Ph. D Thesis by SHAFIQ UR RAHMAN

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

DEPARTMENT OF PHARMACY UNIVERSITY OF PESHAWAR, PESHAWAR, PAKISTAN 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

DEPARTMENT OF PHARMACY UNIVERSITY OF PESHAWAR, PESHAWAR, PAKISTAN 2015

Acknowledgements

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

Compassionate AlMighty 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 IndoPak 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 G as Chromatography Mass Spectrometry ...... 35 2.4.6 GCMS 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 LiebermannBurchard 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 CHCl 3 fraction ...... 47 2.11 .2 Isolation of compound from butanol fraction ...... 53 2.12 Characterization of isolated compounds...... 55 2.12 .1 Character ization of hexadecanoic acid (compound 1)...... 55 2.12.2 Characterization of βsitosterol (c ompound 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 (c ompound 5) ...... 59 2.12.6 Characterization of govanic acid (compound 6)...... 60 2.12.7 Characterization of 20hydroxy ecdysone and 5,20dihydroxy ecdysone 61 (compound 7 and 8) ...... 2.12.8 Characterization of 5, 20hydroxy ecdysone (compound 8)...... 62 2.12.9 Characterization of borassoside E (compound 9)...... 63 2.12 .10 Characterization of govanoside A (c ompound 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 Sm ooth muscle relaxant activity ...... 71 2.13 .1.11 βGlucu ronidase inhibitory activity ...... 72 2.13.1.12 αChymotrypsin 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 Antiinflammatory 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 nhexane 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 comp ound 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 elucidati on 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 a ctivity 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. MeOHExt and fractions...... 130 3.5 .1.4 Anticancer activity ...... 133 3.5.1.4.1 Anticancer activity of Cr. MeOHExt 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 Antiinflammatory activity of Cr. MeOHExt and fractions...... 136 3.5.1.5.2 Antiinflammatory activity of isolated compounds...... 136 3.5 .1.6 Anti leishmanial activity of Cr. Me OH Ext and fractions ...... 139 3.5.1.7 Insecticidal activity of Cr. MeOHExt and fractions...... 140 3.5.1.8 Brine shrimp cytotoxic activity of Cr. MeOHExt and fractions...... 143 3.5.1.9 Muscle relaxant (Spasmolytic) activity of Cr. MeOHExt...... 146 3.5 .1.10 Antiglycation activity of Cr. MeOH Ext and frac tions ...... 149 3.5 .1.11 βGlucu ronidase inhibitory activity of Cr. MeOH Ext and fractions ...... 150 3.5.1.12 αChymotrypsin inhibitory activity of Cr. MeOHExt 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 Antiinflammatory activity of Cr. MeOHExt and fractions ...... 155 3.5.2.3 Analgesic activity of Cr. MeOHExt and fractions ...... 161 3.5 .2.3.1 Tonic visceral chemical induced nociceptio n...... 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 the ir 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 20hydroxyecdysone 61 Table 2.9 Characterization of 5,20dihydroxyecdysone 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. gov anianum 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 nHexfr of T. govanianum rhizomes 91 Table 3.4 1HNMR and 13 CNMR data of compound 1 93 Table 3.5 1HNMR and 13 CNMR data of compound 2 95 Table 3.6 1HNMR and 13 CNMR data of compound 3 97 Table 3.7 1HNMR and 13 CNMR data of compound 4 99 Table 3. 8 1HNMR and 13 CNMR data of compound 5 102 Table 3. 9 1HNMR and 13 CNMR data o f compound 6 105 Table 3.1 0 1HNMR and 13 CNMR data of compound 7 109 Table 3.1 1 1HNMR and 13 CNMR data of compound 8 111

Table 3.1 2 1HNMR and 13 CNMR data of compound 9 115 Table 3.1 3 1HNMR and 13 CNMR data of comp ound 10 120 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 Anti fungal 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 Anti cancer activity of T. govanianum rhizome s Cr. MeOH Ext, fractions and reference drug (doxorubicin) against cancer cells 135 Table 3.19 Anticancer activity of compounds isolat ed 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 IC 50 values (g/mL) of extract and fractions of T. govanianum rhizomes 151 Table 3. 27 αChymotrypsin inhibitory activity of Cr. MeOH Ext and fractions 152 Table 3. 28 Thymidine phosphorylase inhibitory activity of isolated compounds 153 Table 3. 29 Acetylch oline steras e inhibitory activity of Cr. MeOH Ext and its fractions 154 Table 3. 30 Acute toxicity of Cr. MeOH Ext of T. govanianum rhizomes 155

Table 3. 31 Anti inflammatory activity Cr. MeOH Ext and fractions of T. govanianum rhizomes against carrageenan induced paw edema in 158 mice Table 3.32 Antinociceptive effect of T. govanianum rhizomes Cr. MeOH Ext and its fractions in tonicvisceral chemical induced 161 nociception Table 3.33 Antinociceptive effect of Cr. MeOHExt 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 3.6 Chemical structure of compound 2 95

Figure 3.7 Chemical structure of compound 3 97

Figure 3. 8 Chemical structure of compound 4 99

Figure 3. 9 Chemical structure of compound 5 102

Figure 3.10 Chemical structure of compound 6 105

Figure 3.11 Linked scan measurements in compound 6 106

Figure 3.12 Major fragmentation and 1H1HCOSY correlations in compound 106 6 Figure 3. 13 Chemical structure of compound 7 109

Figure 3. 14 Chemical structure of compound 8 111

Figure 3.15 Chemical structure of compound 9 116

Figure 3.16 Key HMBC correlations in compound 9 116

Figure 3.17 Chemical structure of compound 10 121

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

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. MeOHExt and verapamil in isolated rabbit jejunum preparations 148

Figure 3.23 Ca ++ concentration response curves (CRCs) of Cr. MeOHExt and verapamil in isolated rabbit jejunum preparations 148

Figure 3.24 A Anti inflammatory effect of Cr. MeOH Ext on carr ageenan induced paw edema 159

Figure 3.24B Antiinflammatory effect of CHLfr on carrageenan induced paw edema 159

Figure 3.24C Antiinflammatory effect of EtOAcfr on carrageenan induced paw edema 160

Figure 3.24 D 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. MeOHExt and fractions after thirty minutes 166

Figure 3.26B Antinociceptive effect of Cr. MeOHExt and fractions after sixty minutes 166

Figure 3.26 C Antinociceptive effect of Cr. MeOH Ext and fractions after ninety minutes 167

Figure 3.26D Antinociceptive effect of Cr. MeOHExt and fractions after one hour and twenty minutes 167

List of Abbreviations

Cr. MeOHExt Crude Methanolic extract nHexfr nhexane fraction CHLfr Chloroform fraction EtOAcfr Ethyl acetate fraction BuOHfr Butanol fraction Aq fr Aqueous fraction WH O World Health Organization NP Natural Products ADHD Attention deficit hyperactivity disorder CVS Cardio vascular system DPPH 2,2diphenyl1picrylhydrazyl BHT Butylated hydroxytoluene MeOD Methanol CDCl 3 Chloroform CC Column Chromatography TLC Thin Laye r Chromatography GCMS Gas Chromatography Mass Spectrometry pp t 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 1HNMR Proton Nuclear Magnetic Resonance 13 CNMR Carbon Nuclear Magnetic Resonance HRFAB MS High Resolution Fast Atomomic Bombardment Mass Spectrometry DMSO Dimethyl sulfoxide MTT 3[4,5dimethylthiazol2yl]2,5diphenyltetrazolium bromide COX Cyclooxygenase AChE Acetylcholinesterase AIDS Acquired Immune Deficiency Syndrome ROS Reactive oxygen species CCB Calc ium channel blocker AGEs Advanced glycation end products

List of Publications from Thesis

1 ShafiqurRahman, 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 ): 577583. Impact factor; 1.05

2 ShafiqurRahman, 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 ): 270275. doi:10.1016/j.steroids.2015.10.013 . Impact factor; 2.63

3 ShafiqurRahman, Muhammad Ismail, Muhammad Khurram and Inam ul Haq."Pharmacognostic and ethnomedicinal studies on Trillium govanianum. " Pakistan Journal of Botany Vol. No. 47(SI) (2015 ): 187192. Impact factor; 0.82

4 ShafiqurRahman, 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

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. MeOHExt) as well as in chloroform fraction (CHLfr), ethyl acetate fraction (EtOAcfr) and butanol fraction (BuOHfr). GC/MS analyses of nhexane fraction ( nHexfr) identified twelve (12) compounds, including 70% unsaturated and 30% saturated fatty acids.

Using different chromatographic techniques, eight compounds from CHLfr and two compounds from BuOHfr were isolated. The chemical structures of isolated compounds were elucidated using latest spectroscopic and spectrometric techniques i.e. 1HNMR, 13 CNMR, COSY, NOESY, HSQC, HMBC, EIMS, FAB, HRFAB,

HREIMS, IR and UV. Among these compounds, two [govanic acid ( 6) and govanoside A ( 10 )] were new, while the rest were [hexadecanoic acid ( 1), βsitosterol

Summary

(2), stigmasterol (3), diosgenin ( 4), pennogenin (5), 20hydroxyecdysone ( 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 IC 50 ± SD (µg/mL) values, significant antiproliferative activity against HeLa cells was observed for CHLfr (0.84 ± 0.16), EtOAcfr (1.41 ±

0.08) and BuOHfr (1.60 ± 0.34). Similarly, all fractions exhibited good cytotoxicity against PC3 cell lines. The isolated compounds, govanoside A (1.74 ± 0.12 against

PC3; 0.51 ± 0.26 against HeLa) and borassoside E (2.34 ± 0.21 against PC3; 0.67 ±

0.22 against HeLa) exhibited significant cytotoxicity compared to standard doxorubicin (1.69 ± 0.28 against PC3; 0.50 ± 0.15 against HeLa). In DPPH free radical scavenging assay, higher scavenging capacity was observed in nHexfr and

CHLfr compared to other fractions, however the scavenging capacity of all fractions was less than ascorbic acid.

In antifungal assay, the Cr. MeOHExt 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. MeOHExt and fractions exhibited moderate antibacterial potentials against the tested gram positive and gram negative bacteria.

Furthermore, the Cr. MeOHExt exhibited good potential against Leishmania major .

Suppression of oxidative burst (OB) was evaluated through luminol enhanced chemiluminescence assay. Based on IC 50 ± SD (µg/mL), the BuOHfr (16.53 ± 7.54)

Summary exhibited significant inhibition of OB for the whole blood followed by Cr. MeOHExt

(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. MeOHExt completely inhibited both spontaneous as well as high K + induced contractions of isolated rabbit jejunum preparations indicating its spasmolytic effect.

The Cr. MeOHExt 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. MeOHExt and fractions were found inactive against the test insects i.e. Tribolium castaneum and Rhyzopertha dominica.

In enzyme inhibition assays, αchymotrypsin 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. MeOHExt (IC 50 ; 140.8 ± 3.8) and BuOHfr (196.2 ± 1.9) exhibited moderate

βglucuronidase and weak acetylcholineterase inhibitions.

In in vivo carrageenan induced paw edema model, significant antiinflammatory effect was observed for Cr. MeOHExt and fractions (50 and 100 mg/kg). Similarly, the Cr.

MeOHExt and fractions significantly attenuated the tonicvisceral 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

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 medicines 1.

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 earth 2. 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 medicines 3.

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 sources 4. Even at the dawn of 21 st century, 11% of the 252 drugs, considered as basic and essential by the WHO were exclusively of flowering plant origin 2.

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 remedies 5.

Chapter 1 Introduction

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 purposes 6.

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 purpose 8.

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 pathogens 9. 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 world 10 .

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

Chapter 1 Introduction raw form or in the form of decoctions, infusions or extracts 11 . 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 antiinflammatory agent and in the treatment of burns and other wounds based on their antihemorrhagic 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 antioxidant, anticancer, hepatoprotective, anti inflammatory, antibacterial and antiviral properties 15 . 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 agents 16 . It is worth to mention, that many saponins have been reported to exhibit significant antiinflammatory, antinociceptive and antipyretic activities as well as many other diverse potentials such as antiallergic and anticancer 17,18 . 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 today 19,20 . 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 .

Chapter 1 Introduction

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 (ethnomedicinal) 22 . In fact numerous drugs have entered the international pharmacopoeias through the study of ethnopharmacology and traditional medicine 23 . 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 anticancer, antiparasitic, antifungal, antibacterial, analgesics, antiinflammatory and antihistaminic compounds 2426 .

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 practice 27 . 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 classes 28 . 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

Chapter 1 Introduction around the 1940s to date, of the 175 small molecules 85 actually being either natural products or their direct derivatives 2. 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 sources 29

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 Antimalarial Reserpine Rauwolfiа serpentinа Sarpagandha Antihypertensive Salicin Salix alba White willow Analgesic Santonin Artemisa maritima Sea wormwood Ascaricide Silymarin Silybum marianum Blessed milk Hepatotonic thistle Teniposide Podophyllum paltatum Mayapple, Anticancer Theophylline Camellia sinensis Tea plant Bronchodialator Tubocurarine Chondodendron Curare Parasympatholytic Tomentosum Yohimbine Pausinystalia johimbe Yohimbe Aphrodisiac

Chapter 1 Introduction

Table 1.2: Natural product derived drugs in market since 2005 29

Year Trade Generic Name/ Classification Therapeutic Uses Name (Active compound) 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

Chapter 1 Introduction

1.4 Biodiversity of IndoPak Subcontinent

The IndoPak 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 homoeopathy 30 . 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) IranoTuranian (45% of species); (ii) SinoHimalayan (10% of species); (iii) SaharoSindian (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 wounds 32 . Nearly 250,000 higher plants species have been reported from around the world, in which nearly 10% are found in the HindukushHimalayas ranges, of which twothird are of medicinal significance 8.

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 pharmaceuticalbased health care. However, it is a common observation that many medicinal plants growing in this country remain taxonomically unidentified and

Chapter 1 Introduction 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 preclinical 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 1846 33 . 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,

Chapter 1 Introduction rarely white, yellow, or red. Seeds sometimes afforded with an scarlet sarcotesta 34,35 .

Schilling and Farmer reported that the Trilliaceae family, which showed an arcto tertiary distribution, encompass of five genera 36 . 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 2locular, equal or longer than the filaments, superior ovary, proximal segment 3locular, 3 or 6lobed, 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,

Chapter 1 Introduction as well as in western North America 37 . Most of the Trillium species are related with the deciduous forests (ancient ArctoTertiary), 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 America 38 . In Pakistan the genus is represented by single species i.e T. govanianum 39 .

1.6.1 Species of genus Trillium 4042

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 .

