyl group at C-4'. Bathochromic shift (+42 nm) 

in band I was observed upon addition of AlCl3 and AlCl3/ HCl 

indicating the presence of –OH group at C-5 and absence of 

orthohydroxylation as well.

PART II 

In addition, bathochromic shift of band II was observed by the 

effect of NaOAc that confirmed the presence of free –OH group at C- 

7. 

The mass spectrum exhibited a molecular ion at m/z 329 (M + -1) 

which is in a good accordance with a molecular formula C17H14O7for 

this compound. Fragments at m/z 212 corresponding to ring A with 

two methoxyl groups and two hydroxyl groups, 118 corresponding to 

ring B with one hydroxyl group were observed in scheme (8). 

The 1 H-NMR spectrum of this material confirmed the presence 

of flavone through the arise of an olefinic proton singlet signal 

localized at δ 6.7 ppm assigned for H-3. The spectrum also showed 

two broad doublets: one at δ 7.99 (2H, J= 8.75 Hz, H-2', 6') and 

another one at δ 7.1 (2H, J= 8.75 Hz, H-3', 5') that are characteristic 

for 4'-substituted B-ring (211). 

Finally, the data confirmed that material "8" is 5, 7, 4'- 

trihydroxy- 6, 8-dimethoxyflavone. Evidently, material "8" was 

tentatively identified as demethoxysudachitin by comparing its 1 H- 

NMR with the reported data (187) . This represents the first time of 

isolation of this compound from the genus Phyllanthus and from 

Phyllanthus atropurpureus Boj. Hort. Maurit. as well. 

HO 

MeO 

OMe 

OH 

O 

O 

Demethoxysudachitin 

- 180 - 

OH

─ MeOH 


- 181 - 



PART II 


---- MeOH+AlCl3+ HCl 




Boj. Hort. Maurit.

PART II 

Fig.(55): Mass spectrum of material "8" of Phyllanthus atropurpureus 




- 182 - 

329

MeO 

m/z 313 

-OH 

m/z 296 

-OH 

OMe 

O 

O 

-OH 

m/z 280 (9.8%) 

m/z 120( 29.4%) 

-OH 

-OH 

HO 

MeO 

m/z 329 (7.8%) 

OMe 

OH 

HO 

MeO 

Scheme (8): Mass fragmentation pattern of material "8" of 


- 183 - 

O 

O 

-H 

M + 330 

OMe 

OH 

O 

C 

m/z 212 (9.8%) 

HO 

O 

-Me 

-Me 

m/z 182 

O 

-CO 

OH 

O 

m/z 154 (5,9%) 

O 

OH 

-H 

+. 

O 

PART II 

HC 

C 

OH 

m/z 121 (29.4%) 

C 

m/z 118 (11.8%) 

m/z 153 (5.9%) 

-OH 

m/z 101 

OH

PART II 


Material "9" occurs as yellow crystals (43 mg), m.p. 254-255 o C 

and Rf values of 0.31 and 0.6 using solvent systems (6 & 9) 

respectively. It is soluble in ethyl acetate, methanol, insoluble in 

benzene and chloroform. It dissolves in dilute solution of alkalies 

producing intense yellow colour, indicating the flavonoidal nature of 

the material. Alcoholic solution developed a yellow colour when a 

few drops of 0.1 M AlCl3 solution was added and gives bluish green 

colour with neutral ferric chloride solution indicating the presence of 

phenolic group. Material "9" gives positive Molisch's test and reduce 

Fehling's solution after acid hydrolysis. 





Table (32): UV Spectral data of material "9". 

Shift reagent 

λ max nm 

Band II Band I 

MeOH 256 354 

MeOH + NaOMe 271 328 (sh.), 406 

MeOH + AlCl3 272 302(sh.), 366 (sh.), 420 

MeOH + AlCl3 + HCl 262 300(sh.),354,400 (sh.) 

MeOH + NaOAc 261 362 

MeOH+NaOAc+ H3BO3 261 371 


The IR spectrum of material "9" (Fig. 58) showed the 

following absorption frequencies: 

Vmax (KBr) cm -1 : 3500-3100, 1651, 1619, 1447 and 1120. 

- 184 -


- 185 - 

PART II 



m/z (% relative abundance): 302 (100), 301 (17.57), 274 (9.46), 273 

(12.16), 258 (2.7), 152 (5.41), 137 (18.91), 136 (9.5), 134 (5.41), 73 

(54.05), 60 (99.32) and 44 (22.97). 

FAB-MS (positive ion mode) (Fig. 60): 465 [M + 1] + 

1 H-NMR and 13 C-NMR Spectral Analysis: 

The 1 H-NMR spectrum of material "9" (Fig. 61, CD3OD, 500 

MHz) and the 13 C-NMR spectrum (Fig. 62, CD3OD, 125 MHz) are 

represented in table (33) 

Table (33): 1 H-NMR (CD3OD, 500 MHz) and 13 C-NMR (CD3OD, 

125 MHz) spectral data of material "9". 

Position of 

hydrogen 

δ (chemical shift) 

Position of 

carbon 

δ (chemical shift) 

C-2 157.17 

C-3 134.17 

C-4 177.95 

C-5 161.6 

H-6 6.09, 1H, d, J = 2 Hz C-6 98.86 

C-7 160 

H-8 6.28, 1H, d, J = 2 Hz C-8 93.56 

C-9 157.46 

C-10 103.94 

C-1' 121. 67 

H-2' 7.61, 1H, d, J =2 Hz C-2' 116.1 

C-3' 144.53 

C-4' 148.5 

H-5' 6.77, 1H, d, J = 9 Hz C-5' 114.59 

H-6' 7.49, 1H, dd, J = 9& 2 Hz C-6' 121.75 

H-1'' 5.13, 1H, d, J = 7.5 Hz C-1'' 103.01 

H-2'' C-2'' 74.31 

H-3" 3.22- 3.35, 3H, m 

C-3'' 76.98 

H-4" 

C-4'' 69.81 

H-5" 3.38, 1H, m C-5'' 76.73 

H-6"a 3.48, dd, J= 12, 6 Hz 

H-6"b 3.61, dd, J= 12, 2 Hz 

C-6'' 61.14

PART II 


The physical characters, colour reaction and UV absorption of 

material "9" suggest the presence of a flavonoidal glycoside skeleton. 

The IR showed absorption at 3500-3100 together with peak at 

1120 cm -1 indicating the presence of several hydroxyl groups. Also it 

showed the presence of carbonyl group at 1651 cm -1 . 

Material "9" was recognized as a flavonol from its UV 

absorption maxima at 256 nm (band II) and 354 nm (band I). A 

bathochromic shift (+ 52 nm) in band I upon addition of NaOMe 

indicating the presence of free hydroxyl group at C-4'. Addition of 

AlCl3 produced a bathochromic shift (+66 nm) in band I indicating the 

presence of ortho-dihydroxy groups and/ or free 5-OH this will 

confirmed upon addition of HCl, where the absorption of AlCl3 was 

reduced indicating the presence of free 5-OH. This also confirmed by 

a bathochromic shift in band I (+ 17 nm) upon the addition of NaOAc/ 

H3BO3 while band II was unaffected by the use of NaOAc indicating 

substituted OH at C-7. 

The mass spectrum (FAB-MS) exhibited a molecular ion at m/z 

465 [M +1] + which is in a good accordance with a molecular formula 

C21H20O12 for this compound. EI-MS spectrum exhibited fragments a 

m/z 302 corresponding to M + -glu., m/z 152 corresponding to ring A 

with two hydroxyl groups and m/z 134 corresponding to ring B with 

two hydroxyl groups were observed in scheme (9). 

- 186 -

- 187 - 

PART II 

The 1 H-NMR spectrum of material "9" showed two doublets at 

6.09, 6.28 ppm with meta coupling (J= 2 Hz) assigned to H-6 and H-8, 

respectively, two doublets at δ 6.77(J=9 Hz), 7.61(J= 2Hz) ppm 

which were assigned to H-5' and H-2', respectively. Proton at δ 7.49 

(dd, J= 9, 2 Hz) was assigned to H-6' this pattern is typical for 

quercetin. The sugar moiety was confirmed to be glucose by the 

appearance of an anomeric proton signal at δ 5.13 (J =7.5 Hz) 

characteristic for glucose in β-glucosidic linkage confirmed by 13 C- 

NMR at δ 103.01 ppm. The effect of NaOAc on band II (no 

bathochromic shift) indicated glycosylation at C-7. 

The 13 C-NMR spectrum of material "8" showed 21 carbon 

resonances as shown in table (33). The 13 C-NMR spectrum of this 

material was compared to that of quercetin -7- O- glucoside 

and all signals were similar. 

(216, 217) 

Extensive analysis of the 1 H- 1 H and 1 H - 13 C correlation spectral 

data of material "9" confirm the assignment of this compound to be 

quercetin -7- O- glucoside 

The glycosidic linkage at C-7 was suggested through the 

chemical shift of the glucose anomeric proton [δ H 5.13 (d, J= 7.5 Hz)] 

and carbon [δ C 103.01] signals. The gHMBCAD correlation achieved 

between the anomeric proton at δ H 5.13 (H-1'') and δ C 160 (C-7) 

support a C-7 location for the sugar unit in the glucosidic linkage. 

