Phytochemical and Biological

PHYTOCHEMICAL AND BIOLOGICAL STUDIES OF SELECTED MEDICINAL PLANTS Farsetia hamiltonii (BRASSICACEAE) AND Neurada procumbens (NEURADACEAE) FROM CHOLISTAN DESERT (PAKISTAN)

Thesis submitted in the fulfilment of the degree of Ph.D. Pharmacy (Pharmaceutical Chemistry)

By Muhammad Munawar Hayat Roll No. 04-PhDC-12

Session 2012-2015

FACULTY OF PHARMACY BAHAUDDIN ZAKARIYA UNIVERSITY MULTAN-PAKISTAN

(In the name of ALLAH, Who is Most Merciful and Most Beneficent)

Al-Quran

Read! With the name of thy Allah, Who created you” (Al Alaq; 1).

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BRIEF CONTENTS

Sr. No. Description Page No.

1 Title i

2 List of contents vii-xi

3 Abstract xv-xvii

4 List of tables xviii

5 List of figures xix-xxi

6 Chapter No.1: Introduction 01

7 Aims and objective of study 14

8 Chapter No.2: Literature review 16

9 Chapter No.3: Material and methods 31

10 Chapter No.4: Results and discussion 49

11 Discussion 98

12 Conclusion 108

13 References 110

14 Appendix (Publications and CV of scholar) 122

LIST OF CONTENTS

Sr. No. Description Page No. 1 Title i 2 Bismillah ii 3 Al-Quran iii 4 Attendance sheet of Viva iv 5 Examiners report for PhD Viva-Voce / Public defense v 6 Brief contents vi 7 List of contents vii-xi 8 Dedication xii 9 Acknowledgements xiii 10 Certificate xiv 11 Abstract xv-xvii 12 List of tables xviii 13 List of figures xix-xxi 14 List of abbreviations xxii Chapter No. 1: Introduction 01 1 Medicinal plants 02 1.1 Cholistan desert 03 1.2 Botanical description of family Brassicaceae 03 1.3 Botanical description of genus Farsetia 04 1.4 Farsetia hamiltonii Royle 06 1.5 Botanical description of family Neuradaceae 09 1.6 Botanical description of genus Neurada 09 1.7 Neurada procumbens L. 10 1.8 Aims and objectives of study 14 Chapter No. 2: Literature Review 16 2 Literature review on selected plants 17 2.1 Literature review on Farsetia hamiltonii Royle 17 2.1.1 Literature survey on the phytochemical studies of family 17 Brassicaceae 2.1.1.1 Flavonoids 17

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Sr. No. Description Page No. 2.1.1.2 Hydroxycinnamic acids 18 2.1.1.3 Vitamins 18 2.1.1.4 Minerals 19 2.1.1.5 Phytoalexins 19 2.1.1.6 Glucosinolates 22 2.1.2 Phytochemical studies of the genus Farsetia 22 2.1.3 Studies on the biological activities of the genus Farsetia 26 2.1.3.1 Cytotoxic activity 26 2.1.3.2 Antimicrobial activity 26 2.1.3.3 Antioxidant activity 26 2.1.4 Folk uses of genus Farsetia 27 2.2 Literature review on Neurada procumbens L. 28 2.2.1 Literature survey on the phytochemical studies family 28 Neuradaceae 2.2.2 Phytochemical studies of Neurada procumbens L. 28 2.2.3 Biological activities of Neurada procumbens L. 29 2.2.3.1 Effect on Blood Pressure 29 2.2.3.2 Antibacterial activity 29 2.2.3.3 Antifungal activity 29 2.2.4 Folk uses of Neurada procumbens L. 30 Chapter No.3: Material and Methods 31 3.1 Plant collection 32 3.2 Chemicals and reagents 32 3.3 Instrumentation 32 3.4 Detection of secondary metabolites 35 3.4.1 Detection of alkaloids 35 3.4.2 Detection of cardiac glycosides 35 3.4.3 Detection of anthraquinones 35 3.4.4 Detection of flavonoids 36 3.4.5 Detection of saponins 36 3.4.6 Detection of terpenoids 36

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Sr. No. Description Page No. 3.4.7 Detection of sterols 36 3.5 Determination of mineral contents 36 3.5.1 Atomic absorption spectroscopy 37 3.6 Extraction 37 3.7 Preparation of reagents 38 3.7.1 Mayer‟s reagent 38 3.7.2 Dragendorff‟s reagent 38 3.7.3 Wagner‟s reagent 38 3.7.4 Hager‟s reagent 38 3.7.5 Godin‟s reagent 38 3.8 Preparation of Solutions 38 3.8.1 Dilute sulphuric acid (10%) 38 3.8.2 Dilute ammonia solution 38 3.8.3 Dilute hydrochloric acid 39 3.8.4 Sodium hydroxide (10M) solution 39 3.8.5 Ferric chloride solution (10%) 39 3.8.6 Ferric chloride solution (3.5%) 39 3.8.7 Gelatin (1%) solution 39 3.8.8 Lead sub-acetate solution 39 3.9 Total flavonoid contents determination 39 3.10 Total phenol contents determination 40 3.11 Gas chromatography-mass spectrophotometry 40 3.12 Biological and enzyme inhibition activities 40 3.12.1 Antioxidant activity 40 3.12.2 Cholinesterase inhibition activities 41 3.12.3 Lipoxygenase inhibition activity 42 3.12.4 Chymotripsin inhibition activity 43 3.12.5 α-glucosidase inhibition activity 43 3.12.6 Urease inhibition activity 43 3.12.7 Tyrosinase inhibition activity 44 3.12.8 Carbonic anhydrase II inhibition activity 44

Sr. No. Description Page No. 3.12.9 Phosphodiesterase I inhibition activity 44 3.12.10 Antibacterial activity 45 3.13 Experiments on isolated tissues 45 3.13.1 Search for spasmolytic activity 46 3.13.2 Search for bronchorelaxant activity 45 3.14 In vivo study 46 3.14.1 Screening for hypoglycaemic activity 46 3.14.1.1 Induction of hyperglycemia 47 3.14.1.2 Administration of plants extracts 47 3.14.1.3 Protocol of study 48 Chapter No. 4: Results and Discussion 49 4.1 Phytochemical screening of secondary metabollites 50 4.2 Results of atomic absorption spectroscopy (AAS) 51 4.3 Extraction yields 51 4.4 Determination of total flavonoid and phenol contents 52 4.5 GC-MS analysis results of dichloromethane and methanol 53 extracts of different parts of Farsetia hamiltonii Royle and Neurada procumbens L. 4.5.1 GC-MS results of dichloromethane extract of aerial part of 53 Farsetia hamiltonii Royle 4.5.2 GC-MS results of methanol extract of aerial part of 58 Farsetia hamiltonii Royle 4.5.3 GC-MS results of dichloromethane extract of root part of 62 Farsetia hamiltonii Royle 4.5.4 GC-MS results of methanol extract of root part of 68 Farsetia hamiltonii Royle 4.5.5 GC-MS results of dichloromethane extract of Neurada 71 procumbens L. 4.5.6 GC-MS results of methanol extract of Neurada 78 procumbens L. 4.6 Biological and enzymatic activities of extracts of selected 83 medicinal plants.

Sr. No. Description Page No. 4.6.1 Antioxidant activity 83 4.6.2 Cholinesterase inhibition activity 84 4.6.3 Lipoxygenase inhibition activity 84 4.6.4 Chymotripsin inhibition activity 85 4.6.5 Alpha glucosidase inhibition activity 85 4.6.6 Urease inhibition activity 86 4.6.7 Tyrosinase inhibition activity 86 4.6.8 Carbonic anhydrase II inhibition activity 87 4.6.9 Phosphodiesterase I inhibition activity 87 4.6.10 Antibacterial activity (%age inhibition) 88 4.7 Biological activities of selected medicinal plants on 89 isolated tissues 4.7.1 Spasmolytic activity 89 4.7.2 Brochorelaxant activity 94 4.8 In vivo hypoglycaemic activity of F. hamiltonii 96 4.9 Discussion 98 4.10 Conclusion 108 5 References 110 6 Appendix (Publications from Ph D work) 122 6.1 Research article-1: Published in Journal of Ethanopharmacology 6.2 Research article-2: Published in Journal of Chemical Society of Pakistan 6.3 Research article-3: Accepted in Biomedical Research Journal 6.4 CV of Ph D scholar

DEDICATION

The study is dedicated to my beloved family, whose support; encouragement and prayers have always been medium of devotion and power for the achievement of this objective.

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ACKNOWLEDGEMENTS all praises for ,ﷻI begin by bowing my heart to ALMIGHTY ALLAH the most merciful, the most beneficent, Who helped and ,ﷻALMIGHTY ALLAH blessed me all the way along till the completion of the research work. Without His grace and mercy, I am nothing. Then I would pay gratitude to our beloved Prophet .who guided the humanity to the right path ,ﷺHazrat MUHAMMAD In the first place with great pleasure, I express my most cordial and humble thanks to my eminent, respected guide and supervisor Prof. Dr. Muhammad Uzair, Chairman, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, B. Z. University, Multan. His real time guide line, supportive attitude and motivation made me able to complete the research work. His nice, humble and motivated behaviour is exemplary for the whole period of lab work. I express my deep gratitude and special thanks to Chairman, Department of Pharmacy and Dean, Faculty of Pharmacy Prof. Dr. Bashir Ahmed Chaudhary for his valuable guidance and enduring support during my research work. I express my most cordial and humble thanks to my eminent, respected guide and teacher Prof. Dr. Shazia Anjum, Department of Chemistry, The Islamia University of Bahawalpur for providing her valuable guidance, inspiration, supervision, keen interest and moral support from the very early stage of my research work till the end of my dissertation work. Her encouraging words were source of constant motivation to act with quality and precision and they enriched my growth as a student. I am indebted to her for her constant support. I am indebted to my FATHER, for his care, support and love. I have no suitable words that can fully describe his everlasting love to me. It is his dream that I get excellence in my studies. I cannot ask for more from my MOTHER as she is simply perfect. I have no suitable words that can fully describe her everlasting prayers and love to me. I remember many sleepless nights with her accompanying me, giving me strength and praying for my success. I would like to thank my whole family especially my wife Rabia Kausar, daughters Rumaisa Hayat, Sumamah Hayat and son Muhammad Abu Bakar Hayat, their love and support in all respects. I acknowledge and heartiously wish to record my special thanks to all my teachers, colleagues, friends and all those who believe in the richness of learning.

Muhammad Munawar Hayat

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CERTIFICATE It is certified that the research work presented by Muhammad Munawar Hayat (Roll No. 04-PhDC-12) in this thesis entitled as “Phytochemical and biological studies of selected medicinal plants Farsetia hamiltonii (Brassicaceae) and Neurada procumbens (Neuradaceae) from Cholistan desert (Pakistan)” was carried out under my supervision and in our opinion, it is fully satisfactory, in scope and excellence for the degree of Doctor of Philosophy (PhD) in the subject of Pharmacy (Pharmaceutical Chemistry) from the Faculty of Pharmacy, Bahauddin Zakariya University, Multan.

Supervisor

Prof. Dr. Muhammad Uzair Chairman, Department of Pharmaceutical Chemistry, Faculty of Pharmcy, Bahauddin Zakariya University, Multan.

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ABSTRACT The PhD dissertation is based on phytochemical and biological studies of selected medicinal plants of Cholistan desert of Punjab, Pakistan namely Farsetia hamiltonii Royle and Neurada procumbens L. The dichloromethane and methanol extracts of Farsetia hamiltonii Royle (aerial & root parts) and Neurada procumbens L (whole plant) were evaluated for phytochemical, enzyme inhibition and biological studies. Medicinal plants of Cholistan desert have been used as traditional medicines for thousands of years being the essence of pharmaceuticals and folk medicines. The powdered plant material of both selected plants was evaluated for secondary metabolites by using different standard procedures/methods and chemical contents by atomic absorption spectroscopy. The crude extracts of both selected medicinal plants were screened for the total flavonoid and phenol contents. The selected medicinal plants Farsetia hamiltonii Royle and Neurada procumbens L. were evaluated for phytochemical constituents using the Gas chromatography mass spectrometry (GC-MS). Farsetia hamiltonii offered fifteen (15) compounds; (1) 12-methyl-E,E-2,13- octadecadien-1-ol, (2) Estra-1,3,5(10)-trien-17β-ol, (3) Oleic acid, (4) 13-Heptadecyn-1- ol, (5) Ethyl iso-allocholate (6) Octadecan, 6-methyl-, (7) Cyclo propane botanic acid, 2- [[2-[[2-[(2-pentyl cyclo propyl)methyl]cyclo propyl]methyl]cyclo propyl]methyl]-, methyl ester, (8) Hexadecanoic acid, methyl ester, (9) 2,4-Nonanedione, (10) Thymol, (11) n-Hexadecanoic acid, (12) Trans-13-Octadecenoic acid, (13) Behenic alcohol, (14) Hexadecanoic acid, 2-hydroxy-1-(hydroxyl methyl) ethyl ester and (15) 3„,8,8‟- trimethoxy-3-piperidyl-2,2‟-binaphthalene-1,1‟,4,4‟-tetrone which were identified by the analysis of four extracts (FHAD, FHAM, FHRD, FHRM) of aerial and root parts of the plant by using the gas chromatography mass spectrometry (GC-MS). Neurada procumbens offered fifteen (15) compounds; (1) 3,7,11,15-tetramethyl- 2-hexadecen-1-ol, (2) 12-methyl-E,E-2,13-octadecadien-1-ol, (3) 13-Heptadecyn-1-ol, (4) n-Hexadecanoic acid, (5) 9,12,15-octadecatrienoic acid, 2,3-dihydroxy propyl ester, (Z,Z,Z)-, (6) Phytol, (7) Trans-13-Octadecenoic acid, (8) Oleic acid, (9) Ethyl iso- allocholate, (10) Pterin-6-carboxylic acid, (11) Ergosta-5,22-dien-3-ol,acetate, (3β,22E)-, (12) 5H-cyclopropa[3,4] benz[1,2-e]azulen-5-one,9,9a-bis (acetyloxy)- 1,1a,1b,2,4a,7a,7b,8,9, 9a-decahydro-2,4a,7b-trihydroxy-3-(hydroxyl methyl)-1,1,6,8- tetra methyl-, [1aR-(1aα,1bβ,2β,4aβ, 7aα, 7bα,8α,9β,9aα]-, (13) Hexadecanoic acid, methyl ester, (14) 7,10-Octadecadienoic acid and (15) 10-Octadecenoic acid methyl ester,

XV which were identified in dichloromethane and methanol extracts (NPD and NPM) by using the GC-MS analysis. The antioxidant activity of methanol extract of N. procumbens was significant with inhibition 75.38±3.16% at concentration 0.5mg/ml and IC50 88.30±1.21µmole which is very close and comparable to the values of Quercetin standard (83.68±3.76% and IC50 16.96±0.14). The anticholinesterase (antiacetylcholinesterase, antibutyrylcholinesterase) activity of dicloromethane and methanol extracts of root part of F. hamiltonii was at moderate level, whereas both aerial part extracts of F. hamiltonii and both extracts of N. procumbens were found inactive for this activity. The antilipoxygenase activity of all four extracts of F. hamiltonii (FHAD, FHAM, FHRD, FHRM) and dicloromethane extract of N. procumbens were active at moderate level, whereas the methanol extract of N. procumbens was found inactive for this activity. The antichymotripsin activity of all four extracts of F. hamiltonii (FHAD, FHAM, FHRD, FHRM) and dicloromethane extract of N. procumbens were prominently active at moderate level, whereas the methanol extract of N. procumbens was found inactive. The anti-alpha glucosidase activity of methanol extracts (aerial and root parts) of F. hamiltonii and both extracts (dicloromethane and methanol) of N. procumbens were highly active and closely comparable to inhibition and IC50 values of standard Acarbose. The methanol extract of aerial part of F. hamiltonii and both extracts of N. procumbens having greater inhibitions (97.54±2.39%, 88.62±2.25%, 95.49±0.65% and 99.45±0.58%, respectively), also having very significant IC50 values (25.32±0.12, 152.95±1.84, 24.32±0.62 and 8.43±0.56, respectively) with respect to inhibition of standard Acarbose

(92.23±0.14% and IC50 value 38.25±0.12). The dicloromethane extracts of aerial and root parts of F. hamiltonii were very low inhibition (%) values and insignificant IC50 values. The antiurease activity of both extracts of aerial part of F. hamiltonii and N. procumbens was moderately active, whereas antityrosinase activity was possessed only by both extracts of N. procumbens, whereas both extracts of aerial part of F. hamiltonii were inactive for this activity. The anticarbonic anhydrase II activity of only methanol extract of N. procumbens was significant with inhibition 73.92±0.41% and IC50 102.31±0.85µmole which is comparable to the values of Acetazolamide standard (89.03±0.03% and IC50 0.10±0.04). Whereas both aerial part extracts (dicloromethane and methanol) of F. hamiltonii and dicloromethane extract of N. procumbens were found inactive for this activity. The XVI antiphosphodiesterase I activity was not exhibited by all extracts of both selected medicinal plants of this study. The antibacterial activity was exhibited significantly by dichloromethane and methanol extracts of aerial part, and only methanol extract of root part of F. hamiltonii. The spasmolytic activity of methanol extract of F. hamiltonii on isolated rabbit jejunum was evaluated and found significantly effective at concentration 0.01-0.3 mg/ml with ED50 value 0.066 mg/ml to the spontaneous rhythmic contractions and also exerted + relaxation K (80mM) induced contraction at concentration 0.01-0.3mg/ml with ED50 value 0.11mg/ml. Likewise, the methanol extract of N. procumbens showed significant spasmolytic effect to the spontaneous rhythmic contractions in isolated rabbit jejunum preparations at concentration 0.01-1.0 mg/ml with ED50 value 0.075 mg/ml and also + exerted relaxation K (80mM) induced contraction at 0.01-0.1mg/ml with ED50 value 0.024 mg/ml. Verapamil was used as standard drug in this activity. Due to calcium antagonist effect that may provide pharmacological rationale for its use to treat diarrhoea and abdominal cramps successfully. The bronchorelaxant effect of the methanol extract of N. procumbens on carbacol (1µM) induced contractions in isolated rabbit tracheal preparation at concentration 0.01- + 0.3 mg/ml with ED50 value 0.624 mg/ml and also exerted relaxation K (80mM) induced contraction at 0.01-0.1mg/ml with ED50 value 0.096 mg/ml, in a dose dependent manner on the isolated tissues of rabbit trachea was determined and the results were very close to the drug Verapamil. The hypoglycaemic activity of methanol extract of F. hamiltonii at two doses 100mg/Kg and 300mg/Kg was scientifically evaluated in an in vivo study on rats by dividing in different five groups. The results were very excellent and significant and proved the hypoglycaemic use of the plant in comparison to standard Glibenclamide drug to justify the floric use of the plant in the treatment of diabetes by Cholistan peoples. The achieved results in the present study depicted that F. hamiltonii offered fifteen (15) compounds, significant alpha-glucosidase inhibition activity, spasmolytic and in vivo hypoglycemic effect which are reported first time. Similarly, N. procumbens offered fifteen (15) compounds, very potent alpha-glucosidase inhibition activity, spasmolytic and brochorelaxant effect which are reported first time. The conducted study scientifically proved and validated the traditional uses of both selected medicinal plants F. hamiltonii Royle and N. procumbens L.

