Screening of Selected Medicinal Plants for Anticancer and Biological Activities

SCREENING OF SELECTED MEDICINAL PLANTS FOR ANTICANCER AND BIOLOGICAL ACTIVITIES

KHUSHNOOD UR REHMAN

DEPARTMENT OF BOTANY FACULTY OF CHEMICAL AND LIFE SCIENCES ABDUL WALI KHAN UNIVERSITY MARDAN

SESSION: 2013-18

SCREENING OF SELECTED MEDICINAL PLANTS FOR ANTICANCER AND BIOLOGICAL ACTIVITIES

KHUSHNOOD UR REHMAN

A Thesis Submitted to the Faculty of Chemical and Life Sciences, Abdul Wali Khan University Mardan in Partial Fulfillment of the Requirement for the Degree of

DOCTOR OF PHILOSOPHY IN BOTANY

DEPARTMENT OF BOTANY FACULTY OF CHEMICAL AND LIFE SCIENCES ABDUL WALI KHAN UNIVERSITY MARDAN SESSION: 2013-18

میں نے جب لکھنا سیکھا تھا پہلے تیرا نام لِکھا تھا

DEDICATION Success always Solicits for two things Exertion and fortune. If I am successful, then my Success is the exertion of my parents and teachers Those they made to fulfill my Wishes in every critical time, Moreover, fortune is due to their prayers. Hence, I dedicate my humble effort, the fruit of my thoughts and study to my teachers and parents. Specially to my supervisor Prof. Dr. Muhammad Hamayun

Author’s Declaration

I Khushnood Ur Rehman hereby state my PhD Thesis titled ―Screening of Selected Medicinal Plants for Anticancer and Biological Activities‖ is my own work and has not been submitted previously by me for taking any degree from this university ―Abdul Wali Khan University Mardan‖ or anywhere else in country/world.

At any time, if my statement is found to be incorrect even after my Graduate the University has the right to withdraw my PhD degree.

Signature______Khushnood Ur Rehman Date June 20, 2019

ACKNOWLEDGEMENT

The first and most important acknowledgement is to almighty Allah Who enabled me to complete this tiresome job. In the beginning, it appeared to me a hard nut to crack but Allah, appeared in grace and led me to the assigned destination.

The author is highly indebted to his supervisor Professor Dr. Muhammad Hamayun Chairman Department of Botany AWKUM who is not less than a hero to me, for his invaluable assistance and guidance.

While feeling the sin qua non I consulted the following personalities and I am obliged for their proud and great education and due to this persuasion, my task become possible.

The generously given moral support by my co supervisor Prof. Dr. Sumera Afzal khan, Director Center of Biotechnology, UOP In a one way or another is a source of inspiration for me. I have no words to thank her, but for her immense help I am very thankful.

I owe a very special debt to Prof. Dr. Sadiq Azam, Prof Dr. Gul jan, Prof Dr. Muhib shah, Prof. Dr. Humaira, Dr. M. Irshad, Dr. Abid Ali khan, Dr. Imtiaz ahmad, Prof. Dr. Sher Wali and Prof. Dr. Barakat Ullah for their immense help to complete this task.

Prof. Dr. Amjad Iqbal, Prof. Dr. Muhammad Ibrar and Prof. Dr. Zahid Ali Butt are those shining stars who enlightened my way to the target and troubled their selves much for my favor.

I am greatly obliged for the AWKUM office and laboratory staff, for their immense help during my research.

At the last but not the least, I acknowledge all my teachers who continuously inspired me for the research work.

Khushnood Ur Rehman

TABLE OF CONTENTS

LIST OF TABLES VI

LIST OF FIGURES VII

LIST OF ABBREVIATIONS X

CHAPTER 1 INTRODUCTION 2

1.0 Orientation of the study 2

1.1 Medicinal Plants 3

1.2 Medicinal Plants as a Safe Source of Medicines 5

1.3 Uses of Medicinal Plants 6

1.4 Medicinal Plants of Pakistan 8

1.5 Medicinal Plants and Modern Medicine 9

1.6 Cancer and Medicinal Plants 13

1.7 Phytochemistry of Medicinal Plants 17

1.8 Phytochemical Constituents of Phytomedicine 19

CHAPTER 2 MATERIALS AND METHODS 33

2.1 Collections and Drying of Selected Plants Materials 33

2.2 Extraction and Fractionation 33

2.3 Fractionation of Dried Filtrate on the Basis of Solubility 34

2.4 Antifungal Activity 34

2.4.1 Media preparation for fungal growth 34

2.4.2 Antibacterial Activity 35

2.4.3 Microorganisms Used 35

2.4.4 Media for Bacterial Culture 35

2.4.5 Preparation of Inoculum 35

2.4.6 Preparation of Agar Plates 35

2.4.7 Extract Preparation for Activity 35

2.4.8 Incubation and Measurement of Zone of Inhibition 36

2.5 Phytotoxicity Activity 36

2.5.1 Bioassay 36

2.6 Anticancer Activity 36

2.6.1 Principle of MTT Assay 36

2.6.2 Media Preparation 37

2.6.3 Defrosting Cells 37

2.6.4 Trypsinisation of Cells 37

2.6.5 Freezing Cells 38

2.6.6 Cell Counting 38

2.6.7 Preparing MTT Solution: 38

2.6.8 Dilution of Plant Extracts 38

2.6.9 MTT Assay Methodology 38

2.6.10 Statistical analysis 39

2.7. Antioxidant Assays 39

2.7.1 DPPH free radicals scavenging activity 39

2.8 Heavy Metals Analysis 39

2.8.1 Acid Digestion of Samples 39

2.9 Proximate Analysis 40

2.9.1 Determination of Moisture 40

2.9.2 Determination of Ash 40

2.9.3 Determination of Curd Fat 40

2.9.4 Determination of Protein 41 i. Digestion 41 ii. Distillation 41

iii. Titration 41

2.9.5 Determination of Carbohydrate 42

2.10 Phytochemical Analysis of Plant Material 42

2.10.1 Test for Alkaloids (Mayer’s reagent) 42

2.10.2 Test for Flavonoids 42

2.10.3 Test for Saponins 43

2.10.4 Test for Glycosides 43

2.10.5 Test for Phenols 43

2.11 Isolation of Compounds 43

2.11.1 Methodology of Column Chromatography 43

2.11.2 Thin layer chromatography 44

2.11.3 Spotting and developing the TLC plate 44

2.12 Identification and Spectral Analysis of Compounds 45

2.12.1 Nuclear Magnetic Resonance Spectroscopy (NMR) Analysis 45

2.13 Anticancer Activity of Pure Isolated Compounds 45

2.13.1 Chemicals and reagents 45

2.13.2 Cell culture 45

2.13.3 MTT assay 46

2.13.4 Measurement of ROS generation 46

2.13.5 Annexin-V-FITC apoptosis detection assay 46

2.13.6 Caspase-3 activity assay 46

2.13.7 Western immunoblotting 46

CHAPTER 3 RESULTS 48

3.1 Antibacterial Activity 48

3.3 Phytotoxicity of Selected Medicinal Plants 80

3.4 Heavy Metal Analysis 87

3.5 Antioxidant Activity 91

3.6 Proximate analysis 95

3.7 Phytochemical Analysis of Selected Medicinal Plants 101

3.8 Anticancer Activity 102

3.9 Results of Isolated Compound 108

3.10 Anticancer Activity of Isolated Compound 113

3.10.1 Adlumidine Reduce the Cell Viability of HCT 116 And PC3 Cells 113

CHAPTER 4 DISCUSSION 118

4.1 Antifungal Activity 118

4.2 Anti-Bacterial Activities 120

4.3. Heavy Metals in Medicinal Plants 124

4.5 Proximate Analysis of Selected Medicinal Plants 127

4.6 Phytotoxicity of Medicinal Plants 129

4.7 Antioxidant Activity 131

4.8 Phytochemical Analysis of Selected Medicinal Plants 133

4.9 Anticancer Activity 135

4.10 Isolation of Compound 139

CONCLUSION 141

FUTURE PROSPECTS 142

REFERENCES 143

LIST OF TABLES

Table 3. 1 Antibacterial potential of the crude extract of A. lebbeck 49 Table 3. 2 Antibacterial Potential of the Crude Extract of M. azedarach 51 Table 3. 3 Antibacterial Potential of the Crude Extract of Hedera helix 53 Table 3. 4 Antibacterial Potential of the Crude Extract of S. flagellaris 55 Table 3. 5 Antibacterial Potential of the Crude Extract of V. jatamansi 57 Table 3. 6 Antibacterial Potential of the Crude Extract of F. cretica 59 Table 3. 7 Antibacterial Potential of the Crude Extract of W. coagulans 61 Table 3. 8 Antibacterial Potential of the Crude Extract of M. oleifera 63 Table 3. 9 Antifungal Potential of the Crude Extract of A. lebbeck 65 Table 3. 10 Antifungal Potential of the Crude Extract of M. azedarach 67 Table 3. 11 Antifungal Potential of the Crude Extract of H. helix 69 Table 3. 12 Antifungal Potential of the Crude Extract of S. flagellaris 71 Table 3. 13 Antifungal Potential of the Crude Extract of V. jatamansi 73 Table 3. 14 Antifungal Potential of the Crude Extract of F. cretica 75 Table 3. 15 Antifungal Potential of the Crude Extract of W. coagulans 77 Table 3. 16 Antifungal potential of the crude extract of M. oleifera 79 Table 3. 17 Phytotoxicity of Selected Medicinal Plants 82 Table 3. 18 Heavy Metal Concentration of Selected Medicinal Plants 87 Table 3. 19 In vitro Antioxidant Activity of Different Extracts of Selected Medicinal Plants 92 Table 3. 20 Proximate Analysis of Different Parts of Selected Medicinal Plants all Values are Mean ± Sem of Three Values. 96 Table 3. 21 Phytochemical Analysis of Selected Medicinal Plants 102 Table 3. 22 Anticancer Activity of Different Fractions of Selected Medicinal Plants At 25 µm 103 Table 3. 23 Anticancer Activity of Different Fractions of Selected Medicinal Plants at 50 μM Concentrations 104 Table 3. 24 Anticancer Activity of Different Fractions of Selected Medicinal Plants at 100 μM Concentrations 106

LIST OF FIGURES

Figure 1 Moringa oleifera Lam. 23 Figure 2 Albizia lebbeck (L.) Benth. 24 Figure 3 Withania coagulans (Stocks) Dunal 25 Figure 4 Melia azedarach L. 26 Figure 5 Hedera helix auct. non Linn 28 Figure 6 Fagonia cretica L. (Virgin's Mantle) 29 Figure 7 Valeriana jatamansi Jones 30 Figure 8 Saxifraga flagellaris Willdenow in C. M. von Sternberg 31 Figure 9 Antibacterial Activity of Albizia lebbeck 49 Figure 10 Antibacterial Activity of Melia azedarach 51 Figure 11 Antibacterial Activity of Hedera helix 53 Figure 12 Antibacterial Activity of Saxifraga flagellaris 55 Figure 13 Antibacterial Activity of Valeriana jatamansi 57 Figure 14 Antibacterial Activity of Fagonia cretica 59 Figure 15 Antibacterial Activity of Withania coagulans 61 Figure 16 Antibacterial Activity of Moringa oleifera 63 Figure 17 Antifungal Activity of Albizia lebbeck 65 Figure 18 Antifungal Activity of Melia azedarach 67 Figure 19 Antifungal Activity of Hedera helix 69 Figure 20 Antifungal Activity of Saxifraga flagellaris 71 Figure 21 Antifungal Activity of Valeriana jatamansi 73 Figure 22 Antifungal Activity of Fagonia cretica 75 Figure 23 Antifungal Activity of Withania coagulans 77 Figure 24 Antifungal Activity of Moringa oleifera 79 Figure 25 Phytotoxicity of 10/20/40 mg against Radicle of L. sativa 83 Figure 26 Phytotoxicity of 10 mg against Radicle of L. sativa 83 Figure 27 Phytotoxicity of 10 mg against Plumule of L. sativa 84 Figure 28 Phytotoxicity of 20 mg against Radicle of L. sativa 84 Figure 29 Phytotoxicity of 20 mg against Plumule of L. sativa 85 Figure 30 Phytotoxicity of 40 mg against Radicle of L. sativa 85