Chapter 1 Introduction

Table 1.3: Species of genus Trillium

No Species with Occurrence Flowering Specific characteristics common Name period 1 Trillium erectum North AprJun • Rhizomes short, thick, America praemorse • Wake robin • Petals typically red, • Red trillium maroon, or dark purple • Petals usually present in same plane as sepals 2 Trillium nivale United MarAprl • Rhizomes stout, short, States praemorse • Snow trillium (U.S.) • Bracts blade bluish • Dwarf white trillium green • Scapes six gonal in cross section 3 Trillium undulatum Wisconsin AprJun • Rhizomes short, (U.S.) horizontal, stout • Painted trillium • Petals with distinctive • Painted lady dark red colour • Bracts are strongly petiolate 4 Trillium pusillum United MarMay • Rhizomes thin, States horizontal, branched • Dwarf trillium • Bracts very short, • Least trillium subsessilepetiolate • Sepals about as large and prominent aspetals, • petals spreading ascendingly 5 Trillium Mountains AprJun • Rhizomes thick and grandiflorum of Virginia. short (North • Petals erected basally • Great white trillium America) • Ovary ovate to • White wakerobin lanceolate, white or rarely pink 6 Trillium ovatum North MarMay • Rhizomes horizontal to America semi erect, short, stout, • Western white praemorse trillium • Bracts sessile

Chapter 1 Introduction

7 Trillium luteum Joseph AprMay • Rhizomes brownish, rivers and horizontal, short, thick, • Yellow trillium elsewhere in not fragile, praemorse • Yellow toadshade Michigan, • Petals oblanceolate to (U.S.) lanceolate, greenish yellow to lemon yellow in color • Flower odor strongly of lemon 8 Trillium petiolatum North AprMay • Rhizomes erect, very America deep often, praemorse • Purple trillium • Petals long lasting • Roundleaved • Ovary, erect to trillium incurved, light maroon to red, purple, or greenish to yellowish, flat, linear to lanceolate 9 Trillium simile North AprMay • Rhizome forming America clumps, stout, • Sweet white trillium praemorse • Petals creamy white in color • Flowers facing upward, odour sweet like apple 10 Trillium lancifolium North FebMay • Rhizome white, America horizontal, very brittle, • Lance leaved trillium internodes elongated • Petals linear to narrowly spatulate 11 Trillium Korea,Japan AprJun • Rhizome stout and kamtschaticm Russia, straight N. America • Stems tufted and China • 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

Chapter 1 Introduction

12 Trillium tschonoskii Bhutan, JulyAug • Rhizome stout, Japan, horizontal Korea and • Stems tufted China • Leaves sessile, rhombic to orbicular or to broadly rhombic • Anthers 3 to 4 mm, shorter than or equal filaments 13 Trillium taiwanense Taiwan, MayJun • Rhizomes creeping, China stout • Stem solitary • Leaves shortly petiolate, ovate to broadly ovate • Stamens short • Anthers 1to 1.5 mm 14 Trillium parviflorum North MarMay • Rhizomes brownish, America horizontal to erect, • Small flowered thick, praemorse, not trillium brittle • Petals linear to linear lanceolate, white, rarely purplish basally 15 Trillium govanianum Bhutan, AprAug • Rhizomes greyish India, Nepal thick. China and • Adventitious roots Pakistan numerous, fibrous • Stem up to 30 cm tall • Leaves shortly petiolate, ovate or ovate to cordate • Fruit red, globose berry

Chapter 1 Introduction

1.6.2 Phytochemical profiling of genus Trilliu m

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 derivatives 4345 . 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 spirost5en3ol C27 H42 O3 (diosgenin) 46

(25 S)spirost5ene3β, C27 H42 O5 44,47 17α,27triol

(25 S)3β,17α C33 H52 O10 dihydroxyspirost5en27 yl βDglucopyranoside 44

(25 S)17α ,27 C33 H52 O10 dihydroxyspirost5en3 β yl βDglucopyranoside 44

Chapter 1 Introduction

(25 S) C45 H72 O19 27[( βD glucopyranosyl)oxy]17α hydroxyspirost5en3β yl O α Lrhamnopyranosyl (1→2) β D glucopyranoside 44

(25 S)27 [( β C33 H52 O10 Dglucopyranosyl)oxy]17 α,27dihydroxyspirost5 en3yl O(4 Oacetyl α L rhamnopyranosyl)(1 →2) β Dglucopyranoside 44

(25 S)27 [( β D C51 H82 O24 glucopyranosyl)oxy] 17α,27 dihydroxyspirost5en3 β Dglucopyranosyl(1 →6) O[ αLrhamnopyranosyl (1 →2)] βDglucopyranoside 44

(25 S)17 α, 27 C41 H64 O15 dihydroxyspirost 5en3βyl O(4Oacetyl α Lrhamnopyranosyl) (1 →2) β Dglucopyranoside 44

Chapter 1 Introduction

(25 S)17 α,27 C39 H62 O14 dihydroxyspirost5en3 β yl O α L rhamnopyranosyl (1 →2) βD glucopyranoside 48

(25 R)17α hydroxyspirost C39 H62 O13 5en3 βyl O α L rhamnopyranosyl (1→2)β Dglucopyranoside 49

(25 R)17α hydroxyspirost C39 H62 O13 5en3 β yl O α L rhamnopyranosyl(1 →4) β Dglucopyranoside 50

(25 R)17α hydroxyspirost C45 H72 O17 5en3βyl OαL rhamnopyranosyl(1 →2) O[αLrhamnopyranosyl (1 →4)] β Dglucopyranoside 49

Chapter 1 Introduction

(25 R)17α hydroxyspirost C45 H72 O17 5en3βyl OαL rhamnopyranosyl(1 →4) O[αLrhamnopyranosyl (1 →4)] β Dglucopyranoside 44,51

(25 R) C51 H82 O21 17αhydroxyspirost5en 3βyl OαL rhamnopyranosyl(1→2) O[OαL rhamnopyranosyl(1→4) aLrhamnopyranosyl (1→4)]αD glucopyranoside 49

(25 R)spirost5en C39 H62 O13 3βyl OαL rhamnopyranosyl(1→2) βDglucopyranoside 49

(25 R)spirost5en3βyl C45 H72 O16 OαLrhamnopyranosyl (1→2)O[αL rhamnopyranosyl(1→4)] βDglucopyranoside 49

Chapter 1 Introduction

(25 R)spirost 5en 3β yl C51 H82 O20 OαLrhamnopyranosyl (1 →2) O[OαL rhamnopyranosyl(1 →4) αLrhamnopyranosyl (1 →4)] β Dglucopyranoside 49

(25 R)26[βD C53 H88 O22 glucopyranosyl]oxy]22 α methoxyfurost5 en3 β yl OαL rhamnopyranosyl(1→2) O[αLrhamnopyranosyl (1→4)]β Dglucopyranoside 44

(25 R)26[β D C47 H78 O19 glucopyranosyl] oxy]17 α hydroxy22β methoxyfurost5en3β yl OαLrhamnopyranosyl (1→2)β Dglucopyranoside 52

Chapter 1 Introduction

(25 R)26 [β D C53 H88 O23 glucopyranosyl]oxy]17 α hydroxy 22amethoxyfurost 5en3β yl OαL rhamnopyranosyl(1 →2) O[αLrhamnopyranosyl (1→4)]β Dglucopyranoside 52

(25 R)26 [β D C45 H70 O18 glucopyranosyl] oxy]3β [( OαL rhamnopyranosyl(1 →2) βDglucopyranosyl) oxy]cholesta5,17diene 16,22dione49

lO[2,3,4triOacetyl C45 H61 AcO αLrhamnopyranosyl 20 (1→2)4OacetylαL arabinopyranosyl] 21Oacetyl epitrillenogenin 53

(25 S)27 C39 H62 O14 hydroxypenogenin [3O

αLrrhamnopyranosyl (1→2)OβD glucopyranoside]53

Chapter 1 Introduction

(25 R)27 C39 H62 O14 hydroxypenogenin 3Oα Lrhamnopyranosyl (1 →2) OβD glucopyranoside 48

penogenin 3OαL C39 H62 O13 rhamnopyranosyl(1→2) OβDglucopyranoside 49

penogenin 3 OβD C45 H72 O18 glucopyranosyl

(1 →6) [OαL[ rhamnopyranosyl(1 →2)] 48 Oβ[[[ Dglucopyranoside

penogenin 3[Oβ[D C33 H52 O9 49 glucopyranoside [

Chapter 1 Introduction

48 ,54 deox ytrillenoside C47 H70 O23

spirost 5ene 3,17 diol C27 H42 O4 (Pennogenin) 46

(10 R,6 E)7,11ddimethyl3 C21 H38 O9

mehyl 3ene 6dodecaene 1,2,10,11tetraol 10Oβ Dglucopyranoside 48

(10 R,6 E)3,7,11 C21 H38 O8

trimethyl1,6ddodecadien

3,10, 11 11triol 10oO glucopyranoside 48

(10 R,6 E)3,7,11 C21 H38 O8

trimethyl1, 66 6dodecadien

3,10,11 10 triol 10O glucopyranoside 48

7,11 dimethyl C27 H46 O12

3m methylene1,6

dodecadien 10 10,11diol 10

oOβD (1 →4) glucopyranosylOβ Dglucopyranoside 55

Chapter 1 Introduction

55 ,56 methyl ferulorate C11 H12 O4

48 astragalin C21 H20 O11

48 βecdysone C27 H44 O7

26 26 OβdDglucopyranosyl C45 H74 O19

(22,[25 R)[furost5eene 3β,17α,22,26tetraol 3O αLrhamnopyranosyl (1 →2) OβD glucopyranoside 49

26dOβaD23 glucopyranosyl C51 H84 O23 (22,25 R)

furost5eene3β,17α, 22,26tetraol 3OαL rhamnopyranosyl

(1→ 42)[OαL

[rhamnopyranosyl(1→04)] OβDglucopyranoside 48

Chapter 1 Introduction

a26 OβDglucopyranosyl C51 H80 O22 17(20) dehydrokryptogenin 3OαLrhamnopyranosyl (1 →2) [OαL rhamnopyranosyl (1→4)]Oβ Dglucopyranoside 48

26OβDglucopyranosyl C45 H70 O18 17(20) dehydrokryptogenin 3OαLrhamnopyranosyl (1→2)Oβ Dglucopyranoside 49

3,4,5,7 C15 H10 O6 tetrahydroxyflavone 45

quercetin 3Orutinoside; C26H28 O16 [3OβL rhamnopyranosyl(1→6) βDglucopyranoside] 45

Chapter 1 Introduction

kaempferol C33 H40 O20 3Oαrhamnosyl(1 →2) O [αrhamnosyl (l →6)] βglucoside 45

phydroxymethyl benzyl C8H10 O2 alcohol 57

3,7,11trimethyl3,9,11 C18 H36 O6 trihydroxyl1,6 dodecadiene glycerol 57

2methyl 3,4 dihydroxy C7H12 O6 hexanedioic acid 57

Chapter 1 Introduction

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 years 58,59 . 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 pains 60 . The anticancer activity of nBuOH extract has also been reported 59 . The rhizomes of T. erectum called beth roots have been used in folk medicine for the treatment of hemorrhages from uterus, urinary tract and lungs 61 . The cytotoxic activity of the isolated compounds (spirostanol saponins and furostanol saponins) from T. erectum against

HL60 leukemia cells has been reported 44 . Dried underground parts of T. tschonoskii were used as a folk medicine to remove carbuncles and to ameliorate pains, etc 62 . The marked inhibitory action against COX2 production in macrophagocytes of the mouse abdominal cavity by isolated compounds has also been reported 38 . 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 carrageenan 63 . T. tschonoskii can improve learning and memory, and these effects were associated with enhancement of antioxidase expression 64 . The antifungal activity of ethanol extract of the rhizomes and above ground portion of T. grandiflorum has also been reported 46 .

Chapter 1 Introduction

Table 1.5: Reported biological activities of genus Trillium

Activity Part Extract/Isolated Source used compounds anti metastatic effect against Rhizome Isolated Trillium tschonoskii colorectal cancer cells 58 compounds antibacterial and anti Rhizome Extracts Trillium tschonoskii oxidant 65 antifungal 46 Rhizome Extracts and Trillium grandiflorum fractions antifungal 46 Rhizome Isolated Trillium grandiflorum compounds cytotoxicity against HL60 Rhizome Isolated Trillium erectum human promyelocytic compounds leukemia cells 44 cytotoxicity against human Rhizome Isolated Trillium tschonoskii lung cancer cells 66 compounds cytotoxicity against Rhizome Isolated Trillium tschonoskii adriamycin resistant breast compound cancer cells 58 cytotoxicity against malignant Rhizome Isolated Trillium tschonoskii sarcoma cells 67 compounds cytotoxicity against malignant Rhizome Extract/fractions Trillium pendulum neuroblastoma 68 cytotoxicity against multi Rhizome Isolated Trillium tschonoskii drug resistance (MDR) compounds hepatocellular carcinoma cells 69 expression of antioxidase of Rhizome Extracts Trillium tschonoskii aging rat induced with haloperidol 70 analgesic, antiinflammatory Rhizome Extract/fractions Trillium tschonoskii and thrombisis effects 63 learning and memory Rhizome Extract/fractions Trillium tschonoskii enhancement effect 64

Chapter 1 Introduction

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 aliments 71 . 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

Chapter 1 Introduction

1.7.1 Plant Morphology

T. govanianum plant is a perennial herb about 1220 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, 0.53.0 cm in diameter, and seeds are abundant, rhombus, with a pulpy lateral appendage.

Flowering periods is from april to august 39,40 .

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 Shangla 39 .

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 disorders7173 . The powdered rhizomes is also used as anthelmintic 74 .

Chapter 1 Introduction

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

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, Germany

Carrageenan Si gma Chemical Co, St L ouis, MO, USA DPPH Waka Ltd. Japan Butylated hydroxytoluene (BHT) SigmaAldrich, Germany Dimethyl Sulfoxide (DMSO) SigmaAldrich, 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

Chapter 2 Materials and Methods

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 modelMPA100, while optical rotations were determined by digital Polarimeter modelJASCO DIP360.

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, modelU3900/3900H (fully automated) was used for UV spectroscopic analysis of isolated compounds.

2.4.2 IR technique

Infrared Spectrometer, model JASCO 302A was used for IR spectroscopic analysis of isolated compounds.

Chapter 2 Materials and Methods

2.4.3 Mass technique

For the mass spectral studies of isolated compounds, the Mass Spectrophotometer modelMAT311A 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 1HNMR and 13 CNMR spectra of isolated compounds, NMR Spectrometer

(Bruker; AMX600, AM400 and AM300) was used. The 1HNMR spectra were

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

o o Polarization Transfer (DEPT) experiments were executed at 90 and 135 for determination of CH 3, CH 2, and CH moieties of isolated compounds.

2.4.5 Gas Chromatography and Gas ChromatographyMass 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 m 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 A, 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 390 75 .

Chapter 2 Materials and Methods

2.4.6 GCMS identification of components

Identification of proximate fatty acid components of the nonpolar fraction ( nhexane) was based on the computer evaluation of mass spectra of sample through NISTbased

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 techniques 76 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; 70230 mesh size, flash silica; 230400 mesh size) was used as a stationary phase. Mobile phase used includes various organic solvents either alone or in combination like, nhexane, 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 precoated 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 preparative thin layer chromatography for purification of isolated compounds.

Chapter 2 Materials and Methods

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 100110 oC, 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.

Chapter 2 Materials and Methods

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 method 77 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 shadedried 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, Laborota4010) fitted with recirculation chiller (Minichiller,

Huber wH1 plus) and a heating bath (B490) at 40 oC, 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. MeOHExt) was reserved, and the

Chapter 2 Materials and Methods remaining extract was further fractionated on the base of their solvent affinity (solid liquid partition) into nhexane ( nHexfr; 81 g), chloroform (CHLfr; 94 g), ethyl acetate (EtOAcfr; 85 g) and butanol (BuOHfr; 105 g) fractions. The remaining fraction, after the above process was considered as aqueous (Aqfr; 107 g) fraction 79 .

The complete process is documented in Scheme 2.1 .

