PHARMACOLOGICAL PROFILE, PHYTOCHEMICAL STUDIES and METAL ANALYSIS of Polygonatum Verticillatum

PHARMACOLOGICAL PROFILE, PHYTOCHEMICAL STUDIES AND METAL ANALYSIS OF Polygonatum verticillatum

Haroon Khan

DEPARTMENT OF PHARMACY UNIVERSITY OF PESHAWAR 2010

PHARMACOLOGICAL PROFILE, PHYTOCHEMICAL STUDIES AND METAL ANALYSIS OF Polygonatum verticillatum

Haroon Khan

This thesis submitted to the University of Peshawar in Partial fulfillment of the degree of Doctor of Philosophy in

Pharmaceutical Sciences

DEPARTMENT OF PHARMACY UNIVERSITY OF PESHAWAR 2010

CERTIFICATE OF APPROVAL

This thesis entitled “Pharmacological Profile, Phytochemical Studies and Metal Analysis of Polygonatum verticillatum”. submitted by Haroon Khan is hereby approved and recommended as partial fulfillment for the award of degree of “Doctor of Philosophy in Pharmaceutical Sciences”.

Prof. Dr. Muhammad Saeed Research Supervisor ______Department of Pharmacy University of Peshawar

Prof. Dr. Fazal Subhan Chairman ______Department of Pharmacy University of Peshawar

External Examiner ______

Department of Pharmacy University of Peshawar 2010

DDeeddiiccaatteedd TToo MMYY PPAARREENNTTSS && EENNTTIIRREE FFAAMMIILLYY

Acknowledgement

I bow my head to ALMIGHTY ALLAH, Who sanctified me with the dynamism, impudence and understanding to effectively accomplish my PhD project, which is certainly a landmark in my life. This is indeed a tough task to acknowledge the contribution of numerous individual by name. However, I am obliged to all my teachers since my school days. It is a great honor and pleasant feeling to express my in-depth gratitude to incomparable and cherished research supervisor Prof. Dr. Muhammad Saeed, Department of Pharmacy, University of Peshawar. Throughout the research project, his keen and incessant participation, precious supervision and untiring efforts enabled me to achieve this task effectively. His guidance and knowledge that he has bestowed on me is an unending source of inspiration, not only for this study but will also enlighten my ways in the days to come. I deeply owe thanks to Prof. Dr. Fazle Subhan, Chairman Department of Pharmacy, University of Peshawar for his keen interest in research activities and providing the required facilities throughout. Similarly, I would like to appreciate the cooperation of all the teaching faculty of the department. Indeed it is my privilege to acknowledge commendable facilitator and great educator, Prof. Dr. Anwarul Hassan Gilani, Aga Khan Medical University, Karachi. His devotion and contributions for the cause of scientific research in Pakistan is a role model. I grateful to Prof. Dr. Iqbal Muhammad Choudhary, Prof. Dr. Ahsana Dar, Dr. Shabana Usman Simjee of HEJ Research Institute of Chemistry, Karachi and Mr. Faridullah Khan, senior scientific officer, PCSIR, Peshawar. My thanks are due to Dr. Murad Ali Khan, Dr. Izhar-Ul-Haq, Dr. Afsar Khan and Dr. Shazia for their help in spectral analysis. I wish to communicate my earnest appreciation to my lab fellows Mr. Ash-Had Haleemi, Mr. Naveed Muhammad, Mr. Asif Khan, Mr. Saeed Ahmad, Mr. Zakiullah, Mr.Waheed, Miss. Rukhsana Ghaffar, Miss. Farah Gul, Miss. Naila Raziq and Miss. Attiqa Naz for their support and teamwork. Sincere expression of deep regards for my beloved Parents and my entire family and all well wishers. Haroon Khan

Summary

The traditional treatment philosophy primarily based on long empirical learning is mostly without scientific evidence. Polygonatum verticillatum has been traditionally used in different communities across the globe, including Pakistan, since time immemorial for multiple purposes such as analgesic, antipyretic, diuretic, sex stimulant, tonic, for the treatment of urinary infections and pulmonary diseases. The present study was designed to scrutinize and provide scientific rationale in the light of modern sophisticated technologies and to accredit the activities of the crude form with the isolated pure chemical entities.

The crude methanolic extract of both Rhizomes and Aerial parts elicited profound antinociceptive activity in different animal models at test doses (50, 100 and 200 mg/kg). The inhibition of pain perception was dose dependent. From mechanistic point of view, the administration of naloxone caused significant (P<0.01) attenuation of antinociceptive activity of Rhizomes, which established the participation of opioid receptors in its central analgesic activity. However, the analgesic effect of the Aerial parts was not antagonized by the naloxone injection thus ruled out the participation of opioid receptors in its pain relieving effect.

Anti-inflammatory profile of the extracts, Rhizomes and Aerial parts was tested in carrageenan induced paw oedema and significant (P < 0.05) attenuation of the inflammatory reflux provoked by carrageenan at test doses (50, 100 and 200 mg/kg) was observed. Brewer’s yeast induced pyrexia test was employed for the assessment of antipyretic character of plant. The intraperitoneal administration of extracts (50, 100 and 200 mg/kg) demonstrated marked and significant (P < 0.05) attenuation of infectious pyrexia during various assessment times (1 - 5 hour).

For Rhizomes, mild diuretic effect was found after oral administration of 300 mg/kg compared to saline. But at 600 mg/kg, the urine discharge was similar to saline. Whilst, Aerial parts exhibited insignificant effect at both test doses. Pretreatment with extracts did not exhibit any reduction in pentylenetetrazole (PTZ) induced convulsions.

The extracts were administered intraperitoneally at doses of 50, 100 and 200 mg/kg and effects were comparable to control (normal saline). The results in the preliminary toxicity test of extracts revealed their absolute safety up to the dose of 2 g/kg i.e. all the tested animals survived.

The extracts and its sequentially partitioned subsequent solvent fractions demonstrated significant antibacterial activity against various pathogenic bacteria both Gram-positive bacteria (Staphylococcus aureus, and Bacillus subtilis) and Gram-negative bacteria (Escherchia coli, Pseudomonas aeruginosa, flexeneri Shigella and Salmonella typhi), used in the assay. The antibacterial activity of Rhizomes was more prominent than Aerial parts. All the Gram-negative bacteria were susceptible to the components of Rhizomes except Pseudomonas aeruginosa. While the Aerial parts exhibit activity only against Salmonella typhi and Shigella flexeneri. However, the antifungal activity of both the extracts was not worth-mentioning.

Less polar fractions possessed marked antimalarial activity against Plasmodium falciparum in-vitro. In phytotoxic assay, the crude form and aqueous extract of Rhizomes showed 100% inhibition against Lemna acquinoctialis Welv while the ethyl acetate fraction of Aerial parts observed absolute growth inhibition in a dose related patron. . Prominent scavenging activity was observed on stable free radical (DDPH) by the extract as well as subsequent fractions of both Rhizomes and Aerial parts, which revealed its antioxidant potential. Nevertheless, the insecticidal, leishmanicidal and Brine shrimps cytotoxic activities were not prominent. Of the tested fractions, only ethyl acetate and chloroform showed significantly cytotoxicity for Rhizomes and Aerial parts respectively.

In enzyme inhibitory assays, reflective lipoxygenase antagonism was observed for the extract and fractions of the Rhizomes and Aerial parts. Urease inhibitory profile was also prominent for extracts with reasonable potency. Nevertheless, cholinesterase (acetylcholinesterase and butyrylcholinesterase) inhibitions were insignificant.

In the isolated tissue experiments, both the extracts of Rhizomes and Aerial parts of the plant displayed marked anti-hyperactivity with different mechanistic

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involvements. Experimental results on rabbit jejunum revealed that the Rhizomes possessed primarily potassium channel opener constituents for the reversal of hyperactivity in rabbit jejunum while the antispasmodic activity of the Aerial parts was mediated through calcium channel antagonism. In the isolated tracheal tissues of rabbits, the extracts of both Rhizomes and Aerial parts antagonized induced contractions through calcium channel blockade.

The extracts and subsequent fractions were also tested for their micronutrients (Fe, Cu, Zn, Cd, Cr, Sb, Ni, Mn, Pb, Co) and macronutrients (Na, K and Ca) accumulation. The results pointed out the accumulation of reasonable concentrations of these nutrients within recommended limit for plants.

Following the principle of bioactivity guided isolation; Rhizomes of the plant were subjected to column chromatography for the isolation of pure moieties. The structures of isolates were elucidated using physical and comprehensive spectral analysis. These techniques were, 1H-NMR, 13C-NMR, DEPT, 1H-1H COSY, NOESY, HMQC, HMBC and mass spectroscopy (EI-MS and HREI-MS). The column chromatography led to the isolation of a new compound named propyl pentadecanoate (1), along with six known compounds but from a purely new source including 2,3-dihydroxypropyl pentadecanoate (2), 2-Hydoxybenzoic acid (3), 5-Hydroxymethyl-2-furaldehyde (4), Diosgenin 5, β- Sitosterol (6) and Santonin (7). Gas chromatography-mass spectrometry (GC-MS) of hexane fractions of both Rhizomes and Aerial parts revealed that the oily composition was primarily based on terpenes and terpenoids with one fatty acid, melissic acid and eicosadienoic in each hexane fraction respectively.

In the light of available literature, the isolated compounds like 2-hydroxybenzioc acid (3), Diosgenin (5), β-Sitosterol (6) and Santonin (7) have established analgesic, antipyretic and antinflammatory activities thus these activities of extracts were consistent with the isolates. Antimalarial activity of Diosgenin (5) and antibacterial activity against plant pathogens while antioxidant activity of 5-Hydroxymethyl-2-furaldehyde (4) have also been reported by different research groups.

The study revealed significant scientific evidences in favour of various ethnobotanical uses of P. verticillatum in the indigenous system of treatment and also opened a new avenue for researchers to isolate new physiologically active components. Further detailed studies on the extracts as well as isolated entities may lead to the discovery of revolutionary new therapeutic molecule(s) for the management of different diseased conditions.

List of isolated compounds

Propyl pentadecanoate (1) (New compound)

2,3-dihydroxypropyl pentadecanoate (2)

2-Hydroxybenzoic acid (3)

5-Hydroxymethyl-2-furaldehyde (4)

Diosgenin (5)

29 CH3 28 21 26 H3C CH3 22 24 18 20 23 25 CH3

CH3 12 17 27 19 11 13 16 CH3 9 14 15 1 2 10 8

3 5 7 4 6 HO

β-Sitosterol (6)

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Santonin (7)

LIST OF ABBREVIATIONS

S. No. Abbreviation Detail

1 P. verticillatum Polygonatum verticillatum 2 TCM Traditional Chinese Medicine 3 WHO World Health Organization 4 HTS High Throughput Screening 5 CCB Calcium channel blockers 6 CRCs Concentration-response curves 7 VP Verapamil 8 GB Glibenclamide 9 CCh Carbachol 10 AA Arachidonic acid 11 AD Alzheimer's disease 12 LOX Lipoxygenase 13 COX Cyclooxygenase 14 PTZ Pentylenetetrazole 15 TLC Thin Layer Chromatography 16 UV Ultra violet 17 GC Gas chromatography

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18 GC-MS Gas chromatography-mass spectrometry 19 CC Column chromatography 20 NaOH Sodium hydroxide

21 CHCl3 Chloroform

22 CDCl3 Deutrated chloroform 22 HCl Hydrochloric acid 23 Rf Relative flow 24 TNF Tumor necrosis factor 25 MIC Minimum inhibitory concentration 26 DMF dimethylformamide 27 DMSO Di Methyl Sulfoxide 28 FBS Foetal Bovine Serum 29 DTNB 5, 5´-dithiobis [2-nitrobenzoic acid]

30 LD50 Lethal Dose 50

31 IC50 Inhibitory concentration 50 32 STD Standard 33 RBCs Red Blood Cells 34 BSA Bovine serum albumin 35 SEM Standard 36 ANOVA Analysis of variance 37 DPPH 1,1-diphenyl-2-picrylhidrazyl 38 TEA Tetraethylammonium 39 GIT Gastrointestinal tract 40 P. falciparum Plasmodium falciparum 41 AChE Acetylcholinesterase 42 BChE Butyrylcholinesterase 43 E.coli Escherichia coli 44 B.subtulis Bacillus subtilus 45 S.flexenari Shigella flexenari

46 S.aureus Staphylococcus aureus 47 P.aeruginosa Pseudomonas aeruginosa 48 S.typhi Salmonella typhi 49 T.longifusus Trichophyton longifusus 50 C.albicans Candida albicans 51 A.flavus Aspergillus flavus 52 M.canis Mycosporum canis 53 F.solani Fusarium solani 54 C.glabarata Candida glabarata 55 T.castaneum Tribolium castaneum 56 S.oryzae Sitophilus oryzae 57 R.dominica Rhyzopertha dominica 58 C.analis Callosobruchus analis 59 SDA Sabouraud Dextrose Agar 60 SDB Sabouraud Dextrose Broth 61 UV Ultraviolet 62 2D-NMR Two Dimensional Nuclear Magnetic Resonance 63 HMQC Heteronuclear Multiple Bond Connectivity 64 NOESY Nuclear Overhauser Effect Correlation Spectroscopy 65 HMBC Heteronuclear Multiple Bond Connectivity 66 MS Mass Spectroscopy 67 1H-NMR Proton Nuclear Magnetic Resonance 68 13C-NMR Carbon Nuclear Magnetic Resonance 69 FAB Fast Atom Bombardment 70 EI-MS Electron Impact Mass Spectrum

71 IP3 inositol-1,4,5-triphosphate 72 DEPT Distortionless Enhancement by Polarization Transfer 73 EI Electron Impact 74 CI Chemical Ionization

75 BB Broad Band 76 ppm Parts Per Million 77 NMR Nuclear magnetic resonance 78 EtOAc Ethyl acetate

LIST OF FIGURES

Entry Caption P. No. 1.1.1 Drugs derived from plants in different stages of clinical trials. Extracted 08 from (Harvey, 2008). 1.1.2 Flow chart showing various steps involved in the drug development from 09 medicinal plants. Extracted from (Fabricant and Farnsworth, 2001). 1.1.3 Flow chart showing toxicity due to herbal products. Extracted from (Saad 10 et al., 2006a). 1.2. Different parts of Polygonatum verticillatum [L.] All. 17 2.1.2 In vitro experimental set-up for the isolated gastrointestinal tissues 60 preparations. 2.2.1 Gas chromatogram of n-hexane fraction of Rhizomes 81 2.2.2 Gas chromatogram of n-hexane fraction of Aerial parts 82 3.1.1.1 Effect of intraperitoneal dosing of Rhizomes [A] and Aerial parts [B] at 91 50,100 and 200 mg/kg) in formalin test in rats. Values are expressed as mean ±S.E.M. [n = 6]. Asterisks designated significant distinction from control. *P < 0.05, **P<0.01 (ANOVA followed by Dunnett’s test). 3.1.1.2 Effect of intraperitoneal administration (50, 100 and 200 mg/kg) at 3rd 99 hour in carrageenan induced hind paw edema Rhizomes [A] and Aerial

parts [B]. One-way ANOVA was operated for judgment of significant differences among groups followed by Dunnett’s multiple comparison post test. A probability of *P < 0.05, **P<0.01 was characterized as significant from control. 3.1.1.3 Effect of oral administration of Rhizomes and Aerial parts (300 and 600 104 mg/kg) in rats. Cumulative urine volume was expressed as per 100 kg body weight after 6 h in rats. Hydrochlorothiazide (HCT; 10 mg/kg p.o.) was standard drug. Values are communicated as mean ±S.E.M. [n = 6]. Asterisks designed significant distinction from control. **P<0.01 (ANOVA followed by Dunnett’s test). 3.1.2.1 Antibacterial activity of Rhizomes against S. aureus [A], E. coli [B], S. 106 typhi [C] and S. flexeneri [D]. Data are mean of three different findings. 3.1.2.2 Antibacterial activity of Aerial parts of the plant against B. subtilis [A], S. 109 t yphi [B] and S. flexeneri [C]. Data are mean of three different findings. 3.1.2.3 Antifungal activity of Rhizomes against M. canis [A] and F. solani [B]. 112 Data are mean of three different findings. 3.1.2.4 Antifungal activity of Aerial parts against Microspoum canis. Data are 114 mean of three different findings. 3.1.2.5 Phytotoxic activity of Rhizomes at 500 µg/mL [A], 50 µg/mL [B] and 119

at 5 µg/mL [C] concentrations. Data are mean of three findings. 3.1.2.6 Phytotoxic activity of Aerial parts at 500 µg/mL [A], 50 µg/mL [B] and 120 at 5 µg/mL [C] concentrations. Data are mean of three readings. 3.1.2.7 1,1-diphenyl-2-picrylhidrazyl (DPPH) scavenging potential of Rhizomes. 127 PR-1 = Crude extract; PR-2 = n-hexane; PR-3= Chloroform; PR-4 = Ethyl acetate; PR-5 = n-butanol; PR-6 = Aqueous. Standard drug = Vitamin C. Negative control = Ethanol. Symbols stand for mean ± S.E.M. (n = 3). 3.1.2.8 1,1-diphenyl-2-picrylhidrazyl (DPPH) antagonistic potential of Aerial 129 parts. A-1 = Crude extract; A-2 = n-hexane; A-3= Chloroform; A-4 =

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Ethyl acetate; A-5 = n-butanol; A-6 = Aqueous. Standard drug = Vitamin C. Negative control = Ethanol. Symbols represent mean ± S.E.M. (n = 3). 3.1.2.9 Inhibitory effect (%) of the Rhizomes and Aerial parts against 131 acetylcholinesterase [A] and butyrylcholinesterase [B]. Data are mean ± S.E.M. of three assays. 3.1.2.10 Lipoxygenase inhibition (%) of Rhizomes and Aerial parts. Data are 133 mean S.E.M. (n=3). 3.2.11 Urease inhibition of Rhizomes and Aerial parts. Data mean ± S.E.M. of 135 three assays. 3.1.3.1 Tracings showing antispasmodic effect of the crude methanol extract of 138 Rhizomes and cromakalim on spontaneously contracting isolated rabbit jejunum preparations. 3.1.3.2 Concentration-response curves showing effect of Rhizomes of 139 Polygonatum verticillatum on spontaneous [A] and Low K+ (25 μM) induced contractions [B] in isolated rabbit jejunum preparations. Data are mean ± SEM of 4-6 different experimental findings. 3.1.3.3 Concentration-response curves posing effect of cromakalim [A] and 140 verapamil [B] on Low K+ (25 mM) and High K+ (80 mM) originated contractions in isolated rabbit jejunum tissues. Data are mean ± SEM of 4-6 different experimental findings. 3.1.3.4 Tracings showing antispasmodic effect of the crude methanol extract of 143 Aerial parts and verapamil of various concentrations on spontaneously contracting isolated rabbit jejunum preparations. 3.1.3.5 Dose-dependent inhibition of Aerial parts [A] and Verapamil [B] on 144 spontaneous and potassium (80 mM)-elicited contractions in isolated rabbit jejunum preparation. Data stand for mean ± SEM of 3-5 different experimental findings. 3.1.3.6 Dose-response curves of Ca++ created in a calcium free while potassium 145 rich medium in the absence and presence of increasing concentrations of Aerial parts [A] and verapamil [B] in the isolated rabbit jejunum

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preparation. Data symbolize mean ± SEM of 3-5 different experimental findings. 3.1.3.7 Dose-dependent inhibition of Rhizomes [A] and Verapamil [B] on 147 spontaneous and potassium (80 mM) dependent contractions in isolated rabbit trachea preparation. Data symbolize mean ± SEM of 3-5 different experimental findings. 3.1.3.8 Dose-response curves of Ca++ created in a calcium free while potassium 148 rich medium in the absence and presence of increasing concentrations of Rhizomes [A] and verapamil [B] in the isolated rabbit jejunum preparation. Data stand for mean ± SEM of 3-5 different experimental findings. 3.1.3.9 Dose-dependent inhibition of Aerial parts [A] and Verapamil [B] on 149 spontaneous and potassium (80 mM) forced contractions in isolated rabbit trachea preparation. Data symbolize mean ± SEM of 3-5 different experimental findings. 3.1.3.10 Dose-response curves of Ca++ build in a calcium free while potassium 150 rich medium in the absence and presence of increasing concentrations of Rhizomes [A] and verapamil [B] in the isolated rabbit jejunum preparation. Data stand for mean ± SEM of 3-5 different experimental findings. 3.2.1 Quantitative estimation of various phytochemical contents in Rhizomes. 155 Data are expressed as mean ± SEM (n=3). 3.2.2 Quantitative estimation of various phytochemical contents in Aerial 157 parts. Data are expressed as mean ± SEM of three different findings. 3.2.3 Structure of compound (1)1. 160 3.2.4 Some typical correlations observed in HMBC spectrum of compound (1). 160 3.2.5 Structure of compound (2) 163 3.2.6 Some typical correlations observed in HMBC spectrum of compound (2) 163 3.2.6 Structure of compound (3) 165 3.2.7 Structure of compound (4) 167

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3.2.8 Some typical correlations observed in HMBC spectrum of compound (4) 168 3.2.9 Structure of compound (5). 171 3.2.10 Some typical correlations observed in HMBC spectrum of compound (5) 171 3.2.11 Structure of compound (6) 174 3.2.12 Structure of compound (7) 177 3.2.13 Some typical correlations observed in HMBC spectrum of compound (7) 177 3.3.1 Cu Concentration in various fractions of Rhizomes and Aerial parts of 183 the plant. Values are mean ± SEM of three different experiments 3.3.2 Ni concentration of Aerial parts of plant. Data are expressed as mean 189 ±SEM of three different findings. 3.3.3 Concentration of macronutrients, Ca [A], Na [B] and K [C] in Rhizomes 190 of the plant. Data are expressed as mean ±SEM of three different findings. 3.3.4 Concentration of macronutrients, Ca [A], Na [B] and K [C] in Aerial 191 parts of the plant. Data are expressed as mean ±SEM of three different findings.

LIST Of TABLES Entry Caption P. No.

1.1.1 Distribution of registered herbal practitioners (Hakims and Homeopathic 05 physicians) in different provinces of Pakistan. MOH Pakistan, 2002- 2003. Extracted from annual report of MOH Pakistan, (MOH, 2005b). 1.1.2. Utilization of herbal products in different regions of the world and 06 expected annual increase (%).Extract from M.Phil thesis (Khan, 2005). 1.2.1 List of compounds isolated from genus Polygonatum 21 2.1.1 List of chemicals used in various assays 42 2.1.2 Reference antimicrobial strains 50 2.1.3.1 Composition of Tyrode’s normal solution 59 2.1.3.2 Composition of Tyrode’s, Ca++-free, K+- normal solution 61 2.1.3.3 Composition of Tyrode’s, Ca++-free, K+- rich solution 62 2.1.3.4 Composition of Kreb’s solution 63 2.3.1 The instrumental conditions maintained for each element on Atomic 84 Absorption spectrometer (Hitachi Polarized─8000 Japan). 2.3.2 The instrumental conditions maintained for each element on Flame 85 Photometer (Jenway PFP7, UK). 2.3.3 The instrumental conditions maintained for each standard element on 85 Atomic Absorption spectrometer (Hitachi Polarized─8000 Japan).

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2.3.4 The instrumental conditions maintained for each element on Flame 87 Photometer (Jenway PFP7, UK). 3.1.1.1 Effect of the crude extract of Rhizomes in writhing induced by acetic 89 acid in mice at 50, 100 and 200 mg/kg, i.p. 3.1.1.2 Effect of Rhizomes of on hot plate (thermal stimuli) test with and 92 without Naloxone in mice at 50, 100 and 200 mg/kg, i.p. 3.1.1.3 Effect of aerial parts of on hot plate (thermal stimuli) test with and 93 without Naloxone in mice at 50, 100 and 200 mg/kg, i.p. 3.1.1.4 Anti-inflammatory effect of the crude extract of Rhizomes in 96 carrageenan stimulated hind paw edema in rats at 50, 100 and 200 mg/kg. 3.1.1.5 Protection (%) of Rhizomes (50, 100 and 200 mg/kg i.p.) in carrageenan 97 induced hind paw edema in rats. 3.1.1.6 Antipyretic effect of the crude extract of Rhizomes in yeast induced 101 pyrexia in mice at 50, 100 and 200 mg/kg. 3.1.1.7 Antipyretic effect of the crude extract of Aerial parts in yeast induced 102 pyrexia in mice at 50, 100 and 200 mg/kg. 3.1.1.8 Effect of acute toxicity test of Rhizomes and Aerial parts in mice at 500, 105 1000 and 2000 mg/kg, p.o. 3.1.2.1 Anti-bacterial activities of Rhizomes using agar well diffusion method. 107 3.1.2.2 Anti-bacterial activities of Aerial parts of in agar well diffusion method. 108 3.1.2.3 Anti-fungal activities of extract Rhizomes and its solvent fractions. 111 3.1.2.4 Antifungal activity of the crude extract of Aerial parts. 113 3.1.2.5 In vitro anti-malarial activity of the Rhizomes against Plasmodium 115 falciparum 3.1.2.6 In vitro anti-malarial activity of the Aerial parts against Plasmodium 115 falciparum 3.1.2.7 Phytotoxic activity of the crude extract and its successive fractions of 118 Rhizomes against the Lemna acquinoctialis Welv.

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3.1.2.8 Phytotoxic activity of Aerial parts of P. verticillatum against the Lemna 121 acquinoctialis Welv. 3.1.2.9 Insecticidal activities of crude extract and various factions of Rhizomes 122 3.1.2.10 In-vitro antileishmanial activity of Rhizomes against Leishmania major 123 3.1.2.11 Brine shrimp cytotoxicity of Rhizomes. 125 3.1.2.12 Brine shrimp cytotoxicity of Aerial parts. 126 3.1.2.13 DPPH scavenging activity of Rhizomes. 128 3.1.2.14 DPPH scavenging activity of Aerial parts 129 3.1.2.15 In vitro quantitative Inhibition of lipoxygenase by crude extract and 134 various fractions of plant 3.1.2.16 In vitro quantitative Inhibition of Urease 135 3.2.1 Preliminary phytochemical (qualitative) tests 157 3.2.2 NMR spectral estimations of Propyl pentadecanoate (1) 159 3.2.3 NMR spectral estimations of 2`,3`- Dihydroxy propyl pentadecanoate 162 (2) 3.3.4 NMR spectral estimations of 2-hydroxybenzoic acid (3) 165 3.2.5 NMR estimations of 5-hydroxymethyl-2-furancarboxyldehyde (4) 167 3.2.6 NMR spectral estimations of Diosgenin (5) 169 3.2. 7 NMR spectral estimations of β-Sitosterol (6) 171 3.2.8 NMR spectral estimations of Santonin (7) 176 3.2.9 Qualitative and quantitative composition of n-hexane fraction of 179 Rhizomes of Polygonatum verticillatum 3.1.10 Qualitative and quantitative composition of n-hexane fraction of Aerial 180 parts of Polygonatum verticillatum 3.3.1 Quantification of micronutrients (ppm) in crude extract of Rhizomes of 185 Polygonatum verticillatum and its subsequent solvent fractions 3.3.2 Quantification of micronutrients (ppm) in crude extract of Aerial parts 186 of Polygonatum verticillatum and its subsequent solvent fractions. 3.3.3 Permissible limits of various metals in plant 188

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CONTENTS S. NO. Caption P. NO. Acknowledgment I Summary II List of abbreviations V11 List of figures XI List of tables XV 1.0 Introduction 1 1.1. Introduction to phytotherapy 1 1.1.1. History of natural products 1 1.1.1.1. Greek period 2 1.1.1.2. Traditional Chinese Medicine (TCM) 2 1.1.1.3. Traditional Indian Medicine (TIM) 3 1.1.1.4. Arabic period 3 1.1.2. Medicinal plant in Pakistan 4 1.1.3. Medicinal plants: Economic perspective 6 1.1.4. Recent scientific development in phytomedicine 7 1.1.5. Future challenges to phytomedicine 9 1.1.6. Quality control of phytomedicine 10 1.2. Genus Polygonatum 13 1.2.1. Ethnobotanical uses 13 1.2.2. Phytochemistry and pharmacological effects. 14 1.2.3. Taxonomic status 17 1.2.4. Polygonatum verticillatum 17 1.2.4.1. Plant distribution 17 1.2.4.2 Plant description 17 1.2.4.3 Plant habitat and cultivation 18 1.2.4.4 Ethanomedical uses 18 1.2.4.4. Phytochemistry 20 2.0 Experimental 2.1. Pharmacolgical Studies 41 2.1.1. In-Vivo Studies 41 2.1.1.1. Drugs and reagents 41 2.1.1.2. Study Animals 41 2.1.1.3. Plant material 43 2.1.1.4. Antinociceptive activities 44 2.1.1.5. Carrageenan induced oedema 45 2.1.1.6. Antipyretic activity (Yeast induced pyrexia) 46 2.1.1.7. Pentylenetetrazole induced convulsion 47 2.1.1.8. Diuretic activity 47 2.1.1.9. Acute toxicity test 48 2.1.2. In-Vitro Studies 48 2.1.2.1. Antimicrobial studies 48 2.1.2.2 In-vitro antimalarial bioassay 50 2.1.2.3 In-vitro phytotoxic bioassay 51 2.1.2.4. In vitro Insecticidal activity 52 2.1.2.5. In vitro leishmanicidal activity 52 2.1.2.6. Brine shrimp cytotoxic assay 53 2.1.2.7. 1,1-diphenyl-2-picrylhidrazyl (DPPH) scavenging activity 54 2.1.2.8. Enzyme inhibition assays 54 2.1.3. Ex-in vivo (Isolated animal tissue) studies 56 2.1.3.1. Bioassay on rabbit jejunum 56 2.1.3.3. Vascular activity 61 2.1.3.4. Guinea-pig atria 62 2.1.2.5. Statistical applications 63 2.2. Phytochemical investigations 63 2.2.1. Experimental settings 63 2.2.1.1. Spectroscopy 63 2.2.1.2. Chromatography 64 2.2.1.3. Spraying Reagents 64 2.2.1.4. Purification of solvents 64 2.2.2. Quantitative and qualitative phytochemical analysis 65 2.2.2.1. Total contents determination (Quantitative) 65 2.2.2.2. Qualitative analysis 68 2.2.3. Compounds Isolation using column chromatography 69 2.2.3.1. Characterization of propyl pentadecanoate (1) 72 2.2.3.2. Characterization of 2,3-dihydroxypropyl pentadecanoate (2) 73 2.2.3.3. Characterization of 2-Hydroxybenzioc acid (3) 74 2.2.3.4. Characterization of 5-Hydroxymethyl-2-furaldehyde (4) 75 2.2.3.5. Characterization of Diosgenin (5) 76 2.2.3.6. Characterization of β-Sitosterol (6) 77 2.2.3.7. Characterization of Santonin (7) 78 2.2.4. Gas chromatography (GC) and Gas chromatography-mass 79 spectrometry (GC-MS) analysis 2.2.4.1. Instrumentation 79 2.2.4.2. Experimental settings 79 2.3. Metals Analysis 83 2.3.1. Experimental settings 83 2.3.1.1. Instruments 83 2.3.1.2. Reagents / Chemicals 83 2.3.1.3 Contamination control 83 2.3.1.4. Sample preparation 85 3 Results and Discussion 3.1 Pharmacological investigation 86 3.1.1. In vivo studies 86 3.1.1.1. Antinociceptive activity 86 3.1.1.2. Effect of Carrageenan induced oedema 92 2.1.1.3. Effect of antipyretic activity (yeast-induced pyrexia) 97 3.1.1.4. Effect of Pentylenetetrazole induced convulsions 100 3.1.1.5. Effect of diuretic activity 100 3.1.1.6. Effect of acute toxicity studies 101 3.1.2.1. Antimicrobial Activities 102 3.1.2.2. Effect of antifungal activities 110 3.1.2.3. Effect of in-vitro antimalarial 114 3.2.1.4. Effect of in vitro phytotoxicity 117 3.1.2.5. Effect of in vitro insecticidal activity 122 3.1.2.6. Effect of in vitro leishmanicidal activity 123 3.1.2.7. Brine shrimp cytotoxic assay 124 3.1.2.8. Effect of 1,1-diphenyl-2-picrylhidrazyl (DPPH) scavenging activity 126 3.1.2.9. Enzyme inhibition assays 130 3.1.3. Ex-in vivo (Isolated animal tissue) studies 136 3.1.3.1 Effect on the rabbit jejunum 136 3.1.3.2. Effect on rabbit trachea 146 3.1.3.3. Effect on rabbit aorta (vascular activity) 152 3.1.3.4. Effect on guinea-pig atria 153 3.2. Phytochemical Studies 153 3.2.1. Quantitative analysis 153 3.2.1.1 Phenol contents 153 3.2.1.2. Alkaloid contents 153 3.2.1.3. Saponin contents 153 3.2.1.4. Flavonoid contents 154 3.2.2. Qualitative analysis 155 3.2.3. Isolation of pure secondary metabolites 157 3.2.3.1. Structure elucidation of Propyl pentadecanoate (1) 158 3.2.3.2 Structural elucidation of 2`,3`-Dihydroxy propyl pentadecanoate (2) 161 3.2.3.3. Structural elucidation of 2-Hydroxy benzoic acid (3) 164 3.2.3.4. Structural elucidation of 5-Hydroxymethyl-2-furanaldehyde (4) 166 3.2.3.5. Structural elucidation of Diosgenin (5) 168 3.2.3.6. Structural elucidation of β-Sitosterol (6) 172 3.2.3.7. Structural elucidation of Santonin (7) 174 Identification of constituents by gas chromatography-mass 3.2.4. 178 spectrometry (GC-MS) 3.3. Metal Analysis 181 3.3.1. Micronutrients 181 3.3.1.1. Iron (Fe) status 182 3.3.1.2. Copper (Cu) status 182 3.3.1.3. Zinc (Zn) status 184 3.3.1.4. Manganese (Mn) status 184 3.3.1.5. Chromium (Cr) status 185 3.3.1.6. Nickel (Ni) status 187 3.3.2. Macronutrients 187 3.3.2.1. Sodium (Na) status 187 3.3.2.2. Potassium (K) status 189 3.3.2.3. Calcium (Ca) status 192 Conclusions 194 References 196

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1.1 Introduction to Phytotherapy

Over the centuries, medicinal plants have been utilized in various cultures of the world as a natural healing tool. Plants as a source of medication in the form of traditional and folklore based on the rich experiences of innumerable healers over centuries, inherited from ancestors, healer-to-healer transfer or developed through personal experiences over time. Modernity or cultural revolutions has not altered the in-depth wisdom of this natural medical paradigm. Consequently, no modern system of medicine can ordinarily lay claim to it. The traditional system of treatment, differing in concept and protocol, exemplify well- developed systems such as Allopathic, Homeopathic, Ayurvedic, Chinese system of treatment (Gurib-Fakim, 2006; Schippmann et al., 2002). Most of the civilized nations have developed their own Materia Medica, compiling details about various plants used for therapeutic purposes. The merging of this human pharmacopoeia of natural origin with the incredible development in the various fields of modern medical sciences indeed provides the foundation for a much needed revolution in the existing healthcare system.