Figure 31 Phytotoxicity of 40 mg against Plumule of L.sativa 86 Figure 32 Heavy Metal Concentration in Selected Medicinal Plants, 88 Figure 33 Lead Concentration in Selected Medicinal Plants, Using Wet Digestion Method 88 Figure 34 Arsenic Concentration in Selected Medicinal Plants, Using Wet Digestion Method 89 Figure 35 Cadmium Concentration in Selected Medicinal Plants, Using Wet Digestion Method 90 Figure 36 Mercury Concentration in Selected Medicinal Plants, Using Wet Digestion Method 90 Figure 37 Zinc Concentration in Selected Medicinal Plants, Using Wet Digestion Method 91 Figure 38 Antioxidant Activity of Selected Medicinal Plants, Using DPPH Assay 93 Figure 39 Antioxidant Activity of DCM Fractions of Selected Medicinal Plants, Using DPPH Assay 93 Figure 40 Antioxidant Activity of Ethyl Acetate Fractions of Selected Medicinal Plants, Using DPPH Assay 94 Figure 41 Antioxidant Activity of Cr. Methanol Fractions of Selected Medicinal plants, Using DPPH Assay 94 Figure 42 Antioxidant Activity of n-Hexane Fractions of Selected Medicinal Plants, Using DPPH Assay 95 Figure 43 Proximate Composition of Selected Medicinal Plant 96 Figure 44 Ash Composition of Selected Medicinal Plants 97 Figure 45 Moisture Composition of Selected Medicinal Plants 98 Figure 46 Protein Composition of Selected Medicinal Plants 99 Figure 47 Carbohydrate Composition of Selected Medicinal Plants 100 Figure 48 Fat Composition of Selected Medicinal Plants 101 Figure 49 Anticancer Activity of Different Fraction of Selected Medicinal Plants Extracts at All Concentrations against (Colon cancer cells--- HT-29). Cell Line Using MTT- Assay 103 Figure 50 Anticancer Activity of Different Fraction of Selected Medicinal Plants Extracts at 25 μM Concentrations against (Colon cancer cells--- HT-29). Cell Line Using MTT- Assay 104 Figure 51 Anticancer Activity of Different Fraction of Selected Medicinal Plants Extracts at 50 μM Concentrations against (Colon Cancer Cells--- ht-29). Cell Line Using MTT- Assay 105 Figure 52 Anticancer Activity of Different Fraction of Selected Medicinal Plants Extracts at 100 μM Concentrations against (Colon cancer cells--- HT-29). Cell Line Using MTT- Assay 106 Figure 53 1H NMR of Compound 109 Figure 54 13C NMR of Compound 110 Figure 55 DEPT 90 of Compound 111

Figure 56 DEPT 135 of Compound 112 Figure 57 Structure of Adlumidine Compound 113 Figure 58 Adlumidine reduce the cell viability of HCT 116 and PC3 cells 114 Figure 59 Adlumidine Induce Apoptosis in HCT 116 Cells 115 Figure 60 Adlumidine Induce ROS Generation in HCT 116 cells 116 Figure 61 Caspase-3 Activity in Adlumidine Based on Spectrophotometric Detection 117 Figure 62 Adlumidine Lead to Caspase-3 Dependent Apoptosis In HCT 116 And PC3 Cells 117

LIST OF ABBREVIATIONS

PBS Phosphate Buffered saline solution

EDTA Ethylenediaminetetraacetic acid

DMEM Dulbeccos Modified Eagle Medium

FBS Foetal Bovine Serum

MTT ((3-(4, 5-Dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide, A tetrazole)

PDA Potato dextrose agar

DMSO Dimethyl sulphoxide

DPPH 2,2-diphenyl-1-picrylhydrazy

GLOBOCAN Global Cancer Observatory

IACR International Association of Cancer Registries

ABSTRACT Plant secondary metabolites as biologically active compounds have a great future in controlling various degenerative diseases. The bioactive compounds certainly play a key role in effective medications. Bioactive compounds can be probed in the medicinal plants through in vitro and in vivo assays. In the past few decades, extensive work has been done to discover effective drugs from natural sources with low side effects. Due to medicinal significance in folk lore, eight medicinal plants (Albizia lebbeck, Melia azedarach, Hedera helix, Saxifraga flagellaris, Valeriana jatamansi, Fagonia cretica, Withania coagulans, and Moringa oleifera) were selected from the local flora to determine their anticancer potential and other biological activities. Initially, the plant extracts were obtained in 70% ethanol and were later fractionized into ethyl acetate, dichloromethane and n-hexane. The crude plant extracts were then used in various bioassays to determine their anti-bacterial, antifungal, phyto-toxic, anti-oxidant and anticancer potential. Also, the plant extracts were tested for proximate composition, heavy metals and phyto-chemicals. After, isolation and purification of compound, the compound of interest were utilized against cancer cell line (HT-29 Colon cancer cell lines). To estimate the antifungal activity against Aspergillus flavus, Alternaria alternate, Fusarium oxysporum, and Polysphondylium pallidum, an agar well diffusion method was adopted. The results showed that the crude methanolic extract of all plants were very effective against the selected fungal species. Melia azedarach, plants extract showed highest zone of inhibition 30.0±0.67 mm to 65.0±0.45 mm , followed by Saxifraga flagellaris, 29.0±0.67 mm to 65.0±0.54 mm, Moringa oleifera, 23.0±0.78 mm to 60.0±0.22 mm and Hedera helix, 0±0.55 mm to 57.0±0.88 mm. Antibacterial activity was also determined with the help of agar well diffusion method. The bacterial strains Streptococcus mutans, Methicillin-Resistant Staphylococcus aureus (MRSA), Staphylococcus aureus (Gram-positive), and Serratia marcescens (Gram- negative), were used. The antibacterial activity revealed that the crude methanolic extract of all plants were effective against the tested bacterial strains. Fagonia cretica plants extract showed highest zone of inhibition ranging from 07.0±0.66 mm to 15.0±0.33 mm (28 to 57.69%), followed by Valeriana jatamansi (25 to 57.14%). To determine the phytotoxic activity of the crude extracts (10, 20, and 40 mg), Lactuca sativa was used as a test plant. The crude extract of all plants at 20 and 40 mg have restricted the radical and plumule growth of L. sativa. The proximate analysis of the selected medicinal plants displayed substantial quantities of basic nutrients, like proteins,

carbohydrate, fat, moisture and ash contents. The ethyl acetate extract of the tested medicinal plant species presented highest DPPH scavenging activity. Moreover, significant amounts of alkaloids, saponins, glycosides, flavonoids and phenols have been noticed in the crude extract of these plants. The MTT bioassay against available cancer cell line (Colon cancer cells--- HT-29) showed that Saxifraga flagellaris has restricted the cancer cell growth by 90%, followed by Fagonia cretica (31%), Moringa oleifera (23%), Valeriana jatamansi (21%), Withania coagulans (16%) and Hedera helix (10%). The potent crude extract of Saxifraga flagellaris was then subjected to the compound isolation by chromatographic techniques and characterized by 1H NMR, 13C NMR, DEPT 90 and DEPT135. The isolated compound was identified as adlumidine with molecular formula C20H17NO6 and molecular weight of 367.357 amu. This compound was reported in Saxifraga flagellaris for the first time. The purified compound, adlumidine reduced the cell viability of HCT 116 and PC3 cells. This compound proved to be toxic against HCT 116 and PC3 cell lines at ≥ 20 µg/ml concentrations. Adlumidine increased early apoptosis to 19.5%, while boosted late apoptosis to 10.4%. Furthermore, adlumidine induced caspase-3- dependent apoptosis in HCT 116 and PC3 cell lines. From the results of present study, it is concluded that Saxifraga flagellaris has potent bioactive compound(s) that can potentially control cancer and other contagious diseases.

CHAPTER 1 INTRODUCTION

1.0 Orientation of the study Plants are the essential component of nature because all the living organisms get benefits from it one way or other. Plants are considered as one of those materials that comfort the human life indispensably on this planet. They provide all the essential supplies to the consumer in the form of nourishment, energy, remedy, timber, outfit, shelter, gums, oil, etc. They are the massive treasures, not only from the global environmental perspective, but also from the medicinal point of view. These natural sources contain extraordinary and miraculous capabilities that make them remedial, ornamental and edible at the same time. All plants ranging from a thorny bush to a tall evergreen tree can play their role in serving the human in different ways. Medicinal plants are of great importance, serving as therapeutic agents since ancient times. Nature is the home of medicinal principles, including plants that are the most valuable and diverse source of phytochemicals since ancient times (Panda and Sonkamble, 2012). Medicinal aromatic plants accompanied the humankind at every pace of history, without having a strict specific definition. Usually medicinal plants (wild or cultivated plants) are used directly or indirectly for curing, based on their traditional and literary records. These plants contain active components that affect the physiological processes of animals and humans (Chopra et al., 1956) Medicinal plant can be defined as any plant that has certain compounds in its parts that can be used to cure diseases or they may act as initiators for the preparation of antimicrobial drugs. The medicinal plants are used for herbal requirements shaped through exposing plant ingredients to different physical or biological processes (Hamayun et al., 2006b). Additionally, plants help people in the form of medicines - to prevent disease, keep better health and cure illnesses. When a plant is labeled as medicinal, it means that the said plant is useful as a medication or a beneficial agent or is a vital component of any medical research. Hence, they signify a group of plants that possess some special assets or qualities that qualify them as drug item and beneficial agents and are used for medicinal purpose (Iqbal and Hamayun, 2004). It is estimated that almost half a million species of plants exist on this planet earth (Begum et al., 2015), but humans and other animals use a moderately small percentage (1 to 10%) of these plants as a food and medicine (Moerman, 1998). More or less eighty thousands plants species have some medicinal properties with 1340 plants are known to have antibiotic properties (Lim, 2012)

1.1 Medicinal Plants Medicinal plants usage runs parallel with the creation of life on the planet earth. Man knows plant uses for remedial purposes, since the beginning of human civilization. For the long period, plants were the sole and valuable resource of natural products for the maintenance of health. These plants were used to treat severe diseases all over the world before the advent of modern drugs. After time long experimentations in the history of medicines, medicinal plants were recognized as an important reliable medicine. Any plant organ, that is leaf, shoot, root, fruit and seed can act as medicinal part. Earlier, there was no knowledge concerning the phytochemicals of plants, but ancient people were of the view, that these plants have astringent power to treat various diseases (Van Wyk and Wink, 2017). The world most primitive manuscript relating the medicinal plants dates back to 60,000 BC and was found at the oldest archaeological remains at Shanidar IV, an archeological site in Iraq (Tomlinson and Akerele, 2015). The record shows that the medicinal plant are serving as curing agents from Neanderthal man (60,000 BC) to the modern man (Wangchuk and Tobgay, 2015). The earliest analgesic ―Alcohol‖ was prepared as far back as 6400 BC by fermenting grapes and berries in warm place (Ortega, 2009). Approximately, 5,000 years old Sumerians clay tablet from Nagpur comprised of 12 methods for remedy preparation discussing 250 different plants, some of them are alkaloids such as poppy, mandrake, and henbane (Petrovska, 2012). Babylonians and Assyrians also recorded such prescriptions on clay slabs. The legendary Chinese Emperor Shen Nung in his pharmacopeia on roots and grasses ―Pen T‘Sao,‖ had mentioned more than 360 drugs, which are still in use such as camphor, ginseng, Rhei, rhisoma, jimson weed, the great yellow gentian, ephedra, Podophyllum and cinnamon bark, etc. Middle East was the seat of culture and civilization and it is considered, that most of the modern plants were first cultivated there. The Egyptians documented pharmaceutical records as wall images of tombs and on Cyperus aquaticusa Papyrus known during 1500BC containing over 800 prescriptions before 3000BC related to 700 plant species (Stojanović et al., 2017, Alam et al., 2017). These Egyptian medicinal record show plant-based drugs revealed different methods of taking in the form of pills, ointments etc. Egyptians mostly contributed in creating a preservation method- mummification and dentistry along with other works (Bojadzievski, 1992). The recorded history of different civilizations strongly supported the use of herbal medicine by inhabitants of those areas. In the history of Indian System of Medicine, period from 800 BC -