Powder rhizomes of T. govanianum (7 Kg)

Extraction with

MeOH

Crude MeOH Extract (512 g) For biological activities (35 g) Fractionation

nhexane Chloroform Ethyl acetate Butanol Aqueous fraction fraction fraction fraction fraction (81 g) (94 g) (85 g) (105 g) (107 g)

Scheme 2.1: Extraction and fractionation of T. govanianum rhizomes

Chapter 2 Materials and Methods

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

34 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 23 min. Finally the section was mounted with

Canada balsam to make them permanent and was examined under Olympus Digital microscope (MICD). The powder drug was also treated on glass slide, mounted with

Canada balsam and was subjected to microscopic examinations 76,80 .

2.9 Physicochemical parameters

The various physicochemical 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,

Chapter 2 Materials and Methods

weight of total ash Percent total ash value = × 100 weight of crude drug taken

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 (ashless), 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 acidinsoluble 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 100105 oC.

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

Chapter 2 Materials and Methods

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 nhexanes 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 nhexane, chloroform, ethyl acetate, butanol were performed according to the recommended standard protocols 81,8385 .

Chapter 2 Materials and Methods

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 bluegreen or violet color indicates the presence of flavonoids in the test sample.

Chapter 2 Materials and Methods

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 bluegreen 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.

Chapter 2 Materials and Methods

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 LiebermannBurchard test

To the plant extract/fraction, few drops of acetic anhydride was added. Concentrated sulphuric acid (H2SO 4) 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 sample 86 .

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

Chapter 2 Materials and Methods acid come beneath the mixture. A brown ring appearance, indicates the presence of the cardiac glycoside 87 .

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.

Chapter 2 Materials and Methods

2.11 Isolation of compounds

2.11.1 Isolation of compounds from CHCl 3 fraction

The chloroform (CHCl 3) 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 chromatography 88 . Using nhexane and EtOAc solvent system as mobile phase in increasing order of polarity, the fraction was further fractionated into eleven sub fractions (CF ACF K) [Scheme 2.2] .

The sub fraction CF B obtained with 2040% chloroform in nhexane were re chromatographed over silica gel eluting with mixture of nhexane and EtOAc in increasing order of polarity yielded five sub fractions (CF B(a) CF B(e)). The sub fraction

CF B(b) obtained with 510% EtOAc/ nhexane 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 nhexane; 13 mg), compound 2 (5% EtOAc in nhexane; 16 mg) and compound 3 (5% EtOAc in nhexane; 11 mg) [Scheme 2.3] .

The sub fraction CF E obtained with 2040% EtOAc in chloroform was re chromatographed over silica gel eluting with mixture of EtOAc and nhexane in increasing order of polarity yielded compound 4 (20% EtOAc in nhexane; 94 mg), compound 5 (20% EtOAc in nhexane; 21 mg) and compound 6 (60% EtOAc in n hexane; 132 mg) [Scheme 2.4] .

The sub fraction CF H obtained with 5% MeOH in EtOAc was rechromatographed over silica gel eluting with mixture of MeOH and EtOAc in increasing order of

Chapter 2 Materials and Methods polarity yielded five sub fractions. The sub fraction CF Hh 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] .

Chapter 2 Materials and Methods

Chloroform fraction (CHL.fr) (62 g)

Column chromatography (CC) with

gradient elution system

HexCHL (0100%) CHLEtOAc (0100%) and EtOAcMeOH (0100%)

hexane

25% MeOH 100% MeOH 100% 100% EtOAc 100% 4080%CHl hexin 2040% EtOAc in CHL

CF CF A CF C CF E CF G CF I K

CHL hex

EtOAc

Ac in

100%CHL 80% EtO 80% % in MeOH 2040% 2040% CHL in 5% EtOAcinMeOH 50 60

CF B CF D CF F CF H CF J

Scheme 2.2: Fractionation of chloroform fraction

Chapter 2 Materials and Methods

CF B (Sub fraction)

Column chromatography (CC) with gradient elution

510% E tOAc in nhexane

CF B(b)

(CC) (gradient elution)

2% E tOAc in nhexane 5% E tOAc in nhexane

Compound 1 Compound 2 Compound 3 (13 mg) (16 mg) (11 mg)

Scheme 2.3: Isolation of compounds from sub fraction (CF B)

Chapter 2 Materials and Methods

CF E (Sub fraction)

Column chromatography (CC) with gradient elution

20% E tOAc in nhexane 60% E tOAc in nhexane

Compound 4 Compound 5 Compound 6 (94 mg) (21 mg) (132 mg)

Scheme 2.4: Isolation of compounds from sub fraction (CF E)

Chapter 2 Materials and Methods

CF H (Sub fraction)

Column chromatography (CC) with gradient elution

5% MeOH in EtOAc

CF Hh (Sub:Sub Fr )

Preparative TLC with

solvent system MeOH : EtOAc (1:9)

Compound 7 Compound 8 (13 mg) (18 mg)

Scheme 2.5: Isolation of compounds from sub fraction (CF H)

Chapter 2 Materials and Methods

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 (BF ABF E). The sub fraction, BF A which was obtained with 10% MeOH in

EtOAc was rechromatographed 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 BF B which was obtained with 20% MeOH in

EtOAc was rechromatographed over silica gel, eluted with mixture of MeOH and

EtOAc in increasing order of polarity yielded sub fractions (BF Ba BF Be ). The sub fraction, BF Bc which was obtained with 30% MeOH in EtOAc when analyzed by TLC and cerric sulphate reagent showed few prominent spots. Further rechromatography 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] .

Chapter 2 Materials and Methods

Butanol fraction (BuOH.fr) (35 g)

Column chromatography (CC) with gradie nt elution

10% MeOH in EtOAc 20% MeOH in EtOAc

BF A BF B (Sub fraction) (Sub fraction) (CC) with gradient elution

510 % MeOH in EtOAc (CC) gradient elution Compound 9 (48 mg)

30% MeOH in EtO Ac 20 % MeOH in EtOAc 10 % MeOH in EtOAc

BF Bc BF Bb BF Ba

(CC)

Compound 10 20% MeOH in EtOAc

(32 mg)

Scheme 2.6 : Isolation of compounds from butanol fraction

Chapter 2 Materials and Methods

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, CF B(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 C16 H32 O2 HR ESIMS ( m/z ) 256.2361 UV activity UV inactive on TLC Melting point 6064 oC Isolated quantity 13 mg Solubility at room temperature Chloroform/Methanol 1 HNMR (CDCl 3; 600 MHz) (Table 3.4) 13 CNMR (CDCl 3; 150 MHz) (Table 3.4)

Chapter 2 Materials and Methods

2.12.2 Characterization of βsitosterol (compound 2)

Compound 2 was isolated and purified as colorless amorphous powder from the chloroform soluble sub fraction, CF B(b) . The compound was identified and characterized through modern spectroscopic data analysis and was confirmed as β sitosterol.

Table 2.3: Characterization of βsitosterol

Parameters Observations Physical state Colorless amorphous powder

Molecular formula C29 H50 O HR ESIMS ( m/z ) 414.3621 UV activity UV inactive on TLC Melting point 135138 oC Isolated quantity 16 mg Solubility at room temperature Chloroform 1 HNMR (CDCl 3; 600 MHz) (Table 3.5) 13 CNMR (CDCl 3; 150MHz) (Table 3.5)

Chapter 2 Materials and Methods

2.12.3 Characterization of stigmasterol (compound 3)

Compound 3 was isolated and purified as colorless amorphous powder from the chloroform soluble sub fraction, CF B(b) . The compound was identified and characterized through modern spectroscopic data analysis and was confirmed as stigmasterol.

Table 2.4: Characterization of stigmasterol

Parameters Observations Physical state Colorless amorphous powder

Molecular formula C29 H48 O HR ESIMS ( m/z ) 412.3624 UV activity UV inactive on TLC Melting point 161168 oC Isolated quantity 11 mg Solubility at room temperature Chloroform 1 HNMR (CDCl 3; 600 MHz) (Table 3.6) 13 CNMR (CDCl 3; 150 MHz) (Table 3.6)

Chapter 2 Materials and Methods

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, CF E. This compound was identified and characterized through modern spectroscopic data analysis and was confirmed as diosgenin.

Table 2.5: Characterization of diosgenin

Parameters Observations Physical state White to off white needles/powder

Molecular formula C27 H42 O3 HR ESIMS ( m/z ) 414.3042

26 o [α] D 124 (in MeOH) UV activity UV inactive on TLC Melting point 204207 oC Isolated quantity 94 mg Solubility at room temperature Chloroform 1 HNMR (CDCl 3; 600 MHz) (Table 3.7) 13 CNMR (CDCl 3; 150 MHz) (Table 3.7)

Chapter 2 Materials and Methods

2.12.5 Characterization of pennogenin (compound 5)

Table 2.6: Characterization of pennogenin

Parameters Observations Physical state White powder

Molecular formula C27 H42 O4 HR ESIMS ( m/z ) 430.2960

26 o [α] D 99.8 (in MeOH) UV activity UV inactive on TLC Melting point 206208 oC Isolated quantity 21 mg Solubility at room temperature Chloroform 1 HNMR (CDCl 3; 600 MHz) (Table 3.8) 13 CNMR (CDCl 3; 150 MHz) (Table 3.8)

Chapter 2 Materials and Methods

2.12.6 Characterization of govanic acid (compound 6)

Compound 6 was isolated and purified as white powder from the chloroform soluble subfraction, CF E. 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

Parameters Observations Physical state White powder

Molecular formula C18 H34 O5 HR ESIMS ( m/z ) 330.4566

26 [α] D 52.8 (in MeOH) UV activity UV inactive on TLC Melting point 7883 oC Isolated quantity 132 mg Solubility at room temperature Methanol 1 HNMR (CD 3OD; 600 MHz) (Table 3.9) 13 CNMR (CD 3OD; 150 MHz) (Table 3.9)

Chapter 2 Materials and Methods

2.12.7 Characterization of 20hydroxyecdysone and 5,20dihydroxyecdysone

(compounds 7 and 8)

The sub fraction, CF H obtained from CHCl 3 soluble fraction was subjected to column chromatography (CC) over silica gel using gradient solvent system ( nhexane /

EtOAc). The sub fraction (CF Hh ) 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, 20hydroxyecdysone ( 7) and 5,20 dihydroxyecdysone (8).

Table 2.8: Characterization of 20hydroxyecdysone

Parameters Observations Physical state White powder

Molecular formula C27 H44 O7 HR ESIMS ( m/z ) 480.5527 UV activity UV active on TLC Melting point 243245 oC Isolated quantity 13 mg Solubility at room temperature Methanol 1 HNMR (CD 3OD; 600 MHz) (Table 3.10) 13 CNMR (CD 3OD; 150 MHz) (Table 3.10)

Chapter 2 Materials and Methods

2.12.8 Characterization of 5, 20hydroxyecdysone (compound 8)

Table 2.9: Characterization of 5,20dihydroxyecdysone

Parameters Observations Physical state White powder

Molecular formula C27 H44 O8 HR ESIMS ( m/z ) 496.5510 UV activity UV active on TLC Melting point 248251 oC Isolated quantity 18 mg Solubility at room temperature Methanol 1 HNMR (CD 3OD; 600 MHz) (Table 3.11) 13 CNMR (CD 3OD; 150 MHz) (Table 3.11)

Chapter 2 Materials and Methods

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 subfraction, 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

Parameters Observations Physical state White to off white amorphous powder

Molecular formula C45 H72 O16 HR FAB + (m/z ) 869.4725

26 o [α] D 47.2 (in MeOH) UV activity UV inactive on TLC Melting point 263266 oC Isolated quantity 48 mg Solubility at room temperature Methanol 1 HNMR (CD 3OD; 600 MHz) (Table 3.12) 13 CNMR (CD 3OD; 150 MHz) (Table 3.12)

Chapter 2 Materials and Methods

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

Parameters Observations Physical state White amorphous powder

Molecular formula C56 H88 O29 HR FAB + (m/z ) 1225.5426

26 o [α] D 139 (in MeOH) UV activity UV inactive on TLC Melting point 276281 oC Isolated quantity 32 mg Solubility at room temperature Methanol 1 HNMR (CD 3OD; 600 MHz) (Table 3.13) 13 CNMR (CD 3OD; 150 MHz) (Table 3.13)

Chapter 2 Materials and Methods

2.13 Biological studies

2.13.1 In vitro biological activities

The following in vitro biological activities were performed on Cr. MeOHExt, its subsequent solvent soluble fractions and isolated compounds.

2.13.1.1 Antibacterial activity

The Cr. MeOHExt 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 method 89 . Cr. MeOHExt 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. MeOHExt its subsequent solvent soluble fractions and isolated compounds was performed with slight modification of previously reported method 90 . Shortly, samples were serially diluted using 20%

Chapter 2 Materials and Methods dimethyl sulfoxide in 0.9% saline and transferred in duplicate to 96well flatbottom 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 × 10 3 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 ×10 4 CFU/mL. The fungal inocula were added to the samples to achieve a final volume of 200 L. 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 (BioTek, 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

g/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. MeOHExt and its subsequent fractions following previously reported method 91 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 g/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

Chapter 2 Materials and Methods 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;

AI − AII Percent DPPH = × 100 AI

AI = absorbance of the reaction (control)

AII = absorbance of the sample.

2.13.1.4 Anticancer activity

The cytotoxic activity of Cr. MeOHExt its subsequent fractions and isolated compounds was determined by the MTT assay, according to previously reported method 92,93 on two cancer cell lines, i.e. HeLa (cervical cancer cells) and PC3

(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 % CO 2, for 24 h, in a flask. Cells were plated (1 × 10 5 cell/mL) in 96well flat bottom plates and incubated for 24 h for cell attachment. Various concentrations of test sample/fractions ranging from 1.2520 µM were added into the well and incubated for

48 h. A 50 µL MTT [3(4, 5dimethylthiazol2yl)2,5diphenyltetrazolium 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

Chapter 2 Materials and Methods using a microplate reader (Spectra Max 340; Molecular Devices, CA, USA). Finally,

IC 50 values were calculated. For positive control, doxorubicin was used.

2.13.1.5 Antiinflammatory activity

The in vitro anti inflammatory potential of Cr. MeOHExt, its subsequent fractions and isolated compounds was determined through Luminolenhanced chemiluminescence assay following well define protocol previously reported 94 .

Briefly, 25 µL of the diluted whole blood HBSS ++ (HanksBalanced 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 carriedout in 96well plate (whitehalf area), incubated (for 15 min.) at 37ºC in thermostatic chamber of luminometer. On completion of incubation, 25 µL each SOZ (serum opsonizedzymosan) 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. MeOHExt 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 logphase of growth promastigotes were centrifuged (2000 rpm) for 10 min, maintaining the same

Chapter 2 Materials and Methods experimental conditions and were washed (three times) with saline. Fresh culture medium was used to obtain parasites final density by dilution (1×10 6 cells mL1).

Medium was added to different 96 wells micro titerplate, 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 IC 50 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, brineshrimp eggs

(Artemia salina ; 1 mg) was added to it and cover the Jar by aluminum foil, to darken it. The Jar was kept at 25 oC 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 control 97 .

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

Chapter 2 Materials and Methods

(3 mL). A 90 mm filter paper was positioned in petri dishes and loaded with test sample (1019.10 g/cm 2). 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 g/cm 2) and acetone were used as positive control and negative control respectively. By comparison results of test sample with positive control percent mortality was calculated 95,96,98.

By using the following formula percentage mortality was determined.