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LIST OF TABLES

Table No. Description Page No. Table-1 Results of phytochemical screening of secondary metabolites 50 Elemental analysis of plant material of F. hamiltonii and N. procumbens by Table-2 51 atomic absorption spectroscopic analysis Table-3 Percentage yield of crude extracts of selected plants 51

Table-4 Results of total flavonoid and phenol contents 52

Phytochemical constituents identified by GC-MS in dichloromethane Table-5 54 extract of aerial part of F. hamiltonii. Phytochemical constituents identified by GC-MS in methanol extract of Table-6 59 aerial part of F. hamiltonii. Phytochemical constituents identified by GC-MS in dichloromethane Table-7 63 extract of root part of F. hamiltonii. Phytochemical constituents identified by GC-MS in methanol extract of Table-8 69 root part of F. hamiltonii. Phytochemical constituents identified by GC-MS in dichloromethane Table-9 72 extract of N. procumbens. Table-10 Phytochemical constituents identified by GC-MS in methanol extract of N. 79 procumbens. Table-11 Results of antioxidant (DPPH) activity 83 Table-12 Results of cholinesterase (AChE & BChE) inhibition activities 84 Table-13 Results of lipoxygenase inhibition activity 84 Table-14 Results of chymotripsin inhibition activity 85 Table-15 Results of alpha glucosidase inhibition activity 85 Table-16 Results of urease inhibition activity 86 Table-17 Results of tyrosinase inhibition activity 86 Table-18 Results of carbonic anhydrase II inhibition activity 87 Table-19 Results of phosphodiesterase I inhibition activity 87 Table-20 Results of antibacterial activity (%age inhibition) 88

Table-21 Results of antibacterial activity (MIC50) 88 Table-22 Results of intra-peritoneal administration of different doses of methanol 97 extract of F. hamiltonii in alloxan induced diabetic rats for four days.

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LIST OF FIGURES

Figure No. Description Page No. Figure-1 Aerial view of F. hamiltonii Royle. 7 Figure-2 Morphological characters and features of F. hamiltonii Royle 8 Figure-3 Aerial view of N. procumbens L. 12 Figure-4 Morphological characters and features of N. procumbens L. 13 Figure-5 Herberium sheet of F. himaltonii Royle 33 Figure-6 Herberium sheet of N. procumbens L. 34 Structures of Lipoxygenase substrates, linoleic, α- linolenic and Figure-7 42 arachidonic acid GC-MS chromatogram of dichloromethane extract of aerial part of Figure-8 53 F. hamiltonii. Mass spectra and structure of 12-methyl-E,E-2,13-octadecadien-1- Figure-9 55 ol. Figure-10 Mass spectra and structure of Estra-1,3,5(10)-trien-17β-ol. 55 Figure-11 Mass spectra and structure of Oleic acid. 56 Figure-12 Mass spectra and structure of 13-heptadecyn-1-ol. 56 Figure-13 Mass spectra and structure of Ethyl iso-allocholate. 57 Figure-14 GC-MS chromatogram of methanol extract of aerial part of F. 58 hamiltonii. Figure-15 Mass spectra and structure of Octadecan, 6-methyl-. 60 Figure-16 Mass spectra and structure of Cyclopropane botanic acid, 2-[[2-[[2- [(2-pentyl cyclo propyl) methyl]cyclo propyl]methyl]cyclo 60 propyl]methyl]-, methyl ester. Figure-17 Mass spectra and structure of Hexadecanoic acid, methyl ester. 61 Figure-18 Mass spectra and structure of Ethyl iso-allocholate. 61 Figure-19 GC-MS chromatogram of dichloromethane extract of root part of F. 62 hamiltonii. Figure-20 Mass spectra and structure of 2,4-nonanedione. 64 Figure-21 Mass spectra and structure of Thymol. 64 Figure-22 Mass spectra and structure of n-hexadecanoic acid. 65 Figure-23 Mass spectra and structure of Trans-13-octadecenoic acid. 65

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Figure No. Description Page No. Figure-24 Mass spectra and structure of Oleic acid. 66 Figure-25 Mass spectra and structure of Behenic alcohol. 66 Figure-26 Mass spectra and structure of Hexadecanoic acid, 2-hydroxy-1- 67 (hydroxyl methyl) ethyl ester. Figure-27 Mass spectra and structure of 3„,8,8‟-trimethoxy-3-piperidyl-2,2‟- 67 binaphthalene-1,1‟,4,4‟-tetrone. Figure-28 GC-MS chromatogram of methanol extract of root part of F. 68 hamiltonii. Figure-29 Mass spectra and structure of Cyclopropane botanic acid, 2-[[2-[[2- 70 [(2-pentyl cyclo propyl)methyl]cyclo propyl]methyl]cyclo propyl]methyl]-, methyl ester. Figure-30 GC-MS chromatogram of dichloromethane extract of N. 71 procumbens. Figure-31 Mass spectra and structure of 3,7,11,15-tetramethyl-2- hexadecen- 73 1-ol. Figure-32 Mass spectra and structure of 12-methyl-E,E-2,13-octadecadien-1-ol. 73 Figure-33 Mass spectra and structure of 13-heptadecyn-1-ol. 74 Figure-34 Mass spectra and structure of n-hexadecanoic acid. 74 Figure-35 Mass spectra and structure of 9,12,15-octadecatrienoic acid, 2,3- 75 dihydroxy propyl ester, (Z,Z,Z)-. Figure-36 Mass spectra and structure of Phytol. 75 Figure-37 Mass spectra and structure of trans-13-octadecenoic acid. 76 Figure-38 Mass spectra and structure of Oleic acid. 76 Figure-39 Mass spectra and structure of Ethyl iso-allocholate. 77 Figure-40 GC-MS chromatogram of methanol extract of N. procumbens. 78 Figure-41 Mass spectra and structure of Pterin-6-carboxylic acid. 80 Figure-42 Mass spectra and structure of ergosta-5,22-dien-3-ol,acetate, 80 (3β,22E)-. Figure-43 Mass spectra and structure of 5H-cyclopropa[3,4] benz [1,2-e] azulen-5- 81 one,9,9a-bis (acetyloxy)-1,1a,1b,2,4a, 7a,7b,8,9, 9a-decahydro-2,4a,7b- trihydroxy-3-(hydroxylmethyl)-1,1,6,8-tetra methyl-, [1aR- (1aα,1bβ,2β,4aβ, 7aα,7bα,8α,9β,9aα]-

Figure No. Description Page No. Figure-44 Mass spectra and structure of Hexadecanoic acid, methyl ester. 81 Figure-45 Mass spectra and structure of 7,10-octadecadienoic acid. 82

Figure-46 Mass spectra and structure of 10-octadecenoic acid, methyl ester. 82

Figure-47 Tracing showing the spasmolytic effect of methanol extract of F. (A) hamiltonii on spontaneously contractions of isolated rabbit jejunum 90 preparations. Figure-47 Tracing showing the influence of methanol extract of F. hamiltonii 90 (B) on K+ (80nM) induced contraction in isolated rabbit jejunum preparation. Figure-48 Effects of methanol extract of F. hamiltonii on spontaneous rhythmic and 91 K+ (80mM) induced contractions in isolated rabbit jejunum preparations. Figure-49 Tracing showing the spasmolytic effect of methanol extract of N. 92 (A) procumbens on spontaneously contractions of isolated rabbit jejunum. Figure-49 Tracing showing the influance of methanol extract of N. procumbens by 92 (B) blocking the calcium channel on isolated rabbit jejunum. Figure-50 Effects of methanol extract of N. procumbens on spontaneous 93 rhythmic and K+ (80mM) induced contractions in isolated rabbit jejunum preparations. Figure-51 Effects of verapamil on spontaneous rhythmic and K+ (80mM) 93 induced contractions in isolated rabbit jejunum preparations. Figure-52 Tracing showing the bronchorelaxant effect of methanol extract of 94 (A) N. procumbens on cabachol (1µM) induced concentrations in isolated rabbit tracheal preparations. Figure-52 Tracing showing the bronchorelaxant effect of methanol extract of 94 (B) N. procumbens on by K+ (80mM) induced contractions in isolated rabbit tracheal preparations. Figure-53 Effects of methanol extract of N. procumbens on carbachol (1µM) 95 and K+ (80mM) induced contractions in isolated rabbit tracheal preparations.

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LIST OF ABBREVIATIONS

13C-NMR Carbon 13 nuclear magnetic resonance spectroscopy 1H-NMR Hydrogen 1 nuclear magnetic resonance spectroscopy AAS Atomic absorption Spectrophotometer AChE Acetyl Cholinesterase BChE Butryl Cholinesterase BP British Pharmacopoeia CAS # Chemical Abstracts Services number CIDS Cholistan Institute of Desert Studies DAG Diacyl glycerol DCM Dichloromethane DPPH 1,1-Diphenyl-2-picrylhydrazyl E.coli Escherchia coli F. hamiltonii Farsetia hamiltonii Royle FHAD Farsetia hamiltonii (DCM aerial part extract) FHRD Farsetia hamiltonii (DCM root part extract) FHAM Farsetia hamiltonii (Methanol aerial part extract) FHRM Farsetia hamiltonii (Methanol root part extract) FCR Folin-ciocalteu reagents GA Galic acid HPLC High performance liquid chromatography IC50 Inhibitory concentration 50 IFD International Federation of Diabetes IP3 Inositol-1,4,5-triphosphate GC-MS Gass chromatography mass spectrometry LOXs Lipoxygenases MIC Minimum inhibitory concentration NIST# National Institute of Standard Technology number NSCL Non small cell lung N. procumbens Neurada procumbens L. NPW-D Neurada procumbens (DCM extract) NPW-M Neurada procumbens (Methanol extract) Prob. Probability RT Retention time SEM Standard error of mean TFC Total flavonoid contents TPC Total phenol contents TLC Thin layer chromatography USP United State Pharmacopoeia QE Quercetin

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CHAPTER NO. 1 INTRODUCTION

1. Medicinal plants

Medicinal plants are traditionally used to treat different ailments. The history of medicinal plants is as old as the history of mankind. The medicinal plants are used for the treatment of different diseases in human beings. According to report of World Health Organization (WHO), “the medicinal plant is as whole or different parts of plant, contains potential bioactive constituents having importance in treatment of diseases” (Brussels, 2001). The plants having medicinal values are important in treating different diseases from the history of mankind and it is believed that the plants have the healing power. The important aspect of herbal medicine is safety whereas the efficacy is second consideration (Evans and Sauders, 2002). Two kinds of chemical constituents are produced by medicinal plants, primary and secondary metabolites. The Primary metabolites like carbohydrates, amino acids, fats which are vital for the growth and reproduction in the plant while, the Secondary metabolites like flavonoids and alkaloids are derived metabolites which perform many vital functions for the survival of plant. It is an understanding that the secondary metabolites are produced due to outer stress or exposure to harsh environment and provides protection to plants as result of mutation (Mann, 1994). Today, about 74% of prescribed drugs available in market are phytomedicines (Mugabe, 1999). The early 19th century is an era of scientific pharmacy; there was discovery and isolation of alkaloids, glycosides, tannins, saponosides, etheric oils, vitamins and hormones. Currently, plant drugs mentioned in pharmacopoeias like British Pharmacopoeia, United State Pharmacopoeia and European Pharmacopoeia are of real medicinal value. Now, the advanced countries like United Kingdom, Russia, Germany and USA have developed separate herbal drug pharmacopoeias (Biljana, 2012). The Holy Quran is everlasting reference book which describes the importance

also used and (ﷺ) of plants in different Surah‟s and our Holy Prophet Muhammad told the effectiveness of different plants species for various types of diseases (Ahmad et al., 2009). Plants have photochemical constituents as primary and secondary metabolites. The latter are important and used as agrochemical, antimicrobial agents, emetics, laxatives, colouring agent, flavouring agent, food additives and many medicinal purposes (Phillipson, 1990). According to the report of World Health Organization (WHO), about 4000 million people in developing countries believe in

3 the efficacy of herbal remedies (Abdelaaty et al., 2009). Natural medicines are preferred by the peoples due to efficacy, safety, low side effect profile and human system acceptability. So, it is evaluated factor that about eighty (80%) population of world in under developed countries use medicinal plants for health care (Newman, 2008). In different pharmacopoeias, about seven thousands (7000) bioactive compounds are mentioned, which are isolated from plants (Harvey, 2004). However, the only 5-15% out of five lac (5, 00,000) plants has been investigated for their phytochemical and therapeutic importance. Now-a-days, scientist and practioners in the world are emphasing the value of herbal drugs to achieve health parameters. The importance of herbal drugs has been further enriched by editing Pakistan Encyclopedia Planta Medica by Prof. Dr. Atta-ur-Rahman and Prof. Dr. Viqar ud din Ahmed, the great scientists of Pakistan. In order to evaluate the pharmacologically active constituents from plants, a systematic way is being carried out by using the modern techniques. In this way, new chemotherapeutic agents are obtained from the nature, which have the importance in combating the threats to humanity. 1.1 Cholistan desert Cholistan is a desert in south of Punjab, Pakistan spread over an area of twenty six thousands (26,000) square kilometres. This is unique naturally occurring bioregion. The environment is typically xeric confronted with multiple stresses and harsh conditions of high temperature (Arshad et al., 2002). Despite of adverse environmental conditions, Cholistan has diverse vegetation including shrubs, herbs, grasses and trees (Naz et al., 2011; Hameed et al., 2002 and Rao and Baber, 1990). The Cholistan flora consists of large number of plants having plant species 138 out of which 64 are medicinal plants (Arshad et al., 2007). The folk reported medicinal plants are extensively used by the natives and traditional herbal practitioners for the treatment of different kinds of diseases (Arshad et al., 2003). 1.2 Botanical description of family Brassicaceae This is a very big family consisting of 350 genera and about 3500 species. The family is well known due to oil producing seeds. The taxonomy of the family is primarily based on characteristics of fruit, so lack of fruit in herbarium specimen produced difficulty in correct identification. The plants of this family are annual to

4 perennial herbs, some are woody or some are shrubby. The leaves are spirally or alternate arranged. Hairs are usually simple or branched, appressed or absent in some species. Flowers are hermaphrodite, hypogynous, actinomorphic, pedicellate and ebracteate. Four sepals, often free with distinct claw and colour is white or yellow. The stamens are often six or rarely four or two or one. The fruit is long (siliqua) or short (silicula), indehiscent or descent, opening from below and seeds attached to replum and false septum. Seeds are many in one or two rows and smooth, granular or reticulated in shape (Jahangir et al., 2009). It is difficult to identify a genus following the key for tribes. Therefore, there is separate key for all the genus irrespective of their tribes is provided separately in flora. Also alternative keys for each genus under their tribes are provided. Five tribes are provided that showed the fruit characters which give help in identifying the generic and specific keys (Nasir and Ali, 1972). Generally, the plants of this family are used as anticancer, antibacterial, antifungal, antidiabetic, antirheumatic and have insecticidal activity (Rizk, 1986). Many plants of this family used as food source and condiment such as cabbage, turnip, radish and mustard. All these contain unusual flavonol glycoside and glucosinolates, which on hydrolysis give culinary flavours characteristics to plants (Tooky et al., 1980). 1.3 Botanical description of genus Farsetia Farsetia is a genus of flowering plant in the Brassicaceae family. It contains the 79 plants out of these 28 are accepted species. About 10 species in South West Asia, Central & North Africa and India. The plants of this genus are under shrubs or perennial, rare annual herbs, often with woody base and shrubby in appearance, much branched, erect or spreading, hoary with bipartite appressed white hair, leaves are linear oblong, sessile, racemes lax, ebracteate, elongated in fruit. Flowers are small or large, pale yellow, mauve, orange to whitish, shortly pedicelled, sepals erect, obtuse, not saccate, hoary. Petals are slightly longer to about twice as long as sepals, oblong or linear, clawed, apex rounded; margin often crimped and limbs are coiled inwards. Stamens six, filaments linear, anthers oblong-linear, lateral nectar glands horseshoe shaped, open towards the inner side, middle absent. Ovary, oblong, 10-40-ovuled, hairy, distinct style, siliquae oblong, compressed narrow to broad or rarely suborbicular, canescent with bipartite appressed hair, bilocular, dehiscent; gynophores obscure to very short but

5 distinct, valves with distinct mid vein, membranous septum with distinct mid vein and obscure reticulate venation, seeds uniseriate to biseriate, orbicular, winged, compressed and redicle accumbent. The fruits are often variable but size of flower, sepals and petals is reliable. The shrubby may deceive in a year old plant and look like annual (Nasir and Ali, 1972). The folk uses of the plants of this genus are used for relief of rheumatic pains, healing abilities, antibiotics, antidiabetic, antispasmodic and taking as a cooling medicine after pounding. This genus has importance regarding flavonoid compound possessing cytotoxic activity (Eman et al., 2013). This is genus of herbs and undershrubs occurring in the mediterranean region eastwards to India, Pakistan and southwards to tropical Africa. The genus Farsetia is represented by three species that grow in Egypt. The most common one is F. aegyptia Turra. The eight flavonoids compounds: (1) Isorhamnetin 3,7-diglycosides, and (2) 3-O-β-L-arabinopyranoside, 7 –

O - [ β – D – glucopyranosyl - (1->2) – α –L-rhamnopyranoside [C33H40O20] are already reported from this genus (NPD, 2012). The constituents (3) Kaempferol, (4) Kaempferoltrioside, (5) Kaempferol – 3 – O - (2⁪- β – D - glucopyranosyl) – α – L – rhamnopyranoside – 7 – O – α – L - rhamnopyranoside, (6) Kaempferol – 3 – 7 – di – O – α -L-rhamnopyranoside, (7) Apigenin-7-O-β-D-glucopyranoside and (8) Apigenin were isolated from the methanol extract of F. aegyptia (Marzouk et al., 2009) and three flavonol di-O-glycosides compounds were identified from the butanol extract of F. aegyptia using LC/UV-DAD technique (Shahat et al., 2005).Some glucosinolates were reported from the leaves of F. aegyptia and F. ramosissima (Victor & Alexander, 1980). According to flora of Pakistan, there are only seven species distributed in the desert of Pakistan including Farsetia hamiltonii. Which are given below. 1. Farsetia hamiltonii 2. Farsetia heliophila 3. Farsetia jacquemontii 4. Farsetia jacquemontii subsp. edgeworthii 5. Farsetia jacquemontii subsp. jacquemontti 6. Farsetia linearis 7. Farsetia ramosissima

1.4 Farsetia hamiltonii Royle It is perennial, shrubby, often woody at base, 10-40 cm long, densely hoary with bipartite appressed hairs, branches erect or subspreading, leaves oblong-linear, very variable in size, 10-40 mm long, 1-5 mm broad (rarely longer and broader), sessile, entire, strigose. Racemes 5-20 having flowers, lax, ebracteate, length of fruit is up to 30 cm. Flowers are 3 mm across, orange-coloured to whitish; pedicels 46 mm long in fruit, ascending, not thickened. The length of Sepals is about 3.5-4 mm and 1.0 mm broad. Petals slightly longer than sepals. Stamens 3 or 4 mm long; anthers c. 1.0 mm long. Siliquae are about 20 mm long and 3-4 mm broad, compressed, usually slightly narrowed towards both the ends; valves often slightly torulose; style 1.5 mm long, thin, tapering into a short, sub-bilobed stigma; seeds uniseriate, about 3 mm in diameter (Figure-1). The flowering period is March to September. It is endemic to the desert area of Pakistan and India (Bhandari, 1995). This plant species closely resembles to Farsetia linearis. The local name in the Punjab is “Farid Booti”or in some areas of desert known as “Lathi” (Figure-3). The medicinal plant Farsetia hamiltonii Royle has reference for folk use as antidiabetic remedy. (Marzouk et al., 2009; Kirtikau and Basu, 1975).