1,000 AD considered as the Golden Era of Indian Medicine. During that time, Ayurveda medicine flourished with its full glory and even now a days, it is considered as the best herbal healthcare system. Indian history of medicine reflects the great advances in surgical methods, especially plastic surgery. Alternative to Ayurveda, the most popular healthcare system among Muslims is Unani-Tib which is deeply rooted in the Greek Mythology. Greeks owed the ideas about medicine and healthcare from Ancients Egyptians and today they are transferring it to the modern man (Cristóbal-Luna et al., 2017). Greeks were the first who regarded medicine as science and freed it from the realm of superstitions and speculation. Of them, most important figure is Hippocrates (460BC), who is mentioned as father of medicine and his work referred as Materia medica documented over 300 medicinal plants. The important medicinal plants, included garlic as vermifuge against parasites of intestine, opium as narcotics, deadly nightshade as cosmetics and pomegranate as astringents. Theophrastus (371-287 BC) classified then known plants into many groups in his manuscript, Historia Plantarium. Successively, Greek philosophers enriched the world of medicine to a great extent with their contributions (Petrovska, 2012). Hippocrates adopted patient centered philosophy (Stewart, 2003). The father of Pharmacognosy ―Discorides‖, a Greek physicians studied many medicinal plants while travelling with the Roman army. In his work, De Materia medica (77 AD), he described 940 drugs out of which 657 are of plant origin. This book has translations in many languages, is one of the most authentic works on drugs, and is still valued in many European countries. Discorides appreciated the domestic plants such as willows, garlic, onions, etc. Another outstanding figure is a pharmacist, Galen (131-200 AD), who provide such a foundation to medicine, later on, which became a magnificent edifice with the work of Arab scholars. The middle ages, addresses the influence of religious traditions on the practice of medicine and eventually with the establishment of many universities (Nikolovski, 1961). Baghdad city was the main site of medical wisdom for all the people irrespective of their traditions and religions. Muslims, Jewish and Christian‘s scholars collaborated in making a treasure of knowledge, by Persian-Mesopotamian, Byzantine-Greek and Indian traditions (Dauti et al., 2014). It was the era when Arab scholars who enlightened the world with their scientific thinking and discoveries; they were the first who run their privately owned pharmacies, such as Ibn-e-Sina (Tupanceski and Kiprijanovska, 2014). Arabs introduced many new plants to the field of medicine that still used throughout the world. With the advancement in technology, herbalism

took a new turn; the simple use of herbal prescriptions from decoctions and/or infusions, tea converted to the isolation of modern drugs. The discovery of alkaloids from plants marked the beginning of scientific pharmacy. Modern medicines also based on ancient folklore, for example, Digitalis purpurea L. has adopted in modern era and was a known drug at the end of 18th century. This great achievement led to the discovery of different modern drugs that paved the way for the development of modern pharmacology. The practice of remedial plants is not new to man. In every civilization and culture, the soothing stuffs of definite medicinal plants were recognized, illustrated and transferred to the successive generations. Thus understandings of one civilization, delivered on to another, which elevated the old assets and learned novel ones (Petrovska, 2012). Diseases in life is not a new thing, these goes side by side, like flowers and thorns, day and night, merriment and distress, bliss and torture. Some diseases are fatal for life such as cancer, but every disease may be curable with the help of medicinal plants. Medicinal uses of the plants explored through the observations and experimentations of the different communities of the world. Treatment of ailments with the help of medicinal plants is constantly a significant element of Islamic instruction, as Islamic philosophy highly favors the thirst of knowledge (Sheikh and Dixit, 2015, Nasr, 1976). The botanical research of the Muslim scholars dates back to second century of Hijri (7th century AD). This not only covers the studies to classify and describe plants, but also covers their healing properties and many other uses. Their keen interest in this field opened the door of the study of medicinal and agricultural aspects of the floral kingdom (Bnouham et al., 2006). The compilation of medicine in Muslim world commenced in 7th century AD by Abdul- Malik Undlasi composed his famous writing ―Tib-e-Nabvi‖. Lately, other Muslim researchers, like Ibne Siena, Ibn e Nafees and Kazim Bin Jaffer has contributed towards Islamic medicines (Ghaznavi, 1987). 1.2 Medicinal Plants as a Safe Source of Medicines The herbal products symbolize safety as compared to synthetic compounds, which are not environment friendly and have side effects. Throughout the ages, plants are valued for medicinal, nutritive, flavoring and aromatic capacities. For a while, the advent of modern synthetic drugs staged the era of modern synthetic medicines, which completely surpassed the herbal medicines. Now the people are coming back to the natural curing substances, as synthetic medicines are unsafe. The use of herbal antimicrobials is because of several side effects. One of the advantages of medicines from medicinal plant is that these are comparatively harmless from their synthetic

alternatives, which make herbal products easy to use and affordable for treatment (Okigbo et al., 2009). The traditional herbal system of medicine continues to be widely practiced due to many reasons even at present in all parts of the world. Some of the reasons include over population, insufficient supply of the medicinal drugs and severe side effects of synthetic drugs. The development in resistance of infectious agents against the currently used modern synthetic drugs, medicinal plants with low side effects are the first choice of users for therapies (Calixto, 2000). In the presence of above-mentioned reasons, herbal products are preferred over the modern synthetic drugs. The synthetic pharmaceuticals contain a solo chemical. While medicinal and aromatic plants extracts apply and multiply their useful properties from end to end. These plants possesses combined activities of a number of chemical compounds affecting one or a number of target sites related with a biological process (Calixto, 2000, Williamson, 2001). World population is increasing day by day with rapid speed and due to poor health hygiene, new infectious agents and diseases have come to the scene. Modern synthetic medicines remain failed in controlling these problems. In such circumstances, only the use of herbal drugs is the best solution. Comparatively, herbal medicines are easily available, effective, chemically balanced, cheap and with no or less harmful effects on human and environment. Numerous herbal remedies used to control infectious diseases. The plant materials, which are part of traditional medicine, are environment friendly, easily accessible in rural areas and comparatively inexpensive than allopathic medicine (Mann et al., 2008). Therefore, more stress rested on the use of herbal drugs. Hence, it is the voice of the day to search for the plants having the miraculous medicinal abilities. Every plant produces useful phytochemicals even the deadly poisonous plants are helpful in controlling diseases when used carefully in specified conditions. Digitalis species, used in the medicines, for curing heart diseases is one of the best examples.

1.3 Uses of Medicinal Plants All over the ages, plants used for remedial purposes along with other uses. The use is in almost any type of disease from minor wound to chronic heart disease. Still people of the world, particularly the developing countries rely continuously on traditional herbal system to deal with various diseases. A nonprofessional in rural may have no knowledge of the different phytochemicals present in these medicinal plants, but they have a belief that these medicinal plants possess healing powers to cure diseases. Several hundred genera of plants utilized for

medicinal purposes, which act as key sources for effective and powerful drugs (Perry and Metzger, 1980, Sofowora, 1982, Ross, 2007). Botany helps the health of masses, ―the medicine man‖ or ―Pinsari‖ called as the first professional botanist known to human race. As for as the western medicine is concerned, the field of medicine has largely rely on synthetic drugs rather than natural (Hebbar et al., 2004, Bauer and Brönstrup, 2014). However, great change have observed from the last decade, i.e. a shift from modern to natural medicinal system. Currently, in European Union, 1400 herbal preparations are in wide use. In Europe and Germany, herbal preparations are of prior importance in healthcare industry. The popularity of herbal preparations is obvious by their increasing importance in cosmetic industry, such as skin tissue regenerators, anti-wrinkling agents or anti-age creams (Hoareau and DaSilva, 1999). According to another study 1/4th prescription drugs used in the thirty-five countries belonging to OECD. In the Eastern Europe, herbal products are either unmodified or slightly altered to make it more effective (Bahadur et al., 2007, Health, 1997). The consumption of remedial plants in unindustrialized nations is a key element for improved health. Beside the collective dependence on medicinal plants in the technologically advanced cultures have planned to extract and improve numerous remedies and active compounds, which has frequent use in rural therapies. Additionally, herbal mixtures have been more predominant to cure minor illnesses and to minimize the growing burdens of economy on individual to keep up their health (De Silva, 1997, Manju et al., 2017, Okigbo et al., 2009). At the moment, the production and processing of medicinal plants is encouraged all over the world in order to get cheaper and effective medicines. Similarly, in emerging nations, like Pakistan and India, most of the people depend on traditional system of medicine instead of modern medicines and healthcare (Begum et al., 2015). Curative plants are triumphed for centuries to treat different diseases that guarantee great medicinal value, efficiency and safety. The universal involvement of medicinal plants in the medicine industry is evident from their widespread use in all the major systems of medicine. These days Ayurveda, Unani and Homoeopathic uses abundant medicinal plants to produce potent herbal remedies (Rahman et al., 2018). Herbal medicines usually been used in the form of fruit or vegetable, flowers or leaves, root or stem, plants parts or their essences to cure infections and maintain healthy well-being (Hamayun et al., 2006b). Some common health problems in our rural areas, such as skin rashes diarrhea, periodic fever, throat infections, diabetes, fungal and microbial infections treated regularly by using numerous herbal and traditional remedies. Any part of the

plant can act as medicinal agent because some effective healing compounds might be abundant in one organ of the plant (Aziz et al., 2017, Ody, 2017). Almost two third of the Pakistani population, particularly in countryside areas relies on the herbal preparations prescribed by their traditional local experts (Hakims) for their well-being (Shinwari and Gilani, 2003). Besides, more than half of the inhabitants from the lower earning world relies on herbal medicine for their major health care necessities (Planta et al., 2000). In China, about 40% of medicines taken from customary folkloric medications. In Thailand, herbal prescriptions are prepared from leguminous plant. Therefore, it is fair to say that the herbal medicines are getting more and more importance in the word than typical pharmacological products (Kamboj, 2000, Jordan et al., 2010, McFarlin et al., 1999). 1.4 Medicinal Plants of Pakistan Almighty Allah has endowed Pakistan with the natural wealth of flora, including chirruping mosses, thriving fields, lush green woodlands, pleasant fruits, potent medicinal plants and matchless biodiversity. It is a God gifted country having over six thousand species of higher plants. There are thirteen plant hotspots from alpine prairies to mangrove forest (Hamayun et al., 2006a, Shinwari, 2010, Baquar, 1989). Near 700 species of the flora are used for remedial purposes and the circle is still expanding with more research down the road (Shinwari and Qaiser, 2011). Pakistan has rich culture and tradition concerning the uses of medicinal plants. Many people are aware about the medicinal uses of plants growing in their region. This ethnic data about the medicinal plants has been transferring from one age group to another. These plants used to cure almost any kind of disease from mild pain to severe diseases (Abbasi et al., 2010, Shah et al., 2013, Bhardwaj and Gakhar, 2005). After independence, most of the Pakistani people were reliant on local medicines and traditional health practices, but these practices are restricted to the distant rural parts due to the shifting tendencies in resources and ethos (Qureshi et al., 2009). Commercially important medicinal plants cultivated for isolation of a number of active constituents while the local communities use some as phytomedicine. However, different systems of Arabian Medicines (Unani), Sanskrit medicines (Ayurveda), used different medicinal plants in their herbal remedies. The systems have largely preserved this precious wealth about the floristic resources yet it is in the threat of being lost, which are main bases of incomes for many like herbal sellers and exporters but in Pakistan the cultivation of these medicinal plants is still not in practice (Shinwari and Gilani, 2003, Hamayun et al., 2006b).