Number of living insects in test Percent motality = 100 − × 100 Number of living insects in control

2.13.1.9 Protein antiglycation activity

For the in vitro antiglycation assay, previously reported method 99,100 was used to determine the antiglycation potential of Cr. MeOHExt, 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 96well 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

96well plate was observed for fluorescence at wavelength of 330440 nm on microtitre plate spectrophotometer (Spectra Max M2, Molecular Devices, USA).

Chapter 2 Materials and Methods

Rutin was used for positive control. The percent inhibition values were calculated by the following formula;

luorescence of test sample Percent Inhibition = 1 − × 100 luorescence of the control group

The samples that exhibit 50% or above percent inhibition, were processed for IC 50 value calculation by using Ezfit software (Perrella Scientific, USA).

2.13.1.10 Smooth muscle relaxant activity

The muscle relaxant (spasmolytic) potential of Cr. MeOHExt was studied on isolated rabbit jejunum preparations in according to the previously reported protocol 101. In an

organ bath filled with Tyrode’ s solution (37°C) and aerated with natural air, rabbit jejunum (11.5 cm) was suspended. Intestinal contractions were recorded with the help of isometric transducer attached with Powerlab Data Acquisition System connected to computer executing Labchart 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 (antispasmodic) potential in isolated rabbit jejunum tissue.

For the study of Ca ++ channel blocking (CCB) effect, previously reported method was followed with slight modification 27 . 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 dosereliant inhibitory contractions. To validate the Ca ++ antagonistic activity of the testing sample, the

Chapter 2 Materials and Methods

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; NaHCO 3, 11.9; EDTANa 2.2H 2O, 0.1; glucose, 5.05; NaH 2PO 4.2H 2O,

0.32 and MgCl 2.6H 2O, 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 retreated 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 βGlucoronidase inhibitory activity

The Cr. MeOHExt, fractions and isolated compounds were screened for β glucuronidase inhibition. The previously reported assay 102 was followed while using p nitrophenyl βDglucuronide as substrate. The enzyme mixture (total volume 250 mL) contained 50 mL of pnitrophenyl glucuronide, 190 mL of acetate buffer, 5 mL enzyme and 5 mL of inhibitor. The assay mixture was incubated at 37 oC for 40 min, the reaction was stopped by the addition of 50 mL of 0.2 M Na 2CO 3, and the absorbance was measured at 405 nm. D saccharic acid 1,4lactone was used as a standard inhibitor. The percent inhibitory activity (%) was calculated using the following formula;

E − S Percent inhibition = × 100 E

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

Chapter 2 Materials and Methods

2.13.1.12 αChymotrypsin inhibitory activity

The Cr. MeOHExt, 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, Nsuccinylphenylalaninepnitroanilide (0.4 mM, final) was added to start the enzyme reaction. The absorbance of released pnitroaniline 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,

OD of test Sample Percent inhibition = 100 − × 100 OD of the Control

The samples that exhibit 50% or above percent inhibition, were processed for IC 50 value calculation by using Ezfit software (Perrella Scientific, USA).

2.13.1.13 Thymidine phosphorylase inhibitory activity

The Cr. MeOHExt and its fractions of T. govanianum were tested for enzyme thymidine phosphorylase inhibition. The assay was performed as previously reported method 104 . TP/PDECGF (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

Chapter 2 Materials and Methods materials for 10 min at 25 oC 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. 7Deazaxanthine was used as the positive control.

2.13.1.14 Acetylcholinesterase inhibitory activity

The Cr. MeOHExt and fractions of T. govanianum rhizomes were tested for acetyl cholineesterase (AChE) inhibitory potential. The assay was carried out according to the previously reported protocol 105,106. The reaction mixture contain 50 mM TrisHcl with pH 8.0, (200 l), BSA buffer (1%), test sample (100 L) keeping final concentration at 100 g/mL. The method based on the hydrolysis of acetyl thiocholine iodide by the respective enzymes and the formation of 5thio2nitrobenzoate 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 g/mL) was used as positive control. The AChE percent inhibition was calculated by below given formula;

A − B Percent AChE inhibition = × 100 A

Where A represent change in absorbance without test sample, while B represent change in the absorbance with test sample.

Chapter 2 Materials and Methods

2.13.2 In vivo biological studies

The Cr. MeOHExt 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 (2535 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. MeOHExt 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 administration 107 .

2.13.2.3 Antiinflammatory activity

The antiinflammatory activity of Cr. MeOHExt and subsequent fractions was performed on mice of either sex (2535 g) following carrageen induced paw edema protocol previously reported 108,109 . The animals were randomly divided in five groups each comprises of six animals. Group I was treated with normal saline (10 ml/kg)

Chapter 2 Materials and Methods 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. MeOHExt (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;

A − B Percent inhibition = × 100 A

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 Tonicvisceral 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 rhizomes 110 . 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. ).

Chapter 2 Materials and Methods

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 method 111 . 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

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 anticancer use (15% and 12.8% from Swat and Dir respectively at

Chapter 3 Results and Discussion 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 infections 112 , infections other than skin infections72,113 , as anthelmintic 114,115 , and other inflammatory disorders 73 . 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 ages 116 . 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

Chapter 3 Results and Discussion different aliments 117 . 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.

Chapter 3 Results and Discussion

Informants for the ethnomedicinal uses of T. govanianum rhizome from different districts of Khyber Pakhtunkhwa

Buner Shangla Swat Dir

9 39 Total 124 81

5 21 Local elderly people 30 32

3 4 Hakims (Traditional healers) 21 10

1 4 Local drug sellers 31 24

0 10 Plant collectors 42 15

Figure 3.1: Informants for the ethnomedicinal uses of T. govanianum rhizomes from different districts of Khyber Pakhtunkhwa

Chapter 3 Results and Discussion

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; 9.2 7.3 1.9 1.2 71 Sexual tonic) GI Disorders (Abdominal spasms) 10.4 14.7 1.7 Skin Infections 6.2 11.1 2.1 112 Infectious diseases (Healing of wounds, 13.4 16.8 2.1 72 ,113 antiseptic, bacterial diarrhea, dysentry) Anthelmintic 15.3 7.2 3.4 114 ,115 Others (backache; fever; inflammation; 14.7 21.6 4.4 73 headache; kidney problems) Plant Information Local name Matarzela Matajarra Matajarai Matajarra Plant parts used Rhizome Rhizome Rhizome Rhizome Availability Abundant Abundant Rare Rare

Chapter 3 Results and Discussion

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

Chapter 3 Results and Discussion the simplest and economic ways for correct identification of the source materials 118 .

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.

Cortex cells

Vascular bundles (Phloem)

Carinal Cambium

Figure 3.3a: Transverse section of T. govanianum rhizome.

Chapter 3 Results and Discussion

Xylem

Sclereids

Trichomes

Fibers

Starch grains

Calcium oxalate crystals

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 solvent 118,119 . 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 ).

Chapter 3 Results and Discussion

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 drugs 76 . 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 nonpolar solvents, which is an indicative of abundance of sugars, and other polar compounds like glycosides, saponins, flavonoids and steroidal glycosides.

Chapter 3 Results and Discussion

Physicochemical parameters of T. govanianum rhizome

Percentage value (W/W %)

nhexanes soluble 1.2

Chloroform soluble 5.8

Ethyl acetate soluble 2.25

Butanol soluble 7.1

Ethanol soluble 13.62 Extractive values Extractive

Methanol soluble 18.75

Water soluble 21.5

Acid insoluble ash 0.8

Acid soluble ash 2.4 Ash values Ash Water soluble ash 4

Total ash 12.5

Loss on drying 14.8

Figure 3.4: Physicochemical parameters of T. govanianum rhizomes.

Chapter 3 Results and Discussion

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

Trillium 44,120 , which includes species traditionally used in the treatment of different diseases by virtue of these phytochemicals 121123 .

Chapter 3 Results and Discussion

Table 3.2: Preliminary phytochemical profile of T. govanianum rhizomes

Phytochemical Qualitative test Cr. MeOHExt nHexfr CHLfr EtOAcfr BuOHfr

Mayer’s test Alkaloids Wagner’s test Glycosides Keller Killiani test + + + + Ferric chloride test + + + Tannins Lead acetate test + + Ferric chloride test + + + Flavonoids Sodium hydroxide test + + + Carbohydrates Molisch’s test + + + + LiebermannBurchard test + + + + + Sterols Salkowski’s test + + + + + Saponins Frothing test + + + +

+ indicates the presence of phytochemicals

Chapter 3 Results and Discussion

3.4.2 GCMS analysis of nhexane fraction

The proximate fatty acid composition of nHexFr 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 9,12octadecadienoic acid methyl ester

(C 19 H34 O2), pentanoic acid5hydroxy2,4ditbutylphenyl ester (C 19 H30 O3), 9 hexadecenoic acid methyl ester (C 17 H32 O2) and cis 13eicosenoic acid (C20 H38 O2) were detected, whereas 2methyl hexadecanoic acid methyl ester (C 18 H36 O2) and ethyl

13methyl tetradecanoate (C 17 H34 O2) represented the saturated fatty acids present at higher concentrations.

GC/MS analysis of nHexfr showed the presence of saturated and unsaturated fatty acids, and thus nHexfr 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.

Chapter 3 Results and Discussion

Table 3.3: Chemical composition of nHexfr of T. govanianum rhizomes

No Chemical Name Formula Molecular Retention time Abundance Weight (min) (%) 1 2,4Decadienal C10 H16 O 152 18.11 2.07 2 Pentanoic acid5hydroxy2,4ditbutylphenyl ester C19 H30 O3 306 23.2 7.83 3 Ethyl 13methyltetradecanoate C17 H34 O2 270 33.59 6.53 4 Hexadecanoic acid methyl ester C17 H34 O2 270 34.35 7.19 5 9Hexadecenoic acid methyl ester C17 H32 O2 268 35.15 9.65 6 2Methyl hexadecanoic acid methyl ester C18 H36 O2 284 35.58 15.00 7 9,12Octadecadienoic acid methyl ester C19 H34 O2 294 36.25 12.50 8 9,12Octadecadienoic acid ethyl ester C20 H36 O2 308 37.58 3.98 9 9,12Hexadecadienoic acid methyl ester C17 H30 O2 266 38.03 13.86 10 9Octadecanoic acid methyl ester C19 H36 O2 296 36.22 2.50 11 cis 13Eicosenoic acid C20 H38 O2 310 39.42 7.27 12 9,12Octadecadienoic acid2hydroxy1(hydroxy methyl) ethyl ester C21 H38 O4 354 43.25 4.53

*In bold, saturated fatty acids

Chapter 3 Results and Discussion

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, CF B(b) of chloroform soluble fraction. The molecular ion peak at m/z 256 in EIMS spectrum was used to calculate its molecular formula as C 16 H32 O2, supported by its HREIMS

(C 16 H32 O2, 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.

1 The HNMR (CDCl 3, 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 C2 methylene protons also appeared as triplet at δ 2.26

(J = 7.1 Hz). The other methylenic . protons appeared as large multiplet between δ

13 1.311.50. The CNMR (CDCl 3, 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 (C1) while the terminal methyl carbon resonated at δ 14.3 (C16). The physical and spectral data of compound 1 was in close resemblance to that of a known compound, hexadecanoic acid previously reported 126 . Thus the compound 1 was characterized as hexadecanoic acid.

Chapter 3 Results and Discussion

1 13 Table 3.4: HNMR and CNMR (CDCl 3, 600 and 150 MHz) chemical shift assignments in compound 1

C No. δ C δ H (J, Hz) 1 178.8 2 34.4 2.26, t (7.1) 315 24.5 – 31.9 1.311.50 br, m 16 14.3 0.87, t (8.1)

Figure 3.5: Chemical structure of compound 1.

Chapter 3 Results and Discussion

3.4.3.2 Structure elucidation of compound 2

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

chloroform . soluble sub fraction, CF B(b) . Its EIMS spectrum exhibited molecular ion

peak at m/z 414, corresponded to the molecular formula, C29 H50 O (calcd; 414.3892) in

HREIMS. The other major fragments peaks observed at m/z 399, 396, 380, and 303

representing a βsitosterol nucleus. The . IR [ spectrum revealed strong absorption at

3450 and 1625 cm 1 for hydroxy and olefinic functionalities. The 1HNMR spectrum

(600 MHz, CDCl 3) revealed characteristic peaks for steroidal nucleus. The two tertiary methyl group protons (CH 3 18 and CH 319) resonated as singlets at δ 0.61 and 0.91, respectively. The olefinic proton (H6) was observed as multiplet at δ 5.30 while the chemical shift of sole H3α proton was observed at δ 3.34 ( J = 4.0 and 9

Hz) confirming the presence of a 3βhydroxyl functionality at position 3 in ring A.

13 The CNMR (150 MHz, CDCl 3) 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 (C18), 19.7 (C19 .), 19.1 (C21 .), 19.3 (C26 [), 19.3 (C27 .) and .11.9 (C29) while chemical shift values for olefinic carbons were reported at δ 140.3 (C5) and 121.1

(C6), respectively. The physical and spectral data of compound 2 was an agreement with the reported data for known compound β sitosterol 127 . Therefore, compound 2 was characterized as βsitosterol.

Chapter 3 Results and Discussion

1 13 Table 3.5: HNMR and CNMR (CDCl 3, 600 and 150 MHz) chemical shift assignments in compound 2

C No. δC δ H (J, Hz) C No. δC δ H (J, Hz) 1 37.4 1.34, 1.14 m 16 26.5 1.60, 1.34 m 2 31.8 1.55, 1.25 m 17 56.4 1.46 m 3 71.1 3.34 m 18 13.4 0.61 s 4 41.3 2.24 dd (7.1, 6.8) 19 19.7 0.90 s 5 140.3 20 36.1 1.56 m 6 121.1 5.30 d (5.3) 21 19.1 0.79 d (6.5) 7 32.0 2.04, 1.78 m 22 34.0 1.32 m 8 31.5 1.42 m 23 33.8 1.36 m 9 50.7 1.40 m 24 45.8 1.52 m 10 36.9 25 27.2 1.81 m 11 21.4 1.46 m 26 19.3 0.82 d (6.5) 12 40.0 1.5, 1.32 m 27 19.3 0.80 d (6.5) 13 42.5 28 23.1 1.58 m 14 56.7 1.40 m 29 11.9 0.75 t (7.0) 15 25.34 1.63, 1.36 m

Figure 3.6: Chemical structure of compound 2.

Chapter 3 Results and Discussion

3.4.3.3 Structure elucidation of compound 3

+ literature. The EI mass spectrum displayed molecular ion peak at m/z 412 [M ] which was in agreement with molecular formula C29 H48 O (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

1 (hydroxyl group) and 1630 cm (cycloalkene).

1 The HNMR, (CDCl 3, 600 MHz) spectrum showed a strong multiplet at δ 3.39 assigned to H3 proton. The H22 and 23 protons showed chemical shift values at δ

5.23 (m) and 5.26 (m), respectively. The H6 protons appeared doublet at δ 121.7 suggesting double bonds in the molecule. The methyl groups protons of H18, 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).

13 The CNMR (CDCl 3, 150 MHz) spectrum exhibited signals for all the twenty nine carbon atoms (Table 3.6). The hydroxy carbon (C3) resonated δ 71.7 while the olefinic carbons at C5, 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, stigmasterol 128 .