The taxonomic hierarchy of Farsetia hamiltonii Royle is given below.

Kingdom: Plantae

Subkingdom: Angiosperms

Phylum: Eudicots

Class: Rosids

Order: Brassicales

Family: Brassicaceae

Genus: Farsetia

Species: Farsetia hamiltonii Royle

Figure-1: Aerial view of Farsetia hamiltonii Royle.

Figure-2: Morphological characters and features of Farsetia hamiltonii Royle.

1.5 Botanical description of family Neuradaceae This family has 3 genera and 10 species which are occurring in mediterranean region including India and South Africa (Willis, 1973). Three genuses in this family are Neurada, Neuradopsis and Grielum. The characteristics of the family are connate carplels within the calyx tube, this feature differenciate from Rosaceae family. In Neuradaceae, gynoecium syncarpous have 10 styles and 10 locules while in Rosaceae gynoecium syncarpous range from 2 to 5 styles. Very unique type of pollen grains having 3-armed aperture at both the poles and each arm is provided with a circular ores. Hence, it is justified to treat as independent family Neuradaceae (Nasir and Ali, 1972). The plants of this family are annual, prostrate, woolly, herb with alternate leaves, petiolate, usually with minute stipules or exstipulate, mostly small flowers, solitary or in pairs. Calyx 5-fid, lobes alternating with 5 spiny bracteoles, small 5 petals, 10 stamens, carples 3-10, slightly united at base and adnate to throat of calyx tube (hypanthium) tapering into slightly exerted, persistant style. Minute stigma, capitates, ovary inferior, 5-10 loculed, having only ovule in each locule. Fruit is dry, woody, flattened, orbicular, spiny or winged, few seeded. Seeds are horizontal, non endospermic. Seeds are retained in hard woody, discoid fruit, perforated by the plumule upwards and by the radical downwards, persists as a collar around the hypocotyls in case of Neurada genus (Nasir and Ali, 1972). 1.6 Botanical description of genus Neurada It is a monotypic genus Neurada L. (Neurada procumbens L.) originally described by Aegypto-arabian. The plants of this genus are annual herb, stem prostrate, unarmed, woolly, leaves are alternate, petiolate, simple, and lobed and stipules are minute or absent. Flowers are small, pedicellate, axillary, solitary or in pairs. Calyx 5-lobed, persistent cup-shaped, shallow, expanded, nearly flat below, contracted above, echinate, forming a conical disk with carpels. Five petals, small slightly exceeding the calyx, greenish-white colour. Stamens are ten, inside throat of calyx tube. Carpels 10, connate at base and adnate to hypantium, tapering into slightly exserted, subulate, spinescent styles, ovule one in each carpet fruit dry indehiscent, discoid or pentagonal, hard, woody, densely echinate above, smooth or wooly beneath seeds curved (Nasir and Ali, 1972).

In the plants of this genus, seeds are produced within hard discoid pericarp, both radicles and plumules perforate the pericarp, grow downwards and upwards respectively. The old spiny hypanthium often persists as a collar round base of the new plants. The plants of Neurada genus are widely distributed in all territories of Egypt and also in North Africa to India and Sudanian region (Zaki et al., 2007). This genus includes three species in sub family Neuradaceae. Previously, this genus is placed in Rosaceae family and order Rosales (Endlicher, 1836; Focke, 1891) but this genus differs from Rosaceae by its connate carpels within calyx tube and aberrant pollen (Purohit and Panigrahi, 1991). This genus also shows some resembles to Malvaceae due to petals inserted on calyx throat (Willis, 1973). Now, this genus is placed in family Neuradaceae under the order Malvales (Takhtajan, 1980; Cronquist, 1981; Dahigren, 1983). A new species Neurada al-eisawi was identified from the southern desert of Jordan, this species is distinguished from the N. procumbens L. by some taxonomic features of the fruit (Barsotti et al., 2000). According to flora of Pakistan, there is only single species N. procumbens distributed in desert of Pakistan (Nasir and Ali, 1972). 1.7 Neurada procumbens L. The local name of this plant is “Chapperi Booti” (Figure-3). It is annual prostrate, woolly canescent herb, length of root is up to 180 mm long having diffused or procumbent branches which are 100-322 mm long. The leaves have size 6-25 x 4- 15 mm, oblong-ovate, pinnatilobed, lobes obtuse, 1.0 to 3.0 on each side, densely hairy vary in size. The simple and branched hairs; bistipulate, stipules minute; petiole are 3 to 8 mm long, densely hairy. Pedicels 3 to 6 mm long in fruit. Receptacle 5.6 mm in diameter with 5 spiny, subulate, bracteoles. Sepals are 5, acute, long or broader than the alternating bracteoles, connivent above, tube flat, spiny, forming a disc with the mature carpels, the outer teeth triangular, the inner lanceolate. The size of petals are 5, 2.0-4.3 x 1.4-2.0 mm, inserted on the throat of the calyx tube, obovate or oblanceolate, white or light greenish, distinctly veined, slightly exserting the sepals. The stamens are ten, 2.0 mm long, filament slender, glabrous, with broadly ovate anthers, 0.5 x 0.5 mm. Carpels are ten, partialy connate, each one ovuled; styles longer than stamens, spinescent, sparingly tomentose, accrescent and hard in fruit; stigma minute, capitellate. Fruit dry, plano-orbicular, diameter is about 8 to 15 mm, convex above, flat below, persisting as a collar around the young roots of the

11 seedlings even after the plant is fully grown. Seeds curved about 2.5 x 1.5 mm in size, non endospermic, horn like with tapering distal end; testa dark brown, shiny (Figure- 4). It is distributed in Afghanistan, Pakistan, North West Indian, Arabia, Palestine, Syria, Iraq, north Iran, North Africa and mediterranean (Alyemeni et al., 2000). It is fairly common species of dry region, particularly on sand dunes. Two varieties of N. procumbens was recognized: (1) N. procumbens L. var. procumbens (2) N. procumbens varstellata (Zohary, 1966). N. procumbens has a reference regarding its folk use in diarrhea, dysentery and respiratory functions (Abdelaaty et al., 2009). Two dihydroflavonol glycoside were isolated and identified form Neurada procumbens are 3-O-α-L-rhamnosyl-(2R,3R)- taxifolin or called astibin and 6-C-(β-D-glucopyranosyl-3,5,7,3',4'-pentahydroxy-8-β- D-glucopyranosyloxyflavan (Afifi et al., 2008).

The taxonomic hierarchy of Neurada procumbens L is given below:

Kingdom: Plantae

Subkingdom: Angiosperms

Phylum: Eudicots

Class: Rosids

Order: Malvales

Family: Neuradaceae

Genus: Neurada

Species: Neurada procumbens L.

Figure-3: Aerial view of Neurada procumbens L.

Figure-4: Morphological characters and features of Neurada procumbens L.

1.8 Aims and objectives of study Herbal medicines are now-a-days increasing their demand due to synergistic and harm effects neutralizing combination (Gilani and Atta-ur-Rahman, 2005). There is also an importance of harmful effects of synthetic molecules, due to which trend is again shifting towards natural products. About seven thousands (7000) medicinal compounds isolated from plants that are used as drug, mentioned in different pharmacopoeias. Up to now, only 5-15% out of 250,000-500,000 plant species have been evaluated for their therapeutically active compounds. So, there is need to evaluate the remaining 85% plant species for biological and phytochemical purposes to serve the humanity. Modern pharmaceutical chemistry has open a new era of natural products to study analytical and structural chemistry. The isolated and purified compounds are subjected to their structure determination by using the advance techniques nuclear magnatic resonanace (1H- NMR, 13C-NMR) and mass spectrometry (GC-MS, EI-MS). Further, the in vitro and in vivo studies are designed to elucidate the actions of these compounds on human body. The pharmaceutical industries are again starting the research on natural products in advance countries. Herbal drugs are preferred by peoples due to their safety profile and less treatment cost, hence interest is increasing towards natural remedies. Our aim is to focus efforts to continue searching the medicinal plants of Cholistan desert for their therapeutic value as per their folk uses. With in-depth analysis at the bench to bedside will take us to use standardized herbal preparation for most prevalent respiratory and gastrointestinal problems in this area. The aim of this study was to select the medicinal plants from Cholistan desert of Pakistan having folk medicinal importance and residents of Cholistan desert are using such plants as remedy to treat different diseases since long. The flora of Cholistan desert has 28 families and 138 species out of which about 64 species have been identified as medicinal plants. There is very little work is reported regarding biological activities and phytochemical analysis of medicinal plants of Cholistan desert. So, in this study two medicinal plants F. hamiltonii and N. procumbens were selected on the history of their folk uses and emphasize is given on photochemical and biological evaluation of selected medicinal plants. On the basis of comprehensive literature review and perusal, there is no reported work regarding phytochemical analysis, enzyme inhibition and biological activities on

F. hamiltonii. The reported cytotoxic flavonoid compounds are isolated from another species of genus Farsetia and studies effect of these flavonoid compounds against non-small cell lung (NSCL) adenocarcinoma, breast carcinoma colon carcinoma and hepatocellular liver carcinoma cell lines. The other selected medicinal plant N. procumbens from Cholistan desert is that whose methanol and aqueous extracts have cytotoxic activity and only two chemical constituents are isolated from the plant. So, there is need to explore the phytochemical and biological potential of both selected medicinal plants. The general objective is phytochemical and biological studies of selected medicinal plants F. hamiltonii and N. procumbens from Cholistan desert of Pakistan. The ethnomedicinal importance of F. hamiltonii and N. procumbens driven us to explore the therapeutically potential of these plant. The objective of present study was to investigate the phytochemical, enzymatic and biological studies including antioxidant, anticholinesterase, antilipoxygenase, antichymotrypsin, anti-α glucosidase, antiurease, antityrosinase, anticarbonic anhydrase I, antiphosphodiesterase II, antibacterial, spasmolytic, bronchorelaxant and hypoglycaemic activities to prove the folk therapeutic uses of F. hamiltonii and N. procumbens for respiratory and gastrointestinal diseases. So, the defined objectives of the study are; Objective 1: To investigate the phytochemical constituents of aerial and root parts of F. hamiltonii. Objective 2: To study the in vitro and in vivo biological / enzyme inhibition / pharmacological properties of aerial and root parts of F. hamiltonii for gastrointestinal and hypoglycaemic disorders. Objective 3: To investigate the phytochemical constituents of whole plant of N. procumbens. Objective 4: To study the in vitro biological / enzyme inhibition / pharmacological properties of whole plant of N. procumbens for gastrointestinal and respiratory disorders.

CHAPTER NO. 2 LITERATURE REVIEW

2. Literature review on selected plants 2.1 Literature review on Farsetia hamiltonii Royle Farsetia hamiltonii Royle is a desert medicinal plant belongs to family Brassicaceae. 2.1.1 Literature survey on the phytochemical studies of family Brassicaceae Many horticultural crops are included in the Brassicaceae family and the profile of different species is well established having significant amounts of bioactive compounds (Jahangir et al., 2009). The plants of the family possess phytochemicals like vitamins and phenolic compounds, which are health promoting (Vallejo et al., 2002, 2003, 2004; Dekker et al., 2000), fiber, phytic acid, soluble sugars (Pedroche et al., 2004), glucosinolates (Fowke et al.,2003), poly phenols, minerals (Heimler et al.,2005), carotenoids and fat (Zakaria-Rungkat et al.,2000). 2.1.1.1 Flavonoids Flavonoids are phenolic compounds having 15 carbons atoms in manner C6- C3-C6 including three-carbon bridge connecting two rings of flavonone skeleton. They are mostly in O-glycosidic forms with sugar such as glucose, glactose, rhamnose, arabinose etc., but they also exist in C-glycosides. About 25% flavonoids exist in freestate. They have isomers with same molecular weight but different aglycone and sugar component (de Rijke et al., 2006). These are present in epidermis of fruits and leaves in high concentrations and are vital secondary metabolites in plants. Flavonols are mostly occurring form of flavonoids. Quercetin (1), Kaempferol (2), Isorhamnetin (3) and Cyanidin (4) are found as O-glycosides, conjugation occurs at 3 position of the C-ring. The substitution positions are at 5, 7, 4‟, 3‟ and 5‟. They are also found in acylated form by different hydroxycinnamic acids (Maria et al., 2011). They have antiviral, antimicrobial, anti-inflammatory, antihepatotoxic, antiosteoporotic, antiallergic, antiulcer, antiproliferative and immunomodelatory activities (Tapiero et al., 2002).

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2.1.1.2 Hydroxycinnamic acids These are characterized by C6-C3 structures and are non-flavonoid phenolic compounds. Their role is as defensive agents in plants and free found in cell wall. The most common compounds found in Brassicaceae species are p-coumaric acid (5), Sinapic acid (6), 3-O-caffeoylquinic acid (7) and Ferulic acid (8) are found in conjugation with other hydroxycinnamic acids or sugars (Maria et al., 2011).

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2.1.1.3 Vitamins The vegetables of family Brassicaceae contain high concentrations of vitamins including ascorbic acid, folic acid, carotenes and tocopherols (Heimler et al., 2005; Kurilich et al., 1999; Verhoeven et al., 1996). The vegetables of Brassicaceae family have different values (μg/g) of vitamins on fresh weight basis is given below.

Ascorbic α – α – β- Carotene Folate Vegetables acid Carotene Tocopherol (μg/g) (μg/g) (μg/g) (μg/g) (μg/g) Broccoli 748.5±62 0.3 8.9 16.2 1.771 Kale 186 0.6 48.6 19.2 – Cauliflower 499±53 - 72±0.5 1.7 0.53 Chinese 253 - 0.1 0.8 0.81 cabbage White 188±13 0.02 0.8 1.7 - cabbage Brussels 158 - 1.4 1.5 - sprouts

2.1.1.4 Minerals The plants of this family are enrich in minerals like calcium (Ca), Iron (Fe) and green leafy vegetables accumulating high levels of P, K, Cl, Ca, Fe, S, Sr, (Miyazawa et al.,2005; Lefsrud et al., 2006). The different minerals values in μg/g among different vegetables of Brassicaceae on fresh weight basis are given below.

Minerals Broccoli Kale Cauliflower Chinese White Brussels (μg/g) cabbage cabbage sprouts Calcium 272.4±20 2860±430 175±17 470±60 440±60 356±13 Iron 8.7±0.5 4±2 5.0±0.3 2±0.3 1.4±0.3 7.6±0.2 Copper 0.94±0.07 0.4±0.2 0.56±0.07 0.4±0.2 0.5±0.5 0.9±0.09 Magnesium 181±8 510±40 145±22 130±30 140±20 207±12 Potassium 2890±70 7120±5170 2210±140 2280±1120 2660±870 4250±250 Znic 9.5±0.3 2.9±0.5 6.4±0.3 2.3±0.4 2±1 5.8±0.4 Sodium 180±6 120±40 192±27 50±20 30±10 107±7 Menganese 1.92±0.09 3±1 1.31±0.07 0.5±1.4 2±1 2.31±0.13

2.1.1.5 Phytoalexins Phytoalexins are compounds produced by the plants of the family Brassicaceae play a role in protection pathogen attack. They have structural diversity in their molecules (Smith, 1996; Grayer and Harborne, 1994). The phytoalexins isolated from crucifers are Brassinin (9), Brassitin (10), 1-methoxybrassinin (11), 4-methoxybrassinin (12), 1- methoxybrassitin (13), 1-methoxybrassenin A (14), 1-methoxybrassenin B (15), Cyclo brassinin (16), Cyclobrassinin sulfoxide (17), Cyclobrassinone (18), Dehydro-4-

20 methoxycyclobrassinin (19), Spirobrassinin (20), 1-methoxyspirobrassinin (21), 1- methoxyspirobrassinol (22), 1-methoxyspirobrassinol methyl ether (23), Dioxibrassinin (24), methyl 1-methoxyinodole-3-carboxylate (25), Brassilexin (26), Sinalexin (27), Brassicanal A (28), Brassicanal B (29), Brassicanal C (30), Camalexin (31), 6-methoxy camalexin (32), 1-methyl camalexin (33). (Pedras et al., 2000).

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2.1.1.6 Glucosinolates Glucosinolates are sulphur containing compounds. They contain cyno and sulphate groups. Glucosinolates are derived from biosynthesis of amino acid and having vital importance in Brassicaceae family. They have a role in defense mechanism of plant against pests and microbes (Zrybko et al., 1997). The most important health-promoting glucosinolates which are reported in this family are Glucoraphanin, Glucoiberin, Glucoalyssin, Glucobrassicanapin, Gluconapin, Glucobrassicin, Neoglucobrassicin, Gluconasturtiin, 3-butenyl glucosinolate (gluconapin), 4-methyl sulﬁnylbutyl glucosinolate (glucoraphanin), 3-indolylmethyl glucosinolate (glucobrassicin), Progoitrin, Glucoiberin, Epiprogoitrin, Glucrafanin, Sinigrin, Gluconapoleiferin, Gluconapin, Glucoalisin, 4-hydroxybrassicin, Glucobrassicin, Glucobrassicanapin, Gluconasturein, Methoxy glucobrassicin, Neoglucobrassicin, 2-propenyl, 3-methyl-sulphinylpropyl and Indol-3-yl-methyl (Vallejo et al.,2004; Iori et al., 2004; Kushad et al., 1999; Kushad et al.,2004; Kusznierewicz et al., 2008).

2.1.2 Phytochemical studies of the genus Farsetia

The several flavonoids compounds: Isorhamnetin 3,7-diglycosides; 3 – O – β – L -arabinopyranoside, 7 – O - [ β – D – glucopyranosyl - (1->2) – α – L - rhamnopyranoside [C33H40O20] are already reported from this genus (NPD, 2012).

Various constituents such as Kaempferol, Kaempferoltrioside, Kaempferol – 3 – O - ( 2 – β – D-glucopyranosyl) – α – L –rhamnopyranoside – 7 – O – α – L - rhamnopyranoside, Kaempferol – 3 – 7 – di –O – α – L -rhamnopyranoside, Apigenin- 7-O-β-D-glucopyranoside and Apigenin were isolated from the methanol extract of F. aegyptia (Marzouk et al., 2009) and three flavonol di-O-glycosides compounds were identified from the butanol extract of F. aegyptia using liquid chromatography technique with detector UV-DAD (Shahat et al., 2005). The phenolic rich fraction of this fraction was further fractionized and isolated a new flavonoid kampferol-8-C-O-β- diglucopyranoside. Further, Friedelin, Betulin, β-amyrin, Scopoletin and Coumarin were isolated from low polar fraction and identified according to its spectral data. The fraction which is phenolic rich was further fractionized and isolated a new flavonoid Kampferol-8-C-O-β-diglucopyranoside. Some glucosinolates were reported from the leaves of F. aegyptia and F. ramosissima (Victor & Alexander, 1980). The level of glucosinolates in F. aegyptia was monitored and identified (Al-Gendy & Lockwood, 2005). The novel flavonoid compound kaempferol-7-8-diglucoside was first time isolated from methanol extract of F. aegyptia along with two other compounds Kaempferol and Qurestin. The following compounds but-3-enylglucosinolate (34), 4-(methylthio) butylglucosinolate (35), Kaempferol-7,8-diglucoside (36), Kaempferol (37), Quercetin (38), Scopoletin (39), Scopoletin-7-glucoside (40), Catechin (41), Gallic acid (42), p-hydroxybenzoic acid (43), Pyrogallic acid (44), p-coumaric acid (45), Phenol (46), Coumarin (47), Isorhamentin (48), Apigenin (49), Betulin (50), Friedelin (51) and β-amyrin (52) were isolated and identified in past from the genus Farsetia.