Traditionally, Arabian medicine or Islamic medicine also known as Unani way of treatment is a part of our lives. In Pakistan most of the population still considers it an effective and easy way to combat diseases. The general practitioners who have the knowledge of medicinal plants and prepare effective recipes from the herbs against the diseases known as ―Hakim (The wise man)‖. In Indo-Pak, the term prevailed and Hakim images as learned man. Traditional Unani medicine greatly depends on medicinal plants (Said and Saeed, 1996, Khare, 2008, Mhaskar et al., 2000) Pakistan is the eighth principal country that depends on the use of medicinal plants to control various diseases. According to an estimation that two thousand medicinal plants species found in Pakistan. In the market 456 medicinal plants species circulated and merchandised for the preparation of 350 classical medicinal formulas (Ahmad and Husain, 2008, Rizvi et al., 2007). About 70-80% of the Pakistani rural areas use corresponding medicine belonging to homeopathic Ayurveda, and Greco-Arab (Unani) dispensary are as old as Indus valley civilization itself and is quiet well known even now. Nuts used as medications, in powder or oil form (Iqbal and Saddiqi, 2011). Exploitation of these herbal plants mostly rests unexplored or even ill-used due to lack of knowledge of even their occurrence so majority of herbal plants are smuggled for drug industry. Northern areas of Pakistan have rich diversity of medicinal plants. Almost three hundred endemic species (4% of the total flora) are present at highlands of Northern Pakistan (Rizvi et al., 2007). There are about forty thousand hakims, one-lac thirty thousand homoeopaths and four hundred and fifty five ―Vaids‖ (Tabib) registered in Pakistan. There are three hundred Unani industrial establishments and about three hundred companies developing homoeopathic medicines (Shinwari and Qaiser, 2011). In Pakistan, plants still used by different people to cure ailments. The compounds, which are responsible for the remedial activities, yet need exploration. Therefore, Pakistani medicinal plant species need thorough studies for their potency against a number of degenerative diseases, especially cancer. 1.5 Medicinal Plants and Modern Medicine A medicinal plant is a plant, which contain active compounds that control or eradicate the diseases (Huang et al., 2008). The great treasure of knowledge of traditional medicine relying on plants before arriving to the modern man, and to take form of modern herbal medicine has passed through many phases and generations. Any primary or secondary metabolites from plant or animal classified as natural products. For normal growth and development, primary metabolites are necessary, while secondary metabolites

are required for biochemical pathways (Martin, 1995). Primary metabolites form secondary metabolites through biosynthetic pathways; these metabolites are in most cases, limited to confined taxonomic groups. Secondary metabolites may not relate to normal plant growth, but play significant ecological roles, when plants are going through biotic or abiotic stress. In addition, the secondary metabolites might be responsible for ecological roles, like pollinators attraction, providing defense against disease-causing insects and microorganisms and encouraging survival under ecological stresses. Other properties of secondary metabolites, includes colors, medicinal value, characteristic aromas and flavors (Balandrin et al., 1985). However, these metabolites produced in rarer quantities, paralleled to primary metabolites. As for as plant secondary metabolites are concerned, these are very diverse and have a medicinal value in the form of alkaloids, flavonoids, phenolic compounds, saponins, steroids, tannins and terpenoids (Mdlolo, 2009). These metabolites are limited to definite plant family, genus, species or subspecies (Adelaja et al., 2008). When the extracted secondary metabolites from plants utilized in bioassays, they might have antiseptic, anti-inflammatory, antimicrobial, and anticancer activities (Neuwinger, 1996). In the last twenty years, half of the drugs introduced into the market to treat various diseases were from medicinal plants (Huang et al., 2008). These disease-curing drugs are very much important, as these have strengthened the impact of biological effects, isolation of different effective compounds as well as their structural determination. However, it would be hard nut to crack, but establishment of new screening methods made it possible to know its efficacy against certain diseases (Sarkar et al., 1996b). Plants medicines have three generations. FIRST GENERATION: These are the plant medicine with simple botanical materials, which works in crude form. By tradition, medicinal plants utilized, cure different diseases. The parts of plants that used to treat different ailment are barks, bulbs, exudates, flowers, fruits, leaves, roots, stems, twigs, and tubers. These plant ingredients still used in preparation of laxatives, extracts, infusions, snuffs, teas and in several other methods. The selection of these medicines is by old traditional practices (Van Wyk and Gericke, 2000). Traditional medicinal plants cure many diseases like cancer, dysentery, Epilepsy, inflammations, malaria, pneumonia, sexually transmitted diseases, ulcers, and wounds (Van Wyk et al., 2008, De Wet et al., 2012). SECOND GENERATION: second generation emerged due to the industrial revolution, which contains the isolation of active constituents from plant extracts by scientific processing. The

second-generation phyto-pharmaceutical were unadulterated purified molecules, but they differed only by their origin from the synthetic remedial agent, for example reserpine from Rauvolfia spp. (Iwu et al., 1999, Hussain et al., 2017b). For centuries, plants are the source of natural products and almost half of drugs derived from medicinal plants. The current research in bioactive material is flourishing due to several reasons; including dire need of new therapeutics, the outstanding variety of chemical structures and biological activities of existing secondary metabolites. The extractions of plant bioactive compounds made it possible, due to novel and profound techniques that lead to structural characterization of active constituents, more precisely (Clark, 1996). THIRD GENERATION: It the formulation centered on well-controlled, double-blinded clinical and toxicological exercises to improve phyto-medicine to improve quality of plant medicines along with stability and efficacy in synthesis of drugs (de Castria et al., 2013, Kimura et al., 2004) Medicinal plants products in any form offer infinite chances for new drugs discovery due to the incomparable availability of biochemical variety (Cowan, 1999, Sasidharan et al., 2011). About, 25% of the medications approved universally were originate from medicinal plants with 121 compounds are in active use. Moreover, 252 medications considered as elementary and approved by the World Health Organization. Significant number of synthetic drugs established and optimized from natural precursors, e.g. drugs acquired from plants sources are digoxin (Digitalis spp.), quinine and quinidine (Cinchona spp). It is estimated that two/third of anticancer and anti- inflammatory drugs present in the marketplace and in experimental trials are of plant based (Rates, 2001). Antibiotics play a key role in treating infectious diseases. The limitation of effective antibiotics reduces day by day as microbes are becoming resistance is all over the world, so therapy failure are increasing. Infectious diseases are the cause of deaths, approximately, one- half of all deaths in worldwide. In 1981, deaths from infections were on fifth position, while it has become the third principal reason of death (58% increase) in 1992 (Pinner et al., 1996). Above hundreds of herbal products are used traditionally in medicines to cures the bacterial diseases (Vidal et al., 1996, Martin et al., 2003). Although, numerous diseases been cured by allopathic methods, but still there is a rising attention in the use of plant natural products by the locals (Ghose et al., 2003). Quinine from cinchona plants and berberine from berberis plants are the examples of highly effective antibiotics (Chopra et al., 1997, Mustapha et al., 2017). Toxins secreted by Staphylococcus aureus and Streptococcus pneumonia can be cured with liposomes, which can be used singly or in combination with other antibiotic against bacterial

agents. Chrysothol, madreporanone and stachytriol isolated from Azorella cryptantha showed strong antimicrobial activities against enterobacteria, E. coli and S. enteritidis (Rosenthal et al., 2016). Catharanthus roseus leaf extracts revealed maximum antibacterial action against E.coli, Streptococcus sp., and Pseudomonas sp., (Pina-Pérez and Pérez, 2017). These infectious agents remain to be a health problem all over the world (Wekesa, 2017). One of the various infectious agents that may cause severe diseases is pathogenic fungi. On one hand it cause diseases in plants and valuable crops, while on other side fungi are responsible for averse/lethal disorders among consumers by secreting allergens and mycotoxins (Rouhi-Boroujeni et al., 2017, Ahmad et al., 2017). Biochemical fungicides often been used to control fungal infections, but this is related to adverse ecological influences, possible human contact to insecticides, and statement of remains on the fruits. Nevertheless, the efficiency of synthetic fungicides been condensed by the recurrent growth of resistance by the pathogens. Therefore, there is a pronounced call for harmless substitute and effective chemotherapeutic mediators (Dahar and Ismail, 2017). Bio-active plant products can be crucial in this regard that can be obtained from species with noteworthy antifungal activities, for example leaf pulp of Aloe vera (Grayer and Harborne, 1994) or barbaloin, a C-glucoside isolated from Aloe vera (Braga et al., 2007). Antifungal activities of Brazilian locally available medicinal plants assessed against Candida albicans and Cryptococcus neoformans. Among the tested plant species, methanolic extract of Vernonia polyanthes remained the most vigorous one (Portillo et al., 2001). and Allium sativum L. can inhibit the growth of Mycosphaerella eumusae causing leaf spot disease of banana as well as from Myroxylon balsamum (balm of Tolu), by different extraction methods. Still, the balm of Tolu used in drug as an antiseptic, anti-parasitic, and cicatrizing mediator (Boller, 1988). Allelopathy is a biological term in which one organism release active biomolecules that effect the growth, existence, improvement, and reproduction of other individual and these biomolecules designated as allelochemicals. Plant allelopathy is the types of relations concerning receptor (the plant which is effected) and donor plants (which effect) and may exert either positive effects (weed control) negative effects (e.g., soil sickness, biological invasion or auto toxicity) (Cheng and Cheng, 2015). Germination of seeds and plant development altered by allelopathy; therefore, germination is important way to study allelopathy. Allelopathy has a significant part in agriculture and disturbs the progress and magnitude of the crops by the exchange of chemicals among crops, weeds and trees. Allelochemicals secreted by unknown plants significantly affect the growth and

development of the native plant species (Sertkaya et al., 2010, Hussain et al., 2010). Allelochemicals can alter the contents of plant growth hormones or make inequalities in many phyto-hormones, which hinder plant growth and development. Phenolics as an allelochemicals can motivate IAA oxidase activity. These obstruct the reaction of POD with IAA, unavoidable GA or IAA to effect endogenous hormone stages (Rauf et al., 2012, Cheng and Cheng, 2015). After evaluation, the allelopathic activities of 239 medicinal species using the sandwich method and 223 species of them found to inhibit the seeds germination, while 17 species found to stimulate lettuce radicle growth. The allelopathic influence after leaf litter leachates on lettuce seed germination and found inhibitory activity carried out by the sandwich method (Fujii et al., 2003). After screening 81 Pakistani medicinal plants and found that plants with allelopathic potentials also have stimulatory effects side by side with inhibition (Gilani et al., 2000). Four different species belonging to Anthemis, Inulaand Lactuca genera collected from Hindukush ranges of Pakistan. These plants studied with the help of two different methods Sandwich and Homogenated Sandwich. It was determined by application of different concentration leaf leachates has very much substantial value (Khan et al., 2009).

