Department of Botany University of Peshawar, Peshawar Pakistan

PHARMACOGNOSTIC EVALUATION OF EHRETIA SERRATA ROXB. AND EHRETIA OBTUSIFOLIA HOCHT.EX.A.DC. FAMILY BORAGINACEAE

ZILL-E-HUMA

DEPARTMENT OF BOTANY UNIVERSITY OF PESHAWAR, PESHAWAR PAKISTAN

2013-14

AUTHOR’S DECLARATION

I Zill-E-Huma hereby stated that my Ph.D. thesis titled “Pharmacognostic evaluation of Ehretia serrata Roxb. and Ehretia obtusifolia Hocht. Ex. A.DC. Family Boraginaceae” is my own work and has not been submitted to previously by me for taking degree from this University “University of Peshawar” or anywhere in the country/world.

At any time if my statement is found to be incorrect even after my graduation the university has the right to withdraw my Ph.D. degree.

ZILL-E-HUMA

Date______

PLAGIARISM UNDERTAKING

I solemnly declare that research work presented in the thesis titled “Pharmacognostic evaluation of Ehretia serrata Roxb. and Ehretia obtusifolia Hocht. Ex. A.DC. Family Boraginaceae” is solely my research work with non-significant contribution from any other person. Small contribution/help wherever taken has been duly acknowledged and that complete thesis has been written by me.

I understand the zero tolerance policy of the HEC and University of Peshawar towards plagiarism. Therefore I as an Author of the above titled thesis declare that no portion of my thesis has been plagiarised and any material used as reference is properly referenced/cited.

I undertake that if I am found guilty of any formal plagiarism in the above titled thesis even after award of PhD degree, the University reserves the right to withdraw/revoke my PhD degree and that HEC and the University has the right to pub

lish my name on the HEC/University Website on which names of students are placed who submitted plagiarized thesis.

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ZILL-E-HUMA

DEDICATION

This Dissertation is dedicated to my PARENTS,

who not only raise and nurtured me but also

taxed himself dearly over the years for my

education and intellectual development.

and

To my beloved brother Mr. ZIA MUHAMMAD,

whose continuous guidance and help are the

source of motivation and strength during

moments of despair and discouragement.

PHARMACOGNOSTIC EVALUATION OF EHRETIA SERRATA ROXB. AND EHRETIA OBTUSIFOLIA HOCHT.EX.A.DC. FAMILY BORAGINACEAE

A Thesis Submitted to the University of Peshawar in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy In Botany By Zill-e-Huma

Graduate study committee 1. Prof. Dr. Ghulam Dastagir Convener. 2. Prof. Dr. Siraj ud Din Member. 3. Dr. Zahir Muhammad Member 4. Dr. Tanvir Burni Member 5. Dr. Rasool Khan Member

DEPARTMENT OF BOTANY UNIVERSITY OF PESHAWAR, PESHAWAR PAKISTAN 2014

PUBLICATION OPTION

I hereby reserve the right of publication, including right to reproduce this thesis in any form for a period

of 5 years from the date of submission.

ACKNOWLEDGEMENT

All praises are meant to Almighty Allah, The Creator, The Guider and the Sustainer, Who provided the courage, strength and vision to carry out this research work. It is a genuine pleasure to express my deep sense of thanks and gratitude to my mentor, philosopher and guide Prof. Dr. Muhammad Ibrar, Botany department, University of Peshawar. His dedication and keen interest above all his overwhelming attitude to help his students had been solely and mainly responsible for completing my work. His firmly advice, meticulous scrutiny, scholarly advice and scientific approach have helped me to very great extent to accomplish this task.

I owe a deep sense of gratitude for my co-supervisor Asst. Prof. Dr. Barkatullah for his keen interest to me at every stage of my research work. His prompt inspiration, timely suggestions with kindness, enthusiasm and dynamism has enabled me to complete my thesis.

I thank profusely to chairman of Botany department Dr. Ghulam Dastagir for his encouragement and support.

I would like to thank to my respected teacher’s i.e Dr. Siraj-u-din, Dr. Zahir Muhammad, Mrs. Tanvir Burni, Mr. Rehman Ullah, Dr. Lal Badshah, Dr. Fazal Hadi and Dr. Sami Ullah for their support and helping attitude.

I would like to thank to Dr. Memrez Khushal Gigyani, Research officer at VRI, Peshawar, for timely providing me all the necessary facilities and administrative support during my research work.

I am also thankful to Dr. Farah khan PCSIR Peshawar, Dr. Sayyar Kazi and Mr. Iftikhar Department of Biotechnology, University of Agriculture, Peshawar for support and help.

I am also grateful to my research fellows Mr. Ghulam Jilani, Mr. Shafqat Ali Khan, Dr. Maryam Ehsan, Mr. Inam Ullah, Mr.Najeeb Ur Rehman, Mubina Tariq, Sidra Haider, Umbreen Khattak, Ulfat Samreen, Naila Innayat, Palwasha Khan, Umbreen, Saima and Saiqa for pacing with me and maintaining a comfortable working atmosphere.

I would also like to acknowledge the contribution of office and para teaching staff of Department of Botany, University of Peshawar for helping me all the way throughout my study.

It is privilege to thank to my sisters, Miss Haleema Begum, Sadia Noreen, Mehreen Zaman and my Brother in Laws Zia Muhammad and Gohar Ali for their kind help and encouragement throughout my study period.

I want to show my deepest love to my mother for playing the role of mother, guider, supporter and mentor in fulfilling of my dreams.

ZILL-E-HUMA

ABSTRACT

The current investigation is carried out to detect the pharmacognostic, physicochemical and pharmacological evaluation of leaf, stem bark and root of Ehretia serrata Roxb. and Ehretia obtusifolia Hochst. Ex A. DC which belongs to the family Boraginaceae.

The morphological study showed that E. serrata is a small size deciduous tree, less than 10 m tall, locally named as puran and punna in Urdu. Its Leaf is simple with oblong shape (5-16 cm long and 2.8-6.5 cm wide), serrulated margins and glabrous surface. E. serrata contains drupe globose type fruit with yellowish green-light color when unripe and black when ripe. Stem bark is irregular with up to 2.5 cm wide, dark brown outer color and whitish golden inner surface color. E. obtusifolia is a deciduous small shrub or small tree up to 6 m tall; straggling or with several branches from the base. The Leaf is oblong-elliptic or oboviate with obtuse apex, entire margins and reticulate venation. Microscopic study of E. serrata showed the presence of conical trichomes and anomocytic type of stomata on both surfaces where as anisocytic type of stomata was observed only on the lower epidermis with stomatal index 36.7μ. Upper surface showed 30.4 μ stomatal index. Other parameter includes palisade ratio (6.4+0.894 per mm2), vein Islet number (19.4+1.79 per mm2), stomata number (21.8+1.78 per mm2) and vein termination number (10.6+1.987 per mm2). A Similar microscopic anatomical study was carried out of leaf of E. obtusifolia and we observed two types of trichomes i.e simple conical trichomes and capitate glandular trichome on both surfaces. Stomatal cluster was observed only on the lower epidermis with three types of stomata i.e anomocytic, anisocytic and hemiparacytic. Other observed parameters were palisade ratio (6.2 + 0.81 per mm2), vein islets number (18.6 + 1.14 per mm2), vein termination number (8.2 + 0.828 mm2), stomatal number (21.8 + 1.78 per mm2) and stomatal index (35.7 + 6.12 μ).

Stem bark anatomy of both experimented plants showed clear layers of cork, cork cambium, phloem elements, medullary rays and tracheids, etc. Fruit anatomy of both plants showed a clear distinction between three layers of pericarps and seeds. Powder drug microscopy of leaf, stem bark and fruits of E. serrata and E. obtusifolia was carried out and observed stomata, epidermal cells, fibers, tracheids and vessels, etc.

iii

Results of qualitative preliminary phytochemical screenings of examining parts of E. serrata and E. obtusifolia are given, presenting the presence of phenol compounds, starch, protein, reducing sugar, steroids, terpenoids, fats, aldehydes, anthraquinones, glycosides, amino acids, saponins, alkaloids, tannins, phlobatannin. Similarly, results of a fluorescence study of leaf, stem bark and fruit of E. serrata and E. obtusifolia are also given. Results of elemental analysis showed the presence of some essential elements in all examined parts of these two plants. The most abundant elements found in all three parts of E. serrata and E. obtusifolia were Mg, Na, and Fe and which may contribute to analgesic and anti-inflammatory effects of these plants. Proximate analysis of both plants showed carbohydrate, proteins, fibers, fats and moisture in fairly large amounts.

The methanolic extracts of E. serrata and E. obtusifolia leaf, stem bark and fruit were non-toxic against Swiss albino mice. Methanolic extract of leaf of E. serrata showed the highest (P<0.01) cytotoxic activity (83.3%) with the LD50 values 15.29 µg/mL at dose level 1000 µg/mL and in E. obtusifolia the fruit methanolic extract showed highest

(P<0.01) (80.0%) cytotoxic activity at 1000 µg/mL with an LD50 value of 16.07 µg/mL. All experimented parts of both plants showed non-significant (P>0.05) Phytotoxic activity at all dose levels of 10, 100 and 1000 µg/mL. The anti-oxidant effect of all parts (leaf, stem bark and fruit) of both plants was dose dependent. High (94.1%) DPPH inhibition was shown by stem bark extract of E. serrata at dose level 1000 µg/mL after 60 min and fruit extract of E. obtusifolia (85.01%) at level 1000 µg/mL after

30 minutes. Based on IC50 (µg/mL) stem bark of E. obtusifolia and leaf of E. serrata showed significant values as compared to ascorbate.

Both plant methanolic extract was not that much more effective against bacterial strains inhibition. The prominent antibacterial zone (7 mm) was observed in the leaf methanolic extract of E. serrata as compared to reference drug (31.5 mm) at dose level 1000 µg/mL. Similarly, different parts of E. obtusifolia tested against various bacteria which showed no significant results and the most prominent (7 mm) anti-bacterial zone was observed at dose level 1000 µg/mL of leaf extract compared to reference drug (30.2 mm).

Toxicological studies showed that both plants are safe for human use. Analgesic effect was found dose dependent and time dependent and all parts of both investigated parts

(P<0.05) showed significant results. The highly significant analgesic effect was shown by all experimented parts (P<0.01) at all dose level 100 mg/kg, 200 mg/kg and 300 mg/kg over 60 minutes in E. Serrata except stem bark. Stem bark of E. obtusifolia showed highly significant (P<0.01) at all dose after 60 minutes.

Anti-inflammatory effect of all three investigated parts of E. serrata and E. obtusifolia were observed and it is noticed that all parts of E. serrata showed high significancy (P<0.01) at all dose i.e 100 mg/kg, 200 mg/kg and 300 mg/kg after 2 and 3 hours. Similar results were shown by E. obtusifolia.

Antispasmodic effects of methanolic extract of leaf stem bark and fruit of E. serrata and E. obtusifolia was tested in swiss albino mice. Stem bark methanolic extract showed highly significant (P<0.01) results at dose of 200 mg/kg and 300 mg/kg. E. obtusifolia causes better relaxation of the smooth muscles at very low concentrations. Leaf extract at dose of 200 mg/kg and 300 mg/kg, fruit at level of 300 mg/kg and stem bark at all dose showed highly significant (P<0.01) results thus providing a scientific proof for its ethnopharmacological use as an antispasmodic drug.

The present study has revealed the enormous and different medicinal properties of E. serrata and E. obtusifolia both can be exploited for therapeutic preparations on a commercial scale.

LIST OF CONTENT S. No Content Page. No Acknowledgment i-ii Abstract iii-v CHAPTER-1 1-19 INTRODUCTION 1.1 Medicinal plants 1 1.2 Pharmacognosy 1 1.2.1 Macroscopy and histology 4 1.2.2 Fluorescence study 5 1.2.3 Phytochemical screening 6 1.2.4 Elemental analysis 7 1.2.5 Nutritional analysis 7 1.3 Pharmacology 8 1.3.1 Acute toxicological study 8 1.3.2 Cytotoxic activity 8 1.3.3 Phytotoxic activity 9 1.3.4 Antioxidant activity 9 1.3.5 Anti-bacterial activity 10 1.3.6 Analgesic activity 10 1.3.7 Anti-inflammatory activity 11 1.3.8 Anti- diarrhoeal activity 11 1.4 Family Boraginaceae 12 1.4.1 Description 12 1.4.2 Distribution 14 1.4.3 Medicinal uses 14 1.5 Genus Ehretia 15 1.5.1 Distribution 15 1.5.2 Description 15 1.5.3 Medicinal uses 15 1.6 E. serrata Roxb. 16

1.6.1 Distribution 16 1.6.2 Description 16 1.6.3 Medicinal uses 16 1.7 E. obtusifolia Hocht.A.DC. 18 1.7.1 Distribution 18 1.7.2 Description 18 1.7.2 Medicinal uses 18 CHAPTER-2 20-48 REVIEW OF LITERATURE 2.1 Genus Ehretia 20 2.2 Pharmacognostic studies 23 2.2.1 Morphology and anatomy 23 2.2.2 Powder drug study 25 2.2.3 Fluorescence analysis and phytochemical screening 26 2.2.4 Elemental analysis and nutritional analysis 28 2.3 Pharmacology 29 2.3.1 Acute toxicological activity 29 2.3.2 Anti-oxidant activity 29 2.3.3 Cytotoxic activity and phytotoxic activity 33 2.3.4 Anti-bacterial activity 34 2.3.6 Analgesic activity 37 2.3.7 Anti-inflammatory activity 38 2.3.8 Anti-diarrhoeal activity 39 CHAPTER-3 MATERIAL AND METHODS 3.1 Collection and preservation 43 3.2 Pharmacognosy 44 3.2.1 Morphological study 44 3.2.2 Macroscopic and organoleptic study 44 3.2.3 Microscopic and anatomical study 44 3.2.4 Leaf surface study 44 3.2.4.i Stomatal number and stomatal index 44

3.2.4.ii Vein islet and vein termination number 45 3.2.4.iii Palisade ratio 45 3.2.5 Powder drug study 46 3.2.6 Fluorescence characters 46 3.2.7 Preparation of methanolic extracts 47 3.2.8 Preliminary qualitative phytochemical screening 47 3.2.8.i Carbohydrates detection test 48 3.2.8.ii Protein and amino acid detection test 48 3.2.8.iii Alkaloids detection test 48 3.2.8.iv Phenol detection test 49 3.2.8.v Fixed oil detection test 49 3.2.8.vi Flavonoids detection test 49 3.2.8.vii Saponin detection test 49 3.2.8.viii Terpenoids detection test 50 3.2.8.ix Tannins detection test 50 3.2.8.x Anthraquinone detection test 50 3.2.8.xi Glycosides detection test 50 3.2.8.xii Phytosterols detection test 51 3.2.8.xiii Reducing sugar detection test 51 3.2.8.xii Starch detection test 51 3.2.9 Elemental analysis 51 3.2.10 Nutritional analysis 54 3.3.10.i Determination of moisture 54 3.3.10.ii Determination of ash 54 3.3.10.iii Determination of crude protein by micro kjeldahl method 55 3.3.10.iv Determination of fats or ether extract 56 3.3.10.v Determination of crude fibers 57 3.3.10.vi Determination of carbohydrates 58 3.11 Biological activities 58 3.11.i Acute toxicological study 58 3.11.ii Cytotoxic activity 59 3.11.iii Phytotoxic activity 60

3.11.iv Anti-oxidant activity 62 3.11.v Antibacterial activity 63 3.11.vi Analgesic activity 64 3.11.vii Anti-inflammatory activity 65 3.11.viii Anti-diarrhoeal activity 66 Objectives of the study 68

CHAPTER-4 RESULTS AND DISCUSSION 4.1 Systematic positions of the plants 69 4.1.i E. serrata 69 4.1.ii E. obtusifolia 70 4.2 Morphological description 71 4.2.i Morphological description of E. serrata 71 4.2..ii Morphological description of E. obtusifolia 72 4.3 Pharmacognosy 72 4.3.1 Macroscopy and organoleptic characteristics 73 4.3.1.i Organoleptic and microscopical characters of E. serrata 73 leaf 4.3.1.ii Organoleptic and microscopical characters of E. serrata 73 fruit 4.3.1.iii Organoleptic and microscopical characters of E. serrata 74 stem bark 4.3.1.iv Organoleptic and microscopical characters of E. 74 obtusifolia leaf 4.3.v Organoleptic and microscopical characters of E. 75 obtusifolia fruit 4.3.vi Organoleptic and microscopical characters of E. 75 obtusifolia stem bark 4.3.2 Microscopic and anatomical study 79 4.3.2.i Transverse section of E. serrata Leaf 79 4.3.2.ii Leaf surface parameters 80 4.3.2.ii.a Stomatal and trichomal study 81

4.3.2.ii.b Vein islet and vein termination number 82 4.3.2.ii.c Palisade ratio 83 4.3.2.iii Transverse section of E. obtusifolia leaf 84 4.3.2.iv Leaf surface parameters 84 4.3.2.iv.a Stomatal and trichomal study 85 4.3.2.iv.b Vein islet and vein termination number 88 4.3.2.iv.c Palisade ratio 89 4.3.2.v Anatomy of E. serrata stem bark 90 4.3.2.vi Anatomy of E. obtusifolia stem bark 90 4.3.2.vii Anatomy of E. serrata fruit 92 4.3.2.viii Anatomy of E. obtusifolia fruit 92 4.3.3 Powder drug study 93 4.3.3.i Powder drug study of E. serrata leaf 93 4.3.3.ii Powder drug study of E. serrata stem bark 93 4.3.3.iii Powder drug study of E. obtusifolia leaf 94 4.3.3.iv Powder drug study of E. obtusifolia stem bark 94 4.3.4 Phytochemical screening 100 4.3.4.i Phytochemical screening of E. serrata leaf 100 4.3.4.ii Phytochemical screening of E. serrata fruit 100 4.3.4.iii Phytochemical screening of E. serrata stem bark 100 4.3.4.iv Phytochemical screening of E. obtusifolia leaf 101 4.3.4.v Phytochemical screening of E. obtusifolia fruit 101 4.3.4.vi Phytochemical screening of E. obtusifolia stem bark 101 4.3.4.vii Comparison of Phytochemical compounds of leaf, stem 102 bark and fruit methanolic extracts of E. serrata and E. obtusifolia 4.3.4.vii.a Leaf 102 4.3.4.vii.b Fruit 102 4.3.4.vii.c Stem bark 102 4.3.5 Fluorescence study 104 4.3.5.i Fluorescence study E. serrata of leaf, fruit and stem bark 104

4.3.5.ii Fluorescence study of E. obtusifolia leaf, fruit and stem 104 bark 4.3.6 Elemental analysis 108 4.3.6.i Elemental analysis of E. serrata leaf, stem bark and fruit 108 4.3.6.ii Elemental analysis of E. obtusifolia leaf, stem bark and 113 fruit 4.3.7 Nutritional analysis 121 4.3.7.i Nutritional analysis of E. serrata leaf 121 4.3.7.ii Nutritional analysis of E. serrata stem bark 121 4.3.7.iii Nutritional analysis of E. serrata fruit 121 4.3.7.iv Nutritional analysis of E. obtusifolia leaf 122 4.3.7.v Nutritional analysis of E. obtusifolia fruit 122 4.3.7.vi Nutritional analysis of E. obtusifolia stem bark 122 4.3.7.vii Comparison of nutritional values of E. serrata and E. 122 obtusifolia leaf, fruit and stem bark 4.3.7.vii.a Leaf 122 4.3.7.vii.b Fruit 123 4.3.7.vii.c Stem bark 123 4.4 Biological activities 125 4.4.1 Acute toxicological study 125 4.4.2 Cytotoxic activity 126 4.4.2.i Cytotoxic activity of E. serrata leaf 126 4.4.2.ii Cytotoxic activity of E. serrata fruit 127 4.4.2.iii Cytotoxic activity of E. serrata stem bark 127 4.4.2.iv Cytotoxic activity of E. obtusifolia leaf 127 4.4.2.v Cytotoxic activity of E. obtusifolia fruit 128 4.4.2.vi Cytotoxic activity of E. obtusifolia stem bark 128 4.4.2.vii Comparison of cytotoxic potency of E. serrata and E. 128 obtusifolia leaf, fruit and stem bark 4.4.2.vii.a Leaf 128 4.4.2.vii.b Fruit 129 4.4.2.vii.c Stem bark 129

4.4.3 Phytotoxic activity 133 4.4.3.i Phytotoxic activity of E. serrata leaf 133 4.4.3.ii Phytotoxic activity of E. serrata fruit 133 4.4.3.iii Phytotoxic activity of E. serrata stem bark 134 4.4.3.iv Phytotoxic activity of E. obtusifolia leaf 134 4.4.3.v Phytotoxic activity of E. obtusifolia fruit 134 4.4.3.vi Phytotoxic activity of E. obtusifolia stem bark 135 4.4.3.vii Comparison of phytotoxic potency of E. serrata and E. 135 obtusifolia leaf, fruit and stem bark 4.4.3.vii.a Leaf 135 4.4.3.vii.b Fruit 135 4.4.3.vii.c Stem bark 135 4.4.4 Anti-oxidant activity 138 4.4.4.i Anti-oxidant activity of E. serrata leaf 138 4.4.4.ii Anti-oxidant activity of E. serrata fruit 138 4.4.4.iii Anti-oxidant activity of E. serrata stem bark 139 4.4.4.iv Anti-oxidant activity of E. obtusifolia leaf 140 4.4.4.v Anti-oxidant activity of E. obtusifolia fruit 140 4.4.4.vi Anti-oxidant activity of E. obtusifolia stem bark 141 4.4.4.vii Comparison of anti-oxidant potency of methanolic 142 extracts of E. serrata and E. obtusifolia 4.4.4.vii.a Leaf 142 4.4.4.vii.b Fruit 142 4.4.4.vii.c Stem bark 142 4.4.5 Anti-bacterial activity 148 4.4.5.i Anti-bacterial activity of E. serrata leaf 148 4.4.5.ii Anti-bacterial activity of E. serrata fruit 149 4.4.5.iii Anti-bacterial activity of E. serrata stem bark 149 4.4.5.iv Anti-bacterial activity of E. obtusifolia leaf 149 4.4.5.v Anti-bacterial activity of E. obtusifolia fruit 150 4.4.5.vi Anti-bacterial activity of E. obtusifolia stem bark 150

4.4.5.vii Comparison of anti-bacterial potency of methanolic 151 extracts E. serrata and E. obtusifolia 4.4.5.vii.a Leaf 151 4.4.5.vii.b Fruit 151 4.4.5.vii.c Stem bark 151 4.4.6 Analgesic activity 157 4.4.6.i Analgesic activity of E. serrata leaf 157 4.4.6.ii Analgesic activity of E. serrata fruit 157 4.4.6.iii Analgesic activity of E. serrata stem bark 158 4.4.6.iv Analgesic activity of E. obtusifolia leaf 158 4.4.6.v Analgesic activity of E. obtusifolia fruit 159 4.4.6.vi Analgesic activity of E. obtusifolia stem bark 159 4.4.6.vii Comparison of analgesic potency of methanolic extracts 160 of E. serrata and E. obtusifolia 4.4.6.vii.a Leaf 160 4.4.6.vii.b Fruit 160 4.4.6.vii.c Stem bark 161 4.4.7 Anti-inflammatory activity 164 4.4.7.i Anti-inflammatory activity of E. serrata leaf 164 4.4.7.ii Anti-inflammatory activity of E. serrata fruit 165 4.4.7.iii Anti-inflammatory activity of E. serrata stem bark 165 4.4.7.iv Anti-inflammatory activity of E. obtusifolia leaf 166 4.4.7.v Anti-inflammatory activity of E. obtusifolia fruit 167 4.4.7.vi Anti-inflammatory activity of E. obtusifolia stem bark 168 4.4.7.vii Comparison of anti-inflammatory potency of methanolic 169 extracts of E. serrata and E. obtusifolia 4.4.7.vii.a Leaf 169 4.4.7.vii.b Fruit 170 4.4.7 Stem bark 170 4.4.8 Anti-diarrhoeal activity 176 4.4.8.i Anti-diarrhoeal activity of E. serrata leaf 176 4.4.8.ii Anti-diarrhoeal activity of E. serrata fruit 176

4.4.8.iii Anti-diarrhoeal activity of E. serrata stem bark 176 4.4.8.iv Anti-diarrhoeal activity of E. obtusifolia leaf 177 4.4.8.v Anti-diarrhoeal activity of E. obtusifolia fruit 177 4.4.8.vi Anti-diarrhoeal activity of E. obtusifolia stem bark 177 4.4.8.vii Comparison of Anti-diarrhoeal potency of leaf, fruit and 178 stem bark methanolic extracts of E. serrata and E. obtusifolia 4.4.6. vii. a Leaf 178 4.4.6.vii.b Fruit 178 4.4.6.vii.c Stem bark 178 CHAPTER-5 DISCUSSION 183

Conclusion 189 Recommendations 192 References 193

LIST OF TABLES Table No. Title Page No. 3.1 Conditions applied for detection of various elements 53 4.1 Organoleptic and macroscopical characteristics of E. serrata 76 leaf 4.2 Organoleptic and macroscopical characteristics of E. serrata 76 stem bark 4.3 Organoleptic and macroscopical characteristics of E. serrata 77 fruit 4.4 Organoleptic and macroscopical characteristics of E. 77 obtusifolia leaf 4.5 Organoleptic and macroscopical characteristics of E. 78 obtusifolia stem bark 4.6 Organoleptic and macroscopical characteristics of E. 78 obtusifolia fruit 4.7 Leaf surface features of E. serrata 83 4.8 Leaf surface features of E. obtusifolia 89 4.9 List of phyto-constituents found in different parts of E. 103 serrata 4.10 List of phyto-constituents found in different parts of E. 103 obtusifolia 4.11 Fluorescence study of leaf, fruit and stem bark of E. serrata 104 4.12 Fluorescence study of leaf, fruit and stem bark of E. 105 obtusifolia 4.13 Elemental analysis of E. serrata 118 4.14 Elemental analysis of E. obtusifolia 118 4.15 Nutritional analysis of leaf, fruit and stem bark of E. serrata 123 and E. obtusifolia 4.16 Cytotoxic activity of methanolic extract of leaf, fruit and 130 stem bark of E. serrata and E. obtusifolia 4.17 Phytotoxic activity of E. serrata and E. obtusifolia leaf, fruit 135 and stem bark

4.18 Percentage of anti-oxidant activity of Ehretia serrata leaf, 144 fruit and stem bark 4.19 Percentage of anti-oxidant activity of E. obtusifolia leaf, fruit 146 and stem bark 4.20 Antibacterial activity of different parts of E. serrata 153 4.21 Antibacterial activity of different parts of E. obtusifolia 153 4.22 Analgesic effect of E. serrata leaf, fruit and stem bark 161 4.23 Analgesic effect of E. obtusifolia leaf, fruit and stem bark 122 4.24 Anti-inflammatory activity of E. serrata 171 4.25 Anti-inflammatory activity of E. obtusifolia 171 4.26 Anti-diarrhoeal activity of E. serrata and E. obtusifolia leaf, 179 fruit and stem bark

LIST OF FIGURES Figure No. Title of figure Page No. 1.1 Fruits and leaves of E. serrata 17 1.2 Stem of E. serrata 17 1.3 Leaves and flowers of E. serrata 17 1.4 Flowers of E. obtusifolia 19 1.5 Fruits and Leaves of E. obtusifolia 19 1.6 Stem of E. obtusifolia 19 4.1 Leaf of E. serrata 75 4.2 Fruit of E. serrata 75 4.3 Stem bark of E. serrata 75 4.4 Leaf of E. obtusifolia 75 4.5 Fruit of E. obtusifolia 75

4.6 Stem bark of E. obtusifolia 75 4.7 Midrib transverse section of leaf of E. serrata 80 4.8 Laminar portion of leaf of E. serrata Roxb. 80 4.9 Anomocytic stomata on upper epidermis 82 4.10 Unicellular non-glandular trichome on lower epidermis 82 4.11 Anomocytic and anisocytic types of stomata on lower surface of 82 E. serrata leaf 4.12 Vein Islets on the surface of E. serrata leaf 83 4.13 Vein termination number on the surface of E. serrata leaf 83 4.14 Transverse section of midrib of E. obtusifolia 85 4.15 Transverse section of laminar portion of leaf E. obtusifolia 85 4.16 Capitate bi-cellular glandular trichomes on both epidermises 87 4.17 Upper epidermal cells 87 4.18 Unicellular simple conical uni-cellular trichomes on upper 87 epidermis 4.19 Septate 3-celled non-glandular conical trichome 87 4.20 Lower leaf epidermal surface of E. obtusifolia 88 4.21 Anomocytic, anisocytic and hemiparacytic types of stomata on 88 lower epidermis of E. obtusifolia leaf

4.22 Vein islets on surface of E. obtusifolia leaf 89 4.23 Vein termination numbers on surface of E. obtusifolia leaf 89 4.24 T.S of Stem bark of E. serrata 91 4.25 T.S of stem bark of E. obtusifolia 91 4.26 Fruit of E. obtusifolia in cross section 92 4.27 Fruit of E. serrata in cross section 92

4.28 Powder drug of E. serrata leaf 96 4.29 Powder drug of E. serrata stem bark 97 4.30 Powder drug of E. serrata leaf 98 4.31 Powder drug of E. obtusifolia stem bark 99 4.32 Plate A: Staphylococcus aureus zone inhibition by effect of E. 152 obtusifolia stem bark at 1000 µg/mL Plate B: Proteus vulgaris zone inhibition by effect of leaves extract of E. obtusifolia at 1000 µg/mL Plate C: Xanthomonas spp zone inhibition by effect of leaves extract of E. serrata at 1000 µg/mL Plate D: Proteus vulgaris zone inhibition by effect of fruit extract of E. obtusifolia at 1000 µg/mL Plate E: Clavibacter michiganensis zone inhibition by effect of fruit extract of E. serrata at 1000 µg/mL 4.33 Plate A: Administration of drug in albino mice intraperitoneally 162 Plate B: Measurement of analgesic effect of drug on analgesometer 4.34 Plate A: Measurement of normal paw size on plethysmometer 172 Plate B: Administration of drug through injection Plate C: Marking of swiss albino mice tail for identification Plate D: Measurement of paw inflammation after specific time period after drug administration on plethysmometer. 4.35 Plate A: Charcoal administration in Albino swiss mice 180 Plate B: Killing of Albino swiss mice Plate C: Dissection of Albino swiss mice Plate D: Charcoal motility measurement in small intestine

LIST OF GRAPHS Graph No. List of graphs Page No. 4.1 Relative standard deviation percentage difference among 119 different element found in E. serrata leaf 4.2 Relative standard deviation percentage difference among 119 different element found in E. serrata fruit 4.3 Relative standard deviation percentage difference among 119 different element found in E. serrata stem bark 4.4 Relative standard deviation percentage difference among 120 different element found in E. obtusifolia leaf 4.5 Relative standard deviation percentage difference among 120 different element found in E. obtusifolia fruit 4.6 Relative standard deviation percentage difference among 120 different element found in E. obtusifolia stem bark 4.7 Comparison of nutritional values of E. serrata and E. 123 obtusifolia leaf 4.8 Comparison of nutritional values of E. serrata and E. 124 obtusifolia fruit 4.9 Comparison of nutritional values of E. serrata and E. 124 obtusifolia stem bark 4.10 Comparison of cytotoxic activity of E. serrata 131 and E. obtusifolia leaf 4.11 Comparison of cytotoxic activity of fruit extract of E.serrata 131 and E. obtusifolia 4.12 Comparison of cytotoxic activity of stem bark extract of 131 E.serrata and E. obtusifolia 4.13 Comparison of phytotoxic activity of E.serrata and E. 137 obtusifolia leaf 4.14 Comparison of phytotoxic activity of E. serrata 137 and E. obtusifolia fruit 4.15 Comparison of phytotoxic activity of E.serrata and E. 137 obtusifolia fruit

4.16 % DPPH inhibition by E. serrata leaf 145

4.17 % DPPH inhibition by E. serrata fruit 145 4.18 % DPPH inhibition by E. serrata stem bark 145 4.19 % DPPH inhibition by E. obtusifolia leaf 147 4.20 % DPPH inhibition by E. obtusifolia fruit 147 4.21 % DPPH inhibition by E. obtusifolia stem bark 147 4.22 Anti-bacterial activity of E. serrata leaf 154

4.23 Anti-bacterial activity of E. serrata fruit 154 4.24 Anti-bacterial activity of E. serrata stem bark 155 4.25 Anti-bacterial activity of E. obtusifolia leaf 155

4.26 Anti-bacterial activity of E. obtusifolia fruit 156 4.27 Anti-bacterial activity of E. obtusifolia stem bark 156 4.28 Comparison of analgesic activity of E. serrata leaf, fruit, stem 163 bark with standard drug 4.29 Comparison of analgesic activity of E. serrata leaf, fruit, stem 163 bark with standard drug 4.30 Anti-inflammatory action of E. serrata after one hour 173 4.31 Anti-inflammatory action of E. serrata after two hours 173 4.32 Anti-inflammatory action of E. serrata after three hours 174 4.33 Anti-inflammatory action of E. obtusifolia after one hour 174 4.34 Anti-inflammatory action of E. obtusifolia after two hours 175 4.35 Anti-inflammatory action of E. obtusifolia after three hours 175 4.36 % Anti-diarrhoeal activity of E. serrata 181 4.37 % Anti-diarrhoeal activity of E. obtusifolia 181 4.38 Comparison of % intestinal transportation of E. serrata and E. 183 obtusifolia

LIST OF ABBREVIATIONS USED IN GRAPHS

ES Ehretia serrata EO Ehretia obtusofolia ESL Ehretia serrata leaf ESF Ehretia serrata fruit ESSB Ehretia serrata stem bark EOL Ehretoa obtusifolia leaf EOF Ehretia obtusifolia fruit EOSB Ehretia obtusifolia stem bark SA Staphylococcus aureus CM Clavibacter michiganensis PV Proteus vulgaris XP Xanthomonas perforans ILT Initial latency time SD Standard dose C.oil Castor oil Atr Atropine

CHAPTER-1

INTRODUCTION

1.1) Medicinal plants

Plants are an important constituent of the universe and human beings have used plants as medicines from the very beginning. Plants have always been a valuable source of drugs and many of the presently available drugs have been derived directly or indirectly from them (Kahrizi et al., 2012; Ahmad et al., 2009).

Medicinal plants include various types of plants used in herbalism having pharmaceutical activities. These medicinal plants are considered as a rich resource of elements which can be used in drug synthesis. They also have a crucial role in the development of human culture. These plants are important for their nutrition and recommended for their medicinal purposes.

Different drugs are synthesized from medicinal plants by using them as raw material. For example laxatives, blood thinners, antibiotics and antimalarial medications contain ingredients from plants. Moreover the active ingredients of Taxol, vincristine and morphine are isolated from foxglove, periwinkle, yew and opium respectively (Hassan., 2012). In past the plant extracts were used for curing various diseases (Wyk & Wink., 2017). Presently up to 50% of all medicines are obtained from extracts of plants (Cragg & Newman, 2005).

1.2) Pharmacognosy

Pharmacognosy is basically the detailed study of medicines originated from natural sources. According to Tyler (1999) “Pharmacognosy is the study of the physical, chemical, biochemical and biological properties of drugs, drug substances or potential drugs or drug substances of natural origin as well as the search for new drugs from natural sources”.

Pharmacognosy is the combination of two Greek words viz Pharmakon (drug) and Gignosco (to acquire the knowledge of). Pharmacognosy is a branch of biosciences, which treat in detail the medicinal and related products of crude or primary types of

drug obtained from plants, animals, and minerals (Thoha et al., 2012). It has also evolved to include phytotherapy and nutraceuticals (Reader, 2007) .

Nowadays pharmacognosy comprises the wide study of all natural products from plants, bacteria, fungi and marine organisms, botanical dietary supplements as well as herbal remedies (Cardellina, 2002; Ashutosh, 2007; Saleem et al., 2010). Pharmacognosy is a field of scientific and organized study of features of crude drugs (physical, chemical, structural and biological) as well as their history, study of methods of cultivation, preservations, collection and preparation for the commercial purposes (Gokhele et al., 2008; Teki & Binzet., 2017).

This field is related to the provision, development and evolution of new medicines. Pharmacognosy is the precise and scientific study of crude drugs obtained from natural sources such as plants, animals, minerals and metals. It is calculated that 90% of the crude drugs evolve from plants while the rest are extracted from other sources (Joy et al., 1998).

There are five customary parameters involved in the pharmacognostic study of crude drugs, i.e. the chemical, physical, botanical, organoleptic and pharmacological parameters. These parameters help in the evaluation and assessment of the distinctive features of crude drugs in three different phases such as identification of drug, isolation of active compounds and screening for biological activities (Parveen et al., 2007). Pharmacognosists often come across with two familiar procedures, adulteration and substitution. Adulteration means the deterioration of any item which is due to inferiority, admixture, damage, complexity, spoilage and substitution. The word ‘substitution’ means ‘placement of one article in place of another article or “sale of completely different article in place of the required or desired requested article such as cottonseed oil sold as olive oil and American saffron sold as Spanish saffron (Selvam, 2015).

Pharmacognosy is closely related to different scientific fields such as Pharmacology, Analytical Chemistry, Microbiology, Plant Tissue Culture, Biotechnology and Genetic Engineering (Rangari, 2002; Balunasa & Kinghornb, 2005).

Pharmacognosy provides methods for synthesis and formulation of drugs by interlinking many traditional systems in the best accepted allopathic form (Chen et al., 2016). The knowledge about various linked technologies such as extraction, refining, plant tissue culture, etc. supports crude drug preparation (Jarald & Jarald, 2007).

Nowadays pharmacognosy interlinks biology, chemistry and medicines by studying various molecules in nature. In pharmacognosy, strategies and methods show advancement in the selection of organism, bioassays procedures, isolation techniques, and structure elucidation (Bruhn & Bohlin, 1997).

Pharmacognosy became advanced with the passage of time due to development of n technologies and procedures and gained the attention of researchers in the field of research (Kinghorn, 2002; Samuelsson, 2004). Pharmacognosy is basically the study of medicinal plants for therapeutic uses (Okigbo et al., 2008).

Ancient people used medicinal plants to cure many diseases in various forms of crude drug, teas, powder and tinctures. In the field of homeopathy, formula of different medicines and method of use of a particular medicine was transferred from one generation to another generation via communication in past (Ahmad et al, 2006; Balick & Cox, 1997; Samuelsson, 2004).

Pharmacognosy includes the study of flavouring agents, raw materials and filtering aid like antibiotics (Kokate, 1997). Pharmacognosy has complete information about herbal products and natural crude drugs (Modi et al., 2010).

The main fields of interest of pharmacognosy are computational chemistry, structural, cellular and molecular biotechnology, bioanalytical and molecular toxicology (Gangwal, 2008). Pharmacognosy is credited for synthesis of many vital compounds to cure various human chronic disorders (Golchin & Fotokian, 2014; Zongo et al., 2013).

In Pakistan there are more than 6000 plant species and 6.6-10% of them are used for medicinal purposes (Sher et al., 2011).

In Pakistan more than 2,000 plants have non-medicinal therapeutic purposes like flavouring, nutrition, spices, perfumery, beverages, dyeing, repellents, cosmetics, and fuel and in many other industrial uses (Khan et al., 2007). Pakistan is one of the most

blessed countries to have medicinal plant resources (Murad et al., 2011). Plants used for different medicinal purposes are documented but there are still numerous plants which need to be reported for their medicinal worth (Mushtaq et al., 2009).

1.2.1) Macroscopy and histology

The first step of getting information about the characteristics of medicinal plant is morphological and histological study (Zongo et al., 2013). Characteristics of crude drug are better evaluated by the study of its microscopical, physical and anatomical features (Rakholiya & chanda, 2012). Macroscopic and organoleptic characteristics are the easiest way to determine the purity and quality of a drug. These are studied by analysing its color, uniformity, odor, taste, size, shape, surface features and texture characteristics (Naghibi et al., 2005). Morphological, anatomical, qualitative and quantitative assessments are important for standardization of crude drugs. Correct identification, verification and excellence assurance of the preliminary resources are important in phytomedicines for herbal medicine effectiveness (Shweta et al., 2012). For identification of herbal drugs quantitative microscopic assessment such as number of stomata, stomatal index, vein islet, veinlet termination number, palisade ratio, trichomes, fibers and vessels are important to carry out (Soni et al., 2011). Apart from physical appearance, anatomy of plants such as the stomatal apparatus variability is considered the most important feature in differentiation of angiosperms (Zhau et al., 2010). Taxonomists, physiologists, morphologists and pharmacognosists have used stomatal variations in plants in systematics of leafy medicinal herbs (Tripathi & Mondal, 2012). The stomatal variations and trichomes are considered the most important anatomical parameter in the classification of plants (Reader, 2007; Gupta et al., 2012). Many workers such as Mbagwu and Edeoga 2006, Ghimare et al., 2012, Janke & Dearmond, 2004 and Tehseen et al., 2010 showed that the leaf surface studies like shape, size, number, type, arrangement and size of epidermal cells, cuticle, stomata, trichomes, and number of vascular bundles may well provide very important tools in solving the taxonomic troubles in flowering plants.

Morphology and histology is the study of contrasting features of medicinal plants, by evaluating cell, cell contents, types of tissues and their arrangements (Kadam et al., 2010; Zongo et al., 2013; Amponash et al., 2016). It is done for standardization of crude drug through morphological, microscopical and physical evaluation, and information

gathered related to the characteristics of the crude drugs (Kinghorn., 2002; Rakholiya & Chanda, 2012).

Macroscopic and organoleptic features accomplish the easiest way to recognize drug as well as find the purity and quality of drug by studying drug color, consistency, odor, taste, size, shape, surface features, texture characteristics and fracture of surface (Naghibi et al., 2005; Gupta et al., 2012).