Botanical survey estimated the identification of 250, 000 to 350, 000 plant species over the planet. However, approximately 35, 000 species have been used in different communities of world for the treatment of various ailments (Jin-Ming et al., 2003). These plants are mostly exercised in unrefined or semi-processed form, often in mixtures; therefore require quality control testing and rigorous clinical trials for scientific rationale (De Smet, 2002; Kinsel and Straus, 2003). Researchers believed that approximately 15% of medicinal plants have been subjected to phytochemical analysis and 6% to biological screening (Harvey, 2000; Newman et al., 2000; Verpoorte, 2000). The rest of plants remained untouched; therefore, this therapeutic modality has tremendous scope in the discovery of new effective therapeutic agents.

1.1. 1. History of medicinal plants

Extensive investigations have revealed that medicinal plants in different shapes, either in crude form or pure molecules isolated from them, represent the most ancient mode of

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medication. Archaeological studies have been provided reasonable evidences that the healing properties of plants were known to peoples in prehistoric time (Halberstein, 2005). Since the medicinal usage of the plants is as old as human civilization, however some of the oldest references that are available in the Artharvaveda, which is the basis of traditional Indian medicine called Ayurvedic medicine (dating back to 2000 BCE). Mesopotamians (1700 BCE) (Goldsby et al., 2001) described the use of clay tablets while the use of Eber Papyrus in Egyptians (1550 BCE) are documented (Ramawat and Merillon, 2008). Other documented data that revealed the medicinal usage of plants are “De Materia Medica,” written by Dioscorides between CE 60 and 78, and “Pen Ts’ao Ching Classic of Materia Medica” written around 200 CE (Ramawat and Merillon, 2008).

1.1.1.1. Greek period

Greek civilization was an epoch of science and philosophy. Greek has made worth mentioning contribution in pharmaceutical sciences especially in phyto-pharmaceutical (Halberstein, 2005). Aristotle has described 500 crude drugs used in the cure of different pathological conditions (Chatard, 1908). Hippocrates (460-337 BC) is considered as the father of Allopathic medicine. He formulated the first scientiﬁc medical paradigm of treatment. He proposed that large number of pathological conditions were due to disturbance in the normal physiology of human systems. The treatment was therefore, based on the causes of the diseases to normalize the imbalance body systems (Sykiotis et al., 2006). He has pointed out nearly 400 samples of medicinal substances from plant origin. Theophrastus (370−287 BC), a student of Aristotle (Scarborough, 1978) has also mentioned 500 crude drugs in his book. Another important name is that of Claudius Galen Pergamum (modern-day Bergama, Turkey: 129−199). He prepared vegetable drugs using different extraction techniques called Galenicals and introduced the concept of pharmaceutical formulation to formulate stable and therapeutically effective drugs (Buerki and Higby, 2007; Newman et al., 2000). He wrote some 300 books on plants.

1.1.1.2. Traditional Chinese Medicine (TCM)

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The Traditional Chinese Medicine (TCM) represents one of the oldest systems of treatment. TCM is unique in theories, treatment and therapies. This effective system of medicine has tremendous importance in the history of medicine and now got global recognition due to evidence basis approach (Patwardhan et al., 2005). This system is nearly free of external influence. Fu His (2953 BC) is being judged as the pioneer of this system (TCM). The prescription of TCM addresses those exogenous factors which are considered to be engaged in the pathology (Kopp et al., 2003). Later the emperors Shen Nung and Hong Ti developed this system more significantly. Chinese pharmacopoeia Pen Tsao enclosed enormous remedies for various medical problems. Crown of written Chinese medicine goes to Shen Nong Ben Cao Jin (22-250 AD). CaoYuan Fang (550–630) wrote a book named “Zhu Bing Yuan Ji Lun” that described aetiology and symptoms of various diseases. Since then the book is considered as a standard reference book for Chinese medical students (Kopp et al., 2003).

Wang Tao (702–772) has an important contribution in TCM. His published work described approximately 600 prescriptions in the name of “Waitai Miyao”. The foundation of his diagnostic philosophy was tongue. During different pathological conditions, the colour and status of tongue changes (Kopp et al., 2003). A great Chinese physician and naturalist, Li Shizen has written a more inclusive pharmacopoeia Ben Ca Gang Mu, which has been published in 1596. It has 1894 prescriptions and is still in exercise as reference and guide for research and schooling in China and several other communities. Importantly, TCM was traditional knowledge that passes through generations, but only in the 1950s, it was formatted in the form of academic educational training (Xu and Yang, 2009).

1.1.1.3. Traditional Indian Medicine (TIM)

Traditional Indian Medicine or Ayurveda (known as mother of all therapies) is considered as the oldest health care system on earth. The descriptions of the system are available in ancient literatures such as ‘Rig-Veda’ and ‘Atharva-Veda’, approximately 5000 years BC (Mukherjee and Wahile, 2006). Ayurveda is a Sanskrit word that literary means knowledge of life. It is a natural healing system consists of a mixture of physiologic and holistic

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medicine. Ayurveda defined man as a matrix of seven basic tissues that works in harmony while disease is the outcome of imbalance in these components (Routh and Bhowmik, 1999).

1.1.1.4. Arabic period

The Arabs have made enormous progress in the field of science and medicine after the fall of the Roman Empire. Scholars from the Islamic world translated books from Greece and Rome. Arabs physicians introduced the concept of diet control and exercise along with medications (Azaizeh et al., 2003; Azaizeh et al., 2006). Arabs are actually the pioneer in the start of basic pharmacy practices. This includes the foundation of drug stores, the job description of physicians as disease diagnosis while pharmacists were deputed for the drug extraction and formulation. Due to this demarcation, the development in each field has started. As a result of this, Jaber Bin Hayan, a Muslim chemist extracted and isolated various chemicals like alcohols, nitric acids, sulphuric acids etc (Azaizeh et al., 2006).

The religion of Islam has set a new breadth to the science of medicine in Arabia. Islam has specified means for a hygienic life style (Saeed, 1978). These principles are primarily focused on AL Quran and Sunnnah and are entitled as Tibb al-Nabi (Qureshi and Ghufran, 2007). In medicine, the Arabs have come out as the successors of Greek. Ali Ibn Rabban Al Tabri (782-855 AD) was a renowned Muslim scientist. His book Firdous Al Hikmat (Chatard, 1908), consists of seven parts in which one is specially focused on drugs and poisons. Abu Ali Al Hussan Ibn Sina (Avicenna, 980-932 AD) is the creator of the Greco-Arabic school of medicine (Guerra, 1979). His book Canon was considered as a textbook on medicine in Europe, which describes more than 1000 drugs. His other book, Kitab Ash- Shifa is considered as a scientific Encyclopedia. Apart from the therapeutic and healing characteristic, the Arabs also described the toxic aspects of various plants. Abu Musa Jabir ben Hayyan has written a very comprehensive book on different plant poisons and antidotes for that named “The Book on Poisons and Antidotes” (Saad et al., 2006a).

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1.1.2. Medicinal plant in Pakistan

In terms of plants biodiversity, Pakistan possesses an incredible status among the developing countries most probably due to varied climatic and edaphic factors. This valuable heritage is scattered throughout the country. However, there is no systemic approach for the growth / cultivation of these plants. Around the country, it has been speculated that approximately 6000 taxa of flowering plants including the Pakistan- occupied Kashmir. On the basis of ethnobotanical investigations, approximately 600-1000 plants have medicinal properties and only 12% are utilized in the management of different pathological conditions (Husain et al., 2008; Rashid et al., 2009). In the local drug markets (pansara), approximately 350-400 species are traded and used by various manufacturers in the formulation of herbal preparations (Shinwari, 2010).

The traditional Unani System of Treatment is the integral part of Pakistani community. The physician in Unani system of treatment is called Hakim / Tabib and the system as Hikmat / Tibb. This system has its roots in ancient Greek system of treatment that was introduced and practiced by Muslims scholars’ long time ago in the United India. The federal government through Unani, Ayurvedic and homeopathic (UAH) Practitioners Act, 1965, regulates the Unani System of Medicine. National Council of Tibb and National council for Homeopathy are established as corporate bodies under section 3 of the said Act to promote and popularize the traditional system of education. The registered Hakims and vaids throughout the country are estimated at 39,584 and 455 respectively. These healthcare professionals are produced by 125 recognized homeopathic medical colleges in the country (MOH, 2005a). However, reasonable number of unregistered Hakims are too involved in the practice; based on the empiric knowledge came from ancestors. Pakistan is aggressively involved in the international trade of medicinal plants. Pakistan ranks ninth larger importer of medicinal plants (11350 tonnes) while tenth larger exporter with (8100 tonnes) expressing the country’s potential (Schippmann et al., 2002). As a salient point, the country’s herbal industry not flourished over the course of time and therefore, mostly stands on the imports of raw materials. The cultivation of medicinal plants is highly

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recommended for sustainable development of herbal industry by adopting modern scientific techniques to meet not only the growing local demand but also to earn foreign exchange from the export of plant oriented products.

Table 1.1.1: Distribution of registered herbal practitioners (Hakims and Homeopathic physicians) in different provinces of Pakistan. MOH Pakistan, 2002-2003. Province Hakims Homeopathic physician Punjab 31,014 55,915 Sindh 6271 20,124 NWFP (Khyber Pakhtoon Khaw) 2019 4,969 Balochistan 280 878 Islamabad 455 489 Total 39,584 82,375

Extracted from annual report of MOH Pakistan, (MOH, 2005b).

1.1.3. Medicinal plants: Economic perspective

On the face of global acceptance in all ages, phytopharmaceuticals are portrayed as cornerstone in the world trade and economics. The total global herbal market of plant- based drugs has been estimated as $ 18 billion in 2005 (Saklani and Kutty, 2008; Willison and Andrews, 2004). Obviously, the international herbal trade market is revolving around China and India. The annual herbal drugs export of China is estimated over 120,000 tonnes followed by India with approximately 32,000 tones. In contrast, Europe is the primary importer of remedial plants and around 400,000 tonnes are imported each year by different European countries to meet the local demand of herbal formulations (Briskin, 2000; De Smet, 2002).

Report in the British parliament expressed the strong faith of English community in natural remedies. British spent 126 million £ in 2002 while visit to the clinics of 50,000

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herbal practitioners (Robson, 2003). The trend of herbal products usage enormously increasing in United State (Briskin, 2000) and the total herbal market was estimated as $ 4.2 billion in 2001. There is reasonable increase in herbal remedies over the years in the American community. The same scenario has been observed in European countries. The sales of only over the counter (OTC) herbal products have estimated almost $ 5 billion. Importantly, Germany and France are the two major stack holders in herbal remedies (De Smet, 2005) more precisely, 50% of Germans have shown confidence in herbal drugs for the healing of diverse diseases. Similarly, the exercise of herbal products as household remedies is very common in different Asian and African countries (Gilani and Atta-ur- Rahman, 2005). In the present era of medicinal engineering, there are many compounds isolated or derived from medicinal plant that holding a significant market share around the world. It is worth mentioning that twenty-six different phytopharmaceuticals launched during 2000-2006, belonging to different therapeutic classes. Nevertheless, five new different compounds have been approved by the Food and Drug Administration (FDA) for clinical use in different diseased areas. A significant number of natural compounds were included in top 35 best selling products of the world (Chin et al., 2006; Saklani and Kutty, 2008).

Table.1.1.2: Utilization of herbal products in different regions of the world and expected annual increase (%). Sales of Herbal Medicine Annual Growth Rates by Region (%) Region (Million US$) Region 1985-91 1991-92 1993-98 European Union 6,000 European Union 10 5 8 Rest of Europe 500 Rest of Europe 12 8 12 Asia 2,300 South East Asia 15 12 12 Japan 2,100 Japan 18 15 15 North America 1,500 India/Pakistan 12 15 15 Total 1 2,400

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Extract from M. Phil thesis (Khan, 2005).

1.1.4. Recent development in phytomedicine

Medicinal plants have played an amazing role in the devolvement of new clinically effective drugs. Though remarkable development has been made in the fields of chemistry such as synthetic, combinatorial, and biotechnological sciences, medicinal plants can still be exploited as an initial point for the synthesis of new compounds with different structural parameters. In the presence of these sophisticated technologies, the plant-derived drugs become more streamlined. The proper utilization of these techniques has already lead to the discovery of some interesting clinically useful molecules (Balunas and Kinghorn, 2005; Rates, 2001). Importantly, 15 compounds of natural origin have been launched during 2000-2003 while the same number of compounds are in the phase ІІІ clinical trials or registration stage of drug development (Butler, 2004). It has been recently estimated that the natural product offer 100 times higher hit rate when compared with synthetic drugs (Lam, 2007).

Figure 1.1.1: Drugs derived from plants in different stages of clinical trials. Extracted from (Harvey, 2008).

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Perfect coordination of numerous fields is crucial in the discovery of phytomedicine. The identification and collection of plant material from specific locality is the job of ethnobotanist (Huie, 2002) . Phytochemist urges to design rapid but efficient method of extraction from plant source. Keeping in view the fork uses, the ethnopharmacologist proposes and screens out the extract in some relevant assay. Based on the fallout of test, the phytochemist subjects the extract to the isolation of pure chemical entities that could be responsible for the activity. Afterward, different clinical trials are carried for the particular molecule. It is bitter truth that only one molecule out of 5000 successfully completes all stages of development and obtain registration for clinical applications (Balunas and Kinghorn, 2005).

1.1.5. Future challenges to phytomedicine

Many challenges are ahead of drugs discovery from medicinal plants. The various components of the team like ethnobotanist, ethnochemist and ethnopharmacologist needs to further strengthen coordination in order to get more fruitful results in terms of effective therapeutic agents for the treatment of many challenging human disorders (Gilani and Atta- ur-Rahman, 2005). The plant-based scientist has to work out and expedite various techniques involved in the drug discovery (Huie, 2002). It has been observed that drug discovery passing through different stages of development takes approximately 10 years and with overall expenditure of $ 800 million. However, the plant based drugs discovery usually needs more time and complex events as compared to other modes of drug discovery. This has created a negative impact on the fate of various ongoing projects and as a result, the pharmaceutical companies withdrew many research projects on medicinal plants (Butler, 2004).

Since natural compounds are highly value added products, it is therefore extremely important to induct new modern techniques in the method of collection and other processes involved in the product development in order to prevent irrelevant wastage of time and

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expenditures. For instance, paclitaxel was isolated from Taxus brevifolia with prominent anticancer properties. Despite of structural conformation and established therapeutic activity, the compound took almost 20 years in marketing approval as TaxolR (McChesney et al., 2007). However, the introduction of new technologies like high-throughput enormously expedites screening of extracts; 100,000 plant extracts can be screened in a period of 1 week while using a 384-well format. The commendable development in spectroscopy and chromatography in addition to the use of cell culture techniques for considerable increase in percentage yield of desirable components of plants, can revolutionize the plant base new drugs (Ramawat and Merillon, 2008).

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Figure 1.1.2: Flow chart showing various steps involved in the drug development from medicinal plants. Extracted from (Fabricant and Farnsworth, 2001).

1.1.5. Quality control of phytomedicine

The advocates of medicinal plants have the opinion that they are always safe because of natural origin. But the inherent safety claims about medicinal plants always appeared controversial. Nevertheless, this principle of safety is absolutely ruled out by the clinical findings on the heavy metals poisoning from different parts of the world (Basgel and Erdemoglu, 2006; Ernst, 2002; García-Rico et al., 2007). Of particular importance is not only the intrinsic plants toxicity but also adulterations, provoking multiorgan toxicity (Saad et al., 2006b). As a result, the Food and Drug Administration (FDA) has pointed out some of the commonly used botanicals for lethal unwanted effects (Board, 2002). Consistency in composition is primarily important for the efficacy and safety of phytomedicine that also

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Figure.1.1.3: Flow chart showing toxicity due to herbal products. Extracted from (Saad et al., 2006b).

related to the overall therapeutic response. Since botanicals are the mixture of different/numerous biological active components, their standardization could rarely be possible because of multiple known reasons (Board, 2002). However, different models for the standardization of phytomedicine is available that need further polishing (Calixto, 2000).

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Legislative bodies are urging to play their role in the quality control of herbal formulations. Under the umbrella of WHO, a recent comprehensive survey report revealed that many countries have established or near to establish national regulatory guidelines for the safe use of herbal remedies (Barnes et al., 2007). The longstanding traditional experience as an evidence of botanical safety is not always logical and acceptable to detect their rare or late outcomes. The dearth of scientific studies on botanicals is rightly criticized by the advocates of orthodox medicines. Particularly to determine the therapeutic value of any drug, the randomized, controlled trials are frequently suggested. The product safety evidence should be mandatory for any botanicals like orthodox synthetic drug in order to prevent the end users from the unwanted effects of these formulations. That is why the new issued guidelines of the Food and Drug Administration permit the approval of herbal mixtures with the evidence of safety and efficacy, even though the active constituents are not identified (Qiu, 2007). Indeed to guarantee the fruitful outcomes from medicinal plants, scientific inquiry is indispensable in the light of modern sophisticate technologies (Newman et al., 2003).

In short, plant base drugs have unmatched chemical diversity and an incredible potential of novelty with different mechanistic templates. Simply, Nature has incorporated best combinatorial chemistry in plants. Various research laboratories are currently involved in phyto-medicines research with some outstanding success over the years. Consequently, several promising new chemical entities of plant origin are in clinical trial phase. However, many folds are still most wanted to explore unseen secrets of their curative potentials and to relieve humanity from dreaded diseases. Indeed the surging waves of pragmatism based on the experimental findings in various research laboratories addressed the healthcare professionals. Ultimately, the modern medical setup recognizing and moving to a system based on the combination of orthodox and natural therapies as a leading science to deal with the wisdom that lies in botanicals. The merging of this natural human pharmacopoeia with the incredible development in the various fields of modern medical sciences indeed provides the foundation for a much needed revolution in the existing healthcare system.

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1.2. Genus Polygonatum

Polygonatum (King Solomon's-seal, Solomon's Seal) a genus of approximately 60 species belongs to family Liliaceae or Convallariaceae. The various species of the genus are widely distributed in the temperate regions of the East Asia. Specifically in China and Japan, approximately 40 different species of Polygonatum have been reported (Szczecinska et al., 2006; Tamura, 1993). Additionally it is also found in India, Korea, Nepal, Afghanistan, Bhutan, Nepal and Russia. Along with Asia, Polygonatum also grows in the moderate climate zones of North America and Europe. Flora of Pakistan indicates the presence of four different species of Polygonatum. These include P. multiflorum, P. geminiflorum, P. cirrhifolium and P. verticillatum. Polygonatum species are widely distributed in various part of the country like Hazara, Chitral, Swat and Kurram agency (Polygonatum, 2010; Stewart, 1972). They are usually wild perennial rhizomatous herbs (Szczecinska et al., 2006).

Table 1.2.1. Various Polygonatum species in Pakistan (Flora of Pakistan) S. No Species Locations 1 P. multiflorum Not specified 2 P. geminiflorum Chitral, Swat, Hazara, Kurram 3 P. cirrhifolium Gilgit 4 P. verticillatum. Chitral, Dir, Swat, Hazara, Gilgit

1.2.1. Ethnobotanical uses

The ethnomedical uses of Solomon’s seal are very old in the treatment of diverse human disorders. The different parts of the Polygonatum species include rhizomes, leaves, fruits and flowers and are edible and Chinese cooked it with meats (Liansheng et al., 1991). The rhizomes are usually used in the form of decoction, steeped in vine or powder for

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therapeutic purpose. The rhizomes of the plants are used as poultice in some skin problems and it has also been used to remove freckles and is considered helpful in tissue repair. The rhizomes of Polygonatum are antiperiodic, antitussive, cardiotonic, demulcent, diuretic, energizer, hypoglycemic, sedative, tonic and are used in the treatment of dry coughs and pulmonary problems, including tuberculosis (Hou and Jin, 2005; Jiangsu, 1977; 1986). The powdered plant materials have been exercised as a snuff to encourage sneezing and consequently clear the bronchial channels.

In the form of infusion, Polygonatum has been advised in the treatment of stomach inflammations, chronic dysentery and related gastrointestinal disorders including digestive aid. Similarly the practice of Polygonatum has been documented in the treatment of various blood disorders (Jiangsu, 1977). The use of Polygonatum for antiaging properties is also available in literature. Some of the additional uses of the Polygonatum including beneficial effects on kidneys and liver, enhances bones strength, prevent gray hair, vision problems, vertigo and ringworms. Polygonatum has been employed as nervine tonic, reducing the mental capabilities due to ageing (Hou and Jin, 2005). In the Traditional Chinese System of treatment, Polygonatum is widely used in the treatment of diabetes. For the symptomatic relief, the decoction of the Polygonatum is administered orally to overcome hyperglycemia. Sometime it is used in combination with other herbs that employed traditionally as antidiabetic in order to get desirable therapeutic effects. The usual recommended dose is 5- 15 g (Hou and Jin, 2005).

1.2.2. Phytochemistry and pharmacological effects

Research groups have been reported variety of compounds from the genus Polygonatum primarily saponins, phyto-hormones, glycosides, flavonoids and alkaloids (Table 1.2.1). These groups of compounds have different type of activities. Researchers have investigated the effects of steroidal glycoside on the insulin action and secretion as well as glucose utilization, isolated from P. odoratum. The results of the animal model in 90% pancreatectomized rats indicated the antihyperglycemic potential of compound by reducing insulin resistance thereby increasing the glucose uptake and utilization. However, the

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compound did not show any effect on the secretion of insulin. The compound possesses significant insulin sensitizer properties and may be effective in the management of diabetic patients suffering from insulin resistance (Choi and Park, 2002). Apart from this, the antidiabetic character of the total flavonoids contents of P. odoratum is also available in literature (Shu et al., 2009). The roots of P. sibiricum exhibited significant α-glycosidase inhibitory activity and thus describes the mechanism of antidiabetic activity of the plant in the traditional Chinese system of treatment (Gao et al., 2008).

The secondary metabolites isolated from the species of Polygonatum have demonstrated antimicrobial activity against different pathogens. Kinganone (new indolizinone) and 3-ethoxymethyl-5,6,7,8-tetrahydro-8-indolizinone were isolated from the rhizome of Polygonatum kingianum. Both Kinganone and 3-ethoxymethyl-5,6,7,8- tetrahydro-8-indolizinone exhibited antibacterial and antifungal activities in the agar diffusion assay (Wang et al., 2003a). Similarly, homoisolflavanone, triterpenoids and steroidal saponins were isolated from the rhizomes of P. odoratum. These compounds showed outstanding antimicrobial activity against the tested bacteria and fungi (Wang et al., 2009a; Wang et al., 2009b). The aqueous extract of Polygonatum was found effective against various human pathogenic bacteria. The bacteria were S. typhi, S. aureus and M. tuberculosis (Hou and Jin, 2005).

Many studies support the role of Polygonatum in the activation of apoptosis (Liu et al., 2009b; Liu et al., 2009c). The lectin isolated from the P. cyrtonema demonstrated outstanding inhibition against MCF-7 cells. The induction of apoptosis was suggested to be caspase-dependent in nature. Furthermore, it has also been shown that the apoptosis was augmented by autophagy (Liu et al., 2009a). The Bcl-2 is a protein with significant anti- apoptotic properties. As a therapeutic modality, the modulation of Bcl-2 concentration is an effective approach to treat cancers. The secondary metabolite, 8-methyl- dihydrobenzopyrone has been isolated from P. odoratum. The compound exhibited prominent anticancer activity in breast cancers by inducing the phosphorylation of Bcl-2. (Rafi and Vastano, 2007). Most of the saponins isolated from the Polygonatum species have cytotoxic activity. In a phytochemical study, 10 different steroidal saponins

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and a glycoside were isolated from P. zanlanscianense. When analyzed in cytotoxic assay (in vitro) against HeLa cells, all the tested saponins exhibited significant activity while the

IC50 was ranges from 3.14─14.57 µg/mL (Jin et al., 2004). The saponins isolated from the rhizomes of P. sibiricum were tested for cytotoxic potential against human breast cancer cells. The result showed moderate activities of the compounds (Ahn et al., 2006).

The antioxidant potential of Polygonatum has been investigated in comparison with Vitamin E, a known antioxidant (Jeon et al., 2004). The results of study on hypercholesterolemic rabbits revealed that the Polygonatum extract interfered with the different physiological factors that support the antioxidant defense system. These include the modulation of thiobarbituric acid-reactive substances (TBARS) and hydrogen peroxide concentrations in liver while, the hepatic enzymatic activities of catalase and total glutathione were significantly enhances. The antioxidant potential was further augmented by sparing high plasma vitamin E concentration (Jeon et al., 2004). The isolation of a very potent antioxidant like quercetin from P. altelobatum (Pao-Lin et al., 1997) providing a strong evidence of the antioxidant potential of Polygonatum.

The traditional claims of Polygonatum in the treatment of inflammation, pyrexia and analgesia have not been evaluated. However, secondary metabolites with well-defined analgesic, antipyretic and anti-inflammatory properties have been isolated from Polygonatum. For instance, salicylic acid has been reported from P. kingianum (Wang et al., 2003b) a historical analgesic, antipyretic and anti-inflammatory agent (Chiabrando et al., 1989). As shown in Table 1.2.1, many steroidal saponins have been isolated from Polygonatum including diosgenin and related compounds. Research on diosgenin and related steroidal saponins showed significant anti-inflammatory activity. These compounds nonspecifically inhibited both cyclooxygenase (cyclooxygenase 1 and 2). However, cyclooxygenase-2 was more prominent (Yu et al., 2008). The algicidal activity of Polygonatum is also reported in literature (Kim et al., 2006). Liquiritigenin and isoliquiritigenin are isolated from P. kingianum (Wang et al., 2003b). Emodin (1,3,8- trihydroxy-6-methylanthraquinone), an anthraquinone derivative has been isolated from P. multiflorum. The compound possesses ameliorating effects on the memory

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consolidation. For this study, specific animal model was used in which cycloheximide- dependent memory consolidation impairment in rats. The result was produced by the

induction of serotonergic 5-HT1A-receptor partial agonist and 5-HT2 receptor antagonist. However, the muscarinic receptor antagonist showed negative activity (Lu et al., 2007).

1.2.3. Taxonomic status

1.2.2. Taxonomic status of Polygonatum

Kingdom Animalia Phylum Platyhelminthes Class Cestoda Order Liliales Genus Polygonatum Family Liliaceae / Convallariaceae. Specie Polygonatum verticillatum

(Polygonatum, 2010)

1.2.4. Polygonatum verticillatum

1.2.4.1. Plant distribution

Polygonatum verticillatum [L.] All. is a perennial flowering herb. It is broadly allocated in different countries of the Asia and Europe and found at height of 1800-4000 m in grassy slopes. In Asia, it is reported from Nepal, Afghanistan, Bhutan, India, Russia and Pakistan. In Pakistan, it is frequently found in Utror, Gabral and Miandam valleys, Chitral, Dir, Swat, Hazara, and Gilgit as reported by Flora of Pakistan (Polygonatum, 2010; Stewart, 1972).

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1.2.4.2. Plant description

The Rhizomes are usually shortly branched and the thickness various from 0.7-1.5 cm. Stem is usually erect and leaves have 4-8 in wholes, sometime alternate near base of stem. Flowers are pendulous; perianth pale purple after dryness. The ripen fruits or berries are mostly red in colour however, dark green orange berries have been found. They remain hanging after the leaves have fallen and containing seeds (Xinqi and Tamura, 2000), 6-8 mm in diameter.

1.2.4.3. Plant habitat and cultivation

P. verticillatum can produce healthy seeds. In early autumn, seeds of the plant are grown in shady areas or forest. Plant is usually sensitive to heat but can tolerate majority of other envirmental conditions. It has been observed that the plant can easily grow in different types of soils (sandy, loamy or clay) with different pH (acidic, alkaline, or neutral). Climatically, the plant favors humidity in air. Mostly prefer oceanic, forest zones of variable degree (full shade or light shade forests) or grassy area on the top of hills for distribution (Tybjerg and Vestergaard, 1992).

1.2.4.4. Ethnomedical uses

P. verticillatum [Nooreallam / Permole (Pakistan) Salam-misri (India), Whorled Solomon's-seal (English)]. Plant has been used in the treatment of multiple ailments. The Rhizomes of the plant are used to cure kidney problems, appetizer and nervine tonic (beneficial effect upon the nervous system in some way). The plant is also used as a substitute of P. cirrhifolium (Sharma et al., 2004), which is a plant diuretic, contains a glycoside of digitalis group, that has been used for the loss of vigor, inflammation and fullness in the abdominal area, building up of fluids in bone joints, skin rashes, bronchitis, diabetes, hypertension; antibacterial and antifungal (Singh, 2006).

It has been reported that the tender parts of P. verticillatum are cooked as vegetable;

Introduction Chapter1 because of high starch contents while the underground parts are giving to animals for strength (Manandhar, 1986). The syrup of fresh rhizomes is used for the treatment of joint pain as well as antipyretic and for burning sensation (anti-inflammatory) and phthisis (Adnan et al., 2006; Singh, 2006). It is also used to promote urine discharge (diuretic) and attenuate painful urination (Ballabh et al., 2008). Additionally the plant has been used as emollient, aphrodisiac, vitiated condition of pitta and vata, appetizer (Alam, 2004). The other recommended folk uses of the plant in the Utror, Gabral and Miandam valleys (District Swat, Pakistan) includes animal feed for milk enhancement and power, sex stimulant and for painful conditions.

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Figure 1.2.1: Different parts of Polygonatum verticillatum [L.] All.

1.2.4.5. Phytochemistry

Phytochemically, lectin has been isolated from it. The fresh raw material (root stocks) of P. verticillatum was purified by affinity chromatography on thyroglobulin-sepharose. Purified lectin (120 mg/kg) were obtained that contain high percentage of asparaginic acid (28%) (Antoniuk, 1993). To the best of our knowledge based on available literature, Polygonatum verticillatum has never been scrutinized to phytochemical investigation for the isolation of therapeutically active entities.

1.3. Aims and objectives

Primary objectives of our research project were to carried out

 Various pharmacological activities of the extracts of both Rhizomes and Aerial parts of the plant.  Bioactivity guided isolation of the pharmacologically active secondary metabolites.

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 Micro and macro nutrients (metal) analysis of extracts of the plant.

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Table 1.2.1: List of compounds isolated from genus Polygonatum S. No. Botanical Chemical structure and IUPAC/Common name Reference sources 1

2-L-pyrrolidon-5-carboxylic acid

(Pao-Lin et P. altelobatum al., 1997) (3R)-5,7-dihydroxy-8-methoxy-3-(4-methoxybenzyl)-6-methylchrom-an-4-one.

(3R)-5,7,8-trihydroxy-3-(4-hydroxybenzyl)-6-methylchroman-4-one.

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2,5-dihydroxy-3-methyl-6-tricosylcyclohexa-2,5-diene-1,4-dione.

(25R)-Spirost-5-en-3β-ol; 3β-Hydroxy-5-spirostene. Diosgenin

22,23-Dihydrostigmasterol, Stigmast-5-en-3-ol, β-Sitosterin. β- Sitosterol.

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Stigmasta-5,22-dien-3β-ol. Stigmasterol.

2-docosyl-3,6-dihydroxy-5-methylcyclohexa-2,5-dione-1,4-dione. O HO CH3

H3C O

3-hydroxy-2-methyl-5-tetracosylhexa-2,5-diene-1,4-dione

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2-(3,4- dihydroxyphenyl)- 3,5,7- trihydroxy- 4H- chromen- 4-one. Quercetin

5-dodecyl-3-hydroxy-2-methylcyclohexa-2,5-diene-1,4-dione. O OH

HO O

2,5-dihydroxy-3-methyl-6-tetracosylhexa-2,5-diene-1,4-dione.

2,5-di- alkyl-3,6-dihydroxy-p-benzoquinone.

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(3β,23S,25R)-23-(α-L-arabinopyranosyloxy)spirost-5-en-3-yl4-O-(6-deoxy- α-L- mannopyranosyl)-d-glucopyranoside. Polypunctoside A.

Urea 2 HO O (Wang et al., O

O 2003a). OH Wang et al., 4', 7-dihydroxy-3'-methoxyisoflavone. 2003b). P. kingianum (Wang et al., 2003b).

(Yu et al.,

(24S,25R)-3β,24-dihydroxy-spirostan-5-en-12- 2009). one-3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside, (Zhang et Kingianoside I.

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al., 2006).

(He-Shui et al., 2009).

(24S, 25R)-3 β,24-di-hydroxy- (Xing-Cong spirostan-5-en-12-one-3-O-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside. et al., 1992). kingianoside H.

(25R)-[(3-O-β-D-glucopyranosyl- (1→2)-β-D-glucopyranosyl-(1→4)-β-D-galactopyranosyl)oxy]-26-[(β-D- glucopyranosyl)oxy]-22x -hydroxyfurost-5-en-12-one. kingianoside E.

2-hydroxybenzoic acid. Salicylic acid

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(25S)-[(3-O-β-D-glucopyranosyl- (1→)-β-D-galactopyranosyl)oxy]-26-[(β-D-glucopyranosyl)oxy]-22x-hydroxyfurost-5-en- 12-one. (25S)-kingianoside-C

(25S)-[(3-O-β-D-glu-copyranosyl- (1→4)-β-D-fucopyranosyl)oxy]-26-[(β-D-glu-copyranosyl)oxy]-22x-hydroxyfurost-5-en- 12-one. (25S)-kingianoside D.