1.6 Cancer and Medicinal Plants When penicillin discovered, it opened new ways to discover new drugs. The medicinal plants are sources and the perception of these medicinal plants as a source of remedies and important instrument for modern drugs, as these are the springs of maximum novel combinations and compounds related to modern drug industries (Ginsberg, 2005). Now a well-known fact that 80% of beneficial drugs compounds and pharmaceutical products derived from medicinal plants (Diallo et al., 1999). Cancer is life-threatening disease and in advance stages leads to death that ruins the complete family economy and structure. The disease is persistent all over the world and increasing day by day. There are 26 types of cancers known according to the GLOBOCAN project of IARC. In twenty large forms all over the world, new cases were almost 10.9 million and deaths caused by cancer were 6.7 million, people those who were suffering from cancer with three years from diagnosis their number were 24.6 million. The cancers which caused most of the deaths, are half a million (1.15 million) from breast cancer, one million (1 million) from colorectal cancer, lung (1.35 million). while the cancer which deaths toll were higher are; liver cancer (598,000 deaths), more than a million (1.18 million deaths) from lung cancer and the notorious stomach cancer

killed 0.7 million people (700,000 deaths). The breast cancer caused highest deaths of all was 4.4 million. However, there are variations in types of cancers by geographical zones, but the key factors, include lifestyle and environment (Parkin et al., 2005). Cancer is prevailing day by day that causes ill health and death worldwide. According to estimation by GLOGOCAN, in 2012 there are 14 million fresh victims registered with 8million expiries, as per estimation, by the end of 2030 there will be 70% increase. Although there is no direct data entry in most of the developing countries, According to (WHO) 34 of 194 countries of UN reported national mortality data of high quality, while 68 other countries provided data of high-quality incidence for the latest work of Cancer incidence in five continents. GLOBOCAN, a project of the (IARC) provides estimates by cancer site using the best available data in each country (Antoni et al., 2016). According to GLOBOCAN, the common types of cancers found in Pakistan are of bladder, breast, cervix uteri, colon rectum, lip and oral cavity. There are 27 types of cancers responsible for deaths in the world. In Pakistani population, both genders contain high ratio of different cancers; of the total 148,041 cancer cases includes; breast (n = 34038, 23%), colorectal (n = 5335, 3.6%), lip and oral cavity cancer killed near thirteen thousand (n = 12761, 8.6%) people, while lung cancer death toll were near seven thousand (n = 6800, 4.6%). The cancer of the WBC also known as NHL or non- Hodgkin lymphoma killed near six thousand (n = 5964, 4%). The death toll of cancer in Pakistani population is 101,113. (Sarwar and Saqib, 2017, Begum, 2018, Maucort‐ Boulch et al., 2018).

The quest for plant extract and therapeutics is not new to man; due to the experience based on many millennia such constituents that form important remedies as well as have developed basic and modern pharmaceutics. Names such as Belladonna, Cinchona Colchicum, Digitalis, opium etc. are very recent. Ancient remedies are the basis of discovery of natural substances having major biological and therapeutic interest. These also include new anticancer drugs. The NCI screened approximately thirty five thousand plants extracts and tested it for anticancer activity in 1960. Which resulted in approximately 2619 compounds isolation (Sarkar et al., 1996a). Later on some more important drugs were discovered e.g. camptothecin, Taxol, vinblastine, vincristine and drugs derived from natural compounds such as navelbine and taxotere and many are to be added in the list against cancer (Srivastava et al., 2005, Gordaliza, 2007). Free radicals present in body known as oxidants, these belong to chemicals containing unpaired electrons. Nature is full of these sorts of chemicals, their role in chemical reactions is important in medicines. These

radicals are present in carcinogens, which induce tumors initiation and promotion (Weitzman and Gordon, 1990). Hydroxyls radicals produced by hydrogen peroxide appear most possible to be involved in the various stages of carcinogenesis. Detoxification of the Hydrogen peroxide is essential from stopping hydroxyl-radical development. Nucleic acid is the most studied in connection with the free radicals. Oxidants are involved in initiation and promotions of carcinogenesis (Hochstein and Atallah, 1988). Free radicals also known as oxidants are chemicals with free or unpaired electrons. These are very common in nature, play very important roles in nature both harmful and beneficial. In medicines, these are very important. There are two very important Reactive species i.e. reactive Oxygen species (ROS) and reactive Nitrogen species (RNS). The ROS/RNS induce and maintain oncogenic stimulation. The production of free radicals may be due to exogenous or endogenous sources. These free radicals may damage DNA of mitochondria and Nucleus as well as damage lipids and proteins by producing oxidative stress (Valko et al., 2006). The body produce free radicles through food breakdown or can be acquired from polluted environment e.g. cigarette smoke etc. The processes that produce free radicals in body are Fenton- Haber-Weiss reaction

3+ 2+ 2+ + Fe /Cu + O2 → Fe /Cu + O2 (1) 2+ + 3+ 2+ Fe /Cu + H2O2 → Fe /Cu + OH + OH (2) O2

+ H2O2 → O2 + OH + OH (3)

The production of free radicals in vivo (Dreher and Junod, 1996, Babbs, 1990, Naidoo et al., 2008).

Due to cellular metabolism or confronting xenobiotic or radiation may produce free radicals. These reactive oxygen species (ROS) or free radical is equivalent to 5% of the total oxygen taken daily (Pryor, 1982, Cowan et al., 1986, Yendala et al., 2017). Human body antioxidant system can control the level of oxidants to a certain level, if it exceed that level, then oxidation reaction starts which is harmful for the body. Excessive oxidants start Cell damage by inducing cellular oxidative stress, which triggers several diseases including cancer (Poprac et al., 2017). AS per believe, the cancer and other chronic disease are contributed by the accumulation of free

radicals, which bind to Cell membrane, proteins and Nucleic acids, promoting cell damage and lipid oxidation (Höhn et al., 2017, Poprac et al., 2017, Kagan, 2018) The substances, which provide protection against free radicles and oxidative damage, known as antioxidants. Antioxidants are scavengers of free radicles being an important part of our diets; these prevent oxidation and repair cell damage. These are natural scavengers of free radicals and protect our body from oxidative harm against free radicals; biological antioxidants are as α- tocopherol, and ascorbic corrosive, carotenoids, and retinoid are extensively studied against natural cancer prevention (Rehman et al., 2018, Khan et al., 2018, Ravi et al., 2018). Techniques like chemical and cell cultures, have shown that antioxidant slow down and even neutralize cancer, but it is still to be investigated further (Aghajanpour et al., 2017) As per study, the cancer metastasis and invasion depends on angiogenesis. Angiogenesis can be prevented by applying plant natural products as current therapeutic have more problems associated with it. Several anticancer compounds of plant origin have been identified (Bahmani et al., 2017). Plant polyphenols are very effective chemo-preventive and anticancer substances. Flavones has anticancer and antioxidant activities. Seventeen derivatives of flavones were tested out using MTT assay, against human cancer lines: brain epithelial (U87), breast (MCF-7), lung (A549), thirteen trihydroxyflavones influenced anticancer activity against at least one tested cancer cell line (Grigalius and Petrikaite, 2017). Antioxidants must be the part of food to neutralize the free radicals in case of enzymatic action insufficient competency (Goze et al., 2009). Colored fruits, fish, teas and vegetables possess many natural antioxidants. These natural products reduce the risk of cancer by 25% or more (Tsang, 2009). Polyphenolic compounds like anthocyanidins, flavonoids, phenolic acids, and tannins, are secondary metabolites produced by plants. These compounds possess notable antioxidants and anticancer properties (Prasad et al., 2009, Cai et al., 2004). Ethno-medicinal plants have inclusive and marvelous involvement in the discovery of new medications against diverse ailments as well as cancer. Recent surveys with plant products have recovered fame for their natural implication and utility of their fundamental variety. So, citations of usually active anticancer plants and assessment of their essential mechanisms could be supportive in searching of novel anticancer drugs(Fritz and Kaina, 2006). The US National Cancer institute (NCI) introduced the quest for novelty of anticancer substitutes from plants in 1957. Since then 35,000 plant species had probed and contain more or less anticancer drugs, such as Camptothecin, Etoposide analogs, , Indicine–N-oxide, Taxol,

Vinblastine, and Vincristine and their derivatives (Suffness and Douros, 1982). Cancer is surrounded by many causes of infections and loss commonly. Plants-derived compounds like; alkaloids, Taxol, and Vinca, upsets microtubule gathering (Ren et al., 2006, Clarke et al., 2006). 1.7 Phytochemistry of Medicinal Plants Phytochemistry is the study of plant metabolites in addition to their end product. Metabolism in any living organism can be distinguished into primary and secondary metabolism. Primary and secondary metabolites are interrelated to one another as secondary metabolites are formed from the first one(Bennett and Wallsgrove, 1994, Rao and Ravishankar, 2002). Primary metabolites are of immense importance and are necessary for the survival of the plant. It includes chlorophyll, lipids, proteins, carbohydrates and nucleotides that play significant role in different processes like photosynthesis, heredity, differentiation, nutrient translocation and growth and development of the plant body. Secondary metabolites do not take part directly in the growth and development of the plants. Their absence does not lead to immediate death of the plant but long term impairment in functionality of different processes. Secondary metabolites include terpenoids, alkaloids, glycosides, and phenols. Elementary primary metabolites are common in all plant kingdom but the definite secondary metabolites are restricted to a single species or group of related species (Taiz and Zeiger, 2006). According WHO, remedial plants are those which hold such constituents in their organs that are therapeutically important and can be used as pharmaceutical drugs. The parts of such plant including leaves, bark, flowers, fruits, grains, rhizome, root, seeds and stem are are rich in medicinally active compounds and are used in combating a variety of health conditions (Huang et al., 2008, Doughari, 2012). A green plant synthesis and preserves a variety of biochemical compounds normally termed as phytochemicals or phytoconstituents (Preethi et al., 2011). In exact sense Phytochemical are chemicals present in plants. The most common phyto-constituents present in plants are; alkaloids, anthraglycosides, arbutin, bitter drugs, coumarins, flavonoids, phenol carboxylic acids, saponins, terpenes and valepotriates (Liu, 2004). These phyto- constituents contributes definite individualities and possessions to plants (Oberdörster et al., 2001, Sharma and Paliwal, 2013). Phytochemicals can be extracted and can be used as precursors for the synthesis of different types of medicines. Drugs isolated from the plants are being widely preferred than synthetic drugs in medicine science. The curative part of a medicinal plant is not simply its woody stem or fresh

green leaves but the quality and quantity of the phytochemicals present in that part of the medicinal plant. The plants for their own benefits produce these phytochemicals. Some of the phytochemicals are used for the growth and development of the plant body, some act as toxins and protect the plants from the attack of parasites and pathogens. These phytochemicals contain certain chemical constituents such as carbohydrates, proteins, derivatives of carbohydrates, gums mucilages, pectins, various forms of glycosides, tannins, phenolic compounds, lipids, fixed and volatile oils, resins and various kinds of alkaloids etc. These phytochemicals are of great importance to man nutritionally and also medicinally (Preethi et al., 2011). Many plants have different phytochemicals, which are important to both humans and animals. These include fruits, herbs, seeds, and vegetables. . Diets containing an abundance of fruits and vegetables are protective against a variety of diseases, particularly cardiovascular diseases. Herbs and spices are accessible sources for obtaining natural antioxidants (Van Duyn and Pivonka, 2000). Bioactive components present in different vegetables and fruits have shown promising effects in prevention of chronic diseases including cancer (Liu, 2003).Plants have been endowed with a marvelous ability to prepare the aromatic substances in a large quantity. Most of these aromatic ingredients contain important phenols or their oxygen substituted derivatives. These aromatic substances are produced as secondary metabolites by the plants. According to a report, approximately 12000 of the secondary metabolites have been successfully isolated. Less than 10% of the total secondary metabolites take part in plant defense strategy and protect them against predation by microorganisms, insects and herbivores (Cai et al., 2004). The plants for a variety of purposes produce secondary metabolites. These metabolites protect the plant from the attack of herbivores, microorganism like bacteria, viruses and fungi and from the harmful effects of climatic factors like too intense temperature, ultraviolet rays and other physiological stress, they are also used in initiation of symbiotic relationship and also help in pollination of flowers. These secondary metabolites are of great importance from commercial and health point of view as they form the basis of many pharmaceutical compounds. Some secondary metabolites, such as terpenoids give plants their sweet or pungent odors, others such as quinines and tannins are responsible for the plant flavor and aroma (for example the terpenoid capsicum from chili pepper). Some of the herbs and spices used by humans to season food for flavor and aroma from ancient times also yield useful medicinal compounds such as ginger, cardamom, coriander etc. (Cowan, 1999). Most of the plant compounds that are found to be medicinally