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(50) (51)

(52)

2.1.3 Studies on the biological activities of the genus Farsetia The literature survey of genus Farsetia revealed that only cytotoxic, antimicrobial and antioxidant activities have been reported. There is very little work already reported, detail is given below. 2.1.3.1 Cytotoxic activity The ethyl alcohol extract of aerial parts of F. aegyptia was investigated in vitro for cytotoxic potential against non-small cell lung adenocarcinoma (A549), hepatocellular liver carcinoma (HepG2), colon carcinoma (HCT116) and breast carcinoma (MCF-7) cell lines. It exhibited cytotoxicity againgt A549 and HepG2. Kampferol-8-C-O-β-diglucopyranoside showed the cytotoxicity against epitheloid cervix carcinoma (Hela) and MCF-7 cell lines (Eman et al., 2013). 2.1.3.2 Antimicrobial activity It is due to increase of microbial resistance factor, the scientists are trying to search out new antimicrobial agents from the natural source like plants (Ullah et al., 2009). The alcohol extract of F. aegyptia is evaluated for antibacterial and antifungal activities which exhibited significant inhibition against Klebsiella pneumonia and no activity against Candida albicans (Atta et al., 2013). The kale leaves (B. oleracea) exhibited inhibition of Gram positive bacterial growth (Bacillus subtilis, Staphylococcus aureus, Enterobacter faecalis) and Gram negative bacterium Maraxella catarrhalis (Maria et al., 2011).

2.1.3.3 Antioxidant activity

It is due to presence of flavonoids or phenol contents, the plants of the family Brassicaceae possess the higher antioxidant activity like quercetin a flavonol compound found in high concentrations in broccoli. A flavonoid compound kaempferol also showed very high antioxidant property. The antioxidants are possible cardioprotective compounds which play a role in reducing the oxidative stress produced from ROS and inhibiting the oxidation of low density lipoproteins (LDL) having importance in cardiac disease atherosclerosis (Podsedek, 2006; 2007; 2008).

2.1.4 Folk uses of genus Farsetia The folk uses of genus Farsetia reported by indigenous communities of Cholistan desert and herbal prescriber are given below.

Sr. No. Species Part used Traditional uses Reference 1 F. hamiltonii Whole Constipation, piles, pain, Shaheen et al., Royle plant abdominal / stomach problem. 2014.

2 F. hamiltonii Aerial part Nerve tonic and Cooling agent Raza et al., Royle 2014.

3 F. hamiltonii Whole Antidiabetic, antispasmodic, Marzouk et Royle plant Rheumatic pain, Cooling agent al., 2009.

4 F. aegyptia Whole Anticancer, antifungal, Kirtikau Basu, Turra plant antibacterial, antidiabetic, 1975. antirheumatic.

2.2 Literature review on Neurada procumbens L.

2.2.1 Literature survey on the phytochemical studies of family Neuradaceae

Neuradaceae is a very small family having 3 genera and 10 species. The most known plant of this family is Neurda procumbens having the medicinal uses. It is rich source of secondary metabolites like flavonoids, glycosides, bounded anthraquinones, terpenes and sterols (Mossa et al., 1983).

2.2.2 Phytochemical studies of Neurada procumbens L.

Two dihydroflavonol glycoside were isolated and identified from N. procumbens are 3-O-α-L-rhamnosyl-(2R,3R)-taxifolin or called astibin and 6-C-(β– D–glucopyranosyl- 3, 5, 7, 3', 4'–pentahydroxy–8–β–D-glucopyranosyloxyflavan (Afifi et al., 2008).

The following compounds seven flavonoids; Taxifolin (1), Taxifolin 3 – O – β -rhamnopyranoside (2), Vitexin (3), Vitexin 2' – O – α - rhamnopyranoside (4), Orientin 2' –O – α - rhamnopyranoside (5), Isoorientin 2' – O – α - rhamnopyranoside (6) and p-hydroxybenzoic acid (7) have been isolated from the whole plant of N. procumbens L. (Marzouk et al., 2014).

(1) (2)

1 2

(3) 3 (4)

(5) (6)

(7)

2.2.3 Biological activities of Neurada procumbens L.

2.2.3.1 Effect on blood pressure The aqueous extract of N. procumbens had effect on blood pressure of rats was studied. On oral administration of extract, the blood pressure of normotensive rats was increased and this increase in blood pressure was may be mediated through α- adrenergic receptors. It was further evaluated that elevation of blood pressure produced due to vasoconstriction effect on the aortic strips of rats in vitro. So, it warns that N. procumbens use may not be safe for persons with cardiovascular diseases (Chen et al., 2004). 2.2.3.2 Antibacterial activity It is currently reported in year 2017 that aqueous methanol (80%) extract of Neurada procumbens showed inhibition zone against Serratia marcescens (10±4.0mm) and Staphylococcus aureus (8±1.7mm) by agar well diffusion method (Oke et al., 2009; Shahat et al., 2017). 2.2.3.3 Antifungal activity The aqueous methanol (80%) extract of Neurada procumbens have antifungal activity and showed inhibition zone against Aspergillus flavus (10±2.0mm) and Fusarium moniliforme (13±3.6mm) by agar well diffusion method (Ndukwe et al., 2004; Shahat et al., 2017).

2.2.4 Folk uses of Neurada procumbens L. The significance of selected medicinal plant N. procumbens used by the local communities of Cholistan desert and others is evaluated below. Part Sr. No. Species Traditional uses Reference used 1 N. procumbens Aerial Nerve tonic and cooling agent Raza et al., L part 2014 2 N. procumbens Whole Anthelmintic, anticonvulsant, Ahmed et L plant Laxative al., 2014. 3 N. procumbens Whole Earache, eczema, remove bladder Ahmed et L plant stone, infection, rheumatism, al., 2014. sexual problem. 4 N. procumbens Whole For diarrhea, dysentery, heart and Abdelaaty L plant respiration problems. et al., 2009. 5 N. procumbens Whole To treat diarrhea, dysentery, Chen et at., L plant tonic for heart problems and 2009. improvement of respiratory functions.

CHAPTER NO. 3 MATERIAL AND METHODS

3.1 Plant collection Farsetia hamiltonii Royle and Neurada procumbens L were collected from the Jamsar, Kanday wala tooba, Kharlan wali, Dukar and Jugait peer areas of Cholistan desert of Bahawalpur Division in April, 2013. The selected medicinal plants were identified and authenticated by research officers of CIDS (Cholistan Institute of Desert Studies), The Islamia University of Bahawalpur, Bahawalpur, Pakistan. The photographs (Figure-1 and 3) were taken. The herbarium sheets of the both plants of size (12.5” × 18.5”) were prepared and deposited at CIDS for the future reference (Figure-5 and 6). The voucher specimen numbers 3470/CIDS/IUB and 3477/CIDS/IUB were allocated to F. hamiltonii Royle and N. procumbens L., respectively.

3.2 Chemicals and reagents In this study, all chemicals and reagents which have been used were of analytical grade such as methanol, dichloromethane, acetone, ethyl acetate, chloroform, ethanol, hydrochloric acid, nitric acid, sulfuric acid, silver chloride, silver nitrate, sodium chloride, potassium chloride, sodium hydroxide, sodium dihydrogen phosphate, folin ciocaltu reagent (FCR), vanadomolybdate reagent, EDTA, erichrome black T, starch, sucrose, quercetin, galic acid, gum acacia, lithium chloride, ninhydrin and vanillin, purchased from renowned firms like Merck, Fisher, Icon, Sigma Aldrich, VWR; which were supplied through their authorized agents / distributor in Pakistan (which are Ayub brothers, Kemisk, New Chemical centre, UH analytical supplies, Lab Master). The aluminum sheets (20×20 cm) of Thin Layer Chromatography and silica gel 60 F254 (Merck, Germany) were used in the study.

3.3 Instrumentation The equipments which were used in this work were rotary evaporator with vacuum pump (Buchi), spectrophotometer (Shimadzo, Japan), atomic absorption spectrophotometer (Agilent, USA), GC-MS (Agilent, USA), microplate reader 96- wells (Synergy HT BioTek), glucometer (Accua Check Performa, Roche, Germany) and Power Lab Data Acquisition system (AD instruments, Sydney, Australia).

Figure-5: Herberium sheet of Farsetia himaltonii Royle (specimen voucher No.3470/CIDS/IUB).

Figure-6: Herberium sheet of Neurada procumbens L. (specimen voucher No. 3477/CIDS/IUB).

3.4 Detection of secondary metabolites The dry powders or plant material of F. hamiltonii Royle and N. procumbens L. had been evaluated for the presence of flavonoids, glycosides, cardiac glycosides, alkaloids, bonded and non-bonded anthraquinones, saponins, terpenes and sterols (Table-1). Further, the total flavonoid and phenol contents are determined (Table-4). 3.4.1 Detection of alkaloids The powdered plant materials (3.0 gram) of F. hamiltonii and N. procumbens were boiled separately in 10 ml dilute hydrochloric acid for one minute, then cooled and wait to allow to settle. The mixtures were filtered separately. Taken 1.0 ml of the filtrate and added 3 drops of dragendroff‟s reagent in it. It was observed that no precipitation occurred. The dilute ammonia solution was used to alkaline the filtrate. Then performed the liquid extraction with chloroform in separating funnel. The chloroform layer was separated and then again extracted with 10 ml of dilute acetic acid in another separating funnel. Then collected the acetic acid layer and divided three portions. In one portion added few drops of dragendroff‟s reagent and in second portion added few drops of Mayer‟s reagent. The turbidity or precipitates produced in two portions were compared with control portion (Brain and Turner, 1975). The results are given in Table-1. 3.4.2 Detection of cardiac glycosides The powdered plant material (5.0 gram) of both plants were boiled in 10 ml of 70% aqueous alcoholic solution for about 2 minutes and filtered immediately. In the filtrate, added double volume of water and added 1.0 ml of lead sub acetate solution. After filtration, liquid-liquid extraction with chloroform was performed in separating funnel. Then collected the chloroform layer and evaporated on water bath. On complete dryness, added 3 ml of ferric chloride (3.5%) in glacial acetic acid. Then, on addition 1.5 ml of sulfuric acid, a brown colour ring at interface show the presence of cardiac glycosides (deoxy sugar) (Brain and Turner, 1975). The results are given in Table-1. 3.4.3 Detection of anthraquinones The free anthraquinones were detected by extracting the 3.0 gram powdered plant material of both plants in 10 ml of hot water for 5 minutes. After cooling, the mixtures were filtered separately. The filtrates were subjected to extraction with carbon tetrachloride by using the separating funnel. The layer of

36 carbon tetrachloride was separated, after washing with water, the 5.0 ml of dilute ammonia solution was added. No pink or cherry red colour appeared in that layer (Brain and Turner, 1975). For detection of bounded anthraquinones (C-glycoside and O-glycoside), powdered plant material of both plants was heated on water bath in a solution of

FeCl3 (10 ml) and dilute HCl (5 ml) for 10 minutes. The mixture was filtered. Further, the same procedure was repeated as for free anthraquinones. The end point is pink to cherry red colour in that layer indicates presence of bounded anthraquinones (Brain and Turner, 1975). The results are given in Table-1. 3.4.4 Detection of flavonoids The powdered plant material (5 gram) of both plants got boiled in 20 ml of 80% ethanol for 10 minutes and added magnesium metal foil. After that added, conc. HCl drop wise and orange red color developed shows the presence of flavonoids (Brain and Turner, 1975). The results are given in Table-1. 3.4.5 Detection of saponins The powdered plant materials (5 gram) of both plants were mixed with water (5 ml) in the test tube. The mixture was vigorously shaken and observed for occurrence of persistent froth (Brain and Turner, 1975). The results are given in Table-1. 3.4.6 Detection of terpenoids The powdered plant material (5.0 gram) of both plants were extracted with 15 ml of chloroform and filtered after 20 minutes. In the filtrate added few drops of conc. sulfuric acid carefully along with the wall of test tube. As result, the reddish brown circle at interface was formed (Ayoola et al., 2008). The results are given in Table-1. 3.4.7 Detection of sterols The powdered plant material (5.0 gram) of both plants were mixed with chloroform and then filtered. Added acetic acid (92 ml) and concentrated sulphuric acid (2 drops) in 1 ml of the filtrate. The solution turned dark green colour (Ravishankara et al., 2002). The results are given in Table-1. 3.5 Determination of mineral contents The dry powder or plant material of aerial part of F. hamiltonii and whole plant of N. procumbens were used for determination of mineral contents by using the technique atomic absorption spectroscopy.

3.5.1 Atomic absorption spectroscopy Atomic absorption spectrometer is an equipment for determination of mineral contents in sample and by using this technique 62 different metals can be analysed. In this technique the free atom (gas) can absorb radiation at specific frequency and the analyte concentration is determined from the amount of absorption. AAS analysis was performed on Agilent atomic absorption spectrophotometer (240 AA) with wave length range 185-900 nm. This equipment having operating range 5-45 °C and attached four ultra AA lamps. The dry powder / plant material of Farsetia hamiltonii (aerial part) and Neurada procumbens were used in AAS analysis. The AAS analysis results showed the presence of six (06) elements (Table-2).The sample was prepared by dissolving the powdered plant material in distilled water for determination of potassium, sodium and magnesium and in 0.1 N-HCl for iron, manganese and zinc. The spectrophotometer was power on and used after few minutes by applying the suitable flame gases. Every mineral content was analysed by specific hollow cathode tube. For construction of standard cure, the standard solutions are used one by one before sample solutions analysis. The results were recorded as per method reported in literature (Scott and Brewer, 1983) comparing with standard and given in Table-2. 3.6 Extraction The selected plants were air-dried in simple shade for two weeks and then, ground in fine powder form by small scale crushing mill. The powder of aerial part of F. hamiltonii was weighed 900 grams, root part was 200 grams and whole plat of N. procumbens was weighed 850 grams were macerated in solvent dichloromethane for 24 hours, the same process was repeated for 72 hours with methanol. The both dichloromethane and methanol extract were converted in solid residue under reduce pressure by using rotary evaporator. Six extracts were prepared which were abbreviated as under during the study. The extraction yields are given in Table-3. (1) Farsetia hamiltonii aerial part extract in dichloromethane (FHAD). (2) Farsetia hamiltonii aerial part extract in methanol (FHAM). (3) Farsetia hamiltonii root extract in dichloromethane (FHRD). (4) Farsetia hamiltonii root extract in methanol (FHRM). (5) Neurada procumbens (whole plant) extract in dichloromethane (NPD). (6) Neurada procumbens (whole plant) extract in methanol (NPM).

3.7 Preparation of reagents For the phytochemical screening of the plants material of Farsetia hamiltonii and Neurada procumbens, the following reagents were prepared.

3.7.1 Mayer’s reagent It is a solution of potassium mercuric iodide which was prepared by mixing solution of 1.36 grams mercuric chloride in water (60 ml) with solution of 5.0 grams potassium iodide in water (20 ml) and 100ml final volume was prepared. 3.7.2 Dragendorff’s reagent It is a solution of potassium bismuth iodide, which was prepared by dissolving 25 gram tartaric acid in 100 ml water and added 2.1 gram of bismuth oxide nitrate to it. This mixture was shaken for one hour and then added in 50 ml of 40% (w/v) potassium iodide solution. The finally formed solution was kept for 24 hours and then used after filtration. 3.7.3 Wagner’s reagent It is a solution of iodine in potassium iodide and was prepared by dissolving 4.0 grams of potassium iodide in 10 ml water. After that, added 2.0 grams of iodine in it. The mixture was shaken until iodine was dissolved completely. The volume of the resultant solution was 100 ml with water. 3.7.4 Hager’s reagent This reagent was obtained by mixing picric acid in 100 ml water till saturation and filtered to remove excess of picric acid. 3.7.5 Godin’s reagent It was prepared by mixing 1% vanillin solution in ethanol and 3% perchloric acid in equal volumes. 3.8 Preparation of solutions 3.8.1 Dilute sulphuric acid (10%)

The conc. sulfuric acid (10 ml) was added to ethanol drop wise till to make final volume up to 100 ml. 3.8.2 Dilute ammonia solution The strong ammonia (375 ml) was diluted with water and made the final volume up to 1000 ml.

3.8.3 Dilute hydrochloric acid The 274 gram of concentrated hydrochloric acid was added in 726 gram water and mixed. 3.8.4 Sodium hydroxide (10M) solution The 40 gram of sodium hydroxide was dissolved in distilled water (100 ml) to make 10 M solution. 3.8.5 Ferric chloride solution (10%)

The 10 gram of ferric chloride salt was added in water and made the volume up to 100 ml with water. 3.8.6 Ferric chloride (3.5%) solution

The 3.5 gram of FeCl3 was dissolved in glacial acetic acid and total volume was made up to 100 ml with it. 3.8.7 Gelatin (1%) solution The 1.0 gram of gelatin was dissolved in 10% sodium chloride solution making volume up to100 ml. 3.8.8 Lead sub-acetate solution The 40 gram of lead acetate was added in 90 ml of carbon dioxide (free water). The pH 7.5 was maintained by solution of sodium hydroxide (10 M). The supernatant layer (lead sub-acetate solution) was collected.

3.9 Total flavonoid contents determination Total flavonoid contents in six extracts of F. hamiltonii and N. procumbens were analyzed by the reported method (Zengin et al., 2010). The plant extract (20 µl) was mixed with 80 µl de-ionized water in in microplate. Sodium nitrous oxide (5%) solution (6 µl) was added followed by the addition of aluminum chloride (10%) solution (16 µl) and sodium hydroxide (4%) solution (68 µl). After 20 minutes incubation, the absorbance value was read at 510 nm. Above mentioned procedure was repeated for quercetin (QE) standard solution for construction of calibration curve. Total flavonoid contents were calculated (mg/ml) by using calibration curve. The contents of the flavonoids in both extracts were expressed in term of quercetin equivalent (mg QE per gram of extract) in Table-4.

3.10 Total phenol contents determination Total phenolic contents in the six extracts of F. hamiltonii and N. procumbens were calculated by folin-ciocalteu reagent (FCR) by using the reported literature method (Ismail et. al., 2004). The 20 µl of extract solution was added with folin- ciocalteu reagent (90 µl, 1:10 ratio made with water) in micro plate reader. Later on added aqueous sodium carbonate (90 µl, w/v) in it. Total phenolic contents were determined at wave length 725 nm. The same above mentioned process was repeated for Gallic acid (GA) standard solution and the calibration curve was developed. On the basis of measured absorbance, the concentration of total phenol contents were quantified (mg/ml) by using calibration curve. The total phenolic contents in both extracts were expressed in term of Gallic acid equivalent (mg of GA per gram of extract) in Table-4.