Morphology, anatomy, qualitative and quantitative phytochemical screenings are step wise pharmacognostic investigations, which standardize the quality of crude drug. Correct identification, standardization and authentication are necessary for reproducible quality and effectiveness of herbal products (Shweta et al., 2012).

Microscopic characters are important for identification and standardization of herbal drugs such as epidermal parenchymal tissues, stomatal size, stomatal number, trichomes types, fibers, vessels and calcium oxalate crystals, index, vein islet, veinlet termination number, palisade ratio and (Soni et al., 2011).

Structure, size, shape and their arrangement on epidermal surfaces of leaves play a vital role in plant systematics (Gupta et al., 2012; Castro-carvalho et al., 2017). Many scientists such as (Mbagwu and Edeoga, 2006); (Ghimare et al., 2012); (Janke & Dearmond, 2004) worked on leaf surface studies like on shape, size, number, type, arrangement and size of epidermal cells, cuticle, stomata, trichomes and the number of vascular bundles. They suggest that these features are important tools in solving the taxonomic issues in flowering plants (Tehseen et al., 2010; Khan et al., 2016).

1.2.2) Fluorescence study

Fluorescence analysis is an essential tool for standardization and characterization of beneficial crude natural drugs. A Fluorescence study is done by observing crude drug powder or extract of medicinal plant dissolved in a solvent and then observed under UV light 254 nm, 365 nm and visible light (Kadam et al., 2012).

Fluorescence study is an important phenomenon in which through UV light various chemical compounds are detected which are present in plant crude product. This

method is an easiest and quick quantitative method for standardization of drug (Nwachukwu et al., 2010; Khan et al., 2011)).

The fluorescence color which is specific for each compound allows the detection of the accurate and precise purpose of pharmaceutical samples (Ozcan et al., 2011).

1.2.3) Phytochemical screening

Many biological and physiological actions of living organism are dependent on organic compounds such as tannins, alkaloids, carbohydrates, terpenoids, steroids and flavonoids (Kavanagh., 1963; Arunkumar & Muthuselvam., 2009). Many medicinally important plants are rich in many bioactive compounds important for curing many diseases (Yadav & Agarwala, 2011; Hegde & Jayaraj, 2015).

Medicinal plants are diverse from each other and specified for a particular action by the presence of chemical ingredients specific for curing a specific disease. Medicinal plants are vital resources of important bioactive chemical compounds such as alkaloids, tannins, flavonoids and phenolic compounds which lead and clues for modern drug design and formation (Doss, 2009; Acharya, 2012).

To treat chronic disease and production of drugs with precise activity, the relationship between the phytochemical compounds and the bioactivity of medicinal plant is very much important (Pandey et al., 2013; Hegde et al., 2015). Evaluation of crude drug is important for the discovery of new sources of affordable modern medicines for treating severe diseases (Badugu, 2012).

Phytochemical screening gives us hints and evidences to the discovery of new valuable Drugs (Khan et al., 2011). Medicinal plants have been used as herbal drugs since times immemorial.

The identified phytochemicals from medicinal plant have important physiological effects on the body showing analgesic, expectorant, emulsifying and antibiotic effects (Savithramma et al., 2011; Gupta et al., 2012).

More than 45,000 plant bioactive chemical compounds are divided into several groups based on their function, physical, chemical nature and physiological action on the human body (Dai & Mumper, 2010).

1.2.4) Elemental analysis

Macro- and microelements in varying amount are important for maintenance of human health. Many of physiological and biological processes are dependent on these elements (Zetic et al., 2001; Hendler & Sheldon, 1990).

Plants are rich in some of the important elements such as Na, Mg, Al, Si, P, S, K, Ca, Ti, Mn, Fe, Cu, Zn, As, Rb, Sr, and Pb (Queralt et al., 2005; Bahadur et al., 2011).

Due to the presence of these elements, some of the medicinal herbs are considered as a rich source of minerals such as Ca, P, K, Mg, Na, Fe and Zn, and are used in ethno medicines (Ozcan., 2005; Okwu, 2006). Some plants like sea buckthorn berries contain K, Na, Ca, Mg, Fe and P and act as a good source of biochemical and mineral element resource (Sabir et al., 2005; Hameed et al., 2008).

These macro and trace elements are vital constituents of many key enzymes essential for biochemical function of life (Haung & Failla, 2000). These minerals are vital for health of body and are used in orthodox medicines (Hameed et al., 2008; Tüzen, 2003). Medicinally important plants and drugs are standardized by the presence of minerals used in curing many diseases (Saxena et al., 2014)

1.2.5) Nutritional analysis

Plants are the most important source of nutrition. Fats, carbohydrates, protein, vitamins, minerals, etc. are very beneficial for maintenance of healthy life (Offer et al., 2015). These play important role in prevention of many diseases such as heart disease, cancer and diabetes, etc (Aruoma, 2003, Wang et al., 2014). Carbohydrates, fats and proteins are considered vital for life (Sultan et al., 2009; Wang et al., 2014).

1.3) Pharmacology

1.3.1) Acute toxicological study

Toxicology deals with the opposed effect of drugs by checking them in different experimental animals such as mice, rats, guinea pigs and rabbits etc., in order to know about their efficiency, effectiveness and its safe nature (Barkatullah et al., 2015; Al- Snafi., 2013). It is very important to know about the presence of toxic phytoconstituents in plants by trial and toxicological assay as some of them even in small amounts, are enough to kill an organism and should not be allowed to use clinically before testing (Uddin et al., 2012). Sometimes herbs which are used traditionally for hundred years as a phyto-medicine may not be considered as reliable drug, since most of the people do not consider phyto-medicines as a drug (Heinrich et al., 2012). This study reveals about the toxicity of drugs and helped in making decision on the use of new drug clinically. Toxicity of drug can be predicted by knowing the metabolism and chemistry of herb (Sharada et al., 1993). Complication produced by misidentification and differences in composition, may cause more or less dose intake, and adulteration (Nelson et al., 2014).

1.3.2) Cytotoxic activity

Cytotoxicity bioassay is a cheap method for detection of antitumor and pesticides compounds due to commercial availability of cheap brine shrimp eggs (Tolulope., 2007; Carballo et al., 2010; Manilal et al., 2016). An Artemia salina (leach) shrimp larva is affected by anticancer, antitumor and pesticidal compounds (Kivack et al., 2001). The aim of this bioassay is to find out the potency of test plant extracts cytotoxicity, which is further used in drugs for treatment of serious diseases like tumor, cancer, etc. Brine shrimp lethality test shows significance of the test, i.e high mortality rate of brine shrimps at low dose show activeness of biochemical compound of plant extracts (Knox et al., 2010). The present study on E. serrata and E. obstusifolia is also a step in that direction.

1.3.3) Phytotoxic activity

The killing of one plant by another plant due to releasing toxin such as heavy metals, pesticides and trace elements, etc called phytotoxicity (Ahmad et al., 2011). This is due to competition among plants for different like minerals, essential elements, sunlight, water, in soil and environment. Control of weeds by using weedicides and drugs synthesized in a laboratory may cause environmental hazards and water pollution (George et al., 2002). Lemna minor phytotoxic bioassay is a cheap and easy way to observe the antitumor compounds. These compounds are important for inhibition of Lemna frond spreading. It fulfils profitable needs of natural, biodegradable herbicides and plant growth stimulants (Morimoto et al., 2009). Allelochemicals are suggested as main phytotoxic chemicals and are to be considered as friendly, natural herbicides for the environment (Pugazhvendan et al., 2012).

1.3.4) Antioxidant activity

There are natural enzymatic and non-enzymatic defensive system in human body to neutralize free radicals and oxidants which are responsible for producing cancer (Kaarteenaho-Wiik & Kinnula, 2004), cardiovascular disease (Singh and Jialal, 2006), neurological disorders (Sas et al., 2007), Alzheimer’s disease (Smith et al., 2000), mild cognitive impairment (Guidi et al., 2006), Parkinson’s disease (Bolton et al., 2000), alcohol induced liver disease (Arteel, 2003), ulcerative colitis (Ramakrishna et al., 1997), aging (Hyun et al., 2016) and atherosclerosis (Upston et al., 2003). Dietary intake antioxidants are important for protection against oxidant and free radicals. Significant evidence proved that foodstuffs having natural antioxidant nutrients may be of chief importance in disease prevention (Hyun et al., 2016). Some major researchers suggested that mixture of antioxidants, rather than single antioxidant, is more effective for long term use (Ramakrishna et al., 1997). Antioxidants are effective with countless benefits in improving life quality by inhibiting or delaying the beginning of degenerative diseases (Upston et al., 2003).

1.3.5) Anti-bacterial activity

Antibacterial drugs are used against various infectious diseases caused by bacteria (Stein, 2005). Now a days major causes of death is infectious microbes that cause

various diseases (McNeil et al., 2001; Shan et al., 2007). With increase in number of strains of microbes, there is an increase in number of antimicrobial drugs for treatment of these microbes (Montefore et al., 1989).

Most of the plants have antimicrobial potency (Santos et al., 2008; Chaves et al., 2013). Syzygium jambolanum, Eclipta alba and Artemisia dubia possess antibacterial activities (Chandrasekaran & Venkatesalu, 2004; Santos et al., 2008; Saddiqe et al., 2010; Pandey et al., 2011 and Haq et al., 2012). In the present study the antibacterial potency of Ehretia serrata and Ehretia obstusifolia is studied.

1.3.6) Analgesic activity

Ache and swelling sensations are the results of various factors which are found unpleasant and disturbing throughout the world (Srineeraja & Lakshmi, 2013). The chemical compounds used for relieving pain are called pain killers or analgesic compounds and there are many plants which are rich sources of analgesic compounds with no or little side effects (Kumar, 2013; Johnson et al., 2010).

Analgesic compounds are famous for relief in pain and are used in pain killing drugs which have effective results (Ripa et al., 2015). Experimental trials on different plants play a role in discovery of plants which have analgesic potency (Onzago et al., 2013). Phytochemicals derived from different parts of numerous plants are considered to be new cause of analgesic, anti-depressant and anti-inflammatory agents (Suleiman et al., 2014).

Many plants are reported for prominent pain killing effect, e.g alkaloid and flavonoid rich fractions of fenugreek seeds (Trigonella foenum-graecum L.) with antinociceptive and anti-inflammatory effects and Cnestis ferruginea (Mandegary et al., 2012 and Okunade, 2002). Lactuca sativa is effective for its analgesic effect (Sayyah et al., 2004) etc. In the present study experimental animals were used to carry out the analgesic activity of Ehretia serrata and Ehretia obtusifolia.

1.3.7) Anti-inflammatory activity

Inflammation is a basic process of white blood cells in the body produce against pathogens to protect body from diseases. But in some diseases the immune system

activates an inflammatory response in the absence of pathogen. Such type of diseases called autoimmune diseases, in these diseases body starts producing white blood cells against its own tissues which act abnormally (Garrido et al., 2004). Anti-inflammatory drugs work against a panel of key enzymes relating to inflammation (Li et al., 2003; Uddin et al., 2014; Rodrigues et al., 2016). Many medicinal plants having varieties of flavonoid molecules possess anti-inflammatory action and inhibit chronic inflammation (Cuellar, 2001; Guo et al., 2008). Some anti-inflammatory drugs work effectively in combined form (Chen et al., 2011). Flavonoid has the anti-inflammatory capacity and has long been utilized in Chinese medicine (Ripa et al., 2015). Especially, some flavonoids were found to inhibit chronic inflammation in several experimental animal models (Gunoz et al., 2005). Many researchers have done work on medicinal plants having flavonoids, not only for establishing anti-inflammatory mechanisms, but also for developing a new class of anti-inflammatory agents (Kim, 2004; Walker & Somoza, 2017).

1.3.8) Anti- diarrhoeal activity

Loose watery stool infection in the gastrointestinal tract is called diarrhoea. It is a dangerous disease and ratios of death due to this disease are mainly 4-9 million per year worldwide. This disease affects all types of sex in all types of health conditions (Suleiman et al., 2008; Suleiman et al., 2015). Diarrhoea is mainly caused by unhygienic water and environment. Newborns and children below 7 are mostly affected (Yakubu & Salimon, 2015). Synthetic drugs like diphenoxylate, loperamide and antibiotics, etc are used, but, these drugs have many side effects (Yadave & Tangpe., 2007). Numerous hakims homeopathic doctors and practitioners, use medicinal plants to treat diarrhoea (Kabuki et al., 2010). Different medicinal plants e.g, Terminalia avicennoides, Annona senegalensis, Vitellaria paradoxa, Ziziphus abyssinica and Anacardium occidentale are suggested by many researchers for curing and controling diarrhoeal and dysentery diseases (Abubakar et al. 2015; Ghai et al. 2009 and Paul & Saha 2012). In the current investigation the anti-diarrhoeal potential of E. serrata and E. obtusifolia is evaluated.

1.4) Family Boraginaceae

Family Boraginaceae consists of 100 genera and 2000 species which are scattered in temperate regions of the world, most importantly in mediterranean and tropical regions. Among these there are only 32 genera and 135 species of the family boraginaceae found in Pakistan in which 5 species of Cordia, Echium and Anchusa are cultivated. Engler divides Family Boranginaceae into 5 sub-families, but the narrower concept is it to divide into two sub-families (Hickey & King, 1988). In comparison with other families the Boraginaceae is considered to be the lowest ranking families, containing few species of medicinal value (Khan et al., 2011).

1.4.1) Description

Habit

The plants of the family are annuals (Arnebia trichodema, Heliotropium indicum, Heliotropium supinum etc.), perennial herb with creeping rhizome (Pulmonaria and Symphytum), shrubs (Ehretia acuminata) tall trees (Cordia dichotoma) or climbers (Tournefortia).

Root

Taproot consisting plants, one of the important genus of the family Alkanna containing a purple color dye in their leaves is called Alkanet having commercial values.

Stem

Erect or prostrate as well as climbing in some species of the family. Stem is covered by tough and stiff hairs with thick base such as in genus Myosotic, Echium and Heliotropium.

Leaf

Leaves are opposite, alternate or simple. Mostly leaves are exstipulate, lanceolate or linear with toothed margin. Apex of leaves is acute or obtuse. In some genera such as Palmonaria and cynoglossum, radical leaves (broader stalked) are also found.

Inflorescence

Inflorescence is usually cymose (scorpoid and helical). Flowers are arranged in a corymb or panicle in Cordia and Ehretia. In some genera like Trichodenta, the flowers are solitary axillary while in Lithospermum the inflorescence is a spike.

Flower

Flowers are actinomorphic and some are zygomorphic (due to oblique corolla tube and unequal size of stamens) bracteolate or bracteate, pentamerous, and hypogynous. Flowers are generally regular or irregular and bisexual, heterostylous.

Calyx

Calyx is 5-lobed, often enlarging in fruit.

Corolla

Corolla is 5 -lobed, having salverform, funnelform or campanulate shape. It is glabrous or with scales (appendages) or a zone of hairs. A nectiferous ring or collar sometimes present at base of corolla tube.

Stamens

Stamens are 5, epipetalous, alternating with the corolla lobes, equal or unequal. Filaments exerted beyond the corolla tube in some cases.

Ovary

Ovary superior, 4-locular. Style gynobasic or terminal, usually simple; stigma entire or by splitting of style, 24-lobed.

Fruit

Fruits are of 2 or 4 nutlets or drupaceous (Verma., 2011).

1.4.2) Distribution

The family Boraginaceae is distributed throughout the tropical, subtropical, and temperate regions of the world. Members of this family are also found in the continents and on many islands such as the Cape Verde Islands and Madagascar. Although Boraginaceae is only a medium-sized family (21 genera and 110 species) in the Flora of Southern Africa (FSA) compared to the largest family Asteraceae (246 genera and 2240 species), with widespread members in South Africa. The members of Boraginaceae occur in the Desert, Forest, Fynbos, Grassland, Nama-Karoo, Savanna, Succulent Karoo and Thicket Biomes of the FSA region. They are therefore found in a variety of habitats, often inhabiting places like road verges and thus are easily collected. 43 native species have been recorded of this family. It is distributed worldwide with particularly high concentrations in western North America and from the Mediterranean into Asia (Khan et al., 2011).

1.4.3) Medicinal Uses

There are numerous plants which are famous for curing many diseases know throughout the world. Some of the species of the family Boraginaceae are well known for isolation and characterization of various chemical constituents such as pyrrolizidine alkaloids, naphthaquinones, flavonoids, terpenoids, triterpenoids and phenols (Tiwari et al., 2011). Some of the species of Boraginaceae are used as antimicrobial, antitumor, antiviral, anti-inflammatory, cardiotonic, contraceptive, antiplatelet, wound healing and prostaglandin inhibitor agent (Sharma, 2009).

1.5) Genus Ehretia

1.5.1) Distribution

The genus Ehretia comprises about 50 species, most of them in the tropics and subtropics of the old world and a few in Central America and the West Indies.The Genus Ehretia is mainly distributed in Africa and tropical Asia which has many biological properties e.g. antidiarrheal, anti-inflammatory, antispasmodic etc (Ali & Nasir, 1989; Retief & Van Wyk, 2001; Weigend et al., 2013).

1.5.2) Description

Ehretia contains plants which are glabrous trees or shrubs. The leaves are alternate. Inflorescence is a cymose type. Flowers are bracteate. Calyx is 5-partite. Corolla is tube like and short, Stamens are 5; anthers are exerted. Ovary is bilocular or incompletely 4. The bilocular contained 2 ovules per locule. Style is terminal, and stigma is capitate.

1.5.3) Medicinal uses

Ehretia is sometimes placed in the small family called Ehretiaceae. Several other African Ehretia species are used in traditional medicine as well as for timber. A root decoction of E. bakeri Britten, a species restricted to Kenya and Tanzania, is used as a cure for gonorrhoea in Kenya. The leaves, fruits, bark and roots of the plants of genus Ehretia are used traditionally as medicines against swelling, cough, itches, diarrhoea, dysentery, fever, cachexia and syphilis (Wang et al., 2008). In China, the species E. thyrsiflora has been used to make Kudingcha, a bitter tea and which has different types of flavonoid glycosides and phenolic acids (Li et al., 2008). E. laevis is used as a medicine in India to treat headache and ulcers and also is reported to have potency as anthelmintic, diuretic, expectorant, demulcent, and astringent properties as well as its inner bark is used as food (Velappan & Thangaraj, 2014).

1.6) Ehretia serrata Roxb.

Family: Boraginaceae (Forget-me-not family)

Synonyms: Ehretia acuminata, Cordia thyrsiflora, Ehretia ovalifolia

Vernacular name: Koda Tree, brown-cedar, kodowood, silky-ash Puna

1.6.1) Distribution

This specie is distributed in sub-Himalayan and the outer Himalayan from Indus eastward to Sikkim. It is frequently planted as a roadside tree, and is sometimes found in hilly areas up to 2000 m hight. Ehretia serrata Roxb. is a native specie in India and Pakistan (Ali & Nasir., 1989). In Pakistan E. serrata Roxb. is well distributed in sub- Himalayan tract from Azad Kashmir westward to Rawalpindi, Islamabad, Murree, Hazara, and Swat. It has been successfully planted in areas around Lahore (Sheikh, 1993).

1.6.2) Description

It is up to ten meters tall deciduous tree. The young shoots are glabrescent and are almost smooth. Leaves are elliptic; oval in shape and oblong, acuminate, serrulate, glabrous, cuneate, with a prominent midrib on under surface with pubescent nerves. Petioles are up to 2.5 cm long. Inflorescence panicle and flowers are white in color and terminal in position and sessile. Calyx is cup shape with 5 sepals, obtuse and ciliate. Corolla is rotate and 5-lobed and oblong, minutely crenulate. Filaments are shorter than lobes, glabrous; basifixed and oblong. Style is 1.5 mm long with 2-fid. Ovary is subglobose or ovoid in shape and glabrous. Drupe globe like in shape, 4-43 mm broad, black when ripe.

1.6.3) Medicinal uses

The juice of the bark is used in the treatment of fevers (Ajaib & Zaheer-UD-Din, 2014). The green and unripe fresh fruit of E. serrata Roxb. is used in a form of pickle (Haq et al., 2010).

Fig 1.1. Fruits and leaves of Ehretia serrata Fig 1.2. Stem of Ehretia serrata

Fig 1.3. Leaves and flowers of Ehretia serrata

1.7) Ehretia obtusifolia A.DC.

Family: Boraginaceae (Forget-me-not family) Synonyms: Ehretia coerulea Gürke Vernacular names: Sandpaper bush, big-leaved puzzle bush

1.7.1) Distribution

E. obtusifolia occurs in almost all countries of eastern and southern Africa and in Madagascar. In Asia it is extended up to Pakistan and north west India (Schmelzer et al., 2008)

1.7.2) Description

It is a deciduous small shrub or small tree which is up to 6 m tall, straggling or with several branches from the base; color of bark is grey. Leaves are alternate and simple; petiolate elliptical, oblong-elliptic or obovate, pubescent-velvety; apex of the leaves are obtuse to acuminate. Inflorescence is corymbose cyme, terminal or on short lateral shoots. Flowers are bisexual, regular, pentamerous, with pleasant smell; pedicelate, calyx lobes and ovate, elliptical or narrowly triangular joined only at the base; corolla is of different colors such as pink, blue, pale lilac or mauve, tubelike, lobes narrowly, each petal is triangular to oblong, acute or obtuse; stamens are unequal, ovary superior, style 3–6 mm long, deeply forked. Fruit is a globose drupe, 5–6 mm in diameter, orange or red, splitting into two 2-seeded pyrenes (Retief & Van wyk, 2001).

1.7.3) Medicinal Uses

In Zimbabwe the leaves of E. obtusifolia are used as an infusion to treat sore throat and are rubbed on the gums of infants suffering from teething pains. In the same country powdered root is added to porridge to treat painful menstruation and infertility in women and an infusion of the roots is taken against retained placenta. A decoction of the roots is taken as a painkiller, specifically against abdominal pain in Zimbabwe and in Tanzania. The wood is used to make pestles for pounding grain in Zimbabwe. In Pakistan the leaves are used as a fodder and the wood as fuel (Coates Palgrave, 1983).

Fig.1.4 Flowers of Ehretia obtusifolia Fig. 1.5. Fruits and Leaves of Ehretia obtusifolia

Fig. 1.6. Stem of Ehretia obtusifolia

CHAPTER-2

REVIEW OF LITERATURE

2.1) Genus Ehretia

Burkill (1985) concluded the effectiveness of E. cymosa as antipyretic, laxative, and pain-killer, for paralysis, epilepsy, convulsions and spasm. Sap from leaves of E. cymosa is a laxative for children. Its bark is reported to be useful for menstrual cycle.

Selvanayagam et al. (1996) reported ehretianone, extracted from the root bark of E. buxifolia as a new quinonoid xanthene, a sterol type compound having potency as an antisnake venom against Echis carinatus.

Iqbal et al. (2005) isolated phenolic compounds i.e methyl 2‐O‐feruloyl‐1a‐O‐ vanillactate, caffeic anhydride and trans 4‐hydroxycyclohexyl‐2‐O‐p‐coumaroyl β‐D‐ glucopyranoside from the AcOEt‐soluble fraction of E. obtusifolia, along with methyl rosmarinate and rosmarinic acid, which are reported for the first time from this species.

Runyoro et al. (2006) studied roots of E. amoena Klotzsch for anti-fungal activity. They claimed the effectiveness of dried powder of this species as an anti-fungal agent. The information was gathered by ethnobotanical study of plants of four different regions of Tanzania.

Thuy et al. (2007) investigated the leaves of E. dentata Couch. growing in Hoa Binh, Vietnam led to the isolation and structural elucidation of a nitril glucoside, ehretioside A1, the flavonol glucoside astragalin, as well as rosmarinic acid and methyl rosmarinat.

Torane et al. (2010) stated that all parts of E. laevis Roxb. are medicinally important. They are used in dropsy, anasaraca, urticaria, cholera, dysentery, etc. They extracted different amino acids from different parts of this specie. These were compared with the standards. Presence of carbohydrates was performed by using paper chromatographic techniques and compared with the standards.

Zara et al., (2012) evaluated antioxidant activities of various solvent extracts of fruits and leaves of E. serrata. The ethyl acetate fraction of leaves contain the highest

flavonoid as well as phenolic content. It is also found to be most active against free radicals and reducing agents when investigated by various assays.

Chien et al. (2012) extracted important compounds i.e ehretiquinone, ehretiolide, ehreticoumarin, ehretilactone A, ehretilactone B , ehretiamide , ehretine, and ehretiate, together with with other familiar compounds from methanolic extract of the root of E. longiflora (Boraginaceae). Among the isolates, ehretiquinone and prenylhydroquinone showed antitubercular activity against Mycobacterium tuberculosis strain.

Sempombe et al. (2014) worked on preliminary in vivo antitrypanosomal activity and cytotoxicity of E. amoena. They confirmed the potency of the plant against trypanosomiasis and 3-cell line panel consisting of TK10 (renal), UACC62 (melanoma) and MCF7 (breast) cancer cells.

Compen and Ynalvez (2015) researched about antimicrobial potential of E. anacua by showing a clear inhibition zone against Staphylococcus aureus. Beside this, it was also tested for antibacterial and antifungal activities using acetone, ethanol, and diethyl ether extracts. E. anacua was also subjected to qualitative and quantitative phytochemical screening to determine exact constituents exhibiting these biological activities.

Gumgumjee and Hajar (2015) investigated the antibacterial activities and unveil the phytochemicals of the bioactive components in the leaves extract of E. abyssinica. Most susceptible bacteria to this extract were Pseudomonas aeruginosa, followed by Staphylococcus aurues, while the most resistant bacteria were Micrococcus. GC-MS analysis revealed that the ethanol leave extract of E. abyssinica contained mainly octadecenamide, lucenin, docosane and nonacosane, cyclopropene, hematoporphyrin, tetratetracontane, dotriacontane, acetic acid nmethylglycine, propyne antimicrobial. All identified compounds are known to have antimicrobial activity.

Sarkodie et al. (2015) studied, the antihyperglycaemic, antioxidant and antimicrobial activities of ethanolic extract of E. cymosa. The extract showed a significant dose independent reduction of the fasting blood glucose level and the rat intestinal sacs had a significant decrease in glucose absorption. The extract showed strong antioxidant activity and also exhibited inhibitory activity against P. aeruginosa, E. coli, B. subtilis and S. aureus.

Rushikesh et al. (2016) explored the ethnobotanical properties of unexplored plant khandu chakka (Ehretia laevis Roxb.). They stated that E. laevis plant is mainly used for wound healing, joint pain and minor fractures by local peoples with promising results.

Ogundajo and Ashafa (2017) studied the bioactive constituents and in vitro antioxidant and antidiabetic potentials of fractions from extract of E. cymosa. The phytochemistry tests of the fractions revealed the presence of tannins, phenols, flavonoids, steroids, terpene, alkaloid, and cardiac glycosides.

Bande and Murarkar, (2018) investigated the potential of leaves extract of E. laevis as coagulant. The blood coagulation time of prothrombin test of both the plasma and blood samples was reduced as compared to the control in this study.

Shukla and Kaur, (2018) documented systematic review of traditional uses bioactive phytoconstituents of Genus Ehretia. The pharmacological studies confirmed that the crude extracts or individual compounds from the genus showed antioxidant, anti- inflammatory, antibacterial, antiarthritic, antitubercular, and antiallergic activities, as well as anti-snake venom property.

Waheed et al. (2018) reported the in vitro antimicrobial activities of the crude extracts of leaves of E. serrata. The methanolic extract and its sub fractions namely n-hexane, chloroform, ethyl acetate, n-butanol and residual water fraction were screened against a range of 30 different bacterial strains and their zones of inhibition (ZI) and minimum inhibitory concentration (MIC) were subsequently evaluated. Methanolic extract has shown activity against all the tested microorganisms such as Azospirillum lipoferum, Escherichia coli, Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Enterococcus sp.

2.2) Pharmacognostic studies

Pharmacognostic study of medicinal plants is done for authentication and identification as well as for standardization of crude drug by observing their different macro and micro morphological parameters. These include powder drug study, phytochemical

screening, elemental and nutritional analysis, and their fluorescence study (Chanda, 2014).

2.2.1) Morphology and anatomy

Dattagupta and Datta (1977) studied the morphology and anatomy of Heliotropium indicum Linn. (Fam. Boraginaceae) and revealed the presence of various types of non- glandular and glandular hairs with ranunculaceous stomata on both surfaces of leaf. They also found the lacunar collenchyma in mid-veins, suberin at trichome bases and alkaloids in leaf extracts.

Dasti et al. (2003) documented the epidermal characters, including 31 species belonging of three tribes of family boraginaceae. The results indicate that the leaves in all the species were hypoamphistomatic and the prevalent type of stomata were anomocytic. Other types such as anisocytic, brachyparacytic, staurocytic and hemiparacytic types were occasional while paracytic, diacytic and polocytic were rare types.

Akcin et al. (2004) carried out work on Alkanna haussknechtii Bornm. which occurs only in the vicinity of Amasya, and a critically endangered specie. The morphological and anatomical properties of the species were investigated. The leaves were ecvifacial and had stomata cells that were anisocytic and anomocytic.

Akcin and Engin, (2005) investigated endemic specie Onosma bracteosum Hausskn. & Bornm. (Boraginaceae) morphologically, anatomically and ecologically. They found equifacial leaves with amphistomic, anisocytic and anomocytic stomata.

Akcin, (2007) investigated morphological and anatomical characteristics of Anchusa leptophylla Roemer & Schultes subsp. leptophylla and A. leptophylla subsp. incana (Ledep) Chamb. (Boraginaceae). The shape of leaves was linear-lanceolate in A. leptophylla subsp. incana, while it was linear in subsp. leptophylla. In anatomical studies, cross-sections of the root, stem, leaf parts, and the surface sections of the leaves of both subspecies were examined. The root was perennial. However, it was noted in A. leptophylla subsp. leptophylla that the periderm layer was thicker than in subsp. incana. The leaves were equifacial and had stomata cells that were anomocytic.

Souza, (2008) analyzed the morphology and anatomy of the Cordia trichotoma (Vell.) Arrab. Ex I. M. Johnst diaspore. The results showed that the perianth had aerenchyma and the ovary was 2- 4- locule forming endothelium and false septum. The fruit was drupoid nuculanium and the seed was exotestal.

Akcin et al. (2010) illustrated the taxonomic significance of morphological and anatomical features of three Anchusa species, Anchusa undulata subsp. hybrida (Ten.) Coutinho, A. azurea Miller var. azurea and A. pusilla Guşul., collected from Northern Turkey. It was determined that the ratio of calyx lobe to the calyx length and the arrangement of the anthers in the corolla tube were important characters in separating the taxa morphologically.

Kumar et al. (2011) documented morphological and histological parameters of Cordia macleodii (Griff.) Hook. f. & Thoms. (Family Boraginaceae). By using that studies bark can easily be identified on the basis of macroscopic features for e.g. dry bark is grayish brown in colour, curved to channel in shape fracture splintery fibrous. On microscopic examination prismatic crystal, stone cells, heterogenous, multiseriate medullary rays, resin cells were observed in the bark.

Binzet, (2012) studied the morphological and anatomical characteristics of Onosma frutescens Lam. and Onosma inexspectata Teppner. O. inexspectata is an endemic species. Both examined species has secondary root structure, and cortex was multilayered and composed of parenchymatical cells. While, xylem was composed of sclerenchymatic cells and tracheary elements in roots of O. frutescens, xylem was composed of tracheal elements in roots of O. inexspectata. Glandular and eglandular trichomes were found on the epidermis of stem of both species.

Daironas et al. (2014) reported anatomical and morphological characters of leaves and stems of the herbs of Onosma caucasica Levin. Ex M. Pop. and Onosma sericea Willd. (Boraginaceae). All results were supported by photographs. The nature of trichomes on the leaf blade has a major diagnostic value in determining the authenticity of herbs the studied species.

Kasem, (2015) studied valuable taxonomic characters of seven species of Heliotropium L. (Heliotropiaceae) belongs to family Boraginaceae in south Saudi Arabia. He

analysed the micromorphological data such as leaf venation, stomata, stem anatomy, trichomes and pollen grain and used obtain data as a taxonomic tool in differentiation between species of this genus by cluster analysis method.

Hadeethi et al. (2016) reported the anatomical features of different parts of Cordia myxa. The findings showed anomocytic stomata with anticlinal walls of epidermal cells and unicellular trichomes on both sides of leaf of studied plant.

Ozkan et al. (2016) worked on the anatomical and morphological properties of Onsoma argentata. Anatomical sections taken from the root, stem and leaves of the plants. The results have been compared with some species of Boraginaceae

2.2.2) Powder Drug Study

Hadeethi et al. (2016) microscopically analysed the dried leaf powder of different parts of Cordia myxa. The results revealed that powder was brown to green in color, microscopy of the powder showed fragments of unicellular trichomes, stomata, fibers, crystal differ in shape like as regular prismatic, druses, sand crystal, and dendritic crystal like as snowflake formation in shape.

Uthiraselvam et al. (2016) has significantly documented anatomical and histochemical studies of Cordia oblique. The results showed that there was concentration of vascular bundles at the central portion of root cortex, calcium oxalate presence was confirmed in the root powder and non-glandular trichome in the leaves.

Yusufoglu et al. (2018) investigated Arnebia hispidissima (A. hispidissima) a member of the family Boraginaceae. The standardization evaluation comprises of powder microscopy. Results revealed that microscopic study of powder of whole plant of A. hispidissima showed different types of trichomes, vessel and fibres.

Thomson et al. (2018) evaluated powder microscopy of Arnebia hispidissima (A. hispidissima) a member of the family Boraginaceae. Microscopic study of powder of whole plant of A. hispidissima showed different types of trichomes, vessel and fibres.

2.2.3) Fluorescence analysis and Phytochemical screening

El-Shazly et al. (1996) separated and isolated 14 pyrrolizidine alkaloids by GLC and GLC-MS in the alkaloid extracts from different parts of Cynoglossum officinale. From Cynoglossum officinale five pyrrolizidine alkaloids were recorded: supinine, amabiline, rinderine, echinatine, and 3′-O-acetylechinatine.

El-Shazly et al. (1998) detected 40 pyrrolizidine alkaloids in the alkaloid extracts of Paracaryum rugulosum, P. intermedium, Anchusa milleri, Gastrocotyle hispida (syn. Anchusa hispida), Anchusa arvensis, Lappula spinocarpos, Trichodesma africanum, Alkanna orientalis, and Alkanna tuberculata (syn. A. tinctoria) which were analyzed by capillary GLC and GLC-MS.

Wollenweber et al. (2002) accumulated flavonoid aglycones for the first time in Nonea species. They exhibit only flavones, one of them being the rare tricetin-4ʹ-methyl ether. Within the subfamily Boraginoideae, exudate flavonoids appear to be a rare character.

Babula et al. (2009) reviewed and discuss phytochemicals found in many species of families Bignoniaceae, Droseraceae, Plumbaginace, Juglandaceae as well as in species of small families, such as Dioncophyllaceae or Acanthaceae. They discuss the presence of interesting compounds derived from naphthalene - 1,4-naphthoquinones and rarely also 1,2-naphthoquinones in these plants and their utilization for medicinal purposes and their occurrence in nature. Moreover, they review analytical techniques using for their analysis.

Özcan, (2008) investigated mature seed samples of twenty-four Boraginaceae taxa collected from their natural habitats in Turkey. These samples were analysed by GC for total oil content and fatty acid composition. Segregation at the generic level by principle-component analysis was accomplished based on nine major fatty acids. The fatty acid patterns, their relative proportions, and quantities of unusual fatty acids as additional biochemical markers seem to be useful in the taxonomy of Boraginaceae at generic and infrageneric levels.

Silva et al. (2004) analysed the aerial parts of Cordia globosa for phytochemical bioconstiuents. They isolated and identified the structure of narigenin-4', 7-dimethyl ether (0.025 g) and eriodictyol (0.015 g). These compounds were the first flavanones aglycones isolated from the genus Cordia belongs to family Boraginaceae.

Sharma et al. (2009) documented review article on presence of various chemical constituents in different species of Cordia belongs to family Boraginaceae. They reported and review the presence of pyrrolizidine alkaloids, naphthaquinones, flavonoids, terpenoids, triterpenoids and phenols in different species of Cordia.

Kumar et al., (2011) tested the Cordia macleodii (Griff.) Hook. f. & Thoms. (Family Boraginaceae). They screened physicochemical, phytochemical, fluorescence study, in examined plants and got valuable results. Physico-chemical values viz. total ash, acid insoluble ash, hexane soluble extractive, alcohol soluble extractive, water soluble extractive were observed. The total ash, acid insoluble ashes were considered to be an important and useful parameter for detecting the presence of inorganic substance like silicate ion.

Ahmad et al. (2014) presented the phytochemical analysis of Heliotropium bacciferum belongs to Boraginaceae family. The crude and numerous fractions of leaves, stem and roots of the plant were investigated for phytochemical analysis. Phytochemical analysis of crude and fractions of the plant revealed the presence of alkaloids, saponins, tannins, steroids, terpenoids, flavonoids, glycosides, and phenols.

Rahman and Hussain, (2015) examined the physicochemical, phytochemical, fluorescence and HPTLC chromatographic profile of the leaves of Cordia dichotoma Linn (CD). Their results showed the presence of steroid, carbohydrate, alkaloid, saponin, cardiac glycosides, flavonoid and phenolic compounds in methanolic extract. The CD fluorescence was seen in UV light and it was of different colour in different solvents.

Jasiem et al. (2016) analysed some species of genus Cordia for presence of phytochemicals. Various compounds like flavonoids, triterpenes, tannins, alkaloids and fatty acids possessing wide range of bioactivities were isolated from different plant parts of Cordia species.

Shoaib et al. (2017) carried out the physicochemical, phytochemical, and high- performance thin layer chromatography (HPTLC) analysis on root bark of Onosma echioides (OE) L., family Boraginaceae. OE is composed of sterols, tannins, alkaloids, flavonoids, glycoside, saponin, and carbohydrates. The fluorescence characteristics of

leaf powder were studied both in visible light and ultraviolet light (254 nm and 365 nm) after treatment with various reagents and got different colours. The HPTLC profile showed several peaks that indicated the presence of various phytochemicals along with its active constituent, i.e., shikonin.

Yusufoglu et al. (2018) evaluated Arnebia hispidissima (A. hispidissima) a member of the family Boraginaceae. The standardization evaluation comprises of fluorescence analysis and TLC profiling. In addition, preliminary phytochemical screening, determination of total phenol and in vitro free radical scavenging activity (DPPH radicals scavenging assay and reducing power activity) were preformed utilizing the methanol extract. Fluorescence analysis showed different colours under visible light. The phytochemical analysis of the methanol extract gave a positive indication for the presence of active compounds including alkaloids, carbohydrates, glycosides, steroids, triterpenoids saponins, phenols, tannins, flavonoids and proteins.

Thomson et al. (2018) investigated powder fluorescence analysis and TLC profiling of Arnebia hispidissima (A. hispidissima) a member of the family Boraginaceae. This study has been made for the preliminary standardization of A. hispidissima plant. Fluorescence analysis showed different colours under visible light, low UV and high UV. TLC of the hexane extract developed 8, 6, 9 and 10 spots with visible light, low

UV, high UV and ninhydrin-H2SO4 spray, respectively. The phytochemical analysis of the methanol extract gave a positive indication for the presence of active compounds including alkaloids, carbohydrates, glycosides, steroids, triterpenoids saponins, phenols, tannins, flavonoids and proteins.

2.2.4) Elemental analysis and nutritional analysis

Amusan et al. (2016) studied the nutritional composition and anti-oxidant potential of Heliotropium indicum L. (Lamiales: Boraginaceae). Results revealed that these indigenous leafy vegetable which are not widely known and consumed are of high nutritional quality and antioxidant potential because of the presence of varying levels of vitamins like ascorbic acid, retinol, tocopherols, riboflavin, thiamine and niacin. This vegetable can contribute significantly to the nutrient requirement of man and could

complement the conventional ones in enhancing food security and sustainable livelihood.

Ozkan et al. (2016) worked on the concentrations of trace elements in Onsoma argentata (Boraginaceae) by using atomic absorption spectrometry. Large amount of sodium (Na), aluminum (Al), potassium (K) and iron (Fe) were present in all flowers, leaf and stem of O. argentata plant.

2.3) Pharmacology

2.3.1) Acute toxicological activity

Hussain and Kakoti, (2013) investigated the acute toxicity of Cordia dichotoma extract in rats. There were no remarkable changes noticed in the histopathological studies after 50 mg/kg body weight of the extracts of Cordia dichotoma, when administered intraperitoneally for 14 days successively. Pathologically, neither gross abnormalities nor histopathological changes were observed. Collectively results demonstrated that the extracts of the leaves of the Cordia dichotoma have a high margin of drug safety.

Chandrakar et al. (2017) studied the acute toxicity of Cordia macleodii Hook f. and found it nontoxic for further research work.

2.3.2) Anti-oxidant activity

Lissi et al. (1999) reported total reactive antioxidant potential (TRAP) of resinous exudates from Heliotropium species, and was evaluated by measuring the bleaching of stable free radicals. The antioxidant capacity of the resinous exudates in Trolox equivalents, evaluated from the bleaching of ABTS derived radical cations, ranged from 2.0M (H. huascoense) to 5.2 M (H. stenophyllum), indicating a very high concentration of phenolic compounds. Considerably smaller values were obtained by measuring the bleaching of DPPH radicals. The ratio between the values obtained employing ABTS derived radicals and DPPH, ranged from 37 (H. megalanthum) to 4.5 (H. chenopodiaceum var. typica). The magnitude of the difference can be considered as an indication of the relative reactivity of the antioxidants present in the exudates.

Modak et al. (2003) isolated a new compound: 4-(3′, 5′-dihydroxynonadecyl) phenol 1 from the resinous exudate of Heliotropium sinuatum (family Boraginaceae). Its antioxidant potency was assessed by quenching measurements with ABTS and DPPH cation radicals. Results revealed the positive antioxidant potency of resinous exudate of Heliotropium sinuatum.

