Ph. D Thesis By SHAFIQ UR RAHMANprr.hec.gov.pk/jspui/bitstream/123456789/7306/1/... · CERTIFICATE...

PHARMACOGNOSTIC STUDIES

ON

TRILLIUM GOVANIANUM WALL. Ex. ROYLE

Ph. D Thesis

By

SHAFIQ UR RAHMAN

DEPARTMENT OF PHARMACY

UNIVERSITY OF PESHAWAR, PESHAWAR, PAKISTAN

2015

PHARMACOGNOSTIC STUDIES

ON

TRILLIUM GOVANIANUM WALL. Ex. ROYLE

SHAFIQ UR RAHMAN

A THESIS SUBMITTED TO THE DEPARTMENT OF PHARMACY,

UNIVERSITY OF PESHAWAR

IN PARTIAL FULFILLMENT FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

IN

PHARMACEUTICAL SCIENCES



2015

CERTIFICATE OF APPROVAL

This thesis, entitled, “Pharmacognostic studies on Trillium govanianum Wall. Ex.

Royle” submitted by Mr. Shafiq ur Rahman to University of Peshawar is hereby approved and recommended as partial fulfillment for the award of Degree of “Doctor

of Philosophy in Pharmaceutical Sciences”. Prof. Dr. Muhammad Ismail __________________________

Research Supervisor

Department of Pharmacy

University of Peshawar

Prof. Dr. Muhammad Saeed __________________________

Chairman

Department of Pharmacy

University of Peshawar

Prof. Dr. Taous Khan _________________________

External Examiner Department of Pharmacy COMSATS Institute of Information Technology, Abbottabad



2015

Acknowledgements

First of all I bow down my head to the Omnipotent, the most Merciful and the

Compassionate Al-Mighty ALLAH, Who gave me the courage and provided me all

the resources to complete this Ph.D. Project. I wish to pay homage to the most perfect

personality of the world Hazrat Muhammad (PBUH), who enlightened our minds to

recognize our Creator. My research work would not have been possible without the

help, support, and guidance of many people to whom I want to convey my cordial

gratitude.

I would like to thank my supervisor, Prof. Dr. Muhammad Ismail, for his guidance,

support, understanding and patience during the entire period of my studies. I am very

thankful for his admirable supervision, continuous encouragement during my Ph.D.

studies.

I am thankful to Prof. Dr. Muhammad Saeed, sitting Chairman, Department of

Pharmacy, University of Peshawar, for his support and encouragement throughout my

research studies. I am also grateful to Meritorious Professor. Dr. Zafar Iqbal (T.I)

and Prof. Dr. Fazal Subhan for their inspiring guidance and support during the

course of this PhD project.

I am thankful to Dr. Muhammad Raza Shah, Dr. Achyut Adhikari, Dr. Itrat Anis,

Dr. Muhammad Ateeq, Dr. Burhan and Mr. Farid, International Centre for

Chemical and Biological Sciences (ICCBS), H.E.J. Research Institute of Chemistry,

University of Karachi, Karachi for their help and facilitation during this long course

of research studies.

I am obliged to Prof. Dr. Jamshaid Ali Khan, Dr. Amir Zada Khan, Dr. Fazal

Nasir, Dr. Inam Ullah, Dr. Muhammad Ismail, Dr. Fazal Khuda, Dr. Gohar Ali

and Dr. ZakiUllah Department of Pharmacy, University of Peshawar for their

support.

I would like to thank Dr. Muhammad Khurram (Chairman), Mr. Shujat Ahmad,

Mr. Asaf Khan, Mr. Abidullah, Mr. Imad Afzal and all Teaching, Clerical and

Laboratory Staff, Department of Pharmacy, Shaheed Benazir Bhutto University,

Sheringal Dir (U) for their cooperation. I feel indebted to Dr. Farman Ali and Dr.

Abdul Khaliq Jan, Department of Chemistry, Shaheed Benazir Bhutto University,

Sheringal Dir (U) for their assistance.

I want to extend special thanks to my dear friends Dr. Saeed Ahmad Khan, Mr.

Arsalan, Mr. Farhad Ullah, Mr. Khalid, Mr. Tahir Ali, Mr. Sajid Khan Sadozai,

Mr. Muhammad Shahid, Mr. Irfan Ullah and Mr. Muzaffar Abbas.

Last but not the least; I am very thankful to my sweet Parents, wife, brother, sisters,

my uncle retired Principal Mr. Fazal Halim, and all relatives for their prayers,

support and kindness throughout my studies.

Shafiq ur Rahman

Table of Contents

List of Tables................................................................................................................ i List of Figures............................................................................................................... iv List of Abbreviations.................................................................................................... vi List of Publications from Thesis................................................................................... vii Summary....................................................................................................................... 1 1. Introduction.......................................................................................................... 4 1.1 Medicinal plants.......................................................................................................... 4 1.2 Plants metabolites........................................................................................................ 5 1.3 Traditional medicines and drug discovery................................................................... 7 1.4 Biodiversity of Indo-Pak Subcontinent........................................................................ 10 1.5 The Family Trilliaceae................................................................................................. 11 1.6 Genus Trillium............................................................................................................. 12 1.6.1 Species of genus Trillium................................................................................................ 13 1.6.2 Phytochemical profiling of genus Trillium...................................................................... 17 1.6.3 Medicinal importance and biological studies of genus Trillium...................................... 28 1.7 Trillium govanianum................................................................................................... 30 1.7.1 Plant Morphology............................................................................................................ 31 1.7.2 Distribution...................................................................................................................... 31 1.7.3 Ethnobotanical Uses......................................................................................................... 31 1.8 Aims and Objectives.................................................................................................... 32 2. Materials and Methods......................................................................................... 33 2.1 Drugs and Chemicals.................................................................................................. 33 2.2 Research centers for experimental studies................................................................... 33 2.3 Physical constants........................................................................................................ 34 2.4 Spectroscopic techniques............................................................................................. 34 2.4.1 UV technique.................................................................................................................... 34 2.4.2 IR technique...................................................................................................................... 34 2.4.3 Mass technique................................................................................................................. 34 2.4.4 Nuclear Magnetic Resonance (NMR) technique.............................................................. 35 2.4.5 Gas Chromatography and Gas Chromatography-Mass Spectrometry.............................. 35 2.4.6 GC-MS identification of components............................................................................... 35 2.5 Chromatographic techniques for isolation and purification of compounds............... 36 2.5.1 Column Chromatography (CC)........................................................................................ 36 2.5.2 Thin layer Chromatography (TLC).................................................................................. 36 2.5.3 Reagents for visualizing the spots.................................................................................. 36 2.5.3.1 Ceric sulphate solution as reagent............................................................................ 37 2.5.3.2 Vanillin solution as reagent...................................................................................... 37 2.6 Ethnomedicinal study.................................................................................................. 37 2.6.1 Site selection.................................................................................................................... 37 2.6.2 Sampling informants and ethnomedicinal data collection................................................. 37 2.7 Plant materials.............................................................................................................. 38 2.7.1 Collection......................................................................................................................... 38 2.7.2 Extraction and fractionation............................................................................................. 38 2.8 Macroscopic and microscopic features of rhizome..................................................... 40 2.9 Physicochemical parameters........................................................................................ 40 2.9.1 Total ash............................................................................................................................ 40 2.9.2 Water soluble ash............................................................................................................. 41 2.9.3 Acid insoluble ash............................................................................................................ 41 2.9.4 Loss on drying................................................................................................................ 41 2.9.5 Extractive values............................................................................................................. 42 2.9.5.1 Methanol soluble extractive value........................................................................... 42 2.9.5.2 Water and other soluble extractive values................................................................ 42 2.10 Phytochemical tests.................................................................................................. 42

2.10.1 Test for alkaloids........................................................................................................... 43 2.10.1.1 Mayer’s test.......................................................................................................... 43 2.10.1.2 Wagner’s test........................................................................................................ 43 2.10.1.3 Hager’s test........................................................................................................... 43 2.10.2 Test for flavonoids............................................................................................. 43 2.10.2.1 Ferric chloride test............................................................................................... . 43 2.10.2.2 Sodium hydroxide test.......................................................................................... 44 2.10.3 Test for tannins.................................................................................................. 44 2.10.3.1 Ferric chloride test............................................................................................... . 44 2.10.3.2 Lead Acetate test................................................................................................... 44 2.10.4 Test for saponins................................................................................................ 44 2.10.5 Test for steroids................................................................................................. 45 2.10.6 Test for sterols................................................................................................... 45 2.10.6.1 Salkowski’s test.................................................................................................... 45 2.10.6.2 Liebermann-Burchard test...................................................................................... 45 2.10.7 Test for glycosides............................................................................................. 45 2.10.8 Test for carbohydrates....................................................................................... 46 2.10.8.1 Molisch’s test....................................................................................................... 46 2.10.8.2 Benedict’test......................................................................................................... 46 2.10.8.3 Fehling’s test........................................................................................................ 46 2.11 Isolation of compounds............................................................................................ 47 2.11.1 Isolation of compounds from CHCl3 fraction............................................................... 47 2.11.2 Isolation of compound from butanol fraction.............................................................. 53 2.12 Characterization of isolated compounds.................................................................. 55 2.12.1 Characterization of hexadecanoic acid (compound 1)................................................. 55 2.12.2 Characterization of β-sitosterol (compound 2)............................................................ 56 2.12.3 Characterization of stigmasterol (compound 3).......................................................... 57 2.12.4 Characterization of diosgenin (compound 4).............................................................. 58 2.12.5 Characterization of pennogenin (compound 5)........................................................... 59 2.12.6 Characterization of govanic acid (compound 6)......................................................... 60 2.12.7 Characterization of 20-hydroxy ecdysone and 5,20-dihydroxy ecdysone

(compound 7 and 8) ................................................................................................... 61

2.12.8 Characterization of 5, 20-hydroxy ecdysone (compound 8)....................................... 62 2.12.9 Characterization of borassoside E (compound 9)....................................................... 63 2.12.10 Characterization of govanoside A (compound 10)..................................................... 64 2.13 Biological studies.................................................................................................... 65 2.13.1 In vitro biological activities......................................................................................... 65 2.13.1.1 Antibacterial activity............................................................................................. 65 2.13.1.2 Antifungal activity................................................................................................ 65 2.13.1.3 Antioxidant activity.............................................................................................. 66 2.13.1.4 Anticancer activity................................................................................................ 67 2.13.1.5 Anti-inflammatory activity................................................................................... 68 2.13.1.6 Anti leishmanial activity....................................................................................... 68 2.13.1.7 Brine shrimp cytotoxicity..................................................................................... 69 2.13.1.8 Insecticidal activity............................................................................................... 69 2.13.1.9 Protein antiglycation activity................................................................................ 70 2.13.1.10 Smooth muscle relaxant activity........................................................................... 71 2.13.1.11 β-Glucuronidase inhibitory activity....................................................................... 72 2.13.1.12 α-Chymotrypsin inhibitory activity....................................................................... 73 2.13.1.13 Thymidine phosphorylase inhibitory activity........................................................ 73 2.13.1.14 Acetylcholinesterase inhibitory activity................................................................. 74 2.13.2 In vivo biological studies.................................................................................. 75 2.13.2.1 Experimental animals............................................................................................ 75 2.13.2.2 Acute toxicity test.................................................................................................. 75 2.13.2.3 Anti-inflammatory activity..................................................................................... 75 2.13.2.4 Analgesic activity.................................................................................................. 76

2.13.2.4.1 Tonic-visceral chemical induced nociception test............................................ 76 2.13.2.4.2 Hot plate test..................................................................................................... 77 3. Results and Discussion....................................................................................... 78 3.1 Ethnomedicinal studies.............................................................................................. 78 3.2 Morphological studies............................................................................................... 83 3.2.1 Macroscopic features................................................................................................... 83 3.2.2 Microscopic features................................................................................................... 83 3.3 Physicochemical studies............................................................................................. 85 3.4 Phytochemical studies................................................................................................. 88 3.4.1 Qualitative Phytochemical screening............................................................................ 88 3.4.2 GCMS analysis of n-hexane fraction............................................................................ 90 3.4.3 Isolation of compounds................................................................................................. 92 3.4.3.1 Structure-elucidation of compound 1..................................................................... 92 3.4.3.2 Structure-elucidation of compound 2..................................................................... 94 3.4.3.3 Structure elucidation of compound 3..................................................................... 96 3.4.3.4 Structure elucidation of compound 4..................................................................... 98 3.4.3.5 Structure elucidation of compound 5..................................................................... 100 3.4.3.6 Structure elucidation of compound 6, a new compound......................................... 103 3.4.3.7 Structure-elucidation of compound 7..................................................................... 107 3.4.3.8 Structure elucidation of compound 8..................................................................... 110 3.4.3.9 Structure elucidation of compound 9..................................................................... 112 3.4.3.10 Structure elucidation of compound 10, a new compound...................................... 117 3.5 Biological studies....................................................................................................... 123 3.5.1 In vitro biological activities.......................................................................................... 123 3.5.1.1 Antibacterial activity............................................................................................. 123 3.5.1.2 Antifungal activity................................................................................................ 126 3.5.1.2.1 Antifungal activity of Cr. MeOH-Ext and fractions....................................... 126 3.5.1.2.2 Antifungal activity of isolated compounds..................................................... 126 3.5.1.3 DPPH free radical scavenging activity of Cr. MeOH-Ext and fractions.............. 130 3.5.1.4 Anticancer activity............................................................................................... 133 3.5.1.4.1 Anticancer activity of Cr. MeOH-Ext and fractions...................................... 133 3.5.1.4.2 Anticancer activity of isolated compounds.................................................. 133 3.5.1.5 Anti-inflammatory activity (Oxidative burst assay)............................................ 136 3.5.1.5.1 Anti-inflammatory activity of Cr. MeOH-Ext and fractions.......................- 136 3.5.1.5.2 Anti-inflammatory activity of isolated compounds...................................... 136 3.5.1.6 Anti leishmanial activity of Cr. MeOH-Ext and fractions.................................... 139 3.5.1.7 Insecticidal activity of Cr. MeOH-Ext and fractions............................................ 140 3.5.1.8 Brine shrimp cytotoxic activity of Cr. MeOH-Ext and fractions.......................... 143 3.5.1.9 Muscle relaxant (Spasmolytic) activity of Cr. MeOH-Ext.................................. 146 3.5.1.10 Antiglycation activity of Cr. MeOH-Ext and fractions......................................... 149 3.5.1.11 β-Glucuronidase inhibitory activity of Cr. MeOH-Ext and fractions.................... 150 3.5.1.12 α-Chymotrypsin inhibitory activity of Cr. MeOH-Ext and fractions.................... 152 3.5.1.13 Thymidine phosphorylase inhibitory activity of isolated compounds.................. 152 3.5.1.14 Acetylcholinesterase inhibitory activity of Cr. MeOH-Ext and fractions............. 153 3.5.2 In vivo biological studies.................................................................................. 155 3.5.2.1 Acute toxicity........................................................................................................ 155 3.5.2.2 Anti-inflammatory activity of Cr. MeOH-Ext and fractions................................. 155 3.5.2.3 Analgesic activity of Cr. MeOH-Ext and fractions................................................ 161 3.5.2.3.1 Tonic-visceral chemical induced nociception............................................... 161 3.5.2.3.2 Thermal induced nociception........................................................................ 162 Concluding Remarks.................................................................................................... 168 References.................................................................................................................... 169

List of Tables

Table 1.1 Important drugs discovered from plants with their ethnomedical

correlations and sources

8

Table 1.2 Natural product derived drugs in market since 2005 9

Table 1.3 Species of genus Trillium 14

Table 1.4 List of phytochemicals isolated from genus Trillium 17

Table 1.5 Reported biological activities of genus Trillium 29

Table 1.6 Taxonomical classification of T. govanianum 30

Table 2.1 Drugs and chemicals used with their sources 33

Table 2.2 Characterization of hexadecanoic acid 55

Table 2.3 Characterization of β-sitosterol 56

Table 2.4 Characterization of stigmasterol 57

Table 2.5 Characterization of diosgenin 58

Table 2.6 Characterization of pennogenin 59

Table 2.7 Characterization of govanic acid (a new compound) 60

Table 2.8 Characterization of 20-hydroxyecdysone 61

Table 2.9 Characterization of 5,20-dihydroxyecdysone 62

Table 2.10 Characterization of borassoside E 63

Table 2.11 Characterization of govanoside A (a new compound) 64

Table 3.1 Informants and therapeutic uses of T. govanianum rhizomes in different districts of Khyber Pakhtunkhwa

82

Table 3.2 Preliminary phytochemical profile of T. govanianum rhizomes 89

Table 3.3 Chemical composition of n-Hex-fr of T. govanianum rhizomes 91

Table 3.4 1H-NMR and 13C-NMR data of compound 1 93








i



Table 3.14 Antibacterial results of Cr. MeOH-Ext and fractions of T. govanianum rhizomes

125

Table 3.15 Antifungal activity of Cr. MeOH-Ext and fractions of T. govanianum rhizomes

128

Table 3.16 Antifungal activity of compounds isolated from T. govanianum rhizomes

129

Table 3.17 DPPH free radical scavenging activity of T. govanianum extract, fractions and standards (ascorbic acid and BHT)

131

Table 3.18 Anticancer activity of T. govanianum rhizomes Cr. MeOH-Ext, fractions and reference drug (doxorubicin) against cancer cells

135

Table 3.19 Anticancer activity of compounds isolated from T. govanianum rhizomes

135

Table 3.20 Anti-inflammatory effect of T. govanianum rhizomes Cr. MeOH-Ext, fractions and isolated compounds

138

Table 3.21 Leishmanicidal activity of Cr. MeOH-Ext and fractions of T. govanianum rhizomes

140

Table 3.22 Insecticidal activity of Cr. MeOH-Ext and its subsequent fractions of T. govanianum rhizomes against an insect Tribolium castaneum

142

Table 3.23 Insecticidal activity of Cr. MeOH-Ext and its subsequent fractions of T. govanianum rhizomes against an insect Rhyzopertha dominica

142

Table 3.24 Brine shrimp cytotoxic activity of Cr. MeOH-Ext and fractions of T. govanianum rhizomes

144

Table 3.25 Antiglycation activity of Cr. MeOH-Ext and fractions 150

Table 3.26 IC50 values (µg/mL) of extract and fractions of T. govanianum

rhizomes

151

Table 3.27 α-Chymotrypsin inhibitory activity of Cr. MeOH-Ext and fractions 152

Table 3.28 Thymidine phosphorylase inhibitory activity of isolated compounds 153

Table 3.29 Acetylcholinesterase inhibitory activity of Cr. MeOH-Ext and its fractions

154

Table 3.30 Acute toxicity of Cr. MeOH-Ext of T. govanianum rhizomes 155

ii

Table 3.31 Anti-inflammatory activity Cr. MeOH-Ext and fractions of T.

govanianum rhizomes against carrageenan induced paw edema in mice

158

Table 3.32 Antinociceptive effect of T. govanianum rhizomes Cr. MeOH-Ext and its fractions in tonic-visceral chemical induced nociception

161

Table 3.33 Antinociceptive effect of Cr. MeOH-Ext and fractions of T.

govanianum rhizomes in thermal induced nociception 165

iii

List of Figures

Figure 1.1 Trillium govanianum plant 30

Figure 3.1 Informants for the ethnomedicinal uses of T. govanianum

rhizomes from different districts of Khyber Pakhtunkhwa 81

Figure 3.2 Trillium govanianum plant and rhizomes 83

Figure 3.3 Transverse section of T. govanianum rhizome 84

Figure 3.4 Physicochemical parameters of T. govanianum rhizomes 87

Figure 3.5 Chemical structure of compound 1 93






Figure 3.11 Linked scan measurements in compound 6 106

Figure 3.12 Major fragmentation and 1H-1H-COSY correlations in compound 6

106




Figure 3.16 Key HMBC correlations in compound 9 116


Figure 3.18 Key-HMBC-correlations-in compound 10 121

Figure 3.19 Key-NOESY-correlations-in compound 10

122

Figure 3.20 DPPH free radical scavenging activity of extract and fractions 132

iv

Figure 3.21 Percent cytotoxic effect of Cr. MeOH-Ext and fractions of T.

govanianum rhizomes

145

Figure 3.22 Inhibitory effects of T. govanianum rhizomes Cr. MeOH-Ext and verapamil in isolated rabbit jejunum preparations

148

Figure 3.23 Ca++ concentration response curves (CRCs) of Cr. MeOH-Ext and verapamil in isolated rabbit jejunum preparations

148

Figure 3.24A Anti-inflammatory effect of Cr. MeOH-Ext on carrageenan induced paw edema

159

Figure 3.24B Anti-inflammatory effect of CHL-fr on carrageenan induced paw edema

159

Figure 3.24C Anti-inflammatory effect of EtOAc-fr on carrageenan induced paw edema

160

Figure 3.24D Anti-inflammatory effect of BuOH-fr on carrageenan induced paw edema

160

Figure 3.25 Antinociceptive effect of T. govanianum rhizomes in tonic-visceral chemical induced nociception

162

Figure 3.26A Antinociceptive effect of Cr. MeOH-Ext and fractions after thirty minutes

166

Figure 3.26B Antinociceptive effect of Cr. MeOH-Ext and fractions after sixty minutes

166

Figure 3.26C Antinociceptive effect of Cr. MeOH-Ext and fractions after ninety minutes

167

Figure 3.26D Antinociceptive effect of Cr. MeOH-Ext and fractions after one hour and twenty minutes

167

v

List of Abbreviations

Cr. MeOH-Ext Crude Methanolic extract

n-Hex-fr n-hexane fraction CHL-fr Chloroform fraction

EtOAc-fr Ethyl acetate fraction BuOH-fr Butanol fraction

Aq-fr Aqueous fraction WHO World Health Organization

NP Natural Products ADHD Attention deficit hyperactivity disorder

CVS Cardio vascular system DPPH 2,2-diphenyl-1-picrylhydrazyl

BHT Butylated hydroxytoluene MeOD Methanol CDCl3 Chloroform

CC Column Chromatography TLC Thin Layer Chromatography

GCMS Gas Chromatography Mass Spectrometry ppt Precipitate UV Ultraviolet spectroscopy IR Infrared spectroscopy

NMR Nuclear Magnetic Resonance NOESY Nuclear Overhauser Effect Spectroscopy

COSY Correlation Spectroscopy HMBC Heteronuclear Multiple Bond Coherence HSQC Heteronuclear Singal Quantum Coherence

HREI-MS High Resolution Electron Ionization Mass Spectrometry 1H-NMR Proton Nuclear Magnetic Resonance

13C-NMR Carbon Nuclear Magnetic Resonance HRFAB-MS High Resolution Fast Atomomic Bombardment Mass Spectrometry

DMSO Dimethyl sulfoxide MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide COX Cyclo-oxygenase

AChE Acetylcholinesterase AIDS Acquired Immune Deficiency Syndrome ROS Reactive oxygen species CCB Calcium channel blocker

AGEs Advanced glycation end products vi

List of Publications from Thesis

1 Shafiq-ur-Rahman, Muhammad Ismail, Muhammad Raza Shah, Marcello Iriti, and

Muhammad Shahid. "GC/MS analysis, free radical scavenging, anticancer and β-glucuronidase inhibitory activities of Trillium govanianum rhizomes". Bangladesh

Journal of Pharmacology Vol. No. 10 (2015): 577-583. Impact factor; 1.05 2 Shafiq-ur-Rahman, Muhammad Ismail, Muhammad Raza Shah, Achyut Adhikari,

Itrat Anis, Malik Shoaib Ahmad, and Muhammad Khurram. "Govanoside A, a new steroidal saponin from rhizomes of Trillium govanianum". Steroids Vol. No. 104 (2015): 270-275. doi:10.1016/j.steroids.2015.10.013. Impact factor; 2.63

3 Shafiq-ur-Rahman, Muhammad Ismail, Muhammad Khurram and Inam ul

Haq."Pharmacognostic and ethnomedicinal studies on Trillium govanianum." Pakistan Journal of Botany Vol. No. 47(SI) (2015): 187-192. Impact factor; 0.82

4 Shafiq-ur-Rahman, Muhammad Ismail, Achyut Adhikari, Muhammad Raza Shah,

Muhammad Khurram, Muhammad Shahid. "Scientific confirmation of anti inflammatory and analgesic uses of Trillium govanianum rhizomes". Journal of

Ethnopharmacology. Submitted. Impact factor; 2.99 vii

Summary

1

Summary

This dissertation describes ethnomedicine based morphological, chemical and

biological evidences of Trillium govanianum rhizome. T. govanianum belongs to the

family Trilliaceae and is mainly distributed in Asia, from Pakistan to Bhutan. The

ethnomedicinal survey in the four Districts of Khyber Pukhtoonkhwa revealed that

highest presumed indications of T. govanianum rhizomes include inflammatory

disorders, cancers, backache, headache, joint pains, kidney problems and

gastrointestinal disorders.

The transverse section of rhizome showed the presence of cortex cells, trichomes,

carinal canal, sclereids, vascular bundles (xylem and phloem), fibers, cambium,

calcium oxalate crystals and starch grains. Extractive values were high for solvents

like water and methanol, which is indicative of abundance of sugars, and other polar

compounds like glycosides and saponins. Phytochemical screening revealed the

presence of steroids, steroidal glycosides, saponins, tannins, and carbohydrates in

crude methanolic extract (Cr. MeOH-Ext) as well as in chloroform fraction (CHL-fr),

ethyl acetate fraction (EtOAc-fr) and butanol fraction (BuOH-fr). GC/MS analyses of

n-hexane fraction (n-Hex-fr) identified twelve (12) compounds, including 70%

unsaturated and 30% saturated fatty acids.

Using different chromatographic techniques, eight compounds from CHL-fr and two

compounds from BuOH-fr were isolated. The chemical structures of isolated

compounds were elucidated using latest spectroscopic and spectrometric techniques

i.e. 1H-NMR, 13C-NMR, COSY, NOESY, HSQC, HMBC, EI-MS, FAB, HR-FAB,

HREI-MS, IR and UV. Among these compounds, two [govanic acid (6) and

govanoside A (10)] were new, while the rest were [hexadecanoic acid (1), β-sitosterol

Summary

2

(2), stigmasterol (3), diosgenin (4), pennogenin (5), 20-hydroxyecdysone (7), 5,20-

dihydroxyecdysone (8), borassoside E (9)] previously known. However, all the

compounds are reported for the first time from this plant species.

In MTT assay, based on IC50 ± SD (µg/mL) values, significant antiproliferative

activity against HeLa cells was observed for CHL-fr (0.84 ± 0.16), EtOAc-fr (1.41 ±

0.08) and BuOH-fr (1.60 ± 0.34). Similarly, all fractions exhibited good cytotoxicity

against PC-3 cell lines. The isolated compounds, govanoside A (1.74 ± 0.12 against

PC-3; 0.51 ± 0.26 against HeLa) and borassoside E (2.34 ± 0.21 against PC-3; 0.67 ±

0.22 against HeLa) exhibited significant cytotoxicity compared to standard

doxorubicin (1.69 ± 0.28 against PC-3; 0.50 ± 0.15 against HeLa). In DPPH free

radical scavenging assay, higher scavenging capacity was observed in n-Hex-fr and

CHL-fr compared to other fractions, however the scavenging capacity of all fractions

was less than ascorbic acid.