3.11 Gas chromatography-mass spectrometry GC-MS analysis is used for identification of chemical constituents present in the sample. It has great importance in drug detection and identification of unknown phytochemical constituents present in the medicinal plants. So, GC-MS analysis included in the study to investigate the unknown constituents of the selected medicinal plants F. hamiltonii and N. procumbens (David et al., 2011; Hites, 2016). GC-MS analysis was performed on GC Agilent system (B 7890) with mass spectrometer detector (MSD-5977A) employing the following condition: Column HP- 5MS, size 30 m × 0.25 mm, 0.25 µ, composed of 100% dimethyl poly siloxane. The source temperature for ionization was set at 250°C. The 2 µl of DCM and methanol extracts of F. hamiltonii (aerial and root pars) and N. procumbens (whole plant) were used in GC-MS analysis. The GC-MS results revealed the presence of thirty three (33) compounds in different six extracts of different parts of F. hamiltonii Royle and N. procumbens L. The name, molecular formula, molecular weight, CAS# and NIST# of the compounds are given in Table no. 5 to 10. 3.12 Biological and enzymatic activities 3.12.1 Antioxidant activity The DPPH assay is very simple, sensitive and reliable method. An antioxidant is used as a hydrogen donor. DPPH• is well known, stable and available nitrogen radicals. It gives maximum and remarkable absorption at 517 nm. DPPH is formed

41 after absorption of hydrogen (H) from and DPPH• disappeared from sample and the colour turns from purple to yellow (Vinha et al., 2012). The inhibition (%) was determined by the formula: Inhibition (% ) =Control value –Test value / Control value This assay was performed by reported method (Baylac and Racine, 2003). The test solution (10 µl) followed by the addition of 100 µM methanol DPPH solution (90 µl) to make volume of 100 µl in 96-wells microplate. The mixture was incubated for about half an hour. The absorbance was reduced which was measured at 517 nm using the equipment plate reader. The standard compound is Quercetin in this assay. The six dilutions of extracts were prepared (0.5, 0.25, 0.125, 0.0625, 0.0313, 0.015 mM) for IC50 value. The results are given in Table-11. 3.12.2 Cholinesterase inhibition activity Cholinesterases (ChE) are group of enzymes which are present in mammals and cause the breakdown of various neurotransmitters by hydrolysing the ester bonds within the molecule. They are classified into two types according to their substrate specificities, the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). AChE is present in red blood cells, muscle (motor end-plate) and cholinergic fibres, existing as mainly membrane bound structure. BChE is present in the liver and plasma, RBCs and many tissues (Rang and Dale, 2007). Acetylcholine (ACh) is product of the choline and acetyl-coenzyme A in the pre-synaptic neurons. BChE has hydrogen bond between Asp-70 and Tyr-332 which is opening site for its activity. The substrate is positively charged which attach to enzyme and form a complex (cation-π complex) with the Tyr-332 aromatic ring. The substrate interacts with the negatively charged Asp-70 at the same time. The process results in the conformational change in the monomers (Radic et al., 1993). The acetylcholinesterase and butyrylcholinesterase inhibition activities were performed with reported method with little modifications (Ellman et al., 1961). The volume of mixture was 100 µl in both assays. The 60 µl buffer of potassium dihydrogen phosphate (100 mM and pH 7.7) and 10 µl of extract (0.5 mM each well) was added. Then, 10 µl of enzyme was added. After mixing the contents and incubation for 10 minutes, value was measured at 405 nm. On mixing the 10 µl DTNB (0.5 mM / well) and 10 µl of 0.5 mM substrate, the reaction was initiated. The value was read at 405 nm after fifteen minutes. The test solutions were assayed at various dilutions (0.5, 0.25, 0.125, 0.0625, 0.0313, 0.015 mM) for the determination

of IC50 values. For determination of percentage inhibition, the same formula and data software were used in all assays of the study. The results are given in Table-12. 3.12.3 Lipoxygenase inhibition activity Lipoxygenases are enzymes having dioxygenase group. They are extensively present in plants, animals and fungi. They catalyze the oxidation of PUFA by using molecular oxygen (Gardner, 1991).

Figure-7: Structures of linoleic, α- linolenic and arachidonic acid.

The hydro peroxide fatty acids (given in figure-7) are metabolized and form physiologically active lipid-breakdown products, i.e., traumatinabscissic acid and jasmonic acid (Vick, 1993). Even though the biological functions of LOXs are not fully understood, but they are valuable in different physiological aspects of plants. Moreover, many plant species LOXs are present as multiple forms, signifying diverse functions within the plant (Fischer et al., 1999). The products of the reaction are conjugated cis, trans hydroperoxy derivatives. They are very reactive and toxic to body cells. They are rapidly metabolized to non-toxic compounds but are physiologically active. The products of LOXs have a valuable moiety in numerous diseases like tumour angiogenesis (Nie et al., 2002), inflammation and bronchial asthma (Steinhilber, 1999). The reported method (Tappel et al., 1953) with little modifications was used. The buffer solution of 100 mM potassium dihydrogen phosphate of pH 8 (140 µl), 0.5 mM extract (20 µl) and 15 µl enzyme lipoxygenase (600 units/well) to made total volume of mixture (200 µl). The mixture was read at 234 nm after incubation for 10 minutes at 25 °C. On addition of 25 µl solution of substrate, the reaction was initiated. The absorbance was measured after 10 minutes at 234 nm. The positive control was Baicalein (0.5 mM per well) solution. The test solutions were assayed at various dilutions ( 0.5, 0.25, 0.125, 0.0625, 0.0313, 0.015 mM) for the determination of IC50

43 values. For determination of percentage inhibition, the same formula and data software were used in all assays of the study. The results are given in Table-13.

3.12.4 Chymotripsin inhibition activity

The reported method (Atta ur Rahman et al., 2001) with partial modification was used in this study. The total volume of mixture (100μl) having 50mM Tris-HCl buffer of pH 7.6 (60 μl), 10 μl testing plant extract and 15 μl of enzyme (0.9 units) enzyme. After incubation for 20 minutes at 37 oC, the mixture was read at 410 nm. The reaction was started by adding 15 μl of N-succinyl phenyl-alanine-p-nitroanilide substrate (1.3 mM). After half an hour, the absorbance was measured at 410 nm by using the standard procedure as lay down in reported method. The positive control was Chymostatin (0.5 mM per well). The percentage (%) inhibition formula and data software were same used in all assays of the study. The results are given in Table-14.

3.12.5 Alpha glucosidase inhibition activity

The reported method with little modification (Pierre et al., 1978) has been used for this assay. The mixture‟s volume was 100 µl including 70 µl phosphate buffer (50 mM and pH 6.8), 10 µl of extract solution dissolved in methanol and in last added 10 µl solution of enzyme (0.057 units). The mixture was incubated for 10 minute at 37ºC and reading was taken at 400 nm. The reaction was started by the adding 10 µl of substrate (0.5 mM) per well. The positive control was Acarbose used at concentration 0.5 mM. The mixture was kept for incubation for 30 minutes. The absorbance was measured at 400 nm. For determination of percentage inhibition, the same formula and data software were used in all assays of the study. IC50 values compounds were calculated for different concentrations by using Enzyme Kinetics software after making suitable dilutions of test compound. The results are given in Table-15.

3.12.6 Urease inhibition activity

Berthelot assay has been used with partial modification (Lois and Ronald, 1963). The 10 µl phosphate buffer solution (pH 7.0), 10 µl of sample solution and 25 µl of enzyme solution (0.1347 units) were added in well. The total volume of mixture was 85 µl. After incubation for five minutes, 40 µl of 20 mM urea solution was added and again incubated. After ten minutes, phenol hypochlorite reagent (115 µl) was added per well (which is fresh mixture of 45 µl phenol

44 reagent and 70 µl of alkali reagent). On incubation at 37ºC for 10 min, absorbance was read at 625 nm and after colour development. For determination of percentage inhibition, the same formula and data software were used in all assays of the study. The results are given in Table-16.

3.12.7 Tyrosinase inhibition activity

The method (Kim et al., 2002) was used for this assay. The 60 μl solution of 100 mM phosphate buffer (pH 6.8), 10 μl extract solution (0.5 mM) and 10 μl (5.0 units) of enzyme were mixed. The volume (100 μl) was made and mixed in plate wells. The resultant were pre incubated for 10 minutes at 37oĊ and measured at 490 nm. After incubation time, 20 μl of L-DOPA (10 mM) was added. The contents were mixed and incubated again for 30 minutes at 37oĊ. The value of absorbance were noted at 490 nm. Kojic acid was used at concentration 0.5 mM as positive control. The results are given in Table-17.

3.12.8 Carbonic anhydrase II inhibition activity

The reported method (Arslan, 2001) has been used for this assay. The HEPES- tris solution (140 µl) and fresh aqueous solution (20 µl) of purified bovine erythrocyte carbonic anhydrase II (0.1 mg/ml) were mixed. The 20 µl of extract (0.5mg/ml) in DMSO solvent and 20 µl solution of 4-nitrophenyl acetate (0.7 mM) substrate were added. After incubation for 15 minutes at 25oC, absorbance value was taken at 400 nm. The positive control was solution of acetazolamide in this assay. The results are given in Table-18.

3.12.9 Phosphodiesterase I inhibition activity

In the assay, 97 µl of tris-(hydroxymethyl)-aminomethane buffer (50 mM), 20 µl solution of magnesium acetate (20 mM), 15 µl PDE snake venom (7.42 mg/1500 µl) were mixed. After incubation for half hour at room temperature, added 20 µl of extract solution (0.5 mg/ml) and reaction was started by addition of 60 µl of bis (p- nitrophenyl) phosphonate (0.33 mM) dissolved in ammonium acetate (20 mM). The absorbance was measured at 410 nm (Khalid et al., 2009). The EDTA was used as standard. The results are given in Table-19.

The inhibition (%) was determined by using formula, i.e., Inhibition (%) =Control value –Test value / Control value × 100).

3.12.10 Antibacterial activity

The reported method (Atta et al., 2001) was used for this activity which is based on the principle that increase in absorbance is due to microbial cell number increases by using microplate reader of 96 wells. The two Gram positive bacteria (Bacillus subtilis, Staphylococcus aureus) and three Gram negative bacteria (Pseudomonas aeruginosa, Salmonella typhi, Escherichia coli) were included in this activity. The microorganisms were kept on stock culture agar medium. The extracts of known concentrations (20 µg per well) in specific solvents were added into wells. The culture after dilution with nutrient broth medium was added into each well (180 µl). The total volume was 200 µl per well. The absorbance was measured at 540 nm and inhibition was calculated by using standard methods. The drug ciprofloxacin was taken as standard. The mean of three results are taken (n=3, ± SEM). The results were given in Table-20 and 21.

3.13 Experiments on isolated tissues

The animals used in the present study were local breed rabbits of both sex, weighing 1.0-1.5 kg and maintained at 25◦C in animal house, Faculty of Pharmacy, Bahauddin Zakariya University, Multan (Pakistan). These were given fresh green fodder and tap water ad libitum. The experiments were performed in accordance with rulings of the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council (NRC, 1996). The animals were subjected to fasting for 12 hours before experiments but provided free access to drinking water. The rabbits were slaughtered by stunning a blow on the back of head.

3.13.1 Search for spasmolytic activity

The isolated rabbit jejunum preparations were used for screening of the spasmolytic activity of the test materials, FHAM, NPM (i.e., methanol extracts of Farsetia hamiltonii and Neurada procumbens) (Gilani et al., 1999). The animals were dissected and jejunum was exised and placed in a petri dish having Tyrode's solution o at 35±2 C and aerated with carbogen (95% oxygen + 5% carbon dioxide). The jejunum was rendered free of attached mesentries carefully by means of sharp scissor and cut into pieces of 2 cm in length. The segments of isolated rabbit jejunum were mounted in isolated tissue organ bath filled with Tyrode's solution (10 ml) and bubbled with carbogen at 37oC. The isolated rabbit jejunum preparation was allowed to be equilibrated for 30 minutes being attached to isotonic transducer under applied tension

46 of 0.50 mg. The spontaneous periodic contractile and relaxant activity of the mounted isolated rabbit jejunum preparations were recorded isotonically by using Power Lab Data Acquisition System.

Verapamil was used as standard drug possessing relaxant effect on spontaneous contractile activity of isolated rabbit jejunum preparation. The test materials as well as standard drugs were applied to the isolated rabbit jejunum preparation in different tissue bath concentrations and response observed was used to construct graphs showing concentrations versus responses to determine the respective

EC50 by using Graphpad software.

3.13.2 Search for bronchorelaxant activity The test materials had been screened for possible bronchorelaxant activity on isolated rabbit tracheal preparations (Gilani et al., 1999). The rabbit body was dissected and trachea was excised, cut into rings of about 2-4 mm width in a manner that each ring may contain two cartilages. The rings were opened by longitudinal cut on ventral side opposite to the smooth muscle layer. Thus, forming tracheal strips with smooth muscle sandwiched in between cartilaginous parts on the edges. The preparation was suspended in a 10 ml tissue bath containing Krebs solution at 37 oC aerated with carbogen. About 1.0 gram tension was implemented to each of tracheal strip and was permitted to be equilibrated for one hour prior to recording of isometric contractions of tracheal preparation via force displacement transducers connected to power lab. The bronchorelaxant effect of the methanol extract of N. procumbens (NPM) was screened on pre-contracted isolated rabbit tracheal preparations with carbachol (1µM) as well as K+ (80 mM). The NPM exerted relaxant effect on carbachol (1.0 µM)- as well as K+(80 mM)-induced contractions in isolated rabbit tracheal preparation when applied in cumulative manner. The experiment was performed in triplicated on each preparation from 5 different animals in order to minimize animal to animal variation in results.

3.14 In vivo study 3.14.1 Screening for hypoglycaemic activity Farsetia hamiltonii Royle possesses folkloric repute for antidiabetic use; hence, was subjected to screening for possible hypoglycaemic effect on alloxan- induced diabetes in vivo rat model.

The Sprague-Dawley rats (♂♀; 200–250 g) were housed as six animals per cage of dimension 47×34×18 cm3; maintained at temperature (25 ± 2oC) and standard level of humidity with exposure of light and dark cycle in the animal house for 12:12 hours in Research Laboratory, Faculty of Pharmacy, Bahauddin Zakariya University, Multan (Pakistan). Animals were provided commercially available animal feed along with tape water ad libitum. The rats were acclimatized to the experimental conditions for 7 days period prior to the study to reduce stress on animals. The adopted protocols/ procedures were executed subsequent to approval by the ethics committee on animal handling, Faculty of Pharmacy, Bahauddin Zakariya University, Multan (Pakistan).

3.14.1.1 Induction of hyperglycaemia Hyperglycaemia had been induced to the 18 hours fasted rats by intraperitoneal injection of alloxan monohydrate at the dosage of 150 mg / kg in two doses, i.e.; 75 mg/ kg, i.e., 1st and 3rd days (Gupta and Singh, 2007). A fresh solution of alloxan was prepared in chilled buffer of citrate (pH 4.5) prior to administration. The rats were orally given 20% dextrose solution 4 hrs after alloxan exposure in order to protect from hypoglycaemia subsequent to excessive release of insulin from necrotic pancreas (Ankur and Shahjad, 2012). Furthermore, 5% glucose was made available to rats in drinking water for next 24hrs (Nandakumar et al., 2011). The occurrence of alloxan-induced hyperglycaemia was assessed at 72 hours by single touch glucometer (Accu-chek Performa, Roche, Germany) and the animals exhibiting fasting blood glucose level ≥ 200 mg/dl were selected for evaluation of hypoglycaemic effect of extract of aerial part of F. hamiltonii Royle (FHAM).

3.14.1.2 Administration of plants extracts The blood glucose level of alloxan induced diabetic rabbits showed diabetes after 5th day of initial dose. The rats were allowed to stabilize for 7 days. Then, the subjected animals were grouped into five and fasting blood glucose levels were measured. On same day, the treatment was started considering it the first day of study (El-Demerdash et al., 2005; Singh et al., 2010; Nandakumar et al., 2011). The fasting blood glucose levels were determined on 1st and 4th days of the study. The treatment protocol designs are given below:

Group I: Normal untreated group (Citrate buffer 4 ml/kg, intra peritoneal (i.p))

Group II: Positive Control group (Alloxan 150 mg/kg + Normal Saline 4 ml/kg, i.p.)

Group III: Standard Control (Alloxan 150 mg/ kg+ Glibenclamide 10 mg/kg, i.p.)

Group IV: FHAM 100 mg/kg (Alloxan 150 mg/kg + FHAM 100 mg/kg, i.p.)

Group V: FHAM 300 mg/kg (Alloxan 150 mg/kg + FHAM 300 mg/kg, i.p.)

3.14.1.3. Protocol of study

Blood glucose levels had been determined using a glucometer in the range of 10–600 mg/dl by using strips capable of measuring glucose level in blood. For the measurement of glucose level, tip of animal tail was pricked with needle and one blood drop was placed on strip. The blood glucose value was read from the screen of glucometer. The strips have very sensitive and critical test area that is integrated with specific chemicals. The chemical reaction started as blood comes in contact with sensitive area of strip that is designated as glucose dye oxidoreductase mediator reaction (PQQ-dependent glucose dehydrogenase mediator reaction,). The colour of test strip area was changed as result of the reaction. The glucometer measures the quantitative change and converts the change into value of blood glucose. The results are given in table-22.

CHAPTER NO. 4 RESULTS & DISCUSSION

4.1. Phytochemical screening of secondary metabolites The powdered plant material of selected medicinal plants F. hamiltonii (aerial and root parts) and N. procumbens (whole plant) were screened for secondary metabolites like alkaloids, flavonoids, anthraquinones (bounded and non-bounded), glycosides, saponins, terpenes and sterols. The results are illustrated in the table-1 given below.

Table-1: Results of phytochemical screening of secondary metabolites. Plant Name Phytoconstituent Status* Alkaloids - Flavonoids + Bounded anthraquinones + F. hamiltonii Non-bounded anthraquinones - (aerial & root part) Glycosides + Cardiac Glycosides + Saponins + Terpenes + Sterols + Alkaloids - Flavonoids + Bounded anthraquinones - N. procumbens Non-bounded anthraquinones - (whole plant) Glycosides + Cardiac Glycosides + Saponins - Terpenes + Sterols + *Present (+), Absent (-)

4.2. Results of Atomic Absorption spectroscopy Table-2: Elemental analysis of plant material of Farsetia hamiltonii and Neurada procumbens by Atomic Absorption Spectroscopy (AAS) analysis. Dry powder / Plant Dry powder / Plant Serial Name of element material of F. material of N. No. hamiltonii (50 mg) procumbens (50 mg) 1 Potassium 0.950mg 1.391mg 2 Sodium 5.064mg 13.035mg 3 Iron 0.006mg 0.032mg 4 Manganese 0.003mg 0.005mg 5 Zinc 0.004mg 0.003mg 6 Magnesium 3.249mg 6.406mg

4.3. Extraction yields The yield of dicloromethane extracts of F. hamiltonii were lower then respective methanol extracts, whereas were higher in case of N. procumbens extracts.

Table-3: Percentage yields of crude extracts of selected medicinal plants.