Assimopoulou and Papageorgiou, (2005) tested organic solvents and olive oil (extracted at room temperature) extracts of Alkanna tinctoria roots for antioxidant potentiality. Results revealed that experimented plant extracts exhibited very good antiradical activity.

Modak et al. (2005) showed relationships between the structural characteristics of flavonoids isolated from the resinous exudate of Heliotropium sinuatum and their antioxidant activity. It has been found that antioxidant activity depends both on substitution pattern of hydroxyl groups of the flavonoid skeleton and the presence of an unsaturation at the C2–C3 bond.

Alali et al. (2007) reported the relative levels of antioxidant activity and the total phenolic content of aqueous and methanolic extracts of a total of 95 plant species. There was a significant linear correlation between antioxidant activity and total phenolic content for aqueous and methanolic extracts, suggesting that phenolic compounds were the predominant antioxidant components in the investigated plant species. Interestingly, a few of the collected plants had high-antioxidant activity yet “low” phenolic content includes Ceratonia siliqua and Viscum cruciatum .Some plants showed low antioxidant activity including Echium judaeum Lacaita. These plants may serve as sources of antioxidants with new chemotypes.

Okusa et al. (2007) worked on methanolic and dichloromethane extracts of Cordia gilletii De Wild (Boraginaceae) root bark. Experimented plant showed the greater antioxidant activity by scavenging the free radical DPPH with IC50 values of 3.2 and 8.1 μg/mL, respectively.

Makari et al. (2008) examined in-vitro antioxidant effects of the hexane and methanolic leaf extracts of Cordia wallichii and Celastrus paniculata. The methanolic extracts of C. wallichii had shown good DPPH (1, 1-diphenyl-2-picryl-hydrazyl) radical

scavenging activity. The DPPH radical scavenging activity of the extract was increased with the increasing concentration. The methanolic extract of C. wallichii was found to be most effective than hexane extract. The Reducing power of extracts was carried out with ascorbic acid as a standard reducing agent. C. wallichii exhibited higher reducing power than C. paniculata.

Qureshi et al. (2009) evaluated ethanolic extract of Cordia macleodii leaves for possible antioxidant potential. Antioxidant activity of the extracts was evaluated by four established, in vitro methods viz. 1, 1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging method, nitric oxide (NO) radical scavenging method, iron chelation method and reducing power method. The extract demonstrated a significant dose dependent antioxidant activity comparable with ascorbic acid.

Hussain et al. (2010) evaluated crude extract, ethyl acetate, n-hexane, chloroform and aqueous fractions of Heliotropium strigosum for antioxidant activity. The plant showed excellent diphenyl picryl hydrazine (DPPH) scavenging activity. Excellent antioxidant activity was shown by ethyl acetate, n-hexane and aqueous fractions, but crude extract and chloroform fractions were lacking in DPPH scavenging activity.

Končić et al. (2010) tested DPPH radical scavenging potency of Moltkia petraea (Tratt.) Griseb (Boraginaceae). The leaf extracts demonstrated superior antioxidant activity than stem methanolic extract.

Li et al. (2010) studied antioxidant activity of Arnebia euchroma (Royle) Johnst Hairy Roots extract. DPPH radical, reducing power, hydroxyl radical and lipid peroxidation of linoleic acid (LA) assays were chosen for antioxidant experiments. The results showed that different solvent extracts of hairy roots have a strong effect on free radical scavenging.

Al-Musayeib et al. (2011) isolated nine compounds from the ethyl acetate soluble fraction of Cordi sinensis, namely protocatechuic acid (1), trans-caffeic acid (2), methyl rosmarinate (3), rosmarinic acid (4), kaempferide-3-O-β-D-glucopyranoside (5), kaempferol-3-O-β-D-glucopyranoside (6), quercetin-3-O-β-D-glucopyranoside (7), kaempferide-3-O-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside (8) and kaempferol-3-O-α-L-rhamno-pyranosyl (1→6)-β-D-glucopyranoside (9), all reported

for the first time from this species. The antioxidant activities of isolated compounds 1– 9 were evaluated by the DPPH radical scavenging test, and compounds 1, 2, 4 and 7–9 exhibited marked scavenging activity compared to the standard BHA.

Bonesi et al. (2011) studied the antioxidant properties of the essential oil extracted from the leaves of Cordia gilletii De Wild (Boraginaceae). The antioxidant activity of C. gilletii essential oil was screened by two in vitro tests: DPPH and beta-carotene bleaching test. The essential oil revealed antioxidant activity with an IC50 value of 75.0 and 129.9 micro g/mL on DPPH radical and beta-carotene decolouration tests, respectively.

Nariya et al. (2013) evaluated possible antioxidant potential of methanolic and butanol extract of Cordia dichotoma bark. In vitro antioxidant activity of methanolic and butanol extract was determined by 1,1, diphenyl–2, picrylhydrazyl (DPPH) free radical scavenging assay. Antiradical activity of methanolic extract was measured by DPPH assay and was compared to ascorbic acid. In the present study three in vitro models were used to evaluate antioxidant activity. The first two methods were for direct measurement of radical scavenging activity and remaining one method evaluated the reducing power. The present study revealed that the C. dichotoma bark has significant radical scavenging activity.

Pilerood et al. (2014) reported antioxidant potency of aqueous and solvent extracts of Iranian origin Borage (Echium amoenum) and Valerian (Valerian officinalis). Samples were analyzed for antioxidant components viz. polyphenols, vitamin C, β carotene, flavonoids, anthocyanins and tannins. Antioxidant assays such as free radical scavenging activity, reducing power and total antioxidant activity were carried out for ethanol, methanol, acetone, 80% methanol and 80% ethanolic extracts. In borage highest and least activity was observed in water and acetone extract respectively in all assays. In Valerian, 80% methanolic extract showed highest activity in reducing power and free radical scavenging activity assay.

Amusan et al. (2016) reported antioxidant potency of Heliotropium indicum L. (Lamiales: Boraginaceae). Results revealed that this vegetable does not show antioxidant activity.

Shameem et al. (2015) analysed antioxidant activity of methanolic Arnebia benthamii extract. The antioxidant activity of the aerial and root part extracts was almost of the same strength with the root part slightly showing a higher scavenging effect in a concentration-dependent manner in superoxide anion and hydroxyl radicals.

Ganie et al. (2012) evaluated in vitro antioxidant activity of methanolic extract of Arnebia benthamii (A. benthamii) whole plant. DPPH radical scavenging and hydroxyl radical scavenging potential of the plant revealed that the extract to be active radical scavenger. Findings of this experiment demonstrated that the methanol extract of A. benthamii has excellent anti-oxidant activities and could be considered as a potential source of lead molecules for pharmaceutical industries.

2.3.3) Phytotoxic and cytotoxic activity

Kundakovic et al. (2006) examined the root extracts of Onosma leptanhtha for their cytotoxic activity. The cytotoxic activity of the extracts and isolated compounds was also assayed against L1210 murine lymphoblastic leukemia cell line, and human fibrosarcoma HT-1080 cells and showed remarkable cytotoxic activity.

Sharker et al. (2009) screened ethanol extract of leaves of Cordia dichotoma (Family- Borginaceae) for its cytotoxic activity. The results showed potent activity of experimented plant extract against the brine shrimp Artemia salina (LC50: 20 µg/mL and LC90: 180 µg/mL).

Rahman et al. (2011) examined methanol extract of the dried roots of Heliotropium indicum Linn. (Boraginaceae) for its possible cytotoxic potency. The crude extract produced the most prominent cytotoxic activity against brine shrimp Artemia salina

(LC50 = 47.86 µg/mL and LC90 =75.85 µg/mL). The obtained results provide a support for the use of this plant in traditional medicine and its further investigation.

Shah et al. (2015) evaluated the cytotoxic and phytotoxic activities of the crude extract of Heliotropium strigosum and its resultant fractions. In brine shrimp toxicology assays, profound cytotoxicity was displayed by ethyl acetate (LD50 8.3 μg/mL) and chloroform

(LD50 8.8 μg/mL) fractions, followed by relatively weak crude methanolic extract of H. strigosum (LD50 909 μg/mL) and n-hexane fraction (LD50 1000 μg/mL). In case of

phytotoxicity activity against Lemna acquinoctialis, highest phytotoxic effect was showed by ethyl acetate fraction (LD50 91.0 μg/mL), while chloroform fraction, plant crude extract and n-hexane, respectively, caused 50%, 30.76 ± 1.1% and 30.7 ± 1.1% inhibitory action at maximum concentration used, that is, 1000 μg/mL. These data indicates that H. strigosum exhibits cytotoxic and phytotoxic potential, which explore its use as anticancer and herbicidal medicine.

Ghori et al. (2016) reviewed ethnobotanical uses of genus Heliotropium (Boraginaceae). They stated that large number of extracts and bioactive constituents of different species of genus Heliotropium revealed significant biological activities such as antimicrobial, antitumor, antiviral, antiinflammatory, wound healing, cytotoxicity and phytotoxicity. Different parts of plants of genus Heliotropium are examined for valuable pharmacognostic properties.

Ghaffari et al. (2016) studied the methanol extract of Heliotropium dasycarpum for Cytotoxic, α-Chymotrypsin and Urease inhibition activities. In vivo brine shrimp lethality bioassay was used for detecting cytotoxicity of experimented plant. Results showed that methanol extract of the plant exhibited significant cytotoxic activity. Out of 30 brine shrimp larvae, 2 (6%), 26 (86%) and 28 (93%) larvae were survived at concentration of 1000 μg/mL, 100 μg/mL and 10 μg/mL respectively with LD50; 215.837.

2.3.4) Antibacterial activity

Tabata et al. (1982) isolated two specific metabolites, echinone and echinofuran from the callus cultures of Echium lycopsis L. Both metabolites were found to inhibit the growth of gram-positive bacteria. Especially, echinone showed an antibacterial activity that is even stronger than that of the biogenetically related compounds such as shikonin and alkannin.

Radha et al. (2003) investigated antimicrobial activity of different extracts (chloroform, ethylacetate, methanol, water) of the plant Heliotropium marifolium Retz. The zone of inhibition of the extracts was compared with that of ciprofloxacin (5 μg/disc) by disc diffusion method. The findings showed potential antimicrobial

properties of the extracts against the organisms tested. The study suggests that the plant is promising for development of phytomedicine for antimicrobial properties. de Carvalho et al., (2004) evaluated essential oil of aerial parts of Cordia verbenacea D.C. (Boraginaceae) for antimicrobial potency. The main constituents of the essential oil, identified by gas chromatography/mass spectrometry (GC/MS), were α-pinene (29.69%), trans-caryophyllene (25.27%) and aloaromadendrene (9.99%). The Gram- positive bacteria and yeast tested were sensitive to the essential oil of Cordia verbenacea D.C., but most of the gram-negative bacteria were resistant.

Kuruüzüm-Uz et al. (2004) investigated the methanol soluble constituents from aerial parts of E. vulgare and their antimicrobial activities. Kaempferol 3-O-neohesperidoside (kaempferol 3-O-a-L-rhamnopyranosyl-(1-2)-b-D-glucopyranoside) ,Uridine, 3-(30 ,40 -dihydroxyphenyl)-(2R)-lactic acid, rosmarinic acid extracted from the aerial parts of experimented plant and test against four strains of bacteria. All four compounds showed no detectable antimicrobial activity.

Hernandez et al. (2007) reported antimicrobial activity of the essential oil, hexane, chloroform and methanol extracts from aerial parts of Cordia curassavica. The oil and extracts exhibited antimicrobial activity against Gram-positive and Gram-negative bacteria and five fungal strains.

Okusa et al. (2007) examined n-hexane, dichloromethane, ethyl acetate, methanol and water extracts of Cordia gilletii De Wild (Boraginaceae) root bark for antimicrobial activity. The methanol extract showed direct antimicrobial activity against all tested microorganisms with minimum inhibitory concentrations (MIC) ranging between 125 and 1000 μg/mL, whereas the ethyl acetate and the dichloromethane extracts showed activity on four and three strains, respectively. 200 μg/mL of n-hexane and dichloromethane extracts decreased the MICs of penicillin and streptomycin 4–64 fold for methicillin-resistant Staphylococcus aureus.

Adelaja et al. (2008) explored the antimicrobial potency of Heliotropium indicum of the family Boraginaceae. The aqueous extracts of the dried leaves of Heliotropium indicum showed dose dependent antibacterial and antifungal effects.

Morteza-Semnani et al. (2009) isolated essential oil from aerial parts of Echium italicum L. (Boraginaceae) and analyzed by means of GC and GC-MS. Twenty-two components were identified in this oil. The major constituents of the essential oil were hexadecanol (27.1 %) and pulegone (8.8 %). The antimicrobial activity of E. italicum oil was studied using the disk diffusion method and determination of minimal inhibitory concentration (MIC) values against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa, Aspergilus niger and Candida albicans. The E. italicum oil exhibited concentration-dependent antimicrobial activity on all microorganisms tested.

Hussain et al. (2010) studied antibacterial properties of crude extract and fractions of Heliotropium strigosum is an important medicinal plant and belongs to the Boraginaceae family.

Končić et al. (2010) evaluated antibacterial activity in aqueous extracts of leaves, stems and flowers of Moltkia petraea (Tratt.) Griseb from two mountainous localities (Sveti Jure and Sniježnica) in Croatia. The aqueous extracts of M. petraea did not show antimicrobial activity against bacteria.

Ahmad et al. (2014) worked on aerial parts of Heliotropium ellipticum. They sequentially extracted parts of experimented plant in petroleum ether, benzene, chloroform, ethanol and alkaloid rich fractions (petroleum ether and chloroform). Various extracts and reference antibiotics (tetracycline / mycostatin) were screened against selected bacteria and fungi and the resultant inhibition zone(s) and the activity index of crude drug were measured. The sequential extracts exhibited weak antimicrobial activity but it is interesting that the alkaloid-rich fractions demonstrated pronounced activity.

2.3.5) Analgesic activity

Ficarra et al. (1995) reported analgesic activity of of different extracts of Cordia francisci, C. martinicensis, C. myxa, C. serratifolia and C. ulmifolia leaves. The results obtained showed that the petroleum ether and alcoholic extracts especially of C. francisci, C. myxa and C. serratifolia leaves have a significant analgesic potentiality.

Ferreira et al. (2004) reported antinociceptive effect of a quinone fraction (QF) isolated from the heartwood of Auxemma oncocalyx Taub. The effect was dose-dependent and long lasting, and QF was less effective orally. The antinociceptive activity of QF was predominantly peripheral and independent of the opioid system.

Tosun et al. (2008) evaluated roots of Onosma species (Boraginaceae) for antinociceptive potency. The chloroform and ethanol (70%) extracts obtained from the roots of Onosma species (Boraginaceae) viz O. aucheranum DC. O. isauricum Boiss. and Heldr. (endemic), O. sericeum Willd., O. tauricum Pallas ex Willd. var. brevifolium DC. (endemic) and O. tauricum Pallas ex Willd. var. tauricum (Syn: O. velenovskyi Davidov). The experimental data demonstrated that O. aucheranum, O. isauricum and O. sericeum displayed remarkable antinociceptive activities.

Sharker et al. (2009) reported crude ethanol extract of leaves of Cordia dichotoma (Family-Borginaceae) for analgesic activity. The extract produced significant writhing inhibition in acetic acid induced writhing in mice at the oral dose of 500 mg/kg body weight respectively (P<0.001), which was comparable to the standard drug diclofenac sodium at the dose of 25 mg/kg of body weight. The overall results tend to suggest the analgesic potency of plant.

Rahman et al. (2011) examined methanol extract of the dried roots of Heliotropium indicum Linn. (Boraginaceae) for its possible analgesic activity. The extract produced significant writhing inhibition in acetic acid-induced writhing in mice at the oral doses of 250 and 500 mg/kg body weight comparable to the standard drug diclofenac sodium at the dose of 25 mg/kg of body weight.

Esfahani et al. (2012) studied anti-nociceptive effects of Alkanna frigida and Alkanna orientalis ethanolic extracts via the formalin test in mouse. Ethanolic extracts of plant root were prepared and were injected intraperitoneally 60 min before formalin-induced nociception at 100, 200 and 400 mg/kg. anti-nociceptive effects were evaluated during the first hour after formalin injection. Both A. frigida and Alkanna orientalis ethanolic extracts possess a remarkable anti-nociceptive effect at each dose (100, 200 and 400 mg/kg) in a dose-dependent manner during the first hour after formalin injection.

2.3.6) Anti-inflammatory activity

Rapisarda et al. (1992) studied the anti-inflammatory activity of different extracts of Cordia francisci Ten., C. myxa L., and C. serratifolia H. B. K. (Boraginaceae) leaves. The results obtained have shown that, at the highest dose tested, the petroleum ether extracts (% edema inhibition with respect to controls ranging from 37% for C. ulmfolia to 91 % for C. francisci) and, at a lower degree, the ethanolic ones (% edema inhibition ranging from 23% for C. martinicensis to 81 % for C. francisci). All investigated Cordia sp. leaves have a significant anti-inflammatory activity.

Sertié et al. (2005) reported anti-inflammatory potential of different extracts of Cordia francisci, C. martinicensis, C. myxa, C. serratifolia and C. ulmifolia leaves. The results obtained showed that the petroleum ether and alcoholic extracts especially of C. francisci, C. myxa and C. serratifolia leaves have a significant anti-inflammatory activity in the rat.

Kundakovic et al. (2006) worked on root extracts of Onosma leptanhtha to evaluate its anti-inflammatory activity. The results revealed that experimented plant showed dose- defendant anti-inflammatory activity. All the isolated compounds proved to be active for inflammation reduction.

Esfahani et al. (2012) investigated the anti-inflammatory effects of Alkanna frigida and Alkanna orientalis ethanolic extracts via the carrageenan-induced paw edema test in rat. Ethanolic extracts of plant root were prepared and were injected intraperitoneally 60 min before carrageenan-induced inflammation at 100, 200 and 400 mg/kg. Results showed that, in contrast to A. orientalis, ethanolic extract of A. frigida significantly decreases carrageenan-induced inflammation at 400 mg/kg, especially 3 h after inflammation induction.

Kuruuzum et al. (2012) documented anti-inflammatory activity of different extracts from the aerial parts and the roots of Anchusa azurea Miller var. azurea (Boraginaceae), as well as their major constituent, rosmarinic acid. The methanolic extract from the aerial parts, its n-butanol fraction, and rosmarinic acid, which was isolated from the n- butanol fraction of the extract, showed significant dose-dependent anti-inflammatory activity.

Gupta and Kaur, (2015) screened anti-inflammatory activity of C. dichotoma G. Forst. leaf extract. To study anti-inflammatory activity, carrageenan-induced rat paw edema method was used. The methanol extract high dose (400 mg/kg) was found to be highly significant as compared to standard drug.

Khan et al. (2015) evaluated the anti-inflammatory activity of crude extract of Heliotropium strigosum and its subsequent solvent fractions in post carrageenan- induced edema at 50, 100, and 200 mg/kg intraperitoneally. The results revealed marked attenuation of edema induced by carrageenan injection in a dose-dependent manner.

2.3.7) Anti-diarrhoeal activity

Asrie et al. (2016) documented the effect of methanolic extract of root and root bark of Cordia africana for treatment of diarrhoea. The antidiarrheal effect of the plant was tested on castor oil-induced diarrhoea in mice. Reduction in the number of diarrheic drops was observed in groups of mice that received 200 mg/kg (P<0.05) and 400 mg/kg (P<0.01) of the extract compared to the negative controls. The percent inhibition of intestinal fluid accumulation was 26.83%, 46.34%, and 53.66% at the doses of 100, 200, and 400 mg/kg of the extract, respectively. Relative to the negative control group, the mean percent of intestinal length moved by the charcoal meal was decreased by 24.41%, 39.89%, and 51.66% in groups of mice given 100, 200, and 400 mg/kg of the plant extract, respectively. The root bark extract of C. africana was found to be effective in preventing castor oil-induced diarrhoea and intestinal motility in a dose-dependent manner. This reveals that the plant material has promising anti-diarrhoeal activity as it is claimed in traditional medical practice.

Al-Snafi, (2016) reviewed the pharmacological and phytochemical studies of on Cordia myxa fruit extract. Results revealed the presence of oil, glycosides, flavonoids, sterols, saponins, terpenoids, alkaloids, phenolic acids, coumarins, tannins, resins, gums and mucilage. Pharmacological studies revealed that Cordia myxa possessed analgesic, anti-inflammatory, immunomodulatory, antimicrobial, antiparasitic, insecticidal, cardiovascular, respiratory, gastrointestinal and protective effects. This review was designed to highlight the chemical constituents and pharmacological effects of C. myxa.

CHAPTER-3

MATERIALS AND METHODS

The following procedures were followed for the pharmacognostic studies of E. serrata and E. obtusifolia.

3.1) Collection and preservation

3.2) Pharmacognosy

3.2.1) Morphological observations

3.2.2) Macroscopic and organoleptic studies

3.2.3) Anatomical studies (T. S of stem and lead)

3.2.4) Leaf surface studies including,

3.2.4.i) Stomatal types, stomatal number and stomatal indices

3.2.4.ii) Vein islet and Vein termination number

3.2.4.iii) Palisade ratio

3.2.5) Powder drug study

3.2.6) Fluorescence characters

3.2.7) Preparation of extracts

3.2.8) Qualitative phytochemical screening

3.2.8.i) Test for carbohydrates detection

3.2.8.ii) Test for protein and amino acids detection

3.2.8.iii) Test for alkaloids detection

3.2.8.iv) Test for phenol detection

3.2.8.v) Test for fixed oil detection

3.2.8.vi) Test for flavoniod detection

3.2.8.vii) Test for saponin detection

3.2.8.viii) Test for terpeniod detection

3.2.8.ix) Test for tannin detection

3.2.8.x) Test for antharquinone detection

3.2.8.xi) Test for glycoside detection

3.2.8.xii) Test for steroiod detection

3.2.8.xiii) Test for reducing sugar detection

3.2.8.xiv) Test for starch detection

3.2.9) Elemental Analysis

3.2.10) Nutritional Analysis

3.2.10.i) Determination of moisture

3.2.10.ii) Determination of ash

3.2.10.iii) Determination of crude protein by micro kjeldahl method

3.2.10.iv) Determination of fats or ether extract

3.2.10.v) Determination of crude fibers

3.2.10.vi) Determination of carbohydrates

3.11) Biological Activities

3.11.i) Acute Toxicological activity

3.11.ii) Cytotoxic activity

3.11.iii) Phytotoxic activity

3.11.iv) Antibacterial activity

3.11.v) Analgesic activity

3.11.vi) Anti-inflammatory activity

3.11.vii) Antidiarrheal activity

3.1) Collection and preservation

Fresh leaves, fruits and stem bark of E. serrata and E. obtusifolia were collected from trees growing in Department of Botany, University of Peshawar. Some of the morphological and ecological studies such as size of tree, phyllotaxes, root type, etc were conducted at the time of collection. Plant samples were identified by the herbarium curator of Department of Botany, University of Peshawar. The plant samples were dried in shade, pasted on Herbarium sheet and given Voucher specimen number, Bot. Khan. 2007 (PUP) and deposited in the herbarium. Stem barks, leaves and fruits were collected in large amounts and were used for the present pharmacognostic research. Microscopic study of fruits, stem barks and leaves of the fresh sample was carried out. Samples were dried in the shade and then grinded in electric grinder into fine powder which was used for extraction. The fine powder of experimented parts was kept in clean, dry and air tight bottles to keep away from pests, molds, insects, pathogens, and harsh conditions and was later used for phytochemical and various pharmacological activities.

3.2) Pharmacognosy

3.2.1) Morphological study

Morphological observations of fruit, leaf, and stem bark of E.serrata and E obtusifolia were carried out by following the method of Wallis, (1985).

3.2.2) Macroscopic and organoleptic study

The microscopic study was carried out by organoleptic method in which the shape, color, size, odor, taste and other specific features of the leaf, fruit and stem bark of E. Serrata and E. obtusifolia were identified.

3.2.3) Microscopic and anatomical study

The microscopic study on the leaf and stem of the E.serrata and E.obtusifolia was conducted according to the Tajuddin et al., (2013 and Solanki et al., (2011). The histological study of the E. serarta and E. obtusifolia (leaf, fruit and stem bark) was carried out by using through transverse section method (Akcin et al., 2011; Johnson, 2000; Okhle et al., 2010). Fresh specimen was used for section cutting. Thin transverse sections of leaf and stem were cut with a sharp blade by hand. Proper staining of fine transverse sections was done and examined under digital camera fitted with Labomid microscope (Fig. 01). Photographs of clear and fine sections were taken.

3.2.4) Leaf surface study

The following features of the leaf surface were studied,

3.2.4.i) Stomatal number and stomatal index

The average number of stomata on a 1mm square area of both upper and lower epidermis of a leaf is called stomatal number. Stomatal index number (or stomatal density) is the percent ratio of stomata to the epidermal cells in a 1mm square area (Evans, 2002; Xavier et al., 2015).

Procedure

The fresh young leaves were plucked for observing the number and type of stomata in them. Both upper and lower epidermis were peeled off by using a sharp blade and washed with water. These experimental pieces were placed on microscope slides and a drop of glycerine was put on these to prevent drying and the sample was covered with a cover slip. After that, these slides were examined under Labomid digital microscope by using 100X magnification. Number of stomata was recorded in the 1 mm2 area. Following formula was used to find out stomatal index.

Stomatal index= Number of stomata/ No. of stomata+Epidermal cells× 100

3.2.4.ii) Veinlet termination and vein islet number

Average number of veinlet terminations in 1 mm2 of leaf surface area from the midrib to the margin of the leaf is called veinlet termination number. Veinlet termination is the ultimate free end of veinlet. Vein islet number is defined as the average number of veinlet enclosing small green area in 1 mm2 leaf surface (Wallis, 1985; Evans, 2002; Omoregie et al., 2015).

Procedure

To calculate the vein termination number and vein islet number, 1-3 mm2 area of leaf lamina located between the margin and midrib of the leaf was taken. These pieces of leaf were boiled in chloral hydrate for 15 minutes till all the chlorophyll from surfaces was dissolved and discoloration occurred. Then these pieces were observed under microscope at 10X magnification. Number of vein islets and vein terminations were counted in 1 mm2 area. For more accuracy five readings were taken and the average was calculated. Photographs of the specimens were also being taken (Evans, 2002; Hedge et al., 2015).

3.2.4.iii) Palisade ratio

The number of palisade cell under single upper epidermal cell is known as palisade ratio (Evans, 2002). It is used as an important tool and parameter for the characterization of leafy drug (Barkatullah et al., 2015).

Procedure

For determination of palisade ratio, small pieces (1-2 mm) of leaf were taken and boiled in chloral hydrate for discoloration. A piece of the leaf was fixed on a slide and covered with a coverslip and then by the slight rotation of fine adjustment, the underlying palisade cells were focused and counted. Only those palisade cells were counted which more than half covered by epidermal cells were. For accuracy, five different readings were taken and the figure then obtained was divided in 4 to obtain palisade ratio.

3.2.5) Powder drug study

Investigation on the powdered drugs (stem bark, fruit, and leaf) of both plants was carried out for the assessment of physical and microscopical characteristics. Physical characteristics included color, odour and taste, while microscopy was carried out to reveal the characteristic structure such as various types of cells, tissues, starch grains and calcium-oxalate crystals, xylem and phloem tissues and stomata etc., according to following the method of Sailor et al. (2010) as given below.

Procedure

The parts used of both plants were shade dried, grounded into fine powder and sieved through sieve no.60. Small portions of each powder were boiled in chloral hydrate solution for clearing and were transferred to microscopic slides. A drop of glycerine was added to prevent drying, covered with a cover slip and observed under a compound microscope in term of various types of cells, tissues etc.

3.2.6) Fluorescence analysis

Fluorescence is the analysis of chemically bioactive compound in crude powder by dipping or dissolving powder in various chemical reagents to make it fluorescent and then observing it under UV light. It is thought to be the simplest methods for qualitative analysis of crude drugs (Nwachukwu et al., 2010). Fluorescence features of the powder were detected according to the method of Ozcan et al., (2011).

Procedure

Small quantity of powder of all three parts (stem bark, leaf and fruit) was dissolved individually in a specific reagent for 5 minutes and examined under visible light and UV light of both the wavelength (256 nm and 360 nm) for fluorescence. The following reagents were used for this study. i) Iodine ii) Ferric chloride (FeCl3) iii) Sulphuric acid iv) Picric Acid v) Ammonia solution vi) Hydrochloric acid vii) NaOH viii) 50% Nitric acid ix) Acetic Acid

3.2.7) Preparation methanolic extracts

1000 g of dried powder of each part of both plants were soaked for 7 days in 6000 mL methanol separately in a round bottom flask. The flasks were shaken many times during this period in order to speed up and complete extraction procedure. The extracts were filtered by evaporating solvents through the rotary vacuum evaporator. The extracts were then kept in petri dishes, and stored in refrigerator for different qualitative and quantitative phytochemical screening and various pharmacological activities (Miliauskas et al., 2004).

3.2.8) Preliminary qualitative phytochemical screening

Phytochemical screening tests were carried out for detection of various bioactive compounds in all the extracts individually. The following tests were conducted;

3.2.8.i) Carbohydrate detection test

 Benedict test: (an alkaline solution containing cupric complex) few drops of benedict reagent were added in methanolic extract and boiled on water bath. Appearance of reddish brown precipitation confirmed the presence of carbohydrates (Evans, 2009).  Fehling test: (Copper sulphate in distilled water) few drops of Fehling’s A and Fehling’s B were added in methanolic extract solution and boiled on spirit lamp. The appearance of red colour in test tube indicated the presence of reducing sugar (Evans, 2009 & Ozcan et al., 2011).

3.2.8.ii) Protein and amino acid detection test

 Ninhydrin method: (Indane 1, 2, 3 trione hydrate) few drops of 0.2% Ninhydrin solution was mixed with the extract and boiled in the test tube. Formations of violet color precipitates indicated presence of amino acid and protein (Edeoga et al., 2005).

 Biuret test: Few drops of CuSO4 were added in the plants' extract and then 2 mL of ethanol and potassium hydroxide was added. Pink color precipitate appearance indicates the presence of proteins (Edeoga et al., 2005 and Hegde et al., 2015)

 Xanthoproteic test: Few drops of concentrated HNO3 were added in 10 mL of methanolic extract, yellow color indicates the presence of protein (Edeoga et al., 2005).

3.2.8.iii) Alkaloids detection test

 Mayer’s test (Potassium mercuric iodide solution): Mayer’s reagent was added dropwise in 30 mL methanolic extracts solution. Creamy white precipitate formation showed the presence of alkaloids (Deore et al., 2015).  Wagner’s test (solution of iodine in potassium iodide): Few drops of Wagner reagent were added in 10 mL of extract solution through a dropper. The appearance of red brown precipitation indicated alkaloid existence (Deore et al., 2015).

3.2.8.iv) Phenol detection test

 Ferric Chloride Test: Few drops of FeCl3 solution were added to 10 mL extract solution. The appearance of bluish black or green color showed presence of phenols (Chavre, 2015).

3.2.8.v) Fixed oil detection test

Each powder of leaf, stem bark and fruit of both Ehretia serrata and Ehretia obtusifolia were kept in filter paper and pressed. Permanent greasy spots appearance on filter paper proved the presence of fixed oil (Onocha et al., 2011; Hegde et al., 2015).

3.2.8.vi) Flavonoid detection test

 Alkaline reagent test: NaOH solution was added to 20 mL of plants' extract solution. Precipitate of yellowish red color showed the presence of flavonoids (Kokate, 1994).  Lead Acetate test: Few drops of lead acetate solution were mixed with the plant extract solution. Yellow color precipitate appearance in solution indicated the presence of flavonoids (Onocha et al., 2011).

3.2.8.vii) Saponin detection test

 Frothing test: Froth formations after shaking powder drug in a test tube, which persisted for a long time indicated the presence of saponin (Chaouche et al., 2011).  Foam test: Mixing of 3 grams of the powder in 20 mL of distilled water and after vigorous shaking, appearance of permanent foam for up to ten minutes indicated the presence of saponin (Tiwari et al., 2011).  Hydrochloric acid test: In this test few drops of HCl were added in the extract solution. Appearance of a pink color indicated the presence of saponin. If the addition of ammonia solution with HCl in the extract solution changes color of the solution into deep violet, then it is indicating the presence of saponin.

3.8.viii) Terpenoid detection test

 Salkowski’s test: 2 mL of extract solution were mixed with several drops of

chloroform and H2SO4, The formation of red color precipitate in bottom of the test tube showed the presence of terpenoids (Harborne, 1998).  Copper acetate test: Small amount of extract was dissolved in distilled water. Few drops of Copper acetate solution were dissolved in the extract solution. The appearance of green color shows the presence terpenoids (Tiwari et al., 2011).

3.2.8.ix) Tannin detection test

 Ferric chloride test: 5 mL of ferric chloride solution was mixed in 10 mL of extract solution. Formation of bluish black precipitate showed the presence of tannin (Somkuwar and Kamble, 2013).  Alkali reagent test: Sodium hydrochloride solution was added to 10 mL of plants' extract. Formation of yellow-red precipitate confirms the presence of tannin (Somkuwar & Kamble, 2013).

3.2.8.x) Anthraquinone detection test

 Borntrager’s Test: Mixture of 10% FeCl2 solution and 10 mL extract solution was heated, 2 mL of pure HCl was added and the mixture was filtered. After cooling, the filtrated mixture was quickly shaken with diethyl ether followed by addition of concentrated ammonia solution. Formation of pink or deep red color aqueous layer indicated the presence of anthraquinone (Niratker & Sailsaja., 2014).

3.2.8.xi) Glycoside detection test

 Borntrager’s test: 1 mL of the extract solution and 5% of H2SO4 (1 mL) were mixed. The mixture was boiled in a water bath and then filtered. The filtrate was then shaken with equal volume of chloroform and was left standing for 5 minutes. Then lower layer of chloroform was shaken with half of its volume with dilute ammonia. The formation of pink red rose coloured ammoniacal layer gave indication of anthraquinone glycosides (Joshi et al., 2013).

3.2.8.xii) Phytosterol detection test

 Salkowski’s Test: Extracts were treated with chloroform and then filtered. The filtrates were treated with few drops of conc. sulfuric acid, shaken and allowed to stand. Appearance of golden yellow color indicated the presence of triterpenes.  Libermann Burchard’s test: Extracts were treated with chloroform and then filtered. The filtrates were first treated with few drops of acetic anhydride. After that the solution was boiled the solution and cooled, to which a few drops of concentrated sulfuric acid were added. The formation of brown ring indicated the presence of phytosterol (Evans, 2009 & Ozcan et al., 2011)

3.2.8. xiii) Reducing sugar detection test

 Fehling's test: Few drops of Fehling's solution were added in extract solution and then heated on a water bath for 2 minutes. The appearance of a reddish- brown precipitate of cuprous oxide indicated the presence of combined, reducing sugar (Somkuwar & Kamble, 2013).

3.2.8. xiv) Starch detection test

1 mL of aqueous extract was added in 10 mL of NaCl saturated solution. After heating, starch reagent was added. A blue-purplish color is a positive test for the presence of starch. The plant powder sample was mixed with distilled water in a test tube, then was shaked well, and filtered. Then 1% of aqueous hydrochloric acid was added to each plant extract then the samples were boiled with the help of hot plate stirrer. Formation of a red colored precipitate confirmed a positive result (Joshi et al., 2013).

3.2.9) Elemental analysis

Atomic absorption spectrophotometer was used for analysis of elements and detection of heavy metals in leaf, stem bark and fruit methanolic extracts of E. serrata and E. obtusifolia.

Requirements

Sulphuric Acid, Hydrogen per Oxide, Perchloric Acid, Hydrochloric Acid, Hydrogen Fluoride, Per Chloric Acid and Nitric Acid, beaker, funnels, flame photometer (Jenway PFP7 UK) and flame atomic absorption spectrophotometer (AAS) (Jeol japan, model no. 460). All the reagents used were from Merck (Darmstadt, Germany). Pb, Cd, Co and Mn are Sigma made and Cu, Zn and Fe are Aldrich made.

Sample preparation

Sample preparation was done by following standard wet digestion procedure (Hseu., 2004). 1 g of respective powder of each experimented part of E. serrata and E. obtusifolia were mixed with 10 mL of concentrated nitric acid and poured in to conical flask and left for 24 hours. After which 4 mL HClO4 were added and after 30 min, each flask was kept on hot plate for evaporation until 1 mL clear solution left behind. After that distilled water was added which raised the volume of mixture in the flask up to 100 mL, which was then filtered by using Whatman # 24 filter paper.

The sample filtrates were kept in an airtight container for elemental analysis by atomic absorption spectrophotometer (Eslami et al., 2007). A flame photometer (Jenway PFP7, UK) and flame atomic spectrophotometer was used for analysis of elements in a sample. The stock solutions were carefully diluted to prepare the calibration standard for each metal (Saeed et al., 2010).

Procedure

An instrument used for elemental analysis was adjusted according to the instructions and warmed up for a few minutes after switching on each elements respective cathode lamp. The standards 2.5, 5 and 10 ppm were used for each element one-to-one and the instrument was adjusted accordingly. The standard working solution of test elements was prepared to make the standard calibration curve. Flame photometer (Jenway PFP7, UK) was used for the determination of elements that was sodium, and flame atomic absorption spectrophotometer (AAS) (Joel Japan, model no. 460). Seven elements (3 macro elements and 4 microelements) were detected, including sodium, magnesium, zinc, iron, silver, copper, and gold. In AAS, cathode lamp was used as radiation source

which provided both sensitivity and selectivity. Only seven elements were detected under the provided atomic absorption spectrophotometer, other was not, probably due to the reason that they did not absorb that wavelength of spectrophotometer. Concentration of test metals was calculated from calibration curves obtained from standards (Butcher, 2016).

Table 3.1. Conditions applied for detection of various elements

Elements Flame type Wavelength Silt width Acetylene Air oxide Cathode lamp (nm) (nm) flow (L/min) flow (L/min) current (mA) Pb Air/acetylene 283.3 0.7H 2.0 17 10 Zn Air/acetylene 213.9 0.7H 2.0 17 15 Mn Air/acetylene 279.5 0.2H 2.0 17 20 Co Air/acetylene 240.7 0.2H 2.0 17 30 Cr Air/acetylene 357.9 0.7H 2.5 17 25 Cu Air/acetylene 324.8 0.7H 2.0 17 15 Fe Air/acetylene 248.3 0.2H 2.3 17 30

Mg Air/acetylene 766.5 0.7H 2.0 17 12 Na Air/acetylene 589 0.2H 2.0 17 0.8 Ni Air/acetylene 232 0.2H 2.0 17 25 Cd Air/acetylene 230.3 0.2H 2.0 17 25

Statistical Analysis

For analysis of the elemental data arithmetic mean and standard deviation was measured by following standard methods of Saeed et al (2010).

3.10) Nutritional analysis

3.10.i) Determination of moisture

Requirements

Electric balance, electric oven, desiccator and petri dishes with lid

Procedure

Two grams of each experimented sample powder were taken in a petri dish and covered with a lid which were then weighed on electric balance (W1) followed by placement in the oven at 105 ⁰C for up to 6 hours. When all the moisture was removed from samples, petri dishes were placed in desiccator for 30 minutes for cooling. After that, petri dishes were again weighed and percentage of moisture was calculated by the following formula (W2) (A.O.A.C, 2000).

% Moisture= (W2-W1/ Weight of the sample) ×100

3.10.ii) Determination of ash

Requirements

Electric balance, muffle furnace, desiccator, crucible, blow pipe and burner

Procedure

2 grams of each of the samples were weighed in a crucible (W1). It was burnt with blow pipe and placed in a muffle furnace at 550 ⁰C for several hours. When the color of the sample was greyish white, then the furnace was turned off. After cooling, the sample was transferred into the desiccator. It was weighed again and was considered as W2. The percentage of Ash was calculated by following formula (A.O.A.C., 2000)

Calculation

% Ash = (W2-W1/ Wt of sample) × 100

W1= Weight of empty crucible

W2= Weight of crucible + Ash

3.10.iii) Determination of crude protein by micro Kjeldahl method

Requirements

Concentrated H2SO4, 4 % Boric acid solution, 0.1 N standard HCl solution, 40% NaOH, catalyst (7 gm K2SO4, 0.5g CuSO4. 5 H2O and 0.3 g HgO).

Indicator i) Methylene blue, 0.2 % in 5 % ethanol. ii) Methyl red, 0.25 % in 95 % ethanol

Mix (i) and (ii) with 3:2 ratio

Apparatus

Pipette, burette, volumetric flask, Kjeldahl flask and digestion and distillation apparatus etc.

Procedure

Protein (% Nx6.25) was determined by the Macro Kjeldahl method. The procedure is given below.

Dried sample of (0.5 g) was taken in digestion flask. Digestion mixture (CuSO4, K2SO4 and FeSO4 in the ratio of 5, 94 and 1 respectively) is mixed with 25 mL of concentrated

H2SO4 and digested in the digestion flask (kjeldatherm) for 6 hours. After cooling the flask, contents of the flask were transferred into a 250 mL flask, then a small amount of distilled water was added to make the volume of the solution rise upto 50 mL. 10 mL of strong alkali was added to make the solution alkaline.

50mL of 4% boric acid solution with 3-5 drops of indicator was transferred to the distillation flask. 60 mL of 32% NaOH solution and 50 mL of water were then added to it. After distillation, it was then collected in a flask for titration.

Titration was done by taking 0.1 N HCl in the burette. Tape of burette was opened and HCl was added drop wise in the contents of the flask, till the solution turn colorless

from pink colour. The reading was noted and the percentage of protein was determined using the following formula (AOAC., 2000).