(25S)-spirostan-5-en-12-one-3-O-β- D-glucopyranosyl(1→4)-β-D-galactopyranoside. 25S)-kingianoside A

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(25S)-spirostan-5-en-12-one-3-O-β- D-glucopy-ranosyl-(1→2)-β-D-glucopyranosyl-(1→4)-β-D-galactopy-ranoside. (25S)-

pratioside D1

(23S,25R)-spirostan-5- en-3β,23-dihy-droxy-12-one-3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)- β-D-galactopyranoside. (25R)-kingianoside G.

(25R)-[(3-O-β-D-glucopyranosyl-(1→4)-β- D-galactopyranosyl)oxy]-26-[(β-D-glucopyranosyl)oxy]-1β,3β,22x,26-tetrahydroxyfurost-5- ene. (25R,22)-hydroxylwattinoside C.

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Gentrogenin 3-O-β-d-glucopyranosyl(1→4)-β-d- fucopyranoside. Kingianoside B.

26-O-β-d-glucopyranosyl-22- hydroxy-25(R)-furost-5-en-12-on-3β,22-diol3-O-β-d-glucopyranosyl (1→4)-β-d- galactopyranoside. Kingianoside C.

Gentrogenin 3-O-β-d- glucopyranosyl(1→4)-β-d-galactopyranoside. Kingianoside A.

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26-O-β-d-glucopyranosyl-22- hydroxy-25(R)-furost-5-en-12-on-3β,22-diol3-O-β-d-glucopyranosyl (1→4)-β-d- fucopyranoside. Kingianoside D. OH HO O HO O HO HOOH HO OH O OH HO HO O O OH O O OH OH HO O OH O (25R)-[(3-O-β-D-glucopyranosyl-(1→2)-β- D-glu-copyranosyl-(1→4)-β-D-galactopyranosyl)oxy]-26-[(β-D-glucopyranosyl)oxy]- 1β,3β,22x,26-tetrahydroxyfurost-5-ene. kingianoside F.

N O O 3-ethoxymethyl-5,6,7,8-tetrahydro-8-indolizinone.

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HO O

O 7-hydroxy-2-(4-hydroxyphenyl)chroman-4-one. Liquiritigenin OH

HO OH

O (E)-1-(2,4-dihydroxyphenyl)-3(4-hydroxyphenyl)prop-2-en- 1-one. Isoliquiritigenin

5-hydroxymethyl-2-furancarboxaldehyde. HMF.

3 P. latifolium O (Kintya et

O al., 1978). HO HO OH O HO OH O O HO O O O O OHO HO O O O HO OH HO 3β-[0- β -D- glucopyranosyl-(1→3)-0-β-D-glucopyranosyl-(1→4)-0- β-d-galactopyranosyl-(1→3)- β -

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D-glucopyranosyloxy]-(25R)-spirost-5-ene. Polygonatoside E'

26-β-D- galactopyranosyl-(1→3)-α-D-glucopyranosyloxy]-(25R)- furost-5-en-22α-ol. Protopolygonatoside E'. 4 P. officinale (Janeczko et al., 1987)

25R-Furost-5-en-3,22,26- triol3-O-[β-D-glucopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-β-D-glucopyranosyl-

(1→4)-β-D-galactopyranoside]-26-O-β-D-glucopyranoside Polyfuroside. 5 (Yang and P. punctatum Yang, 1-(2,5-dioxoimidazolidin-4-yl)urea. Allantoin

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2006).

(3β,23S,25R)-23-[(2-O-acetyl- α-L- arabinopyranosyl)oxy]spirost-5-en-3-yl4-O-

(6-deoxy- α-L-mannopyranosyl)-D-glucopyranoside. Polypunctoside B

(3β,23S,25R)-23-[(3-O-acetyl-α-L- arabinopyranosyl)oxy]spirost-5-en-3-yl4-O-

(6-deoxy- α-L-mannopyranosyl)-D-glucopyranoside. Polypunctoside C.

(3β,22x,25R)-3-{[2-O-(6-deoxy-α-L- mannopyranosyl)-β-D-glucopyranosyl]-oxy}-22-hydroxyfurost-5-en-26-yl β-D-

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glucopyranoside.

(3β,23S,25R)-23-[(4-O-acetyl-α-L- arabinopyranosyl)oxy]spirost-5-en-3-yl4-O-

(6-deoxy- α-L-mannopyranosyl)-D-glucopyranoside. Polypunctoside D.

β-D-Glucopyranoside, (3β,25R)- spirost-5-en-3-yl O-6-deoxy-α-L-mannopyranosyl-(1→2)-O-[6-deoxy-α-L- mannopyranosyl-(1→4)]-;(25R)-3β-[2-O,4-O-Bis(α-L-rhamnopyranosyl)-β-D- glucopyranosyloxy]spirosta-5-ene. Dioscin.

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OH HO O HO OH O

HO O O OH HO O O HO OH O H3C O HO HOHO 26-O-β-D-glycopyranosyl-22- hydroxyfurost-5-ene-3β,26-diol-3-O-β-diglucorhamnoside. Protodioscin.

2-O-α-L-rhamnopyranosyl-β-D- glucopyranoisede. Prosapogenin A of dioscin. 6 P. sibiricum Ahn et al., 2006).

(Long- 6,7-Dihydro-3-hydroxymethyl-8(5H)a-indolizinone.[Polygonatine A].

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Rusun et al., 2005).

3-(ethoxymethyl)-6,7-dihydroindolizin-8(5H)-one [Polygonatine B] (Jin et al., 2004).

3-(butoxymethyl)-6,7-dihydroindolizin-8(5H)-one. [Kinganone] (Yi-Fen et

O al., 2003). HO H O HO H HO O O OH OH H O HO H O H O OH

OH OHOH (25R,S)-spirost-5-en-3β-o-l3-O-β-D- glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside.

O H H HO OH OH O HO HO O O H R O O 4 O HO O Ac OH

OH OHOH (25S)-spirost-5-en-3β-ol3-O-β-D- glucopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-β-D-glucopyranosyl-(1→4)-2-O-acetyl- β-D-galactopyranoside.

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(25R)-26-O-β-D- glucopyranosyl-furost-5,22(23)-dien-3β,26-diol-3-O-α-L-rhamnopyranosyl-(1→3)-β-D- glucopyranosyl-(1→4)-[α-L-rhamnopyr-anosyl-(1→2)]-β-D-glucopyranoside. H OH O C O O HO OH O HO O O HO O OHHO O H

H3C O HO OH OH 22α-(propionyloxy)-furost-5-en-3β,20α- diol-3-O-β-D-glucopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside. Polygonoide B.

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O OAc HO H O HO OH OH HO O O R O O H 4 O HO O OH OH

OH OHOH (25S)-1-O-acetylspirost-5-ene-1β,3β- diol3-O-β-D-glucopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-β-D-glucopyranosyl- (1→4)-β-D-galactopyranoside.Neosibiricoside B.

(23S,24R,25R)-1-O-acetylspirost-5-ene- 1β,3β,23,24-tetrol3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)-β-D- fucopyranoside. Neosibiricoside A. 7 P. odoratum Morita et al., 1976).

(Qin et al., 9,19-cyclolart-25-en-3β,24(R)-diol

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2003).

(Wang et al., 3-(4-hydroxy-benzyl)-5,7-dihydroxy-6-methyl-chroman-4-one. 2009).

(Qian et al., 2010).

(Zhang et 3-O-β-D-glucopyranosyl-(1→2)-[β- al., 2010) D-xylopyranosyl-(1→3)]-β-D-glucopyranosyl-1→4)-galactopyranosyl-25(S)-spirost- 5(6),14(15)-dien-3β-ol.

3-O-β-D-glucopyranosyl(1!2)- [β-D-xylopyranosyl-(1!3)]-β-D-glucopyranosyl-(1!4)-galactopyranosyl-25(S)–spirost-5(6)- en-3β,14α-diol.

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3-(4-hydroxy-benzyl)-5,7-dihydroxy-6-methyl-8-methoxy- chroman-4-one

CH3 HO O H

H3C OH O 3-(4-hydroxy-benzyl)-5,7-dihydroxy-6,8-dimethyl-chroman-4- one.

CH3 HO O CH3

H3C OH O OH 3-(4-methoxy-benzyl)-5,7-dihydroxy-6,8-dimethyl-chroman- 4-one

OCH3 HO O CH3

H3C OH OH O 3-(4-methoxy-benzyl)-5,7-dihydroxy-6-methyl-8-methoxy- chroman-4-one.

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5,7-dihydroxy-3-(2-hydroxy-4-methoxybenzyl)-8-methoxy- 6-methylchroman-4-one Ophiopogonanone E.

5,7-dihydroxy-3-(4-methylchroman)-4-one. Methylophiopogonanone B.

5,7-dihydroxy-6-methyl-8-methoxy-3-(4′- methoxybenzyl)chroman-4-one.

(E)-7-O-β-D-glucopyranoside-5-hydroxy-3- (4′-hydroxybenzylidene)chroman-4-one. CH3 HO O OH

H3C OH O (E)-5,7-dihydroxy-6,8-dimethyl-3-(4′-

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hydroxybenzylidene)chroman-4-one. CH3

HO O OCH3

H3C OH O OH (±)-5,7-dihydroxy-6,8-dimethyl-3-(2′-hydroxy-4′- methoxybenzyl-)chroman-4-one. CH3

HO O OCH3

H3C OH OH O 5,7-dihydroxy-6,8-dimethyl-3(R)-(3′-hydroxy-4′- methoxybenzyl)chroman-4-one. OH OH OH OH OH O HO O O OH O OH O O OH

HO OH O OH OH 8-(3-(4,5-dihydroxy-6-(hydroxylmethyl)-3- (3,4,5-trihydroxy-6-hydroxymethyl)tetrahydro-2H-pyran-2-ylaxy)tetrahydro-2H-pyran-2- yloxy)-4,5-dihyroxymethyl)tetrahydro-2H-pyran-2-yl)-5,7-dihyroxy-2)-(4-hydroxyphenyl)- 4H-chromen-4-one. Polygonatiin.

OH HO (25 R and S)-spirost-5-en-3β,14α-diol. Neoprazerigenin A.

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8 P. (Jin et al., zanlanscianense 2004)

(25S)-spirost-5-ene-3β,27-diol27-O-β-D- glucopyranosyl-3-O-[-L-rhamnopyranosyl-(1→4)]-β-D-glucopyranoside.Polygonatoside D.

(6R,9R)-9-hydroxy-4-megastigmen-3-one9-O-β-D- glucopyranosyl-(1→6)-β-D-glucopyranoside

Isonarthogenin 3-O-β-D-glucopyranosyl-(1→2)-β- D-glucopyranosyl-(1→4)-β-D-galactopyranoside

Gracillin

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(25S)-3β,27-dihydroxyspirost-5-en- 12-one27-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside. Polygonatoside B.

(25S)-3β,27-dihydroxy-spirost-5-en- 12-one27-O-β-D-glucopyranosyl-3-O-β-D-glu-

copyranosyl-(1→4)-β-D-fucopyranoside, Polygonatoside A.

(23S,25S)-3β,23,27- trihydroxyspirost-5-en-12-one3-O-β-D-glucopyranosyl-(1→4)-β-D-fucopyranoside. Polygonatoside C.

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2.1. Pharmacological Studies

General Note

Various pharmacological and chemical investigations were executed at the Department of Pharmacy, University of Peshawar, Natural Product Research Division, Department of Biological and Biomedical Sciences, The Aga Khan University Medical College, Karachi, Pakistan, and International Center for Chemical and Biological Sciences (ICCBS), H.E.J. Research Institute of Chemistry, University of Karachi, Pakistan, while metal analysis was performed at PCSIR Laboratories Peshawar.

2.1.1. In-Vivo Studies

2.1.1.1. Drugs and reagents

The chemicals used in various studies are illustrated in table 2.1.1. Sterile normal saline was used as control in all studies and the crude methanol extract used in various studies were prepared in normal saline. Stock solutions of compounds were formulated in normal saline or distilled water apart from cromakalim, and glibenclamide, as they were prepared in dimethyl sulfoxide (10%) and further dilutions were made in fresh distilled water/saline. The solvents used for solubility devoid of any effect in different assays.

2.1.1.2. Study Animals

Swiss Albino mice (19-27 g), Wistar rats (210-270 g), adult local rabbits (1.2-1.6 kg), guinea-pigs (490-540 g) of both sex were used in different experimental paradigms. Rodents were housed at the “Animal House” of the Aga Khan University (AKU) or H.E.J research institute, Karachi University, Karachi. They were fed laboratory diet ad libitum, standard animal diet and permitted open contact to drinking water under standard experimental setup i.e. control temperature (23-25 0C), relative humidity 60 ± 10% and light/dark cycles (12/12 hour). Experiments were performed according to ethical principles established in 1979 for laboratory animals at the service of mankind Lyons, France and

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approved by the Ethical Committee of either A.K.U or H.E.J Research Institute, University of Karachi, Karachi.

Table 2.1.1. Chemicals used in various assays Chemicals Source Chemicals Source Glucose Silca gel Ceric sulphate Formalin Tri-chloroacetic acid Acetic acid Sodium bicarbonate Hydrochlorothiazide Magnesium sulfate Merck, Diazepam Calcium chloride Darmstadt, Acetic anhydride Magnesium chloride Germany Morphine sulphate Potassium dihydrogen Aluminum chloride phosphate Sodium dihydrogen phosphate Ammonium Sigma hydroxide Chemical Ethylenediamine tetraacetic Sigma Chemical Acetylcholine Co, St acid Co, St Louis, Louis, MO, Folin-Denis reagent MO, USA Carbachol (CCh) USA potassium chloride Verapamil HCl Aspirin Reckitt & Paracetamol Colman, Pakistan Yeast Vahine Pentylenetetrazole Professional, France DDPH Waka Ltd. Japan Naloxone HCl Glibenclamide RBI, Natick, MA, Dragendorff's reagent USA

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Cromakalim Tocris Ellisviille, ferric chloride MO, USA

. 2.1.1.3. Plant material

2.1.1.3.1. Collection

Polygonatum verticillatum All was collected from District Swat (Gabral and Miandam valleys), Khyber Pukhtoonkhawa, Pakistan, in July-Aug 2007. The botanical identity of the plant material was done by the Taxonomy Department of PCSIR Laboratories Peshawar and a specimen with catalogue No: 9970 (PES) was submitted to the herbarium of PCSIR Laboratories Peshawar.

2.1.1.3.2. Extraction

Plant was air-dried under shade for approximately two months. The whole plant was divided into two portions i.e. Rhizomes (underground parts) and Aerial parts. The Rhizomes of P. verticillatum (8 kg) and Aerial parts (10 kg) was cleaned of adulterants and ground to fine powder and extracted by maceration with methanol at room temperature for 14 days with occasional shaking (Khan et al., 2007). The methanol soluble residue was filtered off by means of a muslin cloth followed by Whatman filter paper No. 1. The filtrates was joint together and solvent freed using a rotary evaporator (R-210, Buchi, Switzerland) accompanied with recirculation chiller (NESLAB instruments) and a heating bath (B-491) at 40oC, yielded a dark greenish semisolid material (2.2 kg, 27.50% w/w) and (2.410 kg, 24.10% w/w) respectively.

2.1.1.3.3. Fractionation

The crude methanol extract of Rhizomes (1.6 kg) was suspended in distilled water for sequential fractionation with hexane, chloroform, ethyl acetate and butanol, yielding hexane (258 g, 16.13% w/w), chloroform (219 g, 13.69% w/w), ethyl acetate (226 g,

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14.13% w/w), butanol (265 g, 16.56% w/w) and aqueous (501 g, 31.31% w/w). Similarly, the crude methanol extract (1.8 kg) of Aerial parts was suspended in distilled water and consecutively fractionalized with different solvents to acquired n-hexane (275 g), chloroform fraction (295 g), ethyl acetate fraction (210 g), n-butanol fraction (317g) and aqueous fraction (445 g). These fractions were then screened for various pharmacological and phytochemical assays.

2.1.1.4. Antinociceptive activities

2.1.1.4.1. Visceral pain model (Acetic acid-induced abdominal constriction)

The peripheral nociceptive activity was investigated by using the acetic acid induced abdominal constriction test (Khan et al., 2010). Briefly, the prescreened animals were divided into groups (n = 6). The writhes were induced by intraperitoneal injection of 1.0% acetic acid (v/v, 0.1 mL/ 10 g body weight). Group I was used as control, received normal saline (10 mL/kg, i.p.); groups II, III and IV were treated with Rhizomes (50, 100 and 200 mg/kg, i.p.) respectively; group V received aspirin (100 mg/kg i.p.), as a standard drug. Similar patron was adopted for the Aerial parts. The number of muscular contractions was counted over a period of 20 minutes after acetic acid injection. The number of writhes in each treated group was compared with control (Saline treated group) and the percent inhibition of the writhes was calculated.

2.1.1.4.2. Formalin test

The method used in our study for the assessment of formalin-induced flinching behaviour in normal rats was described previously (Dubuisson and Dennis, 1977; Khan et al., 2010; Tjolsen et al., 1992). In this method, 0.05 mL of formalin (2.5% formaldehyde) was injected into the plantar surface of the right hind paw, 30 minutes after treating the animals with the extracts (50, 100 and 200 mg/kg i.p.). Nociceptive behavior was quantified as rat walking or can stand on injected paw; paw partially elevated; total elevation of injected paw, injected paw licking or biting. Formalin injection induced a stereotyped response

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lasting (0-5 minutes) followed, by prolonged tonic phase II lasting (25-30 minutes). Morphine (5 mg/kg s.c.) was used as a standard drug.

2.1.1.4.3. Thermal nociception (hot plate test)

Thermal nociception is one of the commonly used test to ascertain central involvement in analgesic activity of compound or extracts. In thermal nociception (hot plate test) mice were screened by placing them on a hot metal plate maintained at 50 ± 0.05°C (Dar et al., 2005; Khan et al., 2010). The mice were treated either with vehicle (10 mL/kg i.p.), Rhizomes and Aerial parts (50, 100 and 200 mg/kg i.p) or morphine (10 mg/kg s.c.) an opioids analgesic as a standard drug. Thermal nociception was estimated by measuring withdrawal response latency in the form of jumping, withdrawal of the paws or the licking of the paws. In the pretreatment session, mice were tested on two separate occasions, each 30 minutes apart and then only those mice were selected for the study, which responded within 15 seconds and which showed comparatively similar results. The response latencies were recorded at 0, 30, 60, 90 and 120 minutes with a cut off period of 30 seconds to avoid damage to the paw in the absence of response.

2.1.1.4.4. Test for opioids involvement

In order to investigate the participation of the opioids system in the antinociceptive effect of extracts, naloxone hydrochloride (2 mg/kg s.c.) a non-selective opioids receptor antagonist was injected, 15 minutes prior to the administration of test samples, as explained above. The hot plate latencies were sequentially measured at 0, 30, 60, 90 and 120 minutes.

2.1.1.5. Carrageenan induced oedema

The carrageenan-activated hind paw assay was performed using a well tested in-vivo previously described protocol (Winter et al., 1962). The test animals were assigned in to five sets (n = 6). Group I entertained normal saline (100 mg/kg) as control. The animals of group II, III and IV injected test extract (50,100 and 200 mg/kg i.p.) respectively. Aspirin (100 mg/kg i.p.) was received by Group V, served as a standard drug. Straight away after

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one hour of injection, acute inflammation was originated in the right hind paw of the rats by sub-plantar injection of phlogiston (01% suspension of carrageenan (0.1 mL) was formulated with 2% gum acacia in normal saline). The paw volume was estimated through plethysmometer (Ugo Basile, Italy) at time interval of one hour up to five hours (1-5 hours) after the carrageenan injection. Statistics was applied on the raw data for the calculation of reduction in rat paw volume (mL) for each group against saline, followed by the measurement of percent reduction in the rat paw using the following formula:

Inhibition (% ) = 1 ─ (dt/dc) × 100

Where “dt” stands for reversal in paw volume in the group treated with drug while, “dc” symbolizes antagonism in the oedema reduction in the control group. (Bukhari et al., 2007; Khan et al., 2009).

2.1.1.6. Antipyretic activity (Yeast induced pyrexia)

Antipyretic activity of the extracts was ascertained by yeast-provoked pyrexia test as described early (Al-Ghamdi, 2001). Normal body temperature of the rodents was traced by inserting digital clinical thermometer about 3-4 cm in the rectum. Pyrexia was induced in animals by subcutaneous injection of 10 mL/kg of 15% suspension of Brewer’s yeast (Saccharomyces cerevisiae) and was kept in their housing cages. Rectal temperature of rats was estimated again after 19 hour of yeast injection as described above. Animals that developed at least 0.5 0C or more increased in body temperature, after 19 hour of yeast treatment were chosen for the experiment. The pre-screened animals were arranged in groups (n = 6) and injected with normal saline (10 mL/Kg) as control, plant materials (50, 100 and 200 mg/kg) or paracetamol (100 mg/Kg) was supplemented as a positive control (Khan et al., 2009; Vimala et al., 1998). After drug treatment, the temperature of each animal using rectal route was again calculated at one hour interval up to 5 hours (1-5 hours). The resulting data was used for the calculation of percentage reduction in rectal temperature. Antipyretic activity was defined as the capacity of test drugs to overturn the yeast originated pyrexia.

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2.1.1.7. Pentylenetetrazole induced convulsion

Normal and healthy Swiss albino mice (19–24 gm) of either sex were practiced in PTZ- induced convulsions test. Briefly, the mice under study were alienated into groups (n = 6). The animals of group I was treated with saline (15 mL/kg i.p.) and this group served as control. Similarly, the animals of group II, III and IV were treated with test extract (50,100,200 mg/kg) intraperitoneally while the animals of group V injected diazepam (7.5 mg/kg i.p.) as reference drug. Following 30 minutes of the injection of the test articles, PTZ in the dose of 90 mg/kg was administered intraperitoneally for the induction of convulsions. Animals of the all groups were keenly observed to the commencement of seizures or hind limb tonic extension in prescribed time (30 minutes). The mortality rate of animals was also recorded and the resulting data were analyzed statistically and compared with control (Nisar et al., 2008).

2.1.1.8. Diuretic activity

The diuretic activity of extracts was determined by method previously established (Jabeen et al., 2009). Male Albino rats (210─270 g) were divided in to four groups (n = 6). The animals were fasted for 24 hour and were fed laboratory diet ad libitum and allowed free access to drinking water. On the day of experiment, the animals of group I was administered with saline (15 mL/kg p.o) and this group served as control. Similarly, the animals of group II, III and IV were administered with Hydrochlorothiazide (10 mg/kg p.o) as standard drug, extract (300 mg/kg and 600 mg/kg p.o) respectively. Same procedure was repeated for Aerial parts. The extracts were dissolved in saline and suitably diluted for administration. Immediately after the drug treatments, the animals were placed in metabolic cages (1 animal in each metabolic cage). Urine was collected in graduated cylinders, while its volume was recorded at 2 hour, 3 hour, and 6 hour. Cumulative volume of urine for each animal under test was measured on the ground of body weight (mL/100 g body weight).

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2.1.1.9. Acute toxicity test

The acute toxicity test for extracts was carried out to evaluate any possible toxicity. Swiss Albino mice (n = 6) of either sex were tested by administering different doses of Rhizomes and Aerial parts by increasing or decreasing the dose, according to the response of animal (Bruce, 1985). The dosing patron was 500, 1000 and 2000 mg/kg p.o., while the control group received only the normal saline (10 mL/kg, p.o). All the groups were observed for any gross effect or mortality during 24 hour.

2.1.2. In-Vitro Studies

2.1.2.1. Antimicrobial studies

2.1.2.1.1. Antibacterial assay

The crude extract and its succeeding solvent fractions were scrutinized against various pathogenic bacteria by agar well diffusion method as explained before (Khan et al., 2008). Shortly, 3 mg/mL of either crude extracts or subsequent solvent fractions was dissolved in dimethyl sulfoxide (DMSO) for the preparation of stock solution. Approximately 45 mL of molten nutrient agar was dispensed on sterilized petri-plates, and was permitted to harden. Bacteria were dispersed on these nutrient agar plates by preparing sterile soft agar accumulating 100 µL of bacterial culture. 6 mm long sterile metallic borer was used for wells digging at suitable distance and spotted for identification. Sample (100 µL) was discharged into each well, and for 24 hour the plates at 37 0C were kept in incubator. The antibacterial activity was estimated in the form of inhibition zone. Standard drug was Imipenem in the assay while DMSO as negative control. Data symbolize mean of the three different readings.

2.1.2.1.2. Antifungal assay

Agar tube dilution method (Khan et al., 2008) was used to estimate antifungal potential of plant and fractions of both Rhizomes and Aerial parts of plant. The sample material (24

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mg/mL) was introduced in sterile dimethyl sulfoxide (DMSO). 4 mL of sabouraud dextrose agar (SDA) was discharged into sterilized screw cap tubes (which were autoclaved at 120 0C for 15 minutes followed by cooling to 15 0C). From the stock solution (66.6 µL), SDA media (non-solidified) was treated in such a way that the final concentration of the plant components was 0.4 mg/mL of SDA. Solidification of the tubes was done at ambient temperature in the slanted position. Seven days old fungi culture was used for inoculation in which a piece (4 mm diameter) was used to inoculate each tube. Other media treated with Miconazol and Amphotericin-B was characterized as standard antifungal while DMSO as negative control (Choudhary et al., 1995).

After one week incubator treatment at 28 ± 10C and relative humidity (40-50 %), percentage inhibition of fungal growth was observed. During incubation period, cultures were examined twice weekly. The visible growth of microorganisms was analyzed in test tubes. Tests were performed in triplicate.

2.1.2.1.3. Microorganisms

The reference bacterial strains in the test are depicted in table 2.1.2.2. The pathogens were preserved on agar angled at 4 0C. Prior to any screening, the bacterial strains were activated on nutrient agar and fungal on Sabouraud glucose agar at 37 0C for 24 hour.

2.1.2.1.4. Minimum Inhibitory Concentration determination (MIC)

10 mg/mL of crude extract and its solvent fractions were dissolved in DMSO. In microplates, extracts were successively diluted with distilled water in a laminar flow cabinet. The equal concentration of a keenly growing culture of the bacteria and fungi under study was introduced to each well and cultures were incubated for 12 hours in 100 % relative humidity at 37 ºC. At the next morning, all wells were supplemented with Tetrazolium violet. Pathogen growth was denoted by a color change (violet color) of the

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culture. Minimum inhibitory concentration (MIC) was symbolized by the lowest concentration of samples that produced absolute growth inhibition. At the test concentration, acetone, as negative control did not exhibited any bacterial effect. Imipinem, Amphotericin B and Miconazol were standard drugs in the assay (Atta-ur-Rehman et al., 2001).

Table 2.1.2 . Reference antimicrobial strains Bacteria Reference bacterial strains Fungi Reference fungal strains E. coli ATCC 25922 T. longifusus (clinical isolate) B. subtilis ATCC 6633 C. albicans ATCC 2091 S. flexeneri (clinical isolate) A. flavus ATCC 32611 S. aureus ATCC 25923 M. canis ATCC 11622 P. aeruginosa ATCC 27853 F. solani ATCC 11712 S. typhi ATCC 19430 C. glaberata ATCC 90030.

2.1.2.2. In-vitro Antimalarial bioassay

The in-vitro antimalarial test of the crude extracts of both the Rhizomes and Aerial parts of P. verticillatum and subsequent solvent fractions was carried out using previously established methods (Makler and Hinrichs, 1993; Makler et al., 1993). For cultural preservation of Plasmodium falciparum, human erythrocytes from infected patients were used. Stock solution of crude extract and solvent fractions (1 mg/mL) were prepared in DMSO (0.1%) which was then diluted with supplemented RPMI-1640 medium. Negative controls contained equal concentration of DMSO. Using 96-well microtiter plates, the total volume (200 µL) was putted into the wells composed of samples and the suspension of P. falciparum-infected RBCs (0.5% hematocrit with 1% parasitemia). At 37 ºC, while using

candle jar, the plates were incubated for 72 hour with 5% CO2. After 24 hours, a blood smear was taken for the calculation of %age inhibition on parasitemia using the following formula

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Later on, LD50 was estimated with the help of EZfit computer program. All tests were replicate of triplicate. Positive controls contained 1 mM chloroquine diphosphate (Sigma).

2.1.2.3. In vitro phytotoxic bioassay

The Lemna minor assay is usually employed for the assessment of phytotoxicity of crude extracts. The test is a multi-character one; can also determine the growth stimulating effects of test material. It has been noted that some drugs support frond propagation and the test might be valuable to distinguish components with positive effects on plant growth. However, it is a primary, comparatively rapid and reliable screening test for weed control.

Briefly, phytotoxicity test (in-vitro) was executed for the crude extract and successive solvent fractions against Lemna acquinoctialis Welv (Atta-ur-Rahman, 1991; Saeed et al., 2010b). For the preparation of medium, different inorganic components were added in 100 mL of distilled water and KOH solution was materialized for the modification of pH (6.0-7.0). The medium at 121 0C was autoclaved for 15 minutes. Plant components (15 mg) were added in ethanol (1.5 mL) used as stock solution. Nine total flasks were utilized in which three were used for each dose such as 1000, 100, and 10 µL of the stock solution corresponding to 500, 50 and 5 ppm respectively. Overnight, the solvent was then omitted in purely sterile environment. Medium (20 mL) was supplemented to each flask. Thereafter, ten plants each holding a rosette of three fronds, were introduced to each flask. One other flask, exhibiting with distilled water as control and standard plant growth inhibitor (Paraquat).

The flasks were closed with cotton and positioned at 30° C in growth cabinet (Fisons Fi Totron 600 H) with relative humidity of 56±10 % and 9000 lux light intensity. The treatment was remained for 12 hours daily for a week. On the day 7, the number of

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fronds in each flask was counted. Results were scrutinized as growth regulation in % age, measured with reference to distilled water.

2.1.2.4. In vitro Insecticidal bioassay

Insecticidal test was performed for the crude extract and its various fractions against different insects (Tribolium castaneum, Sitophilus oryzea, Rhyzopertha dominica, and Callosobruchus analis) adopting method already mentioned in literature (Saeed et al., 2010b; Siddiqui et al., 2003; Siddiqui et al., 2004). Briefly, test material (200 mg) was dissolved in 3 mL of methanol act as stock solution. Utilizing micropipette, the extracts (1572.7 µg/cm2) was introduced on the filter paper of suitable dimension (9 cm or 90 mm) on petri plate. They were kept as such overnight (12 hour) to eliminate the solvent.

On the following day, 10 active and healthy insects of test species of identical size and age were supplemented to every plate. Same was done in case of negative control (methanol) and reference drug (Permethrin 393.17 µg/cm2). Subsequently, under controlled conditions (27 °C for 24 hours) the plates were kept in incubator in growth chamber with 50 % relative humidity. Mean of three different experiments were tabulated. For calculation of percentage mortality (%) of plant extracts, the number of survived insects was numbered in every plate by means of the following formula.

2.1.2.5. In vitro leishmanicidal bioassay

Leishmania major (DESTO) promastigotes were cultured at 22–25 0C in RPMI-1640. The medium was supplemented with 10 % deactivated (56 0C for 30 minutes) fetal bovine serum. Promastigotes culture in the logarithmic phase of growth was centrifuged for 10 minutes at 2000 revelation per minute and cleaned with saline thrice in the identical

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conditions (Aliya, 2005; Habtemariam, 2003). Fresh culture medium was used for the dilution of parasites to get final density of 106 cells per mL. Using 96-well micro titer plate, medium (180 L) was introduced in 1st row while 100 L of medium was supplemented in others wells. Test extracts (20 L) was introduced in medium and diluted in-sequence. All wells were supplemented with 100 L of parasite culture. One row received medium for DMSO as control while one each received medium for Amphotericin B, Pantamidine, as standard drugs. The plate was kept in incubator at 21-22C for 3 days. Later on, the numbers of survived parasites were numbered microscopically in Neubauer chamber. Results are the replicates of three different experiments. Concentrations causing 50 % inhibition (IC50) were calculated by a Windows based EZ-Fit 5.03 Perrella Scientific Software (Saeed et al., 2010b).

2.1.2.6. Brine shrimp cytotoxic bioassay

Brine shrimp (Artemia salina, leach) is a simple, economical and effective test for the assessment of cytotoxicity of compounds or extracts. Briefly, eggs were developed in a thin rectangular plastic bowl (22 x 32 cm) that contained formulated seawater using commercial salt mixture and water obtained after double distilled. A perforated device was utilized for the construction of asymmetrical partition in the plastic dish. About 50 mg of eggs were spread into the big partition of dish that got gloomy. The small partition was unlocked to normal light and incubated at 37 0C. After two days, nauplii from the lighter side were gathered by a pipette. Simples were prepared by putting 20 mg of each in solvent (2 mL dimethylformamide). Three variable concentrations (500, 50 and 5 µg/mL corresponding to 1000, 100 and 10 µg/mL) from this stock solution were shifted to 9 vials (three for each concentration were used for every sample under investigation) and one vial having dimethylformamide (2 mL) was treated as control. The solvent was permitted to eliminate in 12 hours.

The shrimp larvae were completely grown after 48 hours. 1 mL of seawater and 10 shrimps were supplemented to every vial and thus 30 shrimps for each dose. The total volume was modified with seawater (5 mL/vial). Next day, the number of survived shrimp

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were calculated through the application of documented method(Meyer et al., 1982). Finney

computer program was employed for the calculation of LD50 values (Finney, 1991).

2.1.2.7. 1,1-diphenyl-2-picrylhidrazyl (DPPH) scavenging activity

1,1-diphenyl-2-picrylhidrazyl (DPPH) is a decolorization spectrophotometeric assay usually employed for the evaluation of antioxidant activity of extracts and compounds. The antioxidant components directly react with DPPH free radicals and the antioxidant capacity is characterized by its potential to decolorize its dark purple color. The oxidized purple color of DPPH becomes colorless in its reduced state while observing its absorption at 517 nm.