In antifungal assay, the Cr. MeOH-Ext was found active against all tested fungal

strains, with maximum activity against Trichophyton rubrum, Microsporum canis,

and Candida albicans. The compounds, govanoside A and borassoside E showed

good to moderate activities against Aspergillus niger, A. flavus, C. albicans, and C.

glabrata strains, while govanic acid exhibited moderate activity for T. rubrum and M.

canis. In antibacterial assay, the Cr. MeOH-Ext and fractions exhibited moderate

antibacterial potentials against the tested gram positive and gram negative bacteria.

Furthermore, the Cr. MeOH-Ext exhibited good potential against Leishmania major.

Suppression of oxidative burst (OB) was evaluated through luminol enhanced

chemiluminescence assay. Based on IC50 ± SD (µg/mL), the BuOH-fr (16.53 ± 7.54)

Summary

3

exhibited significant inhibition of OB for the whole blood followed by Cr. MeOH-Ext

(30.81 ± 7.02), which indicates their immune suppressive potentials. Among the

tested compounds, pennogenin (05.00 ± 0.84) showed significant suppression of OB

compared to the standard drug, Ibuprofen (11.23 ± 1.91). However, borassoside E

(31.51 ± 6.62) showed moderate activity.

The Cr. MeOH-Ext completely inhibited both spontaneous as well as high K+ induced

contractions of isolated rabbit jejunum preparations indicating its spasmolytic effect.

The Cr. MeOH-Ext relaxed the high K+ induced contractions in an analogous pattern

to standard Ca++antagonist verapamil, representing its calcium channel blocking action.

In insecticidal assay, the Cr. MeOH-Ext and fractions-were found inactive against the

test insects i.e. Tribolium-castaneum and Rhyzopertha dominica.

In enzyme inhibition assays, α-chymotrypsin and thymidine phosphorylase, were not

inhibited by test samples. Therefore, it was assumed that these enzymes are not the

pharmacological target of T. govanianum rhizomes extract and fractions. However,

the Cr. MeOH-Ext (IC50; 140.8 ± 3.8) and BuOH-fr (196.2 ± 1.9) exhibited moderate

β-glucuronidase and weak acetylcholineterase inhibitions.

In in vivo carrageenan induced paw edema model, significant anti-inflammatory effect

was observed for Cr. MeOH-Ext and fractions (50 and 100 mg/kg). Similarly, the Cr.

MeOH-Ext and fractions significantly attenuated the tonic-visceral chemical induced

and thermal induced nociception in experimental mice.

Results of this study strongly support the ethnomedicinal uses of T. govanianum

rhizomes in treatment of cancers, inflammatory disorders, fungal infections and

gastrointestinal disorders which are further endorsed by the isolated compounds.

Chapter 1 Introduction

4

1. Introduction

1.1 Medicinal plants

In the current era, it is extremely desired to discover effective remedies, for diseases,

which are potent, with least adverse effects, and cost effective. Discovering such

products, medicinal plants and herbal medicines can be the best choice as plants are

known to produce a wide range of bioactive molecules, making them a rich source of

different types of medicines1.

Medicinal plants are known to be used by mankind as a source of medicines since

immemorial times. These plants are source of valuable medicines that are used to

prevent diseases, maintain health and cure ailments. In one way or other, they benefit

almost every living being on this planet earth2. They are used to be the basis of

sophisticated traditional medicine systems for long time, and are still at service of

mankind by providing new medicines3.

Natural products obtained from plants have played remarkable role in the

improvement of health care system. According to the World Health Organization

(WHO) estimate about 80% of world population rely on natural sources for their

primary health care need while the remaining 20% of the population uses integrated

natural sources4. Even at the dawn of 21stcentury, 11% of the 252 drugs, considered as

basic and essential by the WHO were exclusively of flowering plant origin2.

At present, the prime pharmacopoeias in the world i.e. European Pharmacopoeia (Ph

Eur 8), United States Pharmacopeia (USP XXXIV), British Pharmacopoeia (BP 2015)

all have mention of plant drugs which heralds the true significance and medicinal

importance of these remedies5.


5

In scientific literature around the world, about 35,000 or more plants species have

been reported, to be used in different human cultures for medicinal purposes6.

Nevertheless, this number could be much higher as knowledge of indigenous use of

medicinal plants mainly passes verbally from one generation to another and largely

remain undocumented. Among the 250,000 reported higher plants species, only 5-

15% have been scrutinized for their bioactive molecules7.

In conclusion, the medicinal plants are an area under focus since their secondary

metabolites encompass a significant number of drugs used in current therapeutics and

their potential as the source of new medicines is beyond any doubts.

1.2 Plants metabolites

The plant primary metabolites like proteins, carbohydrates, lipids and vitamins etc.

are synthesized as a consequence of photosynthesis by green plants, and are involved

in the development, reproduction and normal growth of the plants. The humans and

other organism utilize these primary metabolites chiefly for their dietary purpose8.

The secondary metabolites like alkaloids, glycosides, tannins, saponins, flavonoids,

terpenoids, volatile oils, phytoestrogens, carotenoids and phenols etc. are synthesized

from primary metabolites by different biosynthetic pathways, and are capitalized in

plant defense mechanisms, to fight off herbivores, pests and pathogens9. These

bioactive metabolites were used by people in different cultures, in a variety of ways in

different traditions in every era in cure of diseases and still prevail in this modern

world10.

These metabolites are present in different parts of the plant like barks, roots,

rhizomes, stems, [ flowers, fruits, seeds [and leaves, which are medicinally used either in


6

raw form or in the form of decoctions, infusions or extracts11. Among the secondary

metabolites terpenoids constitute the largest class of secondary metabolites that are

grouped together on basis of their common biosynthetic origin i.e. from acetyl CoA or

glycolytic intermediates. Some nitrogenous terpene derivatives possess potent anti-

hypertensive property. The antimicrobial and insecticidal properties of terpenoids

have led to their utilization as pesticides and fungicides in agriculture and

horticulture12,13. Tannins (polyphenols with multi facet chemistry) are useful as an

anti-inflammatory agent and in the treatment of burns and other wounds based on

their anti-hemorrhagic and antiseptic potentials. In particular, tannins rich recipes are

used as antihelmintics, antioxidants, and antimicrobials14.

Flavonoids consist of a large group of polyphenolic compounds having a benzo

pyrone structure with potent anti-oxidant, anti-cancer, hepatoprotective, anti-

inflammatory, antibacterial and antiviral properties15. Saponins are steroid or

triterpene glycosides widely distributed in the plant that possess hemolytic properties

and poisonous effects against fishes. Crude drugs containing saponins that have less

irritating effects on oral administration are generally used as expectorant and

antitussive agents16. It is worth to mention, that many saponins have been reported to

exhibit significant anti-inflammatory, antinociceptive and antipyretic activities as well

as many other diverse potentials such as antiallergic and anti-cancer17,18. Similarly

alkaloids are one of the most diverse groups of plant secondary bioactive metabolites

and comprise substances possessing remarkable range of pharmacological activities.

Many alkaloids have been reported to be used for hundreds of years in medicine and

some are still important drugs today19,20. In fact million of hidden recipes are present

in medicinal plants, by virtue of which these plants are capitalized for treatment and

preventions of various diseases21.


7

1.3 Traditional medicines and drug discovery

There are various approaches that how plants are selected as a potential candidate for

drug discovery; these approaches includes random selection for phytochemical

screening or random selection followed by biologic assay, the most common

approach, frequently used is based on capitalization of knowledge from traditional

system of medicine (ethno-medicinal)22. In fact numerous drugs have entered the

international pharmacopoeias through the study of ethnopharmacology and traditional

medicine23. Some of the important drugs discovered through ethnomedicinal approach

are given in Table 1.1. Research on medicinal plants, which are used traditionally for

the treatment of systemic and topical infections, has shown that they contain varieties

of anti-cancer, antiparasitic, antifungal, antibacterial, analgesics, anti-inflammatory

and antihistaminic compounds24-26.

From centuries, China and India exercising plants based traditional system of

medicine. According to a report of WHO, plants based traditional system still

continue to play an essential role in health care. At least 119 bioactive chemical

substances derived from plant species from 1959 to 1980 have been considered as

important drugs and are still in practice27. Amongst these drugs, 74% were discovered

from plants used in traditional system of medicine through bioassay guided isolation.

It has been documented that during 1940s to 2007, 155 drug molecules were

discovered, in which 73% were non synthetic with 47% being either natural product

derivatives or natural products. In U.S.A, during 2005 to 2007 thirteen new natural

product derived drugs were approved, amongst these five were novel members of new

classes28. Up to 50% of the approved drugs during the last 30 years are either directly

or indirectly from natural products and in the area of cancer, over the time frame from


8

around the 1940s to date, of the 175 small molecules 85 actually being either natural

products or their direct derivatives2. From 2005 to date natural products or natural

products derived marketed drugs are tabulated in Table 1.2.

Table 1.1: Important drugs discovered from plants with their ethnomedical

correlations and sources29

Drug B. Source Common Name Therapeutic uses

Atropine Atropa belladonna Deadly nightshade Parasympatholytic

Caffeine Camellia sinensis Tea plant CNS stimulant

Cocaine Erythroxylum coca Coca Local anesthetic

Codeine Papaver somniferum Opium Poppy Analgesic

Colchicine Colchicum autumnale Autumn crocus Gouty arthritis

Digoxin Digitalis purpurea Foxglove Cardiac stimulant

Emetine Cephaelis ipecacuanha Ipecacuanha Emetic

Ephedrine Ephedra sinicа Ma Huang Sympathomimetic

Glycyrrhizin Glycyrrhizia glabra Liquorice Antiulcer

Hyoscamine Hyoscamus niger Henbane Anticholinergic

Lobeline Lobelia inflata Astmaweeed Respiratory stimulant

Morphine Papaver somniferum Opium Poppy Analgesic

Nimbidin Azadirachta indica Neem Antiulcer

Noscapine Papaver somniferum Opium Poppy Analgesic, anti tussive

Papain Carica papaya Papaya Mucolytic

Physostigmine Physostigma venenosum Calabar bean Para sympathomimetic

Pilocarpine Pilocarpus jaborandi Jaborandi Para sympathomimetic,

Quinine Cinchona succirubra Peruvian bark Anti-malarial

Reserpine Rauwolfiа serpentinа Sarpagandha Anti-hypertensive

Salicin Salix alba White willow Analgesic

Santonin Artemisa maritima Sea wormwood Ascaricide

Silymarin Silybum marianum Blessed milk thistle

Hepatotonic

Teniposide Podophyllum paltatum Mayapple, Anticancer

Theophylline Camellia sinensis Tea plant Bronchodialator

Tubocurarine Chondodendron

Tomentosum

Curare Parasympatholytic

Yohimbine Pausinystalia johimbe Yohimbe Aphrodisiac


9

Table 1.2: Natural product derived drugs in market since 200529

Year Trade

Name

Generic Name/

(Active compound)

Classification Therapeutic Uses

2005 Prialt® Ziconotide NP Pain

2005 Flisint® Fumagillin NP Antiparasitic

2005 Sativex® Tetrahydrocannabinol NP Pain

2005 Tygacil® Tigecycline Semi synthetic NP Antibacterial

2005 Doribax® Doripenem NP derived Antibacterial

2006 Chantix® Varenicline NP derived Nicotine dependence

2006 Byetta® Exenatide NP Diabetes

2007 Yondelis ® Trabectedin NP Oncology

2007 Vyuanse® Lisdexamfetamine NP derived ADHD

2007 Altabax® Retapamulin Semi synthetic NP Antibacterial

2007 Ixempra® Ixabepilone Semi synthetic NP Oncology

2008 Zeftera® Ceftobiprolemedocaril Semi synthetic NP Antibacterial

2008 Relistor® Methylnaltrexone NP derived Constipation

2009 Vibativ® Telavancin Semi synthetic NP Antibacterial

2009 Istodax ® Romidepsin NP Cancer

2009 Javlor® Vinflunine Semi synthetic NP Cancer

2009 Remitch® Nalfurafine Semi synthetic NP Pruritis

2010 Javtena® Cabazitaxel Semi synthetic NP Cancer

2010 Gilenya® Fingolimod NP derived Multiple sclerosis

2010 Halaven® Eribulin NP derived Cancer

2010 Mepact® Mifamurtide NP derived Cancer

2010 Zuacta® Zucapsaicin NP derived Pain

2011 Dificid® Fidaxomicin NP Antibacterial

2011 Natroba® Spinosad NP Antiparastic

2012 Picato® Ingenolmebutate NP Actinic Keratosis

2012 Forxiga® Dapagliflozin NP derived Type 2 diabetes

2012 Synribo® Omacetaxinmepesucinate NP Oncology

2012 Kyprolis® Carfilzomib NP derived Oncology

2012 Synriam® Arterolane/piperaquine NP derived Antimalerial

2012 Desyne® Novolimus Semi synthetic NP CVS surgery

2013 Invokana® Canagilflozin NP derived Type 2 Diabetes

NP = Natural Product


10

1.4 Biodiversity of Indo-Pak Subcontinent

The Indo-Pak subcontinent has unique distinction, utilizing allopathic or modern

medicines as well as other six known systems of medicine i.e. ayurveda, unani,

siddha, yoga, naturopathy and homoeopathy30. The geography of Pakistan indicates

that it covers an area of 796,095 sq. km, lies between 60° 55’ to 75° 30’ east longitude

and 23° 45’ to 36° 50’ north latitude. Pakistan has a diverse climatic zones and

biodiversity because of wide ranging altitude from 0 to 8611 m. In Pakistan

approximately 6,000 species of higher plants have been reported, out of these 600 to

700 plant species are capitalized for medicinal purposes. Pakistan has four phyto-

geographical regions: (i) Irano-Turanian (45% of species); (ii) Sino-Himalayan (10%

of species); (iii) Saharo-Sindian (9.5% of species); and (iv) Indian element (6% of

species)31.

In Pakistan, the local population of different areas has centuries old knowledge,

regarding traditional uses of plants available in their respective localities. From

generation to generation this indigenous knowledge of plants has been transferred.

These plants are used to treat a range of ailments from headache to stomachic and

from cuts to wounds32. Nearly 250,000 higher plants species have been reported from

around the world, in which nearly 10% are found in the Hindukush-Himalayas ranges,

of which two-third are of medicinal significance8.

Furthermore, there is widespread interest in advancing traditional health systems to

fulfill basic health care needs. This is especially true in this country, as prices of

modern medicines are much higher, and governments find it more difficult to meet the

cost of pharmaceutical-based health care. However, it is a common observation that

many medicinal plants growing in this country remain taxonomically unidentified and


11

there are many more of them, which have not been phytochemically examined.

Furthermore, no attention has yet been paid to characterize them from the

pharmacognostic point of view. Thus, it is expected that the number of medicinal

plants growing or available in Pakistan may be more than what has so far been

reported. It is also important that the countless herbs found in Pakistan should be used

for promotion of health and for fighting diseases. Thus, medicinal plants of Pakistan

hold good promise as potential sources for new drug development. In order to develop

useful drugs from these medicinal plants, efforts should be made to identify them

scientifically, phytochemically, biologically and followed by standardized pre-clinical

studies so as to establish the authenticity of their claimed therapeutic potentials.

1.5 The Family Trilliaceae

The family has been recognized as distinct by Lindley since 184633. Steven Elliott

wrote “This family is an attractive one; A spiral of leaves at the peak of a stem,

sustaining solitary flower, it enclose and covers numerous species”. Family

Trilliaceae includes perennial herbs possessing characteristics underground rhizomes,

slender to stout, frequently creeping, unbranched, occasionally erect, monopodial.

Aerial stems are simple, frequently glabrous, and sometimes pubertal. Foliage leaves

3–22 in a pseudo whorl at top of stem, petiolate to sessile, thinner to broadly ovate, at

the bottom rounded, or sometimes cordate or narrowing, sometimes multicolored,

glabrous or pubescent along core veins on axial surface. Flowers are bisexual, and

frequently solitary. Perianth fragments are persistent, in two whorls. Stamens as

numerous as the perianth fragments; usually anthers are longer than the filaments.

Ovary superior, 1 to 10, locular, Carpels are 3 to 10, ovules numerous, styles are 3 to

5. Fruit are fleshy capsule or a berry, usually maroon, green, blackish or dark purple,


12

rarely white, yellow, or red. Seeds sometimes afforded with an scarlet sarcotesta34,35.

Schilling and Farmer reported that the Trilliaceae family, which showed an arcto-

tertiary distribution, encompass of five genera36. Out of these, three exhibit an

extensive distribution.

• Paris from Iceland to Japan

• Daiswa from Eastern Asia

• Trillium from Eastern Asia and North America

1.6 Genus Trillium

Trillium is the most important genus of Trilliaceae. The genus consists of perennial

herbs with characteristics rhizomes that are horizontal or erect, semi erect, branched

or faintly unbranched, compressed to shortened, elongated to bulky and fleshy, distal

end pointed or premorse, the apex bears large terminal shoot/bud. Stem has leaf-

sheaths and brown scales at the base. Leaves are three located at the top of the main

stem. Flowers are some totally to partly pedicellate, sessile, or syncarpous. Sepals are

separate, green, light maroon, or possessing maroon spotings, ovate to oblong, or

lanceolate, irregular with bracts. Petals are characteristically 3, erect or ovate to linear,

scattering, or recurved, discrete, red, white, yellow, pink, green, or mixture of all

these colors. Stamens are 6 in numbers, irregular in 2 whorls of 3, incurved, erect, or

divergent. Anthers are 2-locular, equal or longer than the filaments, superior ovary,

proximal segment 3-locular, 3- or 6-lobed, some axile, some parietal or a blend of

both, the distal part forms stigmas, stigmas often persistent, occasionally connate,

sessile or with very little style, subulate to linear. Filaments generally short basally

extended. Seeds are numerous and fruit is a berry. The genus Trillium contains about

forty eight interrelated species in eastern North America and temperate eastern Asia,


13

as well as in western North America37. Most of the Trillium species are related with

the deciduous forests (ancient Arcto-Tertiary), which have continued with remarkable

changes in geographical ranges since the early Tertiary period in the northern

hemisphere. At present, each species of Trillium is limited to one of three

geographical areas: western Asia, eastern and eastern North America38. In Pakistan

the genus is represented by single species i.e T. govanianum39.

1.6.1 Species of genus Trillium40-42

Genus Trillium comprises of more than twenty species, and is mainly distributed in

North America and Asia. Some of its important species with specific characteristics

are shown in Table 1.3.


14

Table 1.3: Species of genus Trillium

No Species with

common Name

Occurrence Flowering

period

Specific characteristics

1 Trillium erectum

• Wake robin

• Red trillium

North America

Apr-Jun • Rhizomes short, thick, praemorse

• Petals typically red, maroon, or dark purple

• Petals usually present in same plane as sepals

2 Trillium nivale

• Snow trillium

• Dwarf white trillium

United States (U.S.)

Mar-Aprl • Rhizomes stout, short, praemorse

• Bracts blade bluish green

• Scapes six gonal in cross section

3 Trillium undulatum

• Painted trillium

• Painted lady

Wisconsin (U.S.)

Apr-Jun • Rhizomes short, horizontal, stout

• Petals with distinctive dark red colour

• Bracts are strongly petiolate

4 Trillium pusillum

• Dwarf trillium

• Least trillium

United States

Mar-May • Rhizomes thin, horizontal, branched

• Bracts very short, subsessilepetiolate

• Sepals about as large and prominent aspetals,

• petals spreading ascendingly

5 Trillium

grandiflorum

• Great white trillium

• White wake-robin

Mountains of Virginia.

(North America)

Apr-Jun • Rhizomes thick and short

• Petals erected basally

• Ovary ovate to lanceolate, white or rarely pink

6 Trillium ovatum

• Western white trillium

North America

Mar-May • Rhizomes horizontal to semi erect, short, stout, praemorse

• Bracts sessile


15

7 Trillium luteum

• Yellow trillium

• Yellow toadshade

Joseph rivers and

elsewhere in Michigan,

(U.S.)

Apr-May • Rhizomes brownish, horizontal, short, thick, not fragile, praemorse

• Petals oblanceolate to lanceolate, greenish yellow to lemon yellow in color

• Flower odor strongly of lemon

8 Trillium petiolatum

• Purple trillium

• Round-leaved trillium

North America

Apr-May • Rhizomes erect, very deep often, praemorse

• Petals long lasting

• Ovary, erect to incurved, light maroon to red, purple, or greenish to yellowish, flat, linear to lanceolate

9 Trillium simile

• Sweet white trillium

North America

Apr-May • Rhizome forming clumps, stout, praemorse

• Petals creamy white in color

• Flowers facing upward, odour sweet like apple

10 Trillium lancifolium

• Lance leaved trillium

North America

Feb-May • Rhizome white, horizontal, very brittle, inter-nodes elongated

• Petals linear to narrowly spatulate

11 Trillium

kamtschaticm Korea,Japan

Russia, N. America and China

Apr-Jun • Rhizome stout and straight

• Stems tufted

• Leaves sessile, broadly rhombic to orbicular or ovate to orbicular

• Anthers 7 to 8 mm and longer than filaments

• Fruit a berry, globose to ovoid


16

12 Trillium tschonoskii

Bhutan, Japan,

Korea and China

July-Aug • Rhizome stout, horizontal

• Stems tufted

• Leaves sessile, rhombic to orbicular or to broadly rhombic

• Anthers 3 to 4 mm, shorter than or equal filaments

13 Trillium taiwanense Taiwan, China

May-Jun • Rhizomes creeping, stout

• Stem solitary

• Leaves shortly petiolate, ovate to broadly ovate

• Stamens short

• Anthers 1to 1.5 mm 14 Trillium parviflorum

• Small flowered trillium

North America

Mar-May • Rhizomes brownish, horizontal to erect, thick, praemorse, not brittle

• Petals linear to linear lanceolate, white, rarely purplish basally

15 Trillium govanianum

Bhutan, India, Nepal China and Pakistan

Apr-Aug • Rhizomes greyish thick.

• Adventitious roots numerous, fibrous

• Stem up to 30 cm tall

• Leaves shortly petiolate, ovate or ovate to cordate

• Fruit red, globose berry


17

1.6.2 Phytochemical profiling of genus Trillium

Literature citing different species of genus Trillium indicates a thorough investigation

for phytochemicals, which has yielded a large number of phytochemicals/secondary

metabolites. The results indicate that the genus is very rich source of biologically

active compounds like steroids, terpenoids, sterols, flavonoids, steroidal glycosides

and saponin derivatives43-45. A list of secondary metabolites/phytochemicals reported

from the genus Trillium is shown in Table 1.4.

Table 1.4: List of phytochemicals isolated from genus Trillium

Chemical Name Chemical Structure Molecular

Formula

spirost-5-en-3-ol (diosgenin)46

C27H42O3

(25S)-spirost-5-ene-3β, 17α,27-triol44,47

C27H42O5

(25S)-3β,17α -dihydroxyspirost-5-en-27-yl β-D-glucopyranoside44

C33H52O10

(25S)-17α ,27-dihydroxyspirost-5-en-3 β -yl β-D-glucopyranoside44

C33H52O10


18

(25S)- 27-[( β-D-

glucopyranosyl)oxy]-17α -hydroxyspirost-5-en-3β -yl

O α - L-rhamnopyranosyl-

(1→2)- β -D-glucopyranoside44

C45H72O19

(25S)-27-[( β - D-glucopyranosyl)oxy]-17 α,27-dihydroxyspirost-5-

en-3-yl O-(4- O-acetyl- α -L-

rhamnopyranosyl)-(1→2)- β -D-glucopyranoside44

C33H52O10

(25S)-27-[( β-D-glucopyranosyl)oxy]-

17α,27- dihydroxyspirost-5-en-3 β -D-glucopyranosyl-(1→6)-O-[ α-L-rhamnopyranosyl-