By comparing the MS, 1 H- and 13 C- NMR data of this material 

with the reported data (216) , we can conclude that this material is

PART II 

5, 3', 4'- trihydroxy flavonol-7-O-glucoside. Evidently, material "9" 

was tentatively identified as quercetin-7-O-glucoside or 

Quercimeritrin. 

This represents the first time of isolation of quercetin-7-O- 

glucoside from the genus Phyllanthus. 

Glu 

O 

OH 

O 

O 

- 188 - 

OH 

OH 

Quercetin-7-O-glucoside 

OH

─ MeOH 




- 189 - 

PART II 


---- MeOH+AlCl3+ HCl 


Boj. Hort. Maurit

PART II 



Fig. (59): EI-MS spectrum of material "9" of Phyllanthus atropurpureus 


- 190 -

- 191 - 

PART II 

Fig. (60): FAB-MS spectrum of material "9" of Phyllanthus atropurpureus 



Boj. Hort. Maurit.

PART II 



Fig. (63): 1 H- 1 H Cosy spectrum of material "9" of Phyllanthus atropurpureus 


- 192 -

- 193 - 

PART II 

Fig. (64): gHSQCAD correlation spectrum of material "9" of Phyllanthus 


Fig. (65): gHMBCAD correlation spectrum of material "9" of Phyllanthus 


PART II 

m/z 301(17.57%) 

HO 

OH 

O 

OH 

HO 

m/z 274 (9.46%) 

- H 

m/z 273 (12.16%) 

OH 

Glu 

- H 

O 

- CO 

OH 

OH 

HO 

OH 

OH 

Scheme (9): Mass fragmentation pattern of material "9" of 

O 

O 


- 194 - 

O 

O 

OH 

OH 

- Glu. 

m/z 302(100%) 

O 

C 

O 

m/z 152 (5.41%) 

OH 

OH 

OH 

OH 

O 

M + 464 

C 

HC 

C 

OH 

m/z 134 (5.41%) 

OH 

OH 

m/z 137 (18.91%) 

OH

Introduction: 

Biological Activities 

A. Antihepatotoxic activity. 

- 195 - 

PART III 

The liver is one of the largest gland and most complex organ in 

the body. It performs multiple functions, including the production of 

the proteins and enzymes, detoxification, metabolic functions, the 

regulation of cholesterol and blood clotting (218) .It contains the highest 

concentration of enzymes involved in phase I oxidation-reduction 

reactions (219) . It is the primary site of biotransformation and 

detoxification of exogenous toxin xenobiotics (220) . 

Unfortunately, the liver is often the most abused organ in the 

body. It is exposed to alcohol, drugs, and a multitude of environmental 

toxins. An overstressed liver can impair detoxification and manifest in 

what may appear to be unrelated symptoms. Eventually, a 

dysfunctional liver can not perform its tasks properly and 

consequently the body becomes subject to toxicity and an overall 

decline in metabolic function (221) . 

Problems associated with liver dysfunction can ultimately lead to 

serious illness such as hepatitis, cirrhosis, fatty liver, alcoholic liver 

disease, and biliary cirrhosis (221) . 

Cirrhosis is a complex disease in which several biological and 

biochemical alteration are combined and no proven effective treatment 

capable of reversing it has been developed (222) .

PART III 

Many plants demonstrate hepatoprotective activity. Some 

Phyllanthus plants were used as remedies for hepatic disorders 

224) . 

- 196 - 

(101, 223 

Phyllanthus contains a lot of tannin and flavonoids which possess 

the antioxidant activity (98) . The aim of the present study is to 

investigate the ability of 75% ethanolic extract of Phyllanthus on 

repairing rat liver damage induced by CCl4 and also the possible 

mechanisms of Phyllanthus antihepatotoxic activity. 

I-Materials: 

A. Experimental animals: 

Material and methods 

Fifty adult male albino rats weighing about 200-250 g each were 

used in the present investigation. The animals were housed in cages 

with wood shaving bedding, and allowed to become acclimatized to 

laboratory conditions for one week before the experiment. 

B. Experimental design: 

The animals were randomly divided into two groups according to 

the following design: 

(1) Group (1) (n=10) : 

This group received liquid paraffin (0.3 ml/Kg, i.p.) for 45 days 

and served as normal control group. 

(2) Group (2) (n=40) : 

This group received carbon tetrachloride 3 times a week for 45 

days in a dose of 25 µl/100 g body weight, i.p. diluted (1:6) with 

liquid paraffin and served as subacute cirrhotic group. 

Then group (2) was subdivided into 4 subgroups which are: 

• Subgroup (A) (n=10): Cirrhotic rats without treatment.

- 197 - 

PART III 

• Subgroup (B) (n=10): Cirrhotic rats received the aerial 

part ethanolic extract of Phyllanthus atropurpureus Boj. 

Hort. Maurit. for 30 days in a dose of 200 mg/kg body 

weight, orally. 

• Subgroup (C) (n=10): Cirrhotic rats received the root 

ethanolic extract of Phyllanthus atropurpureus Boj. Hort. 

Maurit. for 30 days in a dose of 200 mg/kg body weight, 

orally. 

• Subgroup (D) (n=10): Cirrhotic rats received the 

silymarin for 30 days in a dose of 100 mg/kg body weight, 

orally. 

C. Drugs and chemicals: 

Table (34): Drugs and chemicals used in antihepatotoxic experiment: 

Drug name Dose Note 

Silymarin 100 mg/kg 

Root extract 200 mg/kg 

Aerial part 

extract 

II-Methods 

200 mg/kg 

The drug was dissolved in normal 

saline 

The drugs was suspended or 

emulsified in 10% gum acacia 

All drug solutions were freshly prepared just before use. 

Induction of liver cirrhosis 

Liver cirrhosis was induced in rats by intraperitoneal injection 

(225) of CCl4 3 times a week for 45 days in a dose of 25µl/100 g body 

weight. CCl4 was freshly diluted by liquid paraffin directly before the 

injection (1:6).

PART III 

Blood sampling and serum preparation 

Blood samples were collected in clean dry test tubes from the 

orbital sinus of fasted rats using heparinized micro capillary tubes 

according to the method of (Riley, 1960 and Sorg &Buchner, 1964) 

(226, 227) . Blood samples were centrifuged directly at 3500 r.p.m for 15 

minutes using Heraeus Sepatech centrifuge (Labofuge 200). Liver 

enzymes (ALT, AST), Proteins (total protein, albumin) and 

antioxidant parameters (Malondialdehyde and glutathione) were 

determined in the collected plasma and serum. 

1. Determination of aspartate aminotransferase (AST): 

Serum AST level was determined by a colorimetric method (228) 

using a diagnostic kit supplied by Plasmatek (Germany). 

Principle: 

The enzyme AST catalyzes the following reaction: 

L-aspartate + 2- oxoglutarate oxalacetate + L-glutamate 

The formed oxalacetate reacts with 2, 4-dinitrophenylhydrazine 

to form oxalacetate hydrazones, which are brown in alkaline medium. 

The product is determined spectrophotometrically at λ505 nm using 1 

cm light path cuvette. 

2. Determination of alanine aminotransferase (ALT): 

Serum ALT level was determined by a colorimetric method (228, 

229) using a diagnostic kit supplied by Plasmatek (Germany). 

Principle 

The enzyme ALT catalyzes the following reaction: 

L-alanine + 2- oxoglutarate pyruvate + L-glutamate 

ALT 

AST 

The formed pyruvate reacts with 2, 4-dinitrophenylhydrazine 

- 198 -

- 199 - 

PART III 

to form pyruvate hydrazones, which are brown in alkaline medium. 

The product is determined spectrophotometrically at λ505 nm using 1 

cm light path cuvette. 

3. Determination of total protein: 

• Biuret method 

Total protein was determined by an enzymatic colorimetric 

method (230) using a diagnostic kit supplied by Biocon (Germany). 

Principle: 

Colorimetric determination of total protein based on the 

principle of Biuret reaction (copper salts in an alkaline medium). 

Protein in serum sample forms a blue coloured complex when treated 

with cupric ion in alkaline solution. The intensity of blue colour is 

proportional to the protein concentration and measured 

spectrophotometrically at λ505 nm. 

4. Determination of serum albumin: 

• Bromcresol green method 

Serum albumin was determined by an enzymatic colorimetric 

method (231, 232) using a diagnostic kit supplied by Biocon (Germany). 

Principle: 

Colorimetric determination of serum albumin by using 

bromcresol green (BCG) at pH 4.2. 

5. Determination of malondialdehyde : 

Malondialdehyde was identified as the product of lipid 

peroxidation that reacts with thiobarbituric acid in acidic medium at 

temperature of 95 o C for 30 minutes to form a pink coloured

PART III 

compound absorbing at 534 nm. Therefore, lipid peroxidation can be 

assayed by recording the increase in absorbance of extracted 

membrane lipids at 534 nm (233) . 

6. Determination of reduced glutathione: 

Glutathione reduce 5, 5' dithiobis (2-nitrobenzoic acid) to 

produce a yellow compound which has absorption at 405 nm that it is 

directly proportional to glutathione concentration (234) . 