The crude methanol extract and successive solvent fractions were tested for potential antioxidant activity on the ground of scavenging action of the stable DPPH free radical (Khan et al., 2005). For preparation of stock solution of DPPH, 5 mL of it was dissolved in 2 mL ethanol. It was reserved in the dark at ambient temperature. Various dilutions of the extracts were made in ethanol and were aliquoatted into a 96-well micro titer plate (Molecular Devices, USA). The reaction mixture was heated in Elisa at 37 °C for 30 minutes and the absorbance was taken at  517 nm. Percentage inhibition of radical scavenging capacity was established by relating the results with control. Ethanol was used as negative control while ascorbic acid (Sigma USA) was used as reference control. All the analysis was executed in triplicate. The concentration of the compound that results 50 %

scavenging on DPPH was estimated as IC50. All the used chemicals were of analytical standard.

2.1.2.8. Enzyme inhibition assays

2.1.2.8.1. Bioassay of cholinesterase inhibition

The established in vitro assay was employed to evaluate the acetylthiocholinesterase (AChE) and butyrylthiocholinesterase (BChE) reversal of extracts (Khan et al., 2007). Acetylthiocholine iodide was act as substrate for AChE while butyrylthiocholine chloride

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for BChE activities in the assay. In the reaction components, there was 150 µL of sodium phosphate buffer (100 mM) (pH 8.0), 10 µL of 5´-dithiobis [2-nitrobenzoic acid], 10 µL (0.2 mM) of solution extracts under test and 20 µL of AChE or BChE solutions. These components were combined together and kept-warm under control conditions (at 25o C for 15 minutes). The addition of acetylthiocholine or butyrylthiocholine (10 µL) initiates the overall reaction. At a wavelength of 412 nm, the hydrolysis of acetylthiocholine and butyrylthiocholine were confirmed by the appearance of yellow 5-thio-2-nitrobenzoate anion. It was produced from the interaction of 5´-dithiobis [2-nitrobenzoic acid] with thiocholine, catalyzed by acetylthiocholine and butyrylthiocholine, respectively during 15 minutes. Test compounds and galanthamine, as standard drug were prepared in ethanol. Finally the rate of enzyme reaction was through the application of Ellman equation.

Where 13,600 is the extinction coefficient of the yellow anion. The inhibitory reactions were performed in 96-well micro-titer plates using Spectra Max-340 (Molecular Devices, CA, USA).

2.1.2.8.2. Bioassay of lipoxygenase inhibition

The lipoxygenase (LOX) inhibitory assay was scrutinized by using different dilutions of the extracts and different solvent fractions (Khan et al., 2007). Type I-B (Soybean) Lipoxygenase 1.13.11.12) and linoleic acid were supplied by Sigma. They were in their original form. Chemicals used in the assay were of analytical scale.

Briefly in the assay, the reaction mixture comprises 160 mL of the sodium phosphate buffer, (0.1mM at pH 7.0), 10 mL of test sample under investigation and 20 mL of LOX solution. The reaction mixtures were kept warm at 258 0C for 5 minutes. Introduction of 10 µL linoleic, act as solution substrate acid and commenced the biochemical reaction. The reaction was assigned into the formation of (9Z, 11E)-13S)-13-hydroperoxyoctadeca-9, 11- dienoate. The absorption was altered and monitored for duration of 10 minutes. 50%

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ethanol was used as solvent for sample preparations while the assays were executed in triplicate.

Baicalein was exercised as standard drug for lipoxygenase while ethanol as control.

The IC50 values were dilutions of drugs resulting into 50% lessening in inhibition against

ethanol. For the calculation of IC50 values, EZ-Fit Enzyme Kinetics program (Perrella Scientific Inc., Amherst, USA) was used.

2.1.2.8.3. Bioassay of urease inhibition

For the study of urease inhibitory potential of extracts, a standard documented assay was used (Khan et al., 2007). Briefly reaction mixtures comprise 25 mL or 40 units per mL of urease solution and 55 mL of buffers possessing urea (2-24 mM for Bacillus pasteurii urease). They were kept warm under controlled conditions (at 30°C for 15 minutes) with 5 µL of the drug substances. In the assay, 96-well plates were used at 560 nm after 10 minutes. All the reactions were repeated thrice in a final composition of 200 mL at pH 6.8 (3 mM sodium phosphate buffer). 7 mg of phenol red was used as indicator in one mL of solution. Thiourea was act as the reference inhibitor of urease while DMSO as negative control. The inhibition (% inhibition) was calculated from following formula

2.1.3. Ex-in vivo (Isolated animal tissue) studies

2.1.3.1. Bioassay on rabbit jejunum

The spasmolytic tendency of the crude extracts was ascertained by means of isolated rabbit jejunum following the standard documented protocol (Arroyo et al., 2004; Gilani et al., 1994). On the day of experiment, rabbit was killed by striking on back of the head. By the process of dissection, the abdomen was opened and jejunum was eliminated out, kept in normal Tyrode’s solution. The composition of Tyrode’s is illustrated in Table 2.1.3. The

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mesenteries were removed and about 2 cm long sections of rabbit jejunum were hanged individually in tissue bath (10 mL) filled with Tyrode’s solution. Of the two ends of section, one was linked to the tissue hook made of metal while the other attacked with a cotton thread to an isotonic Bioscience transducer, coupled with a Student Oscillograph.

The tissue baths were exposed with carbogen (mixture of 95% O2 and 5 % CO2). The thermostat controlled temperature was 37 0C and a pH (7.4) was maintained. Each of the tissues was maintained at resting tension of 1 g and that was reserved constant during the whole experiment (Gilani et al., 1994).

A Harvard Transducer (Harvard Apparatus, Holliston, Mass) attached with a Harvard Student Oscillograph (Harvard Apparatus) was used for the measurement of intestinal responses isotonically. Prior to the introduction of any compound, every tissue was permitted to adjust for approximately 30 minutes. Following equilibration time, each tissue set up was then stabilize with a sub-maximal dose of acetylcholine (0.3 µM) using interval of 3 minutes in anticipation of steady responses. Under the prescribed experimental setup, rabbit jejunum elicits spontaneous regular contractions; permit the evaluation of relaxant (antispasmodic) action straightforwardly even in the absence of an agonist. The inhibitory effects of tested drugs were defined by the change (%) in jejunum spontaneous contractions.

2.1.3.1.1. Test for Ca++ channel blocking (CCB) activity

The tested extracts and drugs were studied against the induced contractions in order to discover the expected mechanism of antispasmodic activity. To study the involvement of Ca++ channel antagonism, high potassium (80 mM) was employed to depolarize the tissue setups as documented in literature (Farre et al., 1991). Usually within 7-10 minutes, stability in the induced contraction was attained; cumulative dosing of the drug materials produced dose-dependent inhibition indicative of Ca++ antagonism (Van-Rossum, 1963). To establish the presence Ca++ blocker agents in the tested drugs, the preparations under investigation was permitted to equilibrate in physiological solution (Tyrode’s solution). To remove Ca++ from the tissues, the solution was afterward substituted with calcium free

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Tyrode’s solution holding EDTA (0.1 mM) for half an hour (Table 2.1.3.2). Subsequently, this solution was again changed with K+-rich and calcium independent Tyrode's solution as shown in Table 2.1.3.3. After approximately 30 minutes of an incubation period, control dose-response curves (CRCs) of Ca++ were achieved. After two cycles, usually the control Ca++ CRCs when showed super-imposable, the tissue was pretreated with tested drug for 1 hour. The CRCs of Ca++ were again plotted while using various doses of the plant extracts and standard drug to calculate the Ca++ blocker activity (Gilani et al., 2000).

2.1.3.1.2. Test for K+ channel opening effect

To evaluate the participation of K+ channel opening activity, the extracts and standard drug were tested against the contraction induced by low potassium (25 mM) (Gilani et al., 2005a; Gopalakrishnan et al., 2004). An agent that selectively relaxes the induced contractions due to low potassium (< 25 mM) is rated as K+ channel openers. This test is effectively used to differentiate Ca++ channel antagonist from K+ channel opener (Hamilton et al., 1986). While applying glibenclamide, a specific antagonist of potassium channel opener (ATP dependent) or tetraethylammonium (TEA), a nonspecific antagonist of K+ channel opener (Frank et al., 1994; Ikeda, 1995), the inhibitory dose response curve of low potassium was observed to characterize the K+ opener activity of extracts. The results as relaxation of the tissues were defined as percent of the control reflux arbitrated by added low potassium concentration.

2.1.3.2. Bioassay on rabbit trachea

Trachea was obtained from guinea-pig and reserved in physiological solution (Kreb’s solution). Rings were formed from tracheal tube approximately 2-3 cm wide. Couple of cartilages was carried by each ring. Rings were unlocked by cutting longitudinally on the ventral side reverse to the smooth muscle. As a result, a strip was formed with central parts have smooth muscle while edges made up of cartilage (Aqel, 1991; Gilani et al., 2005b). The strips were holed in tissue bath (20 mL) carrying Kreb’s solution (pH 7.4), at 37ºC and supplemented with carbogen. Tracheal strips were maintained at approximate tension of 1 g

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constantly during the course of experiment. The tissues were granted an hour to equilibrate prior to the introduction of drug substances. The CCh (1 µM) was used for the stabilization.

2.1.3.2.1. Assessment of Ca++ antagonist

To determine the possible mechanism involved, like jejunum, the isolated tracheal tissues were treated with high potassium (80 mM). The blockade of high K+ induced contraction indicates Ca++ antagonist activity (Gilani et al., 2006; Khan and Gilani, 2006b).

2.1.3.2.2. Assessment of cholinergic activity

To assess the contribution of any cholinergic activity, carbachol (carbamylcholine chloride, CCh) a cholinergic agonist was used to contraction. The blockade of CCh induced contraction indicates cholinergic potential (Gilani et al., 2008; Khan and Gilani, 2006a).

Table 2.1.3.1: Composition of Tyrode’s normal solution Entry Chemicals g/L m mol/L 1 NaCl 8.00 136.9 2 KCl 0.2 2.7

3 MgCl2.6H2O 0.1 0.5

4 NaH2PO4.2H2O 0.05 0.32 5 Glucose 1.00 5.05

6 CaCl2 0.20 1.8

7 NaHCO3 1.00 11.9

2.1.3.3. Vascular activity

The thoracic aorta isolated from rabbit was prepared in the form of rings (2-3 mm wide). Rings were climbed on tissue baths (20 mL) individually having Kreb’s solution, at a temperature 37 ºC and supplement with carbogen (Gilani, 1991; Sahin and Bariskaner,

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hour to equilibrate. PE (1 µM) was employed for the stability of tissues. Activity of the drug materials was first ascertained on the resting base line of the isolated tissue to observe any possibility of vasoconstriction followed by a test for capacity to relaxation potential after contractions induced with phenylephrine and/or potassium. The blockage of phenylephrine

Figure 2.1.2: In vitro experimental set-up for the isolated gastrointestinal tissues preparations.

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Dependent contraction indicates the blockade of Ca++ entrance through the calcium channels receptor-operated (Cao et al., 2005; Wang et al., 2001). The test material was than studied against PE (1 µM)-evoked peaks in the Ca++-free Kreb’s solution (Calcium removed while EDTA 0.1 mM supplemented to guarantee (Hashimoto et al., 1986) removal of extracellular Ca++) to observe its effect on the intracellular stores. As in the Ca++-free medium, PE operate through activation of 1-adrenergic receptors and then the resulting ++ conversion of phosphatidylinositol to inositol-1,4,5-triphosphate (IP3). This discharges Ca from the sarcoplasmic reticulum resulting in a tonic contraction.

Table 2.1.3.2: Composition of Tyrode’s, Ca++-free, K+- normal solution

Entry Chemicals g/L m mol/L 1 NaCl 8.00 136.90 2 KCl 0.20 2.70

3 MgCl2.6H2O 0.10 0.50

4 NaH2PO4.2H2O 0.05 0.32 5 Glucose 1.00 5.05

6 NaHCO3 1.00 11.90

7 EDTA-Na2.2H2O 0.037 0.1

2.1.3.4. Guinea-pig atria

Right atrium segregated from the guinea-pig was accumulated in a tissue bath of 20 mL carrying Kreb’s solution, at prescribed conditions (32 ºC and supplemented with carbogen). The tissue was permitted to beat impulsively attributed to pacemaker under resting tension of 1 g throughout. This tissue set up can be used to study the effect of drugs on both force and rate of contractions (Gilani and Cobbin, 1987; Tanaka et al., 1996). Approximately 45 minutes was given as equilibrium period prior to the introduction of drug components. The outcome on heart rate was calculated by raising the pace of chart recorder.

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Control effects of acetylcholine (1 M) were attained at least in triplicate. The responses induced by drug in both force and rate of atrial contractions were calculated as the percentage change in base-line results straight away prior to the addition of test substance. In the tissues, changes in the tension were measured through force-displacement transducer (model FT-03) coupled with Grass Model 7 Polygraph.

Table 2.13.3: Composition of Tyrode’s, Ca++-free, K+- rich solution Entry Chemicals g/L m mol/L 1 NaCl 5.32 91.03 2 KCl 3.72 50.0

3 MgCl2.6H2O 0.10 0.50

4 NaH2PO4.2H2O 0.05 0.32 5 Glucose 1.00 5.05

6 NaHCO3 1.00 11.90

7 EDTA-Na2.2H2O 0.037 0.1

Table 2.1.3.4: Composition of Kreb’s solution Entry Chemicals g/L m mol/L 1 NaCl 6.92 118.2 2 KCl 0.35 4.70

3 MgSO4.7H2O 0.29 1.20

4 KH2PO4 0.16 1.20 5 Glucose 2.10 11.70

6 NaHCO3 2.10 25.0

7 CaCl2 0.28 2.50

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2.1.2.5. Statistical applications

Results of the experiments were expressed as mean ± standard error of mean (SEM). Student t-test was used to analyze data between groups and analysis of variance (ANOVA) among groups followed by Dunnet's test for multiple comparisons. P < 0.05 was considered significant difference in each case. CRCs were analyzed by non-linear regression using GraphPad program (GraphPad, San Diego, CA, USA).

2.2. Phytochemical Investigations

2.2.1. Experimental Settings

2.2.1.1. Spectroscopy

1 Proton Magnetic Resonance ( H-NMR) spectra were measured in MeOD and CDCl3 at 400 MHz, 500 MHz, or 600 MHz on Bruker AC-300, AM-300, AM-400 or AMX-500 nuclear Magnetic Resonance Spectrometer with Aspect-3000 data systems at a digital resolution of 32 K. TMS was used as an internal standard. The 13C-NMR spectra were recorded in

MeOD and CDCl3 at 300, 400, or 600 MHz on the same instruments. The other related techniques like COSY, HMQC, NOESY and HMBC spectra were measured through Bruker spectrophotometers at 400,500 and 600 MHz.

Mass spectrometric analysis were include electron impact mass spectrum (EIMS) and high resolution fast atom bombardment mass spectrum (HRFABMS) and GCMS spectrum measurements were performed on Varian Mat 312 and Jeol JMS -600H with GC and Jeol JMS HX 110 mass spectrometer.

2.2.1.2. Chromatography

Column chromatography was performed on silica gel 60 (E.Merck), mesh size 230-70. Recoated silica gel GF-254 preparative plates (20 x 20, 0.5 mm thick) (E. Merck) were employed for preparative chromatography. Purification test of the samples was done on the

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similar TLC plates.

Silica gel 60 (70-230 mesh, E. Merck) or flash silica gel 60(230-400 mesh) was

utilized in column chromatography. TLC was carried out on pre-coated Kieselgel 60, F254 aluminum sheets (Merck). Spots were made visible by UV light and ceric sulphate. Purity of the purified compounds was evaluated and compared on TLC plates.

2.2.1.3. Spraying Reagents

Ceric sulphate [Ce(SO4)2] reagent was used to visualize isolated compounds. Ceric sulphate (0.1 g) was prepared by dissolving tri-chloroacetic acid (0.1 g) in 4 mL of distilled water. The solution was heated while the turbidity of the mixture was omitted with the

addition of concentrated H2SO4 in the form of drops.

2.2.1.4. Purification of solvents

Various commercial grade organic solvents were used in phytochemical studies. The purification of these different solvents including n-hexane, chloroform, ethyl acetate, n- butanol, ethanol, methanol etc. was done with the application of distillation apparatus. The same distillation technique was adopted for the purification of the tape water to offered distilled water.

2.2.2. Quantitative and qualitative phytochemical analysis

The extracts of both Rhizomes and Aerial parts of plant were subjected to total contents determination and various preliminary phytochemical tests.

2.2.2.1. Total contents determination (Quantitative)

2.2.2.1.1. Total phenol content determination

The total phenol concentration of extracts and its subsequent solvent fractions were determined by method described previously (Khan et al., 2008). Briefly, extracts (10 mg)

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was treated with Folin-Denis reagent (5 mL), sodium carbonate (20%, 10 mL). Dilution was occurred with distilled water by a factor 100. After filtration, the solution mixture was kept at ambient temperature for 10 minutes. For the calculation of absorbance of extracts against blank at 770 nm, Spectronic 20D (Milton Roy) was employed. The total phenol content of each extract and solvent fraction was measured by comparing with a standard curve (tannic acid) constructed.

2.2.2.1.2. Total saponin content determination

Saponin contents were determined for the crude extract and solvent fractions according to documented method (Khan et al., 2010). Briefly, 2 g of test samples were taken in a beaker and 50 mL of petroleum ether was added and heated gently on water-bath to 40 0C for 5 minutes with regular shaking. The petroleum ether was filtered and repeated the operation twice with further 50 mL of pet ether. The marc obtained was extracted with 4 × 60 mL of methanol on gentle heating. The methanol layer was concentrated to approximately 25 mL on water-bath and 150 mL of dry acetone was added to precipitate the saponins, which was followed by filtration and drying in oven at 100 0C for constant weight.

2.2.2.1.3. Determination of flavonoids content

To determine the flavonoids content, 10 g of the crude extract and successive fractions of the plant material were continually extracted with 10 mL of the aqueous methanol (80%) at

ambient temperature. The resulting solutions were filtered using filter paper (Whatman No. 42.). The filtered was then taken in to a crucible. Solvent was evaporated over water bath and weighted (Boham and Kocipai, 1994).

2.2.2.1.4. Total alkaloid contents

The total alkaloid contents of extracts and subsequent solvent fractions of Polygonatum verticillatum was estimated by using method developed previously (Khan et al., 2010). Briefly, 2 g of each was defatted by extraction with petroleum ether, heated gently on water

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bath to 40 0C for 5 minutes with regular shaking. The marc obtained was acidified with 100 mL of 20% acetic acid in ethanol and allowed to extract for 4 hours. The resulting solution was filtered, concentrated and then basified with concentrated ammonium hydroxide to pH 9 followed by precipitation. The final weight of precipitated mass was designated as the total alkaloid contents.

Scheme. 2.2.1: Maceration and fractionation of the Rhizomes of P. verticillatum.

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Scheme 2.2.2: Maceration and fractionation of Aerial parts of P. verticillatum.

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2.2.2.2. Qualitative analysis

2.2.2.2.1. Test for glycosides

For the estimation of glycoside contents of the Rhizomes and Aerial parts of the plant, the extracts were hydrolyzed with hydrochloric acid by putting over water bath for few hours. 1 mL of pyridine solution was introduced to the hydrolyzed, pursued by the introduction of few drops of sodium nitroprusside preparation. The solution was then made alkaline with sodium hydroxide solution. The accumulation of glycosides was attributed to the change of pink to red colour (Kumar et al., 2009).

2.2.2.2.2. Test for steroids

1 mL of the extracts was putted in 10 mL of CHCl3. An equal quantity of sulphuric acid

(conc.) was introduced via sides of the test tube. Red color appeared at upper layer (CHCl3) while the lower layer (H2SO4) displayed yellow with green fluorescence and thus confirmed the steroidal presence (Kumar et al., 2009).

2.2.2.2.3. Test for anthraquinones

Each extracts approximately 0.5 g was boiled with 10% HCl for few minutes on a water bath. It was filtered and allowed to cool. Equal volume of CHCl3 was added to the filtrate.

Few drops of 10% NH3 were supplemented to the blend and heat. Formation of rose-pink colour indicates the presence of anthraquinones (Kumar et al., 2009).

2.2.2.2.4. Test for amino acids

Test extracts (1 mL) added to few drops of Ninhydrin reagent in a test tube. The presence of amino acids was designated to the appearance of purple colour (Kumar et al., 2009).

2.2.2.2.5. Terpenoids (Salkowski test)

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0.2 g of the extract was treated with 2 mL of CHCl3 followed by the carful introduction 3

mL of H2S04 (concentrated) to form a layer. On the interface, a reddish brown colour was appeared to indicate positive conclusion for the existence of terpenoids (Ayoola et al., 2008).

2.2.2.2.6. Test for tannins

Approximately 0.5 g of each extract in 10 mL of distilled water was boiled in a test tube.

After filtration, few drops of FeCl3 (0.1%) were introduced. The presence of brownish green was attributed to tannins (Ayoola et al., 2008).

2.2.3. Compounds Isolation using column chromatography

Sample (Chloroform + Ethyl acetate extracted fraction) of the methanol extract of Rhizomes was subjected to column chromatography for the isolation of pure chemical entities. Salary of the sample (80 g) was made with the application of silica gel (column grade). The sample was loaded in glass column over silica gel for adsorption act as stationary phase. The column was started initially with 100% n-Hexane as eluent (mobile phase). Polarity of mobile phase was enhanced gradually using chloroform gradient with regular monitoring of the isolate status over TLC until the eluent composition reached to 100% chloroform. The similar fractions were combined. As a result, we collected 12 subfractions (P1-P12).

Later on, methanol was introduced in the mobile phase composition for elution of more polar components. Initially methanol/chloroform (0.5%) composition was used to boost the polarity. The methanol concentration was amplified watchfully in anticipation of 12% (methanol: chloroform; 12: 88). With this treatment, the loaded sample was almost exhausted. After combing the similar fractions, finally we collected 12 subfractions (M1- M9).

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CHCl3 + EtAc soluble fraction 80 g

Loaded in column over silica

12 Subfractions (P1-P12) were from 9 Subfractions (M1-M9) were obtained using hexane-chloroform obtained using methanol-chloroform gradient gradient

Subfraction P7 Subfraction P9 Subfraction P-11

Subfraction P10

-Sitosterol 6 Propyl pentadecanoate (6 mg) 1(8 mg)

Salicylic Acid 3 (16 mg) SubfractionM2 Subfraction M3 2`,3`- Dihydroxy propyl pentadecanoate 2 (6 mg) Subfraction M5 HMF 4 (12 mg)

Santonin 7 Diosgenin 5 (12 mg) (17 mg)

Scheme 2.2.3. Isolation of compounds from chloroform + ethyl acetate soluble fractions Rhizomes of Polygonatum verticillatum.

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2.2.3.1. Characterization of propyl pentadecanoate (1)

The sub-fraction P7 was treated in column chromatography using silica gel. During elution with Ethyl acetate: hexane; (10:90) propyl pentadecanoate (1), as colorless amorphous compound was afforded (Scheme 2.2.3).

Physical state:

Colorless amorphous compound

UV activity:

UV active on TLC

Rf value:

0.56 (ethyl acetate)

Molecular formula:

C18H36O2

Isolated quantity:

11 mg

Solubility:

Soluble in methanol at room temperature

1H-NMR (MeOH):

See table 3.2.2.

13C-NMR (MeOH):

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See table 3.2.2

HREI-MS (m/z):

284.233

2.2.3.2. Characterization of 2,3-dihydroxypropyl pentadecanoate (2)

Subfraction P10 was re-chromatographed via silica gel in column chromatography. Elution with ethyl acete:hexane (45:55) led to the isolation of 2,3-dihydroxypropyl pentadecanoate (2), as white amorphous compound.

Physical state:

White amorphous powder

UV activity:

UV active over TLC

Rf value:

0.34 (ethyl acetate)

Molecular formula:

C18H36O4

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Isolated quantity:

6 mg

Solubility:

Soluble in methanol at room temperature

1 H-NMR (CDCl3):

See table 3.2.3

13 C-NMR (CDCl3):

See table 3.2.3

HREI-MS (m/z):

316.265

2.2.3.3. Characterization of 2-Hydroxybenzioc acid (3)

2-Hydroxybenzoic acid (3) was isolated as colorless crystals when the subfraction P9 was purified through column chromatography (illustrated in scheme 2.2.3.) while eluted with ethyl acetate: hexane (9:1).

Physical state:

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Colorless crystals

UV activity:

UV active over TLC

Rf value:

0.39 (ethyl acetate:hexane; 3:97)

Molecular formula:

C7H6O3

Isolated quantity:

16 mg

Solubility:

Soluble in methanol at room temperature

1H-NMR (600 MHz, MeOD):

See table 3.2.4.

13C-NMR (600 MHz, MeOD):

See table 3.2.4.

HREI-MS (m/z):

138

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2.2.3.4. Characterization of 5-Hydroxymethyl-2-furaldehyde (4)

Purification of subfraction M3 over silica gel in column chromatography using mobile phase as ethyl acetate:hexane (65:35) lead to the isolation of 5-Hydroxymethyl-2- furaldehyde (4) as yellowish oil (illustrated in scheme 2.2.3).

Physical state:

Yellowish oil

UV activity:

UV active over TLC

Rf value:

0.57 (ethyl acetate: hexane; 3:7)

Molecular formula:

C6H6O3

Isolated quantity:

25 mg

Solubility:

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Soluble in methanol at room temperature

1 H-NMR (400 MHz, CDCl3):

See table 3.2.5

13 C-NMR (400 MHz, CDCl3):

See table 3.2.5

HREI-MS (m/z):

126.021

H H

3 4 O 2 5 OH 2 1 O 1 H H H

2.2.3.5. Characterization of Diosgenin (5)

The subfraction M5 was re-chromatographed via silica gel and eluted with methanol: chloroform; (2:98) resulted into white amorphous powder, Diosgenin 5 (17 mg).

Physical state:

Colorless amorphous powder

UV activity:

UV inactive over TLC (ceric sulphate active)

Rf value:

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0.37 (Methanol: Chloroform; 01:99)

Molecular formula:

C27H42O3

Isolated quantity:

17 mg

Solubility:

Methanol-soluble at room temperature

1H-NMR (600 MHz, MeOD):

See table 3.2.6

13C-NMR (600 MHz, MeOD):

See table 3.2.6

HREI-MS (m/z):

414.214

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2.2.3.6. Characterization of β-Sitosterol (6)

Subfraction P11 was re-chromatographed over silica gel. While eluting with chloroform: hexane; (8:2) β-Sitosterol (6) was isolated as colorless amorphous powder.

Physical state:

Colorless amorphous powder

UV activity:

UV inactive over TLC

Rf value:

0.57 (Ethyl acetate)

Molecular formula:

C29H50O

Isolated quantity:

7 mg

Solubility:

Methanol-soluble at room temperature.

1 H-NMR (500 MHz, CDCl3):

See table 3.2.7

13 C-NMR (CDCl3, 125 MHz):

See table

HREI-MS (m/z):

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414.3845

2.2.3.7. Characterization of Santonin (7)

Subfraction M3 was isolated through column chromatography over silica gel using methanol: ethyl acetate (0.5:99.5) mobile phase guided to the isolation of santonin (7).

Physical state:

Light yellowish powder

UV activity:

UV active on TLC

Rf value:

0.46 (Ethyl acetate)

Molecular formula:

C15H18O3

Isolated quantity:

12 mg

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Solubility:

Chloroform-soluble at room temperature

1 H-NMR (600 MHz, CDCl3):

See table 3.2.8

13 C-NMR (CDCl3, 125 MHz):

See table 3.2.8

HREI-MS (m/z):

246.145

2.2.4. Gas chromatography (GC) and Gas chromatography-mass spectrometry (GC-MS) analysis

2.2.4.1. Instrumentation

Gas chromatography followed by GC Mass is a significant analytical tool for the assessment of composition of fixed oils or oily fractions. In this context, the n-hexane fraction of both Rhizomes and Aerial parts of the plant were analyzed for the chemical composition of known active constituents applying gas chromatography attached with

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flame ionization detector (FID) (Falodun et al., 2009; Siddiqui et al., 2004). GC was coupled with mass spectrometer as gas chromatography-mass spectrometry (GC-MS). GC analysis was executed on Shimadzu GC17-A system. GC-MS was supported with Jeol JMS-600H GC and Jeol JMS HX 110 quadruple mass spectrometer. Less polar capillary column DB-5 (Optima-5) was used, coated on fused silica having the dimensions 30 m, 0.25 mm internal diameter and 0.25 mm coating thickness. Column was coated with 5% phenylmethylsiloxane and 95% polydimethylsiloxane.

2.2.4.2. Experimental settings

Test sample (1.0 μL) was injected in AOC-20i auto-sampler onto the GC system at 250 °C in split mode being the split ratio as 40:1. During analysis, the initial GC temperature was tuned to 50 °C for 60 seconds and 80 °C for 3 minutes ramped with 10 °C/minutes until the final temperature 300 °C was achieved. The carrier gas nitrogen was passed at velocity of 35 cm/second while inlet pressure during experiment was adjusted to be 99.31 KPa. The FID type detector was adjusted at 280 °C utilizing hydrogen gas (carrier) at the flow rate of 55 mL per minute while the air flow rate was 400 mL/minute.

For GC-MS analysis, the gas chromatograph was combined with Jeol JMS-600H GC and Jeol JMS HX 110 quadruple mass spectrometer. The mass spectrometer was set in the EI mode with 70 eV (ionization energy) of while the GC experimental conditions were unchanged. As a carrier gas, helium was used at an operating temperature of 250 °C. Qualitative naming of the compounds was done on the comparison / matching of their relative retention times (RT) and mass spectra with the data available at mass spectral search databases (NIST 1998 and GC-MS Library Shimadzu, 1996). In case of quantitative analysis of individual components (% composition), the relative amounts of peak area of each constituent was calculated against the total peak area.

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Figure 2.2.1. Gas chromatogram of n-hexane fraction of Rhizomes.

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Figure 2.2.2. Gas chromatogram of n-hexane fraction of Aerial parts.

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2.3. Metals Analysis 2.3.1. Experimental settings 2.3.1.1. Instruments

Flam Atomic Absorption Spectrometer (Hitachi Polarized Z─8000 Japan) was used for the analysis of Fe, Cu, Zn, Co, Cr, Cd, Sb, Pb, Mn and Ni. The instrumental conditions maintained for each test metal on Atomic Absorption spectrometer is summarized in Table 2.3.1 and 2.3.3. The macro-elements like Na, K and Ca were analyzed using Flame Photometer (Jenway PFP7, UK). The instrumental conditions maintained for each test metal on Flame Photometer is summarized in Table 2.3.2 and 2.3.4.

2.3.1.2. Reagents / Chemicals

Different reagents exercised in the analysis were of analytical scale include nitric acid, perchloric acid, standard test metals, Fe, Cu, Zn, Co, Cr, Cd, Sb, Pb, Mn, Ni, Na, K and Ca (Sigma Aldrich). Dilutions were prepared with deionzed water (Deionizer B 114) and or distilled water.

Stock solutions of standards test metals (Fe, Cu, Zn, Co, Cr, Cd, Sb, Pb, Mn, Ni, Na, K and Ca) containing 1000 ppm of each metal, were used. Appropriate dilution of each metal from stock solutions were prepared for the construction of standards calibration.

2.3.1.3. Contamination control

Standard experimental conditions were followed throughout the analysis. In order to prevent contamination, all the glassware were soaked in chromic acid for 24 hours and thoroughly washed with deionized water and or distilled water. They were dried in oven and stored in dust free environment without touching their inside.

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Table 2.3.1: The instrumental conditions maintained for each element on Atomic Absorption spectrometer (Hitachi Polarized─8000 Japan) S. No Metals λ (nm) Slit width Lamp Burner Oxidant (Air) Acetylene (nm) current Height Kg/cm2 Kg/cm2 (mA) (mm) 1 Fe 248.3 0.2 10.0 7.5 1.60 0.30 2 Cu 324.8 1.3 7.5 7.5 1.60 0.30 3 Zn 23.8 1.3 10.0 7.5 1.60 0.20 4 Cd 228.8 1.3 7.5 7.5 1.60 0.25 5 Cr 359.3 1.3 7.5 7.5 1.60 0.40 6 Ni 232.0 1.3 10.0 10.0 1.60 0.25 7 Pb 283.3 1.3 7.5 7.5 1.60 0.30 8 Mn 279.6 1.4 7.5 7.5 1.60 0.30 9 Co 250.0 0.2 10.0 10.0 1.60 0.30 10 Sb 217.6 0.2 12.5 7.5 1.60 0.60

2.3.1.4. Sample preparation

Test sample (1 g) was supplemented in conical flask and 10 mL of conc. HNO3 (67%) was added. The solution was kept overnight (24 hour) at room temperature followed by the

addition of 4 mL of HClO4 (67%). The resulting solution was concentrated on hotplate at 60 0C until a clear solution of approximately 1 mL was left. The solution was supplemented with deionized/double distilled water after cooling, filtered through Whatman (# 42) filter paper. Latter on final volume (100 mL) was made with deionized water served as stock solution (Saeed et al., 2010a; Saeed et al., 2010c). The sample was then analyzed in triplicate by flame atomic absorption spectrophotometer (Polarized Zeeman Hitachi 2000) and flame photometer (Jenway PFP7, UK). The materials of all the reference metals were

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obtained from Merck (Darmstadt, Germany). Calibration standard of each metal were practiced by suitable dilution of the stock solutions. All chemicals utilized in the study were of analytical scale (Hussain et al., 2006).