(1→2)]- β-D-glucopyranoside44

C51H82O24

(25S)-17α, 27-dihydroxyspirost-

5-en-3β-yl O-(4-O-acetyl- α -L-rhamnopyranosyl)-

(1→2)- β - D-glucopyranoside44

C41H64O15


19

(25S)-17α,27-dihydroxyspirost-5-en-3 β-

yl O- α -L-rhamnopyranosyl-

(1→2)- β-D-glucopyranoside48

C39H62O14

(25R)-17α -hydroxyspirost-5-en-3 β-yl O- α -L-rhamnopyranosyl-

(1→2)-β- D-glucopyranoside49

C39H62O13

(25R)-17α -hydroxyspirost-5-en-3 β -yl O- α -L-

rhamnopyranosyl-(1→4)-β -D-glucopyranoside50

C39H62O13

(25R)-17α-hydroxyspirost- 5-en-3β-yl O-α-L-

rhamnopyranosyl-(1→2)-O-[α-L-rhamnopyranosyl-

(1→4)]-β- D-glucopyranoside49

C45H72O17


20

(25R)-17α-hydroxyspirost- 5-en-3β-yl O-α-L-


(1→4)]-β- D-glucopyranoside44,51

C45H72O17

(25R)- 17α-hydroxyspirost-5-en-

3β-yl O-α-L-rhamnopyranosyl-(1→2)-

O-[O-α-L-rhamnopyranosyl-(1→4)-

a-L-rhamnopyranosyl-(1→4)]-α-D-

glucopyranoside49

C51H82O21

(25R)-spirost-5-en- 3β-yl O-α-L-

rhamnopyranosyl-(1→2)-β-D-glucopyranoside49

C39H62O13

(25R)-spirost-5-en-3β-yl O-α-L-rhamnopyranosyl-

(1→2)-O-[α-L-rhamnopyranosyl-(1→4)]-

β-D-glucopyranoside49

C45H72O16


21

(25R)-spirost-5-en-3β-yl O-α-Lrhamnopyranosyl-

(1→2)-O-[O-α-L-rhamnopyranosyl-(1→4) -α-Lrhamnopyranosyl-


C51H82O20

(25R)-26-[β-D-glucopyranosyl]oxy]-22 α -

methoxyfurost-5- en-3 β -yl O-α-L-



C53H88O22

(25R)-26-[β -D-glucopyranosyl]

oxy]-17 α -hydroxy-22β -methoxyfurost-5-en-3β -yl O-α-L-rhamnopyranosyl-

(1→2)-β- D-glucopyranoside52

C47H78O19


22

(25R)-26-[β -D-glucopyranosyl]oxy]-17 α -

hydroxy-22amethoxyfurost- 5-en-3β -yl O-α-L-



C53H88O23

(25R)-26-[β -D-glucopyranosyl]

oxy]-3β -[(O-α-L-rhamnopyranosyl-(1→2)-

β-D-glucopyranosyl) oxy]-cholesta-5,17-diene-

16,22-dione49

C45H70O18

l-O-[2,3,4-tri-O-acetyl- α-L-rhamnopyranosyl- (1→2)4-O-acetyl-α-L-

arabinopyranosyl]- 21-O-acetyl-

epitrillenogenin53

C45H61AcO20

(25S)-27-hydroxypenogenin- [3-O-

α-L-rrhamnopyranosyl-(1→2)-O-β-D-

glucopyranoside]53

C39H62O14


23

(25R)-27- hydroxypenogenin 3-O-α-

L-rhamnopyranosyl-(1→2)-O-β-D-

glucopyranoside48

C39H62O14

penogenin 3-O-α-L-rhamnopyranosyl-(1→2)-O-β-D-glucopyranoside49

C39H62O13

penogenin 3-O-β-D-glucopyranosyl- (1→6)-[O--α-L[-

rhamnopyranosyl-(1→2)]-O-β-[[[D-glucopyranoside48

C45H72O18

penogenin 3-[O-β-[D-glucopyranoside[

49

C33H52O9


24

deoxytrillenoside48,54

C47H70O23

spirost-5-ene-3,17-diol (Pennogenin)46

C27H42O4

(10R,6E)-7,11-ddimethyl-3-mehyl3ene--6-dodecaene-

1,2,10,11-tetraol 10-O-β - D-glucopyranoside48

C21H38O9

(10R,6E)-3,7,11- trimethyl-1,6-ddodecadien-

3,10,1111-triol 10-oO-glucopyranoside48

C21H38O8

(10R,6E)-3,7,11- trimethyl-1,666-dodecadien-

3,10,1110-triol 10-O-glucopyranoside48

C21H38O8

7,11-dimethyl- 3-mmethylene-1,6-

dodecadien10-10,11-diol 10-oO-β-D-

(1→4)glucopyranosyl-O-β -D-glucopyranoside55

C27H46O12


25

methylferulorate55,56

C11H12O4

astragalin48

C21H20O11

β-ecdysone48

C27H44O7

2626-O-β-dD-glucopyranosyl (22,[25R)-[furost-5-eene-

3β,17α,22,26-tetraol 3-O-α-L-rhamnopyranosyl-

(1→2)-O-β-D-glucopyranoside49

C45H74O19

26-dO-β-aD-23glucopyranosyl (22,25R)-

furost-5-eene-3β,17α, 22,26-tetraol 3-O-α-L-

rhamnopyranosyl- (1→ 42)-[O-α-L-

[rhamnopyranosyl-(1→04)]-O-β-D-glucopyranoside48

C51H84O23


26

a26-O-β-D-glucopyranosyl 17(20)-

dehydrokryptogenin 3-O-α-L-rhamnopyranosyl-

(1→2)-[O-α-L-rhamnopyranosyl-

(1→4)]-O-β- D-glucopyranoside48

C51H80O22

26-O-β-D-glucopyranosyl 17(20)-

dehydrokryptogenin 3-O-α-L-rhamnopyranosyl-

(1→2)-O-β- D-glucopyranoside49

C45H70O18

3,4,5,7-tetrahydroxyflavone45

C15H10O6

quercetin 3-O-rutinoside; [3-O-β-L-

rhamnopyranosyl-(1→6)-β-D-glucopyranoside]45

C26H28O16


27

kaempferol 3-O-α-rhamnosyl-(1→2)-

O- [α-rhamnosyl- (l→6)]-β-glucoside45

C33H40O20

p-hydroxymethyl benzyl alcohol57

C8H10O2

3,7,11-trimethyl-3,9,11-trihydroxyl-1,6-

dodecadiene glycerol57

C18H36O6

2-methyl-3,4 dihydroxy-hexanedioic acid57

C7H12O6


28

1.6.3 Medicinal importance and biological studies of genus Trillium

A number of studies indicate that plant species of Trillium have been extensively used

as a remedy for various diseases. The reported biological/pharmacological activities

of different species (Table 1.5) indicate potentials in crude extracts, solvent fractions

and isolated pure compounds. Trillium tschonoskii has been traditionally used in

China for at least one thousand years58,59. Rhizomes of this plant species have been

used in folk medicine as medicinal herbs for treatment of hypertension, neurasthenia,

giddiness, headache, removing carbuncles, and ameliorating pains60. The anticancer

activity of n-BuOH extract has also been reported59. The rhizomes of T. erectum

called beth roots have been used in folk medicine for the treatment of hemorrhages

from uterus, urinary tract and lungs61. The cytotoxic activity of the isolated

compounds (spirostanol saponins and furostanol saponins) from T. erectum against

HL-60 leukemia cells has been reported44. Dried underground parts of T. tschonoskii

were used as a folk medicine to remove carbuncles and to ameliorate pains, etc62. The

marked inhibitory action against COX-2 production in macrophagocytes of the mouse

abdominal cavity by isolated compounds has also been reported38. It has also been

described that the ethanol extracts, ethyl acetate extracts and butanol extracts of T.

tschonoskii. significantly suppress the edema of rat hind paw swelling elicited by

injection of carrageenan63. T. tschonoskii can improve learning and memory, and

these effects were associated with enhancement of anti-oxidase expression64. The

antifungal activity of ethanol extract of the rhizomes and above ground portion of T.

grandiflorum has also been reported46.


29

Table 1.5: Reported biological activities of genus Trillium

Activity Part

used

Extract/Isolated

compounds

Source

anti metastatic effect against colorectal cancer cells58

Rhizome Isolated compounds

Trillium tschonoskii

antibacterial and anti oxidant65

Rhizome Extracts Trillium tschonoskii

antifungal46 Rhizome Extracts and fractions

Trillium grandiflorum

antifungal46 Rhizome Isolated compounds

Trillium grandiflorum

cytotoxicity against HL-60 human promyelocytic leukemia cells44


Trillium erectum

cytotoxicity against human lung cancer cells66



cytotoxicity against adriamycin resistant breast cancer cells58

Rhizome Isolated compound


cytotoxicity against malignant sarcoma cells67



cytotoxicity against malignant neuroblastoma68

Rhizome Extract/fractions Trillium pendulum

cytotoxicity against multi drug resistance (MDR) hepatocellular carcinoma cells69



expression of anti-oxidase of aging rat induced with haloperidol70

Rhizome Extracts Trillium tschonoskii

analgesic, anti-inflammatory and thrombisis effects63

Rhizome Extract/fractions Trillium tschonoskii

learning and memory enhancement effect64

Rhizome Extract/fractions Trillium tschonoskii


30

1.7 Trillium govanianum

The medicinal plant Trillium govanianum (Fig. 1.1) belongs to family Trilliaceae, and

is used in the traditional system of medicine in subcontinent for different aliments71. It

was selected for detailed scientific study following a thorough literature survey of

their ethnomedicinal uses and reported data. The taxonomical position of T.

govanianum is given in Table 1.6.

Figure 1.1: Trillium govanianum plant.

Table 1.6: Taxonomical classification of T. govanianum

Kingdom Plantae

Sub Kingdom Tracheobionta

Class Liliopsida

Sub class Liliidae

Order Liliales

Family Trilliaceae

Genus Trillium

Species Govanianum


31

1.7.1 Plant Morphology

T. govanianum plant is a perennial herb about 12-20 cm tall. The plant can be

identified by its three leaves in one whorl at the summit of the stem and a solitary,

flower in the center. Leaves are broadly ovate, acute and conspicuously stalked.

Rhizomes are thick. Adventitious roots are numerous and fibrous. Flower is one and

terminal. Stamens are 6, shorter than the perianth and in 2 whorls, filaments are long

about 4 mm. Basifixed anthers are about 5 mm long. Fruit is a red, 0.5-3.0 cm in

diameter, and seeds are abundant, rhombus, with a pulpy lateral appendage.

Flowering periods is from april to august39,40.

1.7.2 Distribution

The T. govanianum is distributed in south Asia, especially in India, Nepal, China,

Pakistan and Bhutan at an altitude of 2700 -4000 m71. In Khyber Pakhtunkhwa the

plant is present at high altitudes in District Dir, Swat and Shangla39.

1.7.3 Ethnobotanical Uses

T. govanianum rhizomes are used in the traditional system of medicine in

subcontinent (Pakistan, India and China) for different ailments. In folk medicine, the

rhizomes is used to cure dysentery, backache, healing of wound, skin boils, menstrual

and sexual disorders71-73. The powdered rhizomes is also used as anthelmintic74.


32

1.8 Aims and Objectives

Due to folkloric knowledge, increased market demand and usage of this plant species,

it is important to provide scientific evidence to its traditional uses, as well as to screen

this valuable herb for phytochemical and potential biological activities. Therefore,

following aims and objectives were set for the present study;

1. Explore the phytochemical constituents of rhizomes, utilizing various

chromatographic, spectrometric and spectroscopic techniques.

2. Evaluate the pharmacognostic features such as physicochemical and

histological characteristics.

3. Perform acute toxicity studies for evaluation of safety profile of the plant

extract.

4. Perform biological activities to find out valid scientific rationale for its

folkloric uses.

5. Investigate potential therapeutic uses, other than folkloric uses, by performing

bioactivity screenings.

Chapter 2 Materials and Methods

33

2. Materials and Methods

2.1 Drugs and chemicals

The chemicals, solvents and drugs consumed in different experimental procedures

were analytical as well as commercial grade (Table 2.1). The commercial grade

solvents were distilled before the start of experiments.

Table 2.1: Drugs and chemicals used with their source

Chemicals/Drugs Source/Supplier

Silica Sigma Chemical Co, St L-ouis, MO, USA

Diclofenac sodium Sigma Chemical Co, St L[ouis, MO, USA

Imipenem Cirin Pharmaceutical, Hattar, Pakistan

Amphotericin B Medinet Pharmaceutical, Karachi, Pakistan

Ibuprofen Allaince Pharmaceutical, Peshawar, Pakistan

Doxorubicin Atco Laboratories, Karachi, Pakistan

Etoposide Atco Laboratories, Karachi, Pakistan

Permethrin Atco Laboratories, Karachi, Pakistan

Ascorbic acid S[igma Aldrich, G-ermany

Carrageenan Si-gma Chemical Co, St L-ouis, MO, USA

DPPH Waka Ltd. Japan

Butylated hydroxytoluene (BHT) Sigma-Aldrich, Germany

Dimethyl Sulfoxide (DMSO) Sigma-Aldrich, Germany

Ceric sulphate Merck, Darmstadt, Germany

Magnesium chloride Me[rck, D[armstadt, Germany

Sodium bicarbonate Mer[ck, D.armstadt, G-ermany

Magnesium sulfate Merc -k, D[[armstadt, Ge-rmany

Calcium chloride Me-rck, D.armstadt, Ger-many

Sodium dihydrogen phosphate Mer-ck, D.armstadt, Ger[many

Potassium dihydrogen phosphate Merck, Darmstadt, Germany


34

2.2 Research centers for experimental studies

Experimental studies were performed in the Department of Pharmacy, University of

Peshawar, H.E.J. Research Institute of Chemistry, International Center for Chemical

and Biological Sciences (ICCBS), University of Karachi, Department of Pharmacy,

Shaheed Benazir Bhutto University, Sheringal, Dir (U) and Institute of Basic Medical

Sciences, Khyber Medical University, Peshawar.

2.3 Physical constants

Melting points of isolated compounds were determined by melting point apparatus

model-MPA-100, while optical rotations were determined by digital Polarimeter

model-JASCO DIP-360.

2.4 Spectroscopic techniques

Most of the spectroscopic studies were carried out through highly sensitive

sophisticated instruments available at H.E.J. Research Institute of Chemistry,

International Center for Chemical and Biological Sciences (ICCBS), University of

Karachi, Karachi.

2.4.1 UV technique

Hitachi Spectrophotometer, model-U-3900/3900H (fully automated) was used for UV

spectroscopic analysis of isolated compounds.

2.4.2 IR technique

Infrared Spectrometer, model- JASCO 302-A was used for IR spectroscopic analysis

of isolated compounds.


35

2.4.3 Mass technique

For the mass spectral studies of isolated compounds, the Mass Spectrophotometer

model-MAT311A linked with computer system of PDP11/34 was used for low

resolution electron impact spectra while Jeol Mass Spectrometer model JMS HX 110

was used for FAB and HR mass spectra.

2.4.4 Nuclear Magnetic Resonance (NMR) technique

For the 1H-NMR and 13C-NMR spectra of isolated compounds, NMR Spectrometer

(Bruker; AMX-600, AM-400 and AM-300) was used. The 1H-NMR spectra were

taken at different MHz i.e. 300, 400, or 600. The Distort-ionless Enhancement by

Polarization Transfer (DEPT) experiments were executed at 90o and 135o

for

determination of CH3, CH2, and CH moieties of isolated compounds.

2.4.5 Gas Chromatography and Gas Chromatography-Mass Spectrometry

GC/MS analysis was carried out on a 6890N Agilent gas chromatograph coupled with

a JMS 600 H JEOL mass spectrometer. The compound mixture was separated on a

fused silica capillary SPBI column, 30 m × 0.32 mm, 0.25 µm film thicknesses, in a

temperature program from 50 to 256°C with a rate of 4°C/minute (min) with 2 min

hold. The injector was at 260°C and the flow rate of the carrier gas (helium) was 1

mL/min. The EI mode of JMS 600 H JEOL mass spectrometer has ionization volt of

70 eV, electron emission of 100 µA, ion source temperature of 250°C and analyzer

temperature of 250°C. Sample was injected manually in split mode. Total elution time

was 90 min. MS scanning was performed from m/z 85 to m/z 39075.


36

2.4.6 GC-MS identification of components

Identification of proximate fatty acid components of the non-polar fraction (n-hexane)

was based on the computer evaluation of mass spectra of sample through NIST-based

AMDIS (automated mass spectral deconvolution and identification software), direct

comparison of peaks and retention times with those for the standard compounds as

well as by following the characteristic fragmentation patterns of the mass spectra of

particular classes of compounds.

2.5 Chromatographic techniques for isolation and purification of compounds

Different chromatographic techniques76 were used for isolation and purification of

compounds from the fractions of T. govanianum rhizomes.

2.5.1 Column Chromatography (CC)

For column chromatography technique, silica gel (column silica; 70-230 mesh size,

flash silica; 230-400 mesh size) was used as a stationary phase. Mobile phase used

includes various organic solvents either alone or in combination like, n-hexane, ethyl

acetate, chloroform, butanol and methanol. Different spots of compounds were made

visible by either UV light (short λ, 254 nm; long λ, 365 nm) or by spraying different

locating reagent. On TLC cards/plates, purity of the isolated compounds were

confirmed.

2.5.2 Thin Layer Chromatography (TLC)

For this technique, silica gel pre-coated cards (PF 0.25, 254 mm) were used. Silica gel

pre coated plates (0.5 mm thickness, 20 x 20 cm) were also applied for pre-parative

thin layer chromatography for purification of isolated compounds.


37

2.5.3 Reagents for visualizing the spots

For visualization or locating the spots of compounds on TLC cards, various spraying

reagents were prepared as per procedure given and sprayed through a suitable spray

gun on TLC cards/plates. The UV light (254 nm and 365 nm) was also used for

visualization of spots on TLC plates/cards.

2.5.3.1 Ceric sulphate solution as reagent

For ceric sulphate reagent preparation, ceric sulphate (0.1 g) was dissolved in distilled

water (4 mL). To avoid any turbidity of solution, heated the solution and sulphuric

acid (few drops) were added. Upon spraying on TLC card/plates and exposure to

heating, the formation of colors indicates the presence of different classes of

compounds.

2.5.3.2 Vanillin solution as reagent

Vanillin solution was prepared by dissolving 1 g of vanillin in 50% phosphoric acid.

The appearance of pink or deep purple color after spraying vanillin solution on TLC

plates and heating up to 100-110oC, confirmed the presence of terpenes and steroids.

2.6 Ethnomedicinal study

2.6.1 Site selection

Four main districts of Khyber Pakhtunkhwa were selected for the study i.e Buner,

Swat, Shangla and Dir, keeping in view the fact that the plant under study is found in

these areas.


38

2.6.2 Sampling informants and ethnomedicinal data collection

The ethnomedicinal survey was carried out from March, 2013 to November, 2013. In

addition to local people who had practical knowledge on medicinal plants, traditional

healers/hakims and pansaries (crude drug and general items sellers) were interviewed

according to reported method77 with slight modifications.

2.7 Plant materials

2.7.1 Collection

Rhizomes of T. govanianum Wall were collected from Kohistan valley (34° 54' and

35° 52' North latitudes and 72° 43' and 73° 57' East longitudes), Dir Upper, Khyber

Pakhtunkhwa, in August, 2013. The plant was identified by Mr. Ghulam Jelani

(Curator), Department of Botany, University of Peshawar. A voucher specimen [No.

Bot. 20092 (PUP)] has been deposited in the herbarium Department of Botany,

University of Peshawar, Pakistan for future reference. The rhizomes were then

washed by water (distilled) and dried at ambient temperature under shade, and then

crushed to powder for analysis.

2.7.2 Extraction and fractionation

The shade-dried rhizomes of T. govanianum (7 Kg) were ground and extracted with

MeOH (40 L) at room temperature, three times for a period of seven days (3 × 40 L)

78. The combined methanolic extract was evaporated to dryness by using a rotary

evaporator (Heidolph, Laborota-4010) fitted with recirculation chiller (Mini-chiller,

Huber w-H1 plus) and a heating bath (B-490) at 40oC, yielded a semi solid brownish

gummy residue as crude methanolic extract (512 g). For screening of different

biological activities about 35 g of extract (Cr. MeOH-Ext) was reserved, and the


39

remaining extract was further fractionated on the base of their solvent affinity (solid-

liquid partition) into n-hexane (n-Hex-fr; 81 g), chloroform (CHL-fr; 94 g), ethyl

acetate (EtOAc-fr; 85 g) and butanol (BuOH-fr; 105 g) fractions. The remaining

fraction, after the above process was considered as aqueous (Aq-fr; 107 g) fraction 79.

The complete process is documented in Scheme 2.1.

Scheme 2.1: Extraction and fractionation of T. govanianum rhizomes

Powder rhizomes of T. govanianum

(7 Kg)

Extraction with MeOH

Crude MeOH Extract (512 g)

Fractionation

For biological activities

(35 g)

Ethyl acetate fraction (85 g)

Aqueous fraction (107 g)

Butanol fraction (105 g)

n-hexane fraction (81 g)

Chloroform fraction (94 g)


40

2.8 Macroscopic and microscopic features of rhizome

Macroscopic appearances of the fresh rhizome and the color, shape, size, surface,

odor and taste of the crude drug were determined. Thin transverse section of the

rhizome was prepared. The material was mounted in center of potato pith and a large

number of transverse cuts were made across the material with the help of a sharp

razor and was kept moist in water. The thin section was selected and staining was

done on glass slide. The staining was carried out by putting the section in safranin for

3-4 min. The section was then gradually dehydrated in 10%, 30%, 50%, and 90% of

alcohols. The dehydrated section was then put into a drop of methylene green and

then washed with absolute alcohol for 2-3 min. Finally the section was mounted with

Canada balsam to make them permanent and was examined under Olympus Digital

microscope (MIC-D). The powder drug was also treated on glass slide, mounted with

Canada balsam and was subjected to microscopic examinations76,80.

2.9 Physicochemical parameters

The various physico-chemical parameters like loss on drying, total ash, water soluble

ash, acid insoluble ash, and extractive values were determined following well

established reported methods76,81,82. Detail procedures of which are given below.

2.9.1 Total ash

For the purpose of total ash determination, crude drug 2 g (air dried) was taken in the

silica dish or platinum (tarred) and ignited upto maximum temperature (not exceeding

450°C), until become carbon free, was cooled then and weighed. Percent total ash was

calculated by using formula,


41

Percenttotalashvalue = weightoftotalash

weightofcrudedrugtaken× 100

2.9.2 Water soluble ash

For the purpose of water soluble ash determination, the ash was mixed with water (25

mL) and boiled for 5 min. On filter paper (ash-less), insoluble matter was collected

and washed continuously with warm water, and then ignite for about 15 min at high

temperature (not exceeding 450°C). From the weight of total ash, weight of the

insoluble matter was subtracted. The water soluble ash (percentage) was calculated

with reference to the air dried drug.

2.9.3 Acid insoluble ash

For the determination of acid insoluble ash, hydrochloric acid (25 mL) was added to

the crucible containing the total ash and boiled for 5 min. The insoluble matter was

collected on the ash less filter paper and washed with hot water until the filtrate is

neutral. The filter paper was transferred to the crucible and ignited to a constant

weight. The residue was to cool in a suitable desiccator for 30 min. The ash was

weighed and percentage of acid-insoluble ash was calculated with reference to air

dried powder.

2.9.4 Loss on drying

For the determination of loss on drying, one gram of dried powder was placed in a

previously dried weighing beaker. The sample was dried in an oven at 100-105oC.

The loss of weight in mg per air dried material was calculated.


42

2.9.5 Extractive values

2.9.5.1 Methanol soluble extractive value

Powder drug (2.0 g) was macerated with 100 mL of methanol in a closed flask for 24

h, shaken frequently during the first 6 hours (h) and allowed to stand for 18 h. The

mixture was then filtered and the methanol was evaporated and allowed the filtrate to

dryness in a tarred shallow dish, and weighed. The percentage of methanol soluble

extractive value was calculated with reference to the air dried drug.

2.9.5.2 Water and other soluble extractive values

The procedure for the determination of extractive values of water, ethanol, butanol,

ethyl acetate, chloroform and n-hexanes was similar to the methanol soluble

extractive value, using the respective solvents instead of methanol.

2.10 Phytochemical tests

For the determination of plant metabolites like alkaloids, tannins, flavonoids,

saponins, sterols and carbohydrates, different qualitative phytochemical tests (color

reactions) of the crude methanolic extract and its subsequent solvents soluble

fractions like n-hexane, chloroform, ethyl acetate, butanol were performed according

to the recommended standard protocols81,83-85.


43

2.10.1 Test for alkaloids

2.10.1.1 Mayer’s test

To the plant extract/fraction solution, few drops of Mayer’s reagent was added. The

appearance of white creamy precipitate (ppt) represents the presence alkaloid contents

in the sample.

2.10.1.2 Wagner’s test

To the plant extract/fraction solution, few drops of Wagner’s reagent was added. The

appearance of reddish brown ppt indicates the presence alkaloid contents in the

sample.

2.10.1.3 Hager’s test

The plant extract/fraction solution was treated with few drops of Hager’s reagent

(saturated solution of picric acid). The appearance of yellow ppt indicates the

presence of alkaloid contents in the sample.

2.10.2 Test for flavonoids

2.10.2.1 Ferric chloride test

To the plant extract/fraction, few drops of 1% ferric chloride solution was added. The

formation of blue-green or violet color indicates the presence of flavonoids in the test

sample.


44

2.10.2.2 Sodium hydroxide test

To the plant extract/fraction, small quantity of distilled water was added and then

filtered. To the filtrate added few drops of 10% sodium hydroxide (NaOH), a yellow

color was produced. The change in color from yellow to colorless after the addition of

few drops of dilute hydrochloric acid indicates the presence of flavonoids in the test

sample.

2.10.3 Test for tannins

2.10.3.1 Ferric chloride test

To the plant extract/fraction, few drops of 1% ferric chloride was added. The

formation of blue-green color indicates the presence of tannins in the test sample86.

2.10.3.2 Lead acetate test

The plant extract/fraction was dissolved in distilled water, heated to boil. After boiling

filtered the solution, and then added lead acetate to the filtrate. The formations of

precipitates represent the presence of tannins in the sample.

2.10.4 Test for saponins

The presence of saponin contents was identified by the simplest frothing test. A

specific quantity of the tested extract/fraction was treated with boiling water, allows

to cool, and is then vigorously stirred in a test tube. The presence of saponins was

confirmed by the appearance and perseverance of the froth.


45

2.10.5 Test for steroids

The plant extract/fraction solution (5 mL) was taken in a test tube and acetic

anhydride (1 mL) was added to it. Change of color to green or blue indicates the

presence of steroidal compounds in the test sample.

2.10.6 Test for triterpenes

2.10.6.1 Salkowski’s test

To the plant extract/fraction, sufficient amount of chloroform and few drops of

concentrated sulphuric acid were added. The mixture was shaked in test tube and

allowed to stand for some time. The appearance of red brown color in the lower layer

indicates the presence of sterols, while the appearance of yellow color in the lower

layer indicates triterpenoids in the test sample.

2.10.6.2 Liebermann-Burchard test

To the plant extract/fraction, few drops of acetic anhydride was added. Concentrated

sulphuric acid (H2SO4) was then added to the test tube containing reaction mixture of

extract and acetic anhydride. Two layers were formed. The green appearance of the

upper layer was the indication of sterols, while deep red color was the indication of

the presence of triterpenoids in the test sample86.

2.10.7 Test for glycosides

The plant extract/fraction aqueous solution (5 mL) was mixed with glacial acetic acid

(2 mL) containing a drop of ferric chloride and added this mixture carefully to

concentrated sulphuric acid (1 mL) in the test tube, so that the concentrated sulphuric


46

acid come beneath the mixture. A brown ring appearance, indicates the presence of

the cardiac glycoside87.

2.10.8 Test for carbohydrates

2.10.8.1 Molisch’s test

To the plant extract/fraction, few drops of Molisch’s reagent were added.

Concentrated sulphuric acid was then added slowly to the sample in the test tube. The

formation of purple to violet color at the junction was the indication of the presence of

carbohydrates in the test sample.

2.10.8.2 Benedict’test

To the plant extract/fraction, few drops of Benedict’s reagent were added in a test

tube and boiled for some time on water bath. The formation of reddish brown

precipitate indicates the presence of reducing sugar in the test sample.

2.10.8.3 Fehling’s test

Few drops of the extract/fraction, were added to equal volume of Fehling’s A and B

and then heated till boiling. The Fehling’s A is the aqueous solution of copper

sulphate and the Fehling’s B reagent is the aqueous solution of potassium tatarate and

sodium hydroxide. A brick red ppt indicates the presence of reducing sugar in the test

sample.


47

2.11 Isolation of compounds

2.11.1 Isolation of compounds from CHCl3 fraction

The chloroform (CHCl3) fraction of T. govanianum rhizomes was selected for

isolation of compounds. Column chromatographic technique was used for separation

of compounds. Slurry was prepared with silica gel and was subjected to column

chromatography88. Using n-hexane and EtOAc solvent system as mobile phase in

increasing order of polarity, the fraction was further fractionated into eleven sub-

fractions (CFA-CFK) [Scheme 2.2].

The sub fraction CFB obtained with 20-40% chloroform in n-hexane were re-

chromatographed over silica gel eluting with mixture of n-hexane and EtOAc in

increasing order of polarity yielded five sub fractions (CFB(a)-CFB(e)). The sub fraction

CFB(b) obtained with 5-10% EtOAc/n-hexane when analyzed on TLC showed few

prominent spots and thus were subjected to further separation processes through

column chromatography with gradient solvent elution system yielded compound 1

(2% EtOAc in n-hexane; 13 mg), compound 2 (5% EtOAc in n-hexane; 16 mg) and

compound 3 (5% EtOAc in n-hexane; 11 mg) [Scheme 2.3].

The sub fraction CFE obtained with 20-40% EtOAc in chloroform was re-

chromatographed over silica gel eluting with mixture of EtOAc and n-hexane in

increasing order of polarity yielded compound 4 (20% EtOAc in n-hexane; 94 mg),

compound 5 (20% EtOAc in n-hexane; 21 mg) and compound 6 (60% EtOAc in n-

hexane; 132 mg) [Scheme 2.4].