Statistical analysis: 

All results are expressed as mean ± standard error of the mean 

(S.E.M). ANOVA and post ANOVA test at p < 0.05 was used to test 

significance of the differences between control and treated 

groups. 

Results and Discussion: 

• A significant reduction of the hepatic glutathione (GSH) level 

was observed in rats administered with CCl4 alone. However, 

treatment with Phyllanthus atropurpureus Boj. Hort. Maurit. root 

extract at dose of 200 mg/kg exhibited a significant increase in the 

plasma levels of glutathione. 

• A significant increase in Malondialdehyde levels was observed 

in CCl4 alone treated rats. However, treatment of the rats with 

Phyllanthus atropurpureus extracts (root & aerial part) at a dose of 

200 mg/kg reduced significantly the MDA elevated by CCl4 treatment, 

also in silymarin treated rats, MDA levels were significantly reduced. 

- 200 -

- 201 - 

PART III 

• The antioxidant activity of extracts of Phyllanthus 

atropurpureus Boj. Hort. Maurit. was comparable to that of silymarin, 

the reference hepatoprotective drug. 

• The results presented in table (35) and Figs. (66 & 67) 

illustrated that : Activities of serum AST & ALT (marker enzymes for 

liver damage) were significantly elevated in CCl4-treated animals 

compared to normal control rats, indicating liver damage as the 

increased cytolysis expressed by the higher levels of serum AST while 

the administration of silymarin at a dose of 100 mg/kg caused a 

significant reduction in ALT and AST levels which is quite similar to 

the significant reduction caused by the oral administration of 

Phyllanthus atropurpureus Boj. Hort. Maurit. extracts at a dose of 

200 mg/kg in CCl4 intoxicated rats. 

• As listed in table (35) and as graphically presented in Figs. 

(68 &69), only oral treatment of cirrhotic rats with ethanolic extract of 

root showed a significant elevation in albumin level while neither root 

extract nor aerial part extract produce any significant change in total 

protein level. 

• Antihepatotoxic activity of Phyllanthus atropurpureus Boj. 

Hort. Maurit. extracts is quite similar to silymarin. Both of them 

improve the parameters of CCl4-induced liver injury including serum 

AST and ALT. Among the extracts tested, root extract showed 

maximum activity as shown in table (35) compared with aerial part 

extract relative to silymarin.

Parameters 

PART III 

Table (35): Effect of total extract of aerial parts and roots of 

Phyllanthus atropurpureus Boj. Hort. Maurit. (200 mg/kg) taken 

orally for 30 days on liver enzymes, plasma protein and antioxidant 

parameters in sub acute male cirrhotic rats. 

Normal rats 

Cirrhotic rats before 

treatment 

Data are expressed as mean ± S.E.M. 

* Significant difference from cirrhotic rats without treatment (after 

30 days) at p

ALT value 

50 

40 

30 

20 

10 

0 

- 203 - 

PART III 

Figs (66, 67): Effect of oral treatment with silymarin (100 mg/kg), total ethanolic 

extract of aerial parts (200 mg/kg) and total ethanolic extract of roots (200 mg/kg) 

for 30 days on ALT and AST levels in adult male cirrhotic rats. 

*Data are expressed as the mean ± S.E.M. *Statistics: ANOVA and post ANOVA test. 

* Significantly different from the corresponding mean values of the cirrhotic rats 

without treatment (after 30 days) (The cirrhotic control group) at p< 0.05 

8 

7 

6 

5 

4 

3 

2 

1 

0 

Normal Cirrhotic after 30 days Silymarin 

Aerial parts extract Root extract 

Aerial parts extract Root extract 

total protein level Normal Cirrhotic after 30 days Silymarin 

* 

* 



for 30 days on total protein and albumin levels in adult male cirrhotic rats. 




AST level 

140 

120 

100 

80 

60 

40 

20 

Albumin level 

Normal Cirrhotic after 30 days 

Silymarin Aerial parts extract 

Root extract 

0 

6 

5 

4 

3 

2 

1 

0 



Root extract 

* 

* 

* 

*

PART III 

Malondialdehyde level 



Root extract 

250 

200 

150 

100 

50 

0 



for 30 days on malondialdehyde and glutathione levels in adult male cirrhotic rats. 




A) Antioxidant activity: 

Discussion 

• The extract of Phyllanthus atropurpureus Boj. Hort. Maurit. 

showed good antioxidant potential and prevented oxidation of 

proteins and lipids. By virtue of its ability to scavenge ROS (Reactive 

Oxygen Species), it probably can modulate transcription factors and 

regulate the levels of antioxidant enzymes. 

• In conclusion the extracts of Phyllanthus atropurpureus Boj. 

Hort. Maurit. act as antioxidant and the anti-lipid peroxidation 

activity of root extract was found to be better than that of aerial part 

extract and that may be attributed to the presence of tannoid in the 

root as reported before (235) . 

* 

* 

* 

- 204 - 

Glutathione level 

3.5 

3 

2.5 

2 

1.5 

1 

0.5 

0 



Root extract 

*

- 205 - 

PART III 

• Potent antioxidant activities of Phyllanthus aerial part may be 

due to the presence of phenolic and polyphenolic compounds, such 

as flavonoids (236) , catachin (237) and hydrolysable tannins, with 

geraniin being the most abundant (76, 80, 238) , ellagic acid (239) and 

lignans (240) . 

• Polyphenolic compounds enhance the stability of low density 

lipoprotein (LDL) to oxidation by scavenging the superoxide anion 

(241) , singlet oxygen (242) and lipid peroxy radicals (243) and stabilizing 

free radicals involved in oxidative processes through hydrogenation 

or complexing with oxidizing species ( 244, 245) . 

• La Casa et al. (246) reported that rutin, a natural flavone, induced a 

significant increase in glutathione activity. Flavonoids can reduce 

macrophage oxidative stress by inhibition of cellular oxygenases 

(such as nicotinamide adenine dinucleotide phosphate), reduced 

form (NADPH) oxidase or by activating cellular antioxidants (such 

as the glutathione system) (247) . 

• The potent anti-peroxidative effect of Phyllanthus protects the 

liver by preventing CCl3 • - induced peroxidative disintegration of 

membranes (248) . 

• The enhancement in hepatic GSH status was associated with 

corresponding decreases in malondialdehyde levels and alanine 

aminotransferases activities, indicating a significant reduction in the 

extent of oxidative hepatocellular damage.

PART III 

B) Hepatoprotective activity: 

1- CCl4: 

• The increased cytolysis expressed by the higher levels of 

serum AST suggests that the enhanced microsomal lipid peroxidation 

in liver is associated with a damage of hepatic tissue, which is in 

agreement with the earlier findings (249) . 

• Carbon tetrachloride (CCl4) is hepatotoxic agent causing 

centrolobular necrosis and associated with fatty liver. 

• CCl4 convert to trichloromethyl free radical (CCl3 • ) by hepatic 

mixed function oxidases. (CCl3 • ) can abstract hydrogen from 

polyunsaturated fatty acids to initiate lipid pre-oxidation, alternatively 

in the presence of oxygen, it forms the more reactive trichloro methyl 

peroxy free radical (CCl3COO • ),(CCl3COO • ) that can participate in 

lipid pre-oxidation, or it can decompose it to phosgene (CCl2O) (250) . 

• Antioxidant and radical scavengers have been used to study 

the mechanism of CCl4 toxicity as well as to protect liver cells from 

CCl4 –induced damage by breaking the chain reaction of lipid 

peroxidation (251) . 

2- Silymarin: 

• Silymarin has been reported to protect liver cells from a wide 

variety of toxins (252, 253) , including CCl4. Hepatoprotective mechanism 

of silymarin may be due to: 

1. Antioxidant activity (254, 255) . 

2. Inhibition of lipid peroxidation (255) . 

3- Aerial part extract: 

The pronounced antihepatotoxic activity (compared to silymarin) 

of ethanolic extract of Phyllanthus atropurpureus Boj. 

- 206 -

- 207 - 

PART III 

Hort. Maurit. in this study is in agreement with that reported about the 

effect of other Phyllanthus species against various chemical liver toxin 

(256-260) 

Conclusion 

It appears that the antioxidant property of ethanolic extracts of 

root and aerial parts of Phyllanthus atropurpureus Boj. Hort. Maurit 

could counteract CCl4 toxicity. Antihepatotoxic activity of 

Phyllanthus atropurpureus Boj. Hort. Maurit. may be due to the 

presence of polyphenolic compounds. Therefore, Phyllanthus 

antihepatotoxic mechanism may involve its antioxidant activity 

against production of ROS. Thus, Phyllanthus atropurpureus Boj. 

Hort. Maurit. can be considered as a new efficient hepatoprotective 

candidate, but more clinical follow-up is needed to test the safety 

variables on other organs.

PART III 

Material and methods 

1. Cell Culture: 

B- Anticancer Activity 

Hepatocarcinoma (HepG2) cells were obtained frozen in liquid 

nitrogen (-180 o C) from the American Type Culture Collection 

(ATCC). The tumor cell lines were maintained in the National Cancer 

Institute, Cairo, Egypt, by serial sub-culturing. HepG2 cells were 

routinely cultured in DMEM (Dulbeco’s Modified Eagle’s Medium). 