Table 2.3.2: The instrumental conditions maintained for each element on Flame Photometer (Jenway PFP7, UK). S. No Metals λ (nm) Slit width Filter Oxidant (Air) Methane (nm) used Kg/cm2 Kg/cm2 1 Na 589.0 0.2 Na filter 1─2 0.0076─0.025 2 K 766.0 2.0 K filter 1─2 0.0076─0.025 3 Ca 422.7 0.7 Ca filter 1─2 0.0076─0.025

Table 2.3.3: The instrumental conditions maintained for each standard element on Atomic Absorption spectrometer (Hitachi Polarized─8000 Japan). S. No Metals Standard concentration Absorbance λ Co-efficient Sensitivity (ppm) (nm) 1 Fe 1 0.0118 0.9987 0.05 5 0.0647 10 0.1257 15 0.1779 20 0.2305 2 Cu 1 0.0079 0.09998 0.03 2 0.0177 4 0.0346 6 0.0517 8 0.0710 3 Zn 1 0.0384 0.09916 0.007 2 0.0648 3 0.0907 4 0.1039 5 0.1351 4 Cd 0.5 0.0879 0.9932 0.01 1 0.1668

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2 0.2928 3 0.3718 4 0.4299 5 Cr 0.5 0.0059 0.9877 0.04 1 0.0137 2 0.0249 3 0.0347 4 0.0396 6 Ni 0.5 0.0063 0.9981 0.05 1 0.0144 2 0.0240 3 0.0374 4 0.0503 7 Pb 0.5 0.0048 0.9956 0.1 1 0.0089 2 0.0183 3 0.0250 4 0.0387 8 Mn 1 0.0097 0.9982 0.03 2 0.1930 4 0.3665 8 0.7304 10 0.8529

9 Co 1 0.0166 0.09970 0.28 2 0.0276 3 0.0467 4 0.0640 10 Sb 2 0.0073 1.0000 0.05 5 0.1149 10 0.0354 20 0.0726

Table 2.3.4: The instrumental conditions maintained for each element on Flame

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Photometer (Jenway PFP7, UK). S. No Metals Standard concentration Emission λ Co-efficient Sensitivity (ppm) (nm) 1 Na 10 589 0.09998 0.03 2 K 10 766 0.09916 0.007 3 Ca 100 622 0.9932 0.01

Pharmacological Investigations

The results of various pharmacological activities are discussed below.

3.1.1. In vivo studies

3.1.1.1. Antinociceptive activity

3.1.1.1.1. Effect of acetic acid-induced abdominal constriction

The extracts were primarily tested in acetic acid provoked abdominal constriction test. The extract of Rhizomes of Polygonatum verticillatum demonstrated a marked and significant (P < 0.01) reduction (15-72%) in the number of writhes induced by acetic acid. The analgesic activity at all tested doses (50,100 and 200 mg/kg i.p.) (Table 3.1.1.1) indicated a dose-dependent relationship in the inhibition of writhing reflux. The attenuation of pain perception of the extract of Rhizomes at 200 mg/kg was quite similar to standard drug aspirin (77% at 100 mg/kg i.p.).

Regarding the results of Aerial parts in peripheral nociceptive test, significant (P <0.01) reduction was recorded in the number of writhes tempted by acetic acid. The antinociceptive activity of Aerial parts at test doses (50,100 and 200 mg/kg) was in a dose- dependent mode (Table 3.1.1.1). The maximum pain relieving effect (65.82%) was estimated at strength of 200 mg/kg.

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3.1.1.1.2. Effect of formalin induced pain

The formalin-induced flinching behaviour was strongly attenuated by Rhizomes in both phases, as compared to control. As shown in (Figure. 3.1.1.1A), the pain relieving potential was more profound and significant (P<0.01) in the first phase of formalin injection as compared to second phase. The antinociceptive effect was found dose dependent in both phases.

By the Aerial parts, formalin-induced flinching behaviour was significantly (P <0.5) attenuated in the first phase at a dosage of 100 and 200 mg/kg, (Figure. 3.1.1.1B). Nevertheless, the flinching behaviour in the late phase was more actively and significantly (P<0.01) attenuated at all test doses (50, 100 and 200 mg/kg). The standard compound was found more resistant to pain stimulus.

3.1.1.1.3. Effect of thermal nociception

The Rhizomes elicited marked analgesic activity in the thermal nociceptive test at 50, 100 and 200 mg/kg. The increase in latency time was dose dependant which was measured at 0

Table 3.1.1.1: Effect of the crude extracts in writhing provoked by acetic acid in mice at 50, 100 and 200 mg/kg, i.p. No. of writhing movements Drugs Doses 1-10 (minutes) % Inhibition 11-20 (minutes) % Inhibition Saline 100 mL/kg 47±1.73 – 78±2.30 – Rhizomes 50 mg/kg 38±2.51* 19.14% 52±3.46** 33.33% 100 mg/kg 21±1.73** 55.31% 37±3.18** 52.56% 200 mg/kg 13±2.30** 72.34% 22±2.30** 71.79% Aspirin 100 mg/kg 11±1.15** 76.59% 17±1.73** 78.20%

Saline 100 mL/kg 44±1.78 – 79±2.23 –

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Aerial 50 mg/kg 39±1.34 11.36% 57±1.79 27.84% parts 100 mg/kg 22±1.79** 50% 40±1.90** 49.36 200 mg/kg 16±1.34** 63.63% 27±2.23** 65.82 Aspirin 100 mg/kg 10±0.89** 77.27% 18±1.34** 77.21

Values are described as mean ± S.E.M. (n=6). The data were analyzed by ANOVA followed by Dunnett’s test. Asterisks indicated statistically significant values from control. *P < 0.05, **P<0.01.

, 30, 60, 90 and 120 minutes (30 minutes gap) after the administration of vehicle, extract of Rhizomes and standard drug morphine (Table 3.1.1.2). The peak protection against thermal pain was achieved at 90 minutes of Rhizomes administration.

The involvement of opioids receptor was assessed by the administration of naloxone (2 mg/kg) subcutaneously, 15 minutes prior to the administration of test samples. The administration of naloxone caused significant (P<0.01) attenuation of anti-nociceptive activity of Rhizomes, establishing the participation of opioids receptor in the central analgesic activity. In the thermal nociceptive test, Aerial parts demonstrated strong pain relieving effect (Table. 3.1.1.3). Analgesia was defined by the increased in latency time (s) which was recorded at 0, 30, 60, 90 and 120 minutes after the administration of vehicle, Aerial parts and standard drug morphine. The protection against thermal induced pain was computed at all test doses (50, 100 and 200 mg/kg) and the response was dose dependent. So far no attenuation observed in the antinociceptive action of Aerial parts after the administration of naloxone thus ruling out the participation of opioids receptor in its analgesic effect. On the other, morphine induced analgesia was perfectly antagonized by the presence of naloxone.

Pain as an unpleasant sensation often accompanied by inflammation is obviously a complex multiage health problem which needs prompt attention as altering physical,

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psychological, and overall quality of life. Such conditions are generally characterized as a protective modality and appear as a significant tool for the diagnosis of different pathological conditions. However, management of chronic pain is considered as a great challenge for most of clinicians while the diagnosis and treatment of acute pain can be a difficult task (John et al., 2008) and ultimately led to the concept of “Fear of Pain”. Pain management for patients with chronic pain is a difficult task because of the risks associated with toxicity due to the drug intervention (Russo, 2008). Non-steroidal anti-inflammatory drugs (NSAIDs) are the most widely prescribed medications for the management of painful conditions, but they are frequently causing gastrointestinal damage (Fiorucci et al., 2001). Ethnobotanicals to treat diseases is a therapeutic modality, which has stood the test of time for the treatment of various ailments (Gilani and Atta-ur- Rahman, 2005).

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Figure 3.1.1.1: Effect of intraperitoneal dosing of Rhizomes [A] and Aerial parts [B] at 50,100 and 200 mg/kg) in formalin test in rats. Values are expressed as mean ±S.E.M. [n = 6]. Asterisks designated significant distinction from control. *P < 0.05, **P<0.01 (ANOVA followed by Dunnett’s test).

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Table 3.1.1.2: Effect of Rhizomes of on hot plate (thermal stimuli) test with and without Naloxone in mice at 50, 100 and 200 mg/kg, i.p. Groups Dose Latency time (mean ± SEM) mg/ 0 min 30 min 60 min 90 min 120 min kg Without naloxane Saline 10 7.10±0.49 7.20±0.13 7.35±0.20 7.40±0.42 7.60±0.49 Rhizomes 50 7.20±0.49 7.35±0.15 7.65±0.15 8.00±0.44 8.00±0.44 100 7.20±0.35 7.40±0.17 8.50±0.44 9.70±0.89 9.10±0.89 200 7.00±0.44 7.40±0.13 11.50±0.44** 13.70±0.84** 12.90±0.84** Morphine 10 7.10±0.40 7.90±0.40 12.70±0.17** 16.10±0.89** 14.36±0.93** With naloxone (2 mg/kg. s.c.) Rhizomes 50 7.30±0.44 7.30±0.58 7.60±0.26 7.50±0.44 7.50±0.89 100 7.40±0.17 7.30±0.31 8.20±0.44 8.10±0.53 8.15±0.51 200 7.40±0.13 7.10±0.89 10.00±0.89* 9.26±0.51** 9.40±1.11* Morphine 10 7.60±0.26 7.10±0.58 7.10±0.40*** 6.85±0.60*** 6.85±0.89***

Without naloxone, data were compared with control (Saline 10 mL/kg). With naloxone (2 mg/kg s.c.), was administered 15 minutes prior to extract or morphine. All the results were compared with their respective test substances in the absence of naloxone. Values are reported as mean ± S.E.M (n=6). The data were analyzed by ANOVA followed by Dunnett’s test. Asterisks indicated statistically significant values from control. *P < 0.05, **P<0.01, ***P<0.001. The independent t-test was used for comparison between 2 groups.

To explore new effective and safe analgesic for the management of different painful conditions, the crude methanol extract of Rhizomes and Aerial parts was screened in various pain models. The extracts of both Rhizomes and Aerial parts showed marked

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antinociceptive activity in various pain models including visceral pain model, formalin test and hot plate test. Acetic acid-induced writhing is a highly sensitive and useful test for analgesic drug development especially peripherally acting analgesics. Acetic acid induces pain by liberating endogenous substances (bradykinin, serotonin, histamine, substance P) (Lu et al., 2007b). In our observation, Rhizomes and Aerial parts significantly (P <0.01) reduced the abdominal constriction response induced by the acetic acid in a dose dependent manner. Therefore, it could be suggested that these extracts might contained pharmacologically active molecule(s) that interfere with the blockade of the effect or the release of endogenous substances that are responsible for the excitation of pain nerve endings.

Table 3.1.1.3: Effect of aerial parts of on hot plate (thermal stimuli) test with and without Naloxone in mice at 50, 100 and 200 mg/kg, i.p. Groups Dose Latency of nociceptive response (mean ± SEM) mg/ 0 min 30 min 60 min 90 min 120 min kg Saline 10 7.10±0.11 7.25±0.44 7.40±0.44 7.10±0.58 7.60±0.67 Without Naloxone Aerial 50 7.25±0.13 7.25±0.33 7.90±0.40 8.90±0.45 9.10±0.76 parts 100 7.35±0.44 7.50 ±0.22 8.50±0.67 10.50±0.89* 9.90±0.89 200 7.35±0.29 7.70±0.31 11.25±0.89** 12.90±0.84** 11.25±0.94* Morphine 10 7.40±0.26 10.15±0.49** 12.65±0.73** 16.75±0.89** 15.55±0.69** With Naloxone (2 mg/kg. s.c.) Aerial 50 7.10±0.49 7.10±0.58 7.90±0.63 8.10±0.89 8.90±0.89 parts 100 7.15±0.65 7.15 ±0.44 8.10±0.45 9.66±1.19 9.55±0.69 200 7.0±0.89 7.0±0.89 10.78±0.89 12.15±0.51 10.90±0.67 Morphine 10 7.95±0.45 7.80±0.58 7.65±0.74*** 7.45±0.89*** 7.50±0.81***

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As writhing test is deficient in specificity and several mechanisms may involve in the abdominal constriction of animals (Andrade et al., 2007). This scarcity can be overcome by using other paradigms. therefore, both extracts were tested in formalin induced pain modal which produced distinct biphasic response (Garrido et al., 2001). From a mechanistic point of view, different analgesics may act differently. Centrally acting drugs such as opioids inhibit both phases equally but peripherally acting drugs (Morteza-Semnani et al., 2004) inhibits only the late phase. We have investigated that both extracts attenuated significantly hyperalgesia produced by formalin injection in both phases therefore; the possible mechanism for this antinociceptive activity of extracts could be attributed to peripherally acting pain mediators with central involvement.

To augment the role of central system in the anti-nociceptive activity of extracts, hot plate test was employed. Hot plate test is selectively used for centrally operating analgesic drugs like morphine, while the peripherally operating analgesics are ineffective in this test (Chen et al., 2008). Extract of both Rhizomes and Aerial parts exhibited marked inhibition on thermally induced hyperalgesia. The Rhizomes and Aerial parts possess significant (P < 0.01) activity. Morphine (10 mg/kg s.c.) being standard drug, showed more potent activity and gradually increased with time up to 120 minutes. Therefore, the result of our study showed the central mediation in the anti-nociceptive activity of Rhizomes. For the assessment of opioids system involvement, a non-selective opioids receptor antagonist, naloxone (2 mg/kg s.c.) was administered. The analgesic activity of Rhizomes was significantly (P < 0.001) antagonized by the administration of naloxone and it is therefore, confirmed the opioids receptors participation in the attenuation of thermal hyperalgesia.

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However, the analgesic activity of Aerial parts was unchanged at all test doses and it is therefore, postulated that the opioids receptors were not participated in the attenuation of thermal hyperalgesia produced by aerial parts.

Of the isolated pure chemical entities from the Rhizomes, 2-hydroxybenzic acid (3) and santonin (7) have proven analgesic activities (Al-Harbi et al., 1994; Doi and Horie, 2010). Additionally santonin also exhibited morphine like activity in hot plate test along with peripheral effect. Our findings on the anti-nociceptive activity of Rhizomes are inconsistent with the said isolated compounds and therefore, can be assigned to them. Phytochemical studies of Rhizomes and Aerial parts showed the presence of therapeutically important chemical groups like saponins, alkaloids, flavonoids, phenols, tannins etc. The antinociceptive potential of these classes are well documented in literature (Khan et al., 2010b; Küpeli et al., 2007; Matsumoto et al., 2005; Takayama, 2004), therefore the analgesic profile of both Rhizomes and Aerial parts could be attributed to these groups. Moreover, to the best of our knowledge on the basis of available literature, Polygonatum verticillatum is the first member of the genus Polygonatum which demonstrated marked anti-nociceptive activity and thus also provided pharmacological foundation for the folk practice of the plant in painful conditions.

3.1.1.2. Effect of Carrageenan induced oedema

Our result showed profound anti-inflammatory capability of both the extracts of Rhizomes and Aerial parts of the plant in carrageenan initiated paw edema model at all test doses in various assessment times. The results are illustrated in Table 3.1.1.4. The methanol extract of Rhizomes demonstrated marked and significant (P < 0.05) anti-inflammatory activity at 100 and 200 mg/kg. Reduction in paw oedema was predominantly dose dependent and remained significant during the assessment time (1-5 hour). The maximum (65.22 %) protection was observed for the Rhizomes after 3 hour of drug administration at the dose of 200 mg/kg. The standard drug, aspirin elicited (76.81%) protection at 100 mg/kg.

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Pretreatment with the methanol extract of Aerial parts of the plant, there was a significant (P < 0.05) attenuation of inflammatory reflux provoked by carrageenan at test doses (50, 100 and 200 mg/kg). In a concentration dependent manner, distinguish protection was observed (Table 3.1.1.4). Although reduction in paw edema was recorded from the starting test dose (50 mg/kg) but maximum susceptibility (60.87%) was found at 3 hour of drug treatment that was remained significant at 4 hour (58.46%) and 5 h (60%) at the dose of 200 mg/kg (Table 3.1.1.5). The patron of anti-inflammatory activity was quite similar to aspirin.

Table 3.1.1.4: Anti-inflammatory effect of the crude extract of Rhizomes in carrageenan stimulated hind paw edema in rats at 50, 100 and 200 mg/kg. Group Dose Increase in paw volume (mean ± SEM) in mL mg/ 1 hour 2 hour 3 hour 4 hour 5 hour kg Saline 10 0.70±0.031 0.69±0.040 0.69±0.049 0.70±0.067 0.72±0.053 Rhizomes 50 0.64±0.045 0.61±0.054 0.55±0.049 0.54±0.053 0.55±0.049 100 0.56±0.036 0.50± 0.049* 0.37±0.058** 0.39±0.062** 0.47± 0.062* 200 0.47±0.040* 0.35±0.067** 0.24±0.045** 0.31±0.053** 0.34±0.062** Aspirin Anti-inflammatory potential of the crude extract of Aerial parts in carrageenan stimulated hind paw edema in rats at 50, 100 and 200 mg/kg. Saline 10 0.70±0.06 0.69±0.07 0.69±0.05 0.65±0.049 0.70±0.04 Aerial 50 0.60.04 0.55±0.06 0.51±0.06 0.52±0.045 0.52±0.07 parts 100 0.50±0.07 0.49±0.06 0.34±0.05** 0.39±0.067** 0.37± 0.05** 200 0.43±0.07** 0.40±0.03** 0.27±0.04** 0.27±0.04** 0.28±0.05** Aspirin 100 0.23±0.040** 0.19±0.017** 0.17±0.031** 0.18±0.022** 0.18±0.036**

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Experimental data are expressed as mean ± S.E.M. for group of at least six animals. One- way ANOVA was utilized as judgment test of significant differences among groups followed by Dunnett’s multiple comparison post test. A probability of *P < 0.05, **P<0.01 was considered significant from control.

The development of an inflammatory response is a complex but well regulated process. Arachidonic acid is a polyunsaturated fatty acid that liberated from cell membrane phospholipids via the hydrolysis by phospholipase A2 enzymes (PLA2). The arachidonic acid is then metabolized by two distinct enzymatic pathways; cyclooxygenase (COX) in to prostaglandins (PGs) and lipoxygenase in to leukotrienes (Maier et al., 2008). PGs are members of the eicosanoid group generated in almost all cells of the human body; the principal mediator of inflammation in most of the inflammatory diseases (Serhan and Levy, 2003). Non-steroidal anti-inflammatory drugs (NSAIDs) stand for the most widely advised medicaments in the management of inflammatory conditions around the globe. Clinical findings revealed that they are useful for the symptomatic relief but do not abrogate the underlying disease process (Miller et al., 2005), and also suffering from serious life threatening side effects (Kearney et al., 2006; Patrono and Rocca, 2009). Secondary metabolite synthesized in the natural laboratory can provide more compact solution of such problems. Active principles of natural origin can act through new/different and or more effective mechanism or multiple mechanisms and can address the safety issues of synthetic compounds that are currently in clinical practice.

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Table 3.1.1.5: Protection (%) of Rhizomes (50, 100 and 200 mg/kg i.p.) in carrageenan induced hind paw edema in rats. Group Dose mg/ kg 1 h 2 h 3 h 4 h 5 h Rhizomes 50 08.57% 11.59% 20.29% 22.85% 23.61% 100 20.00% 27.53% 46.37% 44.29% 34.72% 200 32.86% 49.27% 65.22% 55.71% 52.78% Aspirin

Protection (%) of Aerial parts (50, 100 and 200 mg/kg i.p.) Aerial 50 14.28% 20.29% 26.09% 20.00% 25.71% parts 100 28.57% 28.99% 50.72% 40.00% 47.14% 200 38.57% 42.03% 60.87% 58.46% 60.00% Aspirin 100 67.14% 72.46% 6.81% 75.71% 75.00%

Oedema activated by carrageenan in rat paw is a well established animal model to elucidate the anti-edematous tendency of natural components. It has been observed that the local oedema is induced by the sub-plantar injection of carrageenan that increased progressively. After 3-5 hour of carrageenan administration, the maximum acute response is characterized by almost 100% greater volume of the injected paw which slowly and gradually decreases in 24 hour. Oedema formation due to carrageenan in the rat paw is the biphasic event during 1-5 hour; the initial phase (1 hour or 1.5 hour) is predominately a non-phagocytic edema followed by a second phase with increased edema formation that remained up to 5 hour (Meckes et al., 2004; Rotelli et al., 2003). Studies have been described the involvement of different mediators in various stages of carrageenan induced oedema. The initial phase (up to 1.5 hour) is attributed to the release of histamine, 5- hydroxytryptamine, bradykinin, platelet activating factor and serotonin.

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The second phase (1.5 up to 5 hour) is characterized by the lipid derived eicosanoids (prostaglandins, leukotrienes, HPETEs, etc.). A comprehensive phagocytic inflammation observed after 3 hour of carrageenan injection with large numbers of neutrophils and tissue edema (Meckes et al., 2004; Nguemfo et al., 2007). Our result showed marked and significant (P < 0.05) anti-inflammatory activity of both the Rhizomes and Aerial parts of the plant in carrageenan activated paw edema assay. This activity could be featured to the inhibitory effect of active principles on the release of various mediators of inflammation like lipoxygenase. As the extracts and fractions of both the Rhizomes and Aerial parts under examination demonstrated marked lipoxygenase inhibitory activity (Table 3.1.2.16) therefore it could be one of the possible contributors in the current anti- inflammatory response. The role of lipoxygenase is also documented in carrageenan provoked oedema (Viji and Helen, 2008).

Bioactivity directed isolation yielded seven different compounds including 2- hydroxybenzioc acid (3), and diosgenin (5), ß-sitosterol (6) and santonin (7). These compounds have registered significant anti-inflammatory activities previously (Al-Harbi et al., 1994; Doi and Horie, 2010; Gupta et al., 1980; Lu et al., 2007a) . The cyclooxygenase inhibitory activity of the diosgenin and related steroidal saponins have already been reported (Yu et al., 2008). Thus these compounds are strong contestant for these results. Phytochemical investigation of extracts and its subsequent solvent fractions also showed considerable quantity of saponins, alkaloids, phenols, flavonoids etc. The current anti- inflammatory activities could also be attributed to these pharmacologically active classes as the potent anti-inflammatory of these compounds are well documented in literature (Barbosa-Filho et al., 2006; Choi et al., 2002; Li et al., 2000).

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Figure 3.1.1.2: Effect of intraperitoneal administration (50, 100 and 200 mg/kg) at 3rd hour in carrageenan induced hind paw edema Rhizomes [A] and Aerial parts [B]. One-way ANOVA was operated for judgment of significant differences among groups followed by Dunnett’s multiple comparison post test. A probability of *P < 0.05, **P<0.01 was characterized as significant from control.

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2.1.1.3. Effect of antipyretic assay

The experimental findings of both the Rhizomes and Aerial parts of the plant in Brewer’s yeast provoked pyrexia are illustrated in Tables 3.1.1.6 and 3.1.1.7. The intraperitoneal administration of Rhizomes (50, 100 and 200 mg/kg) demonstrated marked and significant (P < 0.05) attenuation of infectious pyrexia during various assessment times (1-5 hours). The antipyretic effect was dose dependent and was very significant (P < 0.01) at 100 and 200 mg/kg after 3 hour of drug administration.

Similarly, the antipyretic sensitivity of the Aerial parts was prominent as compared to control. The significant attenuation was observed after at 2nd hour of drug administration at 100 mg/kg. The overall activity was dose dependent and maximum protection was exhibited at 200 mg/kg started from the 1st hour and remained significant up to 5 hour. The standard drug, paracetamol produced more dominant effect.

Fever or pyrexia symbolizes a controlled elevation in normal body temperature of human. Body temperature is normalized by the mediation of thermo-susceptible neurons positioned in the anterior hypothalamus. These neurons are vulnerable not only to changes in blood temperature but also to cold and warm receptors situated in skin and muscle and thus uphold an appropriate equilibrium between the heat generation and loss. Therefore, hypothalamus normalizes the set point of body temperature and sustained which usually bump-up in pyrexia (Devi et al., 2003). Fever may be elicited by an infection, malignancy or rheumatologic disorders. These mediators of fever could induce the incursion of endogenous pyrogens discharged from leukocytes into the central nervous system, particularly the organum vasculosum laminae terminalis to endorse PGs liberation. PGs afterward could react with the preoptic/anterior hypothalamus to alter the activity of heat responsive neurons in the hypothalamus (Takayama, 2004).

The intraperitoneal administration of extracts significantly attenuated rectal temperature of yeast induced febrile rat right from the 1 hour of administration and remained significant till the 5 hour at 200 mg/kg. The antipyretic activity was dose

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Table 3.1.1.6: Antipyretic effect of the crude extract of Rhizomes in yeast induced pyrexia in rats at 50, 100 and 200 mg/kg.

Drugs Doses Rectal temperature (0C) Before After yeast After drug administration yeast 0 hour 19 hour 1 hour 2 hour 3 hour 4 hour 5 hour Saline 100 36.05±0.06 37.33±0.02 37.30±0.09 37.30±0.06 37.30±0.08 37.28±0.09 37.28±0.05 ml/kg Rhizomes 50 36.10±0.04 37.37±0.11 37.10±0.08 37.00±0.06 36.92±0.09* 37.20±0.16 37.00±0.08 mg/kg 100 36.17±0.05 37.39±0.08 36.96±0.06* 36.90±0.09* 36.77±0.09** 36.77±0.09* 36.78±0.06** 200 36.10±0.06 37.35±0.06 36.81±0.06** 36.74±0.13** 36.62±0.08** 36.70±0.13** 36.74±0.13** PRA 100 36.15±0.05 37.54±0.08 36.40±0.06** 36.38±0.06** 36.25±0.07** 36.30±0.09** 36.28±0.09**

Values are reported as mean ± S.E.M of at least six animals. The data were analyzed by ANOVA followed by Dunnett’s test. Asterisks indicated statistically significant values from control. *P < 0.05, **P<0.01. PRA = Paracetamol.

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Table 3.1.1.7: Antipyretic effect of the crude extract of Aerial parts in yeast induced pyrexia in rats at 50, 100 and 200 mg/kg. Drugs Doses Rectal temperature (0C) Before yeast After yeast After drug administration 0 hour 19 hour 1 hour 2 hour 3 hour 4 hour 5 hour Saline 100 35.95±0.13 37.20±0.08 37.18±0.08 37.18±0.09 37.18±0.05 37.15±0.05 37.15±0.06 mL/kg Aerial 50 35.90±0.08 37.10±0.06 37.08±0.08 37.00±0.08 36.95±0.06 36.95±0.06 37.00±0.08 parts mg/kg 100 36.10±0.08 37.45±0.09 36.95±0.08 36.77±0.06** 36.67±0.08** 36.68±0.13** 36.72±0.09* 200 36.25±0.11* 37.60±0.10* 36.81±0.06* 36.47±0.08** 36.42±0.08** 36.49±0.08** 36.50±0.13** PRA 100 36.25±0.8 37.50±0.06 36.60±0.06** 36.41±06** 36.37±0.06** 36.35±0.06** 36.33±0.08**

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dependent. The Non-steroidal anti-inflammatory drugs (NSAID) are broadly exercised by the physician for the control of febrile conditions of various origins. The well published mechanism for NSAID is the inhibition of prostaglandins via cyclooxygenase pathway (Blandizzi et al., 2009). For that reason, it is assumed that the secondary metabolites from these extracts might contain active principle(s) with cyclooxygenase inhibitory activity. The cyclooxygenase inhibitory activity of secondary metabolites isolated from the Rhizomes i.e. 2-hydroxybenzoic acid (3), diosgenin (5), ß-sitosterol (6) and santonin (7) have registered antipyretic activity (Al-Harbi et al., 1994; Gupta et al., 1980; Lisina et al., 2008; Yu et al., 2008). Additionally, the extracts of both Rhizomes and Aerial parts possess therapeutically effective groups like alkaloids, saponins, flavonoids etc. therefore, the role of these groups cannot be ignore in the current antipyretic show.

3.1.1.4. Effect of Pentylenetetrazole-induced convulsions

Pentylenetetrazole (90 mg/kg) provoked mioclonic, clonic and tonic and tonic–clonic seizures (HLTE) convulsions in almost all control mice. Pretreatment with crude methanol extract of both Rhizomes and Aerial parts of the plant did not exhibit any reduction in PTZ induced convulsions. In both cases, the extracts were injected intraperitoneally at strength of 50, 100 and 200 mg/kg and effects were comparable to control (normal saline). Hence our finding ruled out the accumulation of any anticonvulsant constituents in both extracts.

3.1.1.5. Effect of diuretic activity

In the traditional system of medicine, Rhizomes are used in combination with other plant based diuretics for the augmentation of urine output. Therefore, both the extracts were also screened for the possible diuretic activity in an established pharmacological paradigm. In our assessment on the diuretic activity of Rhizomes, mild diuretic activity was computed followed oral administration of 300 mg/kg compared to saline (Figure 3.1.1.3). While in contrast, the urine discharge was similar to saline at 600 mg/kg. Urine output was calculated per 100 kg of test animal after 6 hour.

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Similarly, mild but insignificant diuretic effect was exhibited by the Aerial parts of the plant at both test doses (300 and 600 mg/kg). The mild diuretic potential of the plant could be the reason for its use in polypharmacy for diuresis. The characterization of secondary metabolites from the plant could be supportive in understanding the real potential of the plant as diuretic.

Figure 3.1.1.3. Effect of oral administration of Rhizomes and Aerial parts (300 and 600 mg/kg) in rats. Cumulative urine volume was expressed as per 100 kg body weight after 6 h in rats. Hydrochlorothiazide (HCT; 10 mg/kg p.o.) was standard drug. Values are communicated as mean ±S.E.M. [n = 6]. Asterisks designed significant distinction from control. **P<0.01 (ANOVA followed by Dunnett’s test).

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3.1.1.6. Effect of acute toxicity studies

The acute toxicity test is usually carried out to assess the safety profile of the extract and to provide a background for its use in other experimental paradigms. The results in the preliminary toxicity studies of both extracts of Rhizomes and Aerial parts revealed their absolute safety up to the dose of 2 g/kg and the entire test animal survived. Similarly the test animals were observed for any gross effect or mortality. Notably almost all the animals were perfectly safe during 24 h assessment time (Table 3.1.1.7). Based on the experimental findings, both the extract were completely safe up to maximum test dose (2000 mg/kg).

Table 3.1.1.8. Effect of acute toxicity test of Rhizomes and Aerial parts in mice at 500, 1000 and 2000 mg/kg, p.o. Group Dose mg/ kg, p.o. Deed Survived Gross effect Mortality (%) Saline 5mL/kg 15 mL/kg, p.o. – All – – 500 – All – – Rhizomes 1000 – All – – 2000 – All – – 500 – All – – Aerial parts 1000 – All – – 2000 – All – –

3.1.2. In-vitro investigations

3.1.2.1. Antimicrobial Activities

3.1.2.1.1. Effect of antibacterial assay

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The methanol extract of the Rhizomes of Polygonatum verticillatum and its sequentially partitioned subsequent solvent fractions demonstrated significant antibacterial sensitivity against various pathogenic bacteria in the assay. Using the agar well diffusion method, both Gram-positive bacteria (Staphylococcus aureus, and Bacillus subtilis) and Gram-negative bacteria (Escherchia coli, Pseudomonas aeruginosa, flexeneri Shigella and Salmonella typhi) were included in test. The results are shown in table 3.1.2.1 and 3.1.2.2.

Food borne infections stand for a major health problem for the health care professionals worldwide. In humans, Shigella species are the causative agents of bacillary dysentery. They invade and colonize the colonic epithelium causing massive inflammation and destruction of colonic mucosa (Sansonetti, 2001). Salmonella is symbolizing the most imperative pathogenic genera accused in food borne bacterial eruptions and diseases (Cetinkaya et al., 2008). Typhoid fever, a systemic infectious disease caused by Salmonella typhi alone is responsible for 600 thousands death per year around the globe (Vollaard et al., 2005). The lethal complications of typhoid fever in untreated patients are gastrointestinal hemorrhage and perforation.

Marked antibacterial activity was found for the crude extract of the Rhizomes of P. verticillatum and its subsequent solvent fractions against the Shigella flexeneri. The sensitive of S. flexeneri was notable to all fractions of the plant in which aqueous fraction (20 mm) was the most dominant in terms of zone of inhibition followed by crude extract (16 mm) and ethyl acetate (15 mm) (Figure 3.1.2.1.D). The MICs in the micro-dilution technique were in the range of 03-30 µg/mL (Table 3.1.2.1). The aqueous being the most potent with MIC of 03 µg/mL. The Aerial parts of the plant observed moderate antibacterial activity against Shigella flexeneri. The maximum inhibitory activity was offered by the chloroform fraction (12 mm) followed by the ethyl acetate fraction (11 mm). Nevertheless, the least polar fraction, n-hexane and the most polar fraction, n-butanol had not carried any inhibitory potential (Figure 3.1.2.2C). Same results in terms of bacterial sensitivity were recorded in micro-dilution assay. The chloroform fraction was the most

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potent with MIC of 08 µg/mL followed by the ethyl acetate fraction with MIC of 15 µg/mL (Figure 3.1.2.1C).

Similar trend of sensitive was observed against S. typhi. In case of Rhizomes, the inhibitory activity was related with polarity i.e. the less polar fraction such as hexane (11 mm), chloroform (10 mm) and ethyl acetate (10 mm) fractions were sensitive and possess significant zone of inhibition, while the remaining fractions were inactive including crude extract (Figure 3.1.2.C). The calculated MICs were ranges from 3-6 µg/mL. The sensitivity of the various fractions of the Aerial parts against S. typhi is depicted in table 3.1.2.2. Ethyl acetate being the most potent inhibiter of S. typhi (13 mm) had MIC of 2

Table 3.1.2.1: Anti-bacterial activities of Rhizomes using agar well diffusion method. Bacterial strains Zones of inhibition (in mm) Std Crude Hexane Chloroform Ethyl Butanol Aqueous acetate Gram-Positive Bacillus subtilis 23 ------S. aureus 27 - - 10 10 11 - Gram-Negative Escherchia coli 20 14 20 20 18 - - P. aeruginosa 29 ------Salmonella typhi 26 - 11 10 10 - - Shigella flexeneri 28 16 14 10 15 14 20

Anti-bacterial activities of Rhizomes of plant using micro-dilution method Bacterial strains MIC (µg/mL) Std Crude Hexane Chloroform Ethyl Butanol Aqueous acetate Gram-Positive Bacillus subtilis 0.22 ------

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S. aureus 0.17 - - 75 80 75 - Gram-Negative Escherchia coli 0.19 40 05 06 1.5 - - P. aeruginosa 0.31 ------Salmonella typhi 0.17 - 03 05 06 - - Shigella flexeneri 0.13 30 50 16 40 05 03

Standard drug (Std) = Imipinem (10 µg/mL).