The sub fraction CFH obtained with 5% MeOH in EtOAc was re-chromatographed

over silica gel eluting with mixture of MeOH and EtOAc in increasing order of


48

polarity yielded five sub fractions. The sub fraction CFHh obtained with 5% MeOH in

EtOAc when analyzed by TLC under UV light showed few prominent spots. Thus this

sub fraction was further subjected to separation process through preparative thin layer

chromatography using mobile phase of MeOH : EtOAc (1 : 9). As a result of this

separation process, compounds 7 (13 mg) and 8 (18 mg) were obtained [Scheme 2.5].


49

Scheme 2.2: Fractionation of chloroform fraction

Chloroform fraction

(CHL.fr)

(62 g)

CFB

CFA CFC

CFD CFH CFJ

CFE

CFF

CFG CFI CFK

100%

n-h

exan

e

100%

CH

L

20-4

0% C

HL

in h

ex

40-8

0% C

Hl i

n he

x

20-4

0% E

tOA

c in

CH

L

60-8

0% E

tOA

c in

CH

L

25%

MeO

H

100%

MeO

H

5% M

eOH

in E

tOA

c

50%

MeO

H in

EtO

Ac

Column chromatography (CC) with

gradient elution system Hex-CHL (0-100%)

CHL-EtOAc (0-100%) and EtOAc-MeOH (0-100%)

100%

EtO

Ac


50

Scheme 2.3: Isolation of compounds from sub fraction (CFB)

CFB

(Sub fraction)

CFB(b)

5-10% EtOAc in n-hexane


gradient elution

Compound 1

(13 mg)

2% EtOAc in n-hexane

(CC) (gradient elution)

Compound 2

(16 mg) Compound 3

(11 mg)



51

Scheme 2.4: Isolation of compounds from sub fraction (CFE)


gradient elution

Compound 4

(94 mg)


Compound 5

(21 mg) Compound 6

(132 mg)


CFE

(Sub fraction)


52

Scheme 2.5: Isolation of compounds from sub fraction (CFH)

CFH

(Sub fraction)


gradient elution

Compound 7

(13 mg) Compound 8

(18 mg)

5% MeOH in EtOAc

CFHh (Sub:Sub Fr)

Preparative TLC with

solvent system MeOH : EtOAc (1:9)


53

2.11.2 Isolation of compounds from butanol fraction

For isolation of compounds from butanol soluble fraction, the fraction was subjected

to column chromatography over silica gel and gradient elution was carried out with

mixtures of EtOAc and MeOH in increasing order of polarity yielded five sub

fractions (BFA-BFE). The sub fraction, BFA which was obtained with 10% MeOH in

EtOAc was re-chromatographed over silica gel and eluted with mixture of MeOH and

EtOAc in increasing order of polarity afforded compound 9 (borassoside E, 48 mg, 5-

10% MeOH in EtOAc). The sub fraction BFB which was obtained with 20% MeOH in

EtOAc was re-chromatographed over silica gel, eluted with mixture of MeOH and

EtOAc in increasing order of polarity yielded sub fractions (BFBa-BFBe). The sub

fraction, BFBc which was obtained with 30% MeOH in EtOAc when analyzed by TLC

and cerric sulphate reagent showed few prominent spots. Further re-chromatography

over silica gel eluted with mixture of MeOH and EtOAc in increasing order of

polarity yielded compound 10 (govanoside A, 32 mg, 20% MeOH in EtOAc)

[Scheme 2.6].


54

Scheme 2.6: Isolation of compounds from butanol fraction

Butanol fraction

(BuOH.fr)

(35 g)

BFBc BFBb

BFB

(Sub fraction)

BFBa


gradient elution

BFA

(Sub fraction)

Compound 9

(48 mg)

10% MeOH in EtOAc

5-10% MeOH in EtOAc

Compound 10

(32 mg)

20% MeOH in EtOAc

10% MeOH in EtOAc

20% MeOH in EtOAc

30% MeOH in EtOAc

(CC) gradient elution

(CC) with

gradient elution

(CC)

20% MeOH in EtOAc


55

2.12 Characterization of isolated compounds

2.12.1 Characterization of hexadecanoic acid (compound 1)

Compound 1 was isolated as white amorphous powder from the sub fraction, CFB(b) of

chloroform soluble fraction. The compound was characterized through modern

spectroscopic data analysis, and was confirmed as hexadecanoic acid.

Table 2.2: Characterization of hexadecanoic acid

Parameters Observations

Physical state white to colorless solid

Molecular formula C16H32O2

HR ESI-MS (m/z) 256.2361

UV activity UV inactive on TLC

Melting point 60-64oC

Isolated quantity 13 mg

Solubility at room temperature Chloroform/Methanol 1H-NMR (CDCl3; 600 MHz) (Table 3.4)

13C-NMR (CDCl3; 150 MHz) (Table 3.4)


56

2.12.2 Characterization of β-sitosterol (compound 2)

Compound 2 was isolated and purified as colorless amorphous powder from the

chloroform soluble sub fraction, CFB(b). The compound was identified and

characterized through modern spectroscopic data analysis and was confirmed as β-

sitosterol.

Table 2.3: Characterization of β-sitosterol


Physical state Colorless amorphous powder

Molecular formula C29H50O

HR ESI-MS (m/z) 414.3621




Solubility at room temperature Chloroform 1H-NMR (CDCl3; 600 MHz) (Table 3.5)

13C-NMR (CDCl3; 150MHz) (Table 3.5)


57

2.12.3 Characterization of stigmasterol (compound 3)



characterized through modern spectroscopic data analysis and was confirmed as

stigmasterol.

Table 2.4: Characterization of stigmasterol


Physical state Colorless amorphous powder

Molecular formula C29H48O

HR ESI-MS (m/z) 412.3624







58

2.12.4 Characterization of diosgenin (compound 4)

Compound 4 was isolated and purified as whit to off white needles/powder from the

chloroform soluble sub fraction, CFE. This compound was identified and

characterized through modern spectroscopic data analysis and was confirmed as

diosgenin.

Table 2.5: Characterization of diosgenin


Physical state White to off white needles/powder


HR ESI-MS (m/z) 414.3042

[α]26 D -124o (in MeOH)







59

2.12.5 Characterization of pennogenin (compound 5)

Compound 5 was isolated and purified as white to off white powder from the

chloroform soluble sub-fraction, CFE through column chromatography. The

compound was identified and characterized through modern spectroscopic data

analysis and was confirmed as pennogenin.

Table 2.6: Characterization of pennogenin


Physical state White powder


HR ESI-MS (m/z) 430.2960

[α]26 D -99.8o (in MeOH)







60

2.12.6 Characterization of govanic acid (compound 6)

Compound 6 was isolated and purified as white powder from the chloroform soluble

sub-fraction, CFE. The compound was identified and characterized as a new fatty acid

through modern spectroscopic data analysis and was given common name, govanic

acid.

Table 2.7: Characterization of govanic acid




HR ESI-MS (m/z) 330.4566

[α]26 D -52.8 (in MeOH)




Solubility at room temperature Methanol 1H-NMR (CD3OD; 600 MHz) (Table 3.9)

13C-NMR (CD3OD; 150 MHz) (Table 3.9)


61

2.12.7 Characterization of 20-hydroxyecdysone and 5,20-dihydroxyecdysone

(compounds 7 and 8)

The sub fraction, CFH obtained from CHCl3 soluble fraction was subjected to column

chromatography (CC) over silica gel using gradient solvent system (n-hexane /

EtOAc). The sub fraction (CFHh) eluted with EtOAc/MeOH (9.5:0.5v/v) solvent

system was subjected to preparative thin layer chromatography (TLC), using

EtOAc/MeOH (9:1) solvent system yield, 20-hydroxyecdysone (7) and 5,20

dihydroxyecdysone (8).

Table 2.8: Characterization of 20-hydroxyecdysone




HR ESI-MS (m/z) 480.5527

UV activity UV active on TLC






62

2.12.8 Characterization of 5, 20-hydroxyecdysone (compound 8)

Table 2.9: Characterization of 5,20-dihydroxyecdysone




HR ESI-MS (m/z) 496.5510

UV activity UV active on TLC






63

2.12.9 Characterization of borassoside E (compound 9)

Compound 9 was isolated and purified as white to off white amorphous powder from

butanol soluble sub-fraction, BFA. This compound was identified and characterized

through modern spectroscopic data analysis and was confirmed as steroidal glycoside

borassoside E.

Table 2.10: Characterization of borassoside E


Physical state White to off white amorphous powder


HR FAB+ (m/z) 869.4725

[α]26 D - 47.2o (in MeOH)







64

2.12.10 Characterization of govanoside A (compound 10)

Compound 10 was isolated and purified as white amorphous powder from the butanol

soluble sub fraction, BFBc. The compound was identified and characterized through

modern spectroscopic data analysis and was confirmed as a new spirostane steroidal

glycoside. The compound was given a name, govanoside A.

Table 2.11: Characterization of govanoside A


Physical state White amorphous powder


HR FAB+ (m/z) 1225.5426

[α]26 D -139o (in MeOH)







65

2.13 Biological studies

2.13.1 In vitro biological activities

The following in vitro biological activities were performed on Cr. MeOH-Ext, its

subsequent solvent soluble fractions and isolated compounds.

2.13.1.1 Antibacterial activity

The Cr. MeOH-Ext and its subsequent solvents soluble fractions of T. govanianum

rhizomes were screened for their antibacterial potential, against different gram

negative (E. coli, S. flexenari, P. aeruginosa and S. typhi) and gram positive bacteria

(B. subtilis and S. aureus), following agar well diffusion method89. Cr. MeOH-Ext or

subsequent solvent fraction (3 mg/mL) was dissolved in dimethyl sulfoxide (DMSO)

for the preparation of stock solution. Molten nutrient agar (approximately 45 mL) was

distributed in sterilized petri plates, and was permitted to harden. Bacterial culture

was dispersed on these nutrient agar plates by preparing sterile soft agar accumulating

100 µL of bacterial culture. Sterile metallic borer was used for well digging (6 mm

long) at suitable distance and spotted for identification. Sample (100 µL) was poured

into each well, and kept in incubator at 37 oC for 24 h. The antibacterial activity was

observed in the form of zone of inhibition (mm), and percent inhibition was

calculated. Standard antibacterial drug (broad spectrum antibacterial) used was

imipenem in the assay while DMSO was used as negative control.

2.13.1.2 Antifungal activity

Antifungal susceptibility testing of Cr. MeOH-Ext its subsequent solvent soluble

fractions and isolated compounds was performed with slight modification of

previously reported method90. Shortly, samples were serially diluted using 20%


66

dimethyl sulfoxide in 0.9% saline and transferred in duplicate to 96-well flat-bottom

microplates. Candida spp. inocula were prepared by picking 1 to 3 colonies from agar

plates and resuspending in ≈4 ml 0.9% sterile saline. The optical density at 630 nm of

the saline suspensions was compared to the 0.5 McFarland standards. The

microorganisms were diluted in broth (RPMI 1640 at pH 4.5) to afford final target

inocula of 5.0 × 103 for Candida spp. The Aspergillus spp. inocula were made by

carefully removing spores from agar slants, transferring to ≈ 4 ml 0.9 % saline, and

filtering through Miracloth (Merck Millipore, USA). The filtrate was diluted

appropriately in 5% Alamar blue (Life technologies, USA)-RPMI 1640 broth (at pH

7.3) to afford a final target inoculum of 4.0 ×104 CFU/mL. The fungal inocula were

added to the samples to achieve a final volume of 200 µL. Negative control (media

only) and positive control (amphotericin B) were included on each test plate. All

organisms were read at 630 nm using BioTek reader (Bio-Tek, USA) prior to and

after incubation (Candida spp. at 25°C for 18 to 24 h; Aspergillus spp. at 25°C for 72

h). The concentration range, used for determination of MIC was from 0.312 to 20

µg/mL. The MIC was defined as the lowest test concentration that allowed no

detectable growth in comparison to controls.

2.13.1.3 Antioxidant activity

DPPH free radical scavenging assay was used for in vitro antioxidant evaluation of

Cr. MeOH-Ext and its subsequent fractions following previously reported method91

with slight modifications. Two mL of 0.1 mM DPPH free radical solution in methanol

were added to 1 mL of different concentrations (1, 10, 30, 50, 100 and 200 µg/mL) of

the fractions or standards in methanol. The solutions were shaken thoroughly on a

vortex (Gyromixer, Pakland Scientific Production, Pakistan) and incubated in the dark


67

at ambient temperature for 30 min. Absorbance was then measured at 517 nm using

UV visible spectrophotometer (Lambda 25, PerkinElmer, USA) against control which

consisted of 0.1 mM DPPH free radical solution without extracts or standards. Blank

consisted of methanol alone. Ascorbic acid and butylated hydroxytoluene (BHT) were

used as standards antioxidants. The percent DPPH free radical scavenging was

calculated using the following formula;

PercentDPPH = AI − AII

AI× 100

AI = absorbance of the reaction (control)

AII = absorbance of the sample.

2.13.1.4 Anticancer activity

The cytotoxic activity of Cr. MeOH-Ext its subsequent fractions and isolated

compounds was determined by the MTT assay, according to previously reported

method92,93 on two cancer cell lines, i.e. HeLa (cervical cancer cells) and PC-3

(prostate cancer cells). For MTT assay, cells were grown in DMEM (Dulbecco’s

modified Eagle medium) and MEM (modified Eagle’s medium) containing 10% FBS

and 2% antibiotic (penicillin and streptomycin) and maintained at 37°C, in 5 % CO2,

for 24 h, in a flask. Cells were plated (1 × 105 cell/mL) in 96-well flat bottom plates

and incubated for 24 h for cell attachment. Various concentrations of test

sample/fractions ranging from 1.25-20 µM were added into the well and incubated for

48 h. A 50 µL MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide;

0.5 mg/mL] aliquot was added to each well 4 h before the end of incubation. Medium

and reagents were aspirated and 100 µL DMSO was added and mixed thoroughly for

15 min to dissolve the formazan crystals. The absorbance was measured at 570 nm


68

using a microplate reader (Spectra Max 340; Molecular Devices, CA, USA). Finally,

IC50 values were calculated. For positive control, doxorubicin was used.

2.13.1.5 Anti-inflammatory activity

The in vitro anti inflammatory potential of Cr. MeOH-Ext, its subsequent fractions

and isolated compounds was determined through Luminol-enhanced

chemiluminescence assay following well define protocol previously reported94.

Briefly, 25 µL of the diluted whole blood HBSS++ (Hanks-Balanced Salt Solution,

containing standard amount of magnesium and calcium chloride) and samples (25 µL)

with different dilutions (1, 10 and 100 µg/mL) were incubated in triplicate. Only cells

and HBSS++ were added to control wells, while HBSS++, cells and testing samples

were added to other wells. The procedure was carried-out in 96-well plate (white-half

area), incubated (for 15 min.) at 37ºC in thermostatic chamber of luminometer. On

completion of incubation, 25 µL each SOZ (serum opsonized-zymosan) and intra-

cellular reactive oxygen species (ROS), detecting probe, (luminal) were supplemented

to well containing testing samples. The intensity of ROS was obtained by mean of

relative light units (RLU) in luminometer. The standard drug ibuprofen was used as

positive control.

2.13.1.6 Anti leishmanial activity

The Cr. MeOH-Ext and its fractions were investigated for leishmanicidal potential

against leishmania major, using previously reported protocol95,96. Promastigotes

of leishmania were cultured in Roswell Park Memorial Institute (RPMI) medium,

augmented with 10% thermally inactivated fetal bovine serum. At log-phase of

growth promastigotes were centrifuged (2000 rpm) for 10 min, maintaining the same


69

experimental conditions and were washed (three times) with saline. Fresh culture

medium was used to obtain parasites final density by dilution (1×106 cells mL-1).

Medium was added to different 96 wells micro titer-plate, tested samples (20 mL) was

diluted serially by adding medium. Parasite culture (100 mL) was added to each well.

First two rows were specified for controls (medium served as negative control, while

Amphotericin B was used as positive control). Loaded plates were incubated (22-

25°C) for consecutive 72 h. Parasites were counted using on an improved Neubaure’s

chambers. The IC50 of tested samples were determined through Windows operating

Ezfit 5.03 Perella Scientific software. The assay was performed as triplicate.

2.13.1.7 Brine shrimp cytotoxicity

In this bioassay technique artificial sea water was taken in a Jar, brine-shrimp eggs

(Artemia salina; 1 mg) was added to it and cover the Jar by aluminum foil, to darken

it. The Jar was kept at 25oC for 24 h, resulted in hatching ample of larvae. Test sample

(20 mg) was liquefied in 2 mL chloroform (10 mg/mL) to prepare stock solution.

From the stock solution, various concentration (10, 100 and 1000 µg/mL) were

prepared. The DMSO was used for the dilution of each concentration and then sea

water (5 mL) was poured to each vial containing ten brine shrimps and kept for 24 h.

For positive control, the drug etoposide was used. The percent mortality was

calculated for tested groups as well as for positive control97.

2.13.1.8 Insecticidal activity

The insecticidal potential of crude extract methanolic extract and subsequent fractions

were determined against Tribolium castaneum and Rhyzopertha dominica. For the

assay first stock solution was prepared by dissolving test sample (200 mg) in acetone


70

(3 mL). A 90 mm filter paper was positioned in petri dishes and loaded with test

sample (1019.10 µg/cm2). In order to evaporate the volatile organic solvent the petri

dishes was left for 24 h. Ten active insects were transferred to each petri dish next

day, and incubated at 27 ± 1oC for 24 h. Permethrin (239.50 µg/cm2) and acetone were

used as positive control and negative control respectively. By comparison results of

test sample with positive control percent mortality was calculated95,96,98.

By using the following formula percentage mortality was determined.

Percentmotality = 100 −Numberoflivinginsectsintest

Numberoflivinginsectsincontrol× 100

2.13.1.9 Protein antiglycation activity

For the in vitro antiglycation assay, previously reported method99,100 was used to

determine the antiglycation potential of Cr. MeOH-Ext, fractions and isolated

compounds. The fructose mediated production of fluorescent AGEs on Human Serum

Albumin (HSA) assay was employed with slight modifications. Test samples were

dissolved in absolute DMSO at 1 mM Concentration. HSA was employed as the

model protein to be glycated at 10 mg/mL concentration with 0.5 M fructose as

glycating agent. Test samples were incubated in triplicates on 96-well plate at various

concentrations with 10 mg/mL HSA, 0.5 M fructose, 0.1 M phosphate buffer (pH 7.4)

containing 0.1 M sodium azide as bactericidal agent and incubated at 37ºC for 7 days.

HSA, fructose, and phosphate buffer were incubated with the same concentration for

positive control and conditions with absolute DMSO. After 7 days of incubation, the

96-well plate was observed for fluorescence at wavelength of 330-440 nm on

microtitre plate spectrophotometer (Spectra Max M2, Molecular Devices, USA).


71

Rutin was used for positive control. The percent inhibition values were calculated by

the following formula;

PercentInhibition = 1 −"luorescenceoftestsample

"luorescenceofthecontrolgroup× 100

The samples that exhibit 50% or above percent inhibition, were processed for IC50

value calculation by using Ez-fit software (Perrella Scientific, USA).

2.13.1.10 Smooth muscle relaxant activity

The muscle relaxant (spasmolytic) potential of Cr. MeOH-Ext was studied on isolated

rabbit jejunum preparations in according to the previously reported protocol101. In an

organ bath filled with Tyrode’-s solution (37°C) and aerated with natural air, rabbit

jejunum (1-1.5 cm) was suspended. Intestinal contractions were recorded with the

help of isometric transducer attached with Power-lab Data Acquisition System

connected to computer executing Lab-chart software. The tissue was equilibrated for

30 min before tricking with any chemical. Suspended tissue was made stabilized by

subsequent exposure to acetylcholine (0.3 µM) solution, following washing

thoroughly with Tyrode’s solution, until responses (sub--[maximal) of even magnitude

were achieved. The pragmatic tone of impulsive rhythmic contraction was used to

test muscle relaxant (anti-spasmodic) potential in isolated rabbit jejunum tissue.

For the study of Ca++ channel blocking (CCB) effect, previously reported method was

followed with slight modification27. In this analysis high potassium (K+, 80 mM) was

implicated to depolarize the tissue. Testing sample was added in cumulative manner

(on achieving induced contraction plateau) to observe dose-reliant inhibitory

contractions. To validate the Ca++ antagonistic activity of the testing sample, the


72

suspended tissue was stabilized in Tyrode’s[solution, later on the solution was

substituted with another solution (similar to Tyrode’s solution, instead of

Ca++ containing EDTA 0.1 mM) to deprive the tissue from Ca++ for 30 min). The

Ca++free solution was superseded with another solution [containing (mM): KCl, 50;

NaCl, 91.03; NaHCO3, 11.9; EDTA-Na2.2H2O, 0.1; glucose, 5.05; NaH2PO4.2H2O,

0.32 and MgCl2.6H2O, 0.50]. By incubating (30 min) at same temperature, CRCs

(control concentration reaction curves) of Ca++ were observed. Constructing, control

CRCs for Ca ++, the suspended tissue was re-treated with test sample for a period of 1

h. The Ca++ CRCs were plotted in the existence of variable concentration of the

sample to monitor the Ca ++antagonist potential.

2.13.1.11 β-Glucoronidase inhibitory activity

The Cr. MeOH-Ext, fractions and isolated compounds were screened for β-

glucuronidase inhibition. The previously reported assay102 was followed while using p

nitrophenyl β-D-glucuronide as substrate. The enzyme mixture (total volume 250 mL)

contained 50 mL of p-nitrophenyl glucuronide, 190 mL of acetate buffer, 5 mL

enzyme and 5 mL of inhibitor. The assay mixture was incubated at 37oC for 40 min,

the reaction was stopped by the addition of 50 mL of 0.2 M Na2CO3, and the

absorbance was measured at 405 nm. D saccharic acid-1,4-lactone was used as a

standard inhibitor. The percent inhibitory activity (%) was calculated using the

following formula;

Percentinhibition = E − S

E× 100

Where ‘‘E’’ is the activity of enzyme without test material and ‘‘S’’ is the activity of

enzyme with test material.


73

2.13.1.12 α-Chymotrypsin inhibitory activity

The Cr. MeOH-Ext, fractions and isolated compounds were tested for enzyme α-

chymotrypsin inhibition following reported protocol103. For the assay enzyme

chymotrypsin (12 units/mL) prepared in Tris–HCl buffer (pH 7.6) was pre incubated

with test samples (prepared in final concentration of 7% DMSO) at 30°C for 25 min.

The substrate, N-succinyl-phenylalanine-p-nitroanilide (0.4 mM, final) was added to

start the enzyme reaction. The absorbance of released p-nitroaniline was constantly

monitored at 410 nm until a significant color change was observed using a microplate

reader and SoftMax Pro software (Molecular Device, CA, USA). Chymostatin was

used as the standard inhibitor.

The percent inhibition was calculated as,

Percentinhibition = 100 −ODoftestSample

ODoftheControl× 100

The samples that exhibit 50% or above percent inhibition, were processed for IC50

value calculation by using Ez-fit software (Perrella Scientific, USA).

2.13.1.13 Thymidine phosphorylase inhibitory activity

The Cr. MeOH-Ext and its fractions of T. govanianum were tested for enzyme

thymidine phosphorylase inhibition. The assay was performed as previously reported

method104. TP/PD-ECGF (E. coli, thymidine phosphorylase (Sigma T6632) activity

was calculated by measuring the absorbance at 290 nm spectro photometrically.

Shortly, total reaction mixture of 200 µL containing 145 µL of potassium phosphate

buffer (pH 7.4), 30 µl of enzyme (E. coli thymidine phosphorylase (Sigma T6632) at

concentration 0.05 and 0.002 U, respectively, were incubated with 5 µL of test


74

materials for 10 min at 25oC in microplate reader. After incubation, pre reading at 290

nm was taken to deduce the absorbance of substrate particles. Substrate (20 µL, 1.5

mM) dissolved in potassium phosphate buffer was immediately added to plate and

continuously read after 10, 20, and 30 min in microplate reader. 7-Deazaxanthine was

used as the positive control.

2.13.1.14 Acetylcholinesterase inhibitory activity

The Cr. MeOH-Ext and fractions of T. govanianum rhizomes were tested for acetyl

cholineesterase (AChE) inhibitory potential. The assay was carried out according to

the previously reported protocol105,106. The reaction mixture contain 50 mM Tris-Hcl

with pH 8.0, (200 µl), BSA buffer (1%), test sample (100 µL) keeping final

concentration at 100 µg/mL. The method based on the hydrolysis of acetyl thiocholine

iodide by the respective enzymes and the formation of 5-thio-2-nitrobenzoate anion

followed by complexation with DTNB to give yellow color compound, which is then

detected with spectrophotometer. The yellow color was measured at 405 nm after 4

min. Galantamine (final conc. 100 µg/mL) was used as positive control. The AChE

percent inhibition was calculated by below given formula;

PercentAChEinhibition = A − B

A× 100

Where A represent change in absorbance without test sample, while B represent

change in the absorbance with test sample.


75

2.13.2 In vivo biological studies

The Cr. MeOH-Ext and its subsequent solvent soluble fractions of T. govanianum

rhizomes were evaluated for various in vivo biological activities. The detailed

procedures for the in vivo biological activities are described below.

2.13.2.1 Experimental animals

BALB/c mice of either sex (25-35 g) used were acclimatized at 25 ± 2°C under a 12 h

dark/light cycle for ten days. Clean and properly dried food was given to the mice and

the water was changed on daily basis. The experimental protocols for this study were

approved by the Ethical Committee of the Department of Pharmacy, University of

Peshawar, Pakistan.

2.13.2.2 Acute toxicity test

The acute toxicity test was carried out to determine the lethal and non lethal doses of

the Cr. MeOH-Ext of T. govanianum rhizomes. The experimental animals (mice)

were divided into six groups, each containing six animals. The extract was

administered in doses of 250, 500, 1000, 1500, 3000 and 6000 mg/kg body weight

(p.o.). The control animals received an equal volume of saline. The mortality rate was

measured 24 h post drug administration107.

2.13.2.3 Anti-inflammatory activity

The anti-inflammatory activity of Cr. MeOH-Ext and subsequent fractions was

performed on mice of either sex (25-35 g) following carrageen induced paw edema

protocol previously reported108,109. The animals were randomly divided in five groups

each comprises of six animals. Group I was treated with normal saline (10 ml/kg)


76

negative control, group II was treated with diclofenac sodium (10 mg/kg) positive

control, the remaining groups (III, IV and V) were treated with T. govanianum

rhizomes Cr. MeOH-Ext (50, 100, and 200 mg/kg, orally) and fractions (25, 50, and

200 mg/kg, orally). After thirty min of administration, carrageenan (1%, 0.05 mL)

was injected subcutaneously in the sub plantar tissue of the right hind paw of each

mouse. For the measurement of inflammation plethysmometer (model; LE 7500 plan

lab S.L) was used, directly after injection of carrageenan and then after an intervals of

1, 2, 3, 4 and 5h. The average paw swelling in samples treated animals as well as

standard was compared with that of control, and the percent inhibition of edema was

determined using the following formula;

Percentinhibition = A − B

A× 100

Where, "A" represent paw edema volume of control and "B" as paw edema volume of

tested group.