Media were supplemented with 10% fetal bovine serum (FBS), 2mM 

L-glutamine, containing 100 units/ml penicillin G sodium, 100 

units/ml streptomycin sulphate, and 250 ng /ml amphotericin B. Cells 

were maintained at sub-confluency at 37ºC in humidified air 

containing 5% CO2. For sub-culturing, monolayer cells were 

harvested after trypsin /EDTA treatment at 37°C. Cells were used 

when confluence had reached 75%. Tested materials were dissolved in 

dimethyl sulphoxide (DMSO). All cell culture materials were obtained 

from Cambrex BioScience (Copenhagen, Denmark). All chemicals 

were from Sigma/Aldrich, USA. All experiments were repeated three 

times. 

2. Tested materials: 

I- Material 1: Robustaside A. 

II- Material 2: Ethanolic extract of aerial parts of Phyllanthus 


III- Material 3: Ethanolic extract of root of Phyllanthus 


- 208 -

3. Anti-tumour activity 

- 209 - 

PART III 

Cytotoxicity of tested materials was measured against HepG2 

cells using Sulphorhodamine-B (SRB) assay (261) . 

Principle 

SRB is a bright pink aminoxantherene dye with two sulphonic 

groups. It is a protein stain that binds to the amino groups of 

intracellular proteins under mildly acidic conditions to provide a 

sensitive index of cellular protein content. 

Chemicals 

Sigma chemical Co., St. Louis, Mo, U.S.A. is the source of the 

chemicals and buffer used. 

•1% acetic acid (to dissolve the unbound SRB dye). 

•0.4% SRB dissolved in 1% acetic acid (protein dye) 

•50% trichloroacetic acid (TCA) (stock solution). 50 μl of it was 

added to 200 μl RPMI-1640 medium well to yield a final 

concentration of 10% used for protein precipitation. 

•10 mM Tris EDTA buffer (PH 10.5) was used for dye solubilization. 

Procedure 

1. Cells were used when 90% confluence was reached in T25 

flasks. Adherent cells were harvested with 0.025% trypsin. Viability 

was determined by trypan blue exclusion using inverted microscope. 

2. Cells were seeded in 96-well plate (104 cells/ well) in fresh 

medium and left 24 hours, at 37 o C under 5% CO2 atmosphere, to 

attach to the wall of the plate.

PART III 

3. After 24 hours, cells were incubated with appropriate 

concentrations of drugs and the volume was completed to 200 μl/ well 

using fresh medium and incubation was continued for 48 hours. 

Control cells were treated with vehicle alone. Triplicate wells 

were prepared for each individual dose. 

4. After 48 hours, the cells were fixed with 50μl cold 10% 

trichloroacetic acid for 1 hour at 4 o C. 

5. Cells were washed 5 times with water and stained for 30 minutes, 

at room temperature, with 50 μl 0.4% SRB. 

6. Excess stain was washed (4 times) with 1% acetic acid. 

7. The plate was air dried and the attached stain was solubilized 

with 100μl/well of Tris EDTA buffer (PH 10.5) for 5 minutes on 

shaker at 1600 r.p.m. 

8. The absorbance (A) of each well was measured at 564 nm using 

ELISA reader. 

Calculation 

The cell surviving fraction = 

Results (Anti-tumor activity): 

Using SRB assay, the effect of the three materials on the 

proliferation of HepG2 cell line was studied after 48 hrs of incubation. 

As shown in Fig. (72), the treatment of HepG2 cells with the 

material 1 (robustaside A) lead to high inhibition in the cell 

proliferation as concluded by the low IC50 values 4.93 µg/ml, which 

revealed a specific strong anti-tumor activity of the compound against 

hepatocellular carcinoma. 

- 210 - 

A of treated cells 

A of control cells

- 211 - 

PART III 

As shown in Fig. (72), the addition of increasing concentrations 

of either material 2 (ethanolic extract of aerial parts) or material 3 

(ethanolic extract of root) in the culture medium of HepG2 cells 

reduced the cell proliferation in a dose dependant manner. Maximum 

concentration of ethanolic extract of aerial parts and ethanolic 

extract of root (10 μg/ml) exhibited only 25% cell mortality, so IC50 

may be more than 10 μg/ml.

PART III 

Fig (72): Anti-tumor activity of Phyllanthus atropurpureus Boj. Hort. 

Maurit. against hepatic cell line. 

- 212 - 

IC50= 4.93 

Material 1 concentration (µg /ml) 

Material 2 concentration (µg /ml) 

Material 3 concentration (µg /ml)

C-Antimicrobial activity 

- 213 - 

PART III 

Cup-plate method (262) was used to detect the preliminary 

antimicrobial activity of different fractions including light petroleum, 

chloroform, ethyl acetate, aqueous and total ethanolic extract of 

leaves, roots and stem. The samples were dissolved in dimethyl 

sulfoxide (DMSO) at concentration of 200 mg/ml. The nutrient agar 

was seeded by about 10 6 microbial cells. 

Gram +ve bacteria (Staphylococcus aureus) and Gram –ve 

bacteria (Escherichia coli) as well as Candida albicans as a fungus 

were used as tested microorganisms. Each cup was filled by about 100 

µl from each extract (200 mg/ml). Ampicillin and gentamycin as well 

as nystatin were used as standards. The plates were incubated 

overnight at 37 o C for bacteria and 30 o C for fungus. Zones of 

inhibition were measured (mm) and are recorded in Table (36). 

Results and Discussion: 

As shown in table (36), the given results revealed that: 

1. Light petroleum fractions show significant antibacterial activity 

than chloroform, ethyl acetate and ethanolic fractions for different 

parts of the plant against E. coli. The light petroleum activity is more 

than that of Ampicillin and Gentamycin. 

2. All plant fractions except the ethanolic fraction of leaves show 

significant antibacterial activity against S. aureus compared to the 

effect of Gentamycin. 

3. Only petroleum ether fraction of leaves exhibit significant 

antifungal activity against Candida albicans compared to Nystatin.

PART III 

Table (36): Antimicrobial activities of different fractions of Phyllanthus 


Diameter of zone of inhibition (mm) 

Material 

Gram –ve 

bacteria 

Gram +ve 

bacteria 

Fungi 

E.coli S.aureus C.albicans 

1-Light petroleum fraction of leaves 16 18 18 

2- Light petroleum fraction of stem 16 16 14 

3- Light petroleum fraction of root 16 22 14 

4-Chloroform fraction of leaves 14 18 14 

5- Chloroform fraction of stem 10 20 - 

6- Chloroform fraction of root 10 20 10 

7-Ethyl acetate fraction of leaves 14 25 10 

8-Ethyl acetate fraction of stem 10 18 12 

9-Ethyl acetate fraction of root 10 16 10 

10-Total ethanolic fraction of leaves 10 12 10 

11-Total ethanolic fraction of stem 10 16 10 

12-Total ethanolic fraction of root 10 20 10 

13-Total ethanolic fraction of aerial 

parts 

10 14 12 

14-Aq. fraction of leaves - 16 - 

15-Aq. fraction of stem - 16 - 

16-Aq. fraction of root - 16 - 

17-Ampicillin (10µg/well) 14 30 - 

18-Gentamycin (10µg/well) 14 14 - 

19-Nystatin (30µg/well) - - 16 

- = No zone of inhibition. 

200 mg/ml DMSO from plant fractions concentration were used. 

100 µl solutions were applied. 

- 214 -

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218. Marsano, L.S.; Mendez, C.; Hill, D.; Barve, S. and Mc-Clain, C.; 

"Diagnosis and treatment of alcoholic liver diseases and its 

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220. Lee,W.M.; "Drug induced hepatotoxicity"; N. Engl. J. Med., 333: 

1118-1127 (1995). 

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- 236 -

- 237 - 

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the suitability of bromcresol green for the determination of serum 

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233. Satoh, K.; Clin. Chim. Acta,, 90: 37 (1978) & Ohkawa, H.; Ohishi 

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234. Beutler, E.; Duron, O. and Kelly, M.B.; "Improved method for the 

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235. Bhattachary, A.; Chatterjee, A.; Ghosal, S. and Bhattachyra, S.K.; 

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480 (1991). 

237. Deckar, E.A.; "The role of phenolic, conjugated linoleic acid, 

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238. Yeap, F. L. and Herbert, W.; "Phyllanthusiin D, an unusual 

hydrolysable tannin from Phyllanthus amarus"; Phytochemistry, 31: 

711-713 (1992). Through C. A. 117: 44534r. 

239. Ishimaru, K.; Yoshimatsu, K.; Kamada, H. and Shimomura, K.; 

"Phenolic constituents in tissues cultures of Phyllanthus niruri"; 


240. Satynarayana, P.; Subrahmanyan, P.; Viswanthan, K. N. and Ward, 

R.S.; "New seco and hydroxyl lignans from Phyllanthus niruri"; J. 

Nat. Prod., 51: 44- 49 (1988). 