Control = DMSO.

(-)= Inactive.

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Figure 3.1.2.1: Antibacterial activity of Rhizomes against S. aureus [A], E. coli [B], S. typhi [C] and S. flexeneri [D]. Data are mean of three different findings.

µg/mL. This patron of activity was followed by the chloroform fraction with reasonable zone of inhibition (12 mm) and MIC (5 µg/mL). However, n-hexane, n-butanol and aqueous fractions were devoid of activity.

Keeping in mind the results of the preliminary screening, the folk use of the Rhizomes of the plant in crude form for the treatment of feverish conditions and dysentery is justified. Moreover, the Aerial parts of the plant support the use of the plant in the traditional system for the said purpose but were comparatively less potent. Escherichia coli is believed to be a major causative agent in urinary tract infections (UTIs). UTIs fluctuate from asymptomatic bacteriuria to devastating acute pyelonephritis. It is estimated that up to 80% of female will be the victim of UTI at least once in their lifetime and up to 50% of will have recurrent episodes (U-Syn and Yong-Hyun, 2008).

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The crude extract and its subsequent solvent fractions of Rhizomes demonstrated prominent antibacterial activity against E. coli (Figure 3.1.2.1B). The less polar fractions like n-hexane (20 mm), chloroform (20 mm) and ethyl acetate (18 mm) exhibited highest zone of inhibition while the polar fractions like water and butanol were insensitive (Table 3.1.2.1). The ethyl acetate fraction possesses predominant sensitivity against E. coli in the micro-dilution technique with MIC of 1.5 µg/mL. However, the overall MICs were estimated from 1.5-40 µg/mL. Contrary to that, the Aerial parts were found tolerant to E. coli. Pseudomonas aeruginosa was the only Gram-Negative pathogen that was completely insensitive to all the fractions of Rhizomes as well as Aerial parts.

S. aureus can be a major causative agent in a wide verity of infections ranging from minor skin infections to postoperative wound infections. The wide spread usage of antibiotics is responsible for the proliferation of genes with increasing resistance to many strains of S. aureus (Chomnawang et al., 2009). The tested fractions of the Rhizomes did not show promising inhibitory activity against the Gram-Positive pathogens. S. aureus was only sensitive to chloroform, ethyl acetate and n-butanol fractions and the MIC was ranges from 75-80 µg/mL. However, the Aerial parts did not express any activity against S. aureus.

B. subtilis was absolutely insensitive to all fractions of Rhizomes. But the Aerial parts of the plant showed moderated activity against B. subtilis (Figure 3.1.2.2A). The comparatively less polar fractions such as n-hexane, chloroform and ethyl acetate showed more zone of inhibition while the more polar fractions like n-butanol and aqueous were inactive. The MICs were ranges from 11-50 µg/mL, ethyl acetate being the more potent fraction with MIC value of 11 µg/mL.

The preliminary phytochemical screening of the Rhizomes and Aerial parts of the plant explained the accumulation of major classes like saponins, phenols, alkaloids, flavonoids, glycosides, terpenoids and tannins. The antimicrobial tendency of these chemical agents is well documented in literature (Bravo et al., 1999; Chakraborty and Brantner, 1999; Yadava and Jharbade, 2008). For that reason, it can be summarized that

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these chemicals groups are responsible for this antibacterial activity. Moreover, triterpenoids (Wang et al., 2009a), steroidal saponins (Wang et al., 2009b) and indolizinone alkaloid (Wang et al., 2003) have formerly been authenticated from the genus with antibacterial activity.

Table 3.1.2.2: Anti-bacterial activities of Aerial parts of in agar well diffusion method. Bacterial strains Zones of inhibition (in mm) Std Crude Hexane Chloroform EtOAc Butanol Aqueous Gram-Positive Bacillus subtilis 37 - 14 11 15 - - S. aureus 26 ------Gram-Negative Escherchia coli 30 ------P. aeruginosa 32 ------Salmonella typhi 30 9 - 12 13 - - Shigella flexeneri 36 10 - 12 11 9

Antibacterial activity of Aerial parts using macro-dilution method Bacterial strains MIC (µg/mL) Std Crude Hexane Chloroform EtOAc Butanol Aqueous Gram-Positive Bacillus subtilis 0.22 40 50 11 - - S. aureus 0.17 ------Gram-Negative Escherchia coli 0.19 ------P. aeruginosa 0.31 - - - - - Salmonella typhi 0.17 07 - 05 02 - -

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Shigella flexeneri 0.13 40 08 15 - 50

Data represent mean of three different experiments.

Standard drug (Std) = Imipenem (10 µg/mL).

Control = DMSO.

(-)= Inactive.

Figure 3.1.2.2: Antibacterial activity of Aerial parts of the plant against B. subtilis [A], S.

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t yphi [B] and S. flexeneri [C]. Data are mean of three different findings.

In Pakistan, infectious diseases stand for the leading cause of morbidity. During 2002- 2003, the total number of patients admitted with infectious disorders were 196049 (17.90%) in 22 major teaching hospitals of the country (MOH, 2005b). Bacterial resistance to the current antimicrobial drugs is a global issue now. Different new sources are tested to overcome the hazards of resistance. In the light of presumption that the plant extracts exercise their antimicrobial effect on sites other than those used by the synthetic drugs therefore minimizes the chances of resistance, both Rhizomes and Aerial parts of the plant offered a significant natural alternative to resistant synthetic drugs for the effective management of various infections caused by these pathogens. The study also validated the use of the plant in folk medicine as antimicrobial.

For the Rhizomes of plant, the bioactivity guided isolation yielded seven different compounds including 5-hydroxymethyl-2-furaldehyde (4). It has got some selective antibacterial activity against plant pathogens and in oral cavity (Espinoza et al., 2008; Rivero-Cruz et al., 2008). However, further studies on the isolated compound are most warrant.

3.1.2.2. Effect of antifungal activities

The methanol extract of the Rhizomes and its subsequent solvent fractions were found to acquire moderate antifungal action against two fungi out of six selected bacterial strains. The sensitive fungi were Microspoum canis and Fusarium solani. In case of M. canis, only chloroform (10%) and n-butanol (10%) fractions described low antifungal activity, while the remaining fractions were inactive (Table 3.1.2.3). The minimum inhibitory concentrations (MICs) were 360 and 350 µg/mL respectively.

The susceptibility of crude extract (40%) and n-hexane (70%) against F. solani was

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comparatively highest (Figure 3.1.2.3). The MICs for both the fractions were 290 and 190 µg/mL respectively. The antifungal profile of Aerial parts of the plant is presented in table 3.1.2.4. It is event from the results that only M. canis was sensitive to only chloroform (50%) and aqueous fractions. The MICs were 60 and 250 µg/mL respectively. Nevertheless, remaining fungi were absolutely resistant to all extracts.

Table 3.1.2.3: Anti-fungal activities of extract Rhizomes and its solvent fractions. Fungal Inhibition of Fungal Growth (%) strains Std Crude Hexane Chloroform EtOAc Butanol Aqueous T. longifusus 70 1 ------C. albicans 110 1 ------A. flavus 20 2 ------M. canis 98.4 1 - - 10 - 10 - F. solani 73.26 1 40 70 - - - - C. glaberata 110.8 1 ------Minimum Inhibitory Concentration (MIC) in antifungal assay Fungal MIC µg/mL strains Std Crude Hexane Chloroform EtOAc Butanol Aqueous T. longifusus 1.41 ------C. albicans 1.81 ------A. flavus 2.32 ------M. canis 1.6 - - 360 - 350 - F. solani 1.11 290 190 - - - - C. glaberata 0.51 ------

1Standandard Drug = Miconazole, 2Standandard Drug = Amphotericin B.

Sample= 400 µg/mL.

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(-)= Inactive.

Control = DMSO.

The phytochemical tests indicated the accumulation of therapeutically valuable classes of secondary metabolites such as alkaloids, saponins, flavonoids, glycosides, terpenoids and tannins. The antimicrobial tendency of these groups is well documented in literature (Bravo et al., 1999; Chakraborty and Brantner, 1999; Yadava and Jharbade, 2008). Therefore, it can be speculated that they are responsible for this antifungal activity. Additionally, triterpenoids (Wang et al., 2009a), steroidal saponins (Wang et al., 2009b) and indolizinone alkaloid (Wang et al., 2003) have previously been isolated from the genus with mild antifungal activity also supported our outcomes. The antifungal activities of the isolated seven compounds from the Rhizomes are not reported so needs to evaluate.

Figure 3.1.2.3: Antifungal activity of Rhizomes against M. canis [A] and F. solani [B]. Data are mean of three different findings.

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Table 3.1.2.4: Antifungal activity of the crude extract of Aerial parts. Fungal strains % Inhibition of Fungal Growth Std Crude Hexane Chloroform EtOAc Butanol Aqueous T. longifusus 70 1 ------C. albicans 110 1 ------A. flavus 20 2 ------M. canis 98.4 1 - - 50 - - 20 F. solani 73.26 1 ------C. glaberata 110.8 1 ------Minimum Inhibitory Concentration (MIC) in anti-fungal of Aerial parts Fungi MIC (µg/mL) Std Crude Hexane Chloroform EtOAc Butanol Aqueous T. longifusus 1.41 ------C. albicans 1.81 ------A. flavus 2.32 ------M. canis 1.6 - - 60 - - 250 F. solani 1.11 ------C. glaberata 0.51 ------

Data represent mean of three different experiments.

Standard Drugs = 1Miconazol, 2Amphotericin B.

Control=DMSO.

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Sample= 400 µg/mL.

(-)= Inactive.

Figure 3.1.2.4: Antifungal activity of Aerial parts against Microspoum canis. Data are mean of three different findings.

3.1.2.3. Effect of in-vitro antimalarial bioassay

The Rhizomes and Aerial parts of the Polygonatum verticillatum showed keen in-vitro antimalarial activity against Plasmodium falciparum. As presented in Table 3.1.2.5, the crude extract and its less polar fractions exhibited notable antimalarial activity against chloroquine resistant Plasmodium falciparum. The maximum potency was exhibited by the

n-hexane fraction (IC50: 2.33 µg/mL) followed by the chloroform fraction (IC50: 4.62

µg/mL) while the crude extract was the least potent (IC50: 21.67 µg/mL). However, the

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polar fractions such as ethyl acetate, n-butanol and aqueous were found inactive in the assay.

The crude methanol extract and its subsequent solvent fractions of Aerial parts of Polygonatum verticillatum provoked significant in-vitro antimalarial activity against Plasmodium falciparum. According to the results shown in Table 3.1.2.6, the crude extract exhibited profound antimalarial sensitivity (IC50: 14.75µg/mL). The fractionation resulted into prominent change in the activity. Consequently, the less polar fractions demonstrated dominant susceptibility against Plasmodium falciparum. The maximum potency was

Table 3.1.2.5: In vitro anti-malarial activity of the Rhizomes against Plasmodium falciparum

Test organism Extracts/Fractions IC50 (µg/mL) Crude methanol extract 21.67 n-Hexane 2.33

Chloroform 4.62 Ethyl acetate >25 n-Butanol >25 Plasmodium falciparum Aqueous >25 Chloroquine diphosphate 0.025

Tested Sample = 1mg.

Incubation period = 72 hours at 37 0C.

Standard drug = Chloroquine diphosphate = 1 mM

Control = DMSO.

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Table 3.1.2.6: In vitro anti-malarial activity of the Aerial parts against Plasmodium falciparum

Test organism Extracts/Fractions IC50 (µg/mL) Crude methanol extract 14.75 n-Hexane 4.86

Chloroform 5.71 Ethyl acetate >25 n-Butanol >25 Plasmodium falciparum Aqueous >25 Chloroquine diphosphate 0.025

Tested Sample = 1 mg.

Incubation period = 72 hours at 37 0C.

Standard drug = Chloroquine diphosphate= 1 mM.

Control = DMSO.

exhibited by the hexane fraction (IC50: 4.86 µg/mL) followed by the chloroform fraction

(IC50: 5.71 µg/mL).

Malaria is one of the oldest and all time challenge for the health care professionals continue to causes large scale morbidity and mortality around the globe. It has been reported that malaria is responsible for 1-2 millions deaths while 300-500 millions of new cases are reported each year (Snow et al., 2005). According to the survey of WHO on malaria, children and pregnant women are equally suffering from the disease while it has negative impact on the economy of prevalent countries (WHO, 2000). Multidrug-resistant Plasmodium falciparum strains are an increasing problem in tropical and subtropical

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endemic zones of the world and the management of such resistant cases has now becomes a great challenge for the physicians.

Like other developing countries of the world, numerous malaria cases are registered in Pakistan each year. According to the survey of Ministry of Health, the total number of confirmed malarial cases reported throughout the country was 101,761. This statistic was based on 20% of the population using public sector health facilities (the remaining 80% using private sector) the number of malaria cases could be at least five times higher and therefore, we may assume more than half a million cases per year. The total number of confirmed falciparum cases reported throughout the country was 31,407 in 2002 (MOH, 2005a). Therefore, new effective, safe and affordable therapies are urgently required for the effective management of various malarial strains including resistant to available antimalarial drugs.

The potential contribution of plants and various compounds isolated or derived from plants in the treatment of malaria is frequently reported in literature (Bhat and Surolia, 2001). Malaria represents as the single most important ailment that has been effectively treated with herbal products from many years. The classical compounds used in the management of malaria such as quinine and artemisinin, were either directly derived from plants or developed using chemical structures of plant based compounds (Muthaura et al., 2007). Therefore, the plants still have the great potential to be explored.

Out of seven compounds isolated from Rhizomes, the antimalarial activity of diosgenin (5) has been investigated (Alvarez et al., 2004). The antimalarial activity against chloroquine resistant P. falciparum in our study could be attributed to diosgenin. Additionally, the other isolated constituents from the plant need similar type of test. Upon phytochemical analysis, the Rhizomes of the plant demonstrated the presence of various pharmacological active chemical groups like saponins, alkaloids, flavonoids, glycosides, phenols, terpenoids and tannins. The antimalarial activities of these groups are frequently reported in literature (Abdel-Sattar et al., 2008; Addae-Kyereme et al., 2001; Monbrison et al., 2006). Thus, the current finding could also be attributed to these groups.

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3.2.1.4. Effect of in vitro phytotoxicity

The crude methanol extract of the Rhizomes and Aerial parts of Polygonatum verticillatum exhibited outstanding phytotoxic activity followed by their subsequent solvent against Lemna acquinoctialis Welv.

In case of Rhizomes, the crude drug showed 90% growth inhibition at 5 µg/mL concentration (Figure 3.1.2.6C). The sensitivity of extract further increased to 100% at 50 and 500 µg/mL. Fractionation made a great difference to the overall activity of the resulting fractions. Upon fractionation, n-hexane, chloroform, ethyl acetate and n-butanol fractions showed only 40%, 50%, 25% and 50% susceptibly respectively at a dose of 500 µg/mL. The aqueous fraction was the most dominant against the growth of Lemna acquinoctialis Welv at all test doses. At the low testing dose (5 µg/mL), the inhibitory activity was 90%. The inhibitory potential was further enhanced and complete growth inhibition was observed at concentration of 50 and 500 µg/mL concentrations (Table 3.1.2.7).

The crude extract of Aerial parts and its various solvent fractions showed marked inhibitory activity against growth of Lemna acquinoctialis Welv (Table 3.2.8). The activity was in a dose related mode and the crude extract illustrated 48.26% at 500 µg/mL (Figure 3.1.2.6). Prominent increased in the inhibitory activity was observed after fractionation. The n-hexane fraction showed 74.13% sensitivity at 500 µg/mL while the chloroform and n-butanol fractions exhibited complete growth inhibition (100%) at 500 µg/mL. The inhibitory activity of ethyl acetate fraction was 79.3% at 500 µg/mL. However, aqueous fraction possesses 68.96% at 500 µg/mL.

Table 3.1.2.7: Phytotoxic activity of the crude extract and its successive fractions of Rhizomes against the Lemna acquinoctialis Welv. Name of Extracts Number of fronds

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5 µg/mL 50 µg/mL 500 µg/mL Control Sample GI (%) Sample GI (%) Sample GI (%) Crude extract 20 02 90 - 100 - 100 n-hexane 20 20 0 15 25 12 40 Chloroform 20 20 0 15 25 10 50 Ethyl acetate 20 20 0 20 0 15 25 n-butanol 20 20 0 15 25 10 50 Aqueous 20 02 90 - 100 - 100

GI= % Growth Inhibition.

Standard drug (Paraquat) = (3.142 µg/mL).

Test samples = (15 mg).

Control = Ethanol.

(-) = inactive.

Interference of weeds obviously reduces the quality and magnitude of agricultural crops and is responsible for incredible economic losses all over the world. It is estimated in USA that weeds grounds a loss of approximately 12% that costing almost US$ 33 billion. Nevertheless, the situation is more alarming in developing states (Pimentel et al., 2001). Synthetic herbicides are extensively used for the control of weeds in agricultural sectors. However, various factors that restricted the use of synthetic herbicides include water and soil pollution, herbicide-resistant weed populations, herbicide scum and injurious outcomes on non-target (Li et al., 2003). In recent times, more stress has been given to the natural

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Figure 3.1.2.5: Phytotoxic activity of Rhizomes at 500 µg/mL [A], 50 µg/mL [B] and at 5 µg/mL [C] concentrations. Data are mean of three findings.

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Figure 3.1.2.6: Phytotoxic activity of Aerial parts at 500 µg/mL [A], 50 µg/mL [B] and at 5 µg/mL [C] concentrations. Data are mean of three readings.

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Table 3.1.2.8: Phytotoxic activity of Aerial parts of P. verticillatum against the Lemna acquinoctialis Welv. Name of Extracts Number of fronds 5 µg/mL 50 µg/mL 500 µg/mL Control Sample GI (%) Sample GI (%) Sample GI (%) Crude extract 19.33 18 6.88 15 22.4 10 48.26 n-hexane 19.33 18 6.88 10 48.26 5 74.13 Chloroform 19.33 17 12.05 9 53.44 - 100 Ethyl acetate 19.33 18 6.88 10 48.26 4 79.3 n-butanol 19.33 15 22.40 9 53.44 - 100 Aqueous 19.33 18 6.88 11 43.09 6 68.96

GI= % Growth Inhibition.

Standard drug (Paraquat) = (3.142 µg/mL).

Test samples = (15 mg).

Control = Ethanol.

(-) = Zero.

allelochemicals from plants for weed control in crop production especially to cope with the problem of weed resistance.

It has been proved that the phytotoxicity of the plant reduced the growth of weeds without any negative effect on the crops growth and overall yield under normal field conditions, rather significant increased has been recorded in corps production (Batish et al., 2007; Saeed et al., 2010). For that reason, it is believed that the phytotoxic metabolite(s) of the Rhizomes and Aerial parts of the plant possibly will be a significant foundation of

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natural herbicides for weeds control in a continuously for better corps production. Thus the isolation of pure herbicidal(s) could lead to more effective natural based weed protection.

Upon bioactivity guided isolation, the Rhizomes of the plant resulted into seven different compound through application of various chromatographic techniques. Among these compounds, 5-hydroxymethyl-2-furaldehyde (4) had reasonable susceptibility against plant pathogens (Espinoza et al., 2008). While ß-sitosterol (6) also showed some phytotoxicity (Khaliq-Uz-Zaman et al., 1998). However, the rest of compounds had not been tested in such type of assays. Therefore, offers a chance to be tested as phytotoxic in phytotoxicity assay. In the mean time, the present findings can be featured to the various classes of phytochemical groups for which they showed positive test.

3.1.2.5. Effect of in vitro Insecticidal activity

The crude methanol extract of Rhizomes and its subsequent fractions were found mostly inactive against tested insects (Table 3.1.2.9). Among the tested fractions, only the hexane fraction exhibited 50% activity against Rhyzopertha dominica. The remaining insects were not susceptible to all other fractions used in the test.

A similar type of result was observed for the Aerial parts of the plant. The results are shown in table 3.1.2.9. Among the tested insect, Rhyzopertha dominica was the only susceptible insect. The n-hexane fraction offered 50% and chloroform fraction 30%. However, the remaining fractions were inactive against all tested insect (Saeed et al., 2010). Based on the outcomes, it can be wind up that both the Rhizomes and Aerial parts of the plant have active principles deficient or impotent as insecticidal.

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Table 3.1.2.9: Insecticidal activities of crude extract and various factions of Rhizomes. Name of Mortality (%) Std Crude Hexane Chloroform Ethyl butanol Aqueous Insect acetate NC SP NC SP NC SP NC SP NC SP NC SP R. dominica 100 - - - 50 ------T. castaneium 100 ------C. analis 100 ------S. oryzae 100 ------Insecticidal activities of crude extract and various fractions of Aerial parts Name of Mortality (%) Std Crude Hexane Chloroform Ethyl Butanol Aqueous Insect acetate NC SP NC SP NC SP NC SP NC SP NC SP R. dominica 100 - - - 50 - 30 ------T castaneium 100 ------C. analis 100 ------S. oryzae 100 ------

NC = Negative control.

SP = Sample (1019.9 μg/cm2).

Std = Standard (Permethrin) = 235.9 μg/cm2.

(-)= No activity.

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3.1.2.6. Effect of in vitro leishmanicidal activity

The crude methanol extract of Rhizomes and Aerial parts of the plant followed by their subsequent solvent fractions were dormant against Leishmania major. On the other hand, the standard drugs showed prominent antileishmanial activity. The results are displayed in table 3.1.2.10. It is reflected from the results that both Rhizomes and Aerial parts of the plant did not possess any consistent(s) with antileishmanial activity.

Table 3.1.2.10: In-vitro antileishmanial activity of Rhizomes against Leishmania major

Test organism Extracts/Fractions IC50 (µg/mL) Crude extract > 100 Hexane > 100

Chloroform > 100 Ethyl acetate > 100 n-Butanol > 100

Aqueous > 100

Leishmania major (DESTO) Amphotericin-B 0.50 ±0.02 Pentamidine 2.56 ±0.02

In-vitro antileishmanial activity of Aerial parts of Polygonatum verticillatum against Leishmania major

Test organism Extracts/Fractions IC50 (µg/mL) Crude extract > 100 Hexane > 100

Chloroform > 100 Ethyl acetate > 100

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n-Butanol > 100 Aqueous > 100 Leishmania major (DESTO) Amphotericin-B 0.50 ±0.02 Pentamidine 2.56 ±0.02

Incubation period was 72 h at 22 0C.

Standard drugs. Amphotericin-B and Pentamidine.

Control= DMSO.

3.1.2.7. Brine shrimp cytotoxic assay

Brine shrimp cytotoxic assay is generally an effective and rapid assay for the study of acute toxicity of extracts. The crude methanol extract of Rhizomes and its subsequent solvent fraction were comparatively not lethal to the brine shrimps at the concentrations of 10, 100 and 1000 µg/mL. The results are depicted in table 3.1.2.11. Apart from the ethyl acetate fraction, which displayed significant toxicity (LD50 492.846 µg/mL) all the remaining fractions were unable to kill the significant number of shrimps therefore, are considered as safe.

Table 3.1.2.11: Brine shrimp cytotoxicity of Rhizomes

Fractions Conc (µg/mL) Shrimps taken Shrimps survived LD50 (µg/mL) Crude extract 1000 30 20

100 30 21 NA

10 30 28

Hexane 1000 30 26 NA

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100 30 29

10 30 30

Chloroform 1000 30 17

100 30 19 NA

10 30 28 Ethyl acetate 1000 30 14

100 30 20 492.846

10 30 26 n-Butanol 1000 30 21

100 30 23 NA

10 30 26 Aqueous 1000 30 24

100 30 19 NA

10 30 17 Etoposide - - - 07.4625

Results are the replicates of three different experiments.

Control= DMF (2 mL).

Sample= 20 mg.

Standard drug= Etoposide.

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Similarly, the crude extract of Aerial parts and its subsequent solvent fractions were subjected to brine shrimp cytotoxic assay and the results are shown in table 3.1.2.12.

Insignificant toxicity was observed for all the test fractions except chloroform (LD50: 1205.07 µg/mL). Keeping in view the results of preliminary cytotoxic study as well as in vivo acute toxicity test, the extracts of both Rhizomes and Aerial parts can easily be declared safe for their additional exercise in animal experimental models for aiming assorted diseases. In other words, both extracts carried reasonable safety.

Table 3.1.2.12: Brine shrimp cytotoxicity of Aerial parts

Fractions Conc (µg/mL) Shrimps taken Shrimps survived LD50 (µg/mL) Crude extract 1000 30 19

100 30 20 NA

10 30 22

Hexane 1000 30 26

100 30 27 NA

10 30 28

Chloroform 1000 30 14

100 30 23 1205.07

10 30 23 Ethyl acetate 1000 30 23

100 30 26 NA

10 30 27

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n-Butanol 1000 30 23

100 30 25 NA

10 30 28 Aqueous 1000 30 23

100 30 25 NA

10 30 25 Etoposide - - - 07.4625

Results are the replicates of three different experiments.

Control= DMF (2 mL).

Sample= 20 mg.

Standard drug= Etoposide.

3.1.2.6 Effect of 1,1-diphenyl-2-picrylhidrazyl (DPPH) 3.1.2.7 scavenge activity

The crude methanol extract of Rhizomes of Polygonatum verticillatum displayed marked scavenging activity against steady free radical 1,1-diphenyl-2-picrylhidrazyl (DPPH). The antioxidant activity was concentration dependent (Figure 3.2.7). In case of crude extract 30%, 55% and 71% activity was observed at 50, 100 and 200 µg/mL respectively while IC50 was 115 µg/mL. The fractionation has made a great deal in the activity. The n-hexane fraction illustrated little action at test concentrations and was 22%,

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33% and 50% at 50, 100 and 200 µg/mL respectively. The IC50 was 200 µg/mL. The scavenging potential was greatly augmented in case of chloroform fraction i.e. 33%, 59%

Figure 3.1.2.7: 1,1-diphenyl-2-picrylhidrazyl (DPPH) scavenging potential of Rhizomes. PR-1 = Crude extract; PR-2 = n-hexane; PR-3= Chloroform; PR-4 = Ethyl acetate; PR-5 = n-butanol; PR-6 = Aqueous. Standard drug = Vitamin C. Negative control = Ethanol. Symbols stand for mean ± S.E.M. (n = 3).

and 76% at 50, 100 and 200 µg/mL respectively and IC50 was 93 µg/mL. The highest activity and potency was recorded for the ethyl acetate fraction 81% at 200 µg/mL while

IC50 was 90 µg/mL. The n-butanol fraction exhibited maximum potential (77%) at 200 µg/mL. Nevertheless, aqueous fraction was least dynamic fraction and activity was 58% at

200 µg/mL while IC50 was 145 µg/mL.

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The results of Aerial parts of parts revealed significant free radical scavenging activities (Figure 3.1.2.8). Similar to Rhizomes, the antioxidant activity was concentration dependent. The crude extract offered reasonable scavenging with IC50 value of 122 µg/mL (Table 3.1.2.14). Upon fractionation, profound change in activity was observed. The n- hexane fraction displayed insignificant activity while the ethyl acetate demonstrated the

maximum potency and IC50 was 137 µg/mL.

Oxidative stress induces free radical formation that is involved in the patho- physiology of various diseases like arteriosclerosis, malaria, rheumatoid arthritis, caner, aging and variety of inflammatory disorders (Moure et al., 2001). Antioxidants are compounds that checked oxidative stress by preventing free radical formation. The marked antioxidant potential of the Rhizomes and Aerial parts of the could be featured to the presences of various pharmacological active chemical groups like saponins, alkaloids, flavonoids, glycosides, phenols, terpenoids and tannins (Addae-Kyereme et al., 2001; Badami and Channabasavaraj, 2007; Moure et al., 2001). Moreover, the distinct antioxidant potential of both the Rhizomes and Aerial parts of the plant augment the antiinflammatory commotion.

Table 3.1.2.13: DPPH scavenging activity of Rhizomes.

Test material Extracts/Fractions IC50 ±SEM (µg/mL) Crude extract 115±2.88 Hexane 200±5.77

Chloroform 93±2.31 Ethyl acetate 90±3.46 n-Butanol 95±2.88 1,1-diphenyl-2-picrylhidrazyl Aqueous 145±5.77 (DPPH) Vitamin-C 24±1.73

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IC50 values are the mean ± S.E.M. of three assays.

Figure 3.1.2.8: 1,1-diphenyl-2-picrylhidrazyl (DPPH) antagonistic potential of Aerial parts. A-1 = Crude extract; A-2 = n-hexane; A-3= Chloroform; A-4 = Ethyl acetate; A-5 = n- butanol; A-6 = Aqueous. Standard drug = Vitamin C. Negative control = Ethanol. Symbols represent mean ± S.E.M. (n = 3).

Table 3.1.2.14: DPPH scavenging activity of Aerial parts

Test material Extracts/Fractions IC50 ±SEM (µg/mL) Crude extract 122±3.55 n-Hexane NA 1,1-diphenyl-2-picrylhidrazyl Chloroform 190±2.88

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(DPPH) Ethyl acetate 137±5.46 n-Butanol 167±2.88 Aqueous 194±4.04 Vitamin-C 24±1.73

IC50 values are the mean ± S.E.M. of three assays.

While considering the isolated seven different compounds from the Rhizomes of the plant, the antioxidant capacity of 5-hydroxymethyl-2-furaldehyde (4) is already available in literature (Kulkarni et al., 2008). Therefore, the antioxidant activity of Rhizomes can be characterized to the accumulation of this compound in extracts. The rest of the compounds may also have such tendency; need to be subject to such assay.

3.1.2.9. Enzyme inhibition assays

3.1.2.9.1. Effect of cholinesterase inhibition

The crude extract of Rhizomes and its subsequent fractions demonstrated insignificant inhibition against acetylcholinesterase. The crude form of the plant displayed 11% (Figure 3.1.2.9A). Upon fractionation, distinct attenuation of acetylcholinesterase was noted. However, none of the fraction possessed significant inhibition. The order of antagonism for various fractions was; n-hexane (19%), chloroform (13%) ethyl acetate (16%), n-butanol (16%) and aqueous (11%).

While testing the crude extract of the Aerial parts of the plant against acetylcholinesterase, similar trend of insignificance for obtained. Only 7% activity was noted for the crude extract. To analyze the overall potential of aerial parts, its subsequent fractionation was also scrutinized. The attenuation of acetylcholinesterase for different fractions of the aerial parts was in the order of: hexane (19%), ethyl acetate (14%), butanol (12%), chloroform (11%) and aqueous (7%).

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In case of butyrylcholinesterase inhibition test, the Rhizomes exhibited similar insignificant trend of activity (Figure 3.1.2.9B). Crude extract observed only 8% inhibition. The attenuation remained insignificant even upon fractionation and all the tested fractions exhibited poor activity. The order of percent inhibition was; n-butanol (16%), ethyl acetate (15%), n-hexane (15%), chloroform (13%) and aqueous (7%).

A similar test was also performed for the study of inhibitory profile of Aerial parts against butyrylcholinesterase. The test revealed low activity for the crude extract (7%). Fractionation of the Aerial parts showed different order of inhibition against butyrylcholinesterase. Ethyl acetate was most active with 17% inhibition, followed by hexane (14%), chloroform (12%), butanol (12%) and aqueous fraction with 7% inhibition.

Figure 3.1.2.9: Inhibitory effect (%) of the Rhizomes and Aerial parts against acetylcholinesterase [A] and butyrylcholinesterase [B]. Data are mean ± S.E.M. of three assays.

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Acetylcholinesterase (AChE) is well discerning for hydrolysis of acetylcholine (ACh), while butyrylcholinesterase (BuChE), a related enzyme that normally located in all regions that involved cholinergic innervations is competent to metabolize numerous different molecules including several neuroactive peptides (Lane et al., 2006). Alzheimer’s disease (AD) is exemplified by the deficiency of cholinergic neurons. Cholinesterase inhibition is therefore considered as a rational approach in the treatment of AD and other related clinical disorders like parkinson’s disease, ageing, myasthenia gravis and glaucoma (Khan et al., 2007; Porcel and Montalban, 2006; Soreq and Seidman, 2001).

Since the current therapeutic modalities available for the treatment of AD are few and suffering from multiple limitations. This intensifies the need for the discovery of new effective therapeutic agents to counteract their deficiencies and provide better options to clinicians. Unfortunately, both the Rhizomes and Aerial parts of plant were insignificant in its inhibitory activity against both cholinesterases.

3.1.2.9.2. Effect of lipoxygenase inhibition assay

The lipoxygenase inhibitory activity of Rhizomes and Aerial parts of the plant is depicted in table 3.1.2.15. It is manifested by the results that the crude extract of Rhizomes

possessed notable activity and IC50 value was 102 µg/mL. Profound change in activity of Rhizomes was recorded upon fractionation. All the fractions had reasonable activity and potency except n-hexane. The ethyl acetate was the most dominant fraction in terms of

potency and IC50 value was 69 µg/mL followed by n-butanol and chloroform fraction with

IC50 value of 76 µg/mL and 79 µg/mL respectively. Nevertheless, aqueous fraction

possessed significant antagonistic activity against lipoxygenase (IC50: 174 µg/mL).

In case of Aerial parts of the plant, crude extract showed significant lipoxygenase inhibition (IC50: 125 µg/mL). After sequential fractionation, the attenuation potential was greatly changed. Of the tested fractions, ethyl acetate was the most potent inhibitor of the

enzyme (IC50: 97 µg/mL) followed by aqueous fraction (IC50: 109 µg/mL). Additionally, the n-butanol and chloroform fractions displayed considerable potency (Table 3.1.2.15).

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However, only n-hexane fraction did exhibit any inhibition. The standard drug used in assay was Baicalein, showed outstanding potency 22.06 µg/mL.

Figure 3.1.2.10: Lipoxygenase inhibition (%) of Rhizomes and Aerial parts. Data are mean S.E.M. (n=3).

Arachidonic acid metabolism through LOX pathway generates various biologically active lipids that are primary mediator of inflammation including leukotrienes. Leukotrienes are characterized as potent mediators of inﬂammation and allergy (Khan et al., 2007; Rioux and Castonguay, 1998). Results of our study indicated marked lipoxygenase activity of both the extracts and fractions of Rhizomes and Aerial parts of the plant. As both Rhizomes and Aerial parts contained various pharmacologically active chemical groups of compounds, therefore this inhibitory activity could be featured to these

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groups. For further exploration of these activities and mechanistic studies, bioactivity guided isolation of secondary metabolite(s) could provide better understanding.