2.13.2.4 Analgesic activity

2.13.2.4.1 Tonic-visceral chemical induced nociception test

For tonic visceral chemical induced nociception, acetic acid induced abdominal

constriction assay was performed for elucidating the peripheral antinociceptive effect

of T. govanianum rhizomes110. The animals were withdrawn from food 2 h before the

start of experiment. All the extract and fractions of T. govanianum rhizomes were

administered orally through an oral gavage tube at doses of 50 and 100 mg/kg.

Diclofenac sodium was used as standard and was orally administered at a dose of 50

mg/kg. After 1 h of treatment, all animals were injected with 1% acetic acid, (i.p.).


77

The number of writhes was counted after 5 min of acetic acid injection and was

continued for 20 min.

2.13.2.4.2 Hot plate test

The central analgesic effect of T. govanianum rhizomes was evaluated by the hot plate

method111. Animals were withdrawn from food 2 h before the start of experiment. All

animals were screened for pre test latency and only those animals having a pre test

latency of <15 second (sec) were selected for the experiment. A cut off time of 30 sec

was set to avoid thermal injury. All the extract and fractions of the rhizomes were

administered orally through an oral gavage tube at doses of 50 and 100 mg/kg.

Tramadol was used as standard and was administered orally at a dose of 30 mg/kg.

After 1 h of extract and 30 min of standard administration, the latency time was

measured at 30, 60, 90 and 120 min using a hot plate (Havard apparatus) maintained

at 54 ± 0.1°C.

Chapter 3 Results and Discussion

78

3. Results and Discussion

3.1 Ethnomedicinal studies

In this study, regarding the medicinal uses of T. govanianum rhizomes, information

was collected from people of four districts of Khyber Pakhtunkhwa. Informants

included plant collectors, local drug sellers, Hakims and local elders having drug

knowledge (Fig. 3.1). From ethnomedicinal survey, it was found that this plant is

abundantly available in District Upper Dir (Kohistan) and District Swat (Kohistan and

mountainous areas) of Khyber Pakhtunkhwa in comparison to District Shangla and

Buner. Furthermore, during field survey, it was observed that a large number of local

people were involved in digging and collection of this plant species for commercial

sale and earning purposes. Majority of the informants in these areas were unaware of

the uses of rhizomes. They were engaged only in the collection and marketing of the

rhizomes as their earning source. Only a limited number (<17% in any category) of

informants knew about the uses of rhizomes (Table 3.1). The Hakims and local

elderly people of District Dir and Swat confirmed the medicinal uses of the rhizomes

in the treatment of cancer, GI disorders, sexual disorders, backache, kidney problems

and as vermicide. The percent information of informants regarding the uses of

rhizomes were higher in district Dir followed by district Swat in comparison to

district Shangla and Buner.

The ethnomedicinal uses of this plant as reported by the informants from the four

districts indicate that highest presumed indication is inflammatory disorders including

backache, headache, general inflammation, joint pains and kidney problems (with

highest 21.6% and 14.7% informants from Dir and Swat having a consensus at this

use) followed by anti-cancer use (15% and 12.8% from Swat and Dir respectively at


79

this use). In case of other indications, applications in infections (16.8% from Swat and

13.4% from Dir); GI disorders (14.7% from Swat and 10.4% from Dir); and sexual

disorders (9.2% from Dir and 7.3% from Swat) came to picture. From this survey, an

interesting finding was the response from people of Swat who appeared to have more

information regarding the uses of this plant followed by the people of district Dir.

This probably is due to the higher educational level in these two districts in

comparison to Shangla and Buner districts. Moreover, highest numbers of informants

(124) were from Swat followed by Dir (81), Shangla (39) and Buner (9) that shows

the level of understanding in these districts (Fig. 3.1). It was also evident from the

survey that local elders were having appreciable information on the plant use, and that

is shared and transferred to other people. These presumed uses are in confirmation to

some recent reports of plants of genus Trillium that have reported impact in sexual

disorders71, skin infections112, infections other than skin infections72,113, as

anthelmintic114,115, and other inflammatory disorders73. However, the use in cancer

needs to be sifted scientifically and if found to have an impact will be of great

significance in cancer treatment research and thus will serve humanity and will also

be a source of great earning for the people associated with the collection and

processing of this plant as well as will generate revenue for our country. However,

scientific conservation of this plant is needed as over collection may endanger this

therapeutically precious plant.

The ethnomedicinal study enables researchers to work with common population to

investigate knowledge based on experiences of ages116. Moreover, the indigenous

plants which is particularly medicinal species even in this modern era, play a key role

in the socioeconomic strengthening of the rural areas, and a variety of locally

produced medicines are still commonly used as household remedies for treating


80

different aliments117. If this medicinal herb is processed, commercialized and sold in

such a way that no conservation strategy is adopted, there is chance of extinction of

this herb from these areas. Therefore, it is necessary for the concerned authorities and

the government to prepare a conservation strategy to safeguard this valuable asset of

this region. There is also need for creating awareness among the local people

regarding the propagation and cultivation methods in order to conserve this valuable

medicinal herb.


81

Figure 3.1: Informants for the ethnomedicinal uses of T. govanianum rhizomes

from different districts of Khyber Pakhtunkhwa

15

24

10

32

81

42

31

21

30

124

10

4

4

21

39

0

1

3

5

9

Plant collectors

Local drug sellers

Hakims (Traditional healers)

Local elderly people

Total

Informants for the ethnomedicinal uses of T. govanianum

rhizome from different districts of Khyber Pakhtunkhwa

Buner Shangla Swat Dir


82

Table 3.1: Informants and therapeutic uses of T. govanianum rhizomes in different Districts of Khyber Pakhtunkhwa

Therapeutic Uses Dir (U)

(%)

Swat

(%)

Shangla

(%)

Buner

(%)

Reported References

Cancer 12.8 15.0 3.8 0.5 - Sexual disorders (Erectile dysfunction; Sexual tonic)

9.2 7.3 1.9 1.2 71

GI Disorders (Abdominal spasms) 10.4 14.7 1.7 - - Skin Infections 6.2 11.1 2.1 - 112 Infectious diseases (Healing of wounds, antiseptic, bacterial diarrhea, dysentry)

13.4 16.8 - 2.1 72,113

Anthelmintic 15.3 7.2 3.4 - 114,115 Others (backache; fever; inflammation; headache; kidney problems)

14.7 21.6 4.4 - 73

Plant Information - Local name Matarzela Matajarra Matajarai Matajarra - Plant parts used Rhizome Rhizome Rhizome Rhizome - Availability Abundant Abundant Rare Rare -


83

3.2 Morphological studies

3.2.1 Macroscopic features

The macroscopic findings of rhizome can serve as diagnostic parameters. The

collected rhizomes were observed grayish to brown in color (Fig. 3.2a and b) while

their internal matrix was slightly whitish in color. The external surface was rough

having striation and fractures. The pieces were 3 to 5 cm long and up to 0.8 to 1.5 cm

thick slightly curved and twisted. The dried powder was slightly whitish in color

having bitter taste and pungent odor.

Figure 3.2a: Trillium govanianum plant.

Figure 3.2b: T. govanianum rhizomes.

3.2.2 Microscopic features

In the current scientific era, although modern and sensitive techniques for evaluation

of the plant drugs are available but still microscopic examination methods are one of


the simplest and economic ways for correct identification of the source materials118.

The transverse section of rhizomes (Fig. 3.3a and b) showed presence of cortex cells,

trichomes, carinal canal, sclereids, vascular bundles (xylem and phloem), fibers,

cambium, calcium oxalate crystals and starch grains. Calcium oxalate crystals were

abundant in rhizome. These histological and morphological studies of the rhizome are

key in rapid identification of T. govanianum rhizome.

Figure 3.3a: Transverse section of T. govanianum rhizome.

Vascular bundles (Phloem)

Cortex cells

Cambium Carinal


85

Figure 3.3b: Transverse section of T. govanianum rhizome.

3.3 Physicochemical studies

In physicochemical studies, different physicochemical parameters were analyzed. The

extractive values are helpful to assess the chemical constituents present in the crude

drugs, and also help in assessment of definite constituents, soluble in a particular

solvent118,119. Ash values of a drug provide an insight into the earthy matter, inorganic

composition and other impurities present along with the crude drug. With respect to

physicochemical parameters obtained from this study, total ash value was determined

to be 12.5%, water soluble ash 4.0%, acid soluble ash 2.4% and acid insoluble ash

0.8% w/w (Fig. 3.4).

Calcium oxalate crystals

Starch grains

Trichomes

Xylem

Sclereids

Fibers


86

Loss on drying of powder rhizomes was 14.8%. Ultimate dryness is not necessary for

the drug, and majority of the drugs contain some percent of moisture contents, but

higher moisture can result in spoilage by microorganisms especially the fungi, and

also chemical reactions such as hydrolysis and oxidation can deteriorate crude

drugs76. Thus it is key element in drug preparation to know the rate and condition at

which moisture is removed. The loss on drying observed was 14.8% w/w, which

shows high proportion of moisture, and it can be assumed that the powder drug has

high moisture content, and it is also likely, that it is highly hygroscopic.

Extractive values (Fig. 3.4) were high for solvents like water (21.5%) and methanol

(18.75%) as compared to non-polar solvents, which is an indicative of abundance of

sugars, and other polar compounds like glycosides, saponins, flavonoids and steroidal

glycosides.


87

Figure 3.4: Physicochemical parameters of T. govanianum rhizomes.

14.8

12.5

4

2.4

0.8

21.5

18.75

13.62

7.1

2.25

5.8

1.2

Loss on drying

Total ash

Water soluble ash

Acid soluble ash

Acid insoluble ash

Water soluble

Methanol soluble

Ethanol soluble

Butanol soluble

Ethyl acetate soluble

Chloroform soluble

n-hexanes soluble

Ash

val

ues

Ext

ract

ive

valu

esPhysicochemical parameters of T. govanianum rhizome

Percentage value (W/W %)


88

3.4 Phytochemical studies

3.4.1 Qualitative phytochemical screening

The preliminary (qualitative) phytochemical tests of T. govanianum rhizomes

revealed the presence of secondary metabolites like steroids, glycosides and saponins

(Table 3.2), and these metabolites have been previously reported in the genus

Trillium44,120 , which includes species traditionally used in the treatment of different

diseases by virtue of these phytochemicals121-123.


89

Table 3.2: Preliminary phytochemical profile of T. govanianum rhizomes

Phytochemical Qualitative test Cr. MeOH-Ext n-Hex-fr CHL-fr EtOAc-fr BuOH-fr

Alkaloids Mayer’s test - - - - -

Wagner’s test - - - - -

Glycosides Keller Killiani test + - + + +

Tannins Ferric chloride test + - + - +

Lead acetate test + - - - +

Flavonoids Ferric chloride test + - + - +

Sodium hydroxide test + - + + -

Carbohydrates Molisch’s test + - + + +

Sterols Liebermann-Burchard test + + + + +

Salkowski’s test + + + + +

Saponins Frothing test + - + + +

+ indicates the presence of phytochemicals


90

3.4.2 GCMS analysis of n-hexane fraction

The proximate fatty acid composition of n-Hex-Fr was carried out by GC/MS

analysis. Twelve compounds were identified by comparison of GC/MS spectra with

the mass library (NIST based AMDIS), as shown in Table 3.3. Unsaturated fatty

acids (70%) were more abundant than saturated fatty acids (30%). Among the

unsaturated fatty acids, high levels of 9,12-octadecadienoic acid methyl ester

(C19H34O2), pentanoic acid-5-hydroxy-2,4-di-t-butylphenyl ester (C19H30O3), 9-

hexadecenoic acid methyl ester (C17H32O2) and cis-13-eicosenoic acid (C20H38O2)

were detected, whereas 2-methyl hexadecanoic acid methyl ester (C18H36O2) and ethyl

13-methyl tetradecanoate (C17H34O2) represented the saturated fatty acids present at

higher concentrations.

GC/MS analysis of n-Hex-fr showed the presence of saturated and unsaturated fatty

acids, and thus n-Hex-fr represents the biologically active compounds with relevant

antibacterial, antifungal and anticancer activities124,125. Therefore the presence of

these fatty acids in T. govanianum rhizomes supports its potential uses as an

antimicrobial and anticancer agent.


91

Table 3.3: Chemical composition of n-Hex-fr of T. govanianum rhizomes

*In bold, saturated fatty acids

No Chemical Name Formula Molecular

Weight

Retention time

(min)

Abundance

(%)

1 2,4-Decadienal C10H16O 152 18.11 2.07 2 Pentanoic acid-5-hydroxy-2,4-di-t-butylphenyl ester C19H30O3 306 23.2 7.83 3 Ethyl 13-methyl-tetradecanoate C17H34O2 270 33.59 6.53 4 Hexadecanoic acid methyl ester C17H34O2 270 34.35 7.19 5 9-Hexadecenoic acid methyl ester C17H32O2 268 35.15 9.65 6 2-Methyl hexadecanoic acid methyl ester C18H36O2 284 35.58 15.00 7 9,12-Octadecadienoic acid methyl ester C19H34O2 294 36.25 12.50 8 9,12-Octadecadienoic acid ethyl ester C20H36O2 308 37.58 3.98 9 9,12-Hexadecadienoic acid methyl ester C17H30O2 266 38.03 13.86 10 9-Octadecanoic acid methyl ester C19H36O2 296 36.22 2.50 11 cis-13-Eicosenoic acid C20H38O2 310 39.42 7.27 12 9,12-Octadecadienoic acid-2-hydroxy-1-(hydroxy methyl) ethyl ester C21H38O4 354 43.25 4.53


92

3.4.3 Isolation of compounds

3.4.3.1 Structure-elucidation of compound 1

Compound 1 was isolated as white amorphous powder from the sub fraction, CFB(b) of

chloroform soluble fraction. The molecular ion peak at m/z 256 in EI-MS spectrum

was used to calculate its molecular formula as C16H32O2, supported by its HREI-MS

(C16H32O2, 256.2404). The other major characteristic peaks were observed at m/z 85,

71 and 57. The successive methylenic losses observed for compound 1, was as a

characteristic pattern for straight chain fatty acids. The IR spectrum indicated the

presence of acid functionality by the strong absorption at 3420 cm-1 along with

carbonyl absorption at 1680 cm-1.

The 1H-NMR (CDCl3, 600 MHz) spectrum helped in the assigning of chemical shifts

values to almost all the protons. The terminal methyl proton resonated at δ 0.87 as

triplet (J = 8.1 Hz) while the C-2 methylene protons also appeared as triplet-at δ 2.26

(J = 7.1 Hz). The other methylenic. protons appeared as large multiplet between δ

1.31-1.50. The 13C-NMR (CDCl3, 150 MHz) exhibited signals for almost all the

carbon atoms, including one methyl, one quaternary and fourteen methylene (Table

3.4). The acidic carbon atom resonated at δ 178.8 (C-1) while the terminal methyl

carbon resonated at δ 14.3 (C-16). The physical and spectral data of compound 1 was

in close resemblance to that of a known compound, hexadecanoic acid previously

reported126. Thus the compound 1 was characterized as hexadecanoic acid.


93

Table 3.4: 1H-NMR and 13C-NMR (CDCl3, 600 and 150 MHz) chemical shift

assignments in compound 1

C No. δ C δ H (J, Hz)

1 2 3-15 16

178.8 34.4 24.5 – 31.9 14.3

- 2.26, t (7.1) 1.31-1.50 br, m 0.87, t (8.1)

Figure 3.5: Chemical structure of compound 1.


94


Compound- 2 was isolated and purified as colorless amorphous powder from the

chloroform. soluble sub fraction, CFB(b). Its EI-MS spectrum exhibited molecular ion

peak at m/z 414, corresponded to the molecular- formula, C29H50O (calcd; 414.3892) in

HREI-MS. The other major fragments peaks observed at m/z 399, 396, 380, and 303

representing a β-sitosterol nucleus. The. IR[ spectrum revealed strong absorption at

3450 and 1625 cm-1 for hydroxy and olefinic functionalities. The 1H-NMR spectrum

(600 MHz, CDCl3) revealed characteristic peaks for steroidal nucleus. The two

tertiary methyl group protons (CH3 -18 and CH3-19) resonated as singlets at δ 0.61

and 0.91, respectively. The olefinic proton (H-6) was observed as multiplet at δ 5.30

while the chemical shift of sole H-3α proton was observed at δ 3.34 (J = 4.0 and 9

Hz) confirming the presence of a 3β-hydroxyl functionality at position 3 in ring A.

The 13C-NMR (150 MHz, CDCl3) spectrum revealed the presence of all the twenty

nine carbon atom signals as three quaternary, nine methine, six methyl and eleven

methylene carbons (Table 3.5). Signals for the methyl carbons were appeared at δ

13.4 (C-18), 19.7 (C-19.), 19.1 (C-21.), 19.3 (C-26[), 19.3 (C-27.) and.11.9 (C-29) while

chemical shift values for olefinic carbons were reported at δ 140.3 (C-5) and 121.1

(C-6), respectively. The physical and spectral data of compound 2 was an agreement

with the reported data for known compound β sitosterol127. Therefore, compound 2

was characterized as β-sitosterol.


95



C No. δC δ H (J, Hz) C No. δC δ H (J, Hz)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

37.4 31.8 71.1 41.3 140.3 121.1 32.0 31.5 50.7 36.9 21.4 40.0 42.5 56.7 25.34

1.34, 1.14 m 1.55, 1.25 m 3.34 m 2.24 dd (7.1, 6.8) - 5.30 d (5.3) 2.04, 1.78 m 1.42 m 1.40 m - 1.46 m 1.5, 1.32 m - 1.40 m 1.63, 1.36 m

16 17 18 19 20 21 22 23 24 25 26 27 28 29

26.5 56.4 13.4 19.7 36.1 19.1 34.0 33.8 45.8 27.2 19.3 19.3 23.1 11.9

1.60, 1.34 m 1.46 m 0.61 s 0.90 s 1.56 m 0.79 d (6.5) 1.32 m 1.36 m 1.52 m 1.81 m 0.82 d (6.5) 0.80 d (6.5) 1.58 m 0.75 t (7.0)



96

3.4.3.3 Structure elucidation of compound 3



characterized through modern spectroscopic methods and comparison with available

literature. The EI mass- spectrum displayed molecular ion peak at m/z 412 [M+] which

was in agreement with molecular-formula C29H48O (calcd; 412.3689). The mass

fragmentation represents characteristics peaks of steroidal nucleus at m/z 55.0, 314,

351, 300, 229, 271 and 213. The IR spectrum showed the strong absorptions at 3329

(hydroxyl group) and 1630 cm-1 (cyclo-alkene).

The 1H-NMR, (CDCl3, 600 MHz) spectrum showed a strong multiplet at δ 3.39

assigned to H-3 proton. The H-22 and 23 protons showed chemical shift values at δ

5.23 (m) and 5.26 (m), respectively. The H-6 protons appeared doublet at δ 121.7

suggesting double bonds in the molecule. The methyl groups protons of H-18, 19, 21,

26, 27 and 29 resonated at δ 1.06, 1.29, 1.12, 0.92, 0.92 and 0.9, respectively (Table

3.6).

The 13C-NMR (CDCl3, 150 MHz) spectrum exhibited signals for all the twenty nine

carbon atoms (Table 3.6). The hydroxy carbon (C-3) resonated δ 71.7 while the

olefinic carbons at C-5, 6 , 22 and 23 appeared at δ 141.5 and 121.7, 138.2 and 129.2,

respectively. All the physical and spectral data showed close resemblance with that

for a known compound, stigmasterol128.


97




1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

36.9 31.3 71.7 42.2 141.5 121.7 32.0 36.0 50.7 37.49 21.6 38.4 42.1 57.3 24.5

1.33, 1.16 m 1.57,1.24 m 3.39 m 2.24 dd (7.1, 6.8) - 5.30 d (5.2) 2.04, 1.72 m 1.45 m 1.40 m - 1.45 m 1.60, 1.35 m - 1.45 m 1.61, 1.35 m

16 17 18 19 20 21 22 23 24 25 26 27 28 29

30.0 54.5 12.0 20.3 33.8 22.1 138.2 129.2 47.6 32.1 22.4 20.1 25.3 12.0

1.56, 1.38, m 1.51 m 1.06 s 1.29 s 1.54 m 1.12 d (6.5) 5.23 m 5.26 m 1.50 mm 1.79 m 0.92 d (6.5) 0.92 d (6.4) 1.60 m 0.93 t (7.0)



98


Compound 4 was isolated and purified as whit to off white needles/powder from the

chloroform soluble sub fraction, CFE. The molecular ion peak was observed at m/z

414.3012, corresponding to the molecular formula of C27H42O3 (calcd; 414.3134) in

HR-EIMS. The molecular ion peak was also supported by positive FAB-MS

spectrum, showed [M+H]+ at m/z 415. The IR spectrum afforded strong absorption at

3450 cm–1 for a hydroxyl group, at 2970 cm–1 for CH3 stretching, at 1600 cm–1 for a

vinylic group and at 1050 cm–1 for a carboxyl group.

The 1H-NMR (CDCl3, 600 MHz) spectrum revealed signals for almost all the protons,

a multiplet was observed at δ 3.57 for the methine proton (H-3) followed by the

double doublet at δ 2.26 (2H, H-4, J = 7.2 and 6.4 Hz) (Table 3.7). A strong doublet

at δ 5.33 was assigned to the olefinic proton (H-6, J = 5.2 Hz), indicative of the sole

double bond in the steroidal skeleton. Furthermore, the methine protons H-16 and H-

17 resonated- at δ 4.38 (1H, q, J. = 15.60Hz) and 1.80 (1H, dd, J = 8.8, 6.00Hz). The

two tertiary methyl group protons appeared as singlets at δ 0.80 (3H, H-18) and 1.04

(3H, H-19) while the two secondary methyl group protons appeared as δ 0.79 (3H, d,

J = 4.2 Hz, H-21) and 0.78 (0.78, d, J = 6.1 Hz, H-27), respectively.

The 13C-NMR and DEPT spectra (CDCl3, 150 MHz) afforded twenty seven peaks for

all carbon atoms i.e, four [methyl, ten methylene, nine methine and four quaternary

(Table 3.7). The methyl-carbons resonated at δ 16.3 (C-18), 19.4 (C-19), 14.5 (C-21)

and 17.1 (C-27) respectively. The spectrum also exhibited characteristic signals for

three carbons at δ 140.8 (C-5), 121.4 (C-6) and 109.3 (C-22), diagnostic for 5-

spirostane type sapogenin129.


99

Chemical shift for β-hydroxyl group carbon atom was observed at δ 71.7 (C-3). All

the other carbon atoms chemical shift values as well as 2D-NMR correlations showed

resemblance with the reported values for a known compound, diosgenin130,131, thus

compound 4 was identified as diosgenin.




1 2 3 4 5 6 7 8 9 10 11 12 13 14

37.2 31.4 71.7 41.6 140.8 121.4 31.6 31.8 50.1 36.6 20.9 39.8 40.2 56.5

1.30, 1.17 m 1.52, 1.35 m 3.57 m 2.26 dd (7.2, 6.4) 1.78 m - 5.33 d (5.2) 2.07, 1.90 m 1.42 m 1.30 m - 1.44 m 1.45, 1.37 m - 1.40 m

15 16 17 18 19 20 21 22 23 24 25 26 27

32.0 80.8 62.1 16.3 19.4 42.3 14.5 109.3 31.4 28.8 30.3 66.8 17.1

1.73, 1.56 m 4.38 q (15.6) 1.80 dd (6.0, 8.8) 0.80 s 1.04 s 2.42 m 0.79 d (7.2) - 1.60, 3.56 m 1.56 m 1.76 m 3.40 t (10.3) 3.45 dd (10.3, 4.2) 0.78 d (6.1)



100


Compound 5 was isolated and purified as white to off white powder from the

chloroform soluble sub fraction, CFE through column chromatography. The

compound was identified and characterized through modern spectroscopic data

analysis. The molecular formula C27H42O4 for compound 5 was established form its

molecular ion peaks at m/z 430 in EI-MS and at m/z 431 [M+H]+ in FAB positive,

which was further confirmed from its HR-EIMS (calcd; 430.3083). The IR spectrum

exhibited absorption bends for hydroxyl functionality at 3571 cm–1, stretching methyl

group at 2871, ring olefinic group at 1620 cm–1 and for C-O functional group at 1057

cm–1.

The 1H-NMR (CDCl3, 600 MHz) spectrum showed similar pattern of chemical shift

values for all the protons to that of diosgenin except the signal at C-17 (Table 3.8). A

methine proton appeared as multiplet at δ 3.27 (H-3), vinylic proton as doublet at δ

5.32 (H-6, J = 5.2 Hz), methine proton H-16 as triplet at δ 3. 80 (1H, t, J= 15.0 Hz)

and methine proton H-20 as double doublet at δ 1.80 (1H, dd, J = 8.8, 6.0 Hz)

respectively. The two tertiary methyl group protons appeared as singlets at δ 0.83

(3H, H-18) and 1.04 (3H, H-19) while the two secondary methyl group protons

appeared as δ 0.89 (3H, d, J = 4.2 Hz, H-21) and 0.87 (0.78, d, J = 6.1 Hz, H-27)

respectively.

The 13C-NMR and DEPT spectra (CDCl3, 150 MHz) exhibited twenty seven peaks for

all the-carbon atoms comprising four methyls, ten methylene, eight methine and five

quaternary (Table 3.8). The spectrum exhibited characteristic signals for all the

carbon atoms, closely resemble to the diosgenin, except with the appearance of a


101

quaternary carbon signal at δ 90.1 (C-17) for hydroxyl group. The methyl carbons

resonated at δ 17.1 (C-18), 19.4 (C-19), 13.6 (C-21) and 17.1 (C-27) respectively. The

other entire carbon atoms chemical shift values as well as 2D-NMR correlations

showed resemblance with the reported values in literature for a known compound

pennogenin132.


102




1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

37.1 31.6 71.7 42.2 140.8 121.3 31.6 31.6 49.6 36.6 20.7 31.9 44.6 52.9 31.2

1.34, 1.17 m 1.55,1.32 m 3.27 m 2.24 dd (7.2, 6.9) 1.98 m - 5.32 d (5.2) 2.05, 1.80 m 1.43 m 1.34 m - 1.44 m 1.50, 1.33 m - 1.40 m 1.72, 1.49 m

16 17 18 19 20 21 22 23 24 25 26 27

90.9 90.1 17.1 19.4 43.7 13.6 110.1 30.7 28.1 30.1 66.8 17.1

3.80, t (15.0) - 0.83 s 1.04 s 2.42 m 0.89 d (7.2) - 1.60, 3.56 m 1.56 m 1.76 m 3.35 t (10.8) 3.45 dd (m) 0.87 d (12.0)



103


Compound 6 was isolated and purified as white powder from the chloroform soluble

sub fraction, CFE. The compound was identified and characterized through modern

spectroscopic data analysis and was confirmed as a trihydroxy fatty acid. In EI-MS

spectrum the molecular ion peak was displayed at m/z 330, while FAB-MS showed

ion peak at m/z 331 [M+H]+. Its molecular formula of C18H34O5 was obtained from

HREI-MS at m/z 330.4566 (calcd; 330.2402). The molecular formula showed two

degrees of unsaturation due to the presence of an olefinic and a carbonyl group in the

molecule.