Part Extract Weight of Weight of Yield Plant name Solvent used used code dry powder extract (%)

16.2 Aerial Dichloromethane FHAD 900 grams 1.80 % grams

14.0 Aerial Methanol FHAM 900 grams 1.55 % Farsetia grams hamiltonii Root Dichloromethane FHRD 200 grams 2.0 grams 1.0 %

Root Methanol FHRM 200 grams 2.5 grams 1.25 %

15.6 Dichloromethane NPD 850 grams 1.83 % Neurada Whole grams procumbens plant 14.0 Methanol NPM 850 grams 1.55 % grams

4.4. Determination of total flavonoid and phenol contents Flavonoids are naturally occurring bioactive compound, which exist in free as well as in glycosidic form (total flavonoid contents). They show diverse structures, differentiated on degree of unsaturation and oxidation of three carbon segment. Their importance of existence is due to their different biological activities like antioxidant activity and aldone reductase inhibitor blocking sorbitol pathway which linked to diabetes associated problems (Tapiero et al., 2002). Phenols or phenol acids are also exist in free as well as in bound form (total phenol content), their existence and content determination is important due to their protective role in carcinogenic diseases, inflammation, atherosclerosis, thrombosis and antioxidant acivity. The results of all extracts are given below.

Table-4: Results of total flavonoid and phenol contents.

Flavonoids Phenolics S.No. Extract code (mg QE/g of (mg GA/g of extract) extract)

1 FHAD 177.59±1.42 -

2 FHAM 42.07±0.78 14.29±0.34

3 FHRD 60.69±0.77 21.84±0.61

4 FHRM 50.34±0.49 -

5 NPD 143.45±1.25 -

6 NPM 200.69±1.97 12.37±0.12

7 Quercetin 980.42±3.11 -

8 Gallic acid - 868.07±1.34

4.5. GC-MS analysis results of dichloromethane and methanol extracts of different parts of F. hamiltonii and N. procumbens. 4.5.1 GC-MS results of DCM extract of aerial part of F. hamiltonii.

Figure-8: GC-MS chromatogram of dichloromethane extract of aerial part of F. hamiltonii.

Table-5: Phytochemical constituents identified by GC-MS in dichloromethane extract of aerial part of F. hamiltonii.

RT Mol. Mol. S. No. Phytoconstituents CAS# NIST# (min) formula weight 12-methyl-E,E-2,13- 1 12.55 C19 H36O 280 - 130904 octadecadien-1-ol Estra-1,3,5(10)-trien- 2529- 2 13.42 C18 H24O 256 254855 17β-ol 64-8 Oleic acid 112-80- 3 14.36 C18H34O2 282 228066 1 13-Heptadecyn-1-ol 56554- 4 14.53 C17H32O 252 36374 77-9

5 17.13 Ethyl iso-allocholate C26H44O5 436 - 43053

Figure-9: Mass spectra and structure of 12-methyl-E,E-2,13-octadecadien-1-ol.

Figure-10: Mass spectra and structure of Estra-1,3,5(10)-trien-17β-ol.

Figure-11: Mass spectra and structure of Oleic acid.

Figure-12: Mass spectra and structure of 13-heptadecyn-1-ol.

Figure-13: Mass spectra and structure of Ethyl iso-allocholate.

4.5.2 GC-MS results of methanol extract of aerial part of F. hamiltonii.

Figure-14: GC-MS chromatogram of methanol extract of aerial part of F. hamiltonii.

Table-6: Phytochemical constituents identified by GC-MS in methanol extract of aerial part of F. hamiltonii.

RT Mol. Mol. S. No. Phytoconstituents CAS# NIST# (min) Formula weight Octadecan, 6-methyl- 10544- 1 6.17 C19H40 268 35803 96-4 Cyclo propane botanic acid, 2-[[2-[[2-[(2-pentyl cyclo 56051- 2 13.17 propyl)methyl]cyclo C25H42O2 374 28143 53-7 propyl]methyl]cyclo propyl]methyl]-, methyl ester Hexadecanoic acid, 112-39- 3 14.29 C17H34O2 270 42975 methyl ester 0

4 14.37 Ethyl iso-allocholate C26H44O5 436 - 43053

Figure-15: Mass spectra and structure of Octadecan, 6-methyl-

Figure-16: Mass spectra and structure of Cyclo propane botanic acid, 2-[[2-[[2- [(2-pentyl cyclo propyl)methyl]cyclo propyl]methyl]cyclo propyl]methyl]-, methyl ester.

Figure-17: Mass spectra and structure of Hexadecanoic acid, methyl ester

Figure-18: Mass spectra and structure of Ethyl iso-allocholate

4.5.3 GC-MS results of dichloromethane extract of root part of F. hamiltonii.

Figure-19: GC-MS chromatogram of dichloromethane extract of root part of F. hamiltonii.

Table-7: Phytochemical constituents identified by GC-MS in dichloromethane extract of root part of F. hamiltonii.

RT Mol. Mol. S. No. Phytoconstituents CAS# NIST# (min) formula Weight 2,4-Nonanedione 6175-23- 1 7.02 C H O 156 343052 9 16 2 1 Thymol 2 8.18 C H O 150 89-83-8 379278 10 14 n-Hexadecanoic acid 3 13.50 C H O 256 57-10-3 335494 16 32 2 Trans-13-

4 14.72 Octadecenoic acid C18H34O2 282 693-71-0 333615

Oleic acid 5 14.97 C H O 282 112-80-1 228066 18 34 2 Behenic alcohol 6 15.51 C H O 326 661-19-8 23377 22 46 Hexadecanoic acid, 2- 23470- 7 17.54 hydroxy-1-(hydroxyl C H O 330 15400 19 38 4 00-0 methyl) ethyl ester 3„,8,8‟-trimethoxy-3- piperidyl-2,2‟- 127611- 8 17.97 C H NO 487 100439 binaphthalene- 28 25 7 84 -1 1,1‟,4,4‟-tetrone

Figure-20: Mass spectra and structure of 2,4-nonanedione.

Figure-21: Mass spectra and structure of Thymol.

Figure-22: Mass spectra and structure of n-hexadecanoic acid

Figure-23: Mass spectra and structure of trans-13-octadecenoic acid

Figure-24: Mass spectra and structure of Oleic acid.

Figure-25: Mass spectra and structure of Behenic alcohol.

Figure-26: Mass spectra and structure of Hexadecanoic acid, 2-hydroxy-1-(hydroxyl methyl) ethyl ester.

Figure-27: Mass spectra and structure of 3„,8,8‟-trimethoxy-3-piperidyl-2,2‟- binaphthalene-1,1‟,4,4‟-tetrone

4.5.4 GC-MS results of methanol extract of root part of F. hamiltonii.

Figure-28: GC-MS chromatogram of methanol extract of root part of F. hamiltonii.

Table-8: Phytochemical constituents identified by GC-MS in methanol extract of root part of F. hamiltonii.

RT Mol. Mol. S. No. Phytoconstituent CAS# NIST# (min) formula weight Cyclo propane botanic acid, 2-[[2-[[2-[(2-pentyl cyclo

1 13.16 propyl)methyl]cyclo C25H42O2 374 56051-53-7 28143 propyl]methyl]cyclo propyl]methyl]-, methyl ester

Figure-29: Mass spectra and structure of Cyclopropane botanic acid, 2-[[2-[[2-[(2- pentyl cyclo propyl) methyl]cyclo propyl]methyl]cyclo propyl]methyl]-, methyl ester.

4.5.5 GC-MS results of dichloromethane extract of N. procumbens.

Figure-30: GC-MS chromatogram of dichloromethane extract of N. procumbents.

Table-9: Phytochemical constituents identified by GC-MS in dichloromethane extract of N. procumbens. RT Mol. Mol. S. No. Phytoconstituents CAS # NIST# (min) Formula Weight 3,7,11,15-tetramethyl-2- 102608- 1 12.57 C20H40O 296 114703 hexadecen-1-ol 53-7 12-methyl-E,E-2,13- 2 12.64 C19 H36O 280 - 130904 octadecadien-1-ol 56554- 3 12.88 13-Heptadecyn-1-ol C17H32O 252 36374 77-9

4 13.42 n-Hexadecanoic acid C16H32O2 256 57-10-3 335494 9,12,15-octadecatrienoic 18465- 5 14.26 acid, 2,3-dihydroxy propyl C21H36O4 352 15957 99-1 ester, (Z,Z,Z)-

6 14.38 Phytol C20H40O 296 150-86-7 375015 Trans-13-Octadecenoic 7 14.56 C18H34O2 282 693-71-0 333615 acid

8 15.46 Oleic acid C18H34O2 282 112-80-1 228066 15.56 9 Ethyl iso-allocholate C26H44O5 436 - 43053 16.06

Figure-31: Mass spectra and structure of 3, 7, 11, 15-tetramethyl-2-hexadecen-1-ol.

Figure-32: Mass spectra and structure of 12-methyl-E,E-2,13-octadecadien-1-ol.

Figure-33: Mass spectra and structure of 13-heptadecyn-1-ol.

Figure-34: Mass spectra and structure of n-hexadecanoic acid.

Figure-35: Mass spectra and structure of 9,12,15-octadecatrienoic acid, 2,3- dihydroxy propyl ester, (Z,Z,Z)-.

Figure-36: Mass spectra and structure of Phytol.

Figure-37: Mass spectra and structure of trans-13-octadecenoic acid.

Figure-38: Mass spectra and structure of Oleic acid.

Figure-39: Mass spectra and structure of Ethyl iso-allocholate.

4.5.6 GC-MS results of methanol extract of N. procumbens.

Figure-40: GC-MS chromatogram of methanol extract of N. procumbens.

Table-10: Phytochemical constituents identified by GC-MS in methanol extract of N. procumbens.

S. RT Mol. Mol. Phytoconstituents CAS# NIST# No. (min) formula Weight 948- 1 6.19 Pterin-6-carboxylic acid C7H5N5O3 207 127363 60-7 Ergosta-5,22-dien-3-ol,acetate, 2458- 2 12.22 C30H48O2 440 141996 (3β,22E)- 53-9 5H- cyclopropa [3, 4] benz [1,2–e] azulen–5- one, 9, 9a-bis (acetyloxy)-1, 1a, 1b, 2, 4a, 7a, 7b, 77573- 3 12.93 8, 9, 9a-decahydro-2, 4a, 7b- C24H32O9 464 67559 19-4 trihydroxy-3-(hydroxyl methyl)- 1,1,6,8-tetra methyl-, [1aR - (1aα,1bβ,2β,4aβ,7aα,7bα,8α,9β,9aα] 112- 4 13.16 Hexadecanoic acid, methyl ester C17H34O2 270 42975 39-0 56554- 5 14.26 7,10-octadecadienoic acid C19 H34O2 294 35764 24-6 13481- 6 14.29 10-octadecenoic acid, methyl ester C19 H36O2 296 36207 95-3

Figure-41: Mass spectra and structure of Pterin-6-carboxylic acid.

Figure-42: Mass spectra and structure of Ergosta-5,22-dien-3-ol,acetate, (3β,22E)-

Figure-43: Mass spectra and structure of 5H-cyclopropa[3, 4]benz[1, 2-e]azulen-5- one, 9,9a-bis(acetyloxy)-1,1a,1b,2,4a,7a,7b,8,9,9a-decahydro-2,4a,7b-trihydroxy-3- (hydroxyl methyl)-1,1,6,8-tetra methyl-,[1aR-(1aα,1bβ,2β,4aβ,7aα,7bα,8α,9β,9aα]-

Figure-44: Mass spectra and structure of Hexadecanoic acid methyl ester.

Figure-45: Mass spectra and structure of 7, 10-octadecadienoic acid

Figure-46: Mass spectra and structure of 10-octadecenoic acid, methyl ester

4.6. Biological and enzymatic activities of extracts of selected medicinal plants. To search out the new bioactive chemical constituents from the natural source, the plants extracts are evaluated for different enzyme inhibition like cholinesterase, lipoxygenase, chymotripsin, alpha-glucosidase, urease, tyrosinase, carbonic anhydrase II and Phosphodiesterase I. The the study the six plant crude extracts from two selected medicinal plants were also evaluated for antioxidant and antibacterial ctivities. The crude extracts of dicloromethane and methanol of selected medicinal plants Farsetia hamiltonii (FHAD, FHAM, FHRD, FHRM) and Neurada procumbens (NPD, NPM) were evaluated for different enzyme inhibition and biological activities. The results of the activities are given in the tables-11 to table-21.

4.6.1 Antioxidant activity Table-11: Results of antioxidant (DPPH) activity. Inhibition (%) Sample IC50 Description of sample Conc. code (µmoles) (0.5mg/ml) FHAD DCM extract of F. hamiltonii 5.52±2.66 Inactive (aerial)

FHAM Methanol extract of F. hamiltonii 19.20±1.21 Inactive (aerial)

FHRD DCM extract of F. hamiltonii (root) 5.52±2.66 Inactive

FHRM Methanol extract of F. hamiltonii 19.20±1.21 Inactive (root)

NPD DCM extract of N. procumbens 1.28±1.66 Inactive

NPM Methanol extract of N. procumbens 75.38±3.16 88.30±1.21

Control Quercetin 83.68±3.76 16.96±0.14

Note: All extracts are soluble in methanol.

4.6.2 Cholinesterase inhibition activity Table-12: Results of cholinesterase (AChE & BChE) inhibition activity. Enzyme AchE BchE

Sample % Inhibition IC µM % Inhibition IC µM Code 50 50

FHAD 21.96 Inactive 21.96 Inactive FHAM 11.59 Inactive 11.59 Inactive FHRD 68.77±0.62 405.41±0.21 68.77±0.62 405.41±0.21 FHRM 72.77±0.71 399.12±0.19 72.77±0.71 399.12±0.19 NPD 38.27±0.48 Inactive 38.27±0.48 Inactive NPM 23.88±0.01 Inactive 23.88±0.01 Inactive Eserine 91.29±1.17 0.04±0.001 91.29±1.17 0.04±0.0001 Note: All extracts are soluble in methanol.

4.6.3 Lipoxygenase inhibition activity Table-13: Results of Lipoxygenase inhibition activity. Inhibition Sample IC Description of sample (%) Conc. 50 code (µmoles) (0.5mg/ml) DCM extract of F. hamiltonii 44.80±0.12 544.80±1.12 FHAD (aerial)

Methanol extract of F. hamiltonii 57.50±0.98 457.50±0.98 FHAM (aerial)

FHRD DCM extract of F. hamiltonii (root) 75.00±1.46 291.70±2.45

Methanol extract of F. hamiltonii 74.00±0.36 323.50±2.19 FHRM (root)

NPD DCM extract of N. procumbens 44.60±0.23 524.60±1.02

NPM Methanol extract of N. procumbens 31.70±0.76 Inactive

Control Baicalein 93.79±1.27 22.47±0.04

Note: All extracts are soluble in methanol.

4.6.4 Chymotripsin inhibition activity Table-14: Results of Chymotripsin inhibition activity. Inhibition IC Sample code Description of sample (%) Conc. 50 (µmoles) (0.5mg/ml) FHAD DCM extract of F. hamiltonii (aerial) 69.64±0.01 333.12±0.01

FHAM Methanol extract of F. hamiltonii (aerial) 76.93±0.01 238.57±0.01

FHRD DCM extract of F. hamiltonii (root) 69.64±0.01 333.12±0.01

FHRM Methanol extract of F. hamiltonii (root) 76.93±0.01 238.57±0.01

NPD DCM extract of N. procumbens 84.38±0.05 115.71±0.03

NPM Methanol extract of N. procumbens 18.60±0.03 Inactive

Control Chymostatin 93.50±0.91 8.24±0.11

Note: All extracts are soluble in methanol.

4.6.5 Alpha glucosidase inhibition activity Table-15: Results of Alpha glucosidase inhibition activity. Inhibition (%) Sample IC Description of sample Conc. 50 code (µmoles) (0.5mg/ml) FHAD DCM extract of F. hamiltonii (aerial) 35.51±1.83 Inactive

FHAM Methanol extract of F. hamiltonii (aerial) 97.54±2.39 25.32±0.12

FHRD DCM extract of F. hamiltonii (root) 16.45±1.13 Inactive

FHRM Methanol extract of F. hamiltonii (root) 88.62±2.25 152.95±1.84

NPD DCM extract of N. procumbens 95.49±0.65 24.32±0.62

NPM Methanol extract of N. procumbens 99.45±0.58 8.43±0.56

Control Acarbose 92.23±0.14 38.25±0.12

Note: All extracts are soluble in methanol.

4.6.6 Urease inhibition activity Table-16: Results of urease inhibition activity. Inhibition (%) Sample IC Description of sample Conc. 50 code (µmoles) (0.5mg/ml) FHAD Dichloromethane extract of F. hamiltonii 56.77±0.27 381.50±0.52

FHAM Methanol extract of F. hamiltonii 63.31±0.46 351.60±0.54

NPD Dichloromethane extract of N. procumbens 57.23±0.50 375.60±0.58

NPM Methanol extract of N. procumbens 61.90±0.41 315.00±2.91

Control Thiourea 82.11±0.14 22.04±0.12

Note: All extracts are soluble in methanol.

4.6.7 Tyrosinase inhibition activity Table-17: Results of tyrosinase inhibition activity. Inhibition (%) Sample IC Description of sample Conc. 50 code (µmoles) (0.5mg/ml) FHAD Dichloromethane extract of F. hamiltonii 32.03±0.45 Inactive

FHAM Methanol extract of F. hamiltonii 49.90±0.23 Inactive

NPD Dichloromethane extract of N. Procumbens 57.23±0.50 321.60±0.45

NPM Methanol extract of N. procumbens 63.92±0.41 305.00±6.91

Control Kojic acid 93.50±0.14 6.04±0.11

Note: All extracts are soluble in methanol.

4.6.8 Carbonic anhydrase II inhibition activity Table-18: Results of Carbonic anhydrase II inhibition activity. Inhibition Sample IC Description of sample (%) Conc. 50 code (µmoles) (0.5mg/ml) Dichloromethane extract of F. 25.92±0.01 Inactive FHAD hamiltonii

FHAM Methanol extract of F. hamiltonii 8.22±0.01 Inactive

Dichloromethane extract of N. NPD 37.43±0.41 Inactive Procumbens

NPM Methanol extract of N. procumbens 73.92±0.41 102.31±0.85

Control Acetazolamide 89.03±0.03 0.10±0.04

Note: All extracts are soluble in DMSO.

4.6.9 Phosphodiesterase I inhibition activity Table-19: Results of Phosphodiesterase I inhibition activity. Inhibition Sample IC50 Description of sample (%) Conc. code (0.5mg/ml) (µmoles) Dichloromethane extract of F. FHAD 2.92±0.01 Inactive hamiltonii

FHAM Methanol extract of F. hamiltonii 26.12±0.01 Inactive

Dichloromethane extract of N. NPD 1.93±0.41 Inactive Procumbens

NPM Methanol extract of N. procumbens 30.92±0.33 Inactive

Control EDTA 69.02±0.02 277.69±2.52

Note: All extracts are soluble in methanol.

4.6.10 Antibacterial activity Table-20: Results of antibacterial activity. Inhibition (%) Conc. (100µg/well) Sample code Bacillus Staphylococcus Pseudomonas Salmonella Escherichia subtilis aureus aeruginosa typhi coli

FHAD 65.17±4.28 60.17±4.28 74.02±1.39 74.10±1.33 68.64±1.36

FHAM 54.70±0.75 56.33±1.50 54.63±0.05 58.13±1.46 51.40±1.15

FHRD 39.33±0.89 34.75±1.42 37.41±1.52 30.57±2.10 40.95±1.86

FHRM 64.44±2.89 70.98±1.57 62.92±2.74 57.81±5.00 62.41±2.14

NPD 40.28±0.60 42.89±1.93 39.62±2.45 40.48±0.10 45.45±3.64

NPM 48.83±3.83 46.86±2.94 43.68±0.19 45.19±1.00 45.91±3.73

Ciprofloxacin 91.23±1.07 91.23±1.07 90.88±0.16 92.65±1.10 91.45±2.19

Note: All extracts are soluble in methanol.