important are secondary metabolites. In contrast to primary metabolites, secondary metabolites show extensive ecological, taxonomic and biochemical differentiation and diversity. The wide molecular diversity of secondary metabolites throughout the plant flora provide a base for the discovery of novel drugs that greatly helps in drug development for treating a variety of ailments, it has been reported that the use of active substances obtained from the plants are found to be more effective than chemical fungicides. These phytochemicals are non-toxic, efficient and biodegradable; many plant species possess the preservative effect. It suggests the presence of anti- oxidative and antimicrobial agents in the plant tissues (Soliman and Badeaa, 2002, Cimanga et al., 2002). 1.8 Phytochemical Constituents of Phytomedicine Nature offer limitless possibilities, there are 12,000 secondary metabolites isolated from plants until now, it is estimated that these are only 10% and a lot more to be discovered. Secondary metabolites are produced for plant protection against insect and microorganisms. Different properties of plants are shaped by these metabolites like; flavor given by capsanin, odor by terpenoids and pigmentation by tannins and quinines. Most important is the medicinal properties are due to secondary metabolites (Peteros and Uy, 2010, Wallace, 2004). Even elements can also be important phytochemicals, relative to complex organic molecules e.g. selenium is an important component of many vegetables and fruits, involved in many metabolic pathways (Brown and Arthur, 2001). In particular, it is a cofactor for enzymes involved in the production of glutathione, a very important antioxidants (Papp et al., 2007). Phytochemicals are plant chemicals; contain a heterogeneous set of constituents. In broad sense these are plant chemicals that defend plants from environmental threats such as drought, pathogenic attack, pollution, stress and UV light exposure are entitled as phytochemicals. In recent times, it is obviously identified that these have their parts in defense of human health, when significantly taken in diet. These phytochemical compounds are also recognized as secondary plant metabolites and have biological properties such as antimicrobial effect, antioxidant activity, platelet aggregation reduction, enzymes modulation and detoxification, modulation of hormone metabolism, immune system stimulation, and anticancer property (Sen et al., 2010). Phytochemicals are not crucial nutrients as these are not required by human for nourishing life, but have key characteristics to stop or to combat most of common diseases.

Classification of Phytochemicals Phytochemical are bioactive compounds produced by medicinal plant in some quantities, are used as originators to prepare modern drugs (Surh, 2003). As for as the exact classification is concerned it is yet require more exertion, but some of the most important phytochemicals are: Alkaloids form the largest group of secondary metabolites. Alkaloids contain one or more nitrogen atoms as a part of their ring structure. These are alkaline in reaction, which turn the red color of litmus paper into blue color. Common alkaloids are colorless but often optically active substances and mostly exist in solid state but some are liquefied at room temperature. These taste intensely bitter and alcohol solubility is high however lightly soluble in water (Yunusov, 1981). They form crystalline salts when react with acids without the production of water. Alkaloid production is the property of any plant organ. These alkaloids are used by plants as a part of defense strategy against herbivores and pathogens and also those plants produce more secondary metabolites which need cross pollination so these are more abundant in dicots than monocot, as their flowers are showy and scented by phytochemicals, to attract insect(Levin, 1976). These are more effective plant substances, which are toxic and have great physiological properties to humans and animals, due to such properties alkaloids are very effective as drugs (Gurib-Fakim, 2006). Alkaloids are more common in dicots than monocots. Alkaloid rich plants are present in families all over plant kingdom but some family are very rich in secondary metabolites. Pharmacologically, alkaloids are valued as anesthetics and CNS stimulants. There are many Plant obtained alkaloids in medical use, take account of the anesthetics morphine isolated from Papaver somniferum, colchicine for gout, L-hyoscyamine as antispasmodic, codeine for coughs, quinidine as anti-arrythmic and quinine as anti-malarial (Hansen et al., 1995, Willaman and Schubert, 1961). Other important alkaloids of plant origin include (+)-tubocurarine used as a muscle relaxant, the antibiotics berberine and sanguinafine are used as antibiotics cocaine, ephedrine, ergotamine, nicotine, nicotine and scopolamine are used as sedative, and the anticancer agent vinblastin (Somit et al., 2013, Bello‐ Ramírez and Nava‐ Ocampo, 2004). These contain different groups of secondary metabolites bonded with uronic acid monosaccharide- or oligosaccharide. Glycosides contain two portions; Glycone and aglycone, saccharide or uronic acid fragment is termed as glycone, and the additional portion of the molecule is called as aglycone. The important subcategories of glycosides are anthraquinone

glycosides, cardiac glycosides, cyanogenic glycosides, glucosinolates and saponins. Besides, flavonoids commonly come about as glycosides. iii) Cardiac glycosides Possess steroidal aglycone. Their influence is restriction of Na+/K+ and ATPase-pumps of Plasma membrane, which are intensively found in and necessory for the activity of cardiac cells. Cardiac glycosides are existing in medicinal plants like Digitalis purpura (foxglove) of Scrophulariaceae (figwort family), Convallaria majalis (lily of the valley), and Convallariaceae (convall family) (Bernhoft, 2010) iv) Cyanogenic glycosides These contain aglycones group derivatives as of amino acids. These compounds mostly can inhibit iodine utilization and effect in hypothyroidism. As these compounds release hydrogen cyanide, extreme poisonous and noxious at high doses. Some Prunus spp. of Rosaceae contain Cyanogenic glycosides (Ganjewala et al., 2010) v) Glucosinolates This group comprises aglycone formed of sulphur-containing, sharp-tasting amino acid. These compounds have impact on cytochrome P450 in different cells and retard liver bio activation of adjacent pro-carcinogens. The phytochemicals are skin irritating and encourage goiter formation. Glucosinolates are very common in family Brassicaceae (Tookey et al., 1980, Kushad et al., 1999). vi) Saponins It means―soap forming‖ these are also found in glycosides forms. Their aglycones may comprise tetracyclic steroids and pentacyclic triterpenoids. However, their structure is different but contain similar functions. The glycon part of saponins is hydrophilic, which contribute emulsifying features like detergents. Other contributions of these compounds are in immune modulation and antineoplastic effects, haemolysis RBC, photosensitization and hepatitis. Example of saponine containing plant is Narthesium ossifragum of the Family Liliaceae (lily family) (Birk and Peri, 1980). vii) Anthraquinone glycosides These are very inadequate plant kingdom. Their influence is stimulation of water absorption and electrolyte discharge. Rumex crispus of family Polygonaceae is very well known example of saponin containg plant (Agarwal et al., 2000).

vii) Flavonoids and proanthocyanidins Flavonoids possess three-ring structure. These contain to phenolic group benzo-γ-pyrone, while the oligomers of these flavonoids are Proanthocyanidins. These universally exist in plants. Both compounds be able to combine with glycone and form glycosides. The Antioxidant activity and other diverse pharmacological activities of these compounds are due to phenol-groups present in them. The group isoflavones found in flavonoids is recognized as Phytohormone. Flavonoids togather with proanthocyanidins are pigments present in almost all plants. Isoflavones are synthesized in large amount by Fabaeceae species (Kumar and Pandey, 2013).

Basic structure of Flavonoid Flavonoids are produced by plants in reaction to microbial infection (Dixon et al., 1983). These have structure reliant actions, as there are almost 5000 types of Flavonoids found in plants.

(1) (2) (1) glycoside (2) aglycone , (Kumar and Pandey, 2013)

Flavonoids are naturally occurring compounds containing different phenolic. The first flavonoid (rutin) was isolated from citrus, in 1930 it was considered as vitamin P. These are classified as Flavones example of these are luteolin, apigenin, and flavone.

Flavonols these compounds contain fisetin, myricetin, kaempferol, and componds like quercetin.

Flavanones include phytochemicals like naringenin, flavanone, and hesperetin (Middleton, 1998). Latin term “flavus” meaning yellow. These compounds give color to flower fruits and leaves, which on one hand attract insects for pollination and on anther, defend plants from UV rays (Gurib-Fakim, 2006).

Antioxidant Activity of Flavonoids: Flavonoids hold numerous biochemical activity, but the most important defined activity of all classes of flavonoids is being antioxidant. This property is due to the functional groups attached with flavonoids. The conformation, orientation, and number and types of functional groups determine the type of antioxidant activity and potential of free radicals scavenging (Heim et al., 2002). The most important is the configuration of hydroxyl on B ring, which determine the scavenging strength to remove free radicals (Cao et al., 1997).

1.9. Review of the Selected Medicinal Plants

Figure 1 Moringa oleifera Lam. It is a large tree, containing sticky bark, Leaves alternate, compound leaves with tripinnately imparipinnate, deciduous; containing ovoid buds and white flowers containg five stamens and oblong ovary. Fruit is a pendulous pod with nine ribs. Common in tropical regions including Khyber pukhtun khwa, Pakistan. Branches are used as fodder and watchmakers use seed oil as lubricant in very fine machinery. Extracts of Moringa oleifera (MO) is used for water treatment. The pH of seed extract 9.6 or even greater. The coagulant proteins were extracted which showed antimicrobial activities of 1.1–4 log decrease. High turbid samples showed activities similar to alum (Ghebremichael et al., 2005). Almost all parts of Moringa oleifera when used in vitro against microorganisms like bacteria dermatophytes, pathogenic helminths and yeast, showed

promising effects by inhibiting their growth (Caceres et al., 1991). Different extracts of the plants showed positive antioxidant results against peroxyl and superoxyle radicals, among all samples indian plants showed highest antioxidant activities up to 66.8%, quercetin and kaempferol belonging to flavonoid group were the major bioactive compounds, moringa leaves containing highest antioxidant activities (Siddhuraju and Becker, 2003). The plant is an important source of minerals an proteins; including caffeoylquinic acid , kaempferol, quercetin, zeatin, and β- sitosterol apart from that in other recipes used as circulatory and cardiac stimulants, possess antibacterial, antidiabetic, antiepileptic, antifungal Anti-hypertensive, anti-inflammatory, antioxidant, antipyretic, antispasmodic, antitumor, antiulcer, Cholesterol lowering, diuretic, and hepatoprotective. There are many uses of different parts of this multipurpose tree (Anwar et al., 2007). It has strong potential to induce apoptosis, thus, it specifies that the leaf extract has chemoprevention potential for cancer and should be carefully considered as a therapeutic agent for cancer (Sreelatha et al., 2011). Furthermore, the MOL extract presented better cytotoxicity for tumor cells than for normal cells, strongly signifying that it could possibly be a perfect anticancer therapeutic contestant regarding the cancer Cells (Jung, 2014). These results reveal that applying methanol leaf extracts of M. oleifera possessed significant radiation protection of 30 days bone marrow chromosomes in mice (Rao et al., 2001).