The IR spectrum also revealed strong absorptions for acid carbonyl (C=O) and

olefinic (C=C) functionalities at 1690 and 1470 cm-1, respectively. The absorption at

3404 cm-1 showed the existence of acid hydroxyl group. The three extra oxygen atoms

in molecule were placed as hydroxyl groups on the basis of 1H-NMR and connectivity

data.

The linked scan measurements of major peaks in mass spectrum also helped in

establishing the chemical structure as a trihydroxy fatty acid. Accordingly, the linked

scanned measurements were supportive in this regards which have been depicted in

Fig. 3.11. M+ at m/z 330 [M]+, 273 (M+ - 57), 245 (M+ -57-28), 223 (M+ -57-28-

24+2H), 205 (M+ -57-28-20-2H-18), 187 (M+ -57-28-20-2H-18-18), 167 (M+ -57-28-

20-2H-18-18-18-2H) and 123 (M+ -57-28-20-2H-18-18-18+2H-44) in EI-MS. The

consecutive loss of three 18 fragments was evident of three OH groups in the

molecule.


104

The 1H-NMR (MeOD, 600 MHz) spectrum revealed signals for all the protons at

various chemical shift values as observed for a known compound, trihydroxy mono

unsaturated fatty acid133, expect the position of double bond in chain at position 10-

11. The two olefinic methine protons -resonated at. δ 5.46 (1H, dd, J- = 11.1, 6.4 Hz, H-

10) and 5.56 (1H, dd, J[ = 11.1, 6.1 Hz, H-11), respectively.

The location of double bond was confirmed from the daughter ion peaks for these left

side chain losses as given. The fragment ion at m/z 57, 169 (cleavage at C-8, 9), 152

(OH loss) due to [C4H9]+, [CH3(CH2)7CH=CH-CHOH]+

, [CH3(CH2)7CH=CH-CH]+

fragment losses as well as at 171 (right side chain, [OH-CH-OH-CH(CH2)4COOH]+),

155 (O loss) and 137 (H2O loss) which were reported due to the possible breakage of

C-7, 8 points in the chain as depicted in Fig. 3.12.

The terminal methyl protons (H-18) resonated at δ 0.90 (t, J = 8 Hz), while the

methylenic protons (H-3, 4, 5, 14, 15, 16 and 17) resonated in range of δ 1.34 to 2.26

respectively. A triplet was assigned to the methylenic protons of H-2 at δ 2.26 (7.1

Hz) as well as for H-12 protons at δ 2.15 (7.1 Hz) while a multiplet was observed for

H-13 at δ 2.10.

The 13C-NMR (MeOD, 150 MHz) spectrum revealed signals for almost all the carbon

atoms including carbonyl quaternary carbon at δ 177.7 (C-1), olefinic carbons at δ

130 and 134.6 (C-10 and 11) along with the hydroxyl bearing carbons at δ 71.7 (C-7),

76.9 (C-8) and 69 (C-9) (Table 3.9). 1H-1H COSY was helpful in assigning the

correlations among the chain protons (Fig. 3.12) while the cis confirmation was

supporting by the coupling constants (11.1 Hz) between H-10 and H-11.


105

Consequently, compound 6 was assigned as 7, 8, 9-trihydroxy-(10Z)-10-octadecenoic

acid. The compound was given a common name as govanic acid.

Table 3.9: 1H-NMR and 13C-NMR (CD3OD, 600 and 150 MHz) chemical shift



1 2 3-5 6 7 8 9 10 11

177.7 34.9 26.1 - 30.6 34.6 71.7 76.9 69.0 130.7 134.6

- 2.26, t (7.1) 1.34-1.61 br, m 1.59 m 3.73 m 3.25 dd 4.48 dd 5.46 dd (11.1, 6.4) 5.56 m (11.1, 6.1)

12 13 14 15 16 17 18

32.7 30.2 28.9 26.9 30.4 23.6 14.4

2.15 t (7.1) 2.10 m 1.59 1.54 1.44 1.41 0.90 t (8.0)



106

Figure 3.11: Linked scan measurements in compound 6 (EIMS spectrum).

Figure 3.12: Major fragmentation in compound 6 with correlations

in 1H-1H-COSY( ).


107


Compound- 7 was obtained as amorphous white powder from the sub fraction, CFHh

eluted with EtOAc in MeOH (9.5 : 0.5) solvent system by preparative thin layer

chromatography. The mass spectrum in HR-EIMS afforded the molecular ion peak at

m/z 480.5521 [M]+ consistent with the molecular formula of C27H44O7 (calcd;

480.3121). The formula mass was also confirmed by FAB-MS (negative) in glycerol

at m/z 479 [M-H+]. The six degrees of unsaturation was determined as four accounted

for the tetracyclic skeleton while one each for carbonyl (C=O) and a vinylic C=C

group. The overall fragmentation pattern was consistent with that observed for

ecdysteroids skeleton reported for many ecdysteroids134.

The IR spectrum showed similar pattern of peaks, common for ecdysteroids. An

intense absorption at 3378 and 2871 cm-1 indicated hydroxyl group and aliphatic C-H

stretch, respectively. The absorption at 3068, 1472, 1055 and 879 cm-1 indicated and

confirmed a vinyl group, while a strong absorbance at 1646 cm-1 indicated the

existence of cyclo hexenone in the molecule. Similarly, absorption at 1380 cm-1

showed the presence of C-O functionality. The UV spectrum showed absorption at

240 nm indicating an α and β unsaturated carbonyl moieties in the molecule.

The 1H-NMR spectrum, showed two secondary methine protons, C-2 and C-3

resonated downfield at � 3.84 and 3.94 suggesting to be spin coupled (J = 3.2 Hz, 1H-

1H- COSY) with each other and further coupled to adjacent methylenic protons of C-1

and C-4 which in turn resonated at � 1.75 and 1.76, respectively, indicating the

presence of –CH2-CH(OH)-CH(OH)-CH2- system that is placed at C-1-C-4 in the


108

molecule (Table 3.10). The signal pattern for all protons was mostly similar to 20-

hydroxyecdysone previously reported in literature135.

The 13C-NMR spectrum (Table 3.10) showed signals for all 27 carbons comprising

five methyl, six methylenic, nine methine and seven quaternary carbons. The side

chain methylene carbon (C-22) resonated on δ 77.9, while signals for the methylenic

carbons C-23 and C-24 were observed at δ 27.3 and 43.8, respectively. The three

methyl carbon signals were observed at δ 21.5 (C-21), 28.9 (C-26) and 29.7 (C-27)

while the quaternary carbon (C-25) of the side chain resonated at δ 71.3 which was

analogue to that of the 20-hydroxy ecdysone for the sixth hydroxyl carbon in the

molecule. The methyl carbons of C-18 and C-19 resonated at � 18.1 and 24.4,

respectively. Signals for the two hydroxyl methine carbons of ring A were observed at

� 68.7 (C-2) and 68.5 (C-3) while C-5 methine carbon resonated at � 50.5. The

carbonyl carbon appeared on � 206.4 (C-6) along with the two vinyllic carbons

showing chemical shift values � 122.1 (C-7) and 168.0 (C-8) in ring B. These data

suggested α,β unsaturated ketone moiety ascribable to 7 en-6-one system in the

steroid nucleus, also supported by HMBC correlations. The signal pattern was mostly

similar to 20-hydroxyecdysone previously reported in literature136. Thus, on the basis

physical and spectral similarities with the reported 20-hydroxyecdysone, compound 7

was identified as 20-hydroxyecdysone.


109




1 2 3 4 5 6 7 8 9 10 11 12 13 14

37.4 68.7 68.5 32.2 50.5 206.4 122.1 168.0 35.1 39.1 21.5 32.5 57.3 85.2

1.75 3.84 m 3.94 dt (11.7, 3.2) 1.76, 2.13 2.40 - 5.80 d (2.6) - 3.14 - 1.42, 1.46 2.13, 1.33 - -

15 16 17 18 19 20 21 22 23 24 25 26 27

31.7 24.4 37.4 18.1 24.4 78.4 21.5 77.9 27.3 43.8 71.3 28.9 29.7

1.61 1.58, 1.55 2.00 0.88 0.99 - 1.13 3.14 1.42 1.37 - 1.19 1.18



110


Compound 8 was obtained as white amorphous powder from the sub fraction, CFHh

by preparative thin layer chromatography (TLC). The mass spectrum in HR-EIMS

afforded the molecular ion peak at m/z 496.5510 [M] + which was in agreement with

the molecular formula of C27H44O8 (calcd; 496.3021). The formula mass was also

confirmed by FAB-MS (positive) in glycerol at m/z 497 [M+H]+. The six degrees of

unsaturation was determined as four accounted for the tetracyclic skeleton while one

each for carbonyl (C=O) and vinylic C=C functional group. The overall fragmentation

pattern was in consistent with that observed for 20-hydroxy ecdysone skeleton

reported for many ecdysteroids134.

The IR spectrum showed similar pattern of peak, observed in the reported 20-hydroxy

ecdysone skeleton135. The UV spectrum showed absorption at 240 nm confirming the

existence of an α and β unsaturated carbonyl moiety in the compound.

The 1H-NMR signal pattern was mostly similar to 5,20-dihydroxy ecdysone

previously reported135,137. The 13C-NMR spectrum in MeOD (BB and DEPT) showed

signals for all 27 carbons comprising five methyl, six methylenic, nine methine and

seven quaternary carbon atoms (Table 3.11). The side chain methylenic carbon (C-

22) resonated on δ 77.8 while signals for the methylenic carbons, C-23 and C-24 were

observed at δ 27.0 and 43.8 respectively. The quaternary carbon (C-25) of the side

chain resonated at δ 71.3 which was analogues to that of the 20-hydroxy ecdysone.

However compound 8 showed a distinct signal for C-5 at 80.3 as compared to C-5 of

20-hydroxy ecdysone (δ 71.3). The same chemical shift value for C-5 has been

reported for 5,20 dihydroxyecdysone135,137. Thus, on the basis physical and spectral


111

similarities with the reported data, compound 8 was identified as

5,20 dihydroxyecdysone.




1 2 3 4 5 6 7 8 9 10 11 12 13 14

36.1 70.2 68.4 32.2 80.3 202.4 120.6 167.5 35.1 39.1 30.2 71.3 57.4 85.3

1.75 3.64 d (3.5) 3.98 1.97, 1.94 - - 5.84 d (2.7) - 3.18 br m - 1.74, 1.72 2.00, 1.78 - -

15 16 17 18 19 20 21 22 23 24 25 26 27

31.5 23.3 43.6 14.8 24.2 78.4 26.1 77.8 27.0 43.8 71.3 28.9 29.6

1.84, 1.60 1.97, 1.54 2.38 0.89 0.99 - 1.24 3.31 1.42 1.38 - 1.19 1.18



112


Compound 9 was isolated and purified as white to off white amorphous powder from

a butanol soluble sub fraction, BFA. The HRFAB-MS-showed-pseudo molecular ion

[M+H]+ at m/z 869.4790 (calcd; 868.4832), which was consistent with the molecular

composition of C45H72O16.

The IR spectrum (KBr) exhibited prominent absorption for hydroxyl functionality at

3410 cm-1, olefinic (endocyclic) absorption at 1420 cm-1 and C-O linkage at 1305 cm-

1 in the skeleton. The 13C-NMR signals at δ 141.8 (C-5), 122.6 (C-6) and 110.6 (C-

22), showed the same basic skeleton of 5-spirostane type sapogenin129.

As 1H and 13C-NMR data (Table 3.12) of compound 9 were mostly similar to

diosgenin, except the difference in the mass and the presence of sugar moieties which

yielded several signals in the region between δC 70-105. The 1H-NMR (600 MHz,

MeOD) spectrum showed the presence of one olefinic proton signal at δ 5.63 as broad

doublet (J = 5.4 Hz), due to the presence of one double bond in compound. The 1H-

NMR also showed the signals for three anomeric protons at δ 4.87 (1H, d, J = 6.9 Hz,

H-1’), 6.27 (1H, s, H-1’’) and 5.60 (1-H, s, H-1’’’, which suggested the presence of

three monosaccharide moieties. Moreover, the 1H-NMR spectrum showed signals for

two tertiary methyl group protons at δ 1.02 (3H, H-18) and 1.08 (3H, H-19) and two

secondary methyl doublets separately at δ 1.08 (3H, J = 7.6 Hz, H-21), and 1.01 (3H,

J = 6.6 Hz, H-27) respectively.

The 13C-NMR and DEPT (150 MHz, CD3OD) spectra showed signals for all the

carbons including sugar moieties and olefinic functionalities. Furthermore, the 1H and

13C connectivities in steroidal skeleton of aglycone and sugar moieties were made


113

through HSQC, HMBC and 1H-1H COSY spectral studies (Fig. 3.15 and 3.16). Total

45 carbon atoms were present comprising of six methyl, eleven methylene, twenty

four methine and six quaternary-carbon atoms. The endocylic olefinic protons at δ

5.63 (H-6) exhibit 3J correlations with carbons at δ 141.8 (C-5) and 38.5 (C-10),

respectively.

The 1H-1H COSY spectrum was helpful in establishing the connections in steroidal

skeleton as follows. H-1 proton at δ 1.0 showed cross peaks with C-2 methylene

protons (δ 1.92 and 2.10), which was correlated with C-3 methine at δ 3.68 which in

turn showed cross peaks with methylene protons at δ 2.68 and 2.78 to confirm the ring

A/B connectivities. Further COSY assignment assessed the connections amongst the

H-6/ H-7, H-8/H-9, H-11/H12, H-15/H-16, and H-20/H-22 accordingly.

The NOESY spectrum was helpful in deducing the stereochemistry in the steroidal

nucleus as H-3 showed cross peaks with H-1α, assigning it α or axial symmetry. The

proton H-8 was correlated with methyl groups at C-18 and C-19, suggesting there

same orientation i.e., β-oriented. Similarly, H-9 and H-14 were found to be α-

oriented, as H-9 showed NOESY cross-peaks with H-1 (axially oriented). The C-3

signal appeared at δ 79.8, which indicated the glycoside linkage with this position.

This connectivity was further confirmed through the- HMBC correlation of anomeric

proton peak at δ 4.87 (H-1′) with C-3; H-3 also showed NOESY cross peaks with H-

1’, which suggested the α-orientation of both these protons.

The connectivity between sugar molecules was inferred on the basis-of HMBC

correlations. H′-1 (δ 4.87) showed HMBC correlations with C-2′ (δ 77.1), C-3′ (δ

76.8), C-4′ (δ 79.3), and C-5′ (δ 78.0), suggesting the connectivity-of glucose moiety


114

with C-3. Furthermore, the HMBC-correlations of H-1′′ (δ 6.27), with C-5′ (δ 78.0)

indicated the C-1′-O-C-1′′ connectivity, i.e. the α-L-rhamnose molecule attached with

C′-4. The nature and connectivity of this rhamnose molecule was inferred through 1H

and 13C-NMR while the HMBC correlations of H-2′ (δ 4.21) with C-1′′′ (δ 102.6),

revealed the connectivity between the glucose and another α-L-rhamnose molecule.

The presence of another α-L-rhamnose was deduced through the HMBC correlations.

The physical and spectral data coincided with the reported data of borassoside E138,

hence the compound was characterized as a known compound, borassoside E.


115




1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

38.0 30.7 79.8 39.5 141.8 122.6 32.4 31.4 51.7 38.5 21.9 41.4 40.9 57.8 33.2 80.0 61.9 16.7 19.8 42.9 17.4 110.6 29.8 28.2 32.8 69.7 17.8

1.10, 1.68 m 1.92, 2.10 m 3.68 m 2.68 dd (11.2, 12.9) 2.78 dd (2.2, 13.6) - 5.63 br 1.52, 1.90 m 1.42 m 1.10 m - 1.44 m 1.10, 1.88 m - 1.12 m 1.56, 1.90 m 4.02 m 1.80 dd (6.0, 8.8) 1.02 s 1.08 s 2.52 m 1.08 d (7.6) - 1.60 m 1.24 m 1.44 m 3.80 br 1.01 d (6.2)

3-O-D-Glc 1’ 2’ 3’ 4’ 5’ 6’ 2’-O-Rha 1’’ 2’’ 3’’ 4’’ 5’’ 6’’ 4’-O-Rha 1’’’ 2’’’ 3’’’ 4’’’ 5’’’ 6’’’

100.4 77.7 76.8 79.3 78.0 62.10 102.2 72.5 73.0 74.2 70.4 19.2 102.6 73.0 72.8 74.6 70.2 18.8

4.87 d (6.9) 4.21 m 3.58 m 3.92 m 4.10 m 3.90, 4.10 6.27 br s 4.66 m 4.35 dd (1.9, 9.4) 4.72 m 4.70 m 1.52 d (6.4) 5.60 br s 3.98 m 4.26 dd (2.2, 9.0) 4.10 m 4.62 m 1.82 d (6.3)


116


Figure 3.16: Key HMBC correlations in compound 9.


117


Compound 10 was isolated and purified as white amorphous powder from the butanol

soluble sub fraction, BFBC. The compound was identified and characterized through

modern spectroscopic data analysis.

The HRFAB-MS showed pseudo molecular ion [M+H]+ at m/z 1225.5426 (calcd;

1224.5490), which was consistent with the molecular composition of C56H88O29.

The 1H-NMR (CD3OD, 600 MHz) and 13C-NMR (CD3OD, 150 MHz) data (Table

3.13) of compound 10 was largely similar to the basic skeleton of diosgenin, and the

difference in compound 10 was the number of sugar moieties. The 1H-NMR spectrum

showed the presence of three olefinic protons signals at δ 5.55 as broad doublet (J =

5.4 Hz), 5.08 as br (s) and 4.98 br (s), which showed the presence of two C=C in

compound 10. Similarly, 1H-NMR also showed the signals for five anomeric protons

at δ 5.41 br s, 5.18 d (J = 2.4 Hz), 4.72 (dd, J = 8.4 Hz), 4.39 (d, J = 7.2 Hz), 4.38 (d,

J = 7.2 Hz), which suggested the presence of five monosaccharides including one

apiose. In addition to this, the 1H-NMR spectrum showed signals for two methyls at δ

0.91 (s) and 1.08 (s) attached to quaternary carbon, and two methyl signals at δ 1.10

(d, J = 6.6 Hz), and 1.24 (d, J = 6.6 Hz), attached to tertiary carbon. The 13C-NMR

spectrum showed signals for sugar moieties and olefinic functionalities. The

attachment of carbon in steroidal skeleton of saponin and sugar moieties was assigned

on the basis of HSQC, HMBC and COSY correlations (Fig. 3.18 and 3.19). The

olefinic proton at δ 5.55 (H-6) showed HMBC correlations to two quaternary carbons

at δ 139.5 and 43.4 attributed to C-5, and C-10, respectively.


118

The HSQC spectrum showed the correlations of protons at δ 3.51 and 3.39 with

carbon at δ 84.5 and 69.2, suggesting the presence of two OH groups in ring A of

steroidal skeleton. The C-C bond connectivity in ring A and B was inferred through

COSY correlations. H-1 (δ 3.51) indicated COSY cross peaks with C-2 methylene

protons (δ 2.09, 1.71), which further showed cross peaks with H-3 (δ 3.39). Similarly,

H-3 showed COSY cross peaks with methylene protons at δ 2.24 and 2.22. This

suggested the connectivity of C-1 to C-4. H-6 (δ 5.55) showed COSY correlations

with H2-7 (δ 1.96, 1.55), which further showed connectivity with C-8 through COSY

cross peaks with H-8 (δ 1.54).

The COSY correlation of H-20 with H-17 (δ 1.82) and H2-21 (δ 3.63, 3.2), along with

HMBC correlations of H-21 (δ 3.63, 3.63) with C-20 (δ 46.6) and C-22 (δ 112.1)

inferred an OH at C-21. Similarly, the HMBC correlations of H-24 (δ 4.26), H2-26 (δ

4.46, 3.71) with C-22 suggested oxygenated nature of ring-F.

The stereochemistry of steroidal skeleton was deduced on the basis of NOESY

correlations (Fig. 3.19). H-1 showed NOESY cross-peaks with H-3, this could be only

possible if both these protons are axially oriented. Therefore, H-1 and H-3 are α-

oriented. H-8 showed NOESY correlations with H3-18 and H3-19, suggesting there

same orientation i.e., β-oriented. Likewise, H-9 and H-14 were found to be α-oriented,

as H-9 displayed NOESY cross-peaks with H-1 (axially oriented).

The HMBC correlations of H-24 (δ 4.26) with C-23 (δ 72.1), C-25, (δ 144.5), and C-

27 (δ 114.0), along with COSY cross peaks of H-24 with proton at δ 3.74 suggested

the position of OH groups at C-23 and C-24, and an exocyclic C=C bond between C-

25/C-27. The C-24 signal appeared at δ 83.3, which indicated the glycoside linkage


119

with this position. This connectivity was further confirmed through the HMBC

correlation of anomeric proton at δ 4.72 (H-1′′′′) with C-24. H-23 showed NOESY

cross peaks with H-20 and H-24, which suggested the β-orientation of these protons.

The coupling constant value of H-23 and H-24 was consistent with literature reported

value of similar structure i.e. 4.2 Hz, which further confirmed an α-OH at C-23 and C-

24.

The connectivity between sugar molecules was inferred on the basis of HMBC

correlations. H′-1 (δ 4.40) showed HMBC correlations with C-1 (δ 84.5), C-2′ (δ

77.3), C-3′ (δ 88.6), and C-5′ (δ 78.1), suggesting the connectivity of glucose moiety

with C-1. Furthermore, the HMBC correlations of H-1′′ (δ 4.38), with C-3′ (δ 88.6)

indicated the C-3′-O-C-1′′ connectivity, i.e. the second glucose molecule attached

with C′-3. The connectivity of an apiose molecule was inferred through the HMBC

correlations of H-1′′′ (δ 5.18) with C-6′′ (δ 67.1), C-2′′′ (δ 77.5), and C-5′′′ (δ 75.0).

The coupling constant (J = 5.18) and 13C-NMR data were similar to the literature

reported139, which indicated the presence of an apiose unit connected to glucose. The

presence of a deoxy β-D-gulose in connection with C-24 was inferred through HMBC

correlation of H-1′′′′ with C-24. Similarly, the presence of an α-L-rhamnose was

deduced through the HMBC correlations of H-1′′′′′ with C-4′′′′ (δ 80.3) and NMR data

similar to the previously reported data53. Compound 10 was structurally characterized

as (1β,3β,23S,24S)-1-[O-β-D-glucopyranosyl (1→3)-O-β-D-glucopyranosyl (1→6)-

O-β-D-apiofuranosyl]-3,23dihydroxyspirosta-5,25-dienyl-24-[O-α-L rhamnopyranosyl

(1→4)-β-D-6-deoxygulopyranoside] (Fig. 3.17). To the best of our knowledge this

compound is not reported previously and is a new compound. A common name was

proposed for compound 10 as govanoside A.


120




1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

84.5 37.5 69.2 43.3 139.5 126.1 32.6 34.2 51.0 43.4 24.8 40.9 41.7 57.9 33.2 84.4 58.8 17.2 15.4 46.6 62.9 112.1 72.1 83.5 144.5 62.1 114.0

3.51 overlap 1.71, 2.09 m 3.39 m 2.22, 2.24 m - 5.55 br d (5.4) 1.55, 1.96 1.54 m 1.38 m - 1.41, 2.46 1.19, 1.69 m - 1.22 m 1.54, 1.98 m 4.56 q (7.2) 1.82 dd (7.8, 6.6) 0.91 s 1.08 s 2.71 dd (7.2, 6.6) 3.52, 3.63 overlap - 3.74 d (4.2) 4.26 d (4.2) - 3.71 d (12.0), 4.46 d (12.0) 4.98 br s 5.08 br s

1-O-β-D-Glc. 1’ 2’ 3’ 4’ 5’ 6’ 3’-O-β-D-Glc. 1’’ 2’’ 3’’ 4’’ 5’’ 6’’ 6’’-O-β-Api. 1’’’ 2’’’ 3’’’ 4’’’ 5’’’ 24-O-6-deoxy-β-D-Gul 1’’’’ 2’’’’ 3’’’’ 4’’’’ 5’’’’ 6’’’’ 4’’’’-O-α-L-Rha 1’’’’’ 2’’’’’ 3’’’’’ 4’’’’’ 5’’’’’ 6’’’’’

100.2 77.3 88.6 69.8 78.1 63.6 105.3 74.8 78.0 71.8 77.5 67.1 112.6 77.5 65.4 80.0 75.0 103.4 70.2 70.9 80.3 70.7 16.1 101.6 72.8 72.1 73.4 69.9 18.7

4.40 d (7.2) 3.50 m 3.66 m 3.37 m 3.25 m 3.60, 3.69 4.38 d (7.2) 3.51 m 3.67 m 3.37 m 3.27 m 3.60, 3.73 5.18 d (2.4) 3.99 d (3.0) 3.35 s - 3.76 s, 3.78 d 4.72 d (8.4) 3.64 m 3.90 m 3.45 m 4.10 m 1.10 d (6.6) 5.37 br s 3.90 m 3.67 m 3.41 m 4.13 (9.6, 6) 1.24 d (6.6)


121

Figure 3.17: Chemical structure of compound 10

Figure 3.18: Key-HMBC-correlations-in Compound 10.


122

Figure 3.19: Key-NOESY-correlations-in compound 10.


123

3.5 Biological studies

3.5.1 In vitro biological activities

3.5.1.1 Antibacterial activity of Cr. MeOH-Ext and fractions

In antibacterial assay, the Cr. MeOH-Ext and its subsequent solvent soluble fractions

were screened against gram positive (B. subtilis and S. aureus) as well as gram

negative (E. coli, S. flexenari, P. aeruginosa and S. typhi) bacteria in order to explore

its antibacterial potential. The inhibition zone of the extract and all the fractions was

compared with a broad spectrum antibacterial drug, Imipenem, (10 µg/disc) and

percent inhibitions were calculated. The antibacterial results as shown in Table 3.14

pointed out that in general, the Cr. MeOH-Ext and its subsequent solvent soluble

fractions are fairly active against some of the tested bacterial strains. All tested

samples, showed antibacterial activity against S. flexenari. Among the test samples n-

Hex-fr was more active with 47% inhibition followed by Cr. MeOH-Ext, CHL-fr and

EtOAc-fr with 40%, 35% and 26% inhibitions, respectively. All fractions except

BuOH-fr were found active against E. coli with maximum activity in EtOAc-fr (33%)

inhibition. Only butanol and aqueous fractions were found active against S. typhi,

with 25% inhibition each. Similarly, Cr. MeOH-Ext, n-hexane and CHL-fr showed

antibacterial activity against P. aeruginosa with 23%, 32% and 18% inhibitions,

respectively. The Cr. MeOH-Ext, CHL-fr, BuOH-fr and Aq-fr also showed

antibacterial potential against B. subtilis and S. aureus with a maximum of 43%

activity in CHL-fr. The Cr. MeOH-Ext exhibited 38% inhibition against S. aureus.