Chapter 3 Results and Discussion

1 13 Table 3.6: HNMR and CNMR (CDCl 3, 600 and 150 MHz) chemical shift assignments in compound 3

C No. δC δ H (J, Hz) C No. δC δ H (J, Hz) 1 36.9 1.33, 1.16 m 16 30.0 1.56, 1.38, m 2 31.3 1.57,1.24 m 17 54.5 1.51 m 3 71.7 3.39 m 18 12.0 1.06 s 4 42.2 2.24 dd (7.1, 6.8) 19 20.3 1.29 s 5 141.5 20 33.8 1.54 m 6 121.7 5.30 d (5.2) 21 22.1 1.12 d (6.5) 7 32.0 2.04, 1.72 m 22 138.2 5.23 m 8 36.0 1.45 m 23 129.2 5.26 m 9 50.7 1.40 m 24 47.6 1.50 m m 10 37.49 25 32.1 1.79 m 11 21.6 1.45 m 26 22.4 0.92 d (6.5) 12 38.4 1.60, 1.35 m 27 20.1 0.92 d (6.4) 13 42.1 28 25.3 1.60 m 14 57.3 1.45 m 29 12.0 0.93 t (7.0) 15 24.5 1.61, 1.35 m

Figure 3.7: Chemical structure of compound 3.

Chapter 3 Results and Discussion

3.4.3.4 Structure elucidation of compound 4

Compound 4 was isolated and purified as whit to off white needles/powder from the chloroform soluble sub fraction, CF E. The molecular ion peak was observed at m/z

414.3012, corresponding to the molecular formula of C27H42 O3 (calcd; 414.3134) in

HREIMS. The molecular ion peak was also supported by positive FABMS spectrum, showed [M+H] + at m/z 415. The IR spectrum afforded strong absorption at

–1 –1 –1 3450 cm for a hydroxyl group, at 2970 cm for CH 3 stretching, at 1600 cm for a vinylic group and at 1050 cm –1 for a carboxyl group.

1 The HNMR (CDCl 3, 600 MHz) spectrum revealed signals for almost all the protons, a multiplet was observed at δ 3.57 for the methine proton (H3) followed by the double doublet at δ 2.26 (2H, H4, J = 7.2 and 6.4 Hz) (Table 3.7). A strong doublet at δ 5.33 was assigned to the olefinic proton (H6, J = 5.2 Hz), indicative of the sole double bond in the steroidal skeleton. Furthermore, the methine protons H16 and H

17 resonated at δ 4.38 (1H, q, J. = 15.6 0Hz) and 1.80 (1H, dd, J = 8.8, 6.0 0Hz). The two tertiary methyl group protons appeared as singlets at δ 0.80 (3H, H18) and 1.04

(3H, H19) while the two secondary methyl group protons appeared as δ 0.79 (3H, d,

J = 4.2 Hz, H21) and 0.78 (0.78, d, J = 6.1 Hz, H27), respectively.

The 13 CNMR 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 (C18), 19.4 (C19), 14.5 (C21) and 17.1 (C27) respectively. The spectrum also exhibited characteristic signals for three carbons at δ 140.8 (C5), 121.4 (C6) and 109.3 (C22), diagnostic for 5 spirostane type sapogenin129 .

Chapter 3 Results and Discussion

Chemical shift for βhydroxyl group carbon atom was observed at δ 71.7 (C3). All the other carbon atoms chemical shift values as well as 2DNMR correlations showed resemblance with the reported values for a known compound, diosgenin 130,131 , thus compound 4 was identified as diosgenin.

1 13 Table 3.7: HNMR and CNMR (CDCl 3, 600 and 150 MHz) chemical shift assignments in compound 4

C No. δC δ H (J, Hz) C No. δC δ H (J, Hz) 1 37.2 1.30, 1.17 m 15 32.0 1.73, 1.56 m 2 31.4 1.52, 1.35 m 16 80.8 4.38 q (15.6) 3 71.7 3.57 m 17 62.1 1.80 dd (6.0, 8.8) 4 41.6 2.26 dd (7.2, 6.4) 18 16.3 0.80 s 1.78 m 19 19.4 1.04 s 5 140.8 20 42.3 2.42 m 6 121.4 5.33 d (5.2) 21 14.5 0.79 d (7.2) 7 31.6 2.07, 1.90 m 22 109.3 8 31.8 1.42 m 23 31.4 1.60, 3.56 m 9 50.1 1.30 m 24 28.8 1.56 m 10 36.6 25 30.3 1.76 m 11 20.9 1.44 m 26 66.8 3.40 t (10.3) 12 39.8 1.45, 1.37 m 3.45 dd (10.3, 4.2) 13 40.2 27 17.1 0.78 d (6.1) 14 56.5 1.40 m

Figure 3.8: Chemical structure of compound 4.

Chapter 3 Results and Discussion

3.4.3.5 Structure elucidation of compound 5

Compound 5 was isolated and purified as white to off white powder from the chloroform soluble sub fraction, CF E through column chromatography. The compound was identified and characterized through modern spectroscopic data analysis. The molecular formula C27 H42 O4 for compound 5 was established form its molecular ion peaks at m/z 430 in EIMS and at m/z 431 [M+H] + in FAB positive, which was further confirmed from its HREIMS (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 CO functional group at 1057 cm –1.

1 The HNMR (CDCl 3, 600 MHz) spectrum showed similar pattern of chemical shift values for all the protons to that of diosgenin except the signal at C17 (Table 3.8). A methine proton appeared as multiplet at δ 3.27 (H3), vinylic proton as doublet at δ

5.32 (H6, J = 5.2 Hz), methine proton H16 as triplet at δ 3. 80 (1H, t, J= 15.0 Hz) and methine proton H20 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, H18) and 1.04 (3H, H19) while the two secondary methyl group protons appeared as δ 0.89 (3H, d, J = 4.2 Hz, H21) and 0.87 (0.78, d, J = 6.1 Hz, H27) respectively.

13 The CNMR and DEPT spectra (CDCl 3, 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

100

Chapter 3 Results and Discussion quaternary carbon signal at δ 90.1 (C17) for hydroxyl group. The methyl carbons resonated at δ 17.1 (C18), 19.4 (C19), 13.6 (C21) and 17.1 (C27) respectively. The other entire carbon atoms chemical shift values as well as 2DNMR correlations showed resemblance with the reported values in literature for a known compound pennogenin 132 .

101

Chapter 3 Results and Discussion

1 13 Table 3.8: HNMR and CNMR (CDCl 3, 600 and 150 MHz) chemical shift assignments in compound 5

C No. δC δ H (J, Hz) C No. δC δ H (J, Hz) 1 37.1 1.34, 1.17 m 16 90.9 3.80, t (15.0) 2 31.6 1.55,1.32 m 17 90.1 3 71.7 3.27 m 18 17.1 0.83 s 4 42.2 2.24 dd (7.2, 6.9) 19 19.4 1.04 s 1.98 m 20 43.7 2.42 m 5 140.8 21 13.6 0.89 d (7.2) 6 121.3 5.32 d (5.2) 22 110.1 7 31.6 2.05, 1.80 m 23 30.7 1.60, 3.56 m 8 31.6 1.43 m 24 28.1 1.56 m 9 49.6 1.34 m 25 30.1 1.76 m 10 36.6 26 66.8 3.35 t (10.8) 11 20.7 1.44 m 3.45 dd (m) 12 31.9 1.50, 1.33 m 27 17.1 0.87 d (12.0) 13 44.6 14 52.9 1.40 m 15 31.2 1.72, 1.49 m

Figure 3.9: Chemical structure of compound 5.

102

Chapter 3 Results and Discussion

3.4.3.6 Structure elucidation of compound 6

Compound 6 was isolated and purified as white powder from the chloroform soluble sub fraction, CF E. The compound was identified and characterized through modern spectroscopic data analysis and was confirmed as a trihydroxy fatty acid. In EIMS spectrum the molecular ion peak was displayed at m/z 330, while FABMS showed

+ ion peak at m/z 331 [M+H] . Its molecular formula of C18 H34 O5 was obtained from

HREIMS 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 1HNMR 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 + 5728), 223 (M + 5728

24+2H), 205 (M + 5728202H18), 187 (M + 5728202H1818), 167 (M + 5728

202H1818182H) and 123 (M + 5728202H181818+2H44) in EIMS. The consecutive loss of three 18 fragments was evident of three OH groups in the molecule.

103

Chapter 3 Results and Discussion

The 1HNMR (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 acid 133 , 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, H11), 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 C8, 9), 152

+ + + (OH loss) due to [C 4H9] , [CH 3(CH 2)7CH=CHCHOH] , [CH 3(CH 2)7CH=CHCH]

+ fragment losses as well as at 171 (right side chain, [OHCHOHCH(CH 2)4COOH] ),

155 (O loss) and 137 (H 2O loss) which were reported due to the possible breakage of

C7, 8 points in the chain as depicted in Fig. 3.12.

The terminal methyl protons (H18) resonated at δ 0.90 (t, J = 8 Hz), while the methylenic protons (H3, 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 H2 at δ 2.26 (7.1

Hz) as well as for H12 protons at δ 2.15 (7.1 Hz) while a multiplet was observed for

H13 at δ 2.10.

The 13 CNMR (MeOD, 150 MHz) spectrum revealed signals for almost all the carbon atoms including carbonyl quaternary carbon at δ 177.7 (C1), olefinic carbons at δ

130 and 134.6 (C10 and 11) along with the hydroxyl bearing carbons at δ 71.7 (C7),

76.9 (C8) and 69 (C9) (Table 3.9). 1H1H 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 H10 and H11.

104

Chapter 3 Results and Discussion

Consequently, compound 6 was assigned as 7, 8, 9trihydroxy(10Z)10octadecenoic acid. The compound was given a common name as govanic acid.

1 13 Table 3.9: HNMR and CNMR (CD 3OD, 600 and 150 MHz) chemical shift assignments in compound 6

C No. δC δ H (J, Hz) C No. δC δ H (J, Hz) 1 177.7 12 32.7 2.15 t (7.1) 2 34.9 2.26, t (7.1) 13 30.2 2.10 m 35 26.1 30.6 1.341.61 br, m 14 28.9 1.59 6 34.6 1.59 m 15 26.9 1.54 7 71.7 3.73 m 16 30.4 1.44 8 76.9 3.25 dd 17 23.6 1.41 9 69.0 4.48 dd 18 14.4 0.90 t (8.0) 10 130.7 5.46 dd (11.1, 6.4) 11 134.6 5.56 m (11.1, 6.1)

Figure 3.10: Chemical structure of compound 6.

105

Chapter 3 Results and Discussion

Figure 3.11: Linked scan measurements in compound 6 (EIMS spectrum).

Figure 3.12: Major fragmentation in compound 6 with correlations in 1H1HCOSY( ).

106

Chapter 3 Results and Discussion

3.4.3.7 Structure elucidation of compound 7

Compound 7 was obtained as amorphous white powder from the sub fraction, CF Hh eluted with EtOAc in MeOH (9.5 : 0.5) solvent system by preparative thin layer chromatography. The mass spectrum in HREIMS afforded the molecular ion peak at

+ m/z 480.5521 [M] consistent with the molecular formula of C27 H44 O7 (calcd;

480.3121). The formula mass was also confirmed by FABMS (negative) in glycerol at m/z 479 [MH+]. 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 ecdysteroids 134 .

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 CH 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 CO functionality. The UV spectrum showed absorption at

240 nm indicating an α and β unsaturated carbonyl moieties in the molecule.

1 The HNMR spectrum, showed two secondary methine protons, C2 and C3 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 C1 and C4 which in turn resonated at 1.75 and 1.76, respectively, indicating the presence of –CH 2CH(OH)CH(OH)CH 2 system that is placed at C1C4 in the

107

Chapter 3 Results and Discussion molecule (Table 3.10). The signal pattern for all protons was mostly similar to 20 hydroxyecdysone previously reported in literature135 .

The 13 CNMR 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 (C22) resonated on δ 77.9, while signals for the methylenic carbons C23 and C24 were observed at δ 27.3 and 43.8, respectively. The three methyl carbon signals were observed at δ 21.5 (C21), 28.9 (C26) and 29.7 (C27) while the quaternary carbon (C25) of the side chain resonated at δ 71.3 which was analogue to that of the 20hydroxy ecdysone for the sixth hydroxyl carbon in the molecule. The methyl carbons of C18 and C19 resonated at 18.1 and 24.4, respectively. Signals for the two hydroxyl methine carbons of ring A were observed at

68.7 (C2) and 68.5 (C3) while C5 methine carbon resonated at 50.5. The carbonyl carbon appeared on 206.4 (C6) along with the two vinyllic carbons showing chemical shift values 122.1 (C7) and 168.0 (C8) in ring B. These data suggested α,β unsaturated ketone moiety ascribable to 7 en6one system in the steroid nucleus, also supported by HMBC correlations. The signal pattern was mostly similar to 20hydroxyecdysone previously reported in literature 136 . Thus, on the basis physical and spectral similarities with the reported 20hydroxyecdysone, compound 7 was identified as 20hydroxyecdysone.

108

Chapter 3 Results and Discussion

1 13 Table 3.10: HNMR and CNMR (CD 3OD, 600 and 150 MHz) chemical shift assignments in compound 7

C No. δC δ H (J, Hz) C No. δC δ H (J, Hz) 1 37.4 1.75 15 31.7 1.61 2 68.7 3.84 m 16 24.4 1.58, 1.55 3 68.5 3.94 dt (11.7, 3.2) 17 37.4 2.00 4 32.2 1.76, 2.13 18 18.1 0.88 5 50.5 2.40 19 24.4 0.99 6 206.4 20 78.4 7 122.1 5.80 d (2.6) 21 21.5 1.13 8 168.0 22 77.9 3.14 9 35.1 3.14 23 27.3 1.42 10 39.1 24 43.8 1.37 11 21.5 1.42, 1.46 25 71.3 12 32.5 2.13, 1.33 26 28.9 1.19 13 57.3 27 29.7 1.18 14 85.2

Figure 3.13: Chemical structure of compound 7.

109

Chapter 3 Results and Discussion

3.4.3.8 Structure elucidation of compound 8

Compound 8 was obtained as white amorphous powder from the sub fraction, CF Hh by preparative thin layer chromatography (TLC). The mass spectrum in HREIMS afforded the molecular ion peak at m/z 496.5510 [M] + which was in agreement with the molecular formula of C27 H44 O8 (calcd; 496.3021). The formula mass was also confirmed by FABMS (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 20hydroxy ecdysone skeleton reported for many ecdysteroids134 .

The IR spectrum showed similar pattern of peak, observed in the reported 20hydroxy ecdysone skeleton 135 . The UV spectrum showed absorption at 240 nm confirming the existence of an α and β unsaturated carbonyl moiety in the compound.

The 1HNMR signal pattern was mostly similar to 5,20dihydroxy ecdysone

135,137 13 previously reported . The CNMR 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, C23 and C24 were observed at δ 27.0 and 43.8 respectively. The quaternary carbon (C25) of the side chain resonated at δ 71.3 which was analogues to that of the 20hydroxy ecdysone.

However compound 8 showed a distinct signal for C5 at 80.3 as compared to C5 of

20hydroxy ecdysone (δ 71.3). The same chemical shift value for C5 has been reported for 5,20 dihydroxyecdysone 135,137 . Thus, on the basis physical and spectral

110

Chapter 3 Results and Discussion similarities with the reported data, compound 8 was identified as

5,20 dihydroxyecdysone.