Calculation

% Protein = (S- B) × 0.5 × 0.014 × 100 Wt. of sample × aliquot of digest taken for distillation

Where as

S= Sample titration reading

B= Blank titration reading

0.14= Atomic weight of Nitrogen (N)

Percent crude protein contents were calculated for all the samples by multiplying the nitrogen content of the sample by 6.25.

Protein (%) = % Nitrogen × 6.25.

3.10.iv) Determination of fat or ether extract

Requirements

Petroleum ether (B.P 40-60 C), Extraction Thimble (Whatman), Water bath, Soxhlet extraction apparatus.

Procedure

First of all, rinsed all the glass apparatus by petroleum ether and dry it in the oven at 102°C and after removing it keep in the desiccator.Weighed 5 gram of grounded and dried sample and placed it in the thimble. Placed the thimble in the soxhlet extractor. 150 mL round bottom flask was took and cleaned followed by filling the flask with 90 mL petroleum ether. After that whole setting was placed on a heating mantle and allowed the petroleum ether to boil. Continued the extraction process for several hours, almost 6 hours. After that removed the condensing unit from extraction unit and allowed the sample to cool down. Finally, the entire lipid removed from the sample.

Collected almost all the solvent after distillation. Placed the sample in the oven and after removing it placed in the desiccator. Take the weight of the sample (W2). Fat percentage was then calculated by the following formula (A.O.A.O, 2000).

Calculation

% Fat (Ether extract) = (X/ Wight of the sample) × 100

Where as

W1 = Weight of the empty thimble

W2= weight of the empty thimble + sample after evaporation of solvent

X= Weight of the fats = W2 - W1

3.10.v) Crude fiber determination

Requirements

Asbestos, petroleum ether, ethyl alcohol, HCl (2%), NaOH (2%), crude fiber extraction apparatus, muffle furnace, suction pump, electric balance, oven, beaker, filter paper, Gooch crucible, funnels, and cotton cloth.

Procedure

2 grams of each sample were mixed with 200 mL of NaOH (2%) and kept it in a water bath for boiling for thirty minutes and then filtration. The residue was transferred into another beaker and 200 mL of HCl were added and boiled again for 30 minutes. After that filtration of the solution was done through cotton cloth and washed quickly with hot water for removal of acid from residue. Then the residue were transferred into crucibles and kept in the oven for 4 hours at 105 ⁰C. Next it was allowed to cool in a desiccator, weighed and named as (W1). Then the crucible was transferred in to muffle furnace and the temperature of muffle furnace was set at 550 ⁰C for four hours. When residue was converted into white color ash it was weighed again and considered as

(W2). Crude fibers were calculated by following formula designed by (A.O.A.O., 2000). This procedure was repeated for each sample to get accurate values.

Calculations:

% Crude fiber = (W2-W1/Weight of sample) × 100

3.3.10.vi) Determination of Carbohydrates

Carbohydrate determined by subtraction of all combined weight of the content from 100 by following (Merrill & Watt, 1955)

Calculation

% Carbohydrates= (Crude fibre+ Crude fat+ Crude protein+ Moisture contents+ Ash)-

100

3.11) Biological activities

The following biological activities were conducted for the determination of pharmacological and therapeutic effects of the methanolic extracts of leaf stem bark and fruit of E. serrata and E. obtusifolia.

3.11.i) Acute toxicity study

The acute toxicological potency of leaf, stem bark and fruit methanolic extracts of E.seerata and E. obtusifolia was determined by following the standard method of Baliga et al. (2004).

Procedure

Swiss albino mice of both sexes weighing 18 to 24 g were experimented taking from Veterinary Research Institute (VRI) Peshawar. The animals were kept in a suitable environment and balanced food in the cages. The experimental procedures relating to the animals were authorized Veterinary Research Institute (VRI) ethical committee (Est-VRI/Dev (02)/Et-15-03) before starting the study and were conducted under the internationally accepted principles for laboratory animal use and care. Drinking water and food were provided throughout the experiment, except for the short fasting period (4 h), where the drinking water was still in free access but no food supply was provided 12 h prior to treatment. The animals were not allowed to take food and water for 4 hours

before the experiment. Then the mice were divided into 19 groups, each having five replicates. The test sample was sequentially extracted with methanol by adding approximately 100 g of the dried sample (in fine powder form) to 400 mL methanol. The extraction was carried out at room temperature by soaking for 7 days with intermittent stirring during the first day. The extracts were filtered through clean muslin cloth and the extraction process was repeated again for a second time by adding another 400 mL of methanol to the sample residue. The filtrate from each extraction was combined and concentrated under vacuum on a rotary evaporator (Buchi, Switzerland) at 40 °C to 50 °C in order evaporate the excess methanol solvent and until a dark green methanol extract was produced. The concentrated extract was poured into glass Petri plates and brought to dryness at 60 °C in the oven until a paste-like mass was obtained. Then paste form extract was sealed in Petri plates and stored at room temperature (RT). Group 1 of mice was treated with normal saline and considered as a control group, while the rest of the group were treated with intra-peritoneal injecting extracts of the fruit, stem bark and leaf of 100, 150 and 200 mg/kg of body weight. After administering the extract, the animals were observed thoroughly for 10-12 hrs for behavioural changes and appearance of any toxic symptoms.

3.11.ii) Cytotoxic activity

The cytotoxic activity of methanolic extracts of leaf, stem bark and fruits of E. serrata and E.obtusifolia was carried out at different dilutions following the procedures of Meyer et al., 1982; Mamoon and Azam, 2011.

Requirements

Extracts of plants parts, Brine shrimps, Artemia salina eggs, Methanol, Sea salt solution (38 g sea salt/l of distilled water with pH 7.5), Hatching tray, Lamp for attraction of brine shrimp larvae, Micro pipette (of different ranges of 10 to 1000 μL), DMSO and vials.

Procedure

38 g sea salt was dissolved in per litter of distilled water which was filtered. It was then poured into a hatching tray. The tray was divided through the perforated partition into

two unequal parts. The major part was lightened with electric bulb. In the smaller part, brine shrimp eggs were sprinkled and were covered with a black paper to provide darkness. After about 48 hrs, the eggs hatched into naupilii larvae, which actively migrated to the larger illuminated part of the tray through the perforated partition. These hatched larvae of naupilii were then used in bioassay. 20mg of each leaf, fruit and stem bark of extracts were dissolved in methanol individually in a test tube. From each stock solution 5, 50 and 500 µL (equivalent to 10, 100 and 100 µg/mL or ppm) were transferred to the empty sterilized vials. For each dilution three replicates were taken and left overnight to evaporate the solvent. It was followed by the addition of 5 mL of seawater and 10 nauplii larvae in each vial which were incubated for 24 hours. For negative and positive controls the vials were filled with brine solution and reference cytotoxic drug respectively. The % mortality was observed in each vial and LD50 were determined.

Statistical analysis

Statistical analysis was done through a Biostate version of percent mortality LD50 (lethal dose of 50% of the population) by following the procedure of Anderson et al. (1991) and Barkatullah et al. (2011).

3.11.iii) Phytotoxic activity

Phytotoxic activity of leaf, stem bark and fruit of methanolic extract of the two plants (E. serrata and E. obtusifolia) was carried out against Lemna minor by following methods of Silva et al. (2012); Bisio et al. (2011); Nazeefullah et al. (2014); Ali et al. (2014).

Requirements

Lemna minor, leaf, root and stem bark extracts of the experimented plants, distilled water, petri plates (9 cm diameter), micropipette (10, 100 and 1000 μL), filter paper, oven, flasks, e- medium

Preparation of the methanolic extract

20 mg extract of leaf, stem bark and fruit of E. serrata and E. obtusifolia dissolved in 2 mL methanol and form stock solutions. From each solution sample 10, 100 and 1000 µL (equivalent to 20, 200 and 2000 µg/mL) solutions added in pre-sterilized petri dishes with three replicates of each and were keep whole night in the sterilized condition to evaporate the solvent.

Preparation of E-media

E-medium was composed of 10 different salts with calculated amounts which provided mineral nutrients for the Lemna minor plant. A particular amount of salts was dissolved in distilled water to raise the volume up to 1000 mL. The composition of E-medium is given below. Three Rosset fronds containing Lemna minor with 20 m of E-medium was added in each petri. The distilled water was taken as positive control and Paraquate (growth inhibitor) as a negative control. Then all petri dishes were kept in an incubator for 7 days by giving 12 hour photoperiod daily. The results were noted on the 7th day by counting damaged fronds and growth inhibition with reference to the negative control by the following formula.

Inhibition % = 100- Number of fronds in test sample × 100 Number of fronds in negative control

Composition of E-medium:

S. No Chemical Name g/L

1) Potassium dihydrogen phosphate (KH2PO4) =0.68 g/L

2) Potassium nitrate (KNO3) = 1.515 g/L

3) Calcium nitrate (Ca(NO2)2.4H2O)= 1.180 g/L

4) Magnesium sulfate (MgSO4.7H2O) =0.492 g/L

5) Boric acid (H3BO3) =0.00286 g/L

6) Manganese chloride (MnCl2.4H2O) =0.00362 g/L

7) Ferric chloride (FeCl2.4H2O) =0.00540 g/L

8) Zinc sulfate (ZnSO4.5H2O) =0.00022 g/L

9) Copper sulfate (CuSO4.5H2O) =0.00022 g/L

10) Sodium molybdate (Na2MO4.2H2O) = 0.00012 g/L

11) Ethylene diamino tetra acetic acid (EDTA) =0.01120 g/L

Statistical Analysis

The phytotoxic results were analysed with the finney computer program to determine

FI50 values with 95% confidence intervals (Saeed et al., 2010; Barkatullah et al., 2011; Bashir et al., 2009 and Onocha and Ali, 2010).

3.11.iv) Anti-oxidant activity

Free radical formation due to different nutritional and medicinal deterioration is controlled by a number of systems called antioxidants. The antioxidant potency of the experimented parts of E. serrata and E. obtusifolia was determined according to the procedure of Lalitha et al. (2013).

Requirements

1, 1-diphenyl-2-picryl hydrazyl (DDPH), Extracts of different parts of the plants scorbic acid, test tubes, spectrophotometer (UV-VIS Shimazdu)

Procedure

The DPPH (1, 1-diphenyl-2-picryl hydrazyl) was used to detect the free radical scavenging potency of different experimented parts of the two plants. DPPH solution of 0.1 Mm was prepared in ethanol. Different concentrations (10, 100 and 1000 µg/mL) of extracts were formed. A mixture of 1 mL DPPH and 3 mL of each extract was formed. Mixture was shaken thoroughly, and before incubation was kept at room temperature in the dark for 30 minutes. Spectrophotometer (UV-VIS) was set on 517

nm and absorbance of these prepared solutions were checked after 30 minutes, 45 minutes and 60 minutes at the set wavelength. Ascorbic acid was used as a standard. The % age of antioxidant potency of extracts was calculated by using following formula.

% Antioxidant potential= (Absorbance of control (nm) – Absorbance of test nm/× 100 Absorbance of control)

3.11.v) Antibacterial activity

The effectivity of natural extracts and pure compounds against various bacteria is noticed by observing the growth of bacteria in the presence of plant samples that are placed in close contact with them. The following activities were carried out to see the antibacterial potential of leaf, stem bark and fruit extracts of E. serrata and E. obtusifolia against various strains of bacteria by using the agar well diffusion method (Balouiri et al., 2016).

Requirements

Nutrient agar, plant extracts, petri dishes, distilled water, autoclave, laminar flow hood, incubator, filter paper, cotton swab, standard antibiotic (Streptomycin), and micropipettes.

Bacterial strains used

Gram-positive: Staphylococcus aureus, Clavibacter michiganensis. Gram-negative: Proteus vulgaris, Xanthomonas spp.

Procedure

To detect the potential of stem bark, leaf and fruit extract of E. serrata and E. obtusifolia against the microorganisms, agar well diffusion method was used. The activity was done in the research laboratory of the Department of Pathology University of Agriculture Peshawar. Nutrient broth, cotton swab, petri dishes and agar media were autoclaved and were transferred in to the laminar flow hood except petri dishes. Petri dishes were first dried in oven and then transferred to laminar flow hood. After that, proper amount of media was poured into petri dishes and were kept in laminar flow for

half an hour to solidify the media in petri dishes and then transferred in the incubator with set temperature of 37 ⁰C for 24 hours in order to check the sterility of media. On the next day, fresh nutrient broth was added in a test tube and small amount of bacterial culture were mixed with it which formed diluted bacterial culture. After that, in sterilized nutrient agar plates wells were made by using a cork borer (6 mm). Wells were uniformly distributed on agar plates with 24 mm distance between the two wells.

Stock solutions were made by dissolving 3 mg of each extract in 1 mL DMSO separately. Wells were loaded differently by 50 µg /mL and 100 µg/mL formed from each stock solution and labelled. DMSO was used as positive control and amoxicillin as negative control. On the next day, through a scale the zone of inhibition was measured. By using following formula zone of inhibition were measured.

% Inhibition= (Zone of inhibition of sample/Zone of inhibition of standard) × 100

3.11.vi) Analgesic activity

The analgesic activity of leaf, stem bark and fruit methanolic extract of E. serrata and E. obtusifolia were carried out using a hot plate method on an albino Swiss mice by following (Okokon et al., 2012; Okokon et al., 2016).

Materials

Albino mice (about 18-24 g in weight), Plant test samples, Diclofenic sodium, Acetic acid, normal saline, syringes.

Experimental animals

Colony bred Swiss mice from Veterinary Research Institute, Peshawar were used in the study. The animals were kept in polypropylene cages and under stable environment and food, with free access to clean drinking water. The experimental procedures relating to the animals were authorized Veterinary Research Institute (VRI) ethical committee (Est-VRI/Dev (02)/Et-15-03) before starting the study and were conducted under the internationally accepted principles for laboratory animal use and care.

Hot plate method

100 mice were used for this study, and they were divided into 20 groups of five mice each. Eighteen groups were used for detection of analgesic activity at a dose level of 100 mg/kg, 200 mg/kg and 300 mg/kg of leaf, fruit and stem bark extracts of both experimented plants and two groups were used for positive and negative control. Diclofenac sodium was used as negative control and normal saline as positive control. All the animals were fasted for 12 hours with suitable clean drinking water provision. Analgesometer was used for this bio-assay. Each of the mouse was placed on a hot plate, kept at 55 ⁰C ± 10 ⁰C and the initial latency time determined with a stop watch was recorded which denotes the time taken from the mice to show a reaction to the pain stimulus. The response to pain stimulus included; raising and licking of hind foot or jumping. The cut off time was fixed for 20 seconds. This served as control pain reaction time. Response of latency of the animal on hot plate was noted down for three hours with 30- minute interval after treatment (Okokon et al., 2016).

3.11.vii) Anti-inflammatory activity

This activity was done by following the method of Hussain et al., (2017). The albino mice were divided into 20 groups. Group 1 and 2 were treated for normal saline (10 mL/kg) and diclofenac sodium (10 mg/kg) respectively to serve positive and negative control. The remaining 18 groups (five mice in each group) were given 100 mg/kg, 200 mg/kg and 300 mg/kg of the methanolic extract of leaf, fruit and stem bark of E. serrata and E. obtusifolia. After ½ an hour, 1% carrageenan was injected subcutaneously in mice in the sub plantar tissue of the right hind paw. The inflammation was calculated by using plethsymometer, just after carrageenan administration. After 1 hour and 90 minutes the inflammation of right hind paw of mice was calculated again through plethysmometer. The average swelling of the foot of testing mice was compared with the foot of mice which received standard drug and with those which received (-) control. Percentage inhibition of edema was calculated using the formula

% inhibition= A-B/A× 100

A= Edema volume of the (-) control

B= Paw edema for the test group

3.11.viii) Anti diarrhoeal activity

Anti-diarrhoeal activity of leaf, stem bark and fruit methanolic extracts of E. serrata and E. obtusifolia were conducted by gastrointestinal motility model using Swiss albino mice by following Gerald et al. (2007), Yakubu & Salimon. (2015).

Requirement

Swiss albino mice of both sexes (18-24 g), plant sample extracts, charcoal, ruler, normal saline water, feeding tubes, syringes and standard drug (atropine sulfate).

Gastrointestinal motility model

This is a standard method used for checking the extract potency on gastrointestinal transportation in mice. The overnight fasted animals were first divided into 21 groups. 1cc= 10 mg/kg saline water was intra peritonealy injected to group 1, castor oil ( causing diarrhoea ) was injected to group 2 which was considered as negative control and Laperomide drug (1 mg/kg) was injected to group 3 and considered as positive control. Remaining 18 groups used for each plant extract with concentration 100, 150 and 200 mg/kg. Animals were kept fastened overnight. Twenty five minutes later after dosage all the animals were fed with charcoal meal through feeding tubes. These mice died after 30 minutes through cervical dislocation and were dissected them (ethically approved) (Akanji & Yakubu, 2000). After the dissection small intestines were removed. The distance travelled by the charcoal meal through small intestine was considered as the percentage of the total length of small intestine according to the following formula.

% Intestinal transportation= Distance travelled by charcoal meal (cm) × 100 Total length of small intestine (cm)

Statistical analysis

Statistical analysis of data was done through one way ANOVA trough SPSS (22) computer software, Means + SEM was calculated through Microsoft excel, Dunnet test was used for comparison between control and tested treatments. The probability P<0.05 was considered as significant.

OBJECTIVES OF THE STUDY

Following are the objectives of the study on the researched plants

• To study the ecological characteristics of the habitats and ecological status of these plants. • To study ethnobotanical uses of the proposed plants and their economic status. • To study the morphological features of the plants and detailed organoleptic evaluation of the experimented parts of these plants. • To study the foliar characters of these plants such as stomatal type, size, number, index, trichomes types, vein islet number, vein termination number and palisade ratio which has special importance in identification, evaluation and detection of adulteration. • To carry out powder drug study of the parts used by the proposed plants in order to prepare monographs and set parameters for the purity and standardization of their powder drugs. • To carry out physicochemical studies of these plants for their fluorescence characteristics and to work out elemental and nutritional compositions of these plants. • To detect various secondary metabolites in leaves, fruits and stem barks of these plants, including protein, carbohydrates, fats, alkaloids, anthraquinone derivatives and tannins etc. • To study the pharmacology of these plants, including Acute toxicity ٭ Cytotoxic activity ٭ Phytotoxic activity ٭ Antibacterial activity ٭ Anti-oxidant activity ٭ Analgesic activity ٭ Anti-inflammatory activity ٭ Antispasmodic activity ٭

CHAPTER-4

RESULTS AND DISCUSSION

4.1) Systematic position of the plants

4.1. i) Ehretia serrata Roxb.

Kingdom: Plantae (Plants)

Subkingdom: Tracheobionta (Vascular plants)

Super division: Spermatophyta (Seed Plants)

Division: Magnoliophyta (Flowering Plants)

Class: Magnoliopsida (Dicotyledons)

Order: Boraginales

Family: Boraginaceae

Subfamily: Ehretioideae

Genus: Ehretia

Specie: Ehretia serrata Roxb.

Common names

English name: Koda, brown-cedar, kodo wood

Local name in Urdu and Punjabi: Punna

4.1.ii) Ehretia obtusifolia A.DC.

Kingdom: Plantae (Plants)

Subkingdom: Tracheobionta (Vascular plants)

Super division: Spermatophyta (Seed Plants)

Division: Magnoliophyta (Flowering Plants)

Class: Magnoliopsida (Dicotyledons)

Order: Boraginales

Family: Boraginaceae

Subfamily: Ehretioideae

Genus: Ehretia

Specie: Ehretia obtusifolia Hochst.ex.A. DC.

Common names

English name: Stamper wood

Local name in Pushtu: Ghada boty

4.2) Morphological description

4.2.i) Morphological description of E. serrata Roxb.

Morphological study was carried out at the time of collection, which showed that the plant is a perennial small sized deciduous tree less than 10 m, locally named as puran and punna in Urdu. The plant is attractive for its delicious fruit and attractive look. It is a tough, hard and fast growing tree, having dense sprays of delicately scented, white flowers in spring. Leaves are elliptic or oval to obovate or oblong-ovate, leaf size is 5- 13 × 4-6 cm, Petiole up to 2.5 cm long. Leaf margin is regularly serrate with teeth hooked upward. The stem bark is a milky, grey, with vertical tissues. Tiny fragrant white flowers occur in panicles which is 8-15 cm long. Flowers are small white bell shaped flowers present in the form of bunches, with 5 spreading pointed petals. Calyx ± cupular and 1.5 mm long; lobes are 5, obtuse and ciliate. Corolla is rotate. Limb is 5- lobed, ± 2.2 mm long, oblong, minutely crenulate. Filaments are shorter than lobes, glabrous; and c. 1.2 mm long, basifixed, oblong. Style is 1.5 mm long, 2-fid. Ovary is subglobose to ± ovoid, and is 1.1 mm long, glabrous. In autumn, these flowers are followed by edible, sweet tasting, and cluster of yellow-orange berries, which are loved and eaten by birds. Drupe is globose, 4-43 mm broad and black when ripe. The tree is remarkable in fruit production as the whole canopy becomes covered with the brightly colored berries. Trees may become deciduous in dry times. The timber of the tree is used for furniture and woodwork. It is a great shade and a bird attracting tree for parks and home gardens. The drupe with striking yellow-orange color after ripening from January to April. The fruit of this plant has diameter of 4 to 5 mm, with four seeds. The fruit is used in pickles due to its sweet and sour taste. Many rainforest birds enjoy the taste of this delicious fruit such as Lewin's Honeyeater, Rose-crowned Fruit-dove, Brown Cuckoo Dove, Wompoo Fruit Dove and Australasian Figbird (Zara et al., 2012).

4.2.ii) Morphological description of E. obtusifolia

Deciduous small shrub or small tree up to 6 m tall; straggling or with several branches from the base; color of bark is gray. Leaves are alternate and simple; petiolate elliptical, oblong-elliptic or obovate, pubescent-velvety; apex of the leaves are obtuse to acuminate. Inflorescence is corymbose cyme, terminal or on short lateral shoots, Flowers are bisexual, regular, pentamerous, with a pleasant smell, pedicelate, calyx

lobes and ovate, elliptical or narrowly triangular, joined only at the base, corolla is of different colors such as pink, blue, pale lilac or mauve, tubelike, lobes narrowly triangular to oblong, acute or obtuse. Stamens are unequal and 5 in number, ovary is superior, style is 3–6 mm long, deeply forked. Fruit is a globose drupe 5–6 mm in diameter, orange or red, splitting into two 2-seeded pyrenes.

4.3) Pharmacognosy

A pharmacognostic study revealed standardization, quality and quantity assessment of crude drugs. Evaluation of fruit, leaf and stem bark extract was carried out for the pharmacognostic study i-e macroscopic and microscopic study, powder drug study, phytochemical analysis, florescence study, nutritional and elemental analysis.

4.3.1) Macroscopy and organoleptic characteristics

Macroscopic and organoleptic (sensory) assessments play important role in standardization, characterization and identification of crude drugs and the only parameters which do not require any instrument and expenses. Macroscopic features include size, color, shape, texture, odor and taste (Rakholiya & Chanda, 2012). These parameters (morphological, microscopical and physical) are considered the easiest and important information for characterization and recognition of crude drugs (Zongo et al., 2013); (Naghibi et al., 2005).

4.3.1.i) Organoleptic and macroscopical characters of E. serrata leaf

The macroscopical and organoleptic evaluation of leaf of Ehretia serrata was carried out and listed in (Table 4.1). Leaf of E. serrata is simple with ovate, oblong shape, dark green upper side and light green lower side (Fig. 4.1). Leaf is 5-16 cm long, and 2.8- 6.5 cm and 2.8-6.5 cm wide with short petiole. Leaf margins are serrulated with acute apex. Surface of the leaf was glabrous with reticulate venation. Leaf base was symmetrical and phyllotaxy is spiral. Leaf has a strong aromatic odor and taste. Texture of the leaf was soft (Table 4.1).

4.3.1.ii) Organoleptic and macroscopical charaters of E. serrata Fruit

Different macroscopical features of E. serrata fruit were also enlisted (Table 4.3). Investigation revealed that E. serrata bears drupe globose type fruit with Yellowish green-light orange colour when unripe which turn black when ripe (Fig. 4.2). Taste and odor of fruit were sweet with pleasant aroma. Diameter of fruit was 4-10 mm with ovoid shape and soft texture. Normally dehiscence is divided into two pyrenes. Two seeds were present in fruit. Insertion of fruit was ramal.

4.3.1.iii) Organoleptic and macroscopical characters of E. serrata stem bark

Stem bark of E. Sheraton was also microscopically investigated and their different characteristics are enlisted in Table 4.2. Stem bark of E. serrata was irregular with 1- 3mm thickness. Outer color of stem bark was dark brown, with ridge and rough texture. Inner surface was whitish golden in color. Odor and taste of stem bark were pleasant and sweet. The fracture was uneven (Fig. 4.3).

4.3.1.iv) Organoleptic and macroscopical characters of E. obtusifolia leaf

Leaf of E. obtusifolia was also examined for organoleptic and macroscopical features and observed characters are enlisted in (Table 4.4). Leaf of E. obtusifolia was oblong- elliptic or obviate with obtuse apex and entire margins (Fig. 4.4). Color of the leaf was dark green on the upper surface and light green on the lower surface, having 2.5-8 cm length and 1.7-4.2 cm width, with a pleasant odor (Lawsonia inermis like smell). Taste of the leaf was slightly sweet. Shape of leaf was oblong-elliptic or obviate with obtuse apex. Leaf margins were entire, undulated with symmetrical leaf base. Leaf surface was pubescent to velvety with reticulated venation and entire margins. Petiole was present and insertion was ramal with spiral phyllotaxy.

4.3.1.v) Organoleptic and macroscopical characters of E. obtusifolia fruit

Different features of Fruit of E. obtusifolia were also macroscopically examined and enlisted (Table 4.6). Fruit type was drupe, globose, with 5-6 mm diameter. Ripened fruit split into two seeded pyrene (Fig. 4.5). Color was orange to red. Fruit of E. obtusifolia contained pleasant odor and sweet taste. The texture was soft.

4.3.1.vi) Organoleptic and macroscopical characters of E. obtusifolia stem bark

Stem bark of E. obtusifolia was also examined macroscopically; the features are enlisted in (Table 4.5). Outer surface of bark was ridged, roughly textured and dark brown in color. Inner surface was whitish golden in color. Thickness was 1-3mm with an uneven fracture. Odor and taste of stem bark were pleasant and sweet (Fig. 4.6).

Fig. 4.1. Leaf of Ehretia serrata Fig. 4.4. Leaf of Ehretia obtusifolia

Fig. 4.2. Fruit of Ehretia serrata Fig. 4.5. Fruit of Ehretia obtusifolia

Fig. 4.3. Stem bark of Ehretia serrata Fig. 4.6. Stem bark of Ehretia obtusifolia

Table 4.1. Organoleptic and macroscopical characteristics of E. serrata leaf

S.No Characteristics Observations 01 Size 5-16 cm in length and 2.8-6.5 cm in width 02 Colour Upper surface brighter green, Lower surface light green 03 Odor Pleasant 04 Taste Lightly sweet 05 Shape Cuneate, oblong 06 Phyllotaxis Spiral 07 Insertion Ramel. Inserted on branches 08 Leaf base Symmetrical 09 Leaf edge Serrulate 10 Petiole Petiole present 11 Leaf shape Elliptic-oblong 12 Incision Toothed margins 13 Venation Reticulate 14 Apex Acute 15 Surface Glabrous 16 Texture Soft

Table 4.2. Organoleptic and macroscopical characteristics of E. serrata stem bark

S.No Characteristics Observations 01 Shape Irregular 02 Outer surface Rough texture, yellowish green in colour and ridged 03 Inner surface Yellowish golden-pale green in colour 04 Odor Pleasant 05 Taste Slight bitter 06 Fracture Uneven 07 Thickness 2-5 mm

Table 4.3. Organoleptic and macroscopical characteristics of E. serrata fruit

S.No Characteristics Observations 01 Kind Drupe globose 02 Dimensions 4-10 mm 03 Shape Globose 04 Insertion Ramal 05 Dehiscence Bivalvate 06 Color Yellowish green-lght orange, black when ripe 07 Taste Sweet 08 Odour Pleasant 09 Texture Soft 10 Marking Smooth 12 Seeds Two

Table 4.4 Organoleptic and macroscopical characteristics of E. obtusifolia leaf

S.No Characteristics Observations 01 Size 2.5-8 cm in length and 1.7-4.2 cm in width 02 Colour Dark green-coloured above and light green beneath 03 Odour Pleasant, Lawsonia inermis like smell 04 Taste faint sweet Shape oblong-elliptical or obovate 05 Phyllotaxis Spiral, alternate 06 Insertion Ramel, Inserted on branches 07 Leaf base Symmetrical Leaf margins Entire 08 Petiole Petiole present 09 Leaf shape Obovate 10 Incision Entire to undulate 11 Venation Reticulate 12 Apex obtuse to sometimes retuse or subacute 13 Surface pubescent to velvety 14 Texture Soft

Table 4.5. Organoleptic and macroscopical characteristics of E. obtusifolia stem bark

S.No Characteristics Observation 01 Shape Irregular 02 Outer surface Dark brown in colour, rough texture, ridged 03 Inner surface Whitish golden in color. 04 Odor Pleasant 05 Taste Sweet 06 Fracture Uneven 07 Thickness 1-3 mm

Table 4.6. Organoleptic and macroscopical characteristics of E. obtusifolia fruit

S.No Characteristics Observations 01 Kind Drupe 02 Dimensions 5-6 mm 03 Shape Globose 04 Insertion Ramal 05 Dehiscence Bivalvate, splitting inti two seeded pyrenes 06 Color Orange or red 07 Taste Sweet 08 Odour Pleasant 09 Texture Soft 10 Marking Smooth 12 Seeds Two seeded pyrenes

4.3.2 ) Microscopic and anatomical study

In the present work leaf anatomy, midrib anatomy, leaf surface values, stomatal study, bark anatomy and fruit anatomy and microscopy of crude powder drug of E.serrata and E. obtusifolia were carried out.

4.3.2.i) Transverse section of E. serrata leaf

Transverse section of leaf E. serrata showed a bifacial arrangement (Fig 4.7; 4.8). Midrib region is clearly distinguished into collenchymatous tissues with vascular bundles between upper and lower epidermis. Upper epidermal cells of were rectangular in shape and compactly arranged. Upper epidermis contains few stomata and covered with thin cuticle and trichomes. Lower epidermis is consisting of stomata and epidermal cells. Epidermal cells are elongated or irregular in shape. Three to five layered collenchymatous cells are followed by both upper and lower epidermises at midrib region. Cortical cells were isodiametric and large thin walled (Fig.4.7). Mesophyll region is clearly divided into two regions i.e palisade and spongy mesophyll cells. The palisade cells were single layered, composed of an elongated rod like compactly arranged cells with a length of 11.11-15.00 µm with a mean value of 13.55±1.54 µm and width of 2.01-3.89 µm with an average of 2.83±0.55 µm. Sizes of spongy mesophyll cells were variable; shapes were rounded, cylindrical, or oval in both species. Lengths of spongy mesophyll cells were from 10.50-12.99 µm with a mean value of 11.77±0.815 µm in E. serrata. Width of spongy cells in E. serrata was in the range from 1.85 to 3.86 µm with average mean 2.57±0.833 µm (Fig. 4.8). In midrib region collenchymatous region is followed by one layer pericycle with spherical shape cells. Vascular bundles are of collateral type and have phloem fibers, xylem vessels, and xylem and phloem parenchymatous cells (Fig. 4.7).

Upper epidermal cellsCollenchymatous cellsPhloem Xylemtissue tissue Lower epidermal cells

Pericycle Non-glandular hair

Fig. 4.7. Midrib transverse section of E. serrata Roxb. leaf Upper epidermal cells Palisade cells

Spongy mesophyll cells Lower epidermal cells

Fig. 4.8. Laminar portion of E. serrata Roxb. leaf

4.3.2.ii ) Leaf surface parameters

Qualitative and quantitative values of leaf are important in classification of plants. Stomata are responsible for gaseous exchange in plants (Zhau et al., 2010). Morphologists, physiologists and taxonomist use somatogenesis as a tool for intragenic systematics and for intraspecific diffentiation (Tripathi & Mondal, 2012). Important surface values of leaf are stomatal number, stomatal index, stomatal types, vein islet number, vein termination number and palisade ratio, these characters are used to differentiate closely related species of the same family (Mbagwu & Edeoga, 2009).

In the present work foliar anatomy of E. serrata leaf was carried out including stomatal studies, vein islet number, vein termination number and palisade ratio.

4.3.2.ii.a ) Stomatal and trichomal study of E. serrata leaf

Based on stomatal distribution researcher describes three kinds of leaf types such as amphistomatic (stomata on both surfaces), epistomatic (stomata on upper surface only) and hypostomatic (stomata on lower surface only). Stoma is formed by two guard cells and guard cells are surrounded by specialised epidermal cells called subsidiary cells. Stomatal study of E. serrata leaf showed their amphistomatic leaf (stomata on both surfaces) natures but stomata on lower epidermis was abundant than upper epidermis. i) Upper epidermis

Epidermis proper consists of epidermal cells, subsidiary cells, stomatal complex, guard cells and trichomes. Epidermal cells were thick walled, polygonal, amoeboid shaped with lobed margins. Simple 1-2 celled, entire non-glandular tapering trichomes with sharp pointed tips were observed on lower epidermis (Fig. 4.9). Stomata on upper epidermis were of anomocytic types. Average lengths of guard cells was 30.9 μm, average width of stomatal guard cells was 17.4 μm, average pore length of stomatal pore was 25.31 μm and average width of stomatal pore was 4.91 μm. Stomatal index on upper epidermis was 30.4 . ii) Lower Epidermis

Lower epidermis proper contained epidermal cells, Subsidiary cells, guard cells, stomatal complex, and trichomes. Epidermal cells are thick walled with deeply wavy margins. Simple 1-2 celled, non-glandular tapering trichomes with sharp pointed tips were observed on lower epidermis (Fig. 4.10). Stomata on lower epidermis were of both anomocytic and anisocytic types (Fig. 4.11). Average length of stomata guard cell was 25.22 μm, average width of stomatal guard cells was 13.52 μm, average pore length of stomatal pore was 23.17 μ and average width of stomatal pore was 3.00 μm. Stomatal index of lower epidermis of E. serrata was 36.7µ.

Fig. 4.9. Anomocytic stomata on upper Fig. 4.10. Unicellular non-glandular epidermis trichome on lower epidermis

Anisocytic stomata

Anomocytic stomata

Epidermal cells

Fig. 4.11.Anomocytic and anisocytic types of stomata on lower surface of E. serrata leaf

4.3.2.ii.b) Vein islet and vein termination number

Vein islet number is defined as mean of veinlet enclosing in 1 mm2 of leaf surface and Veinlet termination number is defined as mean of terminated veinlets in 1 mm2 of leaf surface, taken from region of midrib to margin of leaf (Wallis, 1985; Evans, 2002; Omoregie et al., 2015).Vein islet and vein termination numbers of leaf are given in Figure (4.12 & 4.13) The range, mean and standard deviation was 15 – 23, 19.4+1.79 and 8 – 14, 10.6+1.98 respectively.

4.3.2.ii.c ) Palisade ratio

Palisade ratio is the average number of palisade cells below each upper epidermal cell. It is an important parameter for determination and characterization of leafy drugs (Evans, 2009; Barkatullah et al. 2015). For the determination of palisade ratio a piece of leaf was cleared by boiling in chloral hydrate solution. Numerical data was documented in the form of range, average ratio and standard deviation after observing it under a microscope (Table. 4.7). The range of palisade ratio was 5-9; average ratio was 6.4 and standard deviation was 0.894 respectively.

Table 4.7. Leaf surface features of E. serrata S.No Features Range Average 01 Stomatal number 15-25 18.4+1.14 02 Stomatal index 20-38 36.0+6.09 03 Vein islet 15-23 19.4+1.79 04 Vein termination number 8-14 10.6+1.987 05 Palisade ratio 5-9 6.4+0.894

Fig. 4.12. Vein islets in the surface of Fig. 4.13. Vein termination number E .serrata leaf in the surface of E. serrata leaf

4.3.2.iii) Transverse section of E. obtusifolia leaf

Microscopic study is important for declaring or investigating the internal histological identification of plant (Nancy & Dengler, 2002). In the present study, the leaf anatomy, leaf surface values, stomatal study E. obtusifolia was performed.

In transverse section of bifacial leaf of E. obtusifolia, upper and lower epidermis plus mesophyll cells in laminar region and in the midrib region the vascular bundles between upper and lower epidermis were present. In the upper epidermis small number of stomata was present and covered with cuticle. In the midrib region upper epidermis was followed by 4-5 layers of oval to round shaped cells collenchymatous cells (Fig. 4.14). In midrib region collenchyma was followed by one layer of pericycle with round shape cells (Fig. 4.14). Vascular bundle was present in the midrib region. Xylem located towards the centre, while phloem was found towards the outer side where as phloem parenchyma was intermixed with xylem vessels. Components of vascular bundles i.e. xyllary fibers, xylem vessels, and xylem and phloem parenchymatous cells were observed (Fig. 4.14). Mesophyll region is clearly divided into two regions i.e. palisade and spongy mesophyll cells (Fig.4.15). Palisade cells were rod like, but slightly shorter than that of the E. serrata and were compactly arranged. Average length of palisade cells was in the range of 11-14 µm with mean value and standard deviation of 12.2 ± 1.2 µm and width of 1.98-2.50 µm with an average mean and standard deviation was of 2.11 ± 0.45 µm (Fig. 4. 15). Spongy mesophyll cells were variable in shape in both the species. Average lengths of spongy mesophyll cells were from 8-11.50 µm with mean value and standard deviation of 9.4 ± 1.14 µm in E. obtusifolia. Width of spongy cells was in the range from 2.01 to 3.50 µm with average mean 2.80 ± 0.822 µm (Fig. 4.15).

Xylem tissue

Phloem tissue Pericycle Collenchymatous cells Upper epidermis

Fig. 4.14. Transverse section of midrib of E. obtusifolia leaf

Upper epidermis Palisade cells Vein Spongy cells Lower epidermis

Fig. 4.15. Transverse section of laminar portion of E. obtusifolia leaf

4.3.2.iv) Leaf surface parameters of E. obtusifolia

Leaf epidermal studies are of enormous importance in finding phylogeny and taxonomy of closely related species, taxonomists and systematics give immense significance to leave epidermal features (Taia, 2005).

Some important characters of leaf epidermis such as stomatal studies, vein islet number, vein termination number and palisade ratio of E. obtusifolia were carried out.

4.3.2.iv.a) Stomatal and trichomal study

Based on stomatal distribution researcher describe three kinds of leaf types such as an amphistomatic leaf (stomata on both surfaces) or on one surface only, either on the upper (epistomatic leaf) or more commonly on lower i.e., hypostomatic leaf. Stoma is guarded by guard cells and guard cells are surrounded by specialised epidermal cells called subsidiary cells.

i) Upper epidermis

Leaves of E. obtusifolia were hypostomatic with no stomata found on upper epidermis. Three types of trichomes were found in upper epidermis a non-glandular, unicellular trichome with thin and smooth cross and lateral walls (Fig. 4.18). The second type of trichome was three celled, non-glandular conical trichome. Third type was vesicular capitate glandular trichomes with simple foot and uni-serriate body which was differentiated into stalk and head (Fig. 4.16). The stalk was 1-2 celled and cylindrical. The lower cell of the stalk was elongated than upper ones with thin and smooth cross walls while lateral walls were thick and smooth. The head was rounded. ii) Lower epidermis

Various types of stomata were found in lower epidermis. Lower epidermis contained subsidiary cells, epidermal cells and trichomes. Both upper and lower epidermal cells were thin walled, irregular with lobed margins. Similar to upper epidermis the same three types of trichomes were found in lower epidermis. Stomata were guarded by a pair of bean shaped guard cell with thick wall toward stoma and thin elastic wall outside. Guard cells contained green granulated chloroplasts (Fig. 4.20). Average lengths of stomatal guard cell were 20.2 μm, average width of stomatal guard cells was 8.89 μm, average pore length of stomatal pore was 18.0 μm and average width of stomatal pore was 2.85 μm.

Fig 4.16. Capitate bi-cellular Fig. 4.17. Upper epidermal cells glandular trichomes

Fig. 4.18. uni-cellular conical Fig. 4.19. 3-celled non trichome on upper epidermis glandular conical trichome

Lower epidermal cells

Stomata on lower epidermis

Fig. 4.20. Lower leaf epidermal surface of E. obtusifolia

Anomocytic stomata

Anisocytic stomata

Hemi paracytic stomata

Fig. 4.21. Anomocytic, anisocytic and hemiparacytic types of stomata on lower epidermis of E. obtusifolia leaf

4.3.2.iv.b) Vein islet and vein termination number

Vein islet (Fig.4.22) and vein termination number (Fig.4.23) was observed under microscope and range, mean and standard deviation was calculated e.g 13 – 22, 18.6 ± 1.14 and 7 – 13, 8.2 ± 0.838 presented in Table.4.8. Some other researchers such as Barkatullah et al. (2015) considered vein islet, vein termination number and palisade ratio as an important parameter for drugs determination.

4.3.2.iv.c) Palisade ratio

Evans (2002) defined palisade ratio as the average number of palisade cells beneath each epidermal cell. Pieces of leaf about 2 mm square were cleared by boiling with chloral hydrate solution. The pieces were observed under microscope. A group of four upper epidermal cells was first focused and then a slight rotation of fine adjustment of the microscope; the underlying palisade cells were focused and counted (Evans, 2002). The range was 5-8; average ratio was 6.2 and standard deviation 0.81 respectively given in Table.4.8.