The n-Hex-fr and EtOAc-fr were found inactive against two gram positive bacteria, S.

aureus and B. subtilis.


124

The global turn down in antibiotic discovery programs by different pharmaceutical

firms and increase of antibiotic resistant micro-organisms, are promptly the scientific

community to look for new or novel and also re-examine old sources of bioactive

chemicals if any, in order to discover potential anti-bacterial compounds. Medicinal

plants are an area under focus, since their secondary metabolites included a

noteworthy number of drugs used in current therapeutics and there is no doubt in their

potential as the source of new drugs140. Keeping in view the fact, the anti-bacterial

screening of T. govanianum rhizomes extract and subsequent fractions was

performed. Our results suggest that the Cr. MeOH-Ext and some of the subsequent

solvent soluble fractions as described above possess moderate antibacterial potential

to some of the tested gram positive as well as gram negative bacterial strains. The

antibacterial potential in the Cr. MeOH-Ext and its successive solvent soluble

fractions might be due to the occurrence of steroids, glycosides, tannins, and saponins.

These medicinally important secondary metabolites exert their antimicrobial action by

virtue of different mechanism141. For example, saponins exerts their antibacterial

action by inhibiting the growth of bacteria and also through leakage of certain

enzymes and proteins from the cell142 that may be the reason for inhibition in present

study. In addition, antibacterial mechanisms for steroids are specifically related with

membrane lipids and cause leakage from the liposomes143. Similarly, the anti-bacterial

action of n-Hex-fr is strongly supported by the presence of certain fatty acids i.e.

octadecanoic acid and hexadecanoic acids (presence suggested from data in (Table

3.3; Page No. 91) possessing antibacterial properties143.


125

Table 3.14: Antibacterial activity of Cr. MeOH-Ext and fractions of T. govanianum rhizomes

Samples Parameters Bacterial -Strains

Gram positive Gram negative

S. aureus B. subtilis E. coli S. flexenari S. typhi P. aeruginosa

Cr. MeOH-Ext Inhibition zone (mm) 10 ± 0.8 3.0 ± 0.3 3.0 ± 0.5 6.0 ±1.2 - 5.0 ± 0.8

Percent inhibition 38 21 17 40 - 23

n-Hex-fr Inhibition zone (mm) - - 4 7 - 7

Percent inhibition - - 22 47 - 32

CHL-fr Inhibition zone (mm) 4.0 ± 1.3 6.0 ± 0.9 5.0 ± 2.2 5.0 ± 0.7 - 4.0 ± 1.1

Percent inhibition 19 43 27 33 - 18

EtOAc-fr Inhibition zone (mm) - - 6.0 ± 1.3 4.0 ± 0.8 - -

Percent inhibition - - 33 26 - -

BuOH-fr Inhibition zone (mm) 4.0 ± 1.1 3.0 ± 1.9 - 3.0 ± 2.1 5.0 ± 0.9 -

Percent inhibition 15 21 - 20 25 -

Aq-fr Inhibition zone (mm) 3.0 ± 0.4 - 4.0 ± 1.3 - 5.0 ± 1.5 -

Percent inhibition 12 - 22 - 25 -

Standard (Imipenem) Inhibition zone (mm) 26.0 ± 0.6 14.0 ± 0.1 18.0 ± 0.7 15.0 ± 1.0 20.0 ± 0.3 22.0 ± 0.5

Zone of growth inhibition are given as mean ± SEM of three independent experiments

Percent inhibition less than 10% , marked as "_"

Blank controls of pure solvents having no activity against the test bacteria


126

3.5.1.2 Antifungal activity

3.5.1.2.1 Antifungal activity of Cr. MeOH-Ext and fractions

In antifungal screening, the Cr. MeOH-Ext and its subsequent solvent soluble

fractions were examined against seven different fungal strains (Candida albicans,

Candida glabrata, Aspergilllus flavus, Aspergillus niger, Aspergillus fumigatus,

Trichphyton rubrum and Microsporum canis). The results (Table 3.15) showed good

to moderate antifungal potential with maximum in Cr. MeOH-Ext and BuOH-fr. The

Cr. MeOH-Ext were found active against all tested strains except A. fumigatus with

maximum inhibition against T. rubrum, (60%), M. canis, (55%), and C. albicans (

40%). The n-Hex-fr exhibited good inhibition (40%) against M. canis. The CHL-fr

showed significant inhibition (70%) against T. rubrum and good (40%) towards M.

canis. The EtOAc-fr was least active with no activity against fungal strains C.

glabrata, A. fumigatus and M. canis. The BuOH-fr was found active against all strains

except T. rubrum with maximum inhibition of 40% against A. niger. The Aq-fr

showed weak inhibition with no activity against test C. albicans and A. niger. The

standard drugs used were amphotericin B and miconazole.

3.5.1.2.2 Antifungal activity of isolated compounds

The isolated compounds [govanoside A and govanic acid (two new compounds),

borassoside E, pennogenin and diosgenin] were screened for their antifungal potential

as the fractions containing them possessed promising antifungal activities. The results

of isolated compounds (Table 3.16) indicated that govanoside A and borassoside E

have good to moderate activities against Aspergillus niger, A. flavus, C. albicans, and


127

C. glabrata strains, while govanic acid exhibited moderate potential against T. rubrum

and M. canis.

Moreover, pennogenin and diosgenin were inactive at the highest test concentration of

20 µg/mL against all tested fungi. In comparison, borassoside E exhibited good

activities (MIC = 2.5-10 µg/mL) against Candida spp. In case of A. niger, govanoside

A exhibited good activity (MIC = 5 µg/mL) while borassoside E was slightly less

active (MIC = 10 µg/mL). As far as A. flavus is concerned Borassoside E had a better

control in comparison to govanoside A. Govanic acid exhibited better result (MIC =

10 µg/mL) for T. rubrum in comparison to M. canis and showed no activity for the

rest of tested fungal strains.

To best of our knowledge, the in vitro antifungal activities of isolated compounds,

govanoside A, borassoside E and govanic acid are reported for the first time in this

study. As govanoside A and borassoside E are steroidal saponins and steroidal

saponins have shown to possess antifungal potentials, our findings are coherent to

earlier findings144,145 in this domain. Similarly, trihydroxy fatty acid have also been

reported for antifungal properties146, which endorse the findings of this study.

As fungal infections are a major cause of morbidity and mortality147, there is need for

the discovery of new antifungal drugs. Therefore, this is a significant finding, though

further detail experiments are required to establish the-exact-mechanism of antifungal

actions of these compounds.


128

Table 3.15: Antifungal activity of Cr. MeOH-Ext and fractions of T. govanianum rhizomes (% inhibition)

Fungal strains

Samples

Cr. MeOH-Ext n-Hex-fr--- CHL-fr EtOAc-fr BuOH-fr [Aq-fr

Percent inhibition

Candida albicans 40 - 20 10 30 -

Candida glabrata 25 - 15 - 25 10

Aspergilllus flavus 20 10 - 20 30 15

Aspergillus niger 10 - - 15 40 -

Aspergillus fumigatus - 10 10 - 30 10

Trichphyton rubrum 60 30 70 10 - 20

Microsporum canis 55 40 40 - 10 15

* Percent inhibition less than 10 is marked as "_".


129

Table 3.16: Antifungal activity of isolated compounds of T. govanianum rhizomes

*Concentration range 0312-20 µg/mL; MIC (minimum inhibitory concentration)

MIC (µg/mL)

Compounds C. glabrata C. albicans A.niger A. fumigatus A. flavus T. rubrum M. canis

Govanoside A 20 5.0 5.0 >20 20 >20 >20

Borassoside E 10 2.5 10 >20 10 >20 >20

Pennogenin >20 >20 >20 >20 >20 >20 >20

Diosgenin >20 >20 >20 >20 >20 >20 >20

Govanic acid >20 >20 >20 >20 >20 10 20

Amphotericin B 2.5 0.6 5.0 5.0 5.0 - -

Miconazole - - - - - 2.5 5.0


130

3.5.1.3 DPPH free radical scavenging activity of Cr. MeOH-Ext and fractions

DPPH free radical scavenging assay is considered as a standard method for the

assessment of the antioxidant potential of natural crude extracts, fractions and pure

compounds91,148,149. Antioxidant potential bearing natural phytochemicals are

effective in reducing the toxic effects in human, due to xenobiotic exposure150. The

Cr. MeOH-Ext and its succeeding solvent soluble fractions were examined for their

antioxidant effect at different concentrations i.e. 1, 10, 20, 50, 100 and 200 µg/mL.

The results (Table 3.17 and Fig. 3.20) indicated that the n-Hex-fr and CHL-fr

possessed relatively higher free radical scavenging capacity as compared to the other

tested fractions. This finding is suggestible due to the presence of certain antioxidant

fatty acids (9,12-octadecadienoic acid and hexadecanoic acid) in n-Hex-fr, and

glycosides, saponins and flavonoids (Table 3.2; Page No. 89) in CHL-fr, as

previously reported in diverse plant species151-154.

The results also demonstrated that the antioxidant potential of Cr. MeOH-Ext as well

as its successive solvent soluble fractions was lower than BHT and ascorbic acid. The

low free radical scavenging capacity of the Cr. MeOH-Ext or its fractions might be

attributed due to the presence of large sized fatty constituents as revealed from their

phytochemical and GC/MS analyses. As DPPH assay is limited by steric accessibility,

thus molecules having small molecular weight, have better access to the DPPH

molecules than larger molecular weight molecules and therefore possess strong free

radical scavenging capacity154. Furthermore, there is also a non-linear relationship

between antioxidant activity and hydrophobicity because an increase of alkyl chain

length results in low scavenging capacity155.


131

Table 3.17: DPPH free radical scavenging activity of T. govanianum extract, fractions and standards

Conc.

(µg/mL)

Percent inhibition (%) ± SEM

n-Hex-fr CHL-fr EtOAc-fr BuOH-fr Cr. MeOH-Ext Ascorbic acid BHT

1 7.61 ± 2.68 2.63 ± 0.56 0.82 ± 0.64 1.35 ± 0.53 1.35 ± 0.02 10.2 ± 4.06 5.19 ± 0.04

10 6.61 ± 2.64 2.99 ± 0.12 1.46 ± 0.64 2.35 ± 0.24 1.84 ± 0.36 41.5 ± 2.24 16.9 ± 3.87

30 11.0 ± 0.08 6.68 ± 2.13 4.25 ± 0.08 3.39 ± 0.51 2.06 ± 0.15 95.8 ± 0.22 39.0 ± 9.12

50 23.0 ± 0.46 33.7 ± 0.75 22.1 ± 4.61 22.3 ± 2.16 21.5 ± 3.91 96.3 ± 0.15 68.7 ± 8.75

100 23.2 ± 0.42 15.8 ± 1.57 7.67 ± 0.53 6.28 ± 0.80 4.32 ± 0.39 96.4 ± 0.01 90.9 ± 0.44

200 20.4 ± 2.13 24.3 ± 1.78 11.0 ± 0.33 11.1 ± 1.88 6.96 ± 1.46 96.5 ± 0.04 93.1 ± 0.03

*Results are mean of three different experiments


132

Figure 3.20: DPPH free radical scavenging activity of extract/fractions or standards

(ascorbic acid and BHT).


133

3.5.1.4 Anticancer activity

3.5.1.4.1 Anticancer activity of Cr. MeOH-Ext and fractions

The anticancer activities of Cr. MeOH-Ext and its subsequent solvent soluble

fractions against two cancer cell lines; HeLa (cervical cancer cells) and PC-3 (prostate

cancer cells), were determined by MTT (3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl

tetrazonium bromide) assay. The Cr. MeOH-Ext and its fractions exhibited significant

cytotoxicity towards both cancer cell lines (Table 3.18). The cytotoxic activity of

CHL-fr towards HeLa cells was slightly lower than that of standard drug doxorubicin,

with IC50 of 0.84 ± 0.16 and 0.34 ± 0.01 µg/mL, respectively. Similarly, this fraction

was also most effective against PC-3 cells (IC50 of 2.70 ± 0.35 µg/mL), though to a

lesser extent than doxorubicin (IC50 = 1.38 ± 0.16). Moreover, the BuOH-fr, although

possessed strong cytotoxic effect against the HeLa cells (IC50 of 1.60 ± 0.34 µg/mL),

but was less effective in inhibiting the PC-3 cells (IC50 of 4.04 ± 0.35 µg/mL). The

EtOAc-fr was also effective towards both cancer cells, with prominent against HeLa

cells (IC50 of 1.41 ± 0.08 µg/mL).

3.5.1.4.2 Anticancer activity of isolated compounds

Keeping in view the significant anticancer potential in the extract and fractions, the

isolated compounds [govanoside A, govanic acid (two new compounds) borassoside

E, diosgenin and pennogenin] from the chloroform and BuOH-fr were tested against

HeLa and PC-3 cell lines for their anticancer effects. The results indicated that

govanoside A and borassoside E exhibited significant cytotoxicity against both cancer

cell lines (Table 3.19). In particular, govanoside A showed significant anticancer

potential against PC-3 and HeLa cells, with IC50 of 1.74 ± 0.12 and 0.51 ± 0.26


134

µg/mL respectively, in comparison to the standard, doxorubicin whose IC50 values

were 1.69 ± 0.28 and 0.50 ± 0.15 µg/mL, towards PC-3 and HeLa cells, respectively.

Govanoside A also showed good anticancer activity with IC50 of 0.67 ± 0.22 µg/mL

against HeLa cells. Pennogenin (IC50 of 9.83 ± 0.37 µg/mL) was found active against

HeLa cells, while diosgenin and govanic acid were found less cytotoxic against both

cell lines with IC50 greater than 30 µg/mL.

So far, a number of anticancer metabolites have been reported in the genus

Trillium53,120,156. These anticancer metabolites include steroidal glycosides and

saponins isolated from Trillium erectum and Trillium tschonoskii44,157. As the

phytochemical analyses of the tested fractions revealed the presence of anticancer

metabolites (steroidal glycosides, flavonoids and saponins), therefore, the potent

anticancer potential in the Cr. MeOH-Ext and its fractions of T. govanianum rhizomes

might be attributed to the presence of these secondary metabolites, which is further

augmented by the pure isolated compounds from these fractions, exhibiting good

anticancer potential. Therefore the rhizomes of this Asian plant species may prove to

be effective in the treatment of cancer.


135

Table 3.18: Anticancer activity of Cr. MeOH-Ext and fractions from T.

govanianum rhizomes

Results are mean ± SEM of three independent experiments; PC-3, human prostate

cancer cells; HeLa, human epithelial carcinoma cells

Table 3.19: Anticancer activity of isolated compounds from T. govanianum

rhizomes

Results are mean ± SEM of three independent experiments

Samples IC50 (µg/mL)

HeLa cells PC-3 cells

Cr. MeOH-Ext 3.14 ± 0.72 6.50 ± 0.52

CHL-fr 0.84 ± 0.16 2.70 ± 0.35

EtOAc-fr 1.41 ± 0.08 5.15 ± 0.34

BuOH-fr 1.60 ± 0.34 4.04 ± 0.35

Doxorubicin 0.34 ± 0.01 1.38 ± 0.16

Compounds IC50 (µg/mL)

PC-3cells HeLa cells

Govanoside A 1.74 ± 0.12 0.51 ± 0.26 Borassoside E 2.34 ± 0.21 0.67 ± 0.22 Pennogenin >30 9.83 ± 0.37 Diosgenin >30 >30 Govanic acid >30 >30 Doxorubicin 1.69 ± 0.28 0.50 ± 0.15


136

3.5.1.5 Anti-inflammatory activity (Oxidative burst assay)

3.5.1.5.1 Anti-inflammatory activity of Cr. MeOH-Ext and fractions-

The in vitro immune suppressive activity, evaluated through suppression of oxidative

burst was performed by luminol enhanced chemiluminescence assay. The results are

presented in Table 3.20. The results showed that the BuOH-fr exhibited significant

inhibition of oxidative burst for the whole blood followed by Cr. MeOH-Ext with IC50

of 16.53 ± 7.54 and 30.81 ± 7.02 µg/mL respectively. The CHL-fr also showed

moderate inhibition with IC50 of 81.64 ± 24.61 µg/mL. Similarly the n-Hex-fr and

EtOAc fraction were found less effective in comparison to other fraction with IC50 of

107 ± 38.40 and 114.81 ± 12.35 µg/mL, respectively. The standard drug used as

positive control was ibuprofen with IC50 of 11.23 ± 1.91 µg/mL.

3.5.1.5.2 Anti-inflammatory activity of isolated compounds

Based on the good results in the Cr. MeOH-Ext and its succeeding solvents soluble

fractions, the isolated compounds borassoside E, diosgenin and pennogenin from

BuOH-fr and CHL-fr were screened for suppression of oxidative burst for the whole

blood. The results are tabulated in Table 3.20. Among the tested compounds,

pennogenin exhibited significant in vitro immune suppressive effect by suppression of

oxidative burst with IC50 of 05.00 ± 0.84 µg/mL in comparison to standard drug

ibuprofen with IC50 of 11.23 ± 1.91 µg/mL. Similarly borassoside E also showed

considerable inhibition for the whole blood with IC50 of 31.51 ± 6.62 µg/mL. The

compound diosgenin was found less active as compare to other tested compounds.


137

Luminol enhanced chemiluminescence assay is based on detection of intracellular

reactive oxygen species (ROS) released by opsonized zymosan activated immune

cells. A determination of chemiluminescence is a proficient and extremely susceptible

assay to investigate various kinds of reactive oxygen species. Thus this method is

suitable for detection of super oxide (free radical anions) in a biological system158.

Inflammation and reactive oxygen species have mutual promotion relationship. ROS

are connected with the inflammatory response and frequently they contribute to the

tissue damaging effects of inflammatory reactions159.

The inflammation can lead to the raise of free radicals. Similarly oxidative stress is

consider to play an imperative role in the pathogenesis of inflammation, not merely

through direct injurious effects, but also by association through molecular

mechanisms160. There is a large amount of evidence indicated that the reactive species

production, such as hydrogen peroxide (H2O2), hypochlorous acid (HOCl), occurs at

the site of inflammation, contributes to tissue damage and potentially promoting

inflammatory processes161,162.

Drugs that inhibit the formation or release of these toxic ROS are effective in

treatment of variety of diseases that involves stimulation of immune cells like AIDS,

rheumatoid arthritis and cancer163. Our study suggests that the Cr. MeOH-Ext and its

fractions considerably inhibit the formation of ROS, which is further confirmed by the

isolated compounds from these fractions, especially, pennogenin and borassoside E

exhibiting significant inhibition of ROS production. Thus, these findings prove

scientifically the traditional use T. govanianum rhizomes in the treatment of various


138

inflammatory diseases. Although studies are in progress, it is necessary to investigate

different mechanisms involved, and also to develop an effective dosage form.

Table 3.20: Anti inflammatory effect of T. govanianum rhizomes Cr. MeOH-Ext,

fractions and isolated compounds

Samples IC50± SD (µg/mL)

Cr. MeOH-Ext 30.81 ± 7.02

n-Hex-fr 107.12 ± 38.40

CHL-fr 81.64 ± 24.61

EtOAc-fr 114.81 ± 12.35

BuOH-fr 16.53 ± 7.54

Pennogenin 05.00 ± 0.84

Borassoside E 31.51 ± 6.62

Diosgenin 53.23 ± 2.71

Ibuprofen (Positive control) 11.23 ± 1.91



139

3.5.1.6 Anti-leishmanial activity of Cr. MeOH-Ext and fractions

The Cr. MeOH-Ext and the fractions were examined for in vitro antiparasitic effect

against promastigotes of Leishmania major (DESCO). The results (Table 3.21)

indicated that the Cr. MeOH-Ext exhibited prominent leishmanicidal potential against

the tested strain, L. major with IC50 of 36.34 ± 2.51 µg/mL. The butanol and aqueous

fractions also showed activity with IC50 of 62.61 ± 3.23 and 94.63 ± 1.84 µg/mL

respectively. The other tested fractions i.e n-Hex-fr, CHL-fr and EtOAc-fr were found

less active with IC50 greater than 100 µg/mL. The standard drug used was

amphotericin B, IC50 of 0.29 ± 0.05 µg/mL.

Leishmaniasis is caused by Leishmania, a genus comprising of protozoan parasites.

The two main types of leishmaniasis are the cutaneous, (skin sores) and visceral,

which involve the internal body organs (liver, spleen, and bone marrow)164.

Leishmaniasis is also considered by the World Health Organization (WHO) as one of

six major infectious diseases, with a high detection rate and ability to produce

deformities, and caused significant morbidity and mortality in different

countries165,166. According to the WHO, the population of eighty eight (88) countries

are threatened by leishmaniasis and about three fifty (350) million people are at risk

from this disease167. At present, a limited number of chemotherapeutic agents are

available for the treatment of this disease; therefore the search for new effective drugs

has become really imperative. Keeping in view these facts, this study was performed,

and as a result it was observed that the Cr. MeOH-Ext possess a good potential for

leishmaniasis and thus can be a promising candidate as an antileishmanial agent.


140

Table 3.21: Leishmanicidal activity against Leishmania major of Cr. MeOH-Ext and

fractions of T. govanianum rhizomes

Samples IC50 ± SD [µg/mL]

Cr. MeOH-Ext 36.34 ± 2.51

n-Hex-fr >100

CHL-fr >100

EtOAc-fr >100

BuOH-fr 62.61 ± 3.23

Aq-fr 94.63 ± 1.84

Amphotericin B 0.29 ± 0.05


Amphotericin B was used as positive control

3.5.1.7 Insecticidal activity of Cr. MeOH-Ext and fractions

The in vitro insecticidal potential of Cr. MeOH-Ext and its subsequent solvent soluble

fractions-was determined against two insects, Tribolium-castaneum and Rhyzopertha-

dominica. The results presented in term of percent mortality are presented in Table

3.22 and 3.23, indicate that all of the test samples were not active against the tested

insects as no mortality was observed in this study.

Currently, synthetic pesticides are largely used for protection of stored grains from

insect168. Most of these species have developed resistance to current insecticides, and

therefore the scientists and academia of the globe are currently trying to isolate

effective compounds from medicinal plants as natural new insecticides. Keeping in

view the search for effective insecticides this screening was performed.


141

Several mechanisms have been reported in the literature by which plant extract exerts

their insecticidal activity. One of the mechanisms for insecticidal activity is blocking

of sterol uptake in the insect gut by plant secondary metabolite saponins169. For the

steroids synthesis, (cholesterol, and insect moulting hormone 20-hydroxyecdysone)

insects requires sterol because they are not able to synthesize sterol structures by

themselves170 and thus get them from their different foodstuff (cholesterol or

phytosterols from plants as precursors). The secondary metabolite saponins form

insoluble complexes with sterols containing foods, there by prevent their absorption.

Similarly, if larvae feed on a food (saponin-rich), the ingested food saponins may

form complex with cholesterol in their body, and thus hinder the biosynthesis of

ecdysteroids necessary for ecdysis171. Moreover, it was also observed and reported in

the literature that the action of saponins, could be opposed by adding of surplus

cholesterol or plant sterols to the diet containing saponins and sapogenins. The

insecticidal activity of saponins also depends upon their sugar moieties attached to

them. So it is expected that glycosylated saponins exert their insecticidal action only

when they are hydrolyzed in the insect gut by enzyme glycosidases169,172.

From this discussion, we postulate that the extract and fractions particularly those

containing saponins that are not very polar, cannot exert their insecticidal action

because these extract/fractions along with saponins are also rich in sterols, steroids

and phytoecdysteroids especially 20-hydroxyecdysone (as we isolated from CHL-fr).

So in the presence of excess of these sterols, steroids, and phytoecdysteroids in the

tested samples dilute the action of saponin and thus the fractions were ineffective

against the tested insets. In addition, those fractions which contain saponins and are

polar, the glycosylated saponins (as we isolated borassoside E, govanoside A from


142

BuOH-fr) could be the reason for its ineffectiveness because it has been reported that

glycosylated saponins are less active than sapogenins against red flour beetles172.

Table-3.22: Insecticidal activity of Cr. MeOH-Ext and its subsequent fractions of T.

govanianum rhizomes against insect Tribolium castaneum

Test Sample

Tribolium castaneum

Total-No.-of

insects-

No. of-survived

insects

No. of-dead

insects

%

Mortality

Cr. MeOH-Ext 10 10 0 0

n-Hex-fr 10 10 0 0

CHL-fr 10 10 0 0

EtOAc-fr 10 10 0 0

BuOH-fr 10 10 0 0

Aq-fr 10 10 0 0

Negative control 10 10 0 0

Positive control* 10 0 10 100

*Permethrin

Table-3.23: Insecticidal activity of Cr. MeOH-Ext and its subsequent fractions of T.

govanianum rhizomes against insect Rhyzopertha dominica

Test Sample

Rhyzopertha dominica

Total No.-of

insects

No. of-survived

insects

No.-of dead

insects

%

Mortality

Cr. MeOH 10 10 0 0

n-Hex-fr 10 10 0 0

CHL-fr 10 10 0 0

EtOAc-fr 10 10 0 0

BuOH-fr 10 10 0 0

Aq-fr 10 10 0 0

Negative control 10 10 0 0

Positive control* 10 0 10 100

*Permethrin


143

3.5.1.8 Brine shrimp cytotoxic activity of Cr. MeOH-Ext and fractions

The results of brine shrimp cytotoxic activity of Cr. MeOH-Ext and fractions are

given in Table 3.24 and Fig. 3.21. Based on LD50 (µg/mL), the cytotoxicity of test

samples was in following order; Aq-fr > BuOH-fr > EtOAc-fr > Cr. MeOH-Ext. The

maximum cytotoxic activity was observed for aqueous fraction and BuOH-fr with

LD50 (µg/mL) of 256 (138-466) and 260 (141-469) respectively. Similarly, EtOAc-fr

and Cr. MeOH-Ext also showed moderate cytotoxicity. The LD50 values for n-Hex-fr

and CHL-fr were found greater than 1000 µg/mL, and thus were considered less

cytotoxic in this study.

The brine shrimp test is an economical and frequently used for detection of cytotoxic

potential173,174. It has been reported that a positive co-relation exists between brine

shrimp cytotoxicity and human nasopharyngeal carcinoma175,176. The findings of this

study suggested significant cytotoxicity against brine shrimp, which is further

validated from its high potential for PC-3 and HeLa cell lines in MTT assay (Table

3.18 and 3.19). Therefore, these results provide a prediction for some potent

anticancer compounds in the extract and fractions, which is up to some extant verified

through isolation of steroids and steroidal glycosides (diosgenin, pennogenin,

borassoside E and govanoside A) that showed anticancer potential.