1 13 Table 3.11: HNMR and CNMR (CD 3OD, 600 and 150 MHz) chemical shift assignments in compound 8

C No. δC δ H (J, Hz) C No. δC δ H (J, Hz) 1 36.1 1.75 15 31.5 1.84, 1.60 2 70.2 3.64 d (3.5) 16 23.3 1.97, 1.54 3 68.4 3.98 17 43.6 2.38 4 32.2 1.97, 1.94 18 14.8 0.89 5 80.3 19 24.2 0.99 6 202.4 20 78.4 7 120.6 5.84 d (2.7) 21 26.1 1.24 8 167.5 22 77.8 3.31 9 35.1 3.18 br m 23 27.0 1.42 10 39.1 24 43.8 1.38 11 30.2 1.74, 1.72 25 71.3 12 71.3 2.00, 1.78 26 28.9 1.19 13 57.4 27 29.6 1.18 14 85.3

Figure 3.14: Chemical structure of compound 8.

111

Chapter 3 Results and Discussion

3.4.3.9 Structure elucidation of compound 9

Compound 9 was isolated and purified as white to off white amorphous powder from a butanol soluble sub fraction, BF A. The HRFABMS showed pseudo molecular ion

[M+H] + at m/z 869.4790 (calcd; 868.4832), which was consistent with the molecular composition of C45 H72 O16 .

The IR spectrum (KBr) exhibited prominent absorption for hydroxyl functionality at

3410 cm 1, olefinic (endocyclic) absorption at 1420 cm 1 and CO linkage at 1305 cm

1 in the skeleton. The 13 CNMR signals at δ 141.8 (C5), 122.6 (C6) and 110.6 (C

22), showed the same basic skeleton of 5spirostane type sapogenin 129 .

As 1H and 13 CNMR 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

1 yielded several signals in the region between δ C 70105. The HNMR (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,

H1’), 6.27 (1H, s, H1’’) and 5.60 (1H, s, H1’’’, which suggested the presence of three monosaccharide moieties. Moreover, the 1HNMR spectrum showed signals for two tertiary methyl group protons at δ 1.02 (3H, H18) and 1.08 (3H, H19) and two secondary methyl doublets separately at δ 1.08 (3H, J = 7.6 Hz, H21), and 1.01 (3H,

J = 6.6 Hz, H27) respectively.

13 The CNMR and DEPT (150 MHz, CD 3OD) spectra showed signals for all the carbons including sugar moieties and olefinic functionalities. Furthermore, the 1H and

13 C connectivities in steroidal skeleton of aglycone and sugar moieties were made

112

Chapter 3 Results and Discussion through HSQC, HMBC and 1H1H 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 (H6) exhibit 3J correlations with carbons at δ 141.8 (C5) and 38.5 (C10), respectively.

The 1H1H COSY spectrum was helpful in establishing the connections in steroidal skeleton as follows. H1 proton at δ 1.0 showed cross peaks with C2 methylene protons ( δ 1.92 and 2.10), which was correlated with C3 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

H6/ H7, H8/H9, H11/H12, H15/H16, and H20/H22 accordingly.

The NOESY spectrum was helpful in deducing the stereochemistry in the steroidal nucleus as H3 showed cross peaks with H1α, assigning it α or axial symmetry. The proton H8 was correlated with methyl groups at C18 and C19, suggesting there same orientation i.e., βoriented. Similarly, H9 and H14 were found to be α oriented, as H9 showed NOESY crosspeaks with H1 ( axially oriented). The C3 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 (H1′) with C3; H3 also showed NOESY cross peaks with H

1’, which suggested the αorientation 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 C2′ ( δ 77.1), C3′ ( δ

76.8), C4′ ( δ 79.3), and C5′ ( δ 78.0), suggesting the connectivity of glucose moiety

113

Chapter 3 Results and Discussion with C3. Furthermore, the HMBC correlations of H1′′ ( δ 6.27), with C5′ ( δ 78.0) indicated the C1′OC1′′ connectivity, i.e. the αLrhamnose molecule attached with

C′4. The nature and connectivity of this rhamnose molecule was inferred through 1H and 13 CNMR while the HMBC correlations of H2′ ( δ 4.21) with C1′′′ ( δ 102.6), revealed the connectivity between the glucose and another αLrhamnose molecule.

The presence of another αLrhamnose was deduced through the HMBC correlations.

The physical and spectral data coincided with the reported data of borassoside E 138 , hence the compound was characterized as a known compound, borassoside E.

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1 13 Table 3.12: HNMR and CNMR (CD 3OD, 600 and 150 MHz) chemical shift assignments in compound 9

C No. δC δ H (J, Hz) C No. δC δ H (J, Hz) 1 38.0 1.10, 1.68 m 2 30.7 1.92, 2.10 m 3ODGlc 3 79.8 3.68 m 1’ 100.4 4.87 d (6.9) 4 39.5 2.68 dd (11.2, 12.9) 2’ 77.7 4.21 m 2.78 dd (2.2, 13.6) 3’ 76.8 3.58 m 5 141.8 4’ 79.3 3.92 m 6 122.6 5.63 br 5’ 78.0 4.10 m 7 32.4 1.52, 1.90 m 6’ 62.10 3.90, 4.10 8 31.4 1.42 m 2’ORha 9 51.7 1.10 m 1’’ 102.2 6.27 br s 10 38.5 2’’ 72.5 4.66 m 11 21.9 1.44 m 3’’ 73.0 4.35 dd (1.9, 9.4) 12 41.4 1.10, 1.88 m 4’’ 74.2 4.72 m 13 40.9 5’’ 70.4 4.70 m 14 57.8 1.12 m 6’’ 19.2 1.52 d (6.4) 15 33.2 1.56, 1.90 m 4’ORha 16 80.0 4.02 m 1’’’ 102.6 5.60 br s 17 61.9 1.80 dd (6.0, 8.8) 2’’’ 73.0 3.98 m 18 16.7 1.02 s 3’’’ 72.8 4.26 dd (2.2, 9.0) 19 19.8 1.08 s 4’’’ 74.6 4.10 m 20 42.9 2.52 m 5’’’ 70.2 4.62 m 21 17.4 1.08 d (7.6) 6’’’ 18.8 1.82 d (6.3) 22 110.6 23 29.8 1.60 m 24 28.2 1.24 m 25 32.8 1.44 m 26 69.7 3.80 br 27 17.8 1.01 d (6.2)

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Chapter 3 Results and Discussion

Figure 3.15: Chemical structure of compound 9.

Figure 3.16: Key HMBC correlations in compound 9.

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Chapter 3 Results and Discussion

3.4.3.10 Structure elucidation of 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.

The HRFABMS showed pseudo molecular ion [M+H] + at m/z 1225.5426 (calcd;

1224.5490), which was consistent with the molecular composition of C56 H88 O29 .

1 13 The HNMR (CD 3OD, 600 MHz) and CNMR (CD 3OD, 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 1HNMR 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, 1HNMR 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 1HNMR 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 13 CNMR 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 (H6) showed HMBC correlations to two quaternary carbons at δ 139.5 and 43.4 attributed to C5, and C10, respectively.

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Chapter 3 Results and Discussion

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 CC bond connectivity in ring A and B was inferred through

COSY correlations. H1 ( δ 3.51) indicated COSY cross peaks with C2 methylene protons ( δ 2.09, 1.71), which further showed cross peaks with H3 ( δ 3.39). Similarly,

H3 showed COSY cross peaks with methylene protons at δ 2.24 and 2.22. This suggested the connectivity of C1 to C4. H6 ( δ 5.55) showed COSY correlations with H 27 ( δ 1.96, 1.55), which further showed connectivity with C8 through COSY cross peaks with H8 ( δ 1.54).

The COSY correlation of H20 with H17 ( δ 1.82) and H 221 ( δ 3.63, 3.2), along with

HMBC correlations of H21 ( δ 3.63, 3.63) with C20 ( δ 46.6) and C22 ( δ 112.1) inferred an OH at C21. Similarly, the HMBC correlations of H24 ( δ 4.26), H 226 ( δ

4.46, 3.71) with C22 suggested oxygenated nature of ringF.

The stereochemistry of steroidal skeleton was deduced on the basis of NOESY correlations (Fig. 3.19). H1 showed NOESY crosspeaks with H3, this could be only possible if both these protons are axially oriented. Therefore, H1 and H3 are α oriented. H8 showed NOESY correlations with H 318 and H 319, suggesting there same orientation i.e., βoriented. Likewise, H9 and H14 were found to be αoriented, as H9 displayed NOESY crosspeaks with H1 ( axially oriented).

The HMBC correlations of H24 ( δ 4.26) with C23 ( δ 72.1), C25, ( δ 144.5), and C

27 ( δ 114.0), along with COSY cross peaks of H24 with proton at δ 3.74 suggested the position of OH groups at C23 and C24, and an exocyclic C=C bond between C

25/C27. The C24 signal appeared at δ 83.3, which indicated the glycoside linkage

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Chapter 3 Results and Discussion with this position. This connectivity was further confirmed through the HMBC correlation of anomeric proton at δ 4.72 (H1′′′′) with C24. H23 showed NOESY cross peaks with H20 and H24, which suggested the βorientation of these protons.

The coupling constant value of H23 and H24 was consistent with literature reported value of similar structure i.e. 4.2 Hz, which further confirmed an αOH at C23 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 C1 ( δ 84.5), C2′ ( δ

77.3), C3′ ( δ 88.6), and C5′ ( δ 78.1), suggesting the connectivity of glucose moiety with C1. Furthermore, the HMBC correlations of H1′′ ( δ 4.38), with C3′ ( δ 88.6) indicated the C3′OC1′′ connectivity, i.e. the second glucose molecule attached with C′3. The connectivity of an apiose molecule was inferred through the HMBC correlations of H1′′′ ( δ 5.18) with C6′′ ( δ 67.1), C2′′′ ( δ 77.5), and C5′′′ ( δ 75.0).

The coupling constant ( J = 5.18) and 13 CNMR data were similar to the literature reported 139 , which indicated the presence of an apiose unit connected to glucose. The presence of a deoxy βDgulose in connection with C24 was inferred through HMBC correlation of H1′′′′ with C24. Similarly, the presence of an αLrhamnose was deduced through the HMBC correlations of H1′′′′′ with C4′′′′ ( δ 80.3) and NMR data similar to the previously reported data 53 . Compound 10 was structurally characterized as (1β,3β,23S,24S)1[OβDglucopyranosyl (1→3)OβDglucopyranosyl (1→6)

OβDapiofuranosyl]3,23dihydroxyspirosta5,25dienyl24[OαL rhamnopyranosyl

(1→4)βD6deoxygulopyranoside] (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.

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1 13 Table 3.13: HNMR and CNMR (CD 3OD, 600 and 150 MHz) chemical shift assignments in compound 10

C No. δC δ H (J, Hz) C No. δC δ H (J, Hz) 1OβDGlc. 1’ 100.2 4.40 d (7.2) 1 84.5 3.51 overlap 2’ 77.3 3.50 m 2 37.5 1.71, 2.09 m 3’ 88.6 3.66 m 3 69.2 3.39 m 4’ 69.8 3.37 m 4 43.3 2.22, 2.24 m 5’ 78.1 3.25 m 5 139.5 6’ 63.6 3.60, 3.69 6 126.1 5.55 br d (5.4) 3’OβDGlc. 1’’ 105.3 4.38 d (7.2) 7 32.6 1.55, 1.96 2’’ 74.8 3.51 m 8 34.2 1.54 m 3’’ 78.0 3.67 m 9 51.0 1.38 m 4’’ 71.8 3.37 m 10 43.4 5’’ 77.5 3.27 m 11 24.8 1.41, 2.46 6’’ 67.1 3.60, 3.73 12 40.9 1.19, 1.69 m 6’’OβApi. 1’’’ 112.6 5.18 d (2.4) 13 41.7 2’’’ 77.5 3.99 d (3.0) 14 57.9 1.22 m 3’’’ 65.4 3.35 s 15 33.2 1.54, 1.98 m 4’’’ 80.0 16 84.4 4.56 q (7.2) 5’’’ 75.0 3.76 s, 3.78 d 17 58.8 1.82 dd (7.8, 6.6) 24O6deoxyβDGul 18 17.2 0.91 s 1’’’’ 103.4 4.72 d (8.4) 19 15.4 1.08 s 2’’’’ 70.2 3.64 m 20 46.6 2.71 dd (7.2, 6.6) 3’’’’ 70.9 3.90 m 21 62.9 3.52, 3.63 overlap 4’’’’ 80.3 3.45 m 22 112.1 5’’’’ 70.7 4.10 m 23 72.1 3.74 d (4.2) 6’’’’ 16.1 1.10 d (6.6) 24 83.5 4.26 d (4.2) 4’’’’OαLRha 25 144.5 1’’’’’ 101.6 5.37 br s 26 62.1 3.71 d (12.0), 2’’’’’ 72.8 3.90 m 4.46 d (12.0) 3’’’’’ 72.1 3.67 m 27 114.0 4.98 br s 4’’’’’ 73.4 3.41 m 5.08 br s 5’’’’’ 69.9 4.13 (9.6, 6) 6’’’’’ 18.7 1.24 d (6.6)

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Chapter 3 Results and Discussion

Figure 3.17: Chemical structure of compound 10

Figure 3.18: Key HMBC correlations in Compound 10.

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Chapter 3 Results and Discussion

Figure 3.19: Key NOESY correlations in compound 10.

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3.5 Biological studies

3.5.1 In vitro biological activities

3.5.1.1 Antibacterial activity of Cr. MeOHExt and fractions

In antibacterial assay, the Cr. MeOHExt 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. MeOHExt 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

Hexfr was more active with 47% inhibition followed by Cr. MeOHExt, CHLfr and

EtOAcfr with 40%, 35% and 26% inhibitions, respectively. All fractions except

BuOHfr were found active against E. coli with maximum activity in EtOAcfr (33%) inhibition. Only butanol and aqueous fractions were found active against S. typhi , with 25% inhibition each. Similarly, Cr. MeOHExt, nhexane and CHLfr showed antibacterial activity against P. aeruginosa with 23%, 32% and 18% inhibitions, respectively. The Cr. MeOHExt, CHLfr, BuOHfr and Aqfr also showed antibacterial potential against B. subtilis and S. aureus with a maximum of 43% activity in CHLfr. The Cr. MeOHExt exhibited 38% inhibition against S. aureus .

The nHexfr and EtOAcfr were found inactive against two gram positive bacteria, S. aureus and B. subtilis .

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Chapter 3 Results and Discussion

The global turn down in antibiotic discovery programs by different pharmaceutical firms and increase of antibiotic resistant microorganisms, are promptly the scientific community to look for new or novel and also reexamine old sources of bioactive chemicals if any, in order to discover potential antibacterial 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 drugs 140 . Keeping in view the fact, the antibacterial screening of T. govanianum rhizomes extract and subsequent fractions was performed. Our results suggest that the Cr. MeOHExt 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. MeOHExt 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 mechanism 141 . For example, saponins exerts their antibacterial action by inhibiting the growth of bacteria and also through leakage of certain enzymes and proteins from the cell 142 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 liposomes 143. Similarly, the antibacterial action of nHexfr 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 properties 143.