241. Robak, J. and Gryglewski, R.J.; "Flavonoids are scavengers of 

superoxide anions"; Biochemical Pharmacology, 37: 837- 841 

(1988). 

242. Husain, S.R.; Cillard, J. and Cillard, P.; "Hydroxyl radical 

scavenging activity of flavonoids"; Phytochemistry, 26: 2489 - 2491 

(1987). 

243. Torel, J.; Cillard, J. and Cillard, P.; "Antioxidant activity of 

flavonoids and reactivity with peroxy radical"; Phytochemistry, 25: 

383-385 (1986). 

244. Lewis, N.G. "Antioxidant in higher plants"; In R.G. Alscher, &J.L. 

Hess (Eds.), Plant phenolics., Boca Raton, CRC Press: 135-169 

(1993). 

245. Shahidi, F. and Wanasusdara, J.P.K. P.D.; "Phenolic antioxidants"; 

Critical Reviews of Food Science and Nutrition, 32: 67-103 (1992). 

246. La Casa, C.; Villegas, I.; Alarcon-de-la-Lastra, C.; Motilva, V. and 

Martin Calero, M.J.; "Evidence for protective and antioxidant 

properties of rutin, a natural flavone, against ethanol induced gastric 

lesion"; J. Ethnopharmacol., 71: 45-53 (2000). 

247. Furhman, B. and Aviram, M.; "Flavonoids protect LDL from 

oxidation and attenuate atherosclerosis"; Current Opinion in 

Lipidology, 12: 41- 48 (2001). 

248. Dhuley, J.N. and Naik, S.R.; "Protective effect of Rhinax, a herbal 

formulation, against CCl4-induced liver injury and survival in rats"; 

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249. Barber, A.A.; "Addendum mechanisms of lipid peroxide formation 

in rat tissue homogenates"; Radiation Research, 3: 33-43 (1963). 

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mechanisms in carbon tetrachloride hepatotoxicity"; J. Free Radic. 

Biol. Med., 1: 27- 38 (1985). 

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105 (2003). 

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alterations in acute CCl4 liver damage"; J. Appl. Toxicol., 10: 275- 

279 (1990). 

- 238 -

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of Silybum marianum L. on lipid peroxidation in rat liver 

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147-154 (1992). 

254. Pietrangelo, A.; Borella, F. and Casalgrandi, G.; "Anti-oxidant 

activity of silybin invivo during long-term iron overload in rats"; 

Gastroenterol., 109: 1941-1949 (1995). 

255. Basage, H.; Poli, G. and Tekkaya, C.; "Free radical scavenging and 

anti-oxidation properties of "silibin" complexes on microsomal lipid 

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256. Liu, J. H. and Meintosh, H.; "Genus Phyllanthus for chronic hepatitis 

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366 (2001). 

257. Xin-Hua, W.; Qngl, C.; Bog, X. and Chua, L.F.; "A comparative 

study of Phyllanthus amarus compound and interferon in the 

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pharmacognostical studies of three Phyllanthus species"; J. 


259. Kumaran, A. and Karunnakaran, R.J.; "In vitro antioxidant activities 

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(1990). 

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Test: Dilution and Disk Diffusion Methods in: Manual of Clinical 

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Tenover, F.C. and Yolken, 6 th ed.; R.H. ASM Press, Washington, 

D.C.: 1327 (1995).

SUMMARY 

- 215 - 

SUMMARY 

Phyllanthus is a widespread tropical genus of the family 

Euphorbiaceae which is characterized by the presence of many 

biological active compounds such as: sterols, terpenes, flavonoids, 

tannins, lignans and polyphenolic compounds beside the presence of 

some alkaloids. 

So, this thesis comprises a phamacognostical study of a plant 

from this family and belong to genus Phyllanthus which was 

cultivated in Egypt, namely Phyllanthus atropurpureus Boj. Hort. 

Maurit. to perform: 

1. Botanical study of its different organs to facilitate the 

identification of the plant in both entire and powdered 

forms. 

2. Isolation and characterization of its chemical constituents. 

3. Biological study of the plant extracts as well as isolated 

compounds. 

I- Macro- and Micromorphological study of different organs was 

carried out to characterize the plant; as a result the following 

characters comprises the main diagnostic features: 

1. The plant is a perennial monoecious shrub with dark olive green 

petiolated leaves showing red, purple or white colourations. 

2. The flower is unisexual, green or purple small axillary one. 

3. The rhizome of the plant is vertical showing dark reddish-brown 

rough, longitudinally wrinkled outer surface, giving 4 main roots 

on its lower end. 

4. Leaves are dorsiventral and its epidermis shows paracytic stomata , 

conical-shaped papillae and no hairs.

- 216 - 

SUMMARY 

5. Short petiole shows parenchymatous cortex with a complete layer 

of subepidermal collenchyma, collenchymatous pericycle and large 

cresent-shaped vascular tissue. 

6. Numerical values of leaves are 64- 86 for stomatal index on lower 

epidermis, 3.63 as vein-islet number, 4.86 for veinlet-termination 

number and 3.0- 3.8 as palisade ratio. 

7. Stem is characterized by the presence of parenchymatous cortex 

with a parenchymatous pericycle showing batches of non-lignified 

pericyclic fibres. 

8. The epidermal cells of sepals, ovary, style, anther and staminal 

column are polygonal, isodiametric or axially elongated, covered 

with thick or smooth cuticle. The cells of stigma are papillosed. 

9. The pollen grains are spherical yellow in colour with three germ 

pores. 

10. The root shows a characteristic diarch primary xylem in the center. 

11. Rhizome shows interrupted bands of lignified sclerides in the 

pericycle. 

12. Cluster crystals of calcium oxalate are present in all organs of the 

plant. 

II- Phytochemical Investigation: 

A- Pharmacopoeial constant of the plant. 

Moisture content, total ash and acid insoluble ash were 

determined and found to be: 15, 8.5 and 1.1% for the aerial part of the 

plant and 10, 6.81 and 1.3% for the root of the plant respectively. 

The successive extractives using selective organic solvents such as 

light petroleum, diethyl ether, chloroform and alcohol were found to 

be: 0.3, 0.11, 0.07 and 13.27% respectively for the aerial parts extracts

- 217 - 

SUMMARY 

and 0.18, 0.02, 0.01 and 4.86 % respectively for the root extracts of 

the plant. 

Preliminary screening proved the probable presence of flavonoids, 

isoflavonoid, tannins, mucilage, sterols and/ or triterpenes. 

B- Fatty Acid Analysis: 

GLC investigation of fatty acid methyl esters together with 

authentic samples revealed the presence of the following fatty acids: 

caprylic, capric, lauric, myristic, palmitic, palmitoleic, margaric, 

stearic, oleic and linoleic. 

GC analysis of unsaponifiable matter revealed the presence of β– 

sitosterol and stigmasterol. 

C- Isolation of the compounds: 

As a result of chromatographic study including column and 

TLC, the following compounds were isolated in pure form. 

1. Mixture of palmitic and stearic acid. 

2. Mixture of β-sitosterol and stigmasterol. 

3. Oleic acid. 

4. Di (3, 4, 5- trihydroxy phenyl) ether (First isolation from 

nature) (New compound). 

5. 5, 6, 8, 4'-tetrahydroxy isoflavone. (First isolation from 

nature) (New compound). 

6. Robustaside A. (First isolation from genus Phyllanthus). 

7. 6'- (3", 4"- dihydroxy cinnamoyl) arbutin. (First isolation 

from nature) (New compound). 

8. Demethoxysudachitin (First isolation from genus 

Phyllanthus).

- 218 - 

SUMMARY 

9. Quercetin-7- O-glucoside (First isolation from genus 

Phyllanthus) 

The characterization, identification and elucidation of their 

structures were done by physical and spectral methods including IR, 

UV, Mass (FAB, EI), 1 H-NMR and 13 C-NMR. 

III- Biological activities: 

1. Pharmacological activities: 

• The alcoholic extract of the aerial parts and roots of the plant 

produced antihepatotoxic activity as they cause a significant 

reduction of liver enzymes which were increased by the 

effect of CCl4 . Thus, these extracts can be considered as a 

new efficient hepatoprotective candidate as it produce an 

effect similar and almost equal to that of silymarin. 

• The alcoholic extract of the aerial parts and roots of the plant 

as well as Roubstaside A were subjected to some 

pathological experiment to reveal its anti-tumor activity. 

Robustaside A was found to produce a specific strong anti- 

tumor activity against hepatocellular carcinoma. 

2. Antimicrobial activity: 

It was found that different extracts of aerial parts and of roots 

exhibited significant antibacterial and antifungal activities against the 

tested organisms: S. aureus (G +ve bacteria), E. coli (G-ve bacteria) 

and Candida albicans (fungi).