Table-21: Results of antibacterial activity (MIC50).

MIC50 (µg/ml)

Sample code Bacillus Staphylococcus Pseudomonas Salmonella Escherichia subtilis aureus aeruginosa, typhi coli

FHAD 13.93±0.84 13.21±1.58 9.69±1.90 9.44±1.09 12.69±0.37

FHAM 13.40±0.34 12.70±0.33 13.48±0.12 12.37±0.12 17.87±0.41

FHRD Inactive Inactive Inactive Inactive Inactive

FHRM 12.83±0.38 11.01±0.43 12.61±0.63 11.69±0.33 12.83±0.10

NPD Inactive Inactive Inactive Inactive Inactive

NPM Inactive Inactive Inactive Inactive Inactive

Ciprofloxacin 7.52±0.67 7.03±0.53 7.58±0.19 7.23±0.71 8.21±0.11

Note: All extracts are soluble in methanol.

4.7. Biological activities of selected medicinal plants on isolated tissues

4.7.1 Spasmolytic activity The crude methanol extract of the aerial part of F. hamiltonii Royle (FH-M.cr) exerted spasmolytic effect on application at concentrations 0.01-0.3 mg/ml (tissue bath concentration) to the spontaneous rhythmic contractions in the isolated rabbit jejunum preparations with EC50 value 0.066 µM (Figure-47 (A); Figure-48); it also relaxed the K+(80 mM)-induced contractions in isolated rabbit jejunum preparations at concentrations 0.01-0.1 mg/ml with EC50 value 0.011 µM (tissue bath concentration) (Figure-47 (B); Figure-48). Similarly, the methanol extract of N. procumbens (NPM) exerted spasmolytic effect on application at 0.01-1 mg/ml mg/ml (tissue bath concentration) to the spontaneous rhythmic contractions in isolated rabbit jejunum preparations with EC50 value 0.075 mg/ml (Figure-49 (A); Figure-50); it also relaxed the K+(80 mM)-induced contractions in isolated rabbit jejunum preparations at 0.01-0.1 mg/ml (tissue bath concentration) with EC50 value 0.024 mg/ml (Figure-49 (B); Figure-50).

Verapamil a standard Ca2+ channel blocking drug also relaxed the spontaneous rhythmic as well as K+(80 mM)-induced contractions in isolated rabbit jejunum preparations with respective EC50 values of 0.35µM and 0.13 µM (Figure-51).

Figure-47 (A). Tracing showing the spasmolytic effect of methanol extract of F. hamiltonii on spontaneous contractions of isolated rabbit jejunum preparations.

Figure-47 (B). Tracing showing influence of methanol extract of F. hamiltoniii on K+(80 mM)-induced contractions in isolated rabbit jejunum preparations.

Figure-48: Effects of methanol extract of F. hamiltonii on spontaneous rhythmic contractions and on K+(80 mM)-induced contractions in isolated rabbit jejunum preparations.

Figure-49 (A). Tracing showing spasmolytic effect of methanol extract of N. procunbens on spontaneous rhythmic contractions in isolated rabbit jejunum preparations.

Figure-49 (B). Tracing showing influence of methanol extract of N. procunbens on K+(80 mM)-induced contractions in isolated rabbit jejunum preparations.

Figure-50. Effects of methanol extract of N. procumbens on spontaneous rhythmic and K+(80 mM)-induced contractions in isolated rabbit jejunum preparations.

Figure-51. Effects of verapamil on spontaneous rhythmic and and K+(80 mM)- induced contractions in isolated rabbit jejunum preparations.

4.7.2. Bronchorelaxant activity The methanol extract of N. procumbens exerted bronchorelaxant effect on carbachol (1µM)-induced contractions in isolated rabbit tracheal preparation at 0.01-0.3 mg/ml (tissue bath concentrations) with EC50 value of 0.624 mg/ml (Figure-52 (A); Figure-53). Whereas, it relaxed K+ (80 mM)-induced contraction at 0.01-0.1 mg/ml

(tissue bath concentration) with EC50 value 0.096 mg/ml (Figure-52 (B); Figure-53).

Figure-52 (A). Tracing showing bronchorelaxant effect of methanol extract of N. procunbens on cabachol (1µM)-induced contractions in isolated rabbit tracheal preparations.

Figure-52 (B). Tracing showing influence of methanol extract of N. procumbens on K+(80 mM)-induced contractions in isolated rabbit tracheal preparations.

Figure-53: Effects of methanol extract of N. procumbens on carbachol (1µM)- and K+(80 mM)-induced contractions in isolated rabbit tracheal preparations.

4.8. In vivo hypoglycaemic activity of F. hamiltonii The blood glucose level in normal control group of rats to whom citrate buffer (4ml/kg) was administered and measured to be 104.17±2.9, 107.17±2.1 and 109.17±1.3 mg/dl on 0, 1st and 4th day respectively. The rats in positive control group were treated with alloxan followed by normal saline (4 ml/kg) the respective values of blood glucose on 0, 1st and 4th day were assessed to be 478.07±6.1, 485.97±3.1 and 468.37±2.7. The animals in standard control group were given glibenclamide (10 mg/kg) subsequent to alloxan treatment, the blood glucose values were found to be 478.07±6.1, 90.87±6.6 and 106.57±4.1, respectively; indicating significant decrease in blood sugar values on 1st and 4th day. The animals of test group A and test group B were given methanol extract of Farsetia hamiltonii Royle at respective doses of 100 and 300 mg/kg following alloxan treatment the blood glucose values on 0, 1st and 4th day of treatment was found to be 483.27±5.0 and 479.87±3.7; 173.27±6.9 and 114.87±1.9; 188.07±3.6 and 107.67±1.2, respectively. Thus, reflecting that significant decrease (P<0.05) in blood glucose level is observed subsequent to treatment with methanol extract of Farsetia hamiltonii Royle in treated group A and Treated group B at respective rate of 100 and 300 mg/kg on day 1st and 4th of treatment in comparisom with animals of positive control group; in a manner comparable to the blood glucose values in standard control group.

Table-22: Results of intra-peritoneal administration of different doses of methanol extract of F. hamiltonii Royle in alloxan induced diabetic rats for four days.

Serial Treatment groups Glucose level (mg/dl) N0. 0 day 1st day 4th day Normal control 1 104.17±2.9 107.17±2.1 109.17±1.3 (Citrate buffer, 4 ml/kg) Positive control 2 476.77±3.9 485.97±3.1 468.37±2.7 (N/S, 4 ml/kg) Standard control 3 478.07±6.1 90.87±6.6 106.57±4.1 (Glibenclamide 10 mg/kg) Test groupA 4 Farsetia hamiltonii Royle 483.27±5.0 173.27±6.9 188.07±3.6 (100 mg/kg) Test groupB 5 Farsetia hamiltonii Royle 479.87±3.7 114.87±1.9 107.67±1.2 (300 mg/kg)

P˂0.001=highly signiﬁcant.

*Farsetia hamiltonii methanol extract treated groups and standard control were compared to positive control and positive control was compared to normal control.

4.9 Discussion Herbal medicine in one guise or other continued to be the preferable source over allopathic medicines. Herbal medicines show compatibility to our physiological system; additionally their low cost make them attractive to traditional healers. The herbal medicines are favoured on the allopathic drugs due to low cost, good safety profile and more compatible towords the human physiology. The knowledge of the traditional healers is also supportive in this regard. (Ahmad et al., 2009; Phillipson, 1990). The present work demonstrated a link between ongoing research and ethno- botanical claims for the folkloric uses of the F. hamiltonii and N. procumbens in treating different illness especially in diabetes, gastrointestinal and respiratory disorders. This research was designed to evaluate the phytochemical and biological studies of selected medicinal plants F. hamiltonii Royle (Brassicaceae) and N. procumbens L. (Neuradaceae) from the Cholistan desert. The study is first detail report regarding phytochemical, chromatographic, spectroscopic, enzymatic and biological (in vitro and in vivo) evaluation of the plant F. hamiltonii and N. procumbens. The study gives a direction to scientists regarding isolation, identification and purification of compounds of therapeutic value to combat with various diseases in human beings. Farsetia hamiltonii Royle (Brassicaceae) is a plant of Cholistan desert having the folk medicinal uses like inflammation, rheumatic problems in joints, diabetes, infections and gastrointestinal diseases. The boiled plant material in water is applied on the wound infections of camel in desert (Arshad et al., 2002). Neurada procumbens L. is a Cholistan desert plant belongs to family Neuradaceae. Traditionally it is used as anthelmintic, anti-convulsant, anti-infective, cooling agent and to increase respiratory functions. It is also used to treat diarrhea, dysentery, eczema, laxative, nerve tonic, rheumatism and sexual problems (Ahmed et al., 2014; Marzouk et al., 2014; Raza et al., 2014). Leaves and fruits are used for medicinal purposes and whole plant is a source of fodder for camel (Rahmatullah et al., 2010). According to Flora of Pakistan, only a single species of genus Neurada found in Pakistan (Nasir & Ali, 1972). After thorough literature survey, it has been found that there is very little phytochemical and biological work on F. hamiltonii and N. procumbens have been done earlier.

The aim of phytochemical investigation is to explore or determine the secondary metabolites and contents in the plants material. The secondary metabolites like flavonoids, bounded anthraquinones, glycosides, cardiac glycoside, saponins, terpenes and sterol are present(+) in plant material of F. hamiltonii whereas alkaloids and free anthraquinones were found absent(-). In case of N. procumbens, the secondary metabolites like flavonoids, glycosides, cardiac glycoside, terpenes and sterol are present (+) whereas alkaloids, bounded & free anthraquinones and saponins were absent (-). The laxative folk use of F. hamiltonii may be due to presence of flavonoids and glycosides (Zengin et al., 2010; Bhargava and Raghupathi, 1994). Thus, the presence of flavonoids and glycosides in F. hamiltonii Royle (Table-1) proves the folk value of the plant regarding its traditional uses by inhibitants of cholistan for gastrointestinal illness like diarrehea. The different values of mineral contents are indicators for physicochemical status of soil of cholistan (Nasim et al., 1996). Atomic absorption spectroscopy was used to determine potassium, sodium, iron, manganese, zinc and magnesium as 0.950, 5.064, 0.006, 0.003, 0.004 and 3.249 mg per 50mg of dry powder F. hamiltonii (Table-2). Total flavonoid and total phenol contents were determined (Zengin et al., 2010; Ismail et al., 2004). Total flavonids contents were evaluated using quercetin equivalents by standard curve equation y=0.4918x+0.1604 (r2=0.997) and expressed in mg of QE/g of extract. The total phenol contents were evaluated as gallic acid equivalents by standard curve equation y=0.0047x+0.0615 (r2=0.998) and expressed in mg of GA/g of extract. Total flavonoid contents in dichloromethane and methanol extracts of F. hamiltonii Royle (aerial part) were 177.59±1.42 and 42.07±0.78, respectively. Total phenol contents were determined 14.29±0.34 only in methanol extract GA/g of the F. hamiltonii. In case of root part extracts of F. hamiltonii, total flavonid contents in dichloromethane and methanol extract were 60.69±0.77 and 50.34±0.49, respectively. Total phenol contents were detected 21.84±0.61 only in dichloromethane extract GA/g of the F. hamiltonii (Table-4). To identify the chemical constituents in the four crude extracts of F. hamiltonii, the GC-MS technique was used in the study. The five (05) compounds were identified in the dichloromethane extract (FHAD), whereas four (04) compounds

100 were identified in methanol extract (FHAM) of aerial part of F. hamiltonii (Table-5 and 6). The eight (08) compounds were identified in dichloromethane extract (FHRD), whereas only one (01) compounds were identified in methanol extract (FHRM) of root part of F. hamiltonii (Table-7 and 8). The three (03) out of eighteen (18) compounds Oleic acid, Ethyl iso- allocholate and Cyclo propane botanic acid, 2-[[2-[[2-[(2-pentyl cyclo propyl) methyl] cyclo propyl] methyl]cyclo propyl]methyl]-, methyl ester are same in four extracts of different parts of same plant F. hamiltonii, in this way fifteen (15) compounds were identified in the plant F. hamiltonii. The fifteen (15) compounds: (1) 12-methyl-E,E-2,13-octadecadien-1-ol, (2) Estra-1,3,5(10)-trien-17β-ol, (3) Oleic acid, (4) 13-heptadecyn-1-ol, (5) Ethyl iso- allocholate, (6) Octadecan, 6-methyl-, (7) Cyclo propane botanic acid, 2-[[2-[[2-[(2- pentyl cyclo propyl) methyl] cyclo propyl]methyl]cyclo propyl]methyl]-, methyl ester (8) Hexadecanoic acid, methyl ester, (9) 2,4-nonanedione, (10) Thymol, (11) n- hexadecanoic acid, (12) trans-13-Octadecenoic acid, (13) Behenic alcohol, (14) Hexadecanoic acid, 2-hydroxy-1-(hydroxyl methyl) ethyl ester and (15) 3„,8,8‟- trimethoxy-3-piperidyl-2,2‟-binaphthalene-1,1‟,4,4‟-tetrone were identified by performing the analysis of four extracts (FHAD, FHAM, FHRD, FHRM) of aerial and root parts of F. hamiltonii by GC-MS technique. The enzyme inhibition in vitro assays for different enzymes (cholinesterase, lipoxygenase, chymotrypsin, α-glucosidase, urease, tyrosinase, carbonic II anhydrase, phosphodiesterase I) for both dichloromethane and methanol extracts of F. hamiltonii were analyzed in triplicate and given with SEM in Table-11 to Table-21. The antioxidant and antibacterial activities were also performed. The products of lipoxygenases play a vital role in many medical disorders such as inflammation, bronchial asthma (Steinhilber, 1999) and tumor angiogenesis (Nie & Honn, 2002). Urease is of medical importance because it is involved for establishing disease state like peptic ulcer, kidney stones, pyelonephritis and other diseases (Fishbein & Gutwein, 1973). So due to the role of urease in the production of certain diseases it is of much importance in pharmaceutical research (Robinson & Wilson, 1939). Urease enzyme is found in most of the organisms. It breaks down the urea to ammonium carbonate and is mostly used for determination of urea (Tarun et al., 2009). Urea catalysis is carried out by urease forming carbonate anions and

101 ammonium cations. Research is going on urea in many laboratories as it has gained much worth in biotechnology and medicine (Mobely et al., 1995). The dichloromethane extract of aerial part of F. hamiltonii (FHAD) exhibited antilipoxygenase, antichymotrypsin, antiurease and antibacterial activities but found inactive for antioxidant, anticholinesterase, anti-alpha glucosidase, antityrosinase, anticarbonic II anhydrase and antiphosphodiesterase I activities The antilipoxygenase activity of FHAD extract was active at moderate level, with inhibition 44.80±0.12% at concentration 0.5mg/ml and IC50 544.80±1.12µmoles and comparable to the values of Baicalein standard (93.79±1.27% and IC50 22.47±0.04µmoles). The antichymotripsin activity of FHAD extract was active at moderate level, with inhibition 69.64±0.01% at concentration 0.5mg/ml and IC50 333.12±0.01µmoles and comparable to the values of Chymostatin standard

(93.50±0.91% and IC50 8.24±0.11µmoles). The antiurease activity of FHAD extract was active at moderate level, with inhibition 56.77±0.27% at concentration 0.5mg/ml and IC50 381.50±0.52µmoles and comparable to the values of Thiourea standard

(82.11±0.14% and IC50 22.04±0.12µmoles). The antioxidant effect of FHAD was not remarkable with inhibition 5.52±2.66% at concentration 0.5mg/ml and not comparable to the values of Quercetin standard (83.68±3.76% and IC50 16.96±0.14µmoles). The anticholinesterase (antiacetylcholinesterase, antibutyrylcholinesterase) activities of FHAD were insignificant. The anti-alpha glucosidase activity of FHAD extract was insignificant and not remarkable with inhibition 35.51±1.83% at concentration 0.5mg/ml and not comparable to the values of Acarbose standard (92.23±0.14 and IC50 38.25±0.12µmoles). The antityrosinase activity of FHAD extract was inactive and not remarkable with inhibition 32.03±0.45% at concentration 0.5mg/ml and not comparable to the values of Kojic acid standard (93.50±0.14% and IC50 6.04±0.11µmoles). The anticarbonic anhydrase II activity of FHAD extract was inactive and not remarkable with inhibition 25.92±0.01% at concentration 0.5mg/ml and not comparable to the values of Acetazolamide standard (89.03±0.03% and IC50 0.10±0.11µmoles). The antiphosphodiesterase I activity was not exhibited by FHAD extract. Significantly, the antibacterial activity of FHAD extract was exhibited inhibition against five strains (Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi, Escherichia coli) at 65.17±4.28%, 60.17±4.28%,

102

74.02±1.39%, 74.10±1.33%, 68.64±1.36% and MIC50 13.93±0.84µg/ml, 13.21±1.58µg/ml, 9.69±1.09µg/ml, 9.44±1.09µg/ml, 12.69±0.37µg/ml which are significant and comparable to standardard ciprofloxacin having inhibition

91.23±1.07%, 91.23±1.07%, 90.88±0.16%, 92.95±1.10%, 91.45±2.19% and MIC50 7.52±0.67µg/ml, 7.03±0.53µg/ml, 7.58±0.19µg/ml, 7.23±0.71µg/ml, 8.21±0.11µg/ml, respectively. The methanol extract of aerial part of F. hamiltonii (FHAM) exhibited antilipoxygenase, antichymotrypsin, anti-alpha glucosidase, antiurease and antibacterial activities but found inactive for antioxidant, anticholinesterase, antityrosinase, anticarbonic II anhydrase and antiphosphodiesterase I activities The antilipoxygenase activity of FHAM extract was active at moderate level, with inhibition 57.50±0.98% at concentration 0.5mg/ml and IC50 457.50±0.98µmoles and comparable to the values of Baicalein standard (93.79±1.27% and IC50 22.47±0.04µmoles). The antichymotripsin activity of FHAM extract was active at good level, with inhibition 76.93±0.01% at concentration 0.5mg/ml and IC50 238.57±0.01µmoles and comparable to the values of Chymostatin standard

(93.50±0.91% and IC50 8.24±0.11µmoles). The anti-alpha glucosidase activity of FHAM extract was highly significant with inhibition 97.54±2.39% at concentration

0.5mg/ml and IC50 25.32±0.12µmoles, more potant than the standard Acarbose

(92.23±0.14 and IC50 38.25±0.12µmoles). The antiurease activity of FHAM extract was active at moderate level, with inhibition 63.31±0.46% at concentration 0.5mg/ml and IC50 351.60±0.54µmoles and comparable to the values of Thiourea standard