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Chapter 3 Results and Discussion

Table 3.14: Antibacterial activity of Cr. MeOHExt 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. MeOHExt 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 nHexfr Inhibition zone (mm) 4 7 7 Percent inhibition 22 47 32 CHLfr 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 EtOAcfr Inhibition zone (mm) 6.0 ± 1.3 4.0 ± 0.8 Percent inhibition 33 26 BuOHfr 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 Aqfr 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

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Chapter 3 Results and Discussion

3.5.1.2 Antifungal activity

3.5.1.2.1 Antifungal activity of Cr. MeOHExt and fractions

In antifungal screening, the Cr. MeOHExt 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. MeOHExt and BuOHfr. The

Cr. MeOHExt 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 nHexfr exhibited good inhibition (40%) against M. canis . The CHLfr showed significant inhibition (70%) against T. rubrum and good (40%) towards M. canis. The EtOAcfr was least active with no activity against fungal strains C. glabrata , A. fumigatus and M. canis . The BuOHfr was found active against all strains except T. rubrum with maximum inhibition of 40% against A. niger. The Aqfr 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

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Chapter 3 Results and Discussion

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 g/mL against all tested fungi. In comparison, borassoside E exhibited good activities (MIC = 2.510 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 findings 144,145 in this domain. Similarly, trihydroxy fatty acid have also been reported for antifungal properties 146, 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.

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Table 3.15: Antifungal activity of Cr. MeOHExt and fractions of T. govanianum rhizomes (% inhibition)

Samples

Fungal strains Cr. MeOHExt nHexfr CHLfr EtOAcfr BuOHfr [Aqfr

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 " ".

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Chapter 3 Results and Discussion

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

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

*Concentration range 031220 µg/mL; MIC (minimum inhibitory concentration)

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Chapter 3 Results and Discussion

3.5.1.3 DPPH free radical scavenging activity of Cr. MeOHExt 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 compounds 91,148,149. Antioxidant potential bearing natural phytochemicals are effective in reducing the toxic effects in human, due to xenobiotic exposure 150. The

Cr. MeOHExt 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 nHexfr and CHLfr 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 (9,12octadecadienoic acid and hexadecanoic acid) in nHexfr, and glycosides, saponins and flavonoids ( Table 3.2; Page No. 89 ) in CHLfr, as previously reported in diverse plant species 151154.

The results also demonstrated that the antioxidant potential of Cr. MeOHExt as well as its successive solvent soluble fractions was lower than BHT and ascorbic acid. The low free radical scavenging capacity of the Cr. MeOHExt 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 capacity 154. Furthermore, there is also a nonlinear relationship between antioxidant activity and hydrophobicity because an increase of alkyl chain length results in low scavenging capacity 155.

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Chapter 3 Results and Discussion

Table 3.17: DPPH free radical scavenging activity of T. govanianum extract, fractions and standards

Percent inhibition (%) ± SEM Conc. (g/mL) nHexfr CHLfr EtOAcfr BuOHfr Cr. MeOHExt 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

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Chapter 3 Results and Discussion

Figure 3.20: DPPH free radical scavenging activity of extract/fractions or standards (ascorbic acid and BHT).

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Chapter 3 Results and Discussion

3.5.1.4 Anticancer activity

3.5.1.4.1 Anticancer activity of Cr. MeOHExt and fractions

The anticancer activities of Cr. MeOHExt and its subsequent solvent soluble fractions against two cancer cell lines; HeLa (cervical cancer cells) and PC3 (prostate cancer cells), were determined by MTT (3(4,5dimethylthazol2yl)2,5diphenyl tetrazonium bromide) assay. The Cr. MeOHExt and its fractions exhibited significant cytotoxicity towards both cancer cell lines (Table 3.18 ). The cytotoxic activity of

CHLfr towards HeLa cells was slightly lower than that of standard drug doxorubicin, with IC 50 of 0.84 ± 0.16 and 0.34 ± 0.01 g/mL, respectively. Similarly, this fraction was also most effective against PC3 cells (IC 50 of 2.70 ± 0.35 g/mL), though to a lesser extent than doxorubicin (IC 50 = 1.38 ± 0.16). Moreover, the BuOHfr, although possessed strong cytotoxic effect against the HeLa cells (IC 50 of 1.60 ± 0.34 g/mL), but was less effective in inhibiting the PC3 cells (IC 50 of 4.04 ± 0.35 g/mL). The

EtOAcfr was also effective towards both cancer cells, with prominent against HeLa cells (IC 50 of 1.41 ± 0.08 g/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 BuOHfr were tested against

HeLa and PC3 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 PC3 and HeLa cells, with IC 50 of 1.74 ± 0.12 and 0.51 ± 0.26

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Chapter 3 Results and Discussion

g/mL respectively, in comparison to the standard, doxorubicin whose IC 50 values were 1.69 ± 0.28 and 0.50 ± 0.15 g/mL, towards PC3 and HeLa cells, respectively.

Govanoside A also showed good anticancer activity with IC 50 of 0.67 ± 0.22 g/mL against HeLa cells. Pennogenin (IC 50 of 9.83 ± 0.37 g/mL) was found active against

HeLa cells, while diosgenin and govanic acid were found less cytotoxic against both cell lines with IC 50 greater than 30 g/mL.

So far, a number of anticancer metabolites have been reported in the genus

Trillium 53,120,156 . These anticancer metabolites include steroidal glycosides and saponins isolated from Trillium erectum and Trillium tschonoskii 44,157. 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. MeOHExt 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.

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Chapter 3 Results and Discussion

Table 3.18: Anticancer activity of Cr. MeOHExt and fractions from T. govanianum rhizomes

Samples IC 50 (g/mL) HeLa cells PC3 cells Cr. MeOHExt 3.14 ± 0.72 6.50 ± 0.52 CHLfr 0.84 ± 0.16 2.70 ± 0.35 EtOAcfr 1.41 ± 0.08 5.15 ± 0.34 BuOHfr 1.60 ± 0.34 4.04 ± 0.35 Doxorubicin 0.34 ± 0.01 1.38 ± 0.16

Results are mean ± SEM of three independent experiments; PC3, human prostate cancer cells; HeLa, human epithelial carcinoma cells

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

IC 50 (g/mL) Compounds PC3cells 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

Results are mean ± SEM of three independent experiments

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Chapter 3 Results and Discussion

3.5.1.5 Antiinflammatory activity (Oxidative burst assay)

3.5.1.5.1 Antiinflammatory activity of Cr. MeOHExt 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 BuOHfr exhibited significant inhibition of oxidative burst for the whole blood followed by Cr. MeOHExt with IC 50 of 16.53 ± 7.54 and 30.81 ± 7.02 g/mL respectively. The CHLfr also showed moderate inhibition with IC 50 of 81.64 ± 24.61 g/mL. Similarly the nHexfr and

EtOAc fraction were found less effective in comparison to other fraction with IC 50 of

107 ± 38.40 and 114.81 ± 12.35 g/mL, respectively. The standard drug used as positive control was ibuprofen with IC 50 of 11.23 ± 1.91 g/mL.

3.5.1.5.2 Antiinflammatory activity of isolated compounds

Based on the good results in the Cr. MeOHExt and its succeeding solvents soluble fractions, the isolated compounds borassoside E, diosgenin and pennogenin from

BuOHfr and CHLfr 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 IC 50 of 05.00 ± 0.84 g/mL in comparison to standard drug ibuprofen with IC 50 of 11.23 ± 1.91 g/mL. Similarly borassoside E also showed considerable inhibition for the whole blood with IC50 of 31.51 ± 6.62 g/mL. The compound diosgenin was found less active as compare to other tested compounds.

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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 system 158 .

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 reactions 159 .

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 mechanisms 160 . 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 processes 161,162 .

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 cancer 163 . Our study suggests that the Cr. MeOHExt 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

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Chapter 3 Results and Discussion 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. MeOHExt, fractions and isolated compounds

Samples IC 50 ± SD ( µg/mL ) Cr. MeOHExt 30.81 ± 7.02 nHexfr 107.12 ± 38.40 CHLfr 81.64 ± 24.61 EtOAcfr 114.81 ± 12.35 BuOHfr 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

Results are mean ± SEM of three independent experiments

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Chapter 3 Results and Discussion

3.5.1.6 Antileishmanial activity of Cr. MeOHExt and fractions

The Cr. MeOHExt 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. MeOHExt exhibited prominent leishmanicidal potential against the tested strain, L. major with IC 50 of 36.34 ± 2.51 µg/mL. The butanol and aqueous fractions also showed activity with IC 50 of 62.61 ± 3.23 and 94.63 ± 1.84 µg/mL respectively. The other tested fractions i.e nHexfr, CHLfr and EtOAcfr were found less active with IC 50 greater than 100 µg/mL. The standard drug used was amphotericin B, IC 50 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 countries 165,166 . 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 disease 167 . 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. MeOHExt possess a good potential for leishmaniasis and thus can be a promising candidate as an antileishmanial agent.

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Chapter 3 Results and Discussion

Table 3.21: Leishmanicidal activity against Leishmania major of Cr. MeOHExt and fractions of T. govanianum rhizomes

Samples IC 50 ± SD [µg/mL] Cr. MeOHExt 36.34 ± 2.51 nHexfr >100 CHLfr >100 EtOAcfr >100 BuOHfr 62.61 ± 3.23 Aqfr 94.63 ± 1.84 Amphotericin B 0.29 ± 0.05

Results are mean ± SEM of three independent experiments

Amphotericin B was used as positive control

3.5.1.7 Insecticidal activity of Cr. MeOHExt and fractions

The in vitro insecticidal potential of Cr. MeOHExt 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.

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Chapter 3 Results and Discussion

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 saponins 169 . For the steroids synthesis, (cholesterol, and insect moulting hormone 20hydroxyecdysone) insects requires sterol because they are not able to synthesize sterol structures by themselves 170 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 (saponinrich), the ingested food saponins may form complex with cholesterol in their body, and thus hinder the biosynthesis of ecdysteroids necessary for ecdysis 171 . 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 glycosidases 169,172 .

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 20hydroxyecdysone (as we isolated from CHLfr).

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

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Chapter 3 Results and Discussion

BuOHfr) could be the reason for its ineffectiveness because it has been reported that glycosylated saponins are less active than sapogenins against red flour beetles 172 .

Table 3.22: Insecticidal activity of Cr. MeOHExt and its subsequent fractions of T. govanianum rhizomes against insect Tribolium castaneum

Tribolium castaneum

Test Sample Total No. of No. of survived No. of dead % insects insects insects Mortality Cr. MeOHExt 10 10 0 0 nHexfr 10 10 0 0 CHLfr 10 10 0 0 EtOAcfr 10 10 0 0 BuOHfr 10 10 0 0 Aqfr 10 10 0 0 Negative control 10 10 0 0 Positive control* 10 0 10 100 *Permethrin

Table 3.23: Insecticidal activity of Cr. MeOHExt and its subsequent fractions of T. govanianum rhizomes against insect Rhyzopertha dominica

Rhyzopertha dominica Test Sample Total No. of No. of survived No. of dead % insects insects insects Mortality Cr. MeOH 10 10 0 0 nHexfr 10 10 0 0 CHLfr 10 10 0 0 EtOAcfr 10 10 0 0 BuOHfr 10 10 0 0 Aqfr 10 10 0 0 Negative control 10 10 0 0 Positive control* 10 0 10 100 *Permethrin

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Chapter 3 Results and Discussion

3.5.1.8 Brine shrimp cytotoxic activity of Cr. MeOHExt and fractions

The results of brine shrimp cytotoxic activity of Cr. MeOHExt and fractions are given in Table 3.24 and Fig. 3.21 . Based on LD 50 (µg/mL), the cytotoxicity of test samples was in following order; Aqfr > BuOHfr > EtOAcfr > Cr. MeOHExt. The maximum cytotoxic activity was observed for aqueous fraction and BuOHfr with

LD 50 (µg/mL) of 256 (138466) and 260 (141469) respectively. Similarly, EtOAcfr and Cr. MeOHExt also showed moderate cytotoxicity. The LD 50 values for nHexfr and CHLfr 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 potential 173,174 . It has been reported that a positive corelation exists between brine shrimp cytotoxicity and human nasopharyngeal carcinoma 175,176 . The findings of this study suggested significant cytotoxicity against brine shrimp, which is further validated from its high potential for PC3 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.

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Table 3.24: Brine shrimp cytotoxic activity of Cr. MeOHExt and fractions of T. govanianum rhizomes

Dose (µg/mL) Total No. of No. of dead Number of survivors Samples LD 50 Std. Drug shrimps shrimps (µg/mL) LD 50 (µg/mL) Cr. MeOHExt 1 10 30 3 27 Etoposide 2 100 30 6 24 720 3 1000 30 16 14 (3641484)* 7.46 nHexfr 1 10 30 30 2 100 30 1 29 > 1000 7.46 3 1000 30 3 27 CHLfr 1 10 30 30 7.46 2 100 30 3 27 > 1000 3 1000 30 14 16 EtOAcfr 1 10 30 2 28 2 100 30 6 24 627 7.46 3 1000 30 18 12 (320 1273) BuOHfr 1 10 30 2 28 2 100 30 13 17 260 7.46 3 1000 30 22 08 (141466) Aqfr 1 10 30 4 26 2 100 30 10 20 256 7.46 3 1000 30 24 06 (138466)

*95% confidence limits in parenthese

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Chapter 3 Results and Discussion

Figure 3.21: Percent cytotoxic effect of Cr. MeOHExt and fractions of T. govanianum rhizomes.

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Chapter 3 Results and Discussion

3.5.1.9 Muscle relaxant (Spasmolytic) activity of Cr. MeOHExt

The results of in vitro effect of Cr. MeOHExt 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. MeOHExt 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. MeOHExt at a dose of

++ (0.10.3 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 (0.030.1 µM) as given in Fig. 3.23 .

Isolated rabbit jejunum is a spontaneously contracting gut preparation 177 , allowing to examine the relaxant effect, without induced contraction. The Cr. MeOHExt 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 blockage 178180. 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 Ltype Ca ++ channels induced smooth muscle contractions, and consequently permitting the inward movement of extracellular Ca ++ which ultimately results a contractile effect 181 . therefore, the agents causing

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Chapter 3 Results and Discussion inhibition of high K + induced contractions are regarded as Ca ++ influx inhibitors 182 . As the Cr. MeOHExt relaxed the high K + induced contractions in a analogous pattern just like standard Ca ++ antagonist verapamil 183 , indicating its calcium Ca ++ antagonist effect.

This effect (Ca ++ antagonist) was further confirmed when the Cr. MeOHExt 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 diarrhea 177 . 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.

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Chapter 3 Results and Discussion

Figure 3.22: Inhibitory effects of T. govanianum rhizomes Cr. MeOHExt and verapamil in isolated rabbit jejunum preparations. Values expressed as mean ± SEM.

Figure 3.23: Ca ++ concentration response curves (CRCs) of Cr. MeOHExt and verapamil in isolated rabbit jejunum preparations. Values expressed as mean ± SEM

(S 1= Cr. MeOHExt).

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Chapter 3 Results and Discussion

3.5.1.10 Antiglycation activity of Cr. MeOHExt and fractions

Antiglycation effect of Cr. MeOHExt 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. MeOHExt (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 amount 184 . 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 compounds 185187 .

Since the Cr. MeOHExt 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 BuOHfrs),

Our observation in this context could be due to the absence or little quantity of these phenolic compounds.

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Chapter 3 Results and Discussion

Table 3.25: Antiglycation activity of Cr. MeOHExt and fractions at dose of 0.5 mg/mL

Samples Percent inhibition IC 50 ± SD (µg/mL)

Cr. MeOHExt 16 nHexfr 4 CHLfr 11 EtOAcfr 3 BuOHfr 6 Aqfr 9 Rutin (Positive control) 96.2 26.4 ± 0.28

3.5.1.11 βglucoronidase inhibitory activity of Cr. MeOHExt and fractions

The Cr. MeOHExt and its fractions were screened for βglucoronidase inhibition.