ﻲﺑﺮﻌﻟا ﺺﺨﻠﻤﻟا 


ﺎﻤﻛ ةرﺎﺤﻟا ﻖﻃﺎﻨﻤﻟا ﻲﻓ ارﺎﺸﺘﻧا ﺔﯿﻨﺒﻠﻟا ﺔﻠﺋﺎﻌﻟا سﺎﻨﺟأ ﺮﺜﻛأ 

ﻦﻣ سﻮﺜﻧﻼﯿﻔﻟا 

ﺲﻨﺟ ﺮﺒﺘﻌﯾ 

،ﺔ ﯿﺛﻼﺜﻟا تﺎ ﻨﯿﺑﺮﺘﻟا ،تﻻوﺮﯿﺘ ﺳﻻا ﻞ ﺜﻣ ﺔ ﻟﺎﻌﻔﻟا ﺔ ﯾﻮﯿﺤﻟا تﺎ ﺒﻛﺮﻤﻟا ﻦ ﻣ ﺪ ﯾﺪﻌﻟا دﻮ ﺟﻮﺑ ﺰ ﯿﻤﺘﯾ 

. تاﺪﯾﻮﻠﻘﻟا 

ﺾﻌﺑ ﺐﻧﺎﺟ ﻰﻟإ 

ﺲﻨ ﺠﻟ ﻊﺑﺎ ﺗ ﺔ ﻠﺋﺎﻌﻟا هﺬ ھ ﻦ ﻣ تﺎ ﺒﻨﻟ 

تﻻﻮﻨﯿﻔﻟا ةﺪﯾﺪﻋ تﺎﺒﻛﺮﻤﻟا و ﺔﻀﺑﺎﻘﻟا داﻮﻤﻟا ،تاﺪﯿﻧﻮﻓﻼﻔﻟا 

ﺔ ﯿﺟﻮﻟﻮﯿﺑو ﺔ ﯾﺮﯿﻗﺎﻘﻋ ﺔ ﺳارد : ﺔ ﺳارﺪﻟا هﺬ ھ لوﺎ ﻨﺘﺗ اﺬ ﮭﻟ و 

و . ﺖ ﯾرﻮﻣ . ترﻮ ھ . جﻮ ﺑ سرﻮ ﺑﺮﯿﺑوﺮﺗا سﻮ ﺜﻧﻼﯿﻓ ﻮ ھو ﺮﺼ ﻣ ﻲ ﻓ عرﺰ ﯾ يﺬ ﻟا 

ﻰ ﻠﻋ و ﺔ ﻠﻣﺎﻜﻟا ﮫ ﯿﺘﻟﺎﺣ ﻲ ﻓ ﮫ ﯿﻠﻋ فﺮ ﻌﺘﻟا ﻞﮭﺴ ﯿﻟ 

. ﻚﻟذ ﻦﻜﻣأ نإ 

. ﺔﻟﻮﺼﻔﻤﻟا تﺎﺒﻛﺮﻤﻟا ﺾﻌﺑ و تﺎﺒﻨﻟا تﺎﺻﻼﺨﻟ 

- 1 - 

ﺔ ﻔﻠﺘﺨﻤﻟا تﺎ ﺒﻨﻟا ءﺎﻀ ﻋﻷ ﺔ ﯿﺗﺎﺒﻧ ﺔ ﺳارد 

سﻮ ﺜﻧﻼﯿﻓ 

. قﻮﺤﺴﻣ ﺔﺌﯿھ 

ﺎﮭﯿﻠﻋ فﺮﻌﺘﻟا و ﺔﯿﺋﺎﯿﻤﯿﻜﻟا ﮫﺗﺎﻧﻮﻜﻣ ﻞﺼﻓ 

تﺎﺒﻨﻠﻟ ﺔﯾﺮﮭﺠﻤﻟا و ﺔﯿﻧﺎﯿﻌﻟا ﺔﺳارﺪﻟا : ﻻوأ 

ﻲﺟﻮﻟﻮﯿﺒﻟا ﺮﺛﻷا ﻢﯿﯿﻘﺗ 

: ﻞﻤﺸﺗ 

ﺔ ﺳارﺪﻟا هﺬ ھ تﺮ ﮭﻇأ ﺪ ﻗ و ﺔ ﻔﻠﺘﺨﻤﻟا تﺎ ﺒﻨﻟا ءﺎﻀ ﻋﻷ ﺔ ﯾﺮﮭﺠﻤﻟا و ﺔ ﯿﻧﺎﯿﻌﻟا ﺔ ﺳارﺪﻟا ﻞﻤﺸ ﺗ و 

ﻲﻧﻮ 

ﺘﯾﺰﻟا ﺮﻀﺧﻷا ﺎﮭﻧﻮﻟ 

ﻲﻓ ﺔﻨﻛاد ﺔﻘﻨﻌﻣ قاروأ تاذ 

ﻊ ﺑرأ 

ﻞﻔﺳﻷا ﮫﻓﺮﻃ ﻦﻣ جﺮﺨﯾ 

. ءﺎﻀﯿﺒﻟا وأ ﺔﯾﺰﻣﺮﻘﻟا 

و ءاﺮﻤﺤﻟا ناﻮﻟﻷا 

. ﺲﻨﺠﻟا ةﺪﯿﺣو 

. 1 

. 2 

. 3 

: ﺔﯿﻟﺎﺘﻟا تاﺰﯿﻤﻤﻟا 

ﻦﻜﺴﻤﻟا ةﺪﯿﺣو ةﺮﻤﻌﻣ ةﺮﯿﺠﺷ تﺎﺒﻨﻟا 

ﺾﻌﺑ ﺮﮭﻈﺗ ﺪﻗ و 

ﻢﺠﺤﻟا ةﺮﯿﻐﺻ نﻮﻠﻟا ﺔﯾﺰﻣﺮﻗ وأ ءاﺮﻀﺧ ةﺮھﺰﻟا 

ﺔﯿﻟﻮﻃ تﺎﺟﺮﻌﺗ ﮫﻟ و ﺮﻤﺤﻣ ﻲﻨﺑ ﺢﻄﺳ وذ ﻲﺳأر موﺰﯾﺮﻟا 

. روﺬﺟ 

ﺪ ﺟﻮﯾ ﻻ و تﺎ ﻤﻠﺣ ﺎ ﮭﺑ ﺎ ﯾﻼﺨﻟا ﺾ ﻌﺑ و ﺎ ﯾﻼﺨﻟا ﺔ ﯾزاﻮﺘﻣ رﻮ ﻐﺛ تاذ ةﺮﻇﺎﻨﺘﻣ ﺮﯿﻏ ﺔﻗرﻮﻟا 

. 

. تاﺮﯿﻌﺷ ﺎﮭﯿﻠﻋ 

ﺔﻨﻨﺠﻠﻣ ﺮﯿﻏ فﺎﯿﻟأ ﮫﺑ ﻲﻤﯿﺸﻧاﺮﺑ ﻞﻜﯿﺴﯾﺮﺑ و ﺔﯿﻤﯿﺸﻧاﺮﺑ ةﺮﺸﻗ دﻮﺟﻮﺑ ﺰﯿﻤﺘﯾ ﺔﻗرﻮﻟا ﻖﻨﻋ 

ﻞ ﻣﺎﻌﻤﻟا 

و ( 3.8-3.0 

) ﺔﯿﺒﺴ ﻨﻟا ﺔ ﯾدﺎﻤﻌﻟا ﺔ ﻤﯿﻘﻟا ﻲ ھو ﺔ ﻗرﻮﻠﻟ ﺔ ﯾدﺪﻌﻟا ﻢﯿ ﻘﻟا ﻦﯿ ﯿﻌﺗ ﻢ ﺗ ﺪ ﻗ 

.( 4.86) 

تﺎﻘﯾﺮﻌﻟا ﺔﯾﺎﮭﻧ دﺪﻋ و ( 3.63) 

ﺔﯿﻗﺮﻌﻟا تاﺮﯾﺰﺠﻟا دﺪﻋ و ( 86-64) 

يﺮﻐﺜﻟا 

. ﺔﻨﻨﺠﻠﻣ ﺮﯿﻏ فﺎﯿﻟأ ﮫﺑ ﻞﻜﯿﺴﯾﺮﺒﻟا و ﺔﯿﻤﯿﺸﻧاﺮﺑ ةﺮﺸﻗ دﻮﺟﻮﺑ ﺰﯿﻤﺘﯾ قﺎﺴﻟا 

ﺔ ﻟوﺎﻄﺘﻣ ،ﺔﻌﻠﻀ ﻣ ﺎ ﯾﻼﺧ ﻦ ﻣ ﻂﯿ ﺨﻟا و ﻚ ﺘﻤﻟا ،ﻢ ﻠﻘﻟا ،ﺾﯿ ﺒﻤﻟا ،تﻼﺒﺴ ﻟا ةﺮﺸ ﺑ نﻮ ﻜﺘﺗ 

. 

تﺎﻤﻠﺣ ﺎﮭﺑ ﻢﺴﯿﻤﻟا ﺎﯾﻼﺧ . ﺔﻘﯿﻗر وأ ﺔﻜﯿﻤﺳ ﺔﻣدﺄﺑ ةﺎﻄﻐﻣ و 

ﺎﯾرﻮﺤﻣ 

. 1 

. 2 

. 3 

. 4 

. 5 

. 6 

. 7 

. 8

. ﺔﯿﺗﺎﺒﻧا بﻮﻘﺛ ثﻼﺛ ﺎﮭﺑ و نﻮﻠﻟا ءاﺮﻔﺻ 

- 2 - 


ﺔﯾوﺮﻛ حﺎﻘﻠﻟا بﻮﺒﺣ 

. ﺰﻛﺮﻤﻟا ﻲﻓ مﺰﺤﻟا ﻲﺋﺎﻨﺛ ﻲﻟوأ ﺐﺸﺧ دﻮﺟﻮﺑ ﺰﯿﻤﺘﯾ رﺬﺠﻟا. 