Table No 4.8. Leaf surface features of E. obtusifolia

S.No Features Range Average 01 Stomatal number 14-25 21.8+1.78 02 Stomatal index 30-38 35.7+6.12 03 Vein islet 13.22 18.6+1.14 04 Vein termination number 7-13 8.2+0.828 05 Palisade ratio 5-8 6.2+0.81

Fig. 4.22.Vein islets on leaf surface of E. Fig. 4.23. Vein termination numbers obtusifolia on leaf surface of E. obtusifolia

4.3.2.v) Anatomy of E. serrata stem bark

Transverse section of E. serrata bark (Fig. 4.24) showed different tissues enlisted as follows

1) Cork consists of a few layers of packed lignified rectangular shaped cork cells (phellem) 2) Single layer of small, elongated or oval shaped cork cambium (phellogen) 3) Secondary cortex (Phelloderm), consisting of many layer parenchymatous cells contain calcium oxalate crystals 4) Secondary phloem 5) Medullary rays

4.3.2.vi) Anatomy of E. obtusifolia stem bark

Bark anatomy of E. obtusifolia (Fig. 4.25) showed the following enlisted tissues

1) Outer layer is cork (phellem) consisting of a few layers of packed lignified rectangular shaped cork 2) Single layer cork cambium (phellogen) consisting of small, elongated oval cork cambium cells 3) Many layered secondary cortex (phelloderm), consisting of parenchymatous cells 4) Secondary phloem, consisting of phloem parenchyma, sieve tubes and phloem fibers 5) Calcium oxalate crystals in phloem parenchyma 6) 2-5 celled wide medullary rays .

Lenticel

Cork (phellem) Cork cambium (Phellogen) Secondary cortex (Phelloderm) Primary Phloem Phloem parenchyma

Secondary phloem

Medullary ray Fig. 4.24. T.S of stem bark of E. serrata

Primary Phloem Phloem parenchyma

Secondary Phloem Cortex Cork cambium (Phellogen) Cork (Phellem)

Medullary rays

Fig. 4.25. T.S of stem bark of E. obtusifolia

4.3.2.vii) Anatomy of E. serrata fruit

E. serrata has a small globose drupe type of fruit. The fruit was almost 4.4 mm broad. Young fruit had green colour, which gradually changed to yellow and become brown after ripening. Cross section of fruit showed two portion i.e. fruit wall (pericarp) and seed. The fruit wall is clearly distinguished into three layers such as epicarp, mesocarp and endocarp. The outermost layer, epicarp is composed of closely arranged thick walled rectangular cells. The middle layer, mesocarp was composed of irregular thin walled fleshy cells. The inner layer of the fruit wall (endocarp) was composed of comparatively larger, thick walled rectangular stone cells. The fruit is dehiscent and has two seeded pyrenes. The seed was non-endospermic and dicotylednous (Fig. 4.27).

4.3.2.viii) Anatomy of E. obtusifolia fruit

E. obtusifolia has globose drupe fruit like that of Ehretia serrata. Fruit of E.obtusifolia was slightly larger than E.serrata (5.5 mm). The fruit on ripening is orange-red. Cross section of fruit showed two portion i.e. fruit wall and seed. The fruit wall is clearly distinguished into three layers; epicarp, mesocarp and endocarp. The outermost layer (epicarp) is made up of slightly thick walled oval shaped cells. The middle layer was thin and made up of medium thin walled fleshy cells. This layer contained large schizogenous and lysoschizogenous oil cavities. The endocarp was composed of slightly cuboid to rectangular stony cells. Fruit contained two seeded pyrenes. Each seed was round to slightly oval in shape and non-endospermic (Fig. 4.26).

Testa Testa Epicarp Mesocarp Epicarp pOil cavity Mesocarp Endocarp Seed Endocarp Cotyledon Seed Cotyledon

Fig. 4.26: Fruit of Ehretia obtusifolia Fig. 4.27: Fruit of Ehretia serrata in cross section in cross section

4.3.3) Powder drug study

Powder microscopy of E. serrata and E. obtusifolia leaf and stem bark were carried out. Presence of various structures in the powder provides a basis for standardization and authentication of crude drug powders. Powder microscopy helps in the identification of the herbal drugs and also in the detection of adulteration in crude drugs (Soni et al., 2011).

4.3.3.i) Powder drug study of Ehretia serrata Leaf

Leaf powder of E. serrata was light green in color with henna like odor and astringent taste. Following components were seen under microscope (Fig. 4.28). i. Wavy walled epidermal cells shaped cells, about 10-15 µm in size ii. Epidermal cell with anomocytic elliptical stomata, of 15-20 µm in length iii. Fragments of epidermis with attached palisade parenchyma cells iv. Vessels cells with attached parenchymatous cells v. Collenchymatous cells with calcium oxalate crystals vi. Isolated starch grain vii. Isolated prismic, rectangular crystals of calcium oxalate. 1.5 µ to 4 µ in diameter viii. Fibers ix. Elongated, tapering single celled trichomes x. Xylem tracheids with spiral walls

4.3.3.ii) Powder drug study of E. serrata stem bark

Stem bark powder of E. serrata was light yellowish golden in colour with fine odour and sweet taste. Common fragments detected in powder drug are enlisted (Fig. 4.29) below i. Cortical thin walled cells with calcium oxalate crystals having 25-40 µm length and 2-4 µm in width ii. Thick walled closely packed cork cells ranging from 30µ to 50 µ in length and 2.5-6 µm in width iii. Elongated fibers up to 170 µm iv. Phloem parenchyma cells v. Sclerieds

vi. Crystals of calcium oxalate with 1.5 - 3 µm in diameter vii. Starch grains, 4 -14 µm in diameter

4.3.3.iii) Powder drug study of E. obtusifolia leaf

Powder of leaf of E. obtusifolia was dark green in color with a pleasant odor and agreeable taste. Many fragments found in leaf powder are enlisted below (Fig. 4.30). i. Thin walled, irregular epidermal cells with 17 µm to 20 µm length and 2-3 µm width ii. Epidermal cells with thick walled stomata having 20-25 µm length and 4-6 µm width iii. Spongy mesophyll cells iv. Palisade cells with attached epidermal cells v. Parenchyma cells vi. Prismed aggregated Calcium oxalate crystals, 2µ to 4µ in diameter vii. Small rounded starch grain viii. Collenchyma’s cells ix. Elongated fibers from vein x. Annular vessels xi. Non-glandular unicellular trichomes xii. Glandular trichomes xiii. Starch grains

4.3.3.iv) Powder drug study of E. obtusifolia stem bark

Bark Powder of E. obtusifolia was a light golden brown in color with a pleasant odor and sweet taste (Fig. 4.31). Following fragments were detected in the bark powder. i. Thick walled cork cells. ii. Thick walled cortical pharenchyma cells iii. Medullary rays fragments iv. Elongated Sclerids v. Pitted Sieve elements vi. Parenchyma with thick walled cells

vii. Sclerieds viii. Elongated fibers with 80 to 95 µm in length ix. Calcium oxalate crystals of various types x. Starch grains

(I) Epidermal cells (II) Epidermal with stomata (III) Epidermal with palisade cells

(IV)Vessel cells with (V) Collanchymatous (VI) Starch grains (VII) Calcium paranchymatous cells cells oxalate crystal

(VIII) Fibers (IX) Simple non-glandular trichome (X) Xylem tracheids

Fig. 4.28. Powder drug study of E. serrata leaf

(I) Cork cells (II) Cortical cells (III) Elongated fibers (IV) Phloem parenchyma

(V) Sclerieds (VI) Calcuim oxalate crystal (VII) Starch grains

Fig. 4.29. Powder drug study of E. serrata stem bark

(I)Epidermal cells (II) Epidermal Cells (III) Spongy mesophyll (IV) Palisade cells with stomata cells with epidermal cells

(V) parenchyma cells (VI) Calcium oxalate(VII) Starch grains (VIII) Collenchyma cells

(IX) Fibers (X) Annular vessels (XI) Non-glandular (XII) Glandular trichome trichome

(XIII) Spiral vessles with parenchyma cells

Fig. 4.30. Powder drug study of E. obtusifolia leaf

(I) Cork cells (II) Cortical cells (III) Medullary rays fragments (IV) Sclerieds

(V) Pitted vessels (VI) Parenchyma cells (VII) Fibers (VIII) Calcium oxalate crystal

(IX) Starch grains

Fig. 4.31. Powder drug study of E. obtusifolia stem bark

4.3.4) Phytochemical screening

The curative potency of medicinal plants, mainly dependent on the presence of secondary bioactive compounds, e.g. alkaloids, anthraquinones, terpenoids and phenolic compounds. In the present work, phytochemical screening was carried out for finding the nature of secondary metabolites in these plants, which have pharmaceutical potentials for pharmaceuticals exploration and public health benefits (Rai et al., 2013)

4.3.4.i) Phytochemical screening of E. serrata leaf

In present work phytochemical screening of E. serrara leaf was carried out to find out the phytoconstituents in its methanolic extract. Phenolic compounds, protein, reducing sugar, steroids, terpenoids, fats, glycosides, amino acids, saponins, alkaloids, tannins, phlobatannin and starch grains were detected in the leaf of E. serrata (table 4.9). Alkaloids and saponins are effective against many diseases and gives protection from pathogens (Fluck, 1973; Sodipo et al., 1991). Due to the presence of alkaloids and saponins the plant can be used to cure various diseases. Similarly tannins play a major curative role in treatments of some human disorders (Asquith & Butler, 1986). Aldehydes and anthraquinone were not detected in the leaves.

4.3.4.ii) Phytochemical screening of E. serrata fruit

Several active constituents of pharmacological importance may be available in a single plant (Rai et al., 2013). In the present investigation the fruit of E. serrata was found to contain phenol compounds, protein, reducing sugar, steroids, terpenoids, fats, glycosides, amino acids, saponins, alkaloids, phlobatannin and starch. Aldehydes, anthraquinone and tannins were not detected in the fruit (Table 4.9)

4.3.4.iii) Phytochemical screening of E. serrata stem bark

Similar to the leaves and fruit, stem bark also contained phenol compounds, protein, reducing sugar, steroids, terpenoids, glycosides, amino acids, saponins, alkaloids, tannins and starch. Fats, aldehydes, anthraquinons and phlobatannins were not detected (Table 4.9).

4.3.4.iv) Phytochemical screening of E. obtusifolia leaf

Leaf of E. obtusifolia was investigated for detection of various bioactive compounds. The leaf contained phenol compounds, protein, reducing sugar, steroids, terpenoids, fats, glycosides, amino acids, saponins, alkaloids, tannins, starch and phlobatannins. Aldehydes and anthraquinones were not detected in the leaf (Table 4.10).

4.3.4.v) Phytochemical screening of E. obtusifolia fruit

Fruit of E. obtusifolia was also investigated for phytochemical screening and found to contain important secondary bioactive constituents such as phenols, proteins, reducing sugar, steroids, terpenoids, fats, glycosides, amino acids, starch, saponin and tannins. Aldehydes, anthraquinones, alkaloids and phlobatannin were not detected during the investigation (Table 4.10).

4.3.4.vi) Phytochemical screening of E. obtusifolia stem bark

In contrast to leaf, and fruit, the stem bark showed the presence of all screened compounds, i.e. phenol compounds, protein, reducing sugar, steroids, terpenoids, glycosides, amino acids, saponin, starch, alkaloids, tannins and phlobatannin. Most of the important active compounds such as fats, aldehydes and anthraquinones were absent (Table 4.10).

All these reports supported the importance of qualitative phytochemical screening of crude drugs, which help in the field of Photochemistry and pharmacology of medicinal plants.

4.3.4.vii) Comparison of phytochemicals of E. serrata and E. obtusifolia leaf, fruit and stem bark

4.3.4.vii.a) Leaf

Both species of the genus Ehretia had the same phyto-constituents which categorized them in the same rank of medicinal value.

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4.3.4.vii.b) Fruit

The comparison of phytochemicals in the species of Ehretia revealed that all the enlisted secondary bioactive compounds were same except of alkaloid and phlobatannin which were detected in the E. serrata but were absent in E. obtusifolia and similarly tannin which present in E. obtusifolia and absent in E. serrata.

4.3.4.vii.c) Stem bark

Both the species had similar phytochemicals but differences were seen in the color of reducing sugar, which were reddish brown in E. serrata and green orange in E. obtusifolia.

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Table No 4.9. List of Phyto-constituents found in different parts of E. serrata

S.No Name of functional group Leaf Fruit Stem bark 1 Phenolic compounds + + + 2 Starch + + + 3 Protein + + + 4 Reducing sugar Light green Red Reddish brown 5 Steroids + + + 6 Terpenoids + + + 7 Fix oil + + - 8 Aldehydes - - - 9 Anthraquinone - - - 10 Glycosides + + + 11 Amino acids + + + 12 Saponins + + + 13 Alkaloids + + + 14 Tannins + - + 15 Phlobatannin + + -

Table 4.10. List of Phyto-constituents found in different parts of E. obtusifolia

S.No Name of functional group Leaf Fruit Stem bark 1 Phenolic compounds + + + 2 Starch + + + 3 Protein + + + 4 Reducing sugar Greenish Red Greenish orange brown 5 Steroids + + + 6 Terpenoids + + + 7 Fats + + - 8 Aldehydes - - - 9 Anthraquinone - - - 10 Glycosides + + + 11 Amino acids + + + 12 Saponins + + + 13 Alkaloids + - + 14 Tannins + + + 15 Phlobatannin + - +

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4.3.5) Fluorescence study

Plants with the presence of different chemical constituents show different color exhibition fluorescence in UV lights and in visible light (Ansari, 2006; Jarald & Jarald, 2007; Reddy & Chaturvedi, 2010). Fluorescence analysis of powder dugs was carried as such and after treating the powder with different solvents for preliminary standardization by using UV light 256 nm, 310 nm, and visible light. Similar qualitative analysis of crude powder drugs was also reported Kadam et al., (2012); and Nwachukwu et al., (2010). This assay helps in the detection of adulteration in crude drugs.

4.3.5.i) Fluorescence study of E. serrata leaf, fruit and stem bark

The fluorescence analysis of leaf, fruit and stem bark was carried out by using UV 256 and UV 300. First the powder was observed in visible light and then they were treated with various reagents such as Iodine, Picric Acid, (FeCl3), NH3 solution, NaOH, HCl,

50% HNO3, acetic acid and H2SO4 and color of solution was noticed under visible and UV light (256 nm and 310 nm). Results are given in Table 4.11. The main purpose of the fluorescence study is to detect adulterants found in powder drugs. This study helps to standardize powder drugs and is useful for authentication of crude drugs.

4.3.5.ii) Fluorescence study of E. obtusifolia leaf, fruit and stem bark

The fluorescent analysis is considered an accurate method for the determination of pharmaceutical samples (Ozcan et al., 2011). The colours of powder drug of leaf, fruit and stem bark seen under visible and ultraviolet (256 nm and UV 310 nm) light after treating them with different reagents like, iodine, picric acid, acetic acid, FeCl3, NH3 solution, NaOH, HCl, 50% HNO3 and H2SO4. The fluorescent compounds were detected by observing different colours exhibited by plants' parts in different solutions and UV lights (Table 4.12).

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Table 4.11. Fluorescence study of E. serrata leaf, fruit and stem bark

S.No Reagents Visible light UV 256 UV 310 1 ESL Powder as such Grass green Dark green Greenish yellow 2 ESL powder +50 Golden brown Yellow Yellowish grey %HNO3 3 ESL powder+Picric acid Yellowish green Pale yellow Dark brown 4 ESL powder +NH3 Green Yellowish green Greenish grey 5 ESL powder+H2SO4 Grass green Yellowish green Dark brown 6 ESL powder+NaOH Purplish yellow Yellowish green Dark brown 7 ESL powder+HCl Dark brown Dark brown Blackish brown 8 ESLpowder+NaOH+ Pale brown Pale brown Greenish brown Ethanol 9 ESL powder+Iodine Pale green Pale green Grey 10 ESL powder+FeCl3 Dark brown Light brown Blackish brown 11 ESB Powder as such Light golden Brown Pinkish brown 12 ESB powder +50 Dark brown Dark brown Dark brown %HNO3 13 ESB powder+Picric acid Yellowish golden Yellow brown Yellow brown 14 ESB powder +NH3 Golden brown Yellowish brown Light brown 15 ESB powder+H2SO4 Yellowish golden Light yellow Dark yellow 16 ESB powder+NaOH Yellowish brown Light yellow Yellow 17 ESB powder+HCl Pinkish brown Dark brown Dark brown 18 ESB powder+NaOH+ Dark brown Maroons brown Brown Ethanol 19 ESB powder+Iodine Golden yellow Dark yellow Dark yellow 20 ESB powder+FeCl3 Dark maroons Dark maroon Black 21 ESF Powder as such Golden brown Dark brown Dark brown 22 ESF powder +50 Pinkish brown Dark brown Maroons %HNO3 23 ESF powder+Picric acid Yellowish golden Yellowish brown Yellowish brown 24 ESF powder +NH3 Pinkish golden Dark brown Light brown brown 25 ESF powder+H2SO4 Yellowish golden Light yellow Light yellow 26 ESF powder+NaOH Light brown Dark brown Dark brown 27 ESF powder+HCl Light pinkish brown Dark brown Reddish brown 28 ESF powder+NaOH+ Light Maroons Dark black Light brownish Ethanol maroons 29 ESF powder+Iodine Yellowish golden Yellowish brown Dark brown 30 ESF powder+FeCl3 Golden brown Dark maroon Dark maroon

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Table 4.12. Fluorescence study of Ehretia obtusifolia leaf, fruit and stem bark

S. No Reagents Visible light UV 256 UV 310 1 EOL powder as such Dark green Dark green Dark Greenish yellow 2 EOL powder +50 Dark brown Yellowish brown Grey %HNO3 3 EOL powder+Picric acid Yellowish dark green Yellowish green Dark blackish brown 4 EOL powder +NH3 Light green Yellowish green Dark green 5 EOL powder+H2SO4 Dark yellowish green Dark greenish Dark green yellow 6 EOL powder+NaOH Pinkish green Dark pale green Dark brown 7 EOL powder+HCl Blackish brown Blackish brown Blackish brown 8 EOLpowder+NaOH+ Yellowish brown Dark brown Greenish brown Ethanol 9 EOL powder+Iodine Yellowish green Greenish yellow Grey green 10 EOL powder+FeCl3 Dark brown Light pinkish Blackish brown brown 11 EOB Powder as such Light whitish golden Brown Purplish brown 12 EOB powder +50 Brown Brown Brown %HNO3 13 EOB powder+Picric acid Yellowish golden Yellow golden Yellow golden brown 14 EOB powder +NH3 Golden brown Yellowish brown Light brown 15 EOB powder+H2SO4 Yellowish light Light golden Dark golden golden 16 EOB powder+NaOH Yellowish pinkish Light pinkish Pinkish yellow brown yellow 17 EOB powder+HCl Pinkish golden Light brown Dark brown 18 EOB powder+NaOH+ Light whitish golden Dark brown Maroons brown Ethanol 19 EOB powder+Iodine Yellowish golden Yellow brown Dark yellow brown 20 EOB powder+FeCl3 Pinkish maroons Dark maroon Blackish brown 21 EOF Powder as such Reddish brown Maroons brown Dark pinkish brown 22 EOF powder +50 Pinkish dark brown Pinkish Dark Dark reddish %HNO3 brown Maroons 23 EOF powder+Picric acid Yellowish brown Dark Yellowish Dark Yellowish brown brown 24 EOF powder +NH3 Pinkish dark brown Blackish brown Dark reddish brown 25 EOF powder+H2SO4 Dark Yellowish Dark brown Dark brown brown 26 EOF powder+NaOH Dark maroon Dark brown Dark brown 27 EOF powder+HCl Light pinkish brown Dark brown Reddish brown 28 EOF powder+NaOH+ Dark reddish brown Purplish black Purplish black Ethanol

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29 EOF powder+Iodine Yellowish brown Yellowish brown Dark brown 30 EOF powder+FeCl3 Reddish brown Dark red Dark maroon

ESL= Ehretia serrata Leaf

ESSB= Ehretia serrata Stem bark

ESF= Ehretia serrata Fruit

EOL= Ehretia obtusifolia Leaf

EOSB= Ehretia obtusifolia Stem bark

EOF= Ehretia obtusifolia Fruit

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4.3.6) Elemental analysis

Herbal medicines are widely used all over the world and these medicines cure many diseases without any serious side effects. Natural products provide significant healings in several diseased ailments. Medicinal plants are those specific species of plants which have important substances and could be used for therapeutic purposes (Parab, 2016) The medicinal values of some plant species used in the homeopathic system may be due to the presence of Ca, Cr, Cu, Fe, Mg, K and Zn (Vartika et al., 2008). Mineral nutrients are usually present in plants in low concentrations and they fluctuate to a great extent, with respect to both space and time and also due to environmental factors such as weather, climate and physico-chemical properties, including soil type, soil pH and erosion (Chaves et al., 2013; Maathuis and Diatloff, 2013).In the present study elemental analysis of E. serrata and E. obtusifolia was carried out using standard procedures and the results are presented in Table 4.13 and 4.14.

4.3.6.i) Elemental analysis of leaf, stem bark and fruit of E. serrata i) Sodium (Na)

The detected concentration of sodium element was very less in all parts of E. serrata. Stem bark had the highest concentration of sodium (8.859+0.1967 ppm) followed by leaf with (7.123+0.102 ppm). The lowest amount of sodium was (6.721+0.0236 ppm) detected in fruit (Table 4.13). Sodium is essential for many physiological processes occurring in living organisms. Like potassium, its acts as an electrolyte in the blood. Sodium is necessary for hydration of the body. Sodium in circumscribed quantity is benign for body where as an inordinate amount of sodium consumption is highly poisonous for health. At the point when some vital processes are taking place, too much of sodium will cause the breakdown of cells (Gbolahan, 2001). Sodium is of great significance for many regulatory systems in the body. No international limit is available, but literature reveals that the minimum recommended intake of sodium must be 2.4 mg/day (Baysal, 2002).

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ii) Magnesium (Mg)

The concentration of magnesium was found to be the highest in the fruits of E. serrata (592.2 + 793.83ppm) followed by the leaf (280.0 + 137.17 ppm) and low in the stem bark (224.5 + 1.87 ppm) (Table 4.13). Magnesium plays an important role in enzymes regulating metabolic processes of proteins, lipids, and carbohydrate metabolism. Magnesium is a central part of the chlorophyll molecule in plants. In plasma and extracellular fluid it helps in maintaining osmotic equilibrium. Mg is required for many essential enzymes-catalysed reactions, especially where reactive species are the magnesium salt, e.g., Mg ATP. Lack of Mg causes muscles irritability and convulsions, as well as excess of Mg leads to disorders of central nervous system (Prasad, 1982). Intracellular deficiency of magnesium leads to impaired function of those enzymes, which utilize high energy phosphate bonds, for example, as in case of glucose metabolism (Stef et al., 2010). The Linus Pauling Institute fortifies the latest RDA for magnesium intake which is 400-420 mg/day for men and 310-320 mg/day for women. Following the Linus Pauling Institute recommendation to take a daily multivitamin/mineral supplement may ascertain an intake of at least 100 mg of magnesium / day.

ii) Nickle (Ni)

Nickel is a heavy metal and plays some role in enzymatic activities. Moderate amount of nickel was observed in all parts of E. serrata. In leaf (0.109 + 0.0479 ppm), in fruit 0.173 + 0.0297 ppm and in stem bark 0.166 + 0.012 ppm nickel calculated (Table 4.13). All detected values were below from the permissible limit (1.63 ppm) set by WHO in eatable plants. Nickel is considered to be a highly mobile element within plants. Ni is accumulated in leaves of plants (Diehl & Mc Grath, 1995). Nickel absorption in the human body is very low so it rarely causes toxicity in human (Onianwa et al., 2000). iii) Iron (Fe)

Iron plays an important role in the formation of blood in haemoglobin as Fe is the central unit of the haemoglobin molecule. In E. serrata, amount of iron was lower than the permissible levels. In fruits, the highest amount of iron accumulation was observed, which was 8.896 + 0.0232 ppm, followed by the leaf which was 7.692 + 0.0202 ppm.

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The lowest concentration of iron was found in stem bark, which was 3.017 + 0.147 ppm, (Table 4.13). The permissible level set by WHO for Iron in edible plants is 20 ppm (Saraf & Samant, 2013).

Iron plays essential role in the oxidation / reduction reaction of different organic compounds found in living organisms such as carbohydrates, protein and fat to control body weight. The largest amount of iron intake is hazardous to health (Critchley, 1986). Iron must be taken in proper amount as low intake cause various health hazards such as gastrointestinal infection, nose bleeding, and myocardial infarction. As iron is the central part of haemoglobin molecule it is also important for the distribution of oxygen to all parts of the body. Low amount of Fe in body leads to excessive menstrual flow and person become anaemic (Sigel, 1978; Reddy et al., 1987). Iron is also used in body tendon and ligament formation. Similarly, some chemical formations in our brain are controlled by the presence or absence of iron. It is also important for maintaining a healthy immune system. iv) Lead (Pb)

Lead is a heavy metal. There is no importance of lead biochemically or physiologically and it acts like toxic pollutant. All our results of lead accumulation in different parts of E. serrata were low from permissible limits set by WHO in different countries. Our results showed that the lead concentration in leaves was 0.005 + 0.0016 ppm, in fruits 0.009 + 0.002 ppm, in stem bark 0.29 + 0.0158 ppm (table 4.13). Lead accumulation causes many disturbances in human body, e.g. high blood pressure, kidney damage, subtle abortion, brain damage, infertility of men, sperm damage in men, children’s mental retardation and disruption of nervous systems (Khan et al., 2011). According to WHO (1992) in China, Malaysia and Thailand the permissible limit for Pb in medicinal plants is 10 ppm (Jabeen et al., 2010). The level of Pb obtained in the present study does not indicate a potential health hazard to users. v) Cadmium (Cd)

The amount of cadmium concentration in the fruit, leaves and stem bark of E. serrata analysed were found to be within the permissible limits. In leaves the lead concentraton was 0.014 + 0.002 ppm, in stem bark 0.006 + 0.002 ppm, and in fruits 0.010 + 0.001

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ppm (Table 4.13). The permissible level suggested by (WHO) for cadmium accumulation in edible plants is 0.21 ppm and for medicinal plants is 0.3 ppm (Jabeen, et al., 2010). This may be due to the low level of cadmium present in the available soil for plant growth. Cadmium is poisonous and has no beneficial functions neither in human body, nor plants (Hussain & Khan, 2010). vi) Chromium (Cr)

Chromium is also a heavy metal. Our results indicated that the concentration of chromium in E. serrata fruits was 0.123 + 0.0086 ppm, followed by leaf (0.201 + 0.0112 ppm) whereas its lowest amount was found in stem bark (0.112 + 0.0058 ppm) (Table 4.13). Chromium plays an important role in the regulation of carbohydrate, nucleic acid, and lipoprotein metabolism and also as insulin action activator (Kaplan et al., 2003). High amount of Cr accumulation may lead to liver, kidney and lung failure (Zayed & Terry, 2003). The chromium concentration studied in three parts of E. serrata and the amounts of chromium found in these examined parts were below the permissible limit. The permissible limits set for chromium in edible plants are 2 ppm (FAO/WHO, 1984). vii) Manganese (Mn)

Manganese plays an important role in the metabolism of food which is linked to the bone formation. The highest concentration of manganese is found in leaf (0.449 + 0.0090 ppm) of E. serrata, followed by fruit (0.422 + 0.0021 ppm) and then stem bark (0.201 + 0.0027 ppm) (Table 4.13). Manganese is required for many biochemical processes occurring in living organism (Guenther & Konieczynski, 2003). Manganese shows the importance in many physiological processes. pharmacological implication of this element is well known such as in the prevention and treatment of diabetes mellitus (Debrah et al., 2011). The permissible limit set by FAO/WHO (1984) for Mn in edible plants is 2 ppm. However, the permissible WHO (2005) limits for Mn in medicinal plants have not yet been set. Its deficiency causes reproductive failure in both male and female (Lokhande et al., 2010).

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viii) Zinc (Zn)

The highest concentration of zinc was found in the fruit of E. serrata (0.601 + 0.0030 ppm) followed by leaf (0.460 + 0.0048 ppm) and then stem bark 0.169 + 0.0060 ppm (Table 4.13). Zinc is an essential component of most enzymes required in metabolism, growth, development and general well- being. Deficiency of zinc leads to heart diseases and several metabolic disorders. Zinc addition (fortification) programs are being studied, in those plant foods which are predominately consumed by people. Fortification of cereal staple foods benefits the whole population, mainly children and pregnant women. Fortification of zinc in food increase height and health (Vitamin: report of FAO/WHO, 1998). ix) Copper (Cu)

The maximum concentration (0.202 + 0.01 ppm) of copper was noticed in the leaf of E. serrata. The moderate amount was noticed in its fruit which was 0.151 + 0.0158 ppm, followed by the lowest quantity (0.034 + 0.0158 ppm) in the stem bark of E. serrata (Table 4.13). The permissible limit set for Cu in edible plants was 3.00 ppm set by FAO/WHO (1984). China and Singapore set the limits of accumulation of Cu for medicinal plants which are 20 ppm and 150 ppm, respectively (WHO, 2005). Copper (Cu) is a vital redox -active co-enzyme that plays an essential role in many metabolic processes. Its higher amount can be toxic, however in limits it is a compulsory component of many enzymes such as cytochrome oxidase, lysyl oxidase and an iron- oxidizing enzyme in the blood (Obi et al., 2006). Our results indicate that the concentration of Cu was well below the permissible limit. x) Cobalt (Co)

Cobalt is an important trace element. In the present research, the highest amount of Co was found in fruits of E. serrata (0.009 + 0.002 ppm) followed by leaf (0.008 + 0.0015 ppm) whereas in stem bark, it was not detected (Table 4.13). High cobalt content in a synergistic action with iron is suggested for treating blood disorders, especially anaemia (Lokhande et al., 2010). Cobalt is an important element which plays an essential role in many physiological processes occurring in living bodies in the form of trace element, in plants it is found in tubercles, helping in nitrogen fixation. Cobalt is found in vitamin

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B12 which is synthesized by animals. Another source of cobalt is cobalamines (Lokhande et al., 2010). The permissible daily intake of Co is 0.13 mg (3 mg of vitamin B12) of which half is absorbed in the intestine. Although Co is found in all human body, it is mainly present in bones, kidney and liver. Deficiency of cobalt and mainly vitamin B12 leads to genetic mutation and failure of gastric mucosa. Vitamin B12 plays an important role in metabolic functions of human body, especially in thyroid metabolism and red blood cells maturation. Vitamin B12 deficiency causes abnormal functioning of red blood cells (Thunus et al., 1994). It is observed that the leaves and fruits of Ehretia serrata will play important role in curing blood related disorders.

4.3.6.ii) Elemental analysis of leaf, stem bark and fruit of E. obtusifolia i) Sodium (Na)

Sodium is an important macronutrient having vital role in central nervous system. In the present study high concentration of Na was detected in the stem bark (5.332 + 0.1036 ppm) followed by leaf (4.987 + 0.0417 ppm) and the lowest amount of sodium was detected in the fruit (4.953 + 0.0596 ppm) of E. obtusifolia (Table 4.14). NaCl (table salt) is an important source of sodium. Sodium plays crucial role in the excitation and transmission of nerve impulse during nerve cell action potential (Saeed et al., 2010; Underwood, 1977). For optimal growth, sodium plays an important role and its deficiency causes muscle cramp dehydration, fatigue, mood changes, hair loss and hypotension. Sodium showed significant role in the distribution of intracellular and extracellular fluid (Morris et al., 2008; Harper et al., 1997). Literature reveals that the daily recommended intake of Na+ is 1-3.8 mg/day (Anonymous, 2001). Hussain et al. (2011) also describe the same role of sodium in some medicinal plants of Khyber Pakhtunkhwa, Pakistan. Similarly Saeed et al. (2010) reported same results for Polygonatum verticillatum. Our results showed resemblance with these reports. ii) Magnesium (Mg)

The concentration of magnesium was the highest in the fruit (280.0 + 137.17 ppm) followed by stem bark (167.1 + 152.35 ppm) and lowest in leaf (43.67 + 20.334 ppm) of E. obtusifolia (Table 4.14). Intracellular deficiency of magnesium leads to impaired

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function of those enzymes, which utilize high energy phosphate bonds as in the case of glucose metabolism (Stef, et al., 2010).

Nickle (Ni)

Nutrient availability in plants depends on the soil in which plant grows (Naser et al., 2009). The present work showed that E. obtusifolia stem bark has a maximum amount (0.176 + 0.0286 ppm) of nickel followed by fruit (0.113 + 0.0438 ppm). The lowest amount of nickel was detected in the leaves (0.075 + 0.0301) (Table 4.14). Nickle is highly accumulated in plants due to its easy availability in soil. In animals its presence in pancreas proves its role in glucose metabolism and insulin production. Its deficiency leads to various liver disorders (Cempel & Janicka, 2002). Its high accumulation also causes various diseases such as skin allergy, liver and kidney disorders, cardiovascular problems, etc. (Denkhaus & Salnikow, 2002; Saeed et al., 2010). The permissible limit of Ni in plants is 1.5 ppm (Markert, 1994). All values were under the standard permissible limits, i.e. 1.5 ppm.

Iron (Fe)

Iron is considered as one of the most important trace elements of human body constituting the core of the haemoglobin molecule (Arredondo & Nunez, 2005). In the current study, iron concentration was noticed the highest in the leaf (6.472 + 0.0414 ppm) followed by stem bark (5.186 + 0.105 ppm) and lowest in the fruit (1.587 + 0.0064 ppm) of E. obtusifolia (Table 4.14). The permissible limit for iron usage is (36-241 ppm). The iron takes part in various metabolic activities occurring in body of a living organism. Gaseous exchange of O2 and CO2, an important step in respiration occurs with the help of the haemoglobin molecule (Sigel, 1978). One of the most common nutrient deficiencies is iron deficiency, especially found in females, due lack of balanced food, and excessive menstrual flow or multiple births. A person becomes anaemic due to Iron deficiency (Beard, 2001). Our body has iron mainly in a form of haemoglobin almost 57.6%.

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Lead (Pb)

Lead (Pb) is considered as a toxic element and causes fatal diseases in organisms at any stage of life. In the present study, lead was found in high concentration (0.21 + 0.0158 ppm) in the fruit, which was lower than the permissible limits. It was followed by leaves (0.14 + 0.0216 ppm) and the lowest amount (0.001 + 0.008 ppm) of lead was detected in the stem bark of E. obtusifolia, indicating its safe nature for health with respect to lead (Table 4.14). Children are affected from lead most easily and it causes many side effects. These mainly include nervous disorders and retardation in development if taken less than 10 μg/dL (Ang & Lee, 2005). Lead toxicity causes nervous, cardiovascular system disorders and anaemia in children (Meridith, 1978; Staessen, 1993). Its harmful effect also leads to reproductive system disorders as stated by researchers Ang & lee, (2005). WHO sets limits for lead which is 10 ppm (1995). The extent of contamination of Pb depends on the traffic densities and environmental pollution (Nasralla and Ali, 1985; Sovljanski et al., 1990). iii) Cadmium (Cd)

The amount of cadmium in the leaf of E. obtusifolia was 0.012 + 0.1058 ppm, followed by stem bark (0.002 + 0.001 ppm) and then in the fruit (0.004 + 0.0015 ppm) (Table 4.14). These findings were within the permissible limits. The permissible level suggested by WHO for cadmium accumulation in edible plants is 0.21 ppm and for medicinal plants is 0.3 ppm (Jabeen et al., 2010). High amount of Cd in kidney leads to high blood pressure and kidney diseases. Accumulation of high quantity of cadmium also causes damage to the central nervous system, inhibition of releasing of acetylcholine in the synapse and activation of choline esterase enzyme, resulting in hyperactivity of the nervous system (Schumacher et al., 1991). The present research plants regarding cadmium content were found safe to use. iv) Chromium (Cr)

Chromium is one of the important trace elements found on earth. It helps in carbohydrate and protein metabolism. The present study reveals that E. obtusifolia leaf contained high amount of chromium (0.123 + 0.0086 ppm) followed by fruit (0.100 + 0.0068 ppm) and the lowest amount of chromium was detected in the stem bark (0.083

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+ 0.0057 ppm) as given in table 4.14. Chromium also acts as a prosthetic group of several enzymes (Anke et al., 1984; Emsley & John, 2001; Zetic et al., 2001). Glucose Tolerance Factor (GTF) which is an important component of glutamic acid, cysteine and niacin required for removal of glucose from the blood. Cr is a vital component of GTF, which activates insulin production (Robenson & Hurly, 1981; Ibrar et al., 2003; Ravi et al., 2011). Cr deficiency leads to improper insulin production which results raise in blood cholesterol and glucose level (Ottaway & Fell, 1986; Zetic et al., 2001). All three parts showed Cr amount below the permissible limit (1.5 ppm), suggesting that plant is safe for use. v) Manganese (Mn)

Mn concentration in the present work was investigated in three parts of E. obtusifolia. Results revealed that the highest concentration (0.295 + 0.0062 ppm) of manganese was found in the fruit, followed by leaves (0.139 + 0.0090 ppm) and stem bark (0.419 + 0.003 ppm) (Table 4.14). Permissible amount of manganese in medicinal plants are 200 mg/kg as set by WHO (1985). All the findings were below the permissible range of Mn for plants (200 ppm). Manganese is also a vital trace element which plays role in glucose metabolisms. This element is necessary for normal functioning of the body and its reproductive system.

Zinc (Zn)

Zinc is an essential non-poisonous micro element mostly stored in human muscles and bones. In E. obtusifolia, maximum Zn was found in fruits (0.420 + 0.0025 ppm), followed by stem bark (0.248 + 0.0020 ppm), and leaves (0.228 + 0.0029 ppm) (Table 4.14). About 300 Zn dependent enzymes and proteins are detected in the human body. This is a vital element which plays role in strengthening and functioning of bones, hormone release and cell signalling (Saeed et al., 2010). Insulin containing Zinc which is an important hormone of human body (Prasad, 1982; Lokhande et al., 2010). Zinc is important for the growth and multiplication of cells, bone metabolism, taste sensation and eye sight (Thunus, 1994). Its deficiency leads to improper glucose metabolism (Ibrar et al, 2003), weight loss (Lokhande et al., 2010), abdominal pain, diarrhea and vomiting (Anonymous, 2001). The daily recommended intake of Zn for a normal adult

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is 15 mg (Prasad, 1982). With the presence of high amount of Zn, E. obtusifolia should be considered important for treating different diseases.

Cobalt (Co)

In the present study, Co contents were found in the leaf (0.001 + 0.008 ppm) and absent in the fruit and stem bark of E. obtusifolia (Table 4.14). Cobalt is an important trace element for nitrogen fixation in plants. Vitamin B12 is an important source of cobalt found in food of animals synthesized by their body (Lokhande et al., 2010). Daily intake recommendation for Co is 0.13 mg (3 mg of vitamin B12). Deficiency of vitamin B12 produces genetic disorders and failure of gastric mucosal lineage. Deficiency of vitamin B12 (Co) also causes disorders related to red blood cells (Thunus et al., 1994). Cobalt with other elements is recommended for treating blood disorders, especially anemia (Lokhande et al., 2010). In the light of the above facts, only the leaves of E.obtusifolia may be useful for treating blood disorders rather than the whole plant. vi) Copper (Cu)

Copper is an essential micronutrient with great health value. Many enzymes are dependent on copper for their normal functioning in the human body (Huang & Failla, 2000). In the present study three parts of E. obtusifolia were examined for copper concentration. The recommended permissible limit for copper is 10 ppm (WHO, 1985). In the present observation the leaf contained (0.200 + 0.015ppm) followed by fruit (0.148 + 0.0158 p.m.) and stem bark (0.123 + 0.105ppm) listed in (Table. 4.14). Cu plays vital role in redox reactions, neurotransmitter metabolism (acetylcholine), energy production, myelin formation and connective tissue formation (Turnlund, 2006; Harris, 1997; Amina et al., 2003). Cu deficiency leads to hypochromic microcytic anaemia, as it affects the distribution of Fe in the body (Arredondo & Nunez, 2005). Recommended daily dose of Cu allowed by recommended dietary allowance is 340–900 μg /day for human beings (Saeed et al., 2010). In the light of the above results, this plant may play some role in to cure mild hypochromic microcytic anaemia.