Figure 2 Albizia lebbeck (L.) Benth. It is a perennial plant, which produce legumes. The plant is medium sized which reaches to 15 m height at maximum and the shade can reach up to 30m in diameter. Bipinnatly compound leaves with oblong leaflets. Fruits may reach to 30 cm containing 15 seeds at maximum produce nonstop jingle in the breeze, retelling women‘s chatter, therefore also known as "women's tongue"

(Orwa et al., 2009). It is a resourceful plant, which is used as fodder, foliage and timber (Fao and Isric, 2010). Albizia lebbeck is found in Pakistan. Compositional studies showed that saponin is important antioxidant component present in pods and seeds. (Seyydnejad et al., 2010). Among twelve medicinal plants Albizia lebbeck ethanol extract showed major antitumor activity (Haque et al., 2000). Cancer is the second chief reason of death all over the globe. Traditional medicines are mostly used for the treatment of cancer, there are 199 anticancer plants including Albizia lebbeck in traditional medicines are used, over seventy plants showed in vitro plant activities out of which 32 plants showed in vivo results and 29 pure anticancer compounds were isolated from different plants (Tariq et al., 2017). A total of sixty five isolates were studied Albizia lebbeck showed positive response in different biological activities (Hussain et al., 2017a)

Figure 3 Withania coagulans (Stocks) Dunal

W. coagulans Dunal is a small shrub up to120 cm tall. The stalk of the leaves is short 1.5 cm broad lamina.. The inflorescence is axillary cymose clusters while berries are maximum12 mm in width, with dark brown seed contain sharp fruity smell.Withania is very important plant in Unani and Ayurvedic system. In Ayurveda, it is famous to treat almost all diseases in Ayurveda. It contain withanolides (steroidal lactones) which are effective against many diseases, which make it multi-purpose medicine (Jain et al., 2012). Different withanolides like flavonoids, phenols etc. may help in identification of plant and prevent adulteration (Prasad et al., 2010). The plant is very

common in Pakistan and India. The fruits are used as coagulants and hence known as ―vegetable rennet‖ (Dymock, 1893). In Pakistan, t the fruits are used to clot milk known as ―paneer‖ so also known as ―Paneer booti‖ (Nawaz et al., 2011). The plant contain various medicinal effects like alterative, diuretic, emetic and sedative, and used in many Unani and Ayurveda prescriptions like blood purification, tooth ache, insomnia and impotency (Bown, 1995). In addition, it is also used as antimicrobial, anti-hyperglycemic, anti-inflammatory, antitumor, and other intestinal infections, cardiovascular, dyspepsia, flatulent colic, hepatoprotective, immuno-suppressive and free radical scavenging (Maurya et al., 2010). Phytochemical screening showed the existence of alkaloids, amino acids, carbohydrates, organic acids, phenolic compounds, proteins, saponins, steroids and tannins (Mathur et al., 2011). Experiment on mice showed that extracts and isolated withanolides 1-3 and 5 induce hypoglycemic effects, which is in conformity with the use of the plant as anti-diabetic agent by local practitioners (Maurya et al., 2008). Withanolides are, C28 steroidal lactones and have shown to some extent anticancer activities and specially against lung cancer, withanolids like Tubocapsanolide may be valuable to prevent lung cancer (Chang et al., 2007). Therefore, in conclusion, W. coagulans, mainly berries contain curative uses, and there is need of more investigation through their biological activities to develop modern drugs (Gupta, 2012, Chen et al., 2011).

Figure 4 Melia azedarach L.

Deciduous trees. Leaves contain three pinnate; leaflets are serrate. Flowers small, inflorescence is panicles. Calyx and petal are 5-lobed. Stamens are 10-12 and in monadelphous condition. Ovary is up to six locular. Fruit is a drupe. Melia azedarach L. belong to family Meliaceae. It is found all over Pakistan. Locally it is known as ―Tora shandi or Bakyana‖ in Punjabi it is known as Dherk. It is favorite of scientist to study because it is effectively used in folk medicines.It used as to resistor pests and diseases. When elm leaf beetle, Xanthogalleruca luteola (Müller) consumed the leaves of Melia, larvae died without molting (Valladares et al., 1997). The ethanolic extracts of seeds and fruits is very effective against pathogenic fungi. These pathogens include Aspergillus Candida albicans flavus, Fusarium monitiform and Microsporum canis (Carpinella et al., 1999). A plant pathogen Aspergillus niger, was tested against different aqueous extract belonging to different plants in which one was Melia azedarach which showed antifungal activity in all concentration, in conclusion it was stated that Melia extract can be utilized to prevent seed borne pathogens (Danish and Robab, 2015). The bark extract of Melia azedarach was treated with Silver nitrate and silver nanoparticles were synthesized, which is believed would help in the improvement of novel biomedical products established on silver nanoparticles (Mehmood et al., 2014). Extract of Melia azedarach flower contain thirty eight phytochemical in which twenty four were reported for the first time from any part of the plant, which contained a polyisoprenoid, aromatics, branched and n-hydrocarbons, fatty acid methyl esters, fatty acids and fatty alcohols (Muhammad et al., 2015). The isomeric mixtures from the plant were examined and it was known that it can be a potential antitumor chemotherapeutic agent as it induce S-phase arrest (Joray et al., 2017).

Figure 5 Hedera helix auct. non Linn It is Perennial climber and can reach up to 30m. Leaves are simple variously lobed, base of the leaf is cordate, while apex is acute, flowers are yellow, Calyx entire, Fruit is berry. Hedera helix, usually recognized as English ivy. A strong, intense, fast growing, climbing vine. In many countries, it is sold as ornament plant. Ivy extracts is the most popular non-antibiotic remedy. It is very effective in acute upper respiratory tract infections (URTIs) by reducing the frequency of cough and symptoms of URTI (Holzinger and Chenot, 2011). Hedera helix leaves extract is effective in the treatment of respiratory infection as it forms intense mucous formation. It contain triterpene saponins compound which is effective in cough, apart from that the extract shows analgesic, anthelmintic, anti-inflammatory, anti-leishmanial, antimicrobial, anti-mutagenic, anti-thrombin, Anti trypanosomial, moluscocidal, spasmolytic/antispasmodic, antioxidant and antitumor, activities (Lutsenko et al., 2010). The α-hederin from the leaf extract of Hedra helix at 0.005 and 0.001 mg/ml is anough to kill Fasciola and Dicrocoelium in vitro as well as in vivo while endured by sheep (Julien et al., 1985). Hedera extract is also effective against skin diseases and have antibacterial action (Brantner and Grein, 1994). The antioxidant and hydrogen peroxide scavenging were determined of the ivy extract and the recommendation is that should be further examined as source of anticancer novel compounds (Romman et al.).The screening of medicinal plants develops new drugs. Polar and non-polar ectracts from, Hedera nepalensi were tested against Escherichia coli, Klebsiella pneumonia, Pseudomonas sp., Staphylococcus aureus, and Staphylococcus epidermidis, which showed moderate antbecterial activity. In all forms of extract,

chloroform extract showed (78%) inhibition of Fusarium oxysporum (Bibi et al., 2015). A saponin, α-hederin compound were tested on mouse B16 cells, it is concluded that it induces cell death (Danloy et al., 1994).

Figure 6 Fagonia cretica L. (Virgin's Mantle) Fagonia cretica also known as Virgin's Mantle has a synonym Fagonia deflexa (Daniel, 2006). The roots are cylindrical tap roots, the plant is up to 30 cm elevated from soil, branched, glandular, while leaves are cauline., trifoliate, narrow elliptic, petiole is about 16 mm long, flowers are polysepalous, polypetalous, purplish, congaing nectars (Beier, 2005). The plant contain Novel compounds, which are effective against diseases like skin lesions and cancer (Qureshi et al., 2016). (Fagonia cretica L. have been previously described in the folk medicines to treat diabetes but further scientific study need to be carried out., the crude extract contain high inhibitory value(IC50 value: 38.1 μg/ml) (Saleem et al., 2014). It is widely used as tea to treat breast cancer; Fagonia is significant source of anticancer drug. With the application of extract time and dose-dependent seizure of the cell cycle occred within five hours and apoptosis within twrnty four hours of the (mutant-p53) human breast cancer cell lines (Lam et al., 2012). The plant has been proved very effective against tumor and cancer. Substantial cytotoxic activities were performed on salt water shrimps at LD-50 118.9 ppm, so it is suggested that the plant contain high anticancer potential (Hussain et al., 2007). Effects triterpenoid saponins I and II were isolated saponin-II decreased the RBC and Leukocytes count (Saeed and Sabir, 2003). The compounds were isolated from Fagonia (Linoleic, lenoleinic, myristic, oleic, palmitic and stearic

acids) and teste for antibacterial activities (Eman et al., 2010). Steroidal saponin glycoside were fractionated from Fagonia and applied which induced apoptosis in cancer cells (Waheed et al., 2012). The major destructive reason throughout and afterward the ischemic/hypoxic affront is the production of free radicals, Fagonia cretica linn contain an extensive series of antioxidants, that are used to treat various disorders, related to gene expression encumbers platelet clump establishment (Rawal et al., 2004, Rawal et al., 2009)

Figure 7 Valeriana jatamansi Jones It is about 50cm tall Perennial herb with thick roots; with numerous persistent petiolate basal leaves, up to 7cm long;; flowers gray or streaked pink, inflorescence corymb; fruit capped with whiskery calyx. Valeriana jatamansi displayed larvicidal action counter to diverse mosquito species. The average lethal concentration (LC50) of extracts, which were effective against mosquito larvae, were 80.60 mg/liter, respectively. The fraction of compounds recorded from extract were 2-butanone,(11.19%), aristolene (5.20%), cadinol (5.32%), caryophyllene oxide (5.55%), cubenol (5.87%), and patchouli alcohol (8.65%) (Dua et al., 2008). Vinutha reported that there are 76 plant among which one is Valeriana jatamansi , are used traditionally for development of mental power (Vinutha et al., 2007). Two novel compounds bakkenolides, valerilactones are two known analogues. were ectracted and isolated from Valeriana jatamansi these compounds presented potent neuroprotective activities neuroblastoma. (Xu et al., 2011). Valeriana jatamansi root extract investigation showed that these comprise considerable quantities of valerenic acid, which possess antioxidant potential of plant extracts. The concentration of

valerenic acid is very important in antioxidant activity.(Negi et al., 2012). Novel Valeriananoids were isolated from Valeriana jatamansi Their structures were interpreted by spectroscopic means as well as X-ray crystallography (Ming et al., 1997). The root of the plant has very much antioxidant potential, 20 compounds were identified from the root extract, which contain 74% essential oils, and considerably greater flavonoids were detected. The plant contain high potential for antioxidant phytochemicals, so it should be commercially cultivated (Bhatt et al., 2012).

Figure 8 Saxifraga flagellaris Willdenow in C. M. von Sternberg The word ―saxifrage‖ came from Dioscorides, the Greek herbalist. ―saxum‖ means rock, and ―fragere‖ means disruption (Webb and Gornall, 1989b). The plant is native to Eurasia. It is also known as spider plant (Webb and Gornall, 1989a). The plant is found in distant clump, with slender rhizome, leaves are cauline with no petiole and elliptic shape, Inflorescence solitary or cyme, sepals erect, glandular and petals yellow not patterned. The plant was collected from and Kashmir after literature review. The plant can be used as medicinal herb and ornament (Zhang, 2013). Dioscorides assumed that Saxifraga eliminate urinary stones (Dioscorides et al., 1959). Because the rhizome bulbils resemble kidney calculi, but this assumption has, no systematic evidence treating this disease. However, Chinese used to cure ear infections with this plant; its common botanic term, ear plant, refers saxifrage to this activity, apart from that it is used in Japanese herbal prescriptions, in Japanese and Tibetan herbal prescription these are used for bleeding, ear infections, eczema, pain, trauma, vomiting etc. Saxifraga spp., have medicinal properties to prevent cancer cells from development, even though the active compounds are still

not known (Zhang, 2013). Saxifrages are also known as London Pride, as these are popular in gardens plants for beauty and capricious flowers (McGregor, 2008). The extract was taken from eight species of genus Saxifraga, which are S. aizoides, S. caespitosa, S. california, S. eschscholtzii, S. ferruginea, S. hirculus, S. integrifolia, and S. michauxii,. The key compounds found in these species were flavonoids, galactosides 3,3′-di-O-methylquercetin, quercetin, myricetin, kaempferole and monogluocosides, along with that 3-O-Methyl, O-methylated flavonols and novel compound O-methylated dihydrokaempferol reported (Miller and Bohm, 1980). Kayani et.al., conducted a survey in which he reported Saxifraga from Abbottabad (Kayani et al., 2014). In ladakh India and Pakistan a system parallel to Unani and Ayurveda is used which is known as the "Amchi system" (medicinal system of Tibet) in which several herbs are mixed together the expert is known as Whim. Saxifraga paste is used in Amchi system to heal wounds and as antiseptic. (Buth and Navchoo, 1988). Barman et.al., (2011) reported that saxifrage contain strong antibacterial (both gram positive and Gram negative ) and antifungal (plant pathogens) activity at 250 μg/ml (Barman et al., 2011). In our research, we selected Albizia lebbeck, Melia azedarach, Hedera helix, Saxifraga flagellaris, Valeriana jatamansi, Fagonia cretica, Withania coagulans, and Moringa oleifera based on their ethno-botanical significance.