10 

. ﻞﻜﯿﺴﯾﺮﺒﻟا ﺔﻘﻄﻨﻣ ﻲﻓ ﺔﻨﻨﺠﻠﻤﻟا ﺔﯾﺮﺠﺤﻟا ﺎﯾﻼﺨﻟا ﻦﻣ تﺎﻋﻮﻤﺠﻣ ﮫﺑ موﺰﯾﺮﻟا. 

11 

. ءﺎﻨﺜﺘﺳا ﻼﺑ تﺎﺒﻨﻟا 

ءاﺰﺟأ ﻞﻛ ﻲﻓ ﺪﺟﻮﺗ ﺔﻌﻤﺠﺘﻤﻟا مﻮﯿﺴﻟﺎﻜﻟا تﻻﺎﺴﻛوا تارﻮﻠﻠﺑ 

ﻲ ﻠﻜﻟا دﺎ ﻣﺮﻟا ،( 

٪15 

تﺎﺒﻨﻠﻟ ﺔﯿﺋﺎﯿﻤﯿﻜﻟا ﺔﺳارﺪﻟا : ﺎﯿﻧﺎﺛ 

: تﺎﺒﻨﻠﻟ ﺔﯾرﻮﺘﺳﺪﻟا ﺖﺑاﻮﺜﻟا ﺪﯾﺪﺤﺗ 

) ﺔ ﺑﻮﻃﺮﻟا ﺔﺒﺴ ﻧ ﻞ ﺜﻣ تﺎ ﺒﻨﻠﻟ ﺔﯾرﻮﺘ ﺳﺪﻟا ﺖ ﺑاﻮﺜﻟا ﺾ ﻌﺑ ﺪ ﯾﺪﺤﺗ ﻢ ﺗ 

ﺎ ﻤﻨﯿﺑ تﺎ ﺒﻨﻟا ﻦ ﻣ ﺔ ﯿﺋاﻮﮭﻟا ءاﺰ ﺟﻸﻟ ( ٪ 1.1) 

ﺾﻣﺎﺤﻟا ﻲﻓ نﺎﺑوﺬﻟا ﻢﯾﺪﻋ دﺎﻣﺮﻟا و ( ٪8.5) 

و ( ٪ 6.81) 

ﻲ ﻠﻜ ﻟا دﺎ ﻣﺮﻟا ،( 

٪10) 

ﺔﺑﻮﻃﺮﻟا ﺔﺒﺴﻧ : ﻲﺗﻷﺎﻛ 

( ٪ 1.3) 

تﺎﺒﻨﻟا روﺬﺠﻟ ﺔﺒﺴﻨﻟﺎﺑ نﻮﻜﺗ 

ﺾﻣﺎﺤﻟا ﻲﻓ نﺎﺑوﺬﻟا ﻢﯾﺪﻋ دﺎﻣﺮﻟا 

: ﻲھ و ﺔﯿﻟﺎﺘﻟا ﺔﺒﻗﺎﻌﺘﻤﻟا تﺎﺼﻠﺨﺘﺴﻤﻟا ﺪﯾﺪﺤﺗ ﻢﺗ ﻚﻟﺬﻛ و 

لﻮ ﺤﻜﻟاو ( ٪0.07 

) مرﻮ ﻓورﻮﻠﻜﻟا ،( 

٪ 0.11) 

ﺮ ﯿﺜﯾﻻا ،( 

٪ 0.3) 

ﻲ ﻟوﺮﺘﺒﻟا ﺮ ﯿﺜﯾﻷا 

: ﻲﻟﺎﺘﻟﺎﻛ ﺔﺒﺴﻨﻟا نﻮﻜﺗ تﺎﺒﻨﻟا روﺬﺠﻟ ﺎﻤﻨﯿﺑ تﺎﺒﻨﻟا ﻦﻣ ﺔﯿﺋاﻮﮭﻟا ءاﺰﺟﻸﻟ ( ٪ 13.27) 

لﻮ ﺤﻜﻟا و ( ٪ 0.01) 

مرﻮ ﻓورﻮﻠﻜﻟا ،( 

٪ 0.02) 

ﺮ ﯿﺜﯾﻻا ،( 

٪ 0.18) 

ﻲ ﻟوﺮﺘﺒﻟا ﺮ ﯿﺜﯾﻻا 

داﻮ ﻤﻟا ،تاﺪ ﯿﻧﻮﻓﻼﻓوﺰﯾﻷا 

. ( ٪4.86) 

،تاﺪ ﯿﻧﻮﻓﻼﻔﻟا دﻮ ﺟو تﺎ ﺻﻼﺨﻟا هﺬﮭﻟ ﻲﻟوﻷا ﺺﺤﻔﻟا ﺖﺒﺛأ ﺪﻗ 

. ﺔﯿﺛﻼﺜﻟا تﺎﻨﯿﺑﺮﺘﻟا 

ﺐﻧﺎﺟ ﻰﻟإ تﻻوﺮﯿﺘﺳﻻا و ﺔﯿﻣﻼﮭﻟا 

داﻮﻤﻟا ،ﺔﻀﺑﺎﻘﻟا 

: ﺔﯿﻨھﺪﻟا ضﺎﻤﺣﻷا ﺔﺳارد 

،ﻚ 

ﻠﻠﯾﺮﺑﺎﻛ 

: ﺔ ﯿﻨھﺪﻟا ضﺎ ﻤﺣﻷا هﺬ ھ ﻰ ﻠﻋ فﺮ ﻌﺘﻟا ﻢ ﺗ ﻞﺋﺎﺴ ﻟا زﺎ ﻐﻟا ﺎﯿﻓاﺮﺟﻮﺗﺎﻣوﺮﻛ 

. 9 

. 12 

. أ 

. ب 

ﻖﯾﺮﻃ ﻦﻋ 

. ﻚﯿﻟﻮﻨﯿﻟ و ﻚﯿﻟوأ ،ﻚﯾرﺎﯿﺘﺳا 

،ﻚﯾﺮﺟرﺎﻣ ،ﻚﯿﻟﻮﯿﺘﻤﻟﺎﺑ ،ﻚﯿﺘﻤﻟﺎﺑ ،ﻚﯿﺘﺳﺮﯿﻣ 

،ﻚﯾرﻮﻟ ،ﻚﯾﺮﺑﺎﻛ 

: تﺎﺒﻛﺮﻤﻟا ﻞﺼﻓ . ج 

ماﺪﺨﺘ ﺳﺎﺑ ﺎ ﯿﻓاﺮﺟﻮﺗﺎﻣوﺮﻛ ﺎﮭﺼ ﺤﻓ و ﺔ ﻔﻠﺘﺨﻤﻟا تﺎ ﺒﻨﻟا تﺎ ﺻﻼﺨﻟ ﺔ ﯿﺋﺎﯿﻤﯿﻜﻟا ﺔ ﺳارﺪﻟا 

ﺖ ﻤﺗ 

تﺎ ﺒﻛﺮﻤﻟا ﻞﺼ ﻓ ﻢ ﺗ 

ﻚ ﻟﺬ ﻟ ﺔ ﺠﯿﺘﻧ و دﻮ ﻤﻌﻟا ﺎ ﯿﻓاﺮﺟﻮﺗﺎﻣوﺮﻛ و ﺔ ﻘﯿﻗﺮﻟا ﺔ ﻘﺒﻄﻟا ﺎ ﯿﻓاﺮﺟﻮﺗﺎﻣوﺮﻛ 

. ﻚﯾرﺎﯿﺘﺳﻻاو 

ﻚﯿﺘﻤﻟﺎﺒﻟا ﺾﻤﺣ ﻦﻣ ﻂﯿﻠﺧ 

. 