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Table 4.13. Elemental Analysis of E. serrata

S.No Fruit Leaf Stem bark Mean SD RSD % Mean SD RSD% Mean SD RSD% (ppm) (ppm) (ppm) Ni 0.173 0.0297 17.21 0.109 0.0479 38.90 0.166 0.012 6.93 Na 6.721 0.0236 0.35 7.123 0.1024 1.44 8.859 0.1967 2.22 Cd 0.010 0.001 10 0.014 0.002 11.29 0.006 0.002 26.3 Cr 0.123 0.0086 7.03 0.201 0.0112 5.59 0.112 0.0058 4.98 Co 0.009 0.002 17.57 0.008 0.0015 19.76 0.000 0.000 0.00 Cu 0.151 0.0158 13.18 0.202 0.01 5.00 0.034 0.0158 4.64 Mg 592.2 793.83 134.0 280.0 137.17 48.99 224.5 1.87 0.83 Pb 0.009 0.002 17.57 0.005 0.0016 31.62 0.29 0.0158 7.53 Fe 8.896 0.0232 0.26 7.692 0.0202 0.26 3.017 0.147 4.88 Mn 0.422 0.0021 0.50 0.449 0.0090 6.45 0.201 0.0027 1.32 Zn 0.601 0.0030 0.50 0.460 0.0048 1.05 0.169 0.0060 3.53

SD= Standard deviation; RSD= Relative standard deviation; PPM= Part per million; Ni= Nickle; Na= Sodium; Cd= Cadmium; Cr= Cromium; Co= Cobalt; Cu= Copper; Mg= Magnesium; Pb= Lead; Fe= Iron; Mn= Manganese, Zn= Zinc

Table 4.14. Elemental analysis E. obtusifolia

S.No Fruit Leaf Stem bark Mean SD RSD % Mean SD RSD% Mean SD RSD% (ppm) (ppm) (ppm) Ni 0.113 0.0438 38.90 0.075 0.0301 39.99 0.176 0.0286 16.24 Na 4.953 0.0596 1.20 4.987 0.0417 43.84 5.332 0.1036 1.94 Cd 0.004 0.0015 39.53 0.012 0.1058 13.18 0.002 0.001 50.0 Cr 0.100 0.0068 6.75 0.123 0.0086 7.03 0.083 0.0057 6.83 Co 0.000 0.000 0.000 0.001 0.008 81.65 0.000 0.000 0.000 Cu 0.148 0.0158 10.54 0.200 0.015 7.91 0.123 0.105 13.18 Mg 280.0 137.17 48.99 43.67 20.334 46.57 167.1 152.35 91.16 Pb 0.21 0.0158 7.53 0.14 0.0216 15.43 0.001 0.008 81.65 Fe 5.186 0.105 2.04 1.587 0.0064 0.41 6.472 0.0414 0.64 Mn 0.295 0.0062 2.09 0.139 0.0090 6.45 0.419 0.003 0.79 Zn 0.420 0.0025 0.59 0.228 0.0029 1.26 0.248 0.0020 0.80

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60 48.99 38.9 40 31.62 20 1.44 11.29 19.76 5.59 0 5 6.45 RSD RSD percentvalue Na 0.26 Mg Ni Cd Cr 1.05 Co Cu Pb Fe Mn Zn Elements

Graph 4.1. Relative standard deviation percentage difference among different elements found in E. serrata leaf

150 134 100 50 0.35 17.21 10 7.03 17.57 0 13.18 17.57 RSD RSD percentvalue Na 0.26 Mg Ni 0.5 Cd Cr 0.5 Co Cu Pb Fe Mn Zn Elements

Graph 4.2. Relative standard deviation percentage difference among different elements found in E.serrata fruit

30 26.35 20

10 2.22 6.93 0.83 4.98 7.53 0 4.64

RSD Percent RSD Value 0 4.88 Na Mg Ni 1.32 3.53 Cd Cr Co Cu Pb Fe Mn Zn Elements

Graph 4.3. Relative standard deviation percentage difference among different elements found in of E. serrata stem bark

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100 81.65 43.8446.57 50 39.99 13.18 7.03 0 7.91 15.43 Na Mg 0.41 6.45 RSD RSD Percentvalue Ni Cd Cr 1.26 Co Cu Pb Fe Mn Zn Elements

Graph 4.4. Relative standard deviation percentage difference among different elements found in E. obtusifolia leaf

60 48.99 38.9 40 39.53 20 1.2 6.75 10.54 0 0 7.53 Na 2.04 2.09 RSD RSD PercentValue Mg Ni Cd Cr 0.228 Co Cu Pb Fe Mn Zn Elements

Graph 4.5. Relative standard deviation percentage difference among different elements found in E. obtusifolia fruit

91.16 100 81.65 80 60 50 40 16.24 13.18 20 6.83

RSD RSD Percntvalue 1.94 0 0.64 0.79 0.8 0 Na Mg Ni Cd Cr Co Cu Pb Fe Mn Zn Elements

Graph 4.6. Relative standard deviation percentage difference among different elements found in E. obtusifolia stem bark

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4.3.7) Nutritional analysis

Food is the basic need of all living organisms, which keeps our body healthy and maintains growth process, reproduction and cell multiplication. A perfect food must have the proper amount of nutrients i-e, protein, carbohydrates, minerals, fats, fibres, water etc. (Adnan et al., 2010). In the present study, the nutritional analyses of leaf, fruit and stem bark of E. serrata an E. obtusifolia are carried out and the results are presented in the table (Table 4.15).

4.3.7.i) Nutritional analysis of E. serrata leaf

E. serrata leaf contained a high amount of carbohydrates (43.1%) followed by crude fibres (15.91%), while moisture content was 15.6%, and protein was 14%. Very little amount of fats (6.39%) and minerals in the form of ash (5%) were also present (table 4.15).

4.3.7.ii) Nutritional analysis of E. serrata fruit

E. serrata fruit contained 42.0% carbohydrates, 11.37% protein, 11% ash, 14.23% moisture, 6.05% fats and 15.35% crude fibres respectively (Table 4.15). Protein is an important nitrogenous biomolecule whereas carbohydrates as a key component of metabolism. Crude fibers on other hand are consequential digestive factor involves in many functions of animals and plant bodies. So this fruit can be a good source of food for birds and animals (Siritientong et al., 2017).

4.3.7.iii) Nutritional analysis of E. serrata stem bark

Stem bark of E. serrata was also analysed for nutritional value. The results showed a high amount of carbohydrates (40.6%) followed by the amount of moisture (33.26%). Same amount of protein (7%), ash (7%) and crude fibre’s (7.54%) were also detected (table 4.15).

4.3.7.iv) Nutritional analysis of E. obtusifolia leaf

E. obtusifolia leaf was also investigated for nutritional content. It was observed that leaf of E. obtusifolia contained 16.68% moisture, 41.0% carbohydrates, 20.90% crude

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fibres, 9.26 % protein, 5% ash and 7.15 % fats (Table 4.15), so this plant can be good source of food for goats and cows etc.

4.3.7.v) Nutritional analysis of E. obtusifolia fruit

Fruit of E. obtusifolia contained the higher amount of carbohydrates (40.2%) followed by moisture amount (26.28%). Amounts of fats, protein, ash and crude fibres were 8.81%, 7%, 8% and 9.72% respectively (Table 4.15).

4.3.7.vi) Nutritional analysis of E. obtusifolia stem bark

Stem bark of E. obtusifolia was analysed for nutrient contents and stem bark contained 41.1% carbohydrates, 18.05% moisture, 11.37% protein, 19.37% crude fiber, 6.75% fats and 3% ash (Table 4.15). Shah et al., (2012) state that protein rich plants had 23% -33% protein, whereas as in the present study protein was not in that range of both species of Ehretia.

4.3.7. vii) Comparison of nutritional value of E. serrata and E. obtusifolia leaf, fruit and stem bark

4.3.7. vii. a) Leaf

Leaf of E. serrata contained higher amount of carbohydrates (43.1%) and protein (14%) as compared to E. obtusifolia leaf. Amount of ash were in the same percentage (5%), in both species. The amount of crude fibers, moisture and fats were in higher amounts in E. obtusifolia than E. serrata (Graph 4.7).

4.3.7.vii.b) Fruits

E. serrata fruits contain high amount of protein (11.37%), ash (11%), carbohydrates (42%) and crude fibres (15.35 %) as compared to E. obtusifolia. Analysis of moisture content revealed the presence of high quantity of moisture (26.28%) and fats (8.805%) in E. obtusifolia as compared to E. serrata (Graph 4.8). Fruits of both the species were rich in nutrients, especially for birds, while the leaves of both the plants can be good forage for cattle’s.

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4.3.7.vii.c) Stem bark

A comparison of both stem barks' nutritional values suggested that the amount of carbohydrates (41.1%), protein (11.37%), fats (6.75%) and crude fibres (19.91%) were in higher amounts in E. obtusifolia as compared to E. serrata and the amount of moisture (33.21%) and ash (7%) were in higher amounts of in E. serrata than E. obtusifolia (Graph 4.9).

Table 4.15. Nutritional analysis of Ehretia serrata and Ehretia obtusifolia leaf, fruit and stem bark

S.No Name Moisture Protein Ash Carbohydrates Fats Crude of Parts content fibber 1 ESL 15.6% 14% 5% 43.1 % 6.39% 15.91% 2 EOL 16.68% 9.26% 5% 41.0% 7.15% 20.90% 3 ESF 14.23% 11.37% 11% 42% 6.05% 15.35% 4 EOF 26.28% 7% 8% 40.2% 8.805% 9.72% 5 ESSB 33.21% 7% 7% 40.6% 4.75% 7.54% 6 EOSB 18.05% 11.37% 3% 41.1% 6.75% 19.73%

50 43.1 45 41 40 35 30 25 20.9 16.68 20 15.6 14 15.91 15 Percent value Percent 9.26 7.15 10 5 5 6.39 5

fats fats

protien protien

crude fibers crude fibers crude

Ash analysis Ash analysis Ash

carbohydrates carbohydrates

moisture content moisture moisturecontent ESL EOLESL EOLESL EOLESL EOLESL EOLESL EOL List of Nutrient

Graph 4.7. Comparison of nutritional values of Ehretia serrata and E. obtusifolia leaf

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45 42 40.2 40 35 30 26.28 25 15.35 20 14.23 11.37 15 11 8 8.805 9.72 10 7 6.05 Percent value Percent 5

fats fats

protien protien

crude fibers crude fibers crude

Ash analysis Ash analysis Ash

carbohydrates carbohydrates

moisturecontent moisturecontent ESF EOF ESF EOF ESF EOF ESF EOF ESF EOF ESF EOF List of Nutrient

Graph 4.8. Comparison of nutritional values of E. serrata and E. obtusifolia fruit

45 40.6 41.1 40 33.21 35 30 25

Percent value Percent 19.73 20 18.05 15 11.375 7 7 7.54 10 4.75 6.75 5 3

fats fats

protien protien

crude fibers crude fibers crude

Ash analysis Ash Ash analysis Ash

carbohydrates carbohydrates

moisture content moisture content moisture ESSB EOSB ESSB EOSB ESSB EOSB ESSB EOSB ESSB EOSB ESSB EOSB List of Nutrient Graph 4.9. Comparison of nutritional values of E. serrata and E. obtusifolia stem bark ESL= Ehretia serrata leaf, ESF= Ehretia serrata fruit, ESSB= Ehretia serrata stem bark EOL= Ehretia obtusifolia leaf, EOF= Ehretia obtusifolia fruit, EOSB= Ehretia obtusifolia stem bark

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4.4) Biological activities

Biological activities were conducted for the determination of pharmacological and the therapeutic potential of methanolic extracts of leaf, fruit, stem bark of E. serrata and E. obtusifolia.

4.4.1) Acute toxicity study

Toxicology deals the negative or poisonous effect of drugs on living organisms and is used to check the safe, effective and efficient nature of drug (Ibrar et al., 2015). Drugs are not permitted to be used clinically before toxicological trial in order to check their safe nature (Uddin et al., 2012). The drugs toxicological assessment is necessary to know about the safe dose administration level and non-toxic nature before their application on human. Therefore, acute toxicological test was performed on methanolic extract of E. serrata and E. obtusfolia leaves, fruits and stem barks by using swiss albino mice and following the standard method of Baliga et al. (2004). Swiss albino mice of both sexes weighing 18 to 24 g were experimented taking from Veterinary Research Institute (VRI) Peshawar. The animals were kept in a suitable environment and balanced food in the cages. The experimental procedures relating to the animals were authorized Veterinary Research Institute (VRI) ethical committee (Est- VRI/Dev(02)/Et-15-03) before starting the study and were conducted under the internationally accepted principles for laboratory animal use and care. Drinking water and food were provided throughout the experiment, except for the short fasting period (4 h), where the drinking water was still in free access but no food supply was provided 12 h prior to treatment. The animals were not allowed to take food and water for 4 hours before the experiment. Then the mice were divided into 19 groups, each having five replicates. Group 1 of mice was treated with normal saline and considered as a control group, while the rest of the group were treated with intra-peritoneal injecting extracts of the fruit, stem bark and leaf of 100, 150 and 200 mg/kg of body weight. After administering the extract, the animals were observed thoroughly for 10-12 hrs for behavioural changes and appearance of any toxic symptoms. No toxicological symptoms were detected in all the groups; neither any behavioural change was noticed in any group of the treated mice after 6 hours. Next the dose was increased up to 1000 mg/kg and then 2000 mg/kg body weights of the mice, but no toxic symptoms or death was detected for the next 12 hours. This proved the non-toxic nature of plants extract

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and revealed the safe nature of these crude drugs for human consumption. The present study confirmed the non-toxic nature of E. serrata and E. obtusifolia, when used orally or intraperitoneally, and it can also be used safely for carrying various bio-assays.

4.4.2) Cytotoxic activity

The brine shrimp (Artemia salina) cytotoxicity assay is considered an effective and cheap method for assessment of toxicity and cytotoxicity. This is an effective way used to detect for cell-line toxicity and anti-tumour activity (Jamal et al., 2017). The brine shrimp assay is also used for isolation of biogenic compounds from plant extracts (Manilal et al., 2016). This bioassay is considered as a bench top method due to easy and inexpensive availability of brine shrimp eggs and its most often used method for detection of antitumor and pesticidal compounds (Carballo et al., 2010). Cytotoxic activity is a first-hand anticancer bioassay to check plant extracts against cancerous growth. The cytotoxic activity of methanolic extracts of leaf, fruit and stem bark of E. serrata and E. obtusifolia was carried out by following the procedures of Meyer et al., (1982) and Mamoon & Azam, (2011).

4.4.2.i) Cytotoxic activity of E. serrata leaf

Percent mortality and LD50 is given in (Table. 4.20). The leaf methanolic extract of E. serrata showed good cytotoxicity with % mortality of brine shrimp were 50.0 + 2.88, 60.0 + 1.20 and 83.3 + 1.45 at doses of 10 µg/mL, 100 µg/mL and 1000 µg/mL respectively. LD50 value was 15.29 µg/mL compared to standard drug (LD50=6.6 µg/ml) (Table 4.16). The cytotoxic activity of the methanolic extract of the leaf was highly dose dependent and at higher dose level (1000 µg/mL) the result was significant (P<0.01) as compared to positive control. Leaf of experimented plant is rich in phenolic compounds and tannins, which are thought to have anti-cancerous properties. The higher amount of dose is preferred to cure cancer effectively.

4.4.2.ii) Cytotoxic activity of E. serrata fruit

The methanolic extract of the fruit of E. serrata exhibited percent cytotoxicity of 43.4 + 1.11, 56.6 + 2.0 and 73.3 + 1.98 at the dose level of 10, 100 and 1000 µg/mL, respectively. The LD50 value was 33.9 µg/mL as compared to standard drug

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(Tamoxifen, LD50 = 6.6 µg/mL) (Table 4.16). Again the cytotoxic effect of fruit extract was dose dependent and 1000 µg/mL dose level showed significant (P<0.05) % mortality (73.3 + 1.98) compared to control (96.66 + 1.52 % mortality).

4.4.2.iii) Cytotoxic activity of E. serrata stem bark

The results showed the percentage of mortality of brine shrimps by using three different doses. The stem bark showed 10 + 0.86 % mortality at 10 µg/mL, 40 + 1.21% at 100

µg/mL and 76.67 + 2.0% at 1000 µg/mL with LD50 188.5 µg/mL as compared to standard drug tamoxifen (LD50=6.6 µg/mL) (Table 4.16). This result also showed dose dependent, and significant (P<0.05) mortality was seen at dose level 1000 µg/mL.

Overall based on the LD50 value, results were not very effective.

4.4.2.iv) Cytotoxic activity of E. obtusifolia leaf

Leaf extract of E. obtusifolia showed mild significant results, 20.0 + 0.97 % mortality at the dose level of 10 µg/mL, 50.0 + 2.10 % mortality at the dose level of 100 µg/mL, and 83.3 + 1.96 % mortality of brine shrimps the dose level of 1000 µg/mL with 80.5 value of LD50 as compared to standard drug (96.6 + 1.52 % mortality with value of

LD50=6.6) (Table 4.15). The results showed dose dependency, and highly significant (P<0.01) mortality rate observed at 1000 µg/mL dose level.

4.4.2.v) Cytotoxic activity of E. obtusifolia fruit

The fruit methanolic extract showed high significant dose dependent cytotoxic activity, 50.0 + 1.89% at 10 µg/mL, 56.6+1.23% at 100 µg/mL, and 80 + 2.02% at dose 1000 µg/mL. It is observed that high dose (1000mg/kg) showed more significant (P<0.01) results. Observed LD50 value was 16.07 µg/mL as compared to standard drug (96.6 +

1.52 % with LD50=6.6 µg/mL) (Table 4.16).

4.4.2.vi) Cytotoxic activity of E. obtusifolia stem bark

Stem bark methanolic extract of E. obtusifolia was also tested for cytotoxic activity. The results are overall non-significant. At 10 µg/mL dose level mortality was 26.6 + 1.89%, at 100 µg/mL it was 30 + 1.99%, and at 1000 µg/mL it was 53.3 + 1.79% with

180.0 value of LD50 compared to standard drug tamaxifen (96.6 + 1.52 % with

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LD50=6.6) (Table. 4.16). It is observed that all doses did not showed any significant (P>0.05) results.

4.4.2. vii) Comparison of cytotoxic potency of E. serrata and E. obtusifolia leaf, fruit and stem bark

4.4.2.vii.a) Leaf

Leaf of E. serrata was more cytotoxic than E. obtusifolia and showed higher percent mortality (83.8 + 1.45%) of Brine shrimps at 1000 µg/mL . Observed LD50 value of E. serrata (15.29 µg/mL) was more significant than E. obtusifolia (80.12 µg/mL).

Comparison of LD50 of standard drug (6.6 µg/mL) with two plants leaf extracts shows more significant relationship with E. serrata (LD50= 15.29 µg/mL) compared to E. obtusifolia (LD50= 80.12 µg/mL) (Graph 4.10).

4.4.2.vii.b) Fruit

By comparing results of methanolic extract of fruits of both species of genus Ehretia, cytotoxic potency of fruit of E. obtusifolia (LD50=16.07 µg/mL) was higher than E. serrata (LD50=33.95 µg/mL). In E.obtusifolia the highest percent mortality was 80.0 + 1.96% at 1000 µg/mL concentration (Graph 4.11).

4.4.2.vii.c) Stem Bark

The stem bark methanolic extract of both plants showed less noticeable cytotoxic effect. Comparable results of both extracts showed that at low dose the effectivity of E. serrata stem bark was low (10 + 0.86%) compared to E. obtusifolia (26.6 + 1.89%) at the dose level of 10 µg/mL. But effectivity of E. serrata increased up to 76.6 + 2.0 % at dose level of 1000 µg/mL more than E. obtusifolia which showed 53.3 + 1.79 % result at the same dose level. LD50 values of both stem bark extracts were almost same (Graph 4.12).

The result of our present work showed that leaf and fruit of E. serrata and E. obtusifolia have significant cytotoxic potential due to the presence of high amount of phenolic compounds in these parts, so it is recommended that advance work should be carried out for the extraction of anticancerous and anti-tumor phytochemicals which are responsible for the cytotoxic effect.

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Table 4.16. Cytotoxic activity of E. serrata and E. obtusifolia leaf, fruit and stem bark S. No Part Used Dose µ/mL Number of Number %Mortality+ LD50 larvae of larvae Standard Error µg/mL survive

1 Standard drug 1 µg/mL 30 01 96.66+1.52 6.6 (tamoxifen) 2 Ehretia serrata 10 µg/mL 30 15 50.0+2.88 15.29 leaf 100 µg/mL 30 12 60.0+1.20 1000 µg/mL 30 05 83.3+1.45** 3 Ehretia serrata 10 µg/mL 30 17 43.4+1.11 33.95 fruit 100 µg/mL 30 13 56.6+2.0 1000 µg/mL 30 08 73.3+1.98* 4 Ehretia serrata 10 µg/mL 30 27 10+0.86 188.5 stem bark 100 µg/mL 30 18 40+1.21 1000 µg/mL 30 07 76.67+2.0* 5 Ehretia 10 µg/mL 30 24 20+0.97 80.12 obtusifolia leaf 100 µg/mL 30 15 50.0+2.10

1000 µg/mL 30 05 80.0+1.96** 6 Ehretia 10 µg/mL 30 15 50.0+1.89 16.07 obtusifolia fruit 100 µg/mL 30 13 56.6+1.23 1000 µg/mL 30 06 80+2.02** 7 Ehretia 10 µg/mL 30 22 26.6+1.89 180.0 obtusifolia stem 100 µg/mL 30 21 30+1.99 bark 1000 µg/mL 30 14 53.3+1.79 Values are reported as mean ± SEM for group of five animals. The data was analyzed by one way ANOVA followed by Dunnett’s test. * Significant at P < 0.05, ** highly significant at P<0.01 and “no symbol” non-significant at P>0.05

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120

100 **

** ** 80

60 % mortality % 40

0 Control (1 ESL (10 EOL ( 10 ESL (100 EOL (100 ESL (1000 EOL (1000 µg/mL) µg/mL) µg/mL) µg/mL) µg/mL) µg/mL) µg/mL)

Graph 4.10. Comparison of cytotoxic activity of E. serrata and E. obtusifolia leaf

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100 ** ** 80 *

60 %mortality 40

0 Control (1 ESF (10 EOF (10 ESF (100 EOF (100 ESF (1000EOF (1000 µg/mL) µg/mL) µg/mL) µg/mL) µg/mL) µg/mL) µg/mL)

Graph 4.11. Comparison of cytotoxic activity E.serrata and E. obtusifolia fruit

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120

100 **

80 *

60 %Mortality

0 Control (1 ESSB (10 EOSB (10 ESSB EOSB ESSB EOFSB µg/mL) µg/mL) µg/mL) (100 (100 (1000 (1000 µg/mL) µg/mL) µg/mL) µg/mL)

Graph 4.12. Comparison of cytotoxic activity of E. serrata and E. obtusifolia stem bark

ESL= Ehretia serrata leaf, ESF= Ehretia serrata fruit, ESSB= Ehretia serrata stem bark, EOL= Ehretia obtusifolia leaf, EOF= Ehretia obtusifolia fruit, EOSB= Ehretia obtusifolia stem bark, SD= Standard drug (Tamoxifen)

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4.4.3) Phytotoxic activity

Phytotoxicity means toxicity showed by a plant on the growth of other plant due to the presence of allochemicals or toxic compounds (Ahmad et al., 2011). Lemna minor phytotoxic bioassay is the common method used to detect the presence of weedicide compounds and accomplishes the need of natural, biodegradable, herbicides as well as plant growth stimulants (Morimoto et al., 2009). There are a lot of side effects of synthetic herbicide and weedicides which cause environmental pollution and spread carcinogenic substances, which leads to various human diseases (Barakatullah et al., 2011). Looking for the solution of these problems, scientists are trying to find and use natural herbicides derived from plant sources which are environment friendly and have no or least toxic effects on human health (Pugazhvendan et al., 2012).

4.4.3.i) Phytotoxic activity of E. serrata leaf

In present study the phytotoxic activity of crude methanolic extract of the leaf of E. serrata was conducted against Lemna minor and the percent frond inhibition and FI50 was recorded. The leaf methanolic extract was used at 10, 100 and 1000 µg/mL dilutions that showed a dose dependent non-significant (P>0.05) frond inhibition, with the percentage of 10 + 0.33, 30 + 0.41 and 40 + 0.42 respectively, with FI50 value of

237.0 µg/mL compared to standard drug (Atrazine, 87.82 + 1.11 with FI50 value 8.8

µg/mL) (Table 4.17). FI50 values are inversely proportional to phytotoxicity of drug, low FI50 value suggested high phytotoxic effect of the experimented drug. Khuda et al. (2012) documented the phytotoxic potential of leaf and root of methanolic extract of Mentha longifolia against Lemna minor and found moderate results.

4.4.3.ii) Phytotoxic activity of E. serrata fruit

The fruit methanolic extract of E. serrata was also tested for its phytotoxicity. The fruit extract also showed non-significant (P>0.05) dose dependent frond inhibition. Frond inhibition were 13.34 + 0.87%, 23.34 + 0.58% and 23.34 + 0.58% at the dose level of

10, 100 and 1000 µg/mL dilutions respectively with the 47088.74 µg/mL value of FI50 compared to standard drug Atrazine (87.82+1.11 with FI50 value 8.8 µg/mL) (Table 4.17). Absence of anthraquinone derivatives might be related with less phytotoxicity of these plant parts.

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4.4.3.iii) Phytotoxic activity of E. serrata stem bark

The phytotoxic activity of methanolic extract of stem bark of E. serrata was also evaluated against Lemna minor. The stem bark also showed slightly higher phytotoxic activity percentage (33.34 + 0.58) at 100 µg/mL dose, compared to other two dose levels, at 10 µg/mL (16.67 + 0.23) and 1000 µg/mL (30.0 + 0.33) doses respectively

(Table 4.17). Based on FI50 the phytotoxic activity of E. serrata stem bark (FI50= 3550.63 µg/mL) was non-significant (P>0.05) in co-relation with standard drug

Atrazine (FI50= 8.87 µg/mL).

4.4.3.iv) Phytotoxic activity of E. obtusifolia leaf

The leaf methanolic extract of E. obtusifolia was tested against Lemna minor. The results were moderate and were dose dependent. Frond inhibition percentages were 13.34 + 0.43 % at 10 µg/mL, 16.67+0.23 % at 100 µg/mL and 23.34 + 0.38 % at 1000 µg/mL compared to standard drug Atrazine (87.82 + 1.11 inhibition). The results were not significant (P>0.05) at all dose levels with FI50 value 70456.0 µg/mL as compared to Atrazine with FI50 (8.87 µg/mL) (Table 4.17).

4.4.3.v) Phytotoxic activity of E. obtusifolia fruit

The fruit methanolic extract of E. obtusifolia was also tested against Lemna minor. Fruit of E. obtusifolia was found to be slightly phytotoxic with 4048.3 value of FI50 and showed 40 + 0.33 % inhibition of fronds at the dose level of 1000 µg/mL. Low phytotoxicity was seen on other dose levels (16.67 + 0.97 % at 10 and 26.76 + 0.56 % at 100 µg/mL) compared to standard drug Atrazine (87.82 + 1.11) (Table 4.16). Overall phytotoxicity of fruit extract based on FI50 was nonsignificant (P>0.05) (FI50= 4048.30

µg/mL) compared to Atrazine (FI50=8.87 µg/mL) (Table 4.17).

4.4.3.vi) Phytotoxic activity of E. obtusifolia stem bark

Methanolic extract of stem bark of E. obtusifolia does not show any significance (P>0.05) pytotoxicity against Lemna minor. Frond inhibition was slightly higher at 1000 µg/mL dose level (23.34 + 0.58 %) followed by dose level of 100 µg/mL (20 +

0.86 %) and least at 10 µg/mL (13.34 + 0.43 %). FI50 value was 90456.0 µg/mL, compared to Atrazine (8.87 µg/mL) (Table 4.17).

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4.4.3. vii) Comparison of phytotoxic potency of Ehretia serrata and Ehretia obtusifolia leaf, fruit and stem bark

4.4.3.vii.a) Leaf

Leaf of E. serrata was more phytotoxic than E. obtusifolia and showed higher percent frond inhibition (40%) at 1000 µg/mL (Graph 4.13).

4.4.3.vii.b) Fruit

By comparing the results, fruit extract of E. obtusifolia showed higher potency as compared to that of E. serrata. The highest percent frond inhibition was 36.67% at 1000 µg/mL concentration in the same plant (Graph 4.14).

4.4.3. vii.c) Stem bark

Phytotoxic activity results of stem bark methanolic extract phytotoxic were also compared and it is observed that stem bark of E. serrata showed a more pronounced effect than E. obtusifolia. Stem bark of E. Serrata showed 33.34 % frond inhibition of L.minor at 1000 µg/mL, which was highest effectivity among the two stem barks extracts (Graph 4.15).

All the results of leaf, fruit and stem bark methanolic extract of E. serrata and E. obtusifolia showed less effectivity against Lemna minor, which means that these are non-phytotoxic. This may be due to the absence of active allochemicals such as anthraquinone in most parts of both plants.

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Table 4.17. Phytotoxic activity of E. serrata and E. obtusifolia leaf, fruit and stem bark S. No Part used Dose Number Total no of %Frond FI 50 (µg/mL) fronds in of fronds inhibition each test in test sample before after dosage dosage 1 Control (Atrazine) 1 µg/mL 04 30 87.82+1.11 8.87 2 Ehretia serrata 10 µg/mL 27 30 10.0+0.33 2397.0773 leaf 100 µg/mL 21 30.0+0.41 1000 µg/mL 18 40.0+0.42 3 Ehretia serrata 10 µg/mL 25 30 16.67+0.65 3550.6389 stem bark 100 µg/mL 20 33.34+0.58 1000 µg/mL 21 30.0+0.33 4 Ehretia serrata 10 µg/mL 26 30 13.34+0.87 4708817.7456 fruit 100 µg/mL 23 23.34+0.58 1000 µg/mL 23 23.34+0.58 5 Ehretia 10 µg/mL 26 30 13.34+0.43 7045699.00174 obtusifolia leaf 100 µg/mL 25 16.67+0.23 1000 µg/mL 23 23.34+0.38 6 Ehretia 10 µg/mL 24 29 20.0+0.86 9045699.00174 obtusifolia stem 100 µg/mL 26 13.34+0.87 bark 1000 µg/mL 23 23.34+0.58 7 Ehretia 10 µg/mL 25 30 16.67+0.97 4048.30362788 obtusifolia fruit 100 µg/mL 28 26.67+0.56

1000 µg/mL 19 40.00+0.33 Values are reported as mean ± SEM for group of five replicates. The data was analyzed by one way ANOVA followed by Dunnett’s test. * Significant at P < 0.05, ** highly significant at P<0.01 and “no symbol” non-significant at P>0.05

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23.34 Control 1 µg/ml ESL 10 µg/ml EOL 10 µg/ml 40 87.82 ESL 100 µg/ml EOL 100 µg/ml 16.67 ESL 1000µg/ml EOL 1000µg/ml 30 10 13.34

Graph 4.13.Comparison of phytotoxic activity of E.serrata and E. obtusifolia leaf

Control (1 µm/mL) 40.04 ESF (10 µg/mL) EOF (10 µg/mL) 87.86 23.32 ESF (100 µg/mL) EOF (100 µg/mL) 26.6 ESF (1000 µg/mL) EOF (1000 (µg/mL) 13.32 23.38 16.68

Graph 4.14 Comparison of phytotoxic activity of E.serrata and E. obtusifolia fruit

control (1 µg/mL) 23.34 ESSB (10 µg/mL) 30 EOSB (10 µg/mL) 87.86 ESSB (100 µg/mL) 13.34 EOSB (100 µg/mL) ESSB (1000 µg/mL) 33.34 EOSB (1000 µg/mL 16.668 20

Graph 4.15. Comparison of phytotoxic activity of E. serrata and E. obtusifolia stem bark

ESL= Ehretia serrata Leaf, EOL= Ehretia obtusifolia leaf, ESF= Ehretia serrata fruit, EOF= Ehretia obtusifolia fruit, ESSB= Ehretia serrata stem bark, EOSB= Ehretia obtusifolia stem bark, Control= Standard drug (Atrazine)

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4.4.4) Anti-oxidant activity

Many disorders like neurodegenerative diseases, cancer and AIDS are due to involvement of free radicals. Antioxidants through their scavenging power are useful for the management of those diseases. DPPH (2,2-diphenyl-1-picrylhydrazyl) stable free radical method is a facile, rapid and sensitive way to survey the antioxidant activity of a specific compound or plant extracts (Koleva et al., 2002; Roleira et al., 2015).

4.4.4.i ) Anti-oxidant activity of E. serrata leaf

Leaf extract of E. serrata showed highly significant (P<0.01) antioxidant activity at 10 µg/mL doses after 30, 60 and 90 minutes, and showing percent inhibition of 72.9 ± 0.62, 70.0 ± 0.57 and 70.2 ± 0.78 respectively, compared to ascorbate which showed percent inhibition of 75.2 ± 0.31 after 30 minutes, 71.3 ± 0.32 after 60 minutes and 70.9±0.35 after 90 minutes. With increasing doses level, the anti-oxidant potency of leaf extract of E. serrata becomes significantly increased (P<0.01) and percent inhibition were 80.5 ± 1.50, 75.03 ± 1.10 and 77.4 ± 0.60 at 100 µg/mL dose, after 30, 60 and 90 minutes respectively, compared to ascorbate, which showed percent inhibition of 85.2 ± 1.12, 80.9 ± 0.32 and 84.1±0.40 after 30 minutes, 60 minutes and 90 minutes respectively. At 1000 µg/mL dose the leaf extract of E. serrata showed significant (P<0.05) results; after 30 minutes (81.94 ± 0.83), 60 minutes (70.9 ± 0.92) and 90 minutes (80.1 ± 0.21), compared to Ascorbate which showed percent inhibition of 95.2 ± 0.81, 92.2 ± 1.10 and 97.1 ± 0.34 after 30 minutes, 60 minutes and 90 minutes respectively. Low IC50 value correlates with high scavenging activity. All results based on IC50, showed significancy at all durations i.e. after 30 minutes (IC50=0.12 µg/mL),

60 minutes (IC50=1.12 µg/mL) and 90 minutes (IC50=1.1 µg/mL) compared to ascorbate with IC50=0.41 µg/mL, IC50= 0.54 µg/mL and IC50=1.96 µg/mL respectively (Table 4.18; Graph 4.16).

4.4.4.ii) Anti-oxidant activity of E. serrata fruit

Fruit extract of E. serrata at 10 µg/mL dose showed highly significant (P<0.01) percent DPPH inhibition after 30 minutes (70.0 ±1.10), which gradually decreased with the passage of time. It became non-significant (P>0.05) showing percent inhibition of 45.1 ± 0.15 after 60 minutes and 45.3 ± 0.15 after 90 minutes, compared to ascorbate which

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showed percent DPPH inhibition of 75.2 ± 0.31 after 30 minutes, 71.3 ± 0.32 after 60 minutes and 70.9 ± 0.35 after 90 minutes. Effectivity was significantly increased (P<0.01) with dose i.e. at 100 µg/mL, percent inhibition was 80.9 ± 0.57 (30 minutes), and then decreased with passage of time to 61.6 ± 0.40 after 60 minutes and 51.9 ± 0.48 after 90 minutes, compared to ascorbate which showed percent inhibition of 85.2 ± 1.12, 80.9 ± 0.32 and 84.1 ± 0.40 after 30 minutes, 60 minutes and 90 minutes respectively. At dose level of 1000 µg/mL, high significancy (P<0.01) was observed, after 30 minutes (90.5 ± 0.35), the anti-oxidant activity was gradually decreased with time, and after 60 and 90 minutes it was (69.2 ± 0.12) and (73.2 ± 0.81), compared to ascorbate with percent inhibition of 95.2 ± 0.81, 92.2 ± 1.10 and 97.1 ± 0.34 after 30 minutes, 60 minutes and 90 minutes respectively. Significancy based on IC50 showed that high effectivity seen after 30 minutes (IC50= 3.37 µg/mL) compared to ascorbate at this time duration (IC50=0.41 µg/mL) than rest of two time durations i.e. 60 minutes

(IC50=63.6 µg/mL) and 90 minutes (IC50=34.8 µg/mL) compared to ascorbate (IC=0.54

µg/mL after 60 minutes, and IC50= 1.96 µg/mL after 90 min) (Table 4.18; Graph 4.17).

4.4.4.iii) Anti-oxidant activity of E. serrata stem bark

The anti-oxidant effect of stem bark methanolic extract of E. serrata at 10 µg/mL was highly significant (P<0.01) after 30 minutes and 60 minutes, and showed percent DPPH inhibition of 70.2 ± 0.52 and 71.0 ± 0.62, compared to control (ascorbate) with 75.2 ± 0.31 and 71.3 ± 0.32 respectively, as well as the percent inhibition which decreased to 27.1 ± 0.10 after 90 minutes compared to ascorbate (70.9 ± 0.35). At dose 100 µg/mL and 1000 µg/mL the results were highly significant (P<0.01) after 30 minutes (74.1 ± 0.48 and 93.2 ± 1.12), and after 60 minutes (74.9 ± 0.31 and 94.1 ± 0.81), compared to control with the values of 85.2 ± 1.12; 95.9 ± 0.81 after 30 minutes and 80.9 ± 0.32; 92.2 ± 0.75 after 60 minutes respectively. The anti-oxidant activity showed by methanolic extract of stem bark after 90 minutes was non-significant, and percent inhibition was shown as 40.0 ± 0.32 and 28.4 ± 0.71, compared to control (ascorbate) which had 84.1 ± 0.40 and 97.1 ± 0.34 respectively (Table 4.17; Graph 4.18).

Significancy based on IC50 showed that stem bark extract showed high inhibition after

60 minutes (IC50= 0.34 µg/mL) compared to ascorbate (IC50=0.54 µg/mL; 60 minutes) followed by 30 minutes with value of IC50=2.2 µg/mL and then 90 minutes (IC50=897.7

µg/mL) compared to ascorbate which showed IC50=2.2 µg/mL after 30 minutes, IC50=

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1.96 µg/mL after 90 minutes (Table 4.18; Graph 4.18).The scavenging activity of E. serrata leaf could be due to the presence of flavonoids and tannins.

4.4.4.iv) Anti-oxidant activity of E. obtusifolia leaf

Methanolic extract of E. obtusifolia leaf showed highly significant (P<0.01) antioxidant potency with percent inhibition of 76.50±0.98 at 10 µg/mL, 70.4 ± 0.23 at 100 µg/mL and 80.9 ± 0.12 at 1000 µg/mL after 30 minutes, compared to control (78.8 ± 0.34 at 10 µg/mL, 72.1 ± 1.12 at 100 µg/mL and 92.9 ± 0.18 at 1000 µg/mL). After 60 minutes the percent inhibition decreased and became non-significant (P>0.05) at 10µg/mL (23.23 ± 0.15) and (47.01 ± 0.23) at 100 µg/mL but slightly increase to (60.0 ± 1.1) at 1000 µg/mL compared to control (70.9 ± 0.39; 80.1 ± 0.46 and 93.1 ± 0.16 at all three doses after 60 minutes respectively). After 90 minutes the scavenging activity percentages of leaf extract of E. obtusifolia tremendously decreased and were non- significant (P>0.05) 22.33 ± 0.15 at 10 µg/mL, 31.08 ± 0.12 at 100 µg/mL and 40.00 ± 0.35 at 1000 µg/mL, compared to ascorbate (71.5 ± 0.46; 86.0 ± 0.35 and 98.6 ± 0.78 percent inhibition at all three doses after 90 minutes) (Table. 4.19; Graph 4.19). After comparison of IC50, the leaf extract showed significant results (IC50=2.5 µg/mL) compared to ascorbate (IC50=2.31 µg/mL) after 30 minutes. While the rest of the two results (IC50=235.6 µg/mL after 60 minutes and IC50= 9090.4 µg/mL after 90 minutes) were nonsignificant, compared to ascorbate (IC50=0.86 µg/mL and IC50= 0.68 µg/mL).

4.4.4.v) Anti-oxidant activity of E. obtusifolia fruit

The fruit extract showed highly significant (P<0.01) results (70.01 ± 0.87) at 10 µg/mL dose, 71.01 ± 0.89 at dose level of 100 µg/mL and 85.33 ± 0.13 at dose level of 1000 µg/mL after 30 minutes, compared to control with values of 78.8 ± 0.34 at 10 µg/mL, 72.1 ± 1.12 at 100 µg/mL and 92.2 ± 0.18 at 1000 µg/mL after 30 minutes. After 60 minutes the percent anti-oxidant potency of fruit extract was (70.0 ± 0.35, 77.0 ± 0.67 and 80.1 ± 1.11) highly significant (P<0.01) at three doses (10 µg/mL, 100 µg/mL and 1000 µg/mL), compared to ascorbate (70.9 ± 0.39, 80.1 ± 0.46 and 93.1 ± 0. 16 respectively at all three doses). After 90 minutes the anti-oxidant potency of fruit extract was non-significant and showed percent scavenging activity of 50.7 ± 0 at dose 10 µg/mL, 65.9 ± 0.68 at 100 µg/mL and 75.00 ± 0.12 at 1000 µg/mL, compared to ascorbate (71.5 ± 0.46, 86.0 ± 0.35 and 98.6 ± 0.78) (Table 4.19; Graph 4.20). After

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comparing IC50 values, E. serrata fruit extract showed significancy (P<0.01) (IC50=1.7

µg/mL and IC50=0.01 µg/mL), compared to ascorbate (IC50=2.31 µg/mL and IC50=0.86

µg/mL) but non-significant (IC50= 9.02 µg/mL), compared to control (IC50=0.68 µg/mL).

4.4.4.vi) Anti-oxidant activity of E. obtusifolia stem bark

Stem bark of E. obtusifolia showed less significant scavenging activity at dose level of 10 µg/mL (58.0 ± 0.81), which is decreasing with increasing dose, at 100 µg/mL (55.0

± 0.51) and 1000 µg/mL (63.0 ± 0.91), it showed significancy (P<0.05) (IC50=0.03 µg/mL) after 30 minutes, compared to ascorbate with percent DPPH inhibition of 78.8 ± 0.34 at 10 µg/mL, 72.1 ± 1.12 at level 100 µg/mL and 92.2 ± 0.18 at 1000 µg/mL

(IC50=2.31 µg/mL). After 60 minutes stem bark extract showed non-significant (P>0.05) percent DPPH inhibition (46.6 ± 0.78, 55.6 ± 0.78 and 70.0 ± 1.12) at 10

µg/mL, 100 µg/mL and 1000 µg/mL dose, with IC50 value of 25.9 µg/mL, compared to ascorbate (70.9 ± 0.39, 80.1 ± 0.46 and 93.1 ± 0.16 with IC50=0.86 µg/mL). After 90 minutes scavenging activity was non-significant at dose level 1000 µg/mL (65.5 ± 0.69) as compared to ascorbate (98.6 ± 0.78), low effectiveness was observed at dose level 10 µg/mL (55.0 ± 1.40) and 100 µg/mL (60.8 ± 0.57), compared to ascorbate (71.5 ±

0.45 and 86.0 ± 0.35). IC50 value was 1.08 µg/mL after 90 minutes, compared to ascorbate (control) with value of IC50=0.68 µg/mL at that time duration (90 minutes) (Table 4.19; Graph 4.21). These results showed the effectivity of stem bark of E. obtusifolia as an anti-oxidant agent.