144

Table 3.24: Brine shrimp cytotoxic activity of Cr. MeOH-Ext and fractions of T. govanianum rhizomes

- Dose (µg/mL) Total No. of-

shrimps

No. of-dead

shrimps

Number of survivors Samples LD50

(µg/mL)

Std.-Drug

LD50--(µg/mL)

Cr. MeOH-Ext 1 10 30 3 27

720 (364-1484)*

Etoposide

7.46 2 100 30 6 24 3 1000 30 16 14 n-Hex-fr

1 10 30 - 30

> 1000

7.46 2 100 30 1 29 3 1000 30 3 27 CHL-fr

1 10 30 - 30 > 1000

7.46 2 100 30 3 27 3 1000 30 14 16 EtOAc-fr

1 10 30 2 28

627 (320- 1273)

7.46 2 100 30 6 24 3 1000 30 18 12

BuOH-fr

1 10 30 2 28

260 (141-466)

7.46 2 100 30 13 17 3 1000 30 22 08

Aq-fr 1 10 30 4 26

256 (138-466)

7.46 2 100 30 10 20 3 1000 30 24 06

*95% confidence limits in parenthese


145

Figure-3.21: Percent cytotoxic effect of Cr. MeOH-Ext and fractions of T.

govanianum rhizomes.


146

3.5.1.9 Muscle relaxant (Spasmolytic) activity of Cr. MeOH-Ext

The results of in vitro effect of Cr. MeOH-Ext on isolated rabbit jejunum are

presented in Fig. 3.22 and 3.23. The results indicated that both spontaneous as well as

high K+ induced contractions of isolated preparations (rabbit jejunum) were

completely inhibited by the Cr. MeOH-Ext at a dose of 5 and 3 mg/mL, comparable

to the standard drug verapamil (calcium channel blocker) which inhibited the high K+

induced and as well as spontaneous contractions at a dose of 3 and 1 µM, respectively

as shown in Fig. 3.22.

Furthermore, in Ca++ channel blocking (CCB) effect, the Cr. MeOH-Ext at a dose of

(0.1-0.3 mg/mL) . caused-rightward shift of the-Ca++concentration response curves

(CRCs) exhibited the suppression of the maximum contraction effect, comparable to

that caused by standard drug verapamil (0.03-0.1 µM) as given in Fig. 3.23.

Isolated rabbit jejunum is a spontaneously contracting gut preparation177, allowing to

examine the relaxant effect, without induced contraction. The Cr. MeOH-Ext when

tested on rabbit jejunum, inhibited high K+ induced and spontaneous contractions in

the rabbit jejunum. It has been reported in different studies, that the

relaxant/spasmolytic activity of medicinal plants is generally mediated through

Ca++channels blockage178-180. Therefore, in order to investigate the CCB mechanism for

spasmolytic effect of T. govanianum rhizomes, the extract was tested on high K+

induced contractions in the jejunum. It is well known, that high K+ (>30 mM) level by

virtue of opening the voltage dependent L-type Ca++channels induced smooth muscle

contractions, and consequently permitting the inward movement of extracellular Ca++

which ultimately results a contractile effect181. therefore, the agents causing


147

inhibition of high K+ induced contractions are regarded as Ca++influx inhibitors182. As

the Cr. MeOH-Ext relaxed the high K+ induced contractions in a analogous pattern just

like standard Ca++antagonist verapamil183, indicating its calcium Ca++antagonist effect.

This effect (Ca++antagonist) was further confirmed when the Cr. MeOH-Ext shifted

the Ca++ concentration response curves to the right with inhibition of the max response,

analogous to the standard drug verapamil.

It has been reported that Ca++ antagonists have beneficial effect in gut disorders, such

as abdominal cramps and diarrhea177. Therefore, the findings (relaxant effect

mediated through Ca++ channel blocking) of this study, rationalize the medicinal use

of this plant in conditions related to hyperactive gut disorders like diarrhea etc and

this also justifies its ethnomedicinal use in diarrhea.


148

Figure 3.22: Inhibitory effects of T. govanianum rhizomes Cr. MeOH-Ext and

verapamil in isolated rabbit jejunum preparations. Values expressed as mean ± SEM.

Figure 3.23: Ca++ concentration response curves (CRCs) of Cr. MeOH-Ext and

verapamil in isolated rabbit jejunum preparations. Values expressed as mean ± SEM

(S1= Cr. MeOH-Ext).


149

3.5.1.10 Antiglycation activity of Cr. MeOH-Ext and fractions

Antiglycation effect of Cr. MeOH-Ext and its fractions of T. govanianum rhizomes

were tested for antiglycation potential. The result presented in Table 3.25, indicated

that all the tested samples exhibit weak antiglycation effect at test concentration of 0.5

mg/mL, with maximum in Cr. MeOH-Ext (16% inhibition).

One of the harmful effects of hyperglycemia is the formation of sugar derived

molecules called advanced glycation end products (AGEs). These AGEs are

heterogeneous group of substances, formed from the reaction (non enzymatic) of

reducing sugars with free amino groups of proteins, nucleic acids and lipids. The

formation of AGEs is highly accelerated in condition like diabetes, where glucose

molecules are available in excess amount184. Thus glycation is also one of the

important factors to be kept in mind, while treating diabetic complications. At present,

a lot of plant extracts, fractions and purified compounds have been tested and verified

for suppression of AGEs formation. Moreover, several scientific reports demonstrate

that antiglycation effect of plant extract and fractions can be attributed to the presence

of phenolic compounds185-187.

Since the Cr. MeOH-Ext and its fractions contains little amount of phenolic

compounds or even deprived of it, and are very rich in steroids and saponins as clear

from the phytochemical tests and isolated compounds (chloroform and BuOH-frs),

Our observation in this context could be due to the absence or little quantity of these

phenolic compounds.


150

Table 3.25: Antiglycation activity of Cr. MeOH-Ext and fractions at dose of 0.5

mg/mL

3.5.1.11 β-glucoronidase inhibitory activity of Cr. MeOH-Ext and fractions

The Cr. MeOH-Ext and its fractions were screened for β-glucoronidase inhibition.

The results are presented in Table 3.26, based on the IC50± SD (µg/mL), the Cr.

MeOH-Ext (140.8 ± 3.8) and BuOH-fr (196.2 ± 1.9) exhibited a moderate level of

enzyme inhibitory activity in comparison to the standard D-saccharic acid-1,4-

lactone, IC50 of 46.7 ± 2.2. The CHL-fr and EtOAc-fr were found less effective in

this study.

β-glucuronides enzyme (present in animal, plants, and bacteria) catalyzes the

hydrolysis of β-glucuronides conjugates of exogenous and endogenous compounds

produced in the body188. Increased level of β-glucuronides in blood has been observed

in liver injury. Over expression of this enzyme may also be related to liver cancer,

arthritis and AIDS. Similarly, β-glucuronidase of intestinal bacteria in human and rats

Samples Percent inhibition IC50 ± SD (µg/mL)

Cr. MeOH-Ext 16

-

n-Hex-fr- 4 - CHL-fr-- 11 - EtOAc-fr- 3 -

BuOH-fr--- 6 -

Aq-fr 9 -

Rutin (Positive control) 96.2 26.4 ± 0.28


151

are connected to colon cancer65. In addition to this β-glucuronidase of bacteria, which

are found in the biliary tract is also associated with gall stone formation189,190. As the

phytochemical analysis (Table 3.2) revealed the presence of steroidal glycosides,

flavonoids and saponins, which explain the moderate β-glucuronidase inhibitory

activity in the extract and fractions. Therefore the rhizomes of T. govanianum may

prove, to be effective in the treatment of various inflammatory disorders, prostate and

cervical cancer and also in the management of liver and colon cancer associated with

an increase activity of β-glucuronidase.

Table 3.26: IC50 values (µg/mL) of extract and fractions of T. govanianum rhizomes

and reference drug against β-glucuronidase

*Results are mean ± SEM of three independent experiments.

D-saccharic acid-1, 4-lactone was used as positive control.

Samples IC50± SD (µg/mL)

Cr. MeOH-Ext 140.8 ± 3.8

CHL-fr >200

EtOAc-fr >200

BuOH-fr 196.2 ± 1.9

D-saccharic acid-1, 4-lactone 46.7 ± 2.2


152

3.5.1.12 α-Chymotrypsin inhibitory activity of Cr. MeOH-Ext and fractions

The Cr. MeOH-Ext and its subsequent fractions were screened for α-chymotrypsin

inhibition. The results (Table 3.27) indicated that none of the tested samples inhibited

the enzyme, therefore it is concluded that, this enzyme is not the pharmacological

target of T. govanianum rhizomes extract and fractions thereof.

Table 3.27: α-Chymotrypsin inhibitory activity of Cr. MeOH-Ext and fractions

*Chymostatin was used as positive control

3.5.1.13 Thymidine phosphorylase inhibitory activity of isolated compounds

The isolated pure compounds from T. govanianum rhizomes were screened for

thymidine phosphorylase inhibition, in order to check their affinity towards this

enzyme. The results of this assay (Table 3.28) revealed that all the tested compounds

were inactive, and none of them exhibit significant inhibition.

Thymidine phosphorylase, is an enzyme involved in the pyrimidine metabolism, is an

angiogenic factor that is over expressed in various cancerous conditions, in which it is

involved in angiogenesis, metastasis and cancer cell growth. It has been reported that

inhibitors of this enzyme suppresses tumor growth by increasing the percentage of

Test sample Concentration (µM) Inhibition (%)

Cr. MeOH-Ext 500 Inactive

n-Hex-fr 500 Inactive

CHL-fr 500 Inactive

EtOAc-fr 500 Inactive

BuOH-fr 500 Inactive

Aq-fr 500 Inactive

Chymostatin 125 98.4


153

apoptotic cells and inhibiting angiogenesis191,192. As the compounds, did not show any

thymidine phosphorylase inhibitory activity, thus indicating that this enzyme is not

the pharmacological target of tested compounds.

Table 3.28: Thymidine phosphorylase inhibitory activity of isolated compounds

*7-Deazaxanthine was used as positive control.

3.5.1.14 Acetylcholinesterase inhibitory activity of Cr. MeOH-Ext and fractions

The Cr. MeOH-Ext and its subsequent fractions were screened for acetylcholineterase

(AChE) inhibition. The results indicated that the extract and fraction exhibited a weak

activity with maximum 19% inhibition in BuOH-fr at test concentration of 500 µg/mL

(Table 3.29). The Cr. MeOH-Ext showed 16% inhibition, and all other fractions were

found less active. The weak inhibition of the tested samples may be attributed to the

presence of steroids and steroidal glycosides as these secondary metabolites possess

AChE inhibitory activity193,194. Thus further confirmation is necessary to isolate

AChE enzyme inhibitory compounds.

Compounds Concentration (mM) Inhibition (%)

Borassoside E 0.5 Inactive

Pennogenin 0.5 Inactive

Diosgenin 0.5 Inactive

7-Deazaxanthine 0.5 99.0


154

Table 3.29: Acetylcholineteras inhibitory activity of Cr. MeOH-Ext and fractions

Samples Concentration Percent inhibition

Cr. MeOH-Ext 250-µg/mL 11

500-µg/mL 16

n-Hex-fr

250-µg/mL -

500-µg/mL -

CHL-fr

250-µg/mL 8

500-µg/mL 11

EtOAc-fr

250-µg/mL 14

500-µg/mL 19

BuOH-fr

250-µg/mL 13

500-µg/mL 19

Aq-fr

250-µg/mL 6

500-µg/mL 10

Galanthamine (positive control)

100-µg/mL 66

200-µg/mL 78


155

3.5.2 In vivo biological studies

3.5.2.1 Acute toxicity

In order to determine the safety profile, the Cr. MeOH-Ext of T. govanianum

rhizomes was tested for toxicity at different concentrations. A dose dependent

increase in percent lethality was observed with the Cr. MeOH-Ext as shown in Table

3.30. Maximum lethality was observed at a dose of 6000 mg/kg while safety was

observed up to the dose of 500 mg/kg. From the LD50 value (2030.42 mg/kg), it was

clear that the Cr. MeOH-Ext was safe to the maximum of dose selected for the study.

Table 3.30: Acute toxicity of Cr. MeOH-Ext of T. govanianum rhizomes

Concentration

(mg/kg )

Total number of mice = 6 %

lethality LD50 (mg/kg) No. of mice

dead

No. of mice

lived

150 0 6 0

2030.42 (1488.84-3069.01)*

500 0 6 0 1000 1 5 16 1500 2 4 33 3000 5 1 83 6000 6 0 100

*95% confidence limit in parentheses

3.5.2.2 Anti-inflammatory activity of Cr. MeOH-Ext and fractions

In carrageenan-induced paw edema model, the anti-inflammatory responses of T.

govanianum rhizomes extract and its succeeding solvent soluble fractions are

presented in Table 3.31 and Fig. 3.24A-D. The results indicate that the Cr. MeOH-

Ext and fractions at dose of 25, 50 and 100 mg/kg body weight, exhibit significant

anti-inflammatory activity comparable to that of control anti-inflammatory drug,

diclofenac.


156

The Cr. MeOH-Ext and its fractions at dose of 100 mg and 200 mg/kg showed an

anti-inflammatory potential, which became significant (P < 0.01) at second phase, 2 h

after the administration of carrageenan and was retained in the second phase with a

maximum percent inhibition of 64.67 ± 4.055a, 63.50 ± 0.500a, 47.50 ± 0.500 aand

72.67 ± 3.930a by Cr. MeOH-Ext, CHL-fr, EtOAc-fr and n-BuOH-fr, respectively.

The extract and its subsequent fractions showed a relatively weak activity in the early

phase of inflammation (0- 1.5 or 2 h). However, it was good in case of BuOH-fr at a

dose of 100 mg/kg.

Carrageenan-induced paw oedema is a valuable model to evaluate the involvement of

mediators concerned in vascular changes related with acute inflammation195. Within

first hour following carrageenan injection, oedema is induced by the release of

mediators i.e. histamine, bradykinin and 5-HT, but not by prostaglandins (PG). These

mediators, following activation of their receptors on endothelial cells, activate

constitutive nitric oxide synthase (cNOS) activation resulting in the production of

nitric oxide (NO). In mice following the intraplantar injection of carrageenan, TNF-

cx, IFN-y as well as cytokines such as IL-1 and IL-2 are produced196. COX-2 is also

induced within 2 h after carrageenan administration197. The NOS and COX pathways

appear to operate together to augment the inflammatory response. The dual inhibition

of PG and NO obtained with NOS inhibitors might be accounted for their marked

anti-inflammatory effect195.

Therefore, from our results we can conclude that the inhibitory effect of Cr. MeOH-

Ext and fractions on carrageenan induced edema inflammation could be due to the

dual inhibition of enzyme cyclo-oxygenase and later inhibition of prostaglandin


157

synthesis. This significant in vivo anti-inflammatory effect of the tested samples were

also endorsed by the in vitro inhibition of ROS in oxidative burst assay (Table 3.20)

by all Cr. MeOH-Ext, subsequent fraction and isolated compounds. Furthermore, the

significant anti-inflammatory potential, particularly of Cr. MeOH-Ext and BuOH-fr

may be attributed to the presence of steroids and steroidal glycosides (saponins). In

phytochemical study of these samples we confirmed the presence of steroids and

saponins, which endorsed these anti-inflammatory findings. Furthermore, it is worthy

to note, that the significant anti-inflammatory, antinociceptive, and antipyretic

activities of plant extract are associated with steroids and saponins133,198-200.


158

Table 3.31: Anti-inflammatory activity Cr. MeOH-Ext and fractions of T. govanianum rhizomes against carrageenan induced

paw edema in mice

Percent inhibition is expressed as mean ± SEM. a = P< 0.001, b = P< 0.01, c = P< 0.05 compared to control

Sample Dose

(mg/kg)

Inhibition (%)

1sth 2

ndh 3

rdh 4

thh 5

thh

Diclofenac 10 27.33 ± 2.7a 47.67 ± 0.8a 67.00 ± 2.5a 70.67 ± 0.6a 74.33 ± 0.6a

Cr. MeOH-Ext

50 8.00 ± 1.7c 18.00 ± 1.7b 22.63 ± 2.3a 32.00 ± 5.2a 34.00 ± 5.2a

100 12.00 ± 1.1b 44.33 ± 4.6a 65.00 ± 4.6a 63.67 ± 1.7a 66.33 ± 4.6a

200 19.00 ± 2.3b 44.67 ± 3.8a 62.67 ± 3.7a 62.67 ± 1.4a 64.67 ± 4.0a

CHL-fr

25 4.00 ± 1.0 8.50 ± 0.5c 21.35 ± 3.5b 35.00 ± 1.0a 42.50 ± 2.5a

50 4.03 ± 2.0b 18.80 ± 1.0b 20.30 ± 2.0b 43.00 ± 1.0a 58.00 ± 1.0a

100 12.12 ± 3.0c 21.50 ± 0.5b 43.50 ± 3.5a 45.00 ± 1.0a 63.50 ± 0.5a

EtOAc-fr

25 3.500 ± 0.5 9.500 ± 0.5c 18.00 ± 3.0b 18.50 ± 1.5b 39.50 ± 0.5a

50 10.50 ± 0.5c 16.00 ± 1.0b 29.50 ± 0.5a 30.50 ± 2.5a 44.50 ± 3.5a

100 9.500 ± 0.5c 22.50 ± 1.5b 30.50 ± 0.5a 33.00 ± 2.0a 47.50 ± 0.5a

BuOH-fr

25 12.94 ± 3.7c 14.33 ± 5.5b 35..25 ± 1.0a 58.00 ± 1.5a 61.00 ± 3.4a

50 18.80 ± 3.4a 46.67 ± 4.9a 68.67 ± 2.3a 66.00 ± 1.7a 69.00 ± 4.3a

100 25.67 ± 1.7a 47.67 ± 2.1a 68.67 ± 1.4a 70.33 ± 2.6a 72.67 ± 3.9a


159

Figure 3.24A: Anti-inflammatory effect of Cr. MeOH-Ext on carrageenan induced

paw edema. a = P< 0.001, b = P< 0.01, c = P< 0.05 compared to control.

Figure 3.24B: Anti-inflammatory effect of CHL-fr on carrageenan induced paw

edema. a = P< 0.001, b = P< 0.01, c = P< 0.05 compared to control.


160

Figure 3.24C: Anti-inflammatory effect of EtOAc-fr on carrageenan induced paw


Figure 3.24D: Anti-inflammatory effect of BuOH-fr on carrageenan induced paw



161

3.5.2.3 Analgesic activity of Cr. MeOH-Ext and fractions

3.5.2.3.1 Tonic-visceral chemical induced nociception

The Cr. MeOH-Ext and its subsequent solvent soluble fractions were tested for tonic-

visceral chemical induced nociception in animal model of mice. The results as shown

in Table 3.32 and Fig. 3.25, indicated that the 50 and 100 mg/kg doses significantly

attenuated the acetic acid induced writhes for n-Hex-fr (P< 0.01, P< 0.001), CHL-fr

(P< 0.01, P< 0.05), EtOAc-fr (P< 0.01), BuOH-fr (P< 0.001, P< 0.05), Aq-fr (P<

0.001), and Cr. MeOH-Ext (P< 0.001, P< 0.01). The antinociceptive activity was

comparable to the standard drug diclofenac, which significantly relieved (P< 0.001)

the tonic visceral chemical induced nociception.

Table 3.32: Antinociceptive effect of T. govanianum rhizomes Cr. MeOH-Ext and its

fractions in tonic-visceral chemical induced nociception

Sample Dose (mg/kg) Number of writhes ± SEM

Saline 10 ml/kg 29.5 ± 2.5

Diclofenac 50 4.5 ± 2.5***

n-Hex-fr 50 7.5 ± 4.5**

100 5.5 ± 3.5***

CHL-fr 50 8.0 ± 1.0**

100 14.5 ± 1.5*

EtOAc-fr 50 10.5 ± 3.5**

100 7.0 ± 4.0**

BuOH-fr 50 5.0 ± 4.0***

100 12.5 ± 4.5*

Aq-fr 50 6.5 ± 3.5***

100 4.0 ± 1.0***

Cr. MeOH-Ext 50 6.5 ± 1.5***

100 7.5 ± 0.5**

*P< 0.05, **P< 0.01, ***P< 0.001 compared to saline treated group, n = 6.


162

Figure 3.25: Antinociceptive effect of T. govanianum rhizomes in tonic-visceral

chemical induced nociception. Values were expressed as mean ± SEM. ANOVA

followed by Dunnett’s post hoc test. *P< 0.05, **P< 0.01, ***P< 0.001 compared to

saline treated group, n = 6. Dic = Diclofenac.

3.5.2.3.2 Thermal induced nociception

The Cr. MeOH-Ext and its subsequent fractions were examined for thermal induced

nociception. The results as shown in Table 3.33, indicated that after 30 minutes

(min), compared to normal saline treatment, significant attenuation of thermal induced

nociception was observed with n-Hex-fr at 50 mg/kg (P< 0.05) and 100 mg/kg (P<

0.01), EtOAc-fr at 100 mg/kg (P< 0.05), BuOH-fr at 100 mg/kg (P< 0.01), and Aq-fr

at 50 and 100 mg/kg (P< 0.01). After 60 min, significant analgesic effect was

observed with Hex-fr at 50 mg/kg (P< 0.01) and 100 mg/kg (P< 0.001), EtOAc-fr at

50 and 100 mg/kg (P< 0.01), BuOH-fr at 100 mg/kg (P< 0.01), Aq-fr at 50 mg/kg (P<

0.01) and 100 mg/kg (P<0.001) and Cr. MeOH-Ext at both doses (P< 0.01) (Fig.

3.26A). Likewise, significant protection against thermal induced nociception after 90

min was observed with all the tested doses of n-Hex-fr (P< 0.01), EtOAc-fr (P< 0.05),


163

Aq-fr (P< 0.05) as well as with100 mg/kg dose of BuOH-fr (P< 0.05) and Cr. MeOH-

Ext (P< 0.01) (Fig. 3.26C). Moreover, the analgesia produced after 120 min was

significant for all the tested doses of n-Hex-fr (P< 0.01), EtOAc-fr (P< 0.05, P< 0.01)

and Aq-fr (P< 0.01), and for only the 100 mg/kg dose of BuOH-fr (P< 0.01) and Cr.

MeOH-Ext (P< 0.05) (Fig. 3.26D).

For evaluating the analgesic potential of drugs, hot plate test is one of the most

common tests used. The mice paws are very sensitive to heat at temperatures, not that

high to damage the skin. The mice responses to heat are jumping, licking or

withdrawal the paws. These responses take prolonged time after administration of

centrally acting analgesic drugs. Thus, the hot plate test model measures the different

response to acute nociceptive or non-inflammatory inputs and is one of the models

normally used for studying central antinociceptive activity201.

In the tonic visceral chemical induced nociception model, the injection of acetic acid

into the peritoneal cavity of mice induces, contraction tracked by extension of the

hind limbs called writhing. This visceral pain model is simple, reliable and rapid for

investigation of peripheral analgesics. In our findings, the significant inhibition of

writhing by extract and fractions suggested peripherally mediated analgesic activity

which is based on the connection of the model with stimulation of peripheral

receptors especially the local peritoneal receptors at the surface of cells lining the

peritoneal cavity202.

The chemical constituent's analyses of T. govanianum rhizomes showed that it is

saponin rich part, and from its fractions (chloroform and butanol) steroids and

saponins have also been isolated in this study. It has been reported in the literature

that saponins are the major chemical constituents in medicinal preparations


164

responsible for most of the anti-inflammatory and analgesic activities. Recent reports

indicate that most saponins can suppress the expression of iNOS and COX-2, thus

resulted in a noticeable lowering of prostaglandin E2 levels203,204. Thus, the findings of

this study are further endorsed by the reported literature.

In conclusion, T. govanianum rhizomes Cr. MeOH-fr and fractions exhibit significant

peripheral and central antinociceptive activities, which support the traditional

analgesic uses of this plant species.


165

Table 3.33: Antinociceptive effect of Cr. MeOH-Ext and fractions of T. govanianum rhizomes in thermal induced nociception

Sample Dose (mg/kg) 30 min 60 min 90 min 120 min

Saline 10 ml/kg 13.3 ± 0.4 13.3 ± 0.5 13.0 ± 1.0 13.6 ± 0.3

Tramadol 30 29.0 ± 1.0*** 28.3 ± 1.7*** 28.4 ± 1.4** 26.0 ± 1.0**

n-Hex-fr 50 23.4 ± 0.7* 25.6 ± 2.3** 29.0 ± 1.0** 26.4 ± 2.1**

100 25.3 ± 0.8** 25.8 ± 1.1*** 29.7 ± 0.2** 25.3 ± 1.6**

CHL-fr 50 17.2 ± 1.5 21.4 ± 1.4 22.6 ± 2.0 20.3 ± 0.4

100 20.3 ± 2.1 19.3 ± 0.1 16.9 ± 2.8 19.0 ± 1.9

EtOAc-fr 50 17.5 ± 0.9 24.3 ± 2.6** 24.2 ± 1.1* 23.9 ± 3.3*

100 22.2 ± 0.1* 23.0 ± 0.9** 23.7 ± 0.6* 25.6 ± 2.5**

BuOH-fr 50 13.7 ± 1.9 18.1 ± 0.6 18.2 ± 4.4 18.2 ± 0.1

100 27.5 ± 1.3** 23.4 ± 0.5** 25.8 ± 4.2* 25.4 ± 2.5**

Aq-fr 50 24.6 ± 3.2** 25.6 ± 2.8** 23.6 ± 0.6* 25.0 ± 0.1**

100 24.8 ± 2.0** 27.1 ± 2.9*** 25.8 ± 1.4* 24.2 ± 1.3**

Cr. MeOH-Ext 50 18.7 ± 3.9 23.9 ± 0.1** 18.4 ± 0.5 16.7 ± 1.0

100 21.3 ± 2.5 25.2 ± 1.2** 26.6 ± 3.4** 22.9 ± 2.7*

Values expressed as mean ± SEM. *P< 0.05, **P< 0.01, ***P< 0.001 compared to saline treated group, n = 6


166

Figure 3.26A: Antinociceptive effect of Cr. MeOH-Ext and fractions after 30 min

* = P< 0.05, ** = P< 0.01, *** = P< 0.001 compared to control.

Figure 3.26B: Antinociceptive effect of Cr. MeOH-Ext and fractions after 60 min



167

Figure 3.26C: Antinociceptive effect of Cr. MeOH-Ext and fractions after 90 min


Figure 3.26D: Antinociceptive effect of Cr. MeOH-Ext and fractions after 120 min


Conclusion

168

Concluding Remarks

Trillium govanianum is an indigenous medicinal herb of Pakistan. The rhizome of this

plant species is used as crude drug in Indo-Pak to cure different ailments. From this

Ph.D. work/project, which is based on ethno-medicinal, phytochemical and biological

investigations of crude drug “rhizomes” we concluded that;

� The rhizomes of T. govanianum are rich source of compounds like steroids,

glycosides and steroidal glycosides (saponins). It also contains trihydroxy fatty

acids and phytoecdysteroids.

� The presence of these phytochemicals and biological testing of crude extract

and its sub-fractions, the crude drug rhizomes validated and proved the

reported folkloric ethnomedicinal uses scientifically.

� The rhizomes of this plant species can be effectively used in the treatment of

cancers, inflammatory disorders, algesia, diarrhoea, abdominal cramps,

bacterial and fungal infections.

� It is recommended that the concerned authorities and Government prepare

conservation strategy to safeguard this valuable asset (T. govanianum herb) of

this region.

Further detail phytochemical and biological studies are required, as the rhizomes of

this plant species possesses great potential for discovery of new lead compounds,

effective in the treatment and management of cancers, inflammatory disorders and

infectious diseases.

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