The results are presented in Table 3.26 , based on the IC 50 ± SD (µg/mL), the Cr.

MeOHExt (140.8 ± 3.8) and BuOHfr (196.2 ± 1.9) exhibited a moderate level of enzyme inhibitory activity in comparison to the standard Dsaccharic acid 1,4 lactone, IC 50 of 46.7 ± 2.2. The CHLfr and EtOAcfr were found less effective in this study.

βglucuronides enzyme (present in animal, plants, and bacteria) catalyzes the hydrolysis of βglucuronides conjugates of exogenous and endogenous compounds produced in the body 188 . Increased level of βglucuronides in blood has been observed in liver injury. Over expression of this enzyme may also be related to liver cancer, arthritis and AIDS. Similarly, βglucuronidase of intestinal bacteria in human and rats

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Chapter 3 Results and Discussion are connected to colon cancer 65 . In addition to this βglucuronidase of bacteria, which are found in the biliary tract is also associated with gall stone formation 189,190 . As the phytochemical analysis ( Table 3.2) revealed the presence of steroidal glycosides, flavonoids and saponins, which explain the moderate βglucuronidase 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 βglucuronidase.

Table 3.26: IC 50 values (g/mL) of extract and fractions of T. govanianum rhizomes and reference drug against βglucuronidase

Samples IC 50 ± SD (µg/mL) Cr. MeOHExt 140.8 ± 3.8

CHLfr >200

EtOAcfr >200 BuOHfr 196.2 ± 1.9 Dsaccharic acid 1, 4lactone 46.7 ± 2.2

*Results are mean ± SEM of three independent experiments. Dsaccharic acid 1, 4lactone was used as positive control.

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Chapter 3 Results and Discussion

3.5.1.12 αChymotrypsin inhibitory activity of Cr. MeOHExt and fractions

The Cr. MeOHExt and its subsequent fractions were screened for αchymotrypsin 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: αChymotrypsin inhibitory activity of Cr. MeOHExt and fractions

Test sample Concentration (µM) Inhibition (%) Cr. MeOHExt 500 Inactive nHexfr 500 Inactive CHLfr 500 Inactive EtOAcfr 500 Inactive BuOHfr 500 Inactive Aqfr 500 Inactive Chymostatin 125 98.4

*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

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Chapter 3 Results and Discussion apoptotic cells and inhibiting angiogenesis 191,192 . 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

Compounds Concentration (mM) Inhibition (%)

Borassoside E 0.5 Inactive

Pennogenin 0.5 Inactive

Diosgenin 0.5 Inactive

7Deazaxanthine 0.5 99.0

*7Deazaxanthine was used as positive control.

3.5.1.14 Acetylcholinesterase inhibitory activity of Cr. MeOHExt and fractions

The Cr. MeOHExt 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 BuOHfr at test concentration of 500 µg/mL

(Table 3.29 ). The Cr. MeOHExt 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 activity 193,194 . Thus further confirmation is necessary to isolate

AChE enzyme inhibitory compounds.

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Chapter 3 Results and Discussion

Table 3.29: Acetylcholineteras inhibitory activity of Cr. MeOHExt and fractions

Samples Concentration Percent inhibition

Cr. MeOHExt 250 µg/mL 11

500 µg /mL 16

nHexfr 250 µg /mL

500 µg/mL

CHLfr 250 µg/mL 8

500 µg/mL 11

EtOAcfr 250 µg/mL 14

500 µg/mL 19

BuOHfr 250 µg/mL 13

500 µg/mL 19

Aqfr 250 µg/mL 6

500 µg /mL 10

Galanthamine 100 µg/mL 66 (positive control) 200 µg/mL 78

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Chapter 3 Results and Discussion

3.5.2 In vivo biological studies

3.5.2.1 Acute toxicity

In order to determine the safety profile, the Cr. MeOHExt of T. govanianum rhizomes was tested for toxicity at different concentrations. A dose dependent increase in percent lethality was observed with the Cr. MeOHExt 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 LD 50 value (2030.42 mg/kg), it was clear that the Cr. MeOHExt was safe to the maximum of dose selected for the study.

Table 3.30: Acute toxicity of Cr. MeOHExt of T. govanianum rhizomes

Total number of mice = 6 Concentration % No. of mice No. of mice LD (mg/kg) (mg/kg ) lethality 50 dead lived 150 0 6 0 500 0 6 0 2030.42 1000 1 5 16 (1488.843069.01)* 1500 2 4 33 3000 5 1 83 6000 6 0 100

*95% confidence limit in parentheses

3.5.2.2 Antiinflammatory activity of Cr. MeOHExt and fractions

In carrageenaninduced paw edema model, the antiinflammatory responses of T. govanianum rhizomes extract and its succeeding solvent soluble fractions are presented in Table 3.31 and Fig. 3.24AD. The results indicate that the Cr. MeOH

Ext and fractions at dose of 25, 50 and 100 mg/kg body weight, exhibit significant antiinflammatory activity comparable to that of control antiinflammatory drug, diclofenac.

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Chapter 3 Results and Discussion

The Cr. MeOHExt and its fractions at dose of 100 mg and 200 mg/kg showed an antiinflammatory 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 ± 4.055 a, 63.50 ± 0.500 a, 47.50 ± 0.500 aand

72.67 ± 3.930 a by Cr. MeOHExt, CHLfr, EtOAcfr and nBuOHfr, 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 BuOHfr at a dose of 100 mg/kg.

Carrageenaninduced paw oedema is a valuable model to evaluate the involvement of mediators concerned in vascular changes related with acute inflammation 195 . Within first hour following carrageenan injection, oedema is induced by the release of mediators i.e. histamine, bradykinin and 5HT, 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, IFNy as well as cytokines such as IL1 and IL2 are produced 196 . COX2 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 antiinflammatory effect 195 .

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 cyclooxygenase and later inhibition of prostaglandin

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Chapter 3 Results and Discussion synthesis. This significant in vivo antiinflammatory 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. MeOHExt, subsequent fraction and isolated compounds. Furthermore, the significant antiinflammatory potential, particularly of Cr. MeOHExt and BuOHfr 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 antiinflammatory findings. Furthermore, it is worthy to note, that the significant antiinflammatory, antinociceptive, and antipyretic activities of plant extract are associated with steroids and saponins 133,198200 .

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Chapter 3 Results and Discussion

Table 3.31: Antiinflammatory activity Cr. MeOHExt and fractions of T. govanianum rhizomes against carrageenan induced paw edema in mice

Dose Inhibition (%) Sample (mg/kg) 1st h 2nd h 3rd h 4th h 5th h

Diclofenac 10 27.33 ± 2.7 a 47.67 ± 0.8 a 67.00 ± 2.5 a 70.67 ± 0.6 a 74.33 ± 0.6 a

50 8.00 ± 1.7 c 18.00 ± 1.7 b 22.63 ± 2.3 a 32.00 ± 5.2 a 34.00 ± 5.2 a

Cr. MeOHExt 100 12.00 ± 1.1 b 44.33 ± 4.6 a 65.00 ± 4.6 a 63.67 ± 1.7 a 66.33 ± 4.6 a

200 19.00 ± 2.3 b 44.67 ± 3.8 a 62.67 ± 3.7 a 62.67 ± 1.4 a 64.67 ± 4.0 a

25 4.00 ± 1.0 8.50 ± 0.5 c 21.35 ± 3.5 b 35.00 ± 1.0 a 42.50 ± 2.5 a b b b a a CHLfr 50 4.03 ± 2.0 18.80 ± 1.0 20.30 ± 2.0 43.00 ± 1.0 58.00 ± 1.0 100 12.12 ± 3.0 c 21.50 ± 0.5 b 43.50 ± 3.5 a 45.00 ± 1.0 a 63.50 ± 0.5 a 25 3.500 ± 0.5 9.500 ± 0.5 c 18.00 ± 3.0 b 18.50 ± 1.5 b 39.50 ± 0.5 a EtOAcfr 50 10.50 ± 0.5 c 16.00 ± 1.0 b 29.50 ± 0.5 a 30.50 ± 2.5 a 44.50 ± 3.5 a 100 9.500 ± 0.5 c 22.50 ± 1.5 b 30.50 ± 0.5 a 33.00 ± 2.0 a 47.50 ± 0.5 a 25 12.94 ± 3.7 c 14.33 ± 5.5 b 35..25 ± 1.0 a 58.00 ± 1.5 a 61.00 ± 3.4 a BuOHfr 50 18.80 ± 3.4 a 46.67 ± 4.9 a 68.67 ± 2.3 a 66.00 ± 1.7 a 69.00 ± 4.3 a 100 25.67 ± 1.7 a 47.67 ± 2.1 a 68.67 ± 1.4 a 70.33 ± 2.6 a 72.67 ± 3.9 a

Percent inhibition is expressed as mean ± SEM . a = P< 0.001, b = P< 0.01, c = P< 0.05 compared to control

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Chapter 3 Results and Discussion

Figure 3.24A: Antiinflammatory effect of Cr. MeOHExt on carrageenan induced paw edema. a = P< 0.001, b = P< 0.01, c = P< 0.05 compared to control.

Figure 3.24B: Antiinflammatory effect of CHLfr on carrageenan induced paw edema. a = P< 0.001, b = P< 0.01, c = P< 0.05 compared to control.

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Chapter 3 Results and Discussion

Figure 3.24C: Antiinflammatory effect of EtOAcfr on carrageenan induced paw edema. a = P< 0.001, b = P< 0.01, c = P< 0.05 compared to control.

Figure 3.24D: Antiinflammatory effect of BuOHfr on carrageenan induced paw edema. a = P< 0.001, b = P< 0.01, c = P< 0.05 compared to control.

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Chapter 3 Results and Discussion

3.5.2.3 Analgesic activity of Cr. MeOHExt and fractions

3.5.2.3.1 Tonicvisceral chemical induced nociception

The Cr. MeOHExt 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 nHexfr (P< 0.01, P< 0.001), CHLfr

(P< 0.01, P< 0.05), EtOAcfr (P< 0.01), BuOHfr (P< 0.001, P< 0.05), Aqfr (P<

0.001), and Cr. MeOHExt ( 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. MeOHExt and its fractions in tonicvisceral 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 *** 50 7.5 ± 4.5 ** nHexfr 100 5.5 ± 3.5 *** 50 8.0 ± 1.0 ** CHLfr 100 14.5 ± 1.5 * 50 10.5 ± 3.5 ** EtOAcfr 100 7.0 ± 4.0 ** 50 5.0 ± 4.0 *** BuOHfr 100 12.5 ± 4.5 * 50 6.5 ± 3.5 *** Aqfr 100 4.0 ± 1.0 *** 50 6.5 ± 1.5 *** Cr. MeOHExt 100 7.5 ± 0.5 **

*P< 0.05, ** P< 0.01, *** P< 0.001 compared to saline treated group, n = 6.

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Chapter 3 Results and Discussion

Figure 3.25: Antinociceptive effect of T. govanianum rhizomes in tonicvisceral 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. MeOHExt 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 nHexfr at 50 mg/kg ( P< 0.05) and 100 mg/kg ( P<

0.01), EtOAcfr at 100 mg/kg ( P< 0.05), BuOHfr at 100 mg/kg ( P< 0.01), and Aqfr at 50 and 100 mg/kg ( P< 0.01). After 60 min, significant analgesic effect was observed with Hexfr at 50 mg/kg (P< 0.01) and 100 mg/kg ( P< 0.001), EtOAcfr at

50 and 100 mg/kg ( P< 0.01), BuOHfr at 100 mg/kg ( P< 0.01), Aqfr at 50 mg/kg ( P<

0.01) and 100 mg/kg ( P<0.001) and Cr. MeOHExt 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 nHexfr ( P< 0.01), EtOAcfr ( P< 0.05),

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Chapter 3 Results and Discussion

Aqfr (P< 0.05) as well as with100 mg/kg dose of BuOHfr ( 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 nHexfr ( P< 0.01), EtOAcfr ( P< 0.05, P< 0.01) and Aqfr ( P< 0.01), and for only the 100 mg/kg dose of BuOHfr ( P< 0.01) and Cr.

MeOHExt (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 noninflammatory inputs and is one of the models normally used for studying central antinociceptive activity 201 .

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 cavity 202 .

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

163

Chapter 3 Results and Discussion responsible for most of the antiinflammatory and analgesic activities. Recent reports indicate that most saponins can suppress the expression of iNOS and COX2, thus

203,204 resulted in a noticeable lowering of prostaglandin E 2 levels . Thus, the findings of this study are further endorsed by the reported literature.

In conclusion, T. govanianum rhizomes Cr. MeOHfr and fractions exhibit significant peripheral and central antinociceptive activities, which support the traditional analgesic uses of this plant species.

164

Chapter 3 Results and Discussion

Table 3.33: Antinociceptive effect of Cr. MeOHExt 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 **

50 23.4 ± 0.7 * 25.6 ± 2.3 ** 29.0 ± 1.0 ** 26.4 ± 2.1 ** nHexfr 100 25.3 ± 0.8 ** 25.8 ± 1.1*** 29.7 ± 0.2 ** 25.3 ± 1.6 **

50 17.2 ± 1.5 21.4 ± 1.4 22.6 ± 2.0 20.3 ± 0.4 CHLfr 100 20.3 ± 2.1 19.3 ± 0.1 16.9 ± 2.8 19.0 ± 1.9

50 17.5 ± 0.9 24.3 ± 2.6 ** 24.2 ± 1.1 * 23.9 ± 3.3 * EtOAcfr 100 22.2 ± 0.1 * 23.0 ± 0.9 ** 23.7 ± 0.6* 25.6 ± 2.5 **

50 13.7 ± 1.9 18.1 ± 0.6 18.2 ± 4.4 18.2 ± 0.1 BuOHfr 100 27.5 ± 1.3 ** 23.4 ± 0.5 ** 25.8 ± 4.2 * 25.4 ± 2.5 **

50 24.6 ± 3.2 ** 25.6 ± 2.8 ** 23.6 ± 0.6 * 25.0 ± 0.1 ** Aqfr 100 24.8 ± 2.0 ** 27.1 ± 2.9 *** 25.8 ± 1.4 * 24.2 ± 1.3 **

50 18.7 ± 3.9 23.9 ± 0.1 ** 18.4 ± 0.5 16.7 ± 1.0 Cr. MeOHExt 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

165

Chapter 3 Results and Discussion

Figure 3.26A: Antinociceptive effect of Cr. MeOHExt and fractions after 30 min * = P< 0.05, ** = P< 0.01, *** = P< 0.001 compared to control.

Figure 3.26B: Antinociceptive effect of Cr. MeOHExt and fractions after 60 min * = P< 0.05, ** = P< 0.01, *** = P< 0.001 compared to control.

166

Chapter 3 Results and Discussion

Figure 3.26C: Antinociceptive effect of Cr. MeOHExt and fractions after 90 min * = P< 0.05, ** = P< 0.01, *** = P< 0.001 compared to control.

Figure 3.26D: Antinociceptive effect of Cr. MeOHExt and fractions after 120 min * = P< 0.05, ** = P< 0.01, *** = P< 0.001 compared to control.

167

Conclusion

Concluding Remarks

Trillium govanianum is an indigenous medicinal herb of Pakistan. The rhizome of this plant species is used as crude drug in IndoPak to cure different ailments. From this

Ph.D. work/project, which is based on ethnomedicinal, 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 subfractions, 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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