4.4.4.vii) Comparison of anti-oxidant action E. serrata and E. obtusifolia leaf, fruit and stem bark

4.4.4.vii.a) Leaf

Leaf extract of E. serrata was highly significant (P<0.01) as compared to leaf extract of E, obtusifolia at all dose levels except, at dose level 10 µg/mL after 30 minutes, E. serrata showed low (72.9 ± 0.62) anti-oxidant activity, compared to E. obtusifolia which showed (76.500 ± 0.98) scavenging effect at the same dose level (10 µg/mL). After 60 minutes highest potency against free radicals was showed by E. serrata (75.0 ± 1.10) compared to E. obtusifolia (47.0 ± 0.23) at dose level 100 µg/mL. After 90

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minutes highest significant scavenging activity was showed by Ehretia serrata (80.0 ± 0.21) at dose level 1000 µg/mL as compared to E. obtusifolia (40.00 ± 0.35). These results revealed that the leaf extract of E, serrata is more potent against free radicals, and showed more anti-oxidant activity than E, obtusifolia. It is suggested that increasing amount of extract and time duration should be more effective and potent (1000 µg/mL after 90 min of E. serrata) against pre radicals.

4.4.4.vii.b) Fruit

Unlike leaf extract, methanolic extract of fruit of E. obtusifolia showed almost the same scavenging potency like that of E. serrata after 30 minutes. Major difference was seen after 60 minutes, E. obtusifolia showed higher anti-oxidant activity than E. serrata, especially at 1000 µg/mL (80.1±1.11). After 90 minutes again scavenging activity of E. obtusifolia enhanced and showed higher percentages (50.7 ± 0.45, 65.9 ± 0.68 and 75.0 ± 0.12) at all dose levels (10 µg/mL, 100 µg/mL and 1000 µg/mL) as compared to E. serrata (45.3 ± 0.15, 51.81 ± 0.48 and 73.2 ± 0.81). So results revealed that E. obtusifolia methanolic fruit extract was potent against free radicals than E. serrata and it is suggested that high dose (1000 µg/mL) after a short time (30 minutes) would be more effective.

4.4.4.vii.c) Stem bark

The anti-oxidant effect of stem bark of E. serrata after two time periods, i.e. 30 minutes (70.2 ± 0.52, 74.1 ± 0.48 and 93.2 ± 1.12) and 60 minutes (71.0 ± 0.62, 74.9 ± 0.31 and 94.1 ± 0.81) at all dose levels (10 µg/mL, 100 µg/mL and 1000 µg/mL) were highly significant than E. obtusifolia. But after 90 minutes the percent anti-oxidant potency of E. serrata decreased (27.1 ± 0.10, 40.0 ± 0.32 and 28.4 ± 0.71) as compared to E. obtusifolia (55.0 ± 1.40, 60.8 ± 0.57 and 65.6 ± 0.69) at all dose levels 10 µg/mL, 100 µg/mL and 1000 µg/mL respectively. Results revealed that stem bark of E. serrata is more effective than E. obtusifolia after short time duration (after 30 and 60 minutes), it might be due to slowing of effectivity of phenolic compounds with the passage of times.

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Table 4.18. Percent anti-oxidant activity by leaf, fruit and stem bark of E. serrata

%DPPH inhibition by ESL %DPPH inhibition by ESF %DPPH inhibition by ESS %DPPH inhibition by Ascorbate (0.002%) 30 min 60 min 90 min 30 min 60 min 90 min 30 min 60 min 90 min 30 min 60 min 90 min µg/mL 10 72.9 **±0.62 70.0**±0.51 70.2 **±0.78 70.0** ±1.10 45.1 ±0.35 45.3 ±0.15 70.2 **±0.5 71.0 **±0.62 27.1 ±0.10 75.2 ±0.31 71.3 ±0.32 70.9 ±0.35 100 80.5 **±1.50 75.0 *±1.10 77.4 **±0.60 80.9** ±0.57 61.6 ±0.40 51.9 ±0.48 74.1** ±0.4 74.9** ±0.31 40.0 ±0.32 85.2 ±1.12 80.9 ±0.75 84.1 ±0.40 1000 81.9* ±0.83 70.9 ±0.92 80.1 ±0.21 90.5**±0.35 69.2 ±0.12 73.2* ±0.81 93.2** ±1.12 94.1**±0.18 28.4 ±0.71 95.9 ±0.81 92.2 ±1.10 97.1 ±0.34

IC50 0.12 1.12 1.1 3.37 63.6 34.8 2.2 0.34 879.7 0.41 0.54 1.96 Values are reported as mean % ± SEM for group pf five replicates. The data was analyzed by one way ANOVA followed by Dunnett’s test. * Significant at P < 0.05, ** highly significant at P<0.01 and “no symbol” non significant at P>0.05

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120 120 100 100 80 80 60 60 40 40

20 30 min 20 30 min %DPPH %DPPH inhibition 0 %DPPH inhibition 0 60min 60min

90 min 90 min

10µg/ml 10µg/ml

100µg/ml 100µg/ml

1000µg/ml 1000µg/ml

1000µg/ml 1000µg/ml Ascorbate ESL Ascorbate ESL Ascorbate ESL Ascorbate ESF Ascorbate ESF Ascorbate ESF Dose Level Dose Level

Graph 4.16. % DPPH inhibition by E. serrata leaf Graph 4.17. % DPPH inhibition by E. serrata fruit

120 100 80 60 40 20 0 30 min

60min %DPPH %DPPH inhibition

90 min

10µg/ml 10µg/ml

100µg/ml 100µg/ml

1000µg/ml 1000µg/ml Ascorbate ESSB Ascorbate ESSB Ascorbate ESSB Dose Level

Graph 4.18. %DPPH inhibition by E. serrata stem bark

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Table 4.19. Percent anti-oxidant activity of E. obtusifoliaof leaf, fruit and stem bark µg/mL %DPPH inhibition by EOSB %DPPH inhibition by EOF %DPPH inhibition by EOL % DPPH inhibition by Ascorbate 30 min 60 min 90 min 30 min 60 min 90 min 30 min 60 min 90 min 30 min 60 min 90 min 10 58.0±0.81 46.6±0.78 55.0±1.40 70.01**±0.87 70.0**±0.35 50.7±0.45 76.5**±0.98 23.2±0.12 22.3±0.15 78.8±0.34 70.9±0.39 71.5±0.46 100 55.1±0.51 55.6±0.78 60.8±0.57 71.0**±0.89 77.0**±0.67 65.9±0.68 78.3**±0.23 47.0±0.23 31.0±0.12 72.1±1.12 80.1±0.46 86.0±0.35 1000 63.0±0.91 70.0±1.12 65.6±0.69 85.3**±0.13 80.1**±1.11 75.0±0.12 80.1*±0.12 60.0±1.1 40.0±0.35 92.9±0.18 93.1±0.16 98.6±0.78

IC50 µg/mL 0.03 0.12 1.08 0.83 1.7 9.02 2.5 235.6 990.4 2.31 0.86 0.68 Values are reported as mean ± SEM for group of five replicates. The data was analyzed by one way ANOVA followed by Dunnett’s test. * Significant at P < 0.05, ** highly significant at P<0.01 and “no symbol” non-significant at P>0.05

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120 120 100 100 80 80 60 60 40 40 20 30 min 20 30 min

0 0 %DPPH %DPPH inhibition %DPPH %DPPH imhibition 60min 60min

90 min 90 min

10µg/ml 10µg/ml 10µg/ml 10µg/ml

100µg/ml 100µg/ml 100µg/ml 100µg/ml

1000µg/ml 1000µg/ml 1000µg/ml 1000µg/ml Ascorbate EOL Ascorbate EOL Ascorbate EOL Ascorbate EOF Ascorbate EOF Ascorbate EOF Dose Level Dose Level

Graph 4.19. % DPPH inhibition by Ehretia obtusifolia leaf Graph 4.20. % DPPH inhibition by Ehretia obtusifolia fruit

120 100 80 60 40 20 30 min 0

% DPPH inhibition 60min

90 min

10µg/ml 10µg/ml

100µg/ml 100µg/ml

1000µg/ml 1000µg/ml Ascorbate EOSB Ascorbate EOSB Ascorbate EOSB Dose level

Graph 4.22. % DPPH inhibition by Ehretia obtusifolia stem bark

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4.4.5) Anti-bacterial activity

Bacteria are causing agents of many fatal diseases which lead to severe illness some time leading to death. However, researchers are struggling for detection of active components and constituents present in plants for their anti-bacterial property in order to reduce illness and mortality caused by microbes around the world (World Health Organization 1998; Hamill et al. 2003; (Maple et al. 1989; Mullingen et al. 1993). The main interest in searching anti-bacterial drugs from plants' mainly due to the least appearance of side effects of these herbal drugs (Davies 1994; Poole 2001; WHO publication 2001; (Ordóñez et al. 2003; Arias et al. 2004). In the present study, the antibacterial potential of various parts of the two species of Ehretia has been investigated.

4.4.5.i) Antibacterial activity of E. serrata leaf

In the present study the antibacterial activity of leaf methanolic extract E. serrata was evaluated against four different types of bacteria, i.e. Staphylococcus aureus, Clavibacter michiganensis, Proteus vulgaris and Xanthomonas spp. The results (Table 4.19) showed 16.07% (5 mm) inhibition against Staphylococcus aureus at 1000 µg/mL concentration and 0 % inhibition at 10 and 100 µg/mL against Staphylococcus aureus. The leaf extract showed 10.48 % (3 mm), and 17.48% (5 mm) inhibition against Clavibacter michiganensis at 100 µg/mL and 1000 µg/mL concentration respectively compared to streptomycin (28.6 mm, 100%). Against Proteus vulgaris the activity was not effective and showed only 7.40% (2 mm) and 14.81% (4 mm) inhibitions compared to standard streptomycin (27.0 mm, 100%). The effectivity of the leaf extract of E. serrata was also tested against Xanthomonas spp and results showed 13.24% (4 mm) inhibition at 100 µg/mL and slightly high (23.17%, 7 mm) inhibition at 1000 µg/mL, compared to streptomycin (30.2 mm, 100%) (Table 4.20; Graph 4.12). Results revealed that the E. serrata leaf methanolic extract is not effective against above mentioned bacteria. It is suggested that compounds present in this extract might be effective against some other types of bacteria.

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4.4.5.ii) Anti-bacterial activity of E. serrata fruit

The fruit methanolic extract was also tested against the four types of bacteria. Fruit extract of E. serrata was ineffective against Proteus vulgaris at all concentration (10, 100,1000 µg/mL), while against Clavibacter michiganensis it showed slightly higher inhibition 13.98% (4 mm) and 20.97% (6 mm) at 100 and 1000 µg/mL concentration respectively compared to streptomycin (28.6 mm, 100%). The extract was slightly effective at higher concentration against Staphylococcus aureus and Xanthomonas spp and showing 16.07% (5 mm) compared to streptomycin (control) (31.1 mm, 100 %) and 19.86 % (6 mm) compared to streptomycin (control) (30.21 mm; 100%) at 1000 µg/mL concentration respectively (Table 4.20; Graph 4.23). Results showed the ineffectiveness of the extract against all above mentioned bacteria.

4.4.5.iii) Anti-bacterial activity of E. serrata stem bark

The results of anti-bacterial activity of stem bark of E. serrata revealed that it was ineffective against Staphylococcus aureus at all concentrations. In the remaining three bacterial strain inhibition zones were noticed only at 1000 µg/mL which was 13.8% (4 mm) inhibition in Clavibacter michiganensis compared to streptomycin (28.6 mm, 100%), 14.81% (4 mm) inhibition in Proteus vulgaris compared to streptomycin (27.0 mm, 100%) and 16.55% (5 mm) zone inhibition in Xanthomonas spp, compared to streptomycin (30.2 mm, 100%) respectively. There was no inhibition zone observed in 10 and 100 µg/mL concentrations, compared to control (streptomycin) (Table 4.20; Graph 4.24). Our results reveal that the stem bark of E. serrata is ineffective against all the mentioned bacteria.

4.4.5.iv) Anti-bacterial activity of E. obtusifolia leaf

In the present study, antibacterial activity of the E. obtusifolia leaf methanolic extract was also examined against four different types of bacteria, i.e. Staphylococcus aureus, Clavibacter michiganensis, Proteus vulgaris, Xanthomonas spp. The antibacterial activity was observed against all strains at only 1000 µg/mL concentration i-e 16.07%, against Staphylococcus aureus, 22.5%, against Clavibacter michiganensis, 22.2% compared inhibition against Proteus vulgaris and more effective 23.7% against Xanthomonas spp compared to streptomycine (Table 4.21; Graph 4.25). Gumgumjee

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and Hajar (2015) investigated the antibacterial activities and unveil the phytochemicals of the bioactive components in the leaves extract of E. abyssinica. All identified compounds are known to have antimicrobial activity.

4.4.5.v) Anti-bacterial activity of E. obtusifolia fruit

Methanolic extract of fuit showed no inhibition against Staphylococcus aureus at all concentrations, but against Clavibacter michiganensis it showed inhibitions at 100 µg/mL and 1000 µg/mL doses (15.26% and 19.08% respectively) compared to streptomycin. Against Proteus vulgaris and Xanthomonas spp the zone inhibitions were seen only at 1000 µg/mL ith the vlue of 19.8% and 15.57% respectively (Table 4.21; Graph 4.26). Overall results showed the ineffectiveness of fruit extract of E. obtusifolia against the above mentioned bacterial strains.

4.4.5.vi) Anti-bacterial activity of E. obtusifolia stem bark

Stem bark extract of E. obtusifolia was also tested against these four strains. Results showed that the stem bark methanolic extract was effective at two concentration levels i-e. 100 and 1000 µg/mL against Staphylococcus aureus and showed 19.8 % and 26.4% inhibition zones compared to standard drug. This methanolic extract showed zone of inhibition against remaining three bacteria at 1000 µg/mL only and the results were 19.08% inhibition against Clavibacter michiganensis, 15.87% inhibition against Proteus vulgaris and 18.69% inhibition against Xanthomonas spp, compared to control (streptomycin) (Table 4.21; Graph 4.27). All extracts showed slight antibacterial activity against the tested strains.

4.4.5.vii) Comparison of anti-bacterial action of E. serrata and E. obtusifolia leaf, fruit and stem bark

4.4.5.vii.a) Leaf

Leaf methanolic extract of E. serrata showed higher potency against all four bacterial strains at 100 and 1000 µg/mL, compared to E. obtusifolia which showed the same activity against above bacteria only at higher doses. The most pronounced results were observed with E. serrata (23.1%) against Xanthomonas spp.

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4.4.5.vii.b) Fruit

Again, fruit of E. serrata was slightly more potent than E. obtusifolia by showing the most pronounced (20.97%) activity against Clavibacter michiganensis at 1000 µg/mL. E. serrata was found ineffective against Proteus vulgaris and E. obtusifolia against Staphylococcus aureus. The remaining antibacterial results of both plants showed similarities with each other.

4.4.5.vii.c) Stem bark

The stem bark of E. obtusifolia showed higher activity against all the four tested bacteria as compared to E. serrata. The largest zone of inhibition was seen at 1000 µg/mL dose in Staphylococcus aureus by E.obtusifolia stem bark which is the causing agent of many fatal diseases (Al-Zoreky., 2009). Whereas E. serrata stem bark was found ineffective against Staphylococcus aureus. Remaining results were similar in both the species. In conclusion all parts of both plants possessed slight antimicrobial activity.

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Plate A Plate B

Plate C Plate D

Plate E Fig. 4.32. Plate A:Staphylococcus aureus; zone inhibition caused by Ehretia obtusifolia stem bark at 1000 µg/mL Plate B: Proteus vulgaris; zone inhibition caused by leaf extract of Ehretia obtusifolia at 1000 µg/mL Plate C: Xanthomonas perfoans; zone inhibition caused by leaf extract of Ehretia serrata at 1000 µg/mL Plate D: Proteus vulgaris; zone inhibition caused by fruit extract of Ehretia obtusifolia at 1000 µg/mL Plate E: Clavibacter michiganensis; zone inhibition caused by fruit extract of Ehretia serrata at 1000 µg/mL

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Table 4.20. Antibacterial activity of different parts of E. serrata

S. NO Bacterial Control Plant parts strains Fruit methanolic extract Leaf Stem bark methanolic methanolic extract extract Streptomycin Zone inhibition/Concentration µg/mL 0.05 mg/kg 10 100 1000 10 100 1000 10 100 1000 1. Staphylococcus 31.1 mm -- -- 5 mm -- -- 5 mm ------aureus (100%) (16.07%) (16.07%) 2. Clavibacter 28.6 mm -- 4 mm 6 mm -- 3 mm 5 mm -- - 4 mm michiganensis (100%) (13.9 (20.97%) (10.48%) (17.48%) (13.8%) 8%) 3. Proteus 27.0 mm ------2 mm 4 mm -- -- 4 mm vulgaris (100%) (7.40%) (14.81%) (14.81%) 4. Xanthomonas 30.2 mm -- -- 6 mm -- 4 mm 7 mm -- -- 5 mm perforans (100%) (19.86%) (13.24%) (23.17%) (16.55%)

Table 4.21. Antibacterial activity of methanolic extracts of E. obtusifolia.

S.NO Bacterial Control Plant parts strains Fruit methanolic extract Leaf Stem bark methanolic methanolic extract extract Streptomycin Concentration µg/mL 0.05 mg/kg 10 100 1000 10 100 1000 10 100 1000 1. Staphylococcus 30.3 mm ------2 mm -- 6 mm 8 mm aureus (100%) (6.60%) (19.8%) (26.4%)

2. Clavibacter 26.2 mm -- 4 mm 5 mm -- -- 6 mm -- -- 5 mm michiganensis (100%) (15.26 (19.08%) (22.5%) (19.08%) %) 3. Proteus 31.5 mm -- -- 6 mm -- -- 7 mm -- -- 5 mm vulgaris (100%) (19.8%) (22.2%) (15.87%) 4. Xanthomonas 32.1 mm -- -- 5 mm -- -- 4 mm -- -- 6 mm perforans (100%) (15.57%) (12.46%) (18.69%)

Zone inhibition of control (Streptomycine) against all bacterial strains taken as 100%, percentages of tests (all doses) were taken with reference to control.

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35 ** ** 30 ** ** 25 20

bacterialactivity - 10

%anti 5 0

Graph 4.22. Anti-bacterial activity of methanolic extract of E. serrata leaf

35 ** ** 30 ** ** 25

%antibacterial activity 5

Graph 4.23. Anti-bacterial activity of methanolic extract of E. serrata fruit

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35 ** ** 30 ** ** 25 20

bacterialactivity - 10

%anti 5 0

Graph 4. 24. Anti-bacterial activity of methanolic extract of E. serrata stem bark

40 35 ** ** ** 30 ** 25 20

bacterialactivity 15 - 10

%anti 5 0

Graph 4.25. Anti-bacterial activity of methanolic extract of E. obtusifolia leaf

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40 35 ** ** ** 30 ** 25 20

bacterialactivity 15 - 10

%anti 5 0

Graph 4.26. Anti-bacterial activity of methanolic extract of E. obtusifolia fruit

40 35 ** ** ** 30 ** 25 20 15 10

%antibacterial activity 5 0

Graph 4.27. Anti-bacterial activity of methanolic extract of E. obtusifolia stem bark

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4.4.6) Analgesic activity

Due to opposed dverse effects, like stomach diseases caused by NSAIDs and tolerance and dependence induced by opiates, the frequent use of these drugs is limited and have not been used in all cases. Scientists are in search of natural analgesic drugs all over the world as alternatives to NSAIDs and opiates. The plant-based analgesic drugs are more effective and cheap due to which traditional medicines are given more preference. According to WHO about 80% of the world population are still dependent on using plant based medicines to fight against different diseases Sukul et al. (2017).

In accordance with the traditional uses of E. serrata and E. obtusifolia the present study was carried out to evaluate the analgesic activity of the methanolic extract of different parts of these plants.

4.4.6.i) Analgesic activity of E. serrata leaf

Leaf extract of E. serrata showed highly significant (P<0.01) analgesic activity at 100 mg/kg dose both after 30 minutes (18.5 + 1.2) and 60 minutes (20.1 + 1.5), compared to diclofenac sodium which showed analgesic activity of 15.02 + 1.1 after 30 minutes and 14.1 + 1.2 after 60 minutes. The analgesic activity shown by leaf of E. serrata at 200 mg/kg was not significant (P>0.05) (11.4 + 1.3) at 30 minutes and highly significant (22.5 + 2.0) after 60 minutes, compared to diclofenac sodium which showed 15.0 + 1.1 after 30 minutes; 14.1 + 1.2 after 60 minutes). At 300 mg/kg dose the leaf extract showed highly significant (P<0.01) result, both after 30 minutes i-e. 14.3 + 1.4 and 14.7 + 1.9 after 60 minutes, with respect to diclofenac sodium (15.0 + 1.1 after 30 min; 14.1 + 1.2 after 60 minutes) (Table 4.22) (Graph 4.28).

4.4.6.ii) Analgesic activity of E. serrata fruit

Fruit extract of E. serrata at 100 mg/kg dose showed highly significant (P<0.01) analgesic activity both after 30 minutes (14.1 + 1.1) and after 60 minutes (22.4 + 4.5), compared to diclofenac sodium (15.0 + 1.1 after 30 minutes; 14.1 + 1.2 after 60 minutes). At 200 mg/kg dose fruit extract of E. serrata showed highly significant (P<0.01) results both after 30 minutes and 60 minutes (15.5 + 2.3; 20.2 + 1.5) respectively, as compared to diclofenac sodium which showed analgesic activity of 15.0 + 1.1 after 30 minutes and 14.1 + 1.2 after 60 minutes. At 300 mg/kg dose, extract

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showed significant (P<0.05) results (13.9 + 1.7) after 30 minutes and highly significant (P<0.01) results (20.8 + 1.7) after 60 minutes, with respect to diclofenac sodium (15.0+1.1 after 30 minutes; 14.1 + 1.2 after 60 minutes) (Table 4.22) (Graph 4.28).

4.4.6.iii) Analgesic activity of E. serrata stem bark

Analgesic effect of stem bark of E. serrata at 100 mg/kg and 200 mg/kg doses were highly significant (P<0.01) i-e 17.1 + 2.0 & 14.6 + 1.8 at 30 minutes, after which the activity began to increase with the passage of time and showed significant result (P<0.01) i-e 21.3 + 2.1 & 17.8 + 2.7 after 60 minutes, as compared to the positive control (diclofenac sodium with values 15.0 + 1.1 after 30 minutes; 14.1 + 1.2 after 60 minutes). With increased dose, significancy increased and stem bark showed highly significant (P<0.01) values at 300 mg/kg (14.2 + 2.4 and 18.3 + 2.5) after 30 and 60 minutes with respect to diclofenac sodium (15.0 + 1.1 after 30 minutes; 14.1 + 1.2 after 60 minutes) (Table 4.22) (Graph 4.28).

4.4.6.iv) Analgesic activity of E. obtusifolia leaf

E. obtusifolia leaf showed non-significant (P>0.05) analgesic activity at 100 mg/kg and 200 mg/kg dose after 30 minutes (10.4 + 1.6, 11.4 + 1.8) and highly significant (P<0.01) values (19.9 + 1.3;17.5 + 1.8) after 60 minutes, compared to diclofenac sodium (15.0 + 1.1 after 30 minutes; 14.1+ 1.2 after 60 minutes) (Table 4.23). The pain killing effect of leaf increased with increasing time and dose. Leaf extract of E. obtusifolia at dose of 300 mg/kg showed highly significant (P<0.01) values both after 30 minutes and 60 minutes (17.4 + 1.2; 25.2 + 1.8) with respect to diclofenac sodium (15.0 + 1.1 after 30 minutes; 14.16 + 1.2 after 60 minutes) (Table 4.22) (Graph 4.29).

4.4.6.v) Analgesic activity of E. obtusifolia fruit

The fruit extract showed highly significant (P<0.01) results (16.0 + 1.1; 20.2 +4.5) at 100 mg/kg both after 30 minutes and 60 minutes. At 200 mg/kg and 300mg/kg doses showed significant (P<0.05) results (13.2 + 1.2, 14.8 + 0.7) after 30 minutes. After 60 minutes at 200 mg/kg level result was highly significant (P<0.01) with value of 14.8 + 4.6 and significant (P>0.05) with value of 12.6 + 2.0 at 300 mg/kg (Table 4.23; Graph 4.29).

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4.4.6.vi) Analgesic activity of E. obtusifolia stem bark

Stem bark of E. obtusifolia showed highly significant (P<0.01) value at dose of 100mg/kg both after 30 minutes and 60 minutes (18.2 + 1.6, 23.7 + 1.3), compared to diclofenac sodium (15.0 + 1.1 after 30 minutes; 14.1+ 1.2 after 60 minutes) as shown in Table.4.22. Stem bark extract showed highly significant (P<0.01) results (13.7 + 1.8, 12.4 + 1.8) at dose of 200 mg/kg and 300 mg/kg after 60 minutes with respect to positive control (diclofenac sodium with value 14.1 + 1.1 after 60 minutes) and significant values (P<0.05) (13.7 + 1.8; 12.456 + 1.2) after 30 minutes, compared to diclofenac sodium (15.02 + 1.1 after 30 minutes) (Table 4.23) (Graph 4.29). The effect of E. obtusifolia against nociception induced by the presence of many phytochemical compounds (phenols) which play a signiﬁcant role in nociceptive processing in both central and peripheral nervous systems (Ferreira et al., 2004).

4.4.7.vii) Comparison of analgesic action of E. serrata and E. obtusifolia leaf, fruit and stem bark

4.4.7.vii.a) Leaf i. After 30 minutes

Leaf extract of E. serrata showed significant analgesic activity at 100 mg/kg and 200 mg/kg doses as compared to E. obtusifolia leaf which showed non-significant analgesic activity at these dose levels after 30 minutes. At dose level 300 mg/kg the leaf extract of both plants' showed highly significant results after 30 minutes. The results revealed the effectiveness of E. serrata higher than that of E. obtusifolia after 30 minutes (Graph 4.28; 4.29). ii. After 60 minutes

Leaf extract of both E. serrata and E. obtusifolia showed highly significant (p<0.01) analgesic activities at 100 mg/kg and 300 mg/kg after 60 minutes. At 200 mg/kg dose E. serrata showed highly significant value (p<0.01), but E. obtusifolia showed significant (P<0.05) values after 60 minutes. Results indicated the overall effectiveness of E. serrata leaf over E. obtusifolia leaf after 60 min (Graph 4.28; 4.29).

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4.4.7.vii.b) Fruit i. After 30 minutes

Fruit extract of E. serrata at 100 mg/kg dose showed significant (P<0.05) analgesic activity but E. obtusifolia showed highly significant (P<0.01) analgesic activity after 30 minutes. Fruits of both plants showed significant results at 200 mg/kg but at 300 mg/kg fruit extract of E. serrata showed significant (P<0.05) but E. obtusifolia showed highly significant (P<0.01) analgesic potency after 30 minutes (Graph 4.28; 4.29). ii. After 60 minutes

Fruit extract of E. serrata at 100 mg/kg, 200 mg/kg and 300 mg/kg doses showed highly significant (P<0.01) analgesic activities after 60 minutes. The Fruit extract of E. obtusifolia showed highly significant (P<0.01) results after 60 minutes. But at dose 200mg/kg and 300mg/kg fruit extract showed only significant (P<0.05) results at 200 mg/kg but non-significant at 300 mg/kg. Overall results showed a high nociceptive effect of the fruit of E. serrata than E. obtusifolia after 60 minutes (Graph 4.28; 4.29).

4.4.7.vii.c) Stem bark i. After 30 minutes

The analgesic effect of stem bark of E. serrata at 100 mg/kg and 200 mg/kg doses were highly significant (P<0.01) after 30 minutes but significant at 300 mg/kg. E. obtusifolia showed highly significant (P<0.01) value of 100 mg/kg but significant (P<0.05) at doses of 200 mg/kg and 300 mg/kg. Overall results revealed that E. serrata showed more significance than E. obtusifolia (Graph 4.28; 4.29).

ii. After 60 minutes

Stem bark extracts of E. serrata and E. obtusifolia showed highly significancy (P<0.01) at all doses i.e 100mg/kg, 200 mg/kg and 300 mg/kg. Results showed that with the passage of time and dose the significancy of E. obtusifolia and E. serrata bark extracts increased (Graph 4.28; 4.29).

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Table 4.22. Analgesic effect of E. serrata leaf, fruit and stem bark S. Treatment Dose Time before Time after dose Time after dose mg/kg dose administration administration No administration (30 minutes) (60 minutes) (0 minutes) 01 Normal saline 9.1±0.4 08.45+2.9 9.31+ 2.4 02 Diclofenic sodium 10 mg/kg 9.2+ 1.0 15.0+1.1** 14.1+1.2** 03 Leaf 100 mg/kg 8.4 +1.2 18.5+1.2** 20.1+1.5** 200 mg/kg 7.8 +1.1 12.6+1.3 22.5+2.0** 300 mg/kg 10.5 +0.6 14.3+1.4** 14.7+1.9** **4.5+22.4 ٭Fruit 100 mg/kg 8.0 +1.5 14.1+1.1 04 ٭*mg/kg 10.0 +1.9 15.5+2.3** 20.2+1.5 200 **1.7+20.8 ٭mg/kg 12.3 +1.3 13.9+1.7 300 05 Stem Bark 100 mg/kg 7.8 +1.5 17.1+2.0** 21.3+2.1** 200 mg/kg 7.7 +1.2 14.6+1.8** 17.8+2.7** 300 mg/kg 10.6 +1.2 14.2+2.4** 18.3+2.5** Values are reported as mean ± SEM for group of five animals. The data were analyzed by one way ANOVA followed by Dunnett’s test. * Significant at P < 0.05, ** highly significant at P<0.01, non-significant at P>0.05.

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Table 4.23. Analgesic effect of E. obtusifolia leaf, fruit and stem bark S.No Treatment Dose Time before dose Time after dose Time after dose mg/kg administration administration administration (0 minutes) (30 minutes) (60 minutes) 01 Normal saline 9.1±0.46 08.4+2.92 9.31+ 2.45 02 Dichlofenic sodium 10mg/kg 9.2+1.0 15.0+1.1** 14.1+1.2** Leaf 100mg/kg 7.8+0.9 10.4+1.6 19.9+1.3** *٭200mg/kg 9.0+1.0 11.4+1.8 17.5+1.8 300mg/kg 7.8+1.0 17.4+1.2** 25.2+1.8** 04 Fruit 100mg/kg 12.2+2.7 16.0+0.7** 20.2+4.4** 200mg/kg 9.5+1.2 13.2+1.2* 14.0+4.6** 300mg/kg 10.3+1.0 14.8+0.7* 12.6+2.0 05 Stem bark 100mg/kg 8.4+1.0 18.2+0.8** 23.7+0.8** 200mg/kg 7.4+1.0 13.7+1.0* 17.4+2.3** 300mg/kg 11.1+0.8 12.4+1.4* 20.5+2.5** Values are reported as mean ± SEM for group of five animals. The data was analyzed by one way ANOVA followed by Dunnett’s test. * Significant at P < 0.05, ** highly significant at P<0.01, non-significant P>0.05.

Plate A Plate B

Fig. 4.33. Plate A: Administration of drug in albino mice intraperitoneally Plate B: Measurement of analgesic effect of drug on analgesometer

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30 ** ** 25 ** ** ** ** ** ** ** 20 ** * ** ** ** ** ** * * ** ** 15 ** ** 10 5

% analgesic activity 0

ESL (200mg/kg)…

ESF (100 ESF mg/kg)… ESF(100 mg/kg)… ESF(200 mg/kg)… ESF(200 mg/kg)… ESF(300 mg/kg)… ESF(300 mg/kg)…

SD(10 mg/kg)30… SD(10mg/kg) 60…

SD(10mg/kg) ILT

ESSB (200 ESSB mg/kg)… ESSB(100 mg/kg)… ESSB(100 mg/kg)… ESSB(100 mg/kg)… ESSB(200 mg/kg)… ESSB(200 mg/kg)… ESSB(300 mg/kg)… ESSB(300 mg/kg)… ESSB(300 mg/kg)…

ESL(100mg/kg) 30… ESL(100mg/kg) 60… ESL(200mg/kg) 30… ESL(300mg/kg) 30… ESL(300mg/kg) 60…

ESL (100mg/kg) ESL ILT ESL(200mg/kg) ILT ESL(300mg/kg) ILT

ESF(100 mg/kg)ILT ESF(300 mg/kg) ILT ESF(200 mg/kg) ILT

Graph 4.28. Comparision of analgesic activity of E. serrata leaf, fruit and stem bark with standard drug

30 ** ** 25 ** ** ** ** 20 ** ** ** ** ** ** ** * * * 15 * 10

% analgesic activity 5

SD(10mg/kg) ILT

EOL (200mg/kg) ILT

EOF (100 mg/kg)ILT

EOL(100 mg/kg) ILT EOL(300 mg/kg) ILT

EOF(200 mg/kg) ILT EOF(300 mg/kg) ILT

SD(10mg/kg) min 60

EOSB(100 mg/kg) ILT EOSB(200 mg/kg) ILT EOSB(300 mg/kg) ILT

SD(10 mg/kg)30 min

EOL(100 mg/kg) 30 min EOL(100 mg/kg) 60 min EOL(200 mg/kg) 30 min EOL(300 mg/kg) 30 min EOL(300 mg/kg) 60 min

EOF(100 mg/kg) 30 min EOF(100 mg/kg) 60 min EOF(200 mg/kg) 30 min EOF(200 mg/kg) 60 min EOF(300 mg/kg) 30 min EOF(300 mg/kg) 60 min

EOL (200 mg/kg) min 60

EOSB(100 mg/kg) min 60 EOSB(200 mg/kg) min 30 EOSB(200 mg/kg) min 60 EOSB(300 mg/kg) min 30 EOSB(300 mg/kg) min 60 EOSB(100 mg/kg) min 30 Graph 4.29. Comparision of analgesic activity of E. obtusifolia leaf, fruit and stem bark with standard drug ILT=Initial latency time S.D= Standard drug (Diclofenac sodium) ESL= E.serrata leaf ESF= E.serrata fruit ESSB= E.serrata stem bark EOL=E.obtusifolia leaf EOF= E.obtusifolia fruit EOSB=E. obtusifolia stem bark

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4.4.7. Anti-inflammatory activity

For searching anti-inflammatory drugs, carrageenan-induced mice paw edema method (Adeyemi et al., 2002) which is widely used by scientists. The anti-inflammatory activity of the leaf, fruit and stem bark methanolic extracts of E. serrata and E. obtusifolia was evaluated by following the methods of Winter et al., (1962) and the results were compiled in tables.

4.4.7.i) Anti-inflammatory activity of E. serrata leaf

The methanolic extract of the Ehretia serrata leaf was tested at three different dose levels, e.g. 100, 200 and 300 mg/kg. The results were observed after 60 minutes, 120 minutes and 180 minutes. The results were co related to positive control and with each other to observe the differences. After 60 minutes inflammations in mice paw were greatly reduced. The extract showed significant (P<0.05) results after 60 minutes, compared to the positive control (diclofenac sodium) (0.5800 + 0.5774) which showed 86 % inflammation reduction. At 100mg/kg and 300 mg/kg the results were (0.1200 + 0.031808; + 0.1200 + 0.01732) 75% and at 200mg/kg the inflammation reduction of the mices paw was (0.1100 + 0.00667) 73% (Graph 4.30). After 120 minutes all the doses (100 mg/kg, 200 mg/kg and 300 mg/kg) showed almost similar highly significant (P<0.01) percent inflammation inhibition i.e (0.1100 + 0.01732; 0.1067 + 0.00333 and 0.1133 + 0.00882) 82 %, 76 % and 78 % respectively in contrast to reference drug, diclofenac sodium (0.5800 + 0.5773; 90%) (Graph 4.31). After 180 minutes the inflammation of the mice’s paw was checked thoroughly and were highly significant (P<0.01) were obtained. The inflammation reduction was greatly maintained at 100 mg/kg, which was (0.0867 + 0.00882) 88 % and 100% both at 200 mg/kg and 300 mg/kg (0.0967 + 0.01453; 0.0967 + 0.00882) respectively, compared to positive control (0.5300 + 0.22189) which showed 100% anti-inflammation (Table 4.23; Graph 4.32). Normal saline (negative control) showed non-significant (P>0.05) anti-inflammatory results at all three time durations i.e after 60 min (0.3000 + 0.14988; 0%), 120 minutes (0.2600 + 0.00365; 12%) and 180 minutes (0.2500 + 0.02266; 19%). The results clearly revealed that anti-inflammatory potency of the leaf extract is dependent on time and dose. High amounts of dose level and long-time duration showed 100% anti- inflammatory results.

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4.4.8.ii) Anti-inflammatory activity of E. serrata fruit

The methanolic extract of fruit E.serrata fruit was also tested at three different doses of 100 mg/kg, 200 mg/kg and 300 mg/kg. The paw percent inflammation inhibition was noticed after different time intervals, i.e. 60 minutes, 120 minutes and 180 minutes. After 60 minutes the results were changed drastically and fruit showed significant (P<0.05) anti-inflammatory action at all three doses which were (0.1200 + 0.00000) 71.00% at 100 mg/kg, (0.1167 + 0.00333) 74% at 200 mg/kg and (0.12000 + 0.01000) 73.00% at 300 mg/kg respectively, as compared to the positive control (diclofenac sodium) (0.5800 + 0.5774) which showed 85 % inflammation reduction (Graph 4.31). After 120 minutes the results were highly significant (P<0.01) at all three doses of 100 mg/kg and 200 mg/kg doses and 300 mg/kg (0.1000 + 0.00577; 96 % and 0.1000 + 0.00577; 88 % and 0.0867 + 0.01333; 100%) respectively, in contrast to reference drug, diclofenac sodium (0.5800 + 0.5773; 90%) (Graph 4.31). After 180 minutes the inflammation inhibition was maintained at all three dose levels which showed a highly significant reduction (P<0.01) of inflammation. At 100 mg/kg and 300 mg/kg doses the effectiveness was outstanding e.g (0.0933 + 0.00577) 100% and (0.0867 + 0.00667) 100%, respectively, and at 200 mg/kg the percent inflammation reduction was (0.0933 + 0.00667) 96% as compared to positive control (0.5300 + 0.22189) which showed 100% anti-inflammation activity (Table 4.23; Graph 4.32). Normal saline (negative control) showed non-significant (P>0.05) anti-inflammatory activity at all three time durations i-e after 60 minutes (0.3000 + 0.14988; 0%), 120 minutes (0.2600 + 0.00365; 12%) and 180 minutes (0.2500 + 0.02266; 19%). Results showed that anti- inflammatory potency of fruit extracts dependent on time and dose and that concentrated dose gives best results. Best anti-inflammatory potency of fruit extracts might be due to the presence of bioactive compounds in it, which showed effective results.

4.4.8.iii) Anti-inflammatory activity of E. serrata stem bark

Stem bark methanolic extracts also showed effectiveness against inflammation. After 60 minutes the activity was more or less similar (P<0.05) i.e. 100 mg/kg showed 73 % (0.1267 + 0.01528), 200 mg/kg exerted 73% (0.1267 + 0.00882) and 300 mg/kg displayed 72 % (0.1233 + 0.00667) of inhibitory effect on inflammation respectively with respect to diclofenac sodium (86%; 0.6000 + 0.5774) (Graph 4.30). The

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effectiveness increased and become highly significant (P<0.01) with the passage of time which were (0.1167 + 0.01202 and 0.1133 + 0.00667) 84% at 100 mg/kg and 86% at 200 mg/kg dose after 120 minutes and (0. 1067 + 0.01856) 89 % at 300mg/kg in contrast to reference drug, diclofenac sodium (0.5800 + 0.5773; 90%) (Graph 4.31). After 3 hours the effectiveness of extracts reached at its peak which was highly significant (P<0.01) (0.1000 + 0.00577) percent inflammation reduction (100 %) at all three doses i-e at 100 mg/kg (0.1000 + 0.00577), at 200 mg/kg (0.1000 + 0.00577) and at 300 mg/kg (0.0967 + 0.01202), compared to positive control (0.5300 + 0.22189) which showed 100% anti-inflammatory activity (Table 4.24) (Graph 4.32). Normal saline (negative control) showed non-significant (P>0.05) anti-inflammatory results at all three time durations i.e. after 60 minutes (0.3000 + 0.14988; 0%), 120 minutes (0.2600 + 0.00365; 12%) and 180 minutes (0.2500 + 0.02266; 19%). It is suggested that high amount of dose and long-time duration are more effective than low amount of dose and short time duration. The results confirmed the presence of anti-inflammatory compounds in the stem bark extract.

4.4.8.iv) Anti-inflammatory activity of E. obtusifolia leaf

Methanolic extracts of E. obtusifolia were also effective in anti-inflammatory property. Methanolic extract was also tested at three doses for three time durations i-e 60 minutes, 120 minutes and 180 minutes. After 60 minutes the effectiveness of methanolic extract increased greatly and showed highly significant (P<0.01) results, by 77% inhibition at 100 mg/kg (0.1167 + 0.00333) and significant result by 70% inhibition at 200 mg/kg (0.1333 + 0.00333; P<0.01) and 70% inhibition at 300 mg/kg (0.1133 + 0.00577) respectively, as compared to the positive control (diclofenac sodium) (0.6000 + 0.5774) which showed a 86% inflammation reduction after 60 minutes (Table 4.25; Graph 4.33). After 120 minutes the inactivity was increased up to some extent and significant (P<0.05) (77%) results were found at two doses 100 mg/kg (0.1167 + 0.00333) and 200 mg/kg (0.1167 + 0.00333) respectively, and highly significant results were seen at 300 mg/kg, which was (0.1000 + 0.01528) 84% in contrast to reference drug, diclofenac sodium (0.5800 + 0.5773; 90%) (Graph 4.34). The effectiveness was maintained after 180 minutes. The anti-inflammatory results after three hours were highly significant (P<0.01) 100 % at all three doses of 100 mg/kg (0.1033 + 0.00882), 200 mg/kg (0.1033 + 0.00333) and 300 mg/kg (0.0867 + 0.00667) as compared to the positive control

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(0.5300 + 0.22189) which showed 100% anti-inflammatory activity (Table 4.24; Graph 4.35). Normal saline (negative control) showed non-significant (P>0.05) anti- inflammatory results at all the three time durations i.e. after 60 minutes (0.3000 + 0.14988; 0%), 120 minutes (0.2600 + 0.00365; 12%) and 180 minutes (0.2500 + 0.02266; 19%). Results revealed the effectiveness of high doses (200 mg/kg and 300 mg/kg) and long-time durations.