(82.11±0.14% and IC50 22.04±0.12µmoles). The antioxidant effect of FHAM was not remarkable with inhibition 19.20±1.21% at concentration 0.5mg/ml and not comparable to the values of Quercetin standard (83.68±3.76% and IC50 16.96±0.14µmoles). The anticholinesterase (antiacetylcholinesterase, antibutyrylcholinesterase) activities of FHAM were inactive. The antityrosinase activity of FHAM extract was less active than 50% with inhibition 49.90±0.23% at concentration 0.5mg/ml and not comparable to the values of Kojic acid standard (93.50±0.14% and IC50 6.04±0.11µmoles). The anticarbonic anhydrase II activity of FHAM extract was inactive and not remarkable with inhibition 8.22±0.01% at concentration 0.5mg/ml and not comparable to the values of

Acetazolamide standard (89.03±0.03% and IC50 0.10±0.11µmoles). The antiphosphodiesterase I activity of FHAM extract was inactive and not remarkable

103 with inhibition 26.12±0.01% at concentration 0.5mg/ml and not comparable to the values of EDTA standard (69.02±0.02% and IC50 277.69±2.52µmoles). Significantly, the antibacterial activity of FHAM extract was exhibited inhibition against five strains (Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi, Escherichia coli) at 54.70±0.75%, 56.33±1.50%,

54.63±0.05%, 58.13±1.46%, 51.40±1.15% and MIC50 13.40±0.34µg/ml, 12.70±0.33µg/ml, 13.48±0.12µg/ml, 12.37±0.12µg/ml, 17.87±0.41µg/ml which are significant and comparable to standardard ciprofloxacin having inhibition

91.23±1.07%, 91.23±1.07%, 90.88±0.16%, 92.95±1.10%, 91.45±2.19% and MIC50 7.52±0.67µg/ml, 7.03±0.53µg/ml, 7.58±0.19µg/ml, 7.23±0.71µg/ml, 8.21±0.11µg/ml, respectively (Table-20 and 21). The research work of this study is published regarding antidiabetic and spasmolytic activities of medicinal plant Farsetia hamiltonii Royle from Cholistan desert Pakistan which is first reported work (Hayat et al., 2014). The inhibition of the spontaneous contractions was observed suggesting a spasmolytic effect. The possible mechanisms for spasmolytic activity were anticholinergic and calcium channel blocking activities. The mechanism of calcium channel blocking was investigated in the study, because it was observed that spasmolytic effect having constituents present in selected plant are usually mediated through a caþþ blocking effect (Gilani et al., 1999, 2000, 2005). High K+ concentration (80 mM) is known to cause contraction of smooth muscles through opening of voltage dependent Ca++ channels and allowing the inﬂux of extracellular Ca++ that produce the contractile effect (Bolton, 1979). A phytoconstituent provoking inhibition of high K+ induced contractions is assumed to be a blocker of calcium channel (Godfraind et al., 1986). Traditional practitioners use F. hamiltonii to treat gastrointestinal problems, to verify scientifically the methanol extract (FHAM) of aerial parts of F. hamiltonii was evaluated for spasmolytic activity on isolated tissues of rabbit jejunum. This extract FHAM showed concentration-dependent (0.01–0.3 mg/ml) relaxation of spontaneous + contractions (Figure-47A) with EC50 value 0.066 mg/ml and high K (80 mM) induced contractions 0.01–0.1 mg/ml (Figure-47B) with EC50 value 0.011 mg/ml (Figure-48). Similarly, verapamil used as standard calcium channel blocker potentiated the relaxation effect of extract FHAM, indicating that methanol extract of F. hamiltonii may contain phytoconstituents with Calcium channel blocking activity. Verapamil on same conditions, relaxed the spontaneous contraction (0.03–1.0 mM) with EC50 value

104

+ 0.35 µM and at high K (80 mM) induced contractions 0.03–0.3 mM with EC50 value 0.13 µM (Figure-48). The pattern of inhibition and comparing our data, it can be emphasized that the methanol extract (FHAM) acts through blockage of calcium influx and exhibited the spasmolytic activity. The Caþþ channel blocking agents are known to be useful as spasmolytic agent to treat diarrhea (Brunton, 1996). Furthermore, the methanol extract aerial part of of F. hamiltonii (FHAM) was first time tested for in vivo hypoglycaemic activity in rats to scientifically confirm the flok antidiabetic use of the plant. After dissolving the methanol extract (FHAM) in normal saline was injected intraperitonealy in alloxan induced diabetic rats at the doses of 100 and 300 mg/kg. At the start, blood glucose level was 104.1±2.9 mg/dl for normal control group, 476.7±3.9 mg/dl was the value of blood glucose of positive control group, and 478.0±6.1 mg/dl was glucose level of standard control group. The glucose level of groups of extract of F. hamiltonii at 100 mg/kg and 300 mg/kg group was observed 483.2±5.0 mg/dl and 479.8±3.7 mg/dl, respectively. On first day; 107.1±2.1 mg/dl was the glucose level of normal control group, 485.9±3.1 mg/dl was value for positive control group, 90.8±6.6 mg/dl was the value of glucose of standard control group, 173.2±6.9 mg/dl was reduced value of blood glucose for the extract of F. hamiltonii 100 mg/kg group, 114.8±1.9 mg/dl was glucose value for 300 mg/kg. On fourth day, 109.1±1.3 mg/dl was the noted glucose values for normal control group, 468.3±2.7 mg/dl was glucose level of positive control group, 106.5±4.1 mg/dl was observed value for standard control group, 188.0±3.6 mg/dl was the calculated amount of glucose for Farsetia hamiltonii 100 mg/kg, while 107.6±1.2 mg/dl was glucose level observed for 300 mg/kg concentration. The glucose level achieved with extract (FHAM) of F. hamiltonii at 300 mg/kg were more significant as compared to 100 mg/kg. So, it is scientifically evaluated that dose dependent antidiabetic activity profile of F. hamiltonii is similar to the glibenclamide. The decreasing patren of blood glucose level is shown in table-22. In this study, Alloxan selectively inhibits the glucokinase, as result glucose induced insulin secretion is stopped resulting in disease IDDM (Lenzen, 2008). The root part of the plant F. hamiltonii Royle was evaluated for antioxidant, anticholinesterase (antiacetylcholinesterase, antibutyrylcholinesterase), antilipoxygenase, antichymotrypsin, anti-alpha glucosidase and antibacterial activities to validate the folk use.

105

The dichloromethane extract of root parts of F. hamiltonii (FHRD) exhibited anticholinesterase inhibition value is same for both antiacetylcholinesterase and antibutyrylcholinesterase which is 68.77±0.62% and IC50 405.41±0.21µmoles, antilipoxygenase (75.00±1.46% and IC50 291.70±2.45µmoles) and antichymotrypsin

(69.64±0.01% and IC50 333.12±0.01µmoles) which are comparable to standards Eserine, Baicalein and Chymostatin respectively at moderate level, whereas antioxidant (5.52±2.66%), anti-alpha glucosidase (16.45±1.13%) and antibacterial activities of FHRD extract were very insignificant and found inactive. The methanol extract of root parts of F. hamiltonii (FHRM) exhibited anticholinesterase (antiacetylcholinesterase, antibutyrylcholinesterase) with good inhibition 72.77±0.71% and IC50 399.12±0.19µmoles, antilipoxygenase

(74.00±0.36% and IC50 323.50±2.19µmoles), antichymotrypsin (76.93±0.01% and

IC50 238.57±0.01µmoles) and anti-alpha glucosidase (88.62±2.25% and IC50 152.95±1.84µmoles) activities. The antibacterial activity is significant against all five strains with inhibition 57-70% and MIC50 ranging between 11 to 12µg/ml at extract strength of 100µg/well. The antioxidant (19.20±1.21%) activity was found inactive (Table-11). N. procumbens is second selected plant of the study, the secondry metabolites flavonoids, glycosides, cardiac glycosides, terpenes and sterols were present in dry plant material of the plant (Table-1) which proves the folk value of the plant regarding its traditional uses by inhibitants of cholistan for gastrointestinal problems. The minerals were determined (Nasim et al., 1996) by using Atomic absorption spectroscopy for potassium, sodium, iron, manganese, zinc and magnesium which were 1.391, 13.035, 0.032, 0.005, 0.003 and 6.406 mg per 50 mg of dry whole plant material of N. procumbens. These minerals play a role in stabilization and other needs of the plants (Table-2). The yield of dichloromethane and methanol extracts of N. procumbens are 1.83% and 1.55%, respectively. Total flavonoid contents (mg QE/g extract) were higher in methanol extract (200.69±1.97) with respect to dichloromethane extract (143.45±1.25) of N. procumbens, whereas total phenol contents in methanol extract were 12.37±0.12mg GA/g of N. procumbens L. (Table-4) and not found in dichloromethane extract. To identify the chemical constituents in the two crude extracts of N. procumbens, the GC-MS technique was used in the study. The nine (9) compounds

106 were identified in dichloromethane extract (NPD) of whole plant of N. procumbens, whereas six (06) compounds were identified in methanol extract (NPM) of whole plant of N. procumbens. (Table-9 and 10). By using the GC-MS analysis, fifteen (15) compounds: (1) 3, 7, 11, 15- tetramethyl-2-hexadecen-1-ol, (2) 12-methyl-E,E-2,13-octadecadien-1-ol, (3) 13- heptadecyn-1-ol, (4) n-hexadecanoic acid, (5) 9,12,15-octadecatrienoic acid, 2,3- dihydroxy propyl ester, (Z,Z,Z)-, (6) Phytol, (7) Trans-13-octadecenoic acid, (8) Oleic acid, (9) Ethyl iso-allocholate, (10) Pterin-6-carboxylic acid, (11) Ergosta-5,22-dien- 3-ol, acetate, (3β,22E)-, (12) 5H-cyclopropa [3, 4] benz [1, 2-e]azulen-5-one, 9, 9a-bis (acetyloxy)-1, 1a, 1b, 2, 4a, 7a, 7b, 8, 9, 9a-decahydro-2, 4a, 7b-trihydroxy-3- (hydroxyl methyl)-1, 1, 6, 8-tetra methyl-, [1aR-(1aα,1bβ,2β,4aβ, 7aα, 7bα, 8α, 9β, 9aα]-, (13) Hexadecanoic acid, methyl ester, (14) 7,10-Octadecadienoic acid and (15) 10-Octadecenoic acid, methyl ester were identified in whole plant material extracts (NPD and NPM) of N. procumbens by performing the analysis. The enzyme inhibition in vitro assays for enzymes like cholinesterase, lipoxygenase, chymotrypsin, α-glucosidase, urease, tyrosinase, carbonic II anhydrase and phosphodiesterase I for the both dichloromethane (NPD) and methanol (NPM) extracts of whole plant of N. procumbens were performed in triplicate and presented with SEM in Table-11 to Table-21. The anti-oxidant and anti-bacterial activities were also performed. The dichloromethane extract of whole plant of N. procumbens (NPD) exhibited moderate antilipoxygenase (44.60±0.23% and IC50 524.60±0.02µmoles), good antichymotrypsin (84.38±0.05% and IC50 115.71±0.03µmoles), excellent anti-alpha glucosidase (95.49±0.65% and IC50 24.32±0.62µmoles), moderate antiurease

(57.23±0.50% and IC50 375.60±0.58µmoles) and moderate antityrosinase

(57.23±0.50% and IC50 321.60±0.45µmoles) activities but antioxidant (1.28±1.66%), anticholinesterase, anticarbonic anhydrase II (37.43±0.41%), antiphosphodiesterase I (1.93±0.41%) and antibacterial activities were not found and inactive. The methanol extract of whole plant of N. procumbens (NPM) exhibited very good antioxidant (75.38±3.16% and IC50 88.30±1.21µmoles), excellent anti-alpha glucosidase (99.45±0.58% and IC50 8.43±0.56µmoles), moderate antiurease

(61.90±0.41% and IC50 315.00±2.91µmoles), moderate antityrosinase (63.92±0.41% and IC50 305.00±6.91µmoles) and very good anticarbonic anhydrase II (73.92±0.41% and IC50 102.31±0.85µmoles) activities but anticholinesterase, antilipoxygenase

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(31.76±3.16%), antichymotrypsin (18.60±0.03%), antiphosphodiesterase I (30.92±0.33%) and antibacterial activities were found inactive. The folk use of the N. procumbens was evaluated by observing the effect of methanol extract (NPM) on isolated tissues of rabbit jejunum and trachea. Spasmolytic activity on rabbit jejunum was established using methanol extract of N. procumbens. The concentration-dependent inhibition of spontaneous contractions (0.01-1.0 mg/ml) + with EC50 value 0.057mg/ml and K (80 mM) induced contractions (0.01-0.1 mg/ml) + with EC50 value 0.024mg/ml were observed. Spontaneous K (80 mM) induced contractions were completely inhibited by standard drug verapamil (Figure-49, A and B). Inhibitory effect of crude methanol extract of N. procumbens was observed on spontaneous contraction of rabbit jejunum that suggests a spasmolytic activity similar to verapamil (Figure-50 and 51). Verapamil was used as standard exhibited relaxation effect on spontaneous contracting jejunum in dose range of 0.03-1.0 µM with EC50 value 0.35 µM and K+ (80 mM) in dose range 0.03-0.3 µM with EC50 value 0.13 µM (Figure-48). The possible mechanism was investigated by using a high K+ (80 mM) was used to cause smooth muscles contractions through opening of calcium channels which results in increase of intra cellular calcium and contractile effect (Bolton, 1979). This result supports the traditional use of N. procumbens in hyperactive gut or in disease diarrhea as calcium antagonist (Sharkey & Wallace, 2011). Justification for the folk use of N. procumbens in respiratory disorders was obtained on the isolated rabbit trachea. The figures 52 (A and B) and 53 show that methanol extract of N. procumbens relaxed tracheal preparation pre contracted with carbachol (1.0 µM) or K+ 80 mM in a concentration dependent fashion similar to standard verapamil. It indicates non-specific brochodilation of trachea mediated through calcium channel blockage. When methanol extract of N. procumbens was applied against carbachol (1.0 µM) and high K+ (80 mM), it produced bronchodilator effect on rabbit trachea and concentration dependent inhibition with respective EC50 value of 0.624 mg/ml (0.01-0.3 mg/ml) and value of EC50 was 0.096 mg/ml (0.01-0.1 mg/ml) (Figure-52 A and B). In summary, the extract coded NPM extract (i.e; methanol extract of whole plant of Neurada procumbens) was very important because it was only found inhibitor of carbonic II anhydrase (73.92±0.41% and IC50 102.31±0.85µmoles) and also only which possess antioxidant activity (75.38±3.16% and IC50 88.30±1.21µmoles) which are very significant and comparable to standards Acetazolamide and Quercetin,

108 respectively. So, this research work provides a new era to scientists to more evaluate the plant N. procumbens with respect to found inhibitor of carbonic anhydrase II enzyme. The DPPH method is a very sensitive method. The anti-oxidant effect is directly corelate to the disappearance of DPPH• in testing material. Due to the formation of DPPH, the color of sample turns from purple to yellow on absorption of hydrogen (Lee et al., 1998). This activiy was performed according to method (Mensor et al., 2001). The results of our study regarding %RSA of methanol extract of Neurada procumbens is 75.38±3.16 (Table-11) which indicates the presence of flavonoids with structure having hydroxyl in molecule can act as proton donating and shows radical scavenging activity (Vinha et al., 2012). However, dichloromethane extract did not show any anti-oxidant activity as it lacks phenol compounds and less concentration of flavonoids constituents as compare to methanol extract (Table-11). By applying GC-MS technique in this study, eighteen (18) compounds have been identified the four extracts of aerial and root parts of F. hamiltonii and fifteen (15) compounds from dichloromethane (NPD) and methanol (NPM) extracts of whole plant of N. procumbens, respectively (see Table-5 to Table-10).

4.10 Conclusion The potential hidden in medicinal plants of Cholistan desert in Pakistan cannot be underestimated. The herbal medicines are used by the residants in rural areas of Pakistan for their health related problems. Due to high cost of allopathic medicines and their many side effects, the educated citizens of Pakistan also prefer the herbal medicines or natural products to to solve health issues. The total thirty three (33) compounds were identified by performing the analysis of six extracts of different parts of two selected medicinal plants Farsetia hamiltonii Royle and Neurada procumbens L in this study. In this research work, it is scientifically proven that methanol extract of Farsetia hamiltonii possess antilipoxygenase, antichymotripsin, antiurease and very significant anti-alpha glucosidase activities. Therapeutic effect of methanol extract (FHAM) of Farsetia hamiltonii Royle (on isolated tissues) is effective as calcium antagonist that proves rationale use of plant in treating diarrhea and abdominal problems. This is first reported in vivo study regarding hypoglycemic effect of methanol extract of Farsetia hamiltonii Royle and scientiﬁcally rationalize folk

109 medicinal use of aerial parts of Farsetia hamiltonii to treat gastrointestinal diseases and diabetes by natives of Cholistan desert. To best of our knowledge, the present investigation on Neurada procumbens L offers rational hypotheses for antidiarrheal and anti-asthmatic medicinal uses of this plant. It was found that methanol extract of Neurada procumbens possess very significant anti-alpha glucosidase, antiurease, antityrosinase, anti-carbonic II anhydrase (only methanol extract), anti-spasmolytic and bronchorelaxant activities in a dose dependent manner. The outcomes of the study are noval and first time published in well reputed journal (appendix). The results scientifically proved the hypothesis and validate the use of the practitioners of the Cholistan and these both selected plants Farsetia hamiltonii Royle and Neurada procumbens L. are valuable in the treatment of the diabetes, gastrointestinal and respiratory diseases in human beings. The study provides a guideline to scientists regarding the study safety and toxicity of therapeutically important compounds that can help in saving humanity from various diseases like diabetes, gastrointestinal and respiratory diseases.

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APPENDIX (Publications from PhD work & CV of Scholar)

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Six (06) Publications from PhD Research Work

1. Hayat et al; 2014: Anti-diabetic and spasmolytic potential of Farsetia hamiltonii Royle from Cholistan desert. Journal of Ethnopharmacology. 156; 347-352. (Published). (Impact Factor 3.05).

2. Hayat MM, Anjum S, Uzair M, Choudhary BA, Nasim FH, Ejaz S and Rashid S. Phytochemical Evaluation of Farsetia hamiltonii Royle from Cholistan Desert. Journal of the Chemical Society of Pakistan. (2015) 37(2); 335-341. (Published). (Impact Factor 0.276).

3. Muhammad Munawar Hayat, Muhammad Uzair. Biological potential and GC-MS analysis of Phytochemicals of Farsetia hamiltonii (Royle). Accepted in Biomedical Research Journal on 29.05.2019, Manuscript No. 0970-938X- 19-241. (Impact Factor 0.219).

4. Muhammad Munawar Hayat, Muhammad Uzair. Phytochemical, enzymatic and biological studies of root extracts of Farsetia hamiltonii Royle. Submitted on 21.01.2019 to Tropical Journal of Pharmaceutical Research, Manuscript No. 20160190.

5. Muhammad Munawar Hayat, Muhammad Uzair. Ethanomedicinal uses, phytochemical and pharmacological aspects of Neurada procumbens (L.) in gastrointestinal and respiratory disorders. Submitted on 11.02.2019 to BMC- Complementary &Alternative Medicines, Manuscript No. BCAM-D-19-00218.

6. Muhammad Munawar Hayat, Muhammad Uzair. Genus Farsetia: Pharmacological And Phytochemical Potentail In Ethanopharmacology, Submitted on 17.05.2019 to Acta Poloniae Pharmaceutica, Manuscript No. APPDR-00507- 2019-01.