Anti-inflammatory potential of compounds purified from the Rhizomes of the plant is already investigated by researchers. These compounds include 2-hydroxybenzioc acid (3), diosgenin (5), ß-Sitosterol (6) and santonin (7). Therefore, the current anti- inflammatory activity of extracts can be attributed to these compounds. The study of these compounds in lipoxygenase pathway could also augment the overall anti-inflammatory response.

Table 3.1.2.15: In vitro quantitative Inhibition of lipoxygenase by crude extract and various fractions of plant

IC50 ±SEM (µg/mL) Extracts/Fractions Rhizomes Aerial parts Crude extract 102± 0.19 125± 0.33 n-Hexane NA NA Chloroform 79 ±0.44 122 ±0.27 Ethyl acetate 69± 0.09 97± 0.77 n-Butanol 76± 0.17 116± 0.69 Aqueous 174±0.32 109±0.09 Baicalein 22.6±0.09 22.6±0.09

IC50 values are the mean ± S.E.M. of three assays.

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3. 1.2.9.3. Effect of urease inhibition assay

The urease inhibition of Rhizomes as well as Aerial parts of the plant is presented in Figure

3.1.2.11. The crude extract of Rhizomes exhibited significant attenuation of enzyme (IC50: 209 µg/mL). When different fractionation were tested for the activity, n-butanol was the

most potent fraction (IC50:169 µg/mL) chased by the chloroform fraction (IC50: 197 µg/mL). However, the n-Hexane and Ethyl acetate fractions were inactive in urease inhibition assay.

Similar trend of inhibition was shown by the Aerial parts of the plant as illustrated in table 3.1.2.17. The crude extract demonstrated significant sensitivity (IC50: 192 µg/mL).

Figure 3.2.11: Urease inhibition of Rhizomes and Aerial parts. Data mean ± S.E.M. of three assays.

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Table 3.1.2.16: In vitro quantitative Inhibition of Urease

IC50 ±SEM (µg/mL) Extracts/Fractions Rhizomes Aerial parts Crude extract 209±0.54 192 ±0.09 n-Hexane NA NA Chloroform 197 ±0.48 NA Ethyl acetate NA 187± 0.77 n-Butanol 169± 0.34 166± 0.69 Aqueous 288±0.48 NA Thiourea 15.06±0.72µM 15.06±0.72µM

IC50 values are the mean ± S.E.M. of three assays.

Control = DMSO. Standard = Thiourea.

After fractionation only the n-butanol and ethyl acetate fractions were susceptible while the n-hexane, chloroform and aqueous fractions were not responsive.

Urease (urea amidohydrolase; E.C.3.5.1.5) is usually found in different bacteria, fungi, algae and plants. Infections induced by these bacteria such as Helicobacter pylori (H. pylori) and Proteus mirabilis usually have high urease activity. Urease is directly engaged in the configuration of infectious stones and the over expression of its activity participates in the pathogenesis of urolithiasis, pyelonephritis, ammonia and hepatic encephalopathy, hepatic coma, urinary catheter encrustation (Zhu-Ping et al., 2007). Urease potentiates the hydrolysis of urea to generate ammonia and carbon dioxide, and to guard the bacteria (Helicobacter pylori) in the acidic surroundings by enhancing the pH. In agriculture, urease over expression grounds for environmental and economic harms (Miroslawa, 2006). Consequently, focusing urease for treating pathogenic disorders of

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urease origin may unbolt a new line of healing for infections rooted by urease producing bacteria.

So for the isolated seven active principles from the Rhizomes of the plant are concern, they did not show any involvement in the urease inhibition. Based on the results of extracts activity, the isolated compounds may have urease antagonistic activity. Apart from this, the pharmacologically active detected groups in these extract are considered to be responsible.

3.1.3. Ex-in vivo (Isolated animal tissue) studies

The results of the extracts on the various isolated animal tissues are presented below.

3.1.3.1. Effect on the rabbit jejunum

The crude methanol extract of Rhizomes and Aerial parts of the plant were tested against isolated rabbit jejunum. The results manifested that the Rhizomes of plant evoked reversal of the spontaneous contractions of rabbit jejunum tissues. The response was in a dose dependent mode and complete attenuation was noted at the concentration of 10 mg/mL (Figure 3.1.3.1). In case of high potassium (80 mM)-induced contractions, the Rhizomes exhibited insignificant relaxation while prominent and complete inhibition was observed against low potassium (25 mM) at maximum test concentration (10 mg/mL) (Figure 3.1.3.2A). Additionally, pretreatment with Rhizomes, the inhibition of low potassium (25 mM) tempted contractions was prevented in the company of glibenclamide (3 µM) (Figure 3.1.3.2B).

Similar patron of activity was shown by the cromakalim pretreatment. Cromakalim exhibited blockade of spontaneous contractions of rabbit jejunum preparations at the dose of 1 mM. When tested against the contraction induced by low potassium (25 mM), it completely relaxes jejunum tissues, devoid of any profound effect on high potassium (80 mM) induced contraction. Additionally, cromakalim also grounded glibenclamide

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responsive relaxation of the contractions provoked by low K+ (25 mM) devoid of any effect on high K+ (80 mM)-induced contractions (Figure 3.1.3.3A). However, verapamil restrained both low potassium (25 mM) and high K+ (80 mM) elicited contractions at a comparable dose range (Figure 3.1.3.3B).

Since the pretreatment of tissues effectively antagonize jejunum spontaneous contractions, thus showing an antispasmodic action. Experimental findings reflect that medicinal plant induced spasmolytic properties mediated through CCB (Bashir et al., 2006; Ghayur and Gilani, 2005; Ghayur et al., 2007; Ghayur et al., 2006). To scrutinize whether the antispasmodic outcome of the Rhizomes is also tracked similar mechanism; the extract was checked against high potassium induced contractions. Though, its weak inhibitory response against high potassium, indicate that the antispasmodic effect is perhaps mediated through some other mechanism(s). For that reason, tested against low potassium elicited contractions, it induced absolute inhibition. The physiological mechanism of potassium channel openers is the attenuation of contractions induced by low K+, while Ca++ blockers equally prevent both low and high K+-induced contractions, and these experiments differentiate K+ channel activation from CCB mechanism (Gilani et al., 2006; Gopalakrishnan et al., 2004; Lawson, 1996).

From mechanistic view point, the K+ channel opening result was augmented, when the reversal of low K+-induced contractions was avoided in the company of glibenclamide, a blocker of ATP-dependent K+ channels (Gilani et al., 2005a; Gilani et al., 2008).

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Figure 3.1.3.1: Tracings showing antispasmodic effect of the crude methanol extract of Rhizomes and cromakalim on spontaneously contracting isolated rabbit jejunum preparations.

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Figure 3.1.3.2: Concentration-response curves showing effect of Rhizomes of Polygonatum verticillatum on spontaneous [A] and Low K+ (25 μM) induced contractions [B] in isolated rabbit jejunum preparations. Data are mean ± SEM of 4-6 different experimental findings.

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Figure 3.1.3.3: Concentration-response curves posing effect of cromakalim [A] and verapamil [B] on Low K+ (25 mM) and High K+ (80 mM) originated contractions in

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isolated rabbit jejunum tissues. Data are mean ± SEM of 4-6 different experimental findings.

Cromakalim, a prototypical KATP channel opener (Khan and Gilani, 2006; Lawson, 1996) initiated related refluxes to that of the Rhizomes extract, excluding that it fabricated no effect on high K+-provoked contractions, while verapamil, a Ca++ antagonist inhibited low and high K+-induced contractions at alike concentration. These results indicate that the spasmolytic effect of the Rhizomes is mediated primarily through ATP-dependent K+ channel activation that augmented by weak CCB mechanisms. Most of the GIT disorders necessitates emergency visit of hospital (Thomas, 1996). Gastrointestinal hypermotality like diarrhoea or involuntary movement like abdominal colic is frequently reported. The record of hospitalized patients in 22 leading teaching hospitals revealed that G.I.T disorders are very common in Pakistani community. During 2002-2003, 101105 (9.23%) patients admitted with various disease of digestive system (MOH, 2005b).

Bioactivity-directed isolation resulted into seven different compounds. From the isolated pure entities, ß-sitosterol only has shown spasmolytic activity and the proposed mechanism was the interference with calcium channel. The remaining these compounds never passed through such test. Preliminary phytochemical outcomes of the Rhizomes showed the accumulation of various chemical groups like saponins, alkaloids, phenol, tannins, terpines etc. The chemical constituents of extract may have profile similar to these groups that involved in the antispasmodic activity mediated via K+ channel activation. For more prominent exploration of mechanistic studies and clinical applications, subjection of isolated molecules to the isolated tissue experiments is highly recommended.

In case of Aerial parts of the plant, the results revealed inhibition of jejunum spontaneous contractions in concentration dependent manner. The absolute attenuation was eminent at 100 mg/mL as shown in Figure 3.1.3.5A. Regarding the results of test against potassium (80 mM) and low K+ (25 mM) originated contractions, the maximal contrite effect of high K+ (80 mM) induced contraction was prevented while insignificant reversal of low potassium (25 mM) induced contractions was observed (data not shown). However,

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verapamil showed marked inhibitory activity against both spontaneous contractions as well as K+ (80 mM) induced contraction (Figure 3.1.3.5B). Additionally the Aerial parts of the plant (01-03 mg/mL) caused rightward shift of the Ca++ CRCs accompanied by repression of the maximum contractile outcome (Figure 3.1.3.6A). This response of extract was similar to that produced by verapamil (0.03-0.1 µM) as shown in Figure 3.1.3.6B.

The effect of the Aerial parts of the plant on the spontaneous contractions as well as high potassium (80 mM) activated contractions in the isolated rabbit jejunum tissues was ++ similar to the attenuation of verapamil, a reference Ca channel blocker (CCBS) (Ghayur et al., 2006). However, verapamil was relatively selective in its antagonistic activity on the + K -provoked contractions, a distinctive feature of CCBS (Gilani et al., 2000; Gilani et al., 2005b). It has been proposed that the relaxation of high K+ (80 mM) induced contraction is not always be considered CCB (Ghayur and Gilani, 2005) rather provide a background for further confirmation. The involvement of Ca++ antagonist like activity was further confirmed when pretreatment of the tissue in Ca++ free but K+ rich medium with the extract of Aerial parts shifted the Ca++ CRCs to the right, alike to that produced by verapamil (standard drug).

Gastrointestinal motility is controlled by numerous physiological mediators which accomplish their contractile outcomes through an eventual increase in cytosolic Ca++ current (Burks, 1987; Ghayur and Gilani, 2005). Hence, agents that are responsible for non-specific inhibition, like Ca++ channel blockers, expected to be more efficiently hold back gut motility (Grasa et al., 2004) and thus, designated a very significant therapeutic class. Keeping in mind the results, it can be postulated that the antispasmodic activity of Aerial parts of the plant is mediated through blockage of Ca++ channels and a member of that beneficial modality.

Preliminary phytochemical testing of the Aerial parts of the plant reflects the presence of pharmacological active chemical groups like saponins, alkaloids, phenols and flavonoids etc. It is therefore, concluded that these chemical modalities may contain pharmacological active secondary metabolites that originated antispasmodic activity via

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Ca++ antagonism. In the light of modern scientific advancement, purification of active principle(s) is crucial for the better understanding of mechanism and clinical utility.

Figure 3.1.3.4: Tracings showing antispasmodic effect of the crude methanol extract of Aerial parts and verapamil of various concentrations on spontaneously contracting isolated rabbit jejunum preparations.

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Figure 3.1.3.5: Dose-dependent inhibition of Aerial parts [A] and Verapamil [B] on spontaneous and potassium (80 mM)-elicited contractions in isolated rabbit jejunum preparation. Data stand for mean ± SEM of 3-5 different experimental findings.

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Figure 3.1.3.6: Dose-response curves of Ca++ created in a calcium free while potassium rich medium in the absence and presence of increasing concentrations of Aerial parts [A]

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and verapamil [B] in the isolated rabbit jejunum preparation. Data symbolize mean ± SEM of 3-5 different experimental findings.

3.1.3.2. Effect on rabbit trachea

The result of the effect on the isolated rabbit tracheal preparations reflects that both Rhizomes and Aerial parts of the plant exhibited anti-hyperactivity. When the crude extract of Rhizomes of the plant was subjected against CCh (1 µM) and potassium (80 mM) forced contractions, prominent attenuation was observed. The effect was in a concentration dependent mode. The extract caused complete relaxation of the K+ (80 mM) activated contraction at a dose of 5 mg/mL. However, the CCh (1 µM) produced contraction was completely inhibited at the concentration of 10 mg/mL (Figure 3.1.3.7A). In case of standard drug, verapamil, similar trend of attenuation was noted. Verapamil evoked relaxation of potassium (80 mM) dependent contraction at a concentration of 3 µM while the CCh (1 µM) forced contraction at a dose of 5 µM (Figure 3.1.3.7B). The extract of Rhizomes possessed marked reduction in the calcium concentration response curves at a dose of 0.03 and 03 mg/mL when constructed in calcium free while K+ rich medium (Figure 3.1.3.8A). Similar right downward shift was observed for the standard verapamil when tested at a dose of 0.01 and 0.1 μM (Figure 3.1.3.8B).

Regarding the effect of the Aerial parts of the plant on the rabbit trachea, prominent prevention of hyperactivity was found. When tested against the contraction induced by K+ (80 mM), the extract offered complete relaxation at a dose of 5 mg/mL in a dose dependent manner. While the CCh (1 µM) induced contraction was inhibited at a concentration of 10 mg/mL (Figure 3.1.3.9A). Standard drug, verapamil followed the same inhibitory principle and blocked the potassium (80 mM) and CCh (1 µM) dependent contractions at a dose of 3 and 5 µM respectively (Figure 3.1.3.9B). When Ca++ dose response curves were created, the extract of Aerial parts showed marked inhibition at the test doses (0.03 and 03 mg/mL) (Figure 3.1.3.10A). The standard compound, verapamil was dominant in its inhibitory

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profile and caused considerable attenuation at the concentration of 0.01 and 0.1 µM (Figure 3.1.3.10B).

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Figure 3.1.3.7: Dose-dependent inhibition of Rhizomes [A] and Verapamil [B] on spontaneous and potassium (80 mM) dependent contractions in isolated rabbit trachea preparation. Data symbolize mean ± SEM of 3-5 different experimental findings.

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Figure 3.1.3.8: Dose-response curves of Ca++ created in a calcium free while potassium rich medium in the absence and presence of increasing concentrations of Rhizomes [A] and verapamil [B] in the isolated rabbit jejunum preparation. Data stand for mean ± SEM of 3-5 different experimental findings.

Figure 3.1.3.9: Dose-dependent inhibition of Aerial parts [A] and Verapamil [B] on spontaneous and potassium (80 mM) forced contractions in isolated rabbit trachea preparation. Data symbolize mean ± SEM of 3-5 different experimental findings.

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Figure 3.1.3.10: Dose-response curves of Ca++ build in a calcium free while potassium rich medium in the absence and presence of increasing concentrations of Aerial parts [A] and verapamil [B] in the isolated rabbit jejunum preparation. Data stand for mean ± SEM of 3-5 different experimental findings.

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Based on the medicinal use of the Polygonatum in the treatment of respiratory disorders, the extracts of both Rhizomes and Aerial parts of Polygonatum verticillatum was tested against isolated trachea preparations. Smooth muscles are the main structural and functional components of viscera. Homeostasis of the physiological system is maintained by the changes in the contraction-relaxation cycle of the smooth muscles (Kuriyama et al., 1998). The hyperactivity of respiratory smooth muscle results in airway constriction, leading to asthma. Ca++ current is characterized as the primary channel for the generation of action potential and contraction of smooth muscles and thus provoked hyperactivity. Ca++ channel blockers are therefore, considered as sensible therapeutic strategy for the reversal of Ca++ induced hyperactivity (Ahmed, 1992; Gilani et al., 2005c).

High K+ (> 30 mM) and carbachol induces contraction of the smooth muscle cells via unlocking of the voltage-dependent calcium channels (L- type Ca++ Channel) and activation of muscarinic receptors respectively thereby increases intracellular Ca++ levels (Khan et al., 2010a). As a result, the invasion of extracellular Ca++ current elicits profound contractile response (Bolton, 1979) and tracheal contraction produces bronchoconstriction. Compounds with their ability to neutralize that induced contraction are named as Ca++ antagonists. The Rhizomes of the plant demonstrated marked inhibition of the maximal contractile response produced by the pretreatment of tissues with K+ (80 mM) and CCh- induced contractions frequently used for the study of Ca++ channel blocker activity in natural substances (Ghayur et al., 2006; Khan and Gilani, 2009). The response was similar to verapamil, a standard Ca++ channel antagonist in dose dependent manner. The involvement of Ca++ antagonist type of activity was further augmented by the marked inhibition of the extract in the Ca++ dose response curves that was constricted in Ca++ free, K+ rich medium. Therefore, it can be assumed that the Rhizomes possess active constituent(s) with prominent CCBs like profile.

Similar type of inhibition of hyperactivity was observed in the Aerial parts of the plant. The extract offered attenuation of K+ (80 mM) and CCh-induced contractions like verapamil, indicating the presence of CCBs components. As K+ induced contraction of

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smooth muscle is considered to be activated by means of Ca++ invasion from extracellular fluid and drugs that inhibits this contraction, are characterized as Ca++ channels antagonist (Ghayur et al., 2007; Ghayur et al., 2006; Khan and Gilani, 2009). From mechanistic point of view in order to confirm the mechanism behind the inhibition of such induced contractions, the extract of Aerial parts was tested in Ca++ dose response curves. The results proved the right ward shift of the curves accompanied by prominent suppression similar to verapamil, a standard Ca++ channel blocker. Therefore, it is event from the results that the extract possesses constituent(s) with CCBs antagonist activity.

Over the years, the prevalence of respiratory disorders increased considerably throughout the world and causing enormous casualties. This multiage problem now effecting 5-10% adults and 10% children each year globally (Lowhagon, 1999; Malhotra, 2000). In Pakistan, 74919 (6.84%) patients were hospitalized in 22 major teaching hospitals with different respiratory disorders during 2002-2003 (MOH, 2005b). The potential therapeutic regimes used in the treatment of respiratory disorders are facing various limitation and short of patient compliance.

During phytochemical investigation of the Rhizomes of the plant, seven different compounds have been elucidated by physical and spectral applications. Only ß-sitosterol showed calcium antagonistic activities. Phytochemical tests of both Rhizomes and Aerial parts of the plant disclosed the presence of saponins, alkaloids, phenols, flavonoids, tannins etc. These different chemical classes of compounds are pharmacological active and the current anti-hyperactivity of both extract can be attributed to these groups. Comprehensive studies of the isolated molecules may be more useful in the elucidation of extract mechanism and lead compounds for clinical use with better patient’s compliance.

3.1.3.3. Effect on rabbit aorta (vascular activity)

The extracts of Rhizomes and Aerial parts were unable to produce any considerable effect on the isolated rabbit aorta (data not mentioned).

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3.1.3.4. Effect on guinea-pig atria

The extract of Rhizomes and Aerial parts of the plant when tested against guinea-pig atria, the Rhizomes of the plant showed mild inhibition of atrial force of contraction (data not shown) while the Aerial parts of the plant was devoid of any effect.

3.2. Phytochemical Studies

3.2.1. Quantitative analysis

3.2.1.1. Phenol contents

The results of total phenol contents revealed that both the Rhizomes and Aerial parts of the plant possess reasonable quantity. As comparison to Aerial parts, the Rhizomes showed prominent dominancy in total phenol contents. The highest concentration for Rhizomes was found in the n-butanol fraction (53 mg/g) followed by ethyl acetate fraction (46 mg/g) (Figure 3.2.1A). In case of Aerial parts, maximum contents were accumulated in the n- butanol (64 mg/10 g) (Figure 3.2.2A).

3.2.1.2. Alkaloid contents

Similarly, in case of alkaloid contents, both the Rhizomes and Aerial parts of the plant contained reasonable quantity. Maximum alkaloid concentration for Rhizomes was accumulated in the chloroform fraction (9 mg/g) followed by ethyl acetate (8 mg/g) (Figure 3.2.1B). For Aerial parts of the plant, chloroform fraction (87 mg/10 g) accumulated highest contents (Figure 3.2.2B).

3.2.1.3. Saponin contents

Regarding the total saponin content, Rhizomes of plant was found very rich. The Aerial parts also possess measurable quantity of saponin but Rhizomes was dominant in comparison (Figure 3.2.1C). The maximum saponin in Rhizomes was exhibited by the

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ethyl acetate and n-butanol fractions 67 mg/g and 66 mg/g respectively. For Aerial parts, ethyl acetate (49 mg/g) was dominant fraction (Figure 3.2.2C).

3.2.1.4. Flavonoid contents

Flavonoid contents were also observed substantially in both Aerial parts and Rhizomes of the plant (Figure 3.2.1D). In case of Aerial parts, the highest contents were found in ethyl acetate fraction (54 mg/g) (Figure 3.2.2D). Similarly, the maximum contents for Rhizomes carried by ethyl acetate (39 mg/g) and n-butanol (39 mg/g) fractions.

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Figure 3.2.1. Quantitative estimation of various phytochemical contents in Rhizomes. Data are expressed as mean ± SEM of three different findings. R-1= Crude extract, R-2= n- hexane, R-3= Chloroform, R-4= Ethyl acetate, R-5= n-butanol, R-6= Aqueous.

3.2.2. Qualitative analysis

The results of both the Rhizomes and Aerial parts of the plant are depicted in table 3.2.1.

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Figure 3.2.2. Quantitative estimation of various phytochemical contents in Aerial parts. Data are expressed as mean ± SEM of three different findings. A-1= Crude extract, A-2= n- hexane, A-3= Chloroform, A-4= Ethyl acetate, A-5= n-butanol, A-6=Aqueous.

Table 3.2.1: Preliminary phytochemical (qualitative) tests S. No Tests Rhizomes Aerial parts 1 Glycosides + + 2 Steroids + + 3 Anthraquinones + ─ 4 Amino acids + + 5 Terpenoids + ─ 6 Tannins + +

3.2.3. Isolation of pure secondary metabolites

Bioactivity-directed isolation resulted into the isolation of seven different compounds including one new compound. Though the remaining compounds are already reported in

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literature but the source is absolutely new. These were including Propyl pentadecanoate (1), 2`,3`-Dihydroxy propyl pentadecanoate (2), 2-Hydroxy benzoic acid (3), 2- Hydroxymethyl-2-furaldehyde (4), Diosgenin (5), ß-Sitosterol (6) and Santonin (7).

3.2.3.1. Structure elucidation of Propyl pentadecanoate (1)

Compound (1) was isolated as colorless amorphous compound over silica in column chromatography. The compound was UV active and soluble in methanol at room temperature. Compound (1) exhibited molecular peak ion at m/z 284.233 in HREI mass spectroscopy corresponding to molecular formula as C18H36O2. The molecular weight for

C18H36O2 was calculated as 284.477.

1H-NMR data of the compound (1) showed a triplet resonating at δ 4.03 t (J = 6.7 Hz) designated to methylene proton of (C-1`) adjacent to O−C=O group. A doublet of triplet was appeared at δ 2.02 dt (J = 8.0 Hz, 7.2 Hz) due to the presence of methylene (C- 2`) proton and similarly a triplet was resonating at δ 0.87 t (J = 7.3 Hz) attributed to methyl proton of (C-3`) and thus together constitute a propyl moiety. A triplet appeared at δ 2.31 t (J = 6.9 Hz) assigned to methylene (C-2) proton and a doublet of triplet resonating at δ 1.67 dt (J = 6.6 Hz) due to methine (C-3) proton adjacent to ester carbon. Presence of aliphatic chain was confirmed by the appearance of multiplet at δ 1.32 due to methine (C-14) proton followed by a triplet at δ 0.90 t (J = 7.1 Hz) due to methyl (C-15) proton.

13C-NMR spectrum of compound (1) explored all the eighteen carbon atoms in compound (1). DEPT-BB experiment of compound (1) displayed presence of two methyl, fifteen methylene and one quaternary carbon atom. The signals noted at δ 69.16 (C-1`), 40.19 (C-2`) and 24.97 (C-3`) suggesting propyl group. The resonance at δ 169.41 due to quaternary (C-1) was assigned to an ester carbonyl. The signal for methyl group of aliphatic chain was appeared at δ 11.43 (C-15) while for methylene group at δ 34.84 (C-2), 26.03 (C-3), 30.40 (C-4―C-13) and 23.73 (C-14). Finally HMBC correlations confirmed H-C connectivities (Figure 3.2.4) in compound (1). While considering all the above assignments compound (1) is elucidated as propyl pentadecanoate.

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Table 3.2.2 : NMR spectral estimations of Propyl pentadecanoate (1) Position 13C [δ] 1H [δ] Multiplicity

1 169.41 ……………… C

2 34.84 2.31 t (J = 6.9 Hz) CH2

3 26.03 1.67 dt (J = 6.6 Hz) CH2

4 30.40 …….. CH2

5 30.40 …….. CH2

6 30.40 …….. CH2

7 30.40 …….. CH2

8 30.40 …….. CH2

9 30.40 …….. CH2

10 30.40 …….. CH2

11 30.40 …….. CH2

12 30.40 …….. CH2

13 30.40 …….. CH2

14 23.73 1.32 m CH2

15 11.43 0.90 t (J = 7.1 Hz) CH3

1’ 69.16 4.03 t (J = 6.7 Hz) CH2

2’ 40.19 2.02 dt (J = 8.0 Hz, 7.2 Hz) CH2

3’ 24.97 0.87 t (J = 7.3 Hz) CH3

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Figure 3.2.3: Structure of compound (1).

Figure 3.2.4: Some typical correlations observed in HMBC spectrum of compound (1).

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3.2.3.2. Structural elucidation of 2`,3`-Dihydroxy propyl 3.2.3.3. pentadecanoate (2)

Compound (2) was isolated as white amorphous compound over silica in column chromatography. Compound (2) was UV active in nature and soluble in methanol at room

temperature. HR-EI mass data exhibited molecular formula C18H36O4 by showing molecular ion peak molecular ion at m/z 316.265 while molecular weight for formula

C18H36O4, was calculated as 316.476.

1H-NMR spectrum of compound (2) showed a triplet resonating at δ 0.89 t (J = 7.0 Hz) due to methyl (C-15) proton and a multiplet appeared at δ 1.30 due to methylene (C- 14) proton assigned to aliphatic chain. A triplet was noted at δ 2.30 t (J = 6.7 Hz) due to methylene (C-2) proton and a doublet of triplet appeared at δ 1.67 dt (J = 6.6 Hz) due to methylene (C-3) proton adjacent to ester moiety. A doublet of doublet was observed at δ 4.03 dd (J = 6.0, 11.7 Hz) for methylene (C-1`) proton suggested the presence of hydroxyl carbon adjacent to ester. Resonance of a multiplet at δ 4.0 and a doublet of doublet at δ 3.89 dd (J = 11.2, 7.0 Hz) confirmed the presence of an oxygenated methine (C-2`) and an oxygenated methylene (C-3`) respectively.

13C-NMR analysis of compound (2) displayed signals for all the eighteen carbon atoms in the compound. The DEPT-BB spectrum described the presence of one methyl, fifteen methylene, one methane and one quaternary carbon atoms

Signals for oxygenated carbons were noted at δ 65.16, 70.25 and 62.73 for methylene (C-1`), methine (C-2`) and methylene (C-3`) respectively. The signal for an ester carbonyl was appeared at δ 170.04 due to a quaternary (C-1). Signals for methylene (C-2) and (C-3) were displayed at δ 34.45 and 26.03 respectively, in continuation the presence of a long aliphatic chain was established by the resonance of methylene (C- 4―C13) at δ 30.56 that ended methyl (C-15) at δ 12.53. Based on all the above mentioned assignments and literature data (Sabudak et al., 2007) compound (2) is declared as 2`,3`- Dihydroxy propyl pentadecanoate.

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Table 3.2.3: NMR spectral estimations of 2`,3`- Dihydroxy propyl pentadecanoate (2) Position 13C [δ] 1H [δ] Multiplicity

1 170.04 ……………… C

2 34.45 2.30 t (J = 6.7 Hz) CH2

3 26.03 1.67 dt (J = 6.6 Hz) CH2

4 30.56 …….. CH2

5 30.56 …….. CH2

6 30.56 …….. CH2

7 30.56 …….. CH2

8 30.56 …….. CH2

9 30.56 …….. CH2

10 30.56 …….. CH2

11 30.56 …….. CH2

12 30.56 …….. CH2

13 30.56 …….. CH2

14 21.73 1.30 m CH2

15 12.53 0.89 t (J = 7.0 Hz) CH3

1’ 65.16 4.03 dd (J = 6.0, 11.7 Hz) CH2 2’ 70.25 4.0 m CH

3’ 62.73 3.89 dd (J = 11.2, 7.0 Hz) CH2

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Figure 3.2.5: Structure of compound (2)

Figure 3.2.6: Some typical correlations observed in HMBC spectrum of compound (2)

3.2.3.3. Structural elucidation of 2-Hydroxy benzoic acid (3)

Compound (3) (2-Hydroxybenzoic acid; Salicylic acid) was isolated as colorless crystals. On TLC, the compound was UV active. It manifested a peak molecular ion at m/z 138 in its

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HREI mass spectroscopy. The molecular formula was computed as C7H6O3 corresponding to the molecular weight as 138.12.

Salicylic acid is a simple compound, in which benzene is attached to a hydroxyl and a carboxyl group. 1H-NMR analysis provides substantial information about the compound. A broad doublet was resonating at δ 6.89 broad d (J = 8.4 Hz) assigned to C-3 methine proton. A signal doublet of triplets appeared at δ 7.42 dt (J = 1.8, 8.4 Hz) due to C-4 methine proton. A triplet was noted at δ 6.85 t (J = 7.8 Hz) designated to C-5 methine proton. A signal of double of doublets at δ 7.83 dd (J = 1.8, 8.4) was characterized for C-6 methine proton confirming a six member ring with alternate double bounded protons.

13C-NMR spectrum showed signals for all the seven carbon atoms in the compound. The DEPT-BB spectrum described the presence of four methine carbons, three quaternary carbons, methine signals were found at δ 118.1 (C-3), 136.6 (C-4), 120.0 (C-5) and 131.5 (C-6) were diagnostic of benzene moiety. The characteristic downfield signal at δ 173.5 (C- 1`) suggested the existence of a carboxylic group in the compound while a signal at δ 163.2 (C-2) confirmed the presence of a hydroxyl group. The described spectroscopic data and various physical parameters, while comparing with the available literature (Jadrijevi and Taka, 2004) suggesting the compound (3) to be 2-hydroxybenzoic acid.

Table 3.3.4: NMR spectral estimations of 2-hydroxybenzoic acid (3) Position 13C [δ] 1H [δ] Multiplicity 1 113.9 …….. C 2 163.2 …….. C 3 118.1 6.89 brd d (J = 8.4 Hz) CH 4 136.6 7.42 dt (J = 1.8, 8.4 Hz) CH 5 120.0 6.85 t (J = 7.8 Hz) CH 6 131.5 7.83 dd (J = 1.8, 8.4) CH 1`(C=O) 173.5 ………. C

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7.83 dd (J = 1.8, 8.4 Hz) H O

H 131.5 1' 173.5 6 6.85 t (J =7.8Hz) 120.0 5 1 113.9 OH

2 136.6 4 163.2 3 H 118.1 OH 7.42 dt (J = 1.8, 8.4 Hz)

H 6.89brdd(J =8.4Hz)

Figure: 3.2.6: Structure of compound (3)

3.2.3.4. Structural elucidation of 5-Hydroxymethyl-2-furanaldehyde (4)

Compound (4) was isolated as dark yellowish oil using silica gel through column chromatography. The compound was UV active in nature as detected on TLC and was found to be soluble in methanol at room temperature. HREI mass spectrum illustrated

molecular ion peak at m/z 126. 021 matching to the molecular formula of C6H6O3. The molecular weight for C6H6O3 was calculated as 126.110.

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1H-NMR spectrum provides valuable information for the structural elucidation of compound (4). A doublet was resonating at δ 7.19 d (J = 3.6 Hz) assigned for C-3 methine proton while, due to methine C-4 proton, a doublet was also recorded at δ 6.50 d (J = 3.6 Hz) proposing a five member ring. A singlet was observed at δ 9.57 (1H) due to methine C- 2`proton suggested a carbonyl group and thus confirmed the presence of 2-furanaldehyde moiety. Similarly, an oxygenated methylene proton was appeared as a singlet at δ 4.70 (2H) designated to C-1`.

13C-NMR spectrum demonstrated compacted structural information for all the six carbon atoms that resonating in the molecule. The DEPT-BB spectrum described the presence of one methylene, three methine and two quaternary carbon atoms

The spectrum showed signals at δ 152.4, 122.7, 110 and 160 resonating due to C-2, C-3, C-4 and C-5 respectively for furan and the signal at δ 177.64 due to C-2` confirmed the 2-furanaldehyde functionality. Resonance appeared at δ 57.7 was assigned to oxygenated methylene C-1`. Considering the physical and spectral results in hand and comparison with the data available in literature (Espinoza et al., 2008), the compound (4) is 5-hydroxymethyl-2-furanaldehyde.

Table 3.2.5 : NMR estimations of 5-hydroxymethyl-2-furancarboxyldehyde (4) Position 13C [δ] 1H [δ] Multiplicity 1 ………… ………… …………. 2 152.40 ………… C 3 122.70 7.19 d (J = 3.6 Hz) CH 4 110.0 6.50 d (J = 3.6 Hz) CH 5 160.0 ……….. C

1` 57.7 4.70 s (2H) CH2

2` (C=O) 177.64 9.57 s (1H) CH

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Figure 3.2.7: Structure of compound (4)

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H H

O 5 2 OH 2 1 1 O H H H

Figure 3.2.8: Some typical correlations observed in HMBC spectrum of compound (4)

3.2.4.5. Structural elucidation of Diosgenin (5)

Compound (5) was isolated as colorless powder. The compound offered solubility in methanol at room temperature. Based on TLC profile, the compound was UV inactive.