4.4.8.v) Anti-inflammatory activity of E. obtusifolia fruit

The fruit extract of E. obtusifolia was tested for inflammation inhibition at three time durations of 60 minutes, 120 minutes and 180 minutes. The effectiveness of the extract increased with time and doses. All the anti-inflammatory results after 60 minutes were variable i-e non-significant (P>0.05) (0.1233 + 0.01202) 64% at 100 mg/kg, significant (P<0.05) (0.1300 + 0.01528) 75 % at 200 mg/kg and highly significant (P<0.01) (0.1067 + 0.0000) 85% at 300 mg/kg, as compared to the positive control (diclofenac sodium) (0.6000 + 0.5774) which showed 86 % inflammation reduction after 60 minutes (Graph 4.33). After 2 hours the effectiveness increased at all three dose levels (100 mg/kg, 200 mg/kg and 300 mg/kg) which were significant (P<0.05) (0.1133 + 0.01453) 74 % at 100 mg/kg and highly significant (P<0.01) (0.1167 + 0.01333) 87 % and (0.1067 + 0.00882) 85 % at 200 mg/kg and 300mg/kg in contrast to reference drug, diclofenac sodium (0.5800 + 0.5773; 90%) (Graph 4.34). After 3 hours the results were highly significant (P<0.01) at all three dose levels e.g (0.0933 + 0.01202) 97 %, (0.1033 + 0.00667) 100 % and (0.0933 + 0.01000) 100 % at 100 mg/kg, 200 mg/kg and 300 mg/kg, respectively as compared to positive control (0.5300 + 0.22189) which showed 100% anti-inflammatory activity (Table 4.25) (Graph 4.35). Normal saline (negative control) showed non-significant (P>0.05) anti-inflammatory results at all three time durations e.g. after 60 minutes (0.3000 + 0.14988; 0%), 120 minutes (0.2600 + 0.00365; 12%) and 180 minutes (0.2500 + 0.02266; 19%). Phytochemical screening of experimented plant confirmed the presence of important anti-inflammatory bioactive constituent.

4.4.8.vi) Anti-inflammatory activity of E. obtusifolia stem bark

The stem bark extract was also tested for anti-inflammatory action. The percent inhibition against inflammation was increasing with time and after 1 hour the results

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were significant (P<0.05) i.e 73% at 100mg/kg (0.1267 + 0.01667), 70% at 200 mg/kg (0.1267 + 0.01333) and highly significant 79% at 300 mg/kg (0.1167 + 0.01453) as compared to the positive control (diclofenac sodium) (0.6000 + 0.5774) which showed 86 % inflammation reduction after 1 hour (Graph 4.33). After 2 hours the effectiveness was increased and the extract showed significant results (P<0.05) (0.1167 + 0.01000) 79% at dose level 100 mg/kg, and highly significant results (P<0.01) (0.1100 + 0.01333) 90% at 200 mg/kg and (0.1100 + 0.00667) 90 % at 300 mg/kg in contrast to the reference drug, diclofenac sodium (0.5800 + 0.5773; 90%) (Graph 4.34). After 3 hours the inflammation reduction was increased and highly significant (P<0.01) i.e (0.1000 + 0.00333) 100%, (0.1000 + 0.01000) 97 % and (0.096 + 0.01453) 100% at all three doses (100mg/kg, 200 mg/kg, 300 mg/kg) respectively as compared to positive control (0.5300 + 0.22189) which showed 100% anti-inflammatory activity (Table 4.25) (Graph 4.35). Normal saline (negative control) showed non-significant (P>0.05) anti-inflammatory results at all three time durations i.e. after 60 minutes (0.3000 + 0.14988; 0%), 120 minutes (0.2600 + 0.00365; 12%) and 180 minutes (0.2500 + 0.02266; 19%).

4.4.8.vii) Comparison of anti-inflammatory action of E. serrata and E. obtusifolia leaf, fruit and stem bark

4.4.8.vii.a) Leaf i) After 60 minutes

After one hour the anti-inflammatory action of both leaf extracts were almost the same. Slight percent changes were noticed among E. serrata and E. obtusifolia leaf extract. E. obtusufolia showed most effective (77%) and E.serrata showed (75%) results after 60 minutes. As a whole, both plant extracts showed significancy (P <0.05) as compared to diclofenac sodium (86%).

After 2 hours

By comparing the effects of both plants after 2 hours, the highest significant (P <0.01) inhibition was (84%) observed in E. obtusifolia at dose of 300 mg/kg and 82% at dose

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of 100 mg/kg in E. serrata. The remaining results were significant (P <0.05) in contrast to control (diclofenac sodium) (90%).

After 3 hours

Inhibition on carrageenan induced paw edema was observed after 3 hours. Results revealed that minimum inhibition was 88% (highly significant P<0.01) showed by E. serrata at dose of 100 mg/kg, and the remaining results were highly significant (P <0.01) i.e 100% at all doses in comparison with positive control (100%).

4.4.8.vii.b) Fruit i) After 1 hour

Maximal paw edema inhibition (highly significant P<0.01) in mice after 60 minutes was (85%) as observed in E. obtusifolia at dose of 300 mg/kg. Rest of all results of E. serrata and E. obtusifolia were significant (P<0.05) as compared to positive control (86%) except at 100 mg/kg dose in E. obtusifolia (non-significant P>0.05). Results showed the potency of both plants against inflammation.

After 2 hours

In case of fruit, E. serrata showed maximum inhibition at all three doses as compared to E. obtusifolia. Maximum inflammation reduction of the fruit of E. serrata was (100 %) at a dose of 300 mg/kg. As a whole the results of both fruit extracts results were highly significant (P<0.01) as compared to positive control (90%).

After 3 hours

Anti-inflammatory activities showed by both plants were dose and time dependent. All the observations were highly significant (P<0.01) at all doses, compared to diclofenac sodium (100%).

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4.4.8.vii.c) Stem bark i) After 1 hour

After one hour maximum (highly significant P<0.01) edema inhibition (79%) was induced by E. obtusifolia at dose of 300 mg/kg. whereas the remaining results were significant (P<0.05) at all doses showed by both plant extracts in contrast to positive control (86%). Results revealed that both plants were effective in inflammation inhibition.

After 2 hours

After two hours both extracts showed highly significant (P<0.01) results as compared with positive control diclofenac sodium. The results revealed the effectiveness of both plants at this time duration. This inflammation inhibition activity showed by the stem bark was due to the presence of major bioactive compounds (anthraquinone, flavonoids, phenols).

After 3 hours

After three hours methanolic extracts of both the experimented plants showed highly significant (P<0.0.1) results at all three doses (100 mg/kg, 200 mg/kg and 300 mg/kg), compared with standard (diclofenac sodium). These results suggested the presence of bioactive compounds in both plants which showed inflammation inhibition in the paw of mice.

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Table 4.24. Anti-inflammatory activity of E. serrata

S.No Treatment Dose Normal Paw Paw volume Paw volume after Paw volume after (mg/kg) Volume (mL) after 1 hr 2 hr 3 hr (Mean ± SEM) 01 Normal saline - 0.1383±0.00477 0.3000±0.14988 0.2800±0.00365 0.2500±0.02266 02 Standard drug 10 0.5300±0 .22189 0.600±.05774** 0.5500±.05773** 0.5300±0.22189** 03 Leaf 100 0.0933± 0.00667 0.1200 ±.031808* 0.1100± .01732** 0.0867 ±0.00882** 200 0.0867 ±0.00882 0.1167±0.00667* 0.1067±0.00333** 0.0967± 0.01453** 300 0.0967± 0.01453 0.1200±0.01732* 0.1133± 0.0088** 0.0967±0 .00882** 04 Fruit 100 0.0933± 0.00667 0.1200±0.00000* 0.1000± 0.00577** 0.0900 ±0.00577** 200 0.0900 ±0.00577 0.1200± 0.00333* 0.1100±0.00577** 0.0900± 0.00667** 300 0.0867 ±0.00882 0.1100±0.01000* 0.0867 ±0.01333** 0.0867 ±0.00667** 05 Stem bark 100 0.1000 ±0.00577 0.1200± 0.01528* 0.1167± 0.01202** 0.1000 ±0.00577** 200 0.10000±0.00577 0.1267± 0.00882* 0.1133±0.00667** 0.10000±0.00577** 300 0.0967±0 .00882 0.1233±0.00667* 0.1067 ±0.01856** 0.0933± 0.01202**

The data of the anti-infllammatory activity of E. serrata are reported as mean ± SEM and were analyzed through ANOVA following Dunnett’s post-hoc, significant at *P<0.05, highly significant at **P<0.01 and non significat P>0.05

Table 4.25. Anti-inflammatory activity of E. obtusifolia

S.No Treatment Dose Normal Paw Paw volume after 1 Paw volume after Paw volume after 3 (mg/kg) Volume (mL) hr 2 hr hr (Mean ± SEM) 01 Normal saline - 0.1383±0.00477 0.3000±0.14988 0.2800±0.00365 0.2500±0.02266 02 Standard drug 10 0.5300±0.22189 0.5800±.05774** 0.0.550±0.57735** 0.5300±0.22189** 03 Leaf 100 0.1033±0.00333 0.1300±0 .00577* 0.1167± 0.0033* 0.1033±.00882** 200 0.1033±.00882 0.1167 ±0.00333* 0.1267 ±0.00667* 0.1033±0.00333** 300 0.0867 ±0.00882 0.1233±0 .00333* 0.1000±0.01528** 0.0867± 0.00667** 04 Fruit 100 0.0900 ±0.01000 0.1233±0 .01202 0.1133± 0.01453* 0.0933 ±0.01202** 200 0.1033±0.00333 0.1300±0 .01528* 0.1167±0.01333** 0.1033± 0.00667** 300 0.0933 ±0.01202 0.1067 ±0.00000** 0.1067± 0.00882** 0.0900 ±0.01000** 05 Stem bark 100 0.1000± 0.01000 0.1267±0 .01333* 0.1167± 0.01000* 0.0967 ±0.00333** 200 0.0967 ±0.00333 0.1167± 0.01333* 0.1100±0 .01333** 0.0967± 0.01000** 300 0.0967 ±0.00333 0.1167± 0.01453** 0.1100 ±0.00667** 0.0967 ±0.01453**

The data of the anti-infllammatory activity of E. obtusifolia are reported as mean ± SEM and were analyzed through ANOVA following Dunnett’s post-hoc, significant at *P<0.05, highly significant at **P<0.01 and non-significant P>0.05

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Plate A Plate B

Plate C Plate D

Fig. 4.34. Plate A: Measurement of normal paw size on plethysmometer Plate B: Adminstration of drug through injection Plate C: Marking of swiss albino mice tail for identification Plate D: Measurement of paw inflammation after specific time period after drug administration on plethysmometer.

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SD (10 mg/kg) ** ESL (100 mg/kg) 85 74.68 75.48 72.6 ESL (200 mg/kg) 74 74.6 72 74 ESL (300 mg/kg) 72 76 ESF (100 mg/kg) ESF (200 mg/kg) ESF (300 mg/kg) ESSB (100 mg/kg) ESSB (200 mg/kg) ESSB (300 mg/kg)

Graph 4.30. Anti-inflammatory action of E. serrata after one hour.

SD (10 mg/kg) ESL (100 mg/kg) 92 ESL (200 mg/kg) 84.28 85.7 84.2 84.28 ESL (300 mg/kg) 85.7 88.56 82.3 ESF (100 mg/kg) 82.84 85.78 ESF (200 mg/kg) ESF (300 mg/kg) ESSB (100 mg/kg) ESSB (200 mg/kg) ESSB (300 mg/kg)

Graph 4.31. Anti-inflammatory action of E. serrata after two hours

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SD (10 mg/kg) ESL (100 mg/kg) 95 ESL (200 mg/kg) 81.12 82.3 83.6 81.12 81.74 ESL (300 mg/kg) 83.76 81.74 ESF (100 mg/kg) 82.34 83.01 ESF (200 mg/kg) ESF (300 mg/kg) ESSB (100 mg/kg) ESSB (200 mg/kg) ESSB (300 mg/kg)

Graph 4. 32. Anti-inflammatory action of E. serrata after three hours

SD (10 mg/kg) EOL (100 mg/kg) 92 83.32 83.8 EOL (200 mg/kg) 84.2 84.4 82.8 EOL (300 mg/kg) 84.74 85.7 EOF (100 mg/kg) 84.74 84.28 EOF (200 mg/kg) EOF (300 mg/kg) EOSB (100 mg/kg) EOSB (200 mg/kg) EOSB (300 mg/kg)

Graph 4.33. Anti-inflammatory action of E.obtusifolia after one hour.

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SD (10 mg/kg) EOL (100 mg/kg) 95 EOL (200 mg/kg) 81.12 81.9 80.58 81.72 EOL (300 mg/kg) 80.58 83.024 83.64 EOF (100 mg/kg) 80.52 82.36 EOF (200 mg/kg) EOF (300 mg/kg) EOSB (100 mg/kg) EOSB (200 mg/kg) EOSB (300 mg/kg)

Graph 4.34. Anti-inflammatory action of E. obtusifolia after two hours.

SD (10 mg/kg) EOL (100 mg/kg) 95 EOL (200 mg/kg) 81.12 81.9 80.58 81.72 80.58 EOL (300 mg/kg) 83.024 83.64 EOF (100 mg/kg) 80.52 82.36 EOF (200 mg/kg) EOF (300 mg/kg) EOSB (100 mg/kg) EOSB (200 mg/kg) EOSB (300 mg/kg)

Graph 4.35. Anti-inflammatory action of E. obtusifolia after three hours.

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4.4.8) Anti-diarrhoeal activity

Anti-diarrhoeal activity of leaf, fruit and stem bark of methanolic extracts of E. serrata and E. obtusifolia was conducted by gastrointestinal motility model using overnight starved swiss albino mice (Sisay et al., 2017)

4.4.8.i) Anti-diarrhoeal activity of Ehretia serrata leaf

In the present bioassay the antidiarrheal potency of the leaf of methanolic extract of E. serrata was tested. Results revealed that the percentages of intestinal transportation of charcoal were 45.1%, 38.5% and 36.5% with mean values of 26.000 + 0.57735, 22.3367 + 0.33168 and 20.7667 + 0.23333 respectively at dose levels of 100 mg/kg, 200mg/kg and 300 mg/kg body weight respectively, compared to Atropine with values 8.333 + 0.666 value. The percent anti-diarrhoeal effect was 54.9, 61.5 and 63.5 with respect to control had 86% anti-diarrheal effect. The results showed that the methanolic extract of the leaf was significant (P<0.05) at higher doses of 200mg/kg and 300 mg/kg (Table 4.26) (Graph 4.36). The presence of many phytochemical constituents such as phenols and tannin might be the reason for the good effect of leaf of the plant.

4.4.8.ii) Anti-diarrhoeal activity of E. serrata fruit

The antidiarrheal potency of the fruit methanolic extract was also tested which gave significant results (P<0.05) at higher doses of 200 mg/kg and 300 mg/kg. The extract showed that percentages of intestinal transportation of charcoal were 52.64, 39.26 and 39.5 % with mean values 30.0300 + 2.50484, 22.7333 + 5.62178 and 23.7000 + 4.97226 at three dose of 100 mg/kg, 200 mg/kg and 300 mg/kg body weight respectively, compared to atropine 14% with 8.333 + 0.666 value (Table 4.26) (Graph 4.36). The percentages of anti-diarrhoeal effect were 47.36 %, 60.74% and 60.5% with respect to atropine which had 86% anti-diarrhoeal effect.

4.4.8.iii) Anti-diarrhoeal activity of E. serrata stem bark

The results were significant (P <0.05) at 100 mg/kg and highly significant (P <0.01) at 200 mg/kg and 300 mg/kg. Stem bark showed i.e 37.21% (22.3333+7.05534) at 100 mg/kg, 25.91% (14.800 + 2.61534) at dose of 200 mg/kg and 20.89% (12.333 + 1.76383) at dose of 300 mg/kg, intestinal transportation of charcoal as compared to

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atropine which showed 14% (8.3333 + 0.6667) transportation of charcoal (Table 4.26). The percentages of anti-diarrhoeal effect were 62.79%, 74.00% and 79.11% with respect to atropine had 86% anti-diarrhoeal effect (Graph 4.36).

4.4.8.iv) Anti-diarrhoeal activity of E. obtusifolia leaf

Leaf extract of E. obtusifolia showed effectiveness against diarrhoea. The methanolic extract showed significant (P<0.05) % intestinal transportation of charcoal at 100 mg/kg which was 35.05% (20.3367 + 2.02732) and highly significant (P<0.01) at dose level of 200mg/kg (15.2000 + 0.11547; 25.80%) and 300 mg/kg (14.000 + 0.00000; 24.13 %) compared to atropine which showed 14% intestinal transportation of charcoal with mean value of 8.333 + 0.6666 (Table 4.26). The percentages of anti-diarrhoeal effects were 64.95%, 74.2% and 75.87% with respect to atropine having 86% anti- diarrhoeal effect (Graph 4.37). The results revealed the presence of some anti-diarrheal compounds in the investigated plant.

4.4.8.v) Anti-diarrhoeal activity of E. obtusifolia fruit

E. obtusfolia fruit showed highly significant (P<0.01) anti diarrhoeal-activity at 300mg/kg body weight. At 100 mg/kg and 200 mg/kg it showed significant (P<0.05) results. Fruit extract decreases transit of charcoal meal through the small intestine in castor oil treated mice. Charcoal motility percentages in small intestines were 43.1, 34.45 and 20.77%, with mean values of 25.0333 + 2.88694, 20.3333 + 1.76383 and 12.4667 + 1.24544 respectively, compared to atropine (14% with mean values 8.333 + 0.6666) (Table 4.26). Percent antidiarrheal effects showed the effectivity of high doses (300 mg/kg; 79.23) with respect to low doses of 200 mg/kg; and 100 mg/kg; 56.9% (Graph 4.37).

4.4.8.vi) Anti-diarrheal activity of E. obtusifolia stem bark

Stem bark of E. obtusifolia was also examined for anti-diarrhoeal potency and highly significant (P<0.01) results were obtained at all doses. Charcoal transportation percentages were 32.27% with mean value 19.3667 + 0.876238 at 100 mg/kg, 30.45% with mean value of 17.9667 + 1.03333 at 200 mg/kg, and 27.00% with mean value of 19.3667 + 0.87623 at 300 mg/kg, compared to atropine, 14% with mean value of 8.3333

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+ 0.66667 (Table 4.26). Percent anti-diarrhoeal effect showed the significance at all doses of 67.73%, 69.55% and 73.00% with respect to Atropine (86%) (Graph 4.37).

4.4.8. Vie) Comparison of anti-diarrhoeal potency of E. serrata and E. obtusifolia leaf, fruit and stem bark

4.4.8.vii.a) Leaf

Leaf extract of E. obtusifolia was more effective than E. serrata. Results revealed that E. obtusifolia showed higher significant (P<0.01) results than E. serrata at all the three doses (Graph 4.38).

4.4.8.vii.b) Fruit

Fruit methanolic extract of both plants are compared with each other for anti-diarrhoeal potency, and it is observed that just like a leaf, fruit extract of E. obtusifolia was more effective (P<0.01) against diarrhoeal disease as compared E. serrata (Graph 4.38).

4.4.8.vii.c) Stem bark

Results of stem bark extracts of two plants were compared and it was observed that unlike leaf and fruit, stem bark of E. serrata showed better performance (P<0.01) against diarrhoea than E. obtusifolia at all doses (Graph 4.38).

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Table 4.26. Anti-diarrhoeal activity of E. serrata and E. obtusifolia leaf, fruit and stem bark

S. No Drug treatment Dose Distance travelled % intenstinal % Anti- by charcoal transportation diarrhoeal effect 01 Normal saline 5 mL 51.333±9.222 87.00 13 02 Atropine 5 mg/kg 8.3333+0.66667** 14 86 (standard) 03 ESL 100 mg/kg 26.000+0.57735 45.1 54.9 200 mg/kg 22.3367+0.33168* 38.5 61.5 300 mg/kg 20.7667+0.23333* 36.5 63.5 04 ESF 100 mg/kg 30.0300+2.50484 52.64 47.36 200 mg/kg 22.7333+5.62178* 39.26 60.74 300 mg/kg 23.7000+4.97226* 39.5 60.5 05 ESSB 100 mg/kg 22.3333+7.05534* 37.21 62.79 200 mg/kg 14.8000+ 2.61534** 25.91 74.0 300 mg/kg 12.3333+1.76383** 20.89 79.11 06 EOL 100 mg/kg 20.3367+2.02732* 35.05 64.95 200 mg/kg 15.2000+0.11547** 25.80 74.2 300 mg/kg 14.0000+0.0000** 24.13 75.87 07 EOF 100 mg/kg 25.0333+2.88694 43.1 56.9 200 mg/kg 20.3333+1.76383* 34.45 65.55 300 mg/kg 12.4667+1.24544** 20.77 79.23 08 EOSB 100 mg/kg 19.36670.876238** 32.27 67.73 200 mg/kg 17.9667+1.03333** 30.45 69.55

300 mg/kg 16.2000+2.61534** 27.00 73.00

The data of the anti-diarrheal activity of E. serrata and E. obtusifolia are reported as mean ± SEM and were analysed through ANOVA following Dunnett’s post-hoc, significant at *P<0.05 and highly significant at **P<0.01 and non-significant at P>0.05.

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Plate A Plate B

Plate C Plate D Fig. 4.35. Plate A: Charcoal administration in albino swiss mice Plate B: Killing of albino swiss mice Plate C: Dissection of albino swiss mice Plate D: Charcoal motility measurement in small intestine

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100 90 ** 80 * ** 70 * * ** 60 * * 50

40 diarrhoeal activity diarrhoeal

- 30 20 10

0 Percent anti Percent

Graph 4.36. Percent anti-diarrhoeal activity of E. serrata

100 90 ** ** 80 ** ** ** ** 70 * * ** 60

50 diarrhoeal activity diarrhoeal - 40 30 20

10 Percent anti Percent 0

Graph 4.37. Percent anti-diarrhoeal activity of E. obtusifolia

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52.64

50 45.1 43.1 39.26 39.5 40 38.5 36.5 37.21 35.05 34.45 32.27 30.45 30 27 25.91 25.8 24.13 20.89 20.77

20 Percentintenstinal transportation

100mg/kg 100mg/kg 200mg/kg 200mg/kg 300mg/kg 300mg/kg 100mg/kg 100mg/kg 200mg/kg 200mg/kg 300mg/kg 300mg/kg 100mg/kg 100mg/kg 200mg/kg 200mg/kg 300mg/kg 300mg/kg ESL EOL ESL EOL ESL EOL ESF EOF ESF EOF ESF EOF ESSB EOSB ESSB EOSB ESSB EOSB Dose Level

Graph 4.38. Comparison of % intenstinal transportation of E. serrata and E. obtusifolia

C.oil= Castor oil

Atr= Atropine

ESL=Ehretia serrata leaves

ESF= Ehretia serrata fruit

ESSB= Ehretia serrata stem bark

EOL= Ehretia obtusifolia leaf

EOF= Ehretia obtusifolia fruit

EOSB= Ehretia obtusifolia stem bark

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CHAPTER-5

DISCUSSION

The current research was divided into three parts i.e pharmacognostic, physicochemical and pharmacological evaluation of leaf, stem bark and root of Ehretia serrata Roxb. and Ehretia obtusifolia Hochst. Ex A. DC which belongs to the family Boraginaceae.

E. serrata is locally known as puran and punna in Pakistan. Morphological study of this plant revealed that it is a small size deciduous tree, less than 10 m tall. Its leaf is simple. Shape of leaf is oblong (5-16 cm long and 2.8-6.5 cm wide) with serrulated margins and glabrous surface. E. serrata contains drupe globose type of fruit. The colour of fruit is yellowish green-light when unripe and black when ripe. Stem bark is irregular with up to 2.5 cm wide, dark brown outer color and whitish golden inner surface color. Ackin et al. (2010) also illustrated the taxonomic significance of morphological and anatomical features of three Anchusa species (Boraginaceae), Anchusa undulata subsp. hybrida (Ten.) Coutinho, A. azurea Miller var. azurea and A. pusilla Guşul., collected from Northern Turkey. E. obtusifolia is a deciduous small shrub or small tree up to 6 m tall; straggling or with several branches from the base. The Leaf is oblong-elliptic or oboviate with obtuse apex, entire margins and reticulate venation. Microscopic study of E. serrata showed the presence of conical trichomes and anomocytic type of stomata on both surfaces where as anisocytic type of stomata was observed only on the lower epidermis with stomatal index 36.7μ. Upper surface showed 30.4 μ stomatal index. Aarthi et al. (2014) studied another specie of this genus i.e Ehretia microphylla. The pharmacognostic results showed the presence of unicellular trichomes and anomocytic stomata in the leaves of E. microphylla which shows resemblance with our results. 2 Other parameter includes palisade ratio (6.4 + 0.894 per mm ), vein Islet number (19.4 + 1.79 per mm2), stomata number (21.8 + 1.78 per mm2) and vein termination number (10.6 + 1.987 per mm2). E. obtusifolia is a deciduous small shrub up to 6 m tall; straggling or with several branches from the base; color of bark is grey. Leaves are alternate and simple; petiolate elliptical, oblong-elliptic or obovate, pubescent-velvety; apex of the leaves are obtuse to acuminate. Fruit is a globose drupe 5–6 mm in diameter, orange or red, splitting into two 2-seeded pyrenes. Anatomical study showed two types of trichomes i.e simple conical trichomes and capitate glandular trichome on both surfaces. Stomatal cluster was observed only on the lower epidermis with three types

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of stomata i.e anomocytic, anisocytic and hemiparacytic. Anomocytic stomata in the family Boraginaceae was previously described by Özörgücü (1991). In addition, Metchalfe & Chalk (1979) pointed out that there were both anisocytic and anomocytic stomata in this family. Other observed parameters were palisade ratio (6.2 + 0.81 per mm2), vein islets number (18.6 + 1.14 per mm2), vein termination number (8.2 + 0.828 mm2), stomatal number (21.8 + 1.78 per mm2) and stomatal index (35.7 + 6.12 μ). It was determined that in some species of family Boraginaceae of very xeric habitats such as A. crispa, stomatal density is higher in the adaxial rather than in the abaxial surface (Selvi & Bigazzi, 2001).

Stem bark anatomy of both experimented plants showed clear layers of cork, cork cambium, phloem elements, medullary rays and tracheids, etc. Fruit anatomy of both plants showed a clear distinction between three layers of pericarps and seeds. Both species have drupes with a four-parted endocarp, which is also confirmed by Gottschling and Hilger (2004) in their report on characterization of a novel fruit type found in Ehretia (Ehretiaceae, Boraginales). Powder drug microscopy of leaf and stem bark of E. serrata and E. obtusifolia was carried out and observed stomata, epidermal cells, fibers, trachieds and vessels, etc. Arthi et al. (2014) also studied the crude powder drug of E. microphylla Lamk aerial parts and the results showed similarity with our results by having abundant unicellular trichomes, foliar epidermal cells with anomocytic (Ranunculaceous) stomata and druses of calcium oxalate crystals which are diagnostic characters to identify the drug.

Results of qualitative preliminary phytochemical screenings of examining parts of E. serrata and E. obtusifolia are given, presenting the presence of phenol compounds, starch, protein, reducing sugar, steroids, terpenoids, fats, aldehydes, anthraquinones, glycosides, amino acids, saponins, alkaloids, tannins, phlobatannin. Zara et al., (2012) researched the ethyl acetate fraction of leaves of E.serrata which also contain the highest flavonoid as well as phenolic content like our results. Ogundajo and Ashafa (2017) studied the bioactive constituent’s E. cymosa leaf extract. The phytochemistry tests of the fractions revealed the presence of tannins, phenols, flavonoids, steroids, terpene, alkaloid, and cardiac glycosides. Similarly, results of a fluorescence study of leaf, stem bark and fruit of E. serrata and E. obtusifolia are also given. Iqbal et al. (2005) also investigated E.obtusifolia for the presence of Phytochemicals and they

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isolated phenolic compounds i.e methyl 2‐O‐feruloyl‐1a‐O‐vanillactate, caffeic anhydride and trans 4‐hydroxycyclohexyl‐2‐O‐p‐coumaroyl β‐D‐glucopyranoside from the AcOEt‐soluble fraction of E. obtusifolia, along with methyl rosmarinate and rosmarinic acid, which are reported for the first time from this species. Presence of these important bioactive compounds in both species give them medicinal importance.

Results of elemental analysis showed the presence of some essential elements in all examined parts of these two plants. The most abundant elements found in all three parts of E. serrata and E. obtusifolia were Mg, Na, and Fe and which may contribute to analgesic and anti-inflammatory effects of these plants. Torane et al. (2010) also analyzed elements found in leaves of E. laevis and found Mg, Ca, K, Na and Fe in abundance which showed similarity with our results.

It is expected that E. serrata and E. obtusifolia consumption does not induce anti- nutritional effects (Oatway et al., 2001; Shamsuddin, 2002), on the contrary it may induce health benefits. Proximate analysis of both plants showed carbohydrate, proteins, fibers, fats and moisture in fairly large amounts. Torane et al. (2010) also justified nutritive claims of the leaves of E. laevis by the nutritional analysis and confirm the presence of various phytoconstituents such as starch, proteins, sugars, alkaloids, steroids and tannins in it. The protein content in all investigated parts of E. serrata and E. obtusifolia (14% d.w in ESL; 9.26% d.w in EOL; 11.37% d.w in ESF; 7% d.w in EOF, 7% d.w in ESSB and 11.37% d.w in EOSB) was higher than for common fruits and vegetables, for example, apple (Malus domestica) has 1.9% d.w., rhubarb (Rheum rhabarbarum) 14.08% d.w., and dragon fruit (Hylocereus undatus) 10.26% d.w. but lower than from E. tinifolia pulp (16.5% d.w.) (USDA, 2009; Ledezma-Solano et al., 2010; Peo-Leon, 2012).

Cytotoxic activity is an effective way used to detect for cell-line toxicity and anti-tumor activity (Jamal et al., 2017). E. serrata leaf extract showed the highest (P<0.01) cytotoxic activity (83.3%) with the LD50 values 15.29 µg/mL at dose level 1000 µg/mL and in E. obtusifolia the fruit methanolic extract showed highest (P<0.01) (80.0%) cytotoxic activity at 1000 µg/mL with an LD50 value of 16.07 µg/mL. Sempombe et al. (2014) also worked on cytotoxicity of another species of genus Ehretia i.e E. amoena. and confirmed the potency of the plant against trypanosomiasis and 3-cell line panel

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consisting of TK10 (renal), UACC62 (melanoma) and MCF7 (breast) cancer cells. All experimented parts of both plants showed non-significant (P>0.05) Phytotoxic activity at all dosese of 10, 100 and 1000 µg/mL. No literature available on this genus for pytotoxic activity on any other specie but Khurm et al. (2016) investigated Heliotropium strigosum Willd. (Family Boraginaceae) for phytotoxicity. Their results showed moderate phytotoxic potential with percent inhibition of 50% at the concentration of 1000 µg/mL. These results revealed that this family has very little phytotoxic potency.

The anti-oxidant effect of all parts (leaf, stem bark and fruit) of both plants was dose dependent. High (94.1%) DPPH inhibition was shown by stem bark extract of E. serrata at dose level 1000 µg/mL after 60 min and fruit extract of E. obtusifolia (85.01%) at level 1000 µg/mL after 30 minutes same like anti-oxidant activity showed by ethanolic extract of E. cymosa (Sarkodie et al. (2015). Based on IC50 (µg/mL) stem bark of E. obtusifolia and leaf of E. serrata showed significant values as compared to ascorbate as of fruits of E. laevis. This antioxidant potential along with good phenolic content tends to pave these plants suggested for domesticate and health supplement which will definitely fragile the disease prone (Sivasankari et al., 2013).

Plants are important source of potentially useful structures for the development of new chemotherapeutic agents. The first step towards this goal is the in vitro antibacterial activity assay (Tona et al., 1998). Many reports are available on the antiviral, antibacterial, antifungal, anthelmintic, antimolluscal and anti-inflammatory properties of plants (Samy and Ignacimuthu, 2000; Palombo and Semple, 2001; Kumaraswamy et al., 2002; Stepanovic et al., 2003). However, not many reports are available on the exploitation of antifungal or antibacterial property of plants for developing commercial formulations for applications in crop protection. In present study, methanolic extract of both plants was not that much more effective against bacterial strains inhibition. The prominent antibacterial zone (7 mm) was observed in the leaf methanolic extract of E. serrata as compared to reference drug (31.5 mm) at dose level 1000 µg/mL. Similarly, different parts of E. obtusifolia tested against various bacteria which showed no significant results and the most prominent (7 mm) anti-bacterial zone was observed at dose level 1000 µg/mL of leaf extract compared to reference drug (30.2 mm). For instance, leaves extract of E. abyssinica showed high antimicrobial activity against

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Pseudomonas aeruginosa, followed by Staphylococcus aurues, while the most resistant bacteria were Micrococcus (Gumgumjee and Hajar, 2015).

Toxicological studies showed that both plants are safe for human use. Analgesic effect was found dose dependent and time dependent and all parts of both investigated parts (P<0.05) showed significant results. The highly significant analgesic effect was shown by all experimented parts (P<0.01) at all dose level 100 mg/kg, 200 mg/kg and 300 mg/kg over 60 minutes in E. Serrata except stem bark. Stem bark of E. obtusifolia showed highly significant (P<0.01) at all dose after 60 minutes. A decoction of the roots is taken as a painkiller, specifically against abdominal pain in Zimbabwe and in Tanzania. These results confirmed the traditional use of both plants to treat headache and toothache in many countries of the world (Velappan & Thangaraj, 2014; Coates Palgrave, 1983).

The pharmacological studies confirmed that the crude extracts or individual compounds from the genus Ehretia showed antioxidant, anti-inflammatory, as well as anti-snake venom property. (Shukla and Kaur, 2018). Anti-inflammatory effect of all three investigated parts of E. serrata and E. obtusifolia were observed and it is noticed that all parts of E. serrata showed high significancy (P<0.01) at all dose i.e 100 mg/kg, 200 mg/kg and 300 mg/kg after 2 and 3 hours. Similar results were shown by E. obtusifolia. Jyothirmai et al. (2016) evaluated the anti-inflammatory potency of E. leavis Roxb. and get similar results as ours.

Ehretianone together with known sterols, exhibited anti snake venom as well anti- diarrhoeal property which is a common bioactive compound of genus Ehretia (Selvanayagam et al., 1996). Antispasmodic effects of methanolic extract of leaf stem bark and fruit of E. serrata and E. obtusifolia was tested in swiss albino mice. Stem bark methanolic extract showed highly significant (P<0.01) results at dose of 200 mg/kg and 300 mg/kg. E. obtusifolia causes better relaxation of the smooth muscles at very low concentrations. Leaf extract at dose of 200 mg/kg and 300 mg/kg, fruit at level of 300 mg/kg and stem bark at all dose showed highly significant (P<0.01) results thus providing a scientific proof for its ethnopharmacological use as an antispasmodic drug. The mixture of α and β-amyrin, baurenol exhibited analgesic activity (51%), anti- inflammatory activity (20%), anti-diarrheal activity (29%) and moderate antimicrobial activity (Villasenor et al., 2004). These results open new gates for new researchers to

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investigate actual cause of antispasmodic potency of both plants. The current research has revealed many and different medicinal properties of E. serrata and E. obtusifolia and both can be exploited for preparations of therapeutic drugs commercialy.

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CONCLUSIONS

● Locally in Punjab E. serrata Roxb. is known as “Puran or punna” and its stem bark juice is extensively used in the treatment of fever. The green and unripe fresh fruit of E. serrata is used in the form of a pickle. This specie is distributed in sub-Himalayan and the outer himalaya from Indus eastward to Sikkim. In Pakistan E. serrata is well distributed in sub-himalayan tract from Azad Kashmir westward to Rawalpindi, Islamabad, Murree, Hazara, and Swat.

● Locally in Khyber pkhtunkhwa E. obtusifolia Hocht.ex.A.DC. is called “Ghada boty” and its leaves are used as an infusion to treat sore throat and are rubbed on the gums of infants suffering from teething pains. In Zimbabwe powdered root is added to porridge to treat painful menstruation and infertility in women and an infusion of the roots is taken against retained placenta, and is used to cure abdominal pain. E. obtusifolia occurs in almost all countries of eastern and southern Africa and in Madagascar. It extends Asia up to Pakistan and northwest India.

● Pharmacognostic evaluation shows that E. serrata leaf is oblong-ovate with serrulate margins. Leaf of E. obtusifolia has obtuse shape and pubescent velvet surface. The transverse section of both species through the midrib region showed bifacial arrangement.

● Leaf surface parameters of E. serrata showed that it has a palisade ratio of 6.4±0.894, vein islets number 19.4±1.79 per mm2, vein termination number 10.6±1.987 per mm2, stomatal number 18.4±1.148 per mm2 and stomatal index 36.0±6.09. Two different types of stomata were observed on leaf surfaces in which anomocytic type was most dominant.

● Leaf of E. obtusifolia is simple and anatomically bifacial. Leaf surface values showed a palisade ratio of 6.2±0.81, vein islets number 18.6±1.14 per mm2, vein termination number 8.2±0.828 per mm2, stomatal number 21.8.±1.78 per mm2 and stomatal index 35.7± 6.12 were calculated.

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● Fruit types of both species are globose drupe type. Transverse section of both species shows two portion i.e. fruit wall and seed. The fruit wall is clearly divided into three layers, epicarp, mesocarp and endocarp. Seeds are enclosed individually by endocarp within single mesocarp and epicarp. The seed was non endospermic and contained elongated embryo.

● Stem bark of E. serrata and E. obtusifolia clearly distinguished into phellem, phellogen and phalloderm. Secondary phloem and phloem rays were also seen clearly in the stem bark of both species.

● Leaf powder of E. serrata appeared light green in colour with Hina like pleasant odour and astringent taste while bark powder of this plant appeared yellowish golden in colour with fine odour and sweet taste. Powder microscopy of the studied parts of this plant showed characteristic fragments.

● E. obtusifolia, leaf powder was dark green in color with a pleasant odor and good taste. Bark powder was light golden brown in color with light, pleasant odor and sweet taste. Both glandular and non-glandular trichomes were present on both plant leaf surfaces.

● Qualitative preliminary phytochemical screenings of leaf, fruit and stem bark methanolic extract of E. serrata showed the presence of phenolic compounds, protein, reducing sugars, tannins (leaf and stem bark), phlobataninnin (leaf and fruit), steroids, glycosides, amino acids, saponins, alkaloids and starch.

● Phytochemical screening of E. obtusifolia showed the presence of phenolic compounds, protein, reducing sugars, tannins (leaf and stem bark), phlobataninnin (leaf and fruit), steroids, glycosides, amino acids, saponin, alkaloids and starch

● Mg, Fe and Na were present in fairly good amounts in all parts of both plants. The detected elements were present in all parts of both plants in permissible limits set by WHO.

● Proximate analysis of both plants showed the presence of carbohydrate, proteins, fibers and fats.

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● Both plants were evaluated for toxicological effects and were found to be nontoxic and safe for human use.

● Both plants have slight cytotoxic activity.

● Both plants did not show phytotoxicity in Lemna minor bioassay.

● Both plants showed significant antioxidant activities.

● E. obtusifolia has some good anti-bacterial potential as compared to E. serrata.

● Both plants showed a maximum analgesic effect at all doses. The results were highly significant and dose dependent.

● The anti-inflammatory effect of both plant parts was found to be dose dependent. Like the standard drug, both plants induced reduction of inflammation in mice paw after 1 hour of dose administration and the reduction of inflammation continued with the passage of time. All the doses showed highly significant reduction in inflammation (P < 0.01) after 3 hours of dose administration. All three doses showed a maximum reduction at 2nd and 3rd hour.

● Leaf, fruit and stem bark of E. serrata and E. obtusifolia showed dose dependent effect and showed the maximum effect (P<0.01) at 300mg/kg dose.

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RECOMMENDATIONS

In the light of the valuable findings in the present research work, the following recommendations are presented for perusal.

● Morphological and anatomical study of these plants should be used for intra generic differentiation. This will help in setting parameters for numerous features of the parts used as crude drug.

● The pharmacognostic study of these plants is helpful in standardization and identification as well as adulteration detection in crude drugs. Macroscopical and microscopical features also possess great value in plant systematics.

● Physicochemical analyses such as florescence study and phytochemical screening of E. serrata and E. obtusifolia was helpful in the characterization of powder drug of different parts of these plants and this should be used in future researches via considering its secondary metabolite composition by phytochemist.

● Determination of trace, micro and macro elements and various nutritional components present in the crude drug of different parts of these plants declared its nutritional importance and are helpful in exploring for future research work as a good source of fodder.

● Pharmacological activities such as cytotoxic, pytotoxic, antibacterial, antioxidant analgesic, anti-inflammatory and antispasmodic activities revealed that these plants are safe for human and other animals and should be recommended for extraction of various active constituents responsible for specific effect. These findings should be used to prepare low-cost, effective tonics for various diseases.

● Various conservation measures should be adapted for long lasting, sustainable use of these valuable medicinal plants, which will give support in raising the economic conditions of the local inhabitants.

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