لوﺮﯿﺘﺳﻮﺘﯿﺳ ﺎﺘﯿﺒﻟا و لوﺮﯿﺘﺳ ﺎﻤﺠﯿﺘﺴﻟا 

ﻦﻣ ﻂﯿﻠﺧ 

: ﺔﯿﻟﺎﺘﻟا 

. 1 

. 2


- 3 - 

. ﻚﯿﻟوﻷا ﺾﻤﺣ 

ﮫ ﻠﺼ ﻓ ﻢ ﺘ ﯾ ةﺮ ﻣ لوﻷ ﺪ ﯾﺪﺟ ﺐ ﻛﺮﻣ) 

ﺮ ﯿﺜﯾا ( ﻞ ﯿﻨﯿﻓ ﻲﺴ ﻛورﺪﯿھ ياﺮ ﺗ -5 

،4 

،3) 

ياد 

ﻦ ﻣ ﮫﻠﺼ ﻓ ﻢﺘ ﯾ ةﺮ ﻣ لوﻷ ﺪ ﯾﺪﺟ ﺐ ﻛﺮﻣ) 

نﻮ ﻓﻼﻓوﺰﯾا ﻲﺴ ﻛورﺪﯿھاﺮﺘﯿﺗ 

. ﮫﯾإ 

.( ﺔﻌﯿﺒﻄﻟا ﻦﻣ 

'4 

, 8 , 6, 

5 

. ( ﺔﻌﯿﺒﻄﻟا 

ﺪﯿﺳﺎﺘﺳﺎﺑور 

ﻢﺘ ﯾ ةﺮ ﻣ لوﻷ ﺪ ﯾﺪﺟ ﺐ ﻛﺮﻣ) 

ﻦﯿﺗﻮ 

ﯿﺑرا ( ﻞﯾﻮﻣﺎﻨﯿ ﺳ ﻲﺴ ﻛورﺪﯿھ ياد " 4 , " 3) 

-'6 

. ( ﺔﻌﯿﺒﻄﻟا 

ﻦﻣ ﮫﻠﺼﻓ 

.( ﺲﻨﺠﻟا اﺬھ ﻦﻣ ﺐﻛﺮﻤﻟا اﺬھ ﻞﺼﻓ ﻢﺘﯾ ةﺮﻣ لوﻷ) 

ﻦﯿﺘﯾﺎﻛوﺪﯿﺳ ﻲﺴﻛﻮﺜﯿﻤﯾد 

.( ﺲﻨﺠﻟا اﺬھ ﻦﻣ 

ﺐﻛﺮﻤﻟا اﺬھ ﻞﺼﻓ ﻢﺘﯾ ةﺮﻣ لوﻷ) 

ﺪﯾزﻮﻛﻮﻠﺟ -وأ 

-7-ﻦﯿﺘﺳراﻮﻛ 

ﺔ ﺻﻼﺧ و ﺔ ﯿﺣﺎﻧ ﻦ ﻣ 

ﮫﺗﺎﯾﻮﺘﺤﻣ و تﺎﺒﻨﻠﻟ يﻮﯿﺤﻟا 

ﺮﯿﺛﺄﺘﻟا : ﺎﺜﻟﺎﺛ 

تﺎ ﺒﻨﻠﻟ 

ﺔ ﯿﺋاﻮﮭﻟا ءاﺰ ﺟﻷا ﻦ ﻣ ﻞ ﻜﻟ 

ﮫ ﺑﺎﺸ ﻣ ظﻮﺤﻠﻣ ﺮﯿﺛﺄﺗ ﺎﮭﻟ نأ ﺚﯿﺣ يﺪﺒﻜﻟا ﻒﯿﻠﺘﻠﻟ ﻂ ﺒﺜﻣ ﺮﯿﺛﺄﺗ 

: ﻲﺟﻮﻟﻮﻛﺎﻣرﺎﻔﻟا ﺮﯿﺛﺄﺘﻟا 

ﺔ ﯿﻟﻮﺤﻜﻟا ﺔ ﺻﻼﺨﻟا ﺮ ﮭﻈﺗ 

ى ﺮﺧ أ ﺔﯿﺣﺎﻧ ﻦﻣ روﺬﺠﻟا 

ﺪ ﺒﻜ ﻟا تﺎ ﻤﯾﺰﻧإ ﺾ ﻔﺧ ﻰ ﻠﻋ ﺔ ﺳارﺪﻟا هﺬھ ﻲﻓ ﺔﻧرﺎﻘﻤﻟا ﻞﺤﻣ ﻦﯾرﺎﻤﻠﯿﺴﻟا ﺐﻛﺮﻣ ﺮﯿﺛﺄﺘﻟ 

تﺎ ﺻﻼﺨﻟا هﺬھ ﺢﺷﺮﯾ ﺎﻤﻣ ﺪﯾارﻮﻠﻛاﺮﺘﺗ نﻮﺑﺮﻜﻟﺎﺑ ﻦﻘﺤﻟﺎﺑ ˝ﺎﯿﺒﯾﺮﺠﺗ 

ﺎﮭﺘﺒﺴﻧ ﺖﻌﻓر 

. ﻦﯾرﺎﻤﻠﯿﺴﻟا 

ﺐﻛﺮﻣ 

ﺮﯿﺛﺄﺘﻟ يوﺎﺴﻣ ﮫﺒﺷ يﺪﺒﻜﻟا ﻒﯿﻠﺘﻠﻟ 

ﻲﺘﻟا 

جﻼﻌﻛ 

و روﺬ ﺠﻟا ﺔ ﺻﻼﺧ و تﺎﺒﻨﻠﻟ ﺔﯿﺋاﻮﮭﻟا ءاﺰﺟﻷا ﻦﻣ ﻞﻜﻟ ﺔﯿﻟﻮﺤﻜﻟا ﺔﺻﻼﺨﻟا عﺎﻀﺧإ ﻢﺗ 

ﻂﺒ ﺜﻤﻟا 

ﺎ ھﺮﯿﺛﺄﺗ ءﻼﺠﺘ ﺳﻻ ﺔ ﯿﺟﻮﻟﻮﺛﺎﺒﻟا برﺎ ﺠﺘﻟا ﺾﻌﺒ ﻟ 

ﺎ ﯾﻼﺨﻟا 

ﻰ ﻠﻋ ﻂﺒ ﺜﻣ و لﺎ ﻌﻓ ﺮﯿﺛﺄ ﺗ ﮫ ﻟ ﮫ ﯾإ ﺪﯿ ﺳﺎﺘﺳﺎﺑور 

ﮫ ﯾإ 

ﺪﯿ ﺳﺎﺘﺳﺎﺑور ﺐ ﻛﺮﻣ 

ﺐ ﻛﺮﻣ نأ ﺪ ﺟو ﺚﯿﺣ ماروﻸﻟ 

. ﺪﺒﻜﻟا ﻲﻓ ﺔﯿﻧﺎﻃﺮﺴﻟا 

نأ ﺔ ﯿﺟﻮﻟﻮﯿﺑوﺮﻜﯿﻤﻟا برﺎ ﺠﺘﻟا ﺾ ﻌﺑ لﻼ ﺧ ﻦﻣ ﺢﻀﺗا : ﻲﺟﻮﻟﻮﯿﺑوﺮﻜﯿﻤﻟا 

ﺮﯿﺛﺄﺘﻟا 

ماﺮ ﺠﻟا ﺔ ﺒﺟﻮﻣ ﺎ ﯾﺮﯿﺘﻜﺒﻟا عاﻮ ﻧأ ﺾ ﻌﺑ ﻰ ﻠﻋ ﻂﺒ ﺜﻣ ﺮﯿﺛﺄ ﺗ ﺔ ﻔﻠﺘﺨﻤﻟا تﺎ ﺒﻨﻟا تﺎ ﺻﻼﺨﻟ 

اﺪ ﯾﺪﻧﺎﻛ) 

تﺎ ﯾﺮﻄﻔﻟا و ( يﻻﻮ ﻛ ﺎﯿ ﺷﺮﯿﺷا) 

م اﺮ ﺠﻟا ﺔﺒﻟﺎ ﺳ ، ( ﺲ ﯾروأ ﺲﻛﻮﻛﻮﻠﯿﻓﺎﺘ ﺳ) 

. 

ﺔﺳارﺪﻟا 

ﻞﺤﻣ ( ﺲﻧﺎﻜﯿﺒﻟأ 

. 3 

. 4 

. 5 

. 6 

. 7 

. 8 

. 9 

. 1 

• 

• 

. 2

2009

2009

ﺝﺮﺳ ﻰﻔﻄﺼﻣ ﻪـﻃ /. ﺩ. 

ﺃ 

ﻖﻳﺯﺎﻗﺰﻟﺍ ﺔﻌﻣﺎﺟ -ﺔﻟﺪﻴﺼﻟﺍ 

ﺔﻴﻠﻛ -ﲑﻗﺎﻘﻌﻟﺍ 

ﺫﺎﺘﺳﺃ 

ﲏﻐﻟﺍ ﺪﺒﻋ ﻲﻠﻋ ﺪﻴﺴﻟﺍ ﻑﺎﻔﻋ /. ﺩ. 

ﺃ 

. ﻖﻳﺯﺎﻗﺰﻟﺍ ﺔﻌﻣﺎﺟ -ﺔﻟﺪﻴﺼﻟﺍ 


ﺫﺎﺘﺳﺃ 

ﺪﻳﺍﺯ ﻱﺩﺎﳍ 

ﺍﺪﺒﻋ ﺔﻳﻭﺍﺭ / ﻡ. 

ﺃ 

. ﻖﻳﺯﺎﻗﺰﻟﺍ ﺔﻌﻣﺎﺟ -ﺔﻟﺪﻴﺼﻟﺍ 


ﺪﻋﺎﺴﻣ ﺫﺎﺘﺳﺃ 

ﲑﻗﺎﻘﻌﻟﺍ ﻢﺴﻗ 

ﺔﻟﺪﻴﺼﻟﺍ ﺔﻴﻠﻛ 

ﻖﻳﺯﺎﻗﺰﻟﺍ ﺔﻌﻣﺎﺟ 

2009

1 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

7 

 

11 

 

2009

In Pharmaceutical Sciences (Pharmacognosy)

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