Molecular formula C27H42O3 was identified via HREI mass spectrum as the molecular ion peak was appeared at m/z 414.214. The molecular weight for C27H42O3 calculated as 414.619.

1H-NMR spectral analysis of compound (5) revealed its steroidal nature. Two singlet resonating at  0.78 and 1.03 due to the C-18 and C-19 quaternary methyl protons respectively inductive of two primary methyl groups. Two doublets were resonating at  0.78 (J = 6.2 Hz) and 0.79 (J = 7.1 Hz) due to C-21 and C-27 secondary methyl protons respectively for two secondary methyl groups. The presence of a double bond is attributed to the resonance observed as broad doublet at  5.33 (J = 4.0) due to C-6 methine proton. It

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can be observed that protons at C-26 are attached with an oxygen atom, and therefore, produced quite distinguished resonance due to the de-shielded effect. As a result, they are clearly removed from the methylene envelope. Moreover, splitting resonance has been observed due to C-26α, proton in the form of a triplet at δ 3.38 t (J = 10.50 Hz) and a double "double-doublet" at δ 3.47 ddd (J = 10.40 Hz) for C-26β proton.

The 13C-NMR spectrum of compound (5) revealed diagnostic signal for all 27 carbon atoms. The multiplicity in the molecule was determined by DEPT experiment and disclosed the occurrence of four methyl, ten methylene, nine methine, and four quaternary carbon atoms.

Resonances for methyl groups were appeared at δ17.5 (C-27), 15.43 (C-21), 20.04 (C-19) and 17.10 (C-18) suggested four methyl groups in the compound. The signal of the C-27 at δ 17.5 is the inductive of R-orientation. The resonance of chemical shift at δ 71.8 assigned to C-3 is the diagnostic of the presence of hydroxyl group. Keeping in view the spectral assignments of the compound in comparison with the data available in literature (Benjamin et al., 1984; Deng-Jyec et al., 2003), it is suggested to be (25 R)-spirost-5-ene- 3β-ol diosgenin.

Table 3.2.6: NMR spectral estimations of Diosgenin (5)

Position I3C [δ] 1H [δ] Multiplicity

1 37.9 …… CH2

2 33.1 …… CH2 3 71.8 …… CH

4 43.3 …… CH2 5 141.6 …… C 6 121.3 5.33 brd d (J = 5.40 Hz) CH

7 32.5 …… CH2 8 32.1 …… CH

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9 50.1 …… CH 10 37.6 …… C

11 21.3 …… CH2

12 40.3 …… CH2 13 40.7 …… C 14 57.3 …… CH

15 31.8 …… CH2 16 79.4 4.33 q (J =7. l0 Hz) CH 17 62.8 ……. CH

18 17.10 0.78 (s) CH3

19 20.04 1.03 (s) CH3 20 42.88 ……. CH

21 15.43 0.78 (J = 7.10 Hz) CH3 22 109.4 …… C

23 33.2 …… CH2

24 29.6 …… CH2 25 31.9 …… CH

26α 67.44 3.38 t (J = 10.50 Hz) CH2 26β …… 3.47 ddd ( J = 10.40 Hz)

27 17.5 0.78 d (J = 6.0 Hz) CH3

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Figure 3.2.9: Structure of compound (5).

Figure 3.2.10: Some typical correlations observed in HMBC spectrum of compound (5)

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3.2.4.5. Structural elucidation of β-Sitosterol (6)

β-Sitosterol (6) was obtained as colorless powder. The compound showed solubility in methanol while UV inactive on TLC. Molecular formula of the compound C29H50O was assigned by HR mass data by showing a molecular peak ion at m/z 414.3845 (calculated for

C29H50O as 414.3855).

1H-NMR estimation of the compound (6) revealed its steroidal nature with six methyl groups. Out of six, two quaternary protons showed resonances at δ 0.67 and 0.99 for C-18 and C-19 respectively, inductive of methyl moiety. A characteristic resonances of three doublets of secondary methyl protons was recorded at δ 0.79 d (J = 6.8 Hz) for (C- 27), 0.83 d (J = 6.8 Hz) for (C-26) and 0.91 d (J = 6.3 Hz) for (C-21). However, a triplet of primary C-29 methyl proton appeared at δ 0.82 t (J = 7.0 Hz). The 3β-hydroxy position in the compound was confirmed by the multiplet resonating at δ 3.65 (1H, m). Presence of unsaturated double bond in the molecule was established by the resonance of a multiplet at δ 5.35 (1H, m) at position C-6.

In 13C-NMR spectrum, signals for all 29 carbons atoms were recorded. Six methyl groups in the molecule were further confirmed by the resonances at δ 12.1 (C-18), 18.7 (C- 19), 21.5 (C-21), 18.9 (C-26), 21.5 (C-27) and 12.6 (C-29). Finally the structure of compound (6) was elucidated as β-Sitosterol, as reported in literature (Moghaddam et al., 2007).

Table 3.2. 7: NMR spectral estimations of β-Sitosterol (6) Position 13C [δ] 1H [δ] 1 37.1 ……… 2 31.6 ……… 3 72.1 3.65 (m) 4 42.4 ……… 5 139.5 ………

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6 120.0 5.35 (m) 7 32.5 ……… 8 35.3 ……… 9 49.8 ……… 10 36.2 ……… 11 22.1 ……… 12 40.3 ……… 13 43.1 ……… 14 56.9 ……… 15 24.7 ……… 16 27.9 ……… 17 54.7 ………

18 (CH3) 12.1 0.67 (s)

19 (CH3) 18.7 0.99 (s) 20 40.1 ………

21 (CH3) 21.5 0.91 d (J = 6.3 Hz) 22 33.8 ……… 23 28.8 ……… 24 50.5 ……… 25 26.7

26 (CH3) 18.9 0.83 d ( J = 6.8 Hz)

27 (CH3) 21.5 0.79 d (J = 6.8 Hz) 28 23.1 ………

29 (CH3) 12.6 0.82 t (J = 7.0 Hz)

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0.82 t (J = 7.0 Hz)

CH3 29 28

0.91 d (J = 6.3 Hz) 21 22 0.83 d (J = 6.8 Hz) H3C CH3 24 26 0.67 (s) 18 20 23 25 CH3 17 CH3 12 27 0.99 (s) 19 11 13 16 0.79 d (J = 6.8 Hz) CH3 14 15 1 9 2 10 8

3.65 (m) 3 5 7 4 6 HO 5.35 (m)

Figure 3.2.11. Structure of compound (6)

3.2.4.6. Structural elucidation of Santonin (7)

Compound (7) was isolated light yellowish powder. The compound was found soluble in chloroform at room temperature. On TLC, it was UV active in nature. Compound 7 showed a molecular ion peak at m/z 246.145. Molecular formula of the compound 7 was

determined as C15H18O3, based on HEEI-mass data.

1H-NMR spectra of compound (7) showed a doublet resonating at δ 6.64 (d, J = 10.0) due to olefinic C-1 methine proton and a doublet appeared at δ 6.18 (d, J = 10.0) due to C-2 methine proton, that resulted in formation of cross peaks. A doublet was resonating at δ 4.74 (d, J = 11.0) corresponding for C-6 methine proton, which exhibited proton- proton spin correlation with C-7 methine proton singling at δ 1.21. The C-7 proton showed cross peaks with the proton of C-8 at δ 1.96 brd (12.5 Hz) and C-11, the latter also had vicinal coupling methyl proton of C-13 at δ 2.37 dq (J = 6.5, 13.5 Hz).

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13C-NMR analysis of compound (7) confirmed signals for all 15 carbon atoms. The multiplicity in compound based on DEPT-BB showed presence of three methyl, two methylene, one methine and five quaternary carbon atoms. C-6 was resonating at δ 80.30, geminal to the lactone moiety. The two olefinic resonances for double bound appeared at δ 154.90 and 125.75 due to C-1 and C-2 carbon atoms, respectively. The oxygen functionality in the compound was confirmed by signals recorded at δ 186.24 and 177.54 due to C-3 and C-12 respectively (Ata and Nachtigall, 2004). Based on various spectroscopic assignments, the structure of compound (7) was assigned as (3S,3aS,5aS,9bS)-3,5a,9-trimethyl-3a,4,5,5a-tetrahydronaphtho[1,2-b]furan-2,8(3H,9bH)- dion (santonin).

Table 3.2.8: NMR spectral estimations of Santonin (7)

Position 13C [δ] 1H [δ] Multiplicity 1 154.90 6.64 d (J = 10.0) CH 2 125.75 6.18 d (J = 10.0) CH 3 186.24 ……… C 4 128.57 ……… C 5 150.98 ……… C 6 81.30 4.74 d (J = 11.0) CH

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7 53.44 1.76 dddd (3,12,12,12) CH

1.21 d (6.5)

8 22.96 1.96 brd (12.5 Hz) CH2

1.65 dddd (3.5, 12.5, 12.5, 12.5)

9 37.75 1.45 ddd (4.0, 13, 13.5) CH2

1.85 brd (J = 13.5) 10 41.29 ……… C 11 40.9 2.37 dq (J = 6.5, 13.5 Hz) CH 12 177.54 ……… C

13 12.41 ……… CH3

14 24.40 ……… CH3

15 10.82 ……… CH3

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Figure 3.2.12: Structure of compound (7)

H H H CH3 14 H H 1 9 10 H 2 8 H 3 5 7 CH3 4 6 13 O 11 H H 12 CH3 O 15

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Figure 3.2.13: Some typical correlations observed in HMBC spectrum of compound (7)

3.2.4. Identification of constituents by gas chromatography-mass spectrometry (GC-MS)

The results of the composition of fixed oils of both Rhizomes and Aerial parts of the plant are illustrated in Table 3.2.9 and 3.3.10 respectively. It is evident from the results that oily composition was primarily based on terpenes and terpenoids with one fatty acid in each hexane fraction. For the Rhizomes, the order of the composition (%) of identified compounds was Carvacrole (3.9905%), Melissic acid (2.8264%), Nonadecane (2.4733%), Linalool (2.0405%), Globulol (1.9711%), β-Humulene (1.8247%), β-Terpinolene (1.4505%) and Spathulenol (1.2735%). In case of Aerial parts, composition was α- Bulnesene (1.5648%), Linalyl acetate (0.4535%), Eicosadienoic (0.3702%), Pentacosane (0.3319%), Piperitone (0.3091%), Docasane (0.1720%), and Calarene (0.1321%).

It is worth mentioning that only a little part of the total composition of the oily fraction was characterized in both cases. The literature study revealed that considerable variations have been found in the composition of fixed oils of plants collected from different regions and of different parts (Marzouki et al., 2008). In our findings, different compounds of terpenes and terpenoids groups with one fatty acid in each fraction were elucidated.

Of the different in vitro tested activities of the hexane fraction of the plant, prominent antimalarial activity against the chloroquine resistant Plasmodium falciparum was observed. The antimalarial activities of hexane fraction of the medicinal plant are already cited in literature (Saiin et al., 2003) but the pure compounds that contribute to the activity still need to be determined. Nevertheless, the antimalarial activity of sesquiterpene (Bischoff et al., 2004) and sesquiterpenoids (Takaya et al., 1998) is available in literature. It is therefore assumed that the antimalarial activity of the hexane fraction of both Rhizomes and Aerial parts of the plant could be attributed to these groups.

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Oily fractions of both Rhizomes and Aerial parts of the plant demonstrated attractive susceptibility against variety of bacterial tested in the assay. The antibacterial activity of fixed oils is reported from different research laboratories (Ashaala et al., 2010; Dung et al., 2008). Our research investigations of the hexane fractions as antibacterial could be attributed to the components of fixed oils.

Table 3.2.9: Qualitative and quantitative composition of n-hexane fraction of Rhizomes of Polygonatum verticillatum Peak No. Compound Retention Time Molecular weight Concentration (min)

1 Linalool 23.327 154.2 [C10H18O] 2.0405 2 N/D 26.948 204.2 2.4601

3 Melissic acid 30.610 252.5 [C30H60O2] 2.8264 4 N/D 32.662 204.3 1.5516

5 Spathulenol 34.020 220.2 [C15H24O] 1.2735

6 β-Terpinolene 37.119 136.2 [C10H16] 1.4505 7 N/D 40.344 282.2 2.6320

8 β-Humulene 43.315 204.2 [C15H24] 1.8247

9 Globulol 44.463 222.4 [C15H26O] 1.9711 10 N/D 47.608 204.4 4.8466

11 Nonadecane 49.030 268.5 [C19H40] 2.4733 12 N/A 52.613 204.2 17.2317

13 Carvacrole 53.878 150.2 [C10H14O] 3.9905 14 N/D 56.812 N/D 17.7966 15 N/D 60.329 N/D 8.3554 16 N/D 63.831 N/D 2.8016 17 N/D 69.113 N/D 18.4418 18 N/D 73.942 N/D 1.4932 19 N/D 75.230 N/D 4.5391

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N/D = Not determined. Data bases used for the elucidation of constituents was performed were: GC-MS Library of Shimadzu Class-5000, ver 2.0 (1996). NIST Mass Spectral Search Program for the NIST/EPA/ss Spectral Library, ver. 16d (06/24/1998). Gaithersburg, MD, USA.

Table 3.1.10: Qualitative and quantitative composition of n-hexane fraction of Aerial parts of Polygonatum verticillatum Peak Compound Retention Time Molecular weight Concentration No. (min) (%) 1 N/D 18.812 N/D 0.1212

2 Docasane 19.298 310.3 [C22H46] 0.1720 3 N/D 21.047 N/D 0.4329 4 N/D 22.197 N/D 0.2537 5 N/D 23.064 N/D 0.7487

6 Pentacosane 23.490 352.4 [C25H52] 0.3319 7 N/D 25.061 N/D 0.4469 8 N/D 25.286 N/D 0.3215

9 Linalyl acetate 25.788 196.2 [C10H20O2] 0.4535

10 α-Bulnesene 27.048 204.3 [C15H24] 1.5648 11 N/D 27.990 N/D 0.4898

12 Eicosadienoic 28.844 308.3 [C20H36O2] 0.3702 13 N/D 29.067 N/D 0.1948 14 N/D 29.686 N/D 0.7196 15 N/D 30.732 270.0 1.9624 16 N/D 32.460 N/D 0.3740 17 N/D 32.644 N/D 0.4329 18 N/D 33.084 N/D 0.2619

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19 N/D 33.695 N/D 0.3738 20 N/D 34.168 256.2 0.7676 21 N/D 35.060 204.2 0.1642 22 N/D 36.139 N/D 0.7037

23 Piperitone 37.119 152.2 [C10H16O6] 0.3051 24 N/D 37.283 308.3 0.9512

25 Calarene 38.216 204.2 [C15H24] 0.1321 26 N/D 38.765 284.2 0.4077 27 N/D 39.765 364.3 0.7412 28 N/D 40.555 N/D 0.3983 29 N/D 41.677 N/D 1.0842 30 N/D 43.470 N/D 3.2137 31 N/D 47.727 N/D 4.9128 32 N/D 49.168 N/D 2.3831 33 N/D 52.650 N/D 14.8762 34 N/D 53.898 N/D 3.7734 35 N/D 56.800 N/D 16.4486 36 N/D 60.453 N/D 9.0349 37 N/D 64.101 N/D 2.9988 38 N/D 69.276 N/D 15.9242 39 N/D 71.902 N/D 3.3017 40 N/D 74.235 N/D 5.7280 41 N/D 79.000 N/D 1.7192

N/D = Not determined. Data bases used for the elucidation of constituents was performed were: GC-MS Library of Shimadzu Class-5000, ver 2.0 (1996). NIST Mass Spectral Search Program for the NIST/EPA/NIH Mass Spectral Library, ver. 16d (06/24/1998). Gaithersburg, MD, USA.

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3.3. Metal Analysis

3.3.1. Micronutrients

The results on micronutrients investigation of the Rhizomes and Aerial parts and their subsequent solvent fractions of Polygonatum verticillatum are depicted in Table 3.3.1.

3.3.1.1. Iron (Fe) status

The results of our findings reflect that the Rhizomes had prominent Fe concentration crude form as well as in fractions. The crude extract contained reasonable concentration (191.65 ppm) of Fe that changed upon fractionation. The n-hexane fraction exhibited the maximum Fe concentration (144 ppm) while the overall concentrations were ranges from 89.20–144 ppm. Similarly, the Aerial parts of the plant appeared as a rich source of Fe. The maximum concentration was found in the crude methanol extract (204 ppm). However, the concentration was varied in subsequent fractions and ranges from 63-204 ppm).

Iron represents the most abundant essential trace element of human body tissues. Its optimal concentration is required for the existence of plants, animals and microorganisms (Arredondo and Nunez, 2005). The world health organization has reported that around 46 % of the world’s children and 48 % of pregnant women are suffering from anemia. The Fe deficiency causes irreversible alterations of brain functions and affects immune response in many ways (Beard, 2001). Most of the body iron is taken by hemoglobin (57.6 %) and non- heme iron complexes (33 %) including ferritin and hemosiderin. Food and Nutrition Board has recommended the daily iron intake as 8 mg/day for male 18 mg/day for female and 27 mg/day during pregnancy (IOM, 2001). None of the tested sample crossed the permissible limits (36–241 ppm) and therefore appeared as a significant source of iron.

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3.3.1.2. Copper (Cu) status

Copper is another essential plant micronutrient that involved in CO2 absorption and ATP formation. Many human body proteins are dependent on copper. Cu is necessary for proper working of immune system (Huang and Failla, 2000). During infections, the generation of interleukin-2 by activated lymphocytic cells is dependent on Cu. Systemic decreased in Cu levels causes cellular iron deficiency (Arredondo and Nunez, 2005). Cu toxicity in infancy is based on improper liver functioning. Cu deficiency affects Fe transport in the body tissues and is responsible for a hypochromic microcytic anemia comparable to that caused by Fe deficiency.

Figure 3.3.1. Cu Concentration in various fractions of Rhizomes of the plant. Values are mean ± SEM of three different experiments.

In case of Rhizomes, crude extract and fractions exceeded the permissible limit (10 ppm) of Cu in plants. The maximum concentration was observed in the n-hexane fraction (48.80 ppm) while the overall concentrations were in the range of 21.51–48.80 ppm. In case of Aerial parts of the plant, the various fractions of the plant exhibited notable amount of Cu and ranges from 0.6–7.43 ppm. It is worth mentioning that all the tested fractions exceeded the permissible limit (Figure 3.3.1) (10 ppm) for plants (Table 3.3.3). The possibility of toxicity enhanced due to accumulation of such a high Cu concentration in various fractions of Rhizomes. The recommended dietary allowance (RDA) for Cu is 340– 900 µg /day.

3.3.1.3. Zinc (Zn) status

In human, Zinc is classified as one of the most copious crucial nutrients. It is institute in all body tissues mostly in muscle and bone (85%), 11% in the skin and the liver while the residual Zn is distributed in other body tissues (Tapiero and Tew, 2003). There are more than 300 body proteins that are zinc dependent. Zinc act as anti-inflammatory, antioxidant; bone resorptive, important for cell signaling, release of hormones and in apoptosis. Zinc

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deficiency in human mostly occurred in pregnancy (Moser-Veillon, 1990) and is characterized by growth failure, improper parturition (dystocia), neuropathy, decreased and cyclic food intake, diarrhea, skin disease, hair loss, bleeding disorder, hypotension, seizers and hypothermia. Acute zinc toxicity causes abdominal pain, nausea, vomiting and diarrhea. Chronic exposure of zinc elicits copper deficiency (IOM, 2001).

The results revealed that the Rhizomes of plant possess marked concentration of Zn (Table 3.3.1). The crude extract of Rhizomes showed highest concentration of Zn (50.11 ppm) but did not exceed the permissible limits. Similarly, the aerial parts of the plants exhibited significant concentration of Zn in the range of 38.8–60 ppm (Table 3.3.2). All the samples had values within the limit (50 ppm) except butanol fraction (60 ppm).

3.3.1.4. Manganese (Mn) status

Manganese is an essential trace element. It plays a pivotal role in the normal growth, skeletal formation and normal reproductive function. Mn intoxication is responsible for Parkinsonism which is usually become progressive and unresponsive and causing permanent injure to neurologic structures (Wang et al., 2008). The permissible limit for plants is estimated as 200 ppm (Table 3.3.3). In the present analysis, the crude extract of Rhizomes of the plant exhibited marked concentration (143.55 ppm) but with in specified limits. However, the concentration considerable reduced upon fractionation. The overall range was from 05–143.55 ppm. Regarding the results of Aerial parts, the crude extract had the highest concentration (7.91 ppm) while the overall range was from 5.20–7.91 ppm.

3.3.1.5. Chromium (Cr) status

Chromium is provisionally considered to be a nutrient because of its metabolic role and is one of the copious elements on the planet (Emsley and John, 2001). It plays important role in the synthesis of fatty acids and cholesterols (IOM, 2001). Chromium plays putative roles in carbohydrate, protein, and lipid metabolism; and it have been proved that it facilitates the action of insulin. Therefore, chromium based supplement are used for weight loss

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Table 3.3.1: Quantification of micronutrients (ppm) in crude extract of Rhizomes of Polygonatum verticillatum and its subsequent solvent fractions

Minerals Crude Hexane Chloroform Ethyl acetate Butanol Aqueous Zinc 50.11±0.58 48.80±0.03 47.10±0.03 40.40±0.02 49.20±0.06 37.70±0.03 Cu 21.51±0.29 48.80±0.06 40.40±0.02 44.00±0.57 41.40±0.03 48.20±0.03 Cr 1.64±0.02 01±00 01±00 1.40±0.01 02±0.06 0.60±0.01 Fe 191.65±0.58 144±2.31 137.2±0.05 124.80±0.03 89.20±0.03 141.60±1.55 Pb ND ND ND ND ND ND Mn 143.55±0.58 08±0.29 10.80±0.01 7.40±0.02 05±0.17 7.60±0.03 Ni 1.02±0.01 0.6±0.01 1.10±0.03 0.30±00 1.40±0.01 0.3±0.01 Sb ND ND ND ND ND ND Cd ND ND ND ND ND ND Co ND ND ND ND ND ND

Macronutrients

Minerals Crude Hexane Chloroform Ethyl acetate Butanol Aqueous Ca 133.32±0.18 90±1.15 180±1.15 170±0.56 190±1.16 120±1.16 Na 234.83±0.02 280±1.73 450±1.73 450±1.15 280±5.77 60±1.15 K 1267.96±0.02 1320±5.77 1250±2.29 1250±2.31 1570±5.77 1600±5.77

sults are mean ± S.E.M of three different experiments. .

(Lukaski et al., 2007). The estimated permissible limit for Cr in plants is 1.5 ppm. The results on Rhizomes of plant reflect significant accumulation of Cr. The highest and beyond permissible concentration was accumulated by the n-butanol fraction (02 ppm). However, the remaining fractions were within the limits. In case of Aerial parts, the results revealed the Cr concentration within recommended range (0.36–01 ppm).

Table 3.3.2: Quantification of micronutrients (ppm) in crude extract of Aerial parts of

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Polygonatum verticillatum and its subsequent solvent fractions

Minerals Crude Hexane Chloroform Ethyl acetate Butanol Aqueous Zn 46.24±0.02 45.4±0.04 38.8±0.05 40.6±0.01 60±0.17 45.6±0.03 Cu 6.40±0.01 4.40±0.01 0.6±0.02 1.6±0.01 7.43±0.02 3.6±0.02 Cr 0.36±0.01 01±0.05 0.8±0.05 0.8±0.02 01±0.05 0.6±0.03 Fe 204±1.15 128.2±1.15 106.8±0.03 115.2±0.11 63±0.06 134±1.15 Pb 0.17±0.02 ND ND ND ND ND Mn 7.91±0.11 7.20±0.05 5.6±0.02 6.4±0.02 5.20±0.05 7.20±0.11 Ni 0.54±0.02 1.80±0.01 1.20±0.01 2.40±0.01 1.2±00 0.2±0.01 Sb ND ND ND ND ND ND Cd ND ND ND ND ND ND Co ND ND ND ND ND ND

Macronutrients (ppm)

Minerals Crude Hexane Chloroform Ethyl acetate Butanol Aqueous Ca 129.42±0.04 100±0.56 140±1.15 140±1.73 140±00 220±1.15 Na 219.34±0.57 120±0.57 140±1.15 120±0.57 560±1.73 400±2.31 K 3250±2.31 2900±4.04 3400±6.92 2500±1.15 3120±2.88 3400±5.19

Results are mean ± S.E.M of three different experiments.

3.3.1.6. Nickel (Ni) status

Nickel is a metallic element that is naturally present in the earth’s crust. Due to its abundance, natural nickel deficiency does not usually occur and dietary deficiency of nickel is rarer because of nickel’s abundance in all types of food. Health related risks of nickel include skin disorders, lung fibrosis, kidney and cardiovascular system poisoning and elevation of neoplastic transformation (Denkhaus and Salnikow, 2002). Nickel is mostly accumulated in the pancreas and shares an imperative role in the production of

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insulin. Nickel deficiency is responsible for liver disorders (Denkhaus and Salnikow, 2002). The permissible limit of Ni in plants is 1.5 ppm (Srivastava et al., 2006).

Our data revealed that the various fractions of Rhizomes of plant accumulated reasonable concentration of Ni and was in the range of 0.30 – 1.40 ppm thus these are within recommended limits. However, the Aerial parts of the plant exceed the permissible limit in some fractions. The n-hexane fraction (1.80 ppm) and ethyl acetate fraction (2.40 ppm) exceeded the permissible limit.

It is worthwhile that the concentration of some the micronutrients were not detectable in both the Rhizomes as well as Aerial parts of the plant. These metals were including Pb, Sb, Cd and Co.

3.3.2. Macronutrients

The results on macronutrients in our investigation of the crude extract and its subsequent solvent fractions of Rhizomes and Aerial parts of the Polygonatum verticillatum are presented in Table 3.3.1 and 3.3.2.

3.3.2.1. Sodium (Na) status

Sodium is very important macronutrient of human body system. The most common dietary source of sodium is common table salt (NaCl). It has got the prime role in the maintenance of normal physiology in all living organisms. A lack of sodium intake is incompatible with survival. An enough ingestion of sodium is vital for optimal growth. Delivery of intracellular and extracellular fluid volumes is sodium dependent. On that ground, a shortage or excess of sodium will amend the overall fluid balance and distribution (Morris et al., 2008). Na depletion is characterized by mood changes, muscle cramps, fatigue, hair loss, hypotension and dehydration (Harper et al., 1997).

We observed marked concentration of Na in various fractions of the Rhizomes of the plant (Figure 3.3.3B). The most dominant concentration was possess by both the

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chloroform and ethyl acetate fractions each (450 ppm). The overall Na accumulation in various fractions of Rhizomes was 60–450 ppm. Similar trend was observed for the Aerial parts of the plant. The n-butanol fraction exhibited the maximum concentration (560 ppm). While the overall Na concentration was ranges from 120–560 ppm (Figure 3.3.4B). There is no international limit which reflects concentration of Na in plants. However, the recommended daily intake of Na is 1–3.8 mg/day (IOM, 2004).

Table 3.3.3: Permissible limits of various metals in plant

S. No Metals Permissible limits (ppm) Authority 1 Zn 50 (Srivastava et al., 2006) 2 Cu 10 (Srivastava et al., 2006) 3 Pb 10 (W.H.O, 1999) 4 Cr 1.5 (Srivastava et al., 2006) 5 Ni 1.5 (Srivastava et al., 2006) 6 Fe 36─241 (Ajasa et al., 2004) 7 Sb Not available in literature 8 Cd 0.3 (W.H.O, 1999) 9 Mn 200 (Srivastava et al., 2006) 10 Co 0.2 (Srivastava et al., 2006) 11 Na 44─614 (Ajasa et al., 2004) 12 Ca 2610─51340 (Ajasa et al., 2004) 13 K 6380─36,600 (Ajasa et al., 2004)

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Figure 3.3.2: Ni concentration of Aerial parts of plant. Data are expressed as mean ±SEM of three different findings.

3.3.2.2. Potassium (K) status

Potassium represents a very important macronutrient of living organism. The concentration of K ions is certainly connected with monitoring of action potentials and intercellular signaling in electrically vigorous cells. The K channels are involved in multiple functions in both excitable and non-excitable cells. These cellular monitoring include balance of membrane potential, signal transduction, insulin secretion, hormone release, regulation of vascular tone, cell volume and immune response (Curran, 1998). There is no international limit which reflects the concentration of potassium in plants. However, the average intake of potassium is 2300 mg/day for adult’s women and 3100 mg/day for adult’s men (IOM, 2001).

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The results of our study on the Rhizomes of the plant revealed that marked concentrations were accumulated in various fractions. The highest K concentration was concentrated in the aqueous (1600 ppm) fraction. The overall accumulation in different

Figure 3.3.3: Concentration of macronutrients, Ca [A], Na [B] and K [C] in Rhizomes of the plant. Data are expressed as mean ±SEM of three different findings.

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Figure 3.3.4: Concentration of macronutrients, Ca [A], Na [B] and K [C] in Aerial parts of the plant. Data are expressed as mean ±SEM of three different findings.

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fractions were ranges from 1250–1600 ppm (Figure 3.3.3C). In case of Aerial parts, the concentration of K was more prominent. The highest concentration was found in both chloroform and aqueous fractions (3400 ppm) each. However, the overall range in various fractions was 2500–3400 ppm (Figure 3.3.4C).

3.3.2.3. Calcium (Ca) status

Calcium symbolized an imperative macronutrients mostly obtained from various dietary sources. Apart from its crucial role in the body’s metabolic process, Ca along with phosphorus is a structural component of bones, teeth, and soft tissues (Shapiro and Heaney, 2003). Binding of the calcium ions on the surface of human growth hormone provides considerable thermodynamic stability to protein by changing the secondary structure of the protein (Saboury et al., 2005). Cellular calcium is involved in various regulatory functions like regulation of muscle and nerve functions, glandular secretions, and blood vessel dilation and contraction. Ca deficiency is responsible for weakness of the bones and thus bones are more prone to fracture. It can produce skeletal muscles spasm and abnormality in heart beat and can even cease functioning of heart. Ca intoxication is rarer but when occur is characterized by hypercalcemia, which causes constipation, kidney stones appetite loss, nausea , vomiting, abdominal pain, confusion, seizures, and even coma (IOM, 1997).

Regarding the results of Ca accumulation in Rhizomes of plant, significant concentrations were observed in different fractions. The maximum concentration was carried by the n-butanol fraction (190 ppm). However, the overall concentration was in the ranges from 90–190 ppm (Figure 3.3.3A). In case of Aerial parts, the tested samples accumulated significant concentration of Ca and were ranges from 100–220 ppm (Figure 3.3.4A). According to the Institute of Medicine, the recommended daily intake of Ca is 1000 mg/day. Based on our results, both Rhizomes and aerial parts of the plant is a rich source of Ca.

Metals being non-biodegradable have the tendency to endure for long time in both aquatic and terrestrial environments. They may be transported via soils to reach ground

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waters or may be taken up by plants, including agricultural crops (Boularbah et al., 2006). The minerals vital to human food fairly accumulated in various parts of plants. As required for their growth, life cycle and as a part of their food chain, plants acquire essential elements from the environment. The plants also have the tendency to accumulate some of the metals which are not linked directly to their survival like Cd, Co and Ag (Ajasa et al., 2004). In human, trace elements play a pivotal role both as preventive and as curative against various diseases. However, contaminations of plants by heavy toxic metals due to any factor could constitute serious health problems because there is a narrow concentration range between the deficiency and toxicity levels of heavy metals in human (En et al., 2003).

It can be concluded on the base of our results that both Rhizomes and Aerial parts of P. verticillatum are an excellent source of micro and macro nutrients. These nutrients are usually required for the normal physiological functioning of human body. Mostly these nutrients were found within the permissible limits for plants with the exception of few fractions. Therefore, our study validated the ethno-botanical use of the plant as tonic and energizer.

Conclusion

Even in the present era of medicinal engineering, plants have incredibly contributed in the discovery of novel drug leads as an infinite source. Plants as a therapeutic modality have a glorious past and present in the alleviation of numerous human ailments. Multifaceted approach has been developed in the recent times using modern technologies like High- throughput screening and plant cell culture accompanied by molecular based pharmacological assays, which obviously expedites the discovery processes. Traditional system of treatment symbolizing century’s old practice based on long empirical learning transferred from generation to generation. Such traditional heritage that enriches our ethnopharmacology needs scientific validation.

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To rationalize the traditional uses of P. verticillatum in Pakistan and various other communities, different experimental paradigms were designed to test the extracts of both Rhizomes and Aerial parts. The undertaken various in vivo pharmacological activities with significant effect were antinociceptive, anti-inflammatory, and antipyretic without acute toxicity. However, insignificant exhibition was observed in anticonvulsant, diuretic test. Profound antibacterial, antimalarial, DDPH based free radical and phytotoxic activities were registered, while antifungal, leishmanicidal, insecticidal, brine shrimps lethality test were not significant. Different enzyme inhibitory activities were also carried out. Promising inhibition was demonstrated against lipoxygenase and urease.

The principles of bioactivity-directed isolation lead to the isolation of seven compounds through the application of different chromatographic and spectroscopic techniques. The isolated plant secondary metabolites included one new compound and six already reported by various research groups. Nevertheless, we reported for the first time from this plant. The pure chemical entities strongly supported the activities of extracts and some need further studies.

Plant biodiversity conservation is obviously a global subject. Conservationist encounters the involvement of multiple factors and also offers different solutions. In this regard, overtrading without cultivation of wild species is counted as the primary factor. In case of P. verticillatum, only Rhizomes of the plant are materialized. Based on the results of Aerial parts of the plant in comparison with Rhizomes, efficacy of both was essentially comparable in most of the activities but marginal differences were observed in potencies. Therefore, both parts can be equally utilized for therapeutic purposes.

On the face of global recognition, FDA as well as Western communities, the birth place of orthodox medicines, are now accepting natural organic therapeutic agents once validated for efficacy, potency and safety. While providing scientific validation of Polygonatum verticillatum in variety of experimental conditions, it could be considered a prominent natural healing agent and subject to further detailed studies as a potential source of new drug leads as well.

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Related publication

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