CHAPTER 2 MATERIALS AND METHODS

The locally available plant species ―Albizia lebbeck, Melia azedarach, Hedera helix, Saxifraga flagellaris, Valeriana jatamansi, Fagonia cretica, Withania coagulans, and Moringa oleifera‖ collected and tested for the anticancer biological activities.

2.1 Collections and Drying of Selected Plants Materials Plants were surveyed, and gathered from diverse areas of Khyber Pakhtunkhwa, Pakistan. The parts of each plant species were used for extraction with 70% ethanol. Plant parts were washed, shade dried, followed by oven drying, grinded to powder. The powder was kept in cool dry places before extraction of the bioactive compound(s).

2.2 Extraction and Fractionation For extraction, cold maceration method was used. About, 1.5 kg powdered material of tested plant was taken and dipped in 70% ethanol (2 L) and kept at 25 ºC for 5 d. After 5 days of incubation, the mixture passed thrice through Whatmann filter paper and a clear filtrate was collected. The filtrate was then evaporated at 40 ºC under vacuum using rotary evaporator. Different parts of selected medicinal plants are used for extraction (Table.2.1).

Table 2. 1 Medicinal plants parts used for extraction S. No Codes Plants names Part used 1 K1 Albizia lebbeck Fruits 2 K2 Melia azedarach Fruits 3 K3 Hedera helix Leaves + stem 4 K4 Saxifraga flagellaris Rhizome 5 K5 Valeriana jatamansi Whole plant 6 K6 Fagonia cretica Whole plant 7 K7 Withania coagulans Fruits 8 K8 Moringa oleifera Fruits

2.3 Fractionation of Dried Filtrate on the Basis of Solubility The dried filtrate was re-dissolved in water (100 ml) and fractioned by using various organic solvents, like n-hexane, ethyl acetate, chloroform and methanol with the help of separating funnel. The aqueous solution of the filtrate then transferred to the separating funnel and supplemented with n-hexane (in a ratio of 2:1). The contents of the funnel then shaken vigorously and left it to stay for a while to get distinct phases of water and n-hexane. The n-hexane is less dense as compared to the water and stayed as an upper layer in the separating funnel. Water layer was first collected in a separate glass beaker or flask leaving the n- hexane layer in a separating funnel. The n-hexane fraction then transferred to a new glass beaker. This process formerly repeated many times until a colorless n-hexane layer attained. The separated n-hexane layer was then concentrated in rotary evaporator and marked as an n-hexane fraction of the plant sample. Similarly, the remaining distilled water (aqueous) layer was subsequently subjected to fractionation with chloroform and ethyl acetate following the same procedure as described earlier.

2.4 Antifungal Activity Fungal strains used

The pathogenic strains, i.e. Aspergillus flavus, Alternaria alternate, Fusarium oxysporum, and Polysphondylium pallidum were obtained from and used in antifungal assays. 2.4.1 Media preparation for fungal growth Dissolved 39 g of potato dextrose agar (PDA) in 1 litre of distilled water and sterilized in autoclave at 15psi (121 ºC) for 15 minutes. Cooled to room temperature and pour into sterilized petriplates to soloidify. The hot PDA medium was transferred in to Petri plates using sterile conditions of the laminar flow hood and left them in the hood to solidify. Agar well diffusion method was followed (Samie et al., 2010). Using the micropipette, 100 μl of the different fungal cultures in sterile distilled water (SDW) was placed over the surface of an agar plate and spread using a sterile inoculation loop. Using a sterile cork borer, holes were made in each of the culture plates. In each well, 75 μl of crude extract or solvent fractioned portion along with control (SDW) of the selected plant was added. The culture plates were then incubated at 37 °C, and the results were observed after 24 hrs depending on the fungal growth. The clear zone around each well was measured in mm, indicating the activity of the plant extract against the fungus. Each test was triplicated and standard deviation was calculated.

2.4.2 Antibacterial Activity We used agar well diffusion method (Perez et al., 1990). The method was implemented for screening of antibacterial activity. In the assay all fractions of selected eight medicinal plants were used. 2.4.3 Microorganisms Used Streptococcus mutans, Methicillin-Resistant Staphylococcus aureus (MRSA), Staphylococcus aureus, (Gram-positive), and Serratia marcescens,, (Gram-negative) bacteria were used for antibacterial activity.

2.4.4 Media for Bacterial Culture Luria Broth, miller (LBM) medium (25 g/l) was liquefied in distilled water and the pH was adjusted to 7.0. After the media was prepared, 100 ml of LB broth was taken in 250 ml flask and autoclaved. Bacterial strains were then inoculated and the flasks were transferred to a shaking incubator operated at at 37 ºC and 150 rpm for an overnight. For the preparation of LB Agar, 25 gm on LB agar was dissolved in one liter of distilled water after adjusting the pH it was autoclaved.

2.4.5 Preparation of Inoculum 6 McFarland turbidity standard [10 colony forming unit (CFU) ml-1] was attained by mixing selected bacterial strains and normal saline together. The inoculums were then introduced to the LB Agar Medium.

2.4.6 Preparation of Agar Plates LB agar was prepared, autoclaved and were added into sterilized Petri plates, the poured ager were then allowed to cool at room temperature. Maintaining high sterile ambiance agar well diffusion method (Perez et al., 1990) was adopted by using sterilized cotton swab, cultures lawn of the selected bacterial strains were introduced on agar plates. With the help of sterilized borer, five wells were tunneled in medium.

2.4.7 Extract Preparation for Activity Crude extracts of and fractions of selected medicinal plants were dissolved in DMSO (20mg/ml) separately. A standard antibiotic solution (2 mg/ml of Cefotaxime) was used as positive control and pure DMSO was used as a negative control.

2.4.8 Incubation and Measurement of Zone of Inhibition Using micropipette, 75-μ l of a fraction was transferred in to a labeled well. One of the well in a plate was spared for cefotaxime as a positive control and dimethyl sulphoxide (DMSO) as negative control. After the samples transferred to the designated wells, the plates were incubated at 37ºC for 24 hrs. The diameter of transparent zones (mm) that represents antibacterial potential of the fraction was measured. Standard deviation was determined, along with mean value after repeating the experiment three times.

2.5 Phytotoxicity Activity 2.5.1 Bioassay For allelopathic studies, sandwich method of Fujii (2003), (Fujii and Hiradate, 2005) was followed with slight modification. In this method, 15 g agar was dissolved in a 1liter of distilled water and was autoclaved. The agar solution was was poured into sterilized Petri plates and left in sterile environment at room temperature to solidify. Dried plant material of each test species at various amounts (10 mg, 20 mg and 40 mg) were placed on the agar plate and covered with another thin layer of agar solution. After complete solidification, ten seeds of lettuce (Lactuca sativa L.) were placed on agar gel in the plate. Each plate was then sealed with the plastic tape and incubated for 72 h at 24 ºC under dark conditions. The lengths of radicle and plumule were recorded in cm. In the control treatment, only agar gel without any plant material was used as a seedbed for lettuce seeds. Each treatment was repeated three times. Means of root and hypocotyl lengths of each medicinal plant were calculated by comparing with control. The normal distribution pattern was evaluated for the 10 mg, 20 mg and 40 mg of plant materials with root and hypocotyl growths of selected medicinal plants.

2.6 Anticancer Activity To check the anticancer activity of selected medicinal plants, cell viability assay was carried. MTT reagent and HT-29 colon cancer cell lines was used to test the anticancer activity. 2.6.1 Principle of MTT Assay This technique is used to find out the cytotoxicity of plant extract and can only be applie to living cells when mitochondrial enzymes are functional. In this technique mitochondrial reductase (mitochondrial enzyme) is involved. When formazan is formed during the reaction, the yellow color of MTT reagents becomes blue. These complexes comprise cell toxicity, deregulation of 36

metabolic development and later reduced appearance in the bioassay. The Mitochondrial enzyme reductase is responsible to form purple formazan by reduction of Yellow MTT reagent ((3-(4, 5- Dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide, A tetrazole) (Mosmann, 1983). Formazan was then dissolved into DMSO and ethanol. 2.6.2 Media Preparation To prepare the media Foetal Bovine Serum (FBS) and Dulbeccos Modified Eagle Medium (DMEM) (Fischer Scientific) were heated at 37 ºC. Then Penicillin and streptomycin (10mg/ml) of each were taken and antibiotic solution were prepared, after this 5ml of antibiotic solution and 50 ml of FBS were added into 500 ml of DMEM. To stabilize the cell culture L-glutamine were taken in 200 mM The prepared media was stored at 2-8 ºC for the utilization in experiment. 2.6.3 Defrosting Cells Media for the cells DMEM was warmed up at 37ºC, just sixty minutes earlier use, Cryo vials possessing cell culture were put in the incubator at 37ºC (Thermo Hera cell 150). These vials containing cell were stored earlier in Liquid Nitrogen. To make the vial surface free from contamination, prior to opening were surface sterilized with the help of 70% Ethanol and were Centrifuged (Centrifuge Thermo scientific legend RT+) at 1000 rpm for five minutes. After removing the supernatant, the PPT was suspended in 10ml of media. Cell culture flasks (25ml) properly labelled with (Date/Cell Type/ Passage number) containing the suspensions were shifted to incubator and were incubated for several hours at 37°C. After each 24 hours the media was refreshed. 2.6.4 Trypsinisation of Cells Trypsinisation is required for individual cell to get optimum ambiance and nutrients, while proceeding with the experiments. These were trypsinized when the confluent reached to 80-100%. Media was removed and Cells were washed thoroughly with 10 ml of PBS (Phosphate Buffer Saline) to remove dead cells. The dead cells do not adhere to flask surface so these were detached. Trypsin-EDTA (2 ml per 25 ml flask) at 37°C for 1-3 min was optimized and was placed into culture flask. The Trypsin-EDTA was then added to Cell masses to break it, after that observation was carried out under inverted microscope (Leica DMIL inverted microscope). Subsequently cells were fully separated from the flask; then added with 8ml arranged media. The Cell masses was broken down with the help of pipette, by the action of taking it up and down. The solution was now divided into two parts and each part was added with 5 ml of DMEM media and was placed

in the incubators at 37°C for 24 hrs. For the removal of trypsin media was emptied and refreshed. 2.6.5 Freezing Cells When the cells were 80-100% confluent they were freeze. Cells were pour out as already explained. After displacing cells. To remove any traces of trypsin 5 ml of media was added. Cells were then centrifuged for 60 seconds at 1000 rpm, the was suspended in a mixture of 90% FCS (Foetal Calf Serum) and 10% DMSO and shifted to cryo vial tube. After that the tubes were placed in freezer at -80°C for 24 h and then transferred to liquid nitrogen. 2.6.6 Cell Counting Trypsinisation, followed by centrifugation (1000 rpm/ minute) resulted in precipitated cells; the pellet was re-suspended in DMEM (1 ml). The cell suspension (10 μl) and trypan blue (90 μl) was then added and the contents were thoroughly mixed with the help of a pipette. To count the cells, 10 μl of cell suspension was placed on cell counter (Haemocytometer) Means were calculated after counting cells in each 5 square of cell counter. Number of cells was calculated using the following equation: Number of cells/ml = Mean of cells x 100 x 10 (dilution factor) 2.6.7 Preparing MTT Solution: Using PBS as a stock solution, the MTT solution (5mg/ml) was designed. According to the need the solution were further fractionized and diluted. Stock solution was diluted to 1:5 in media (RPMI 1640). 2.6.8 Dilution of Plant Extracts The extracts of each selected medicinal plants were then dissolved in DMSO, to prepare a stock solution and were further fractionized into 10/50/100 μg/ml. the dilution was carried out by the addition of DMEM, as per requirement after calculating the amounts. 2.6.9 MTT Assay Methodology A 96-well plates was used to seed the cells (5,000 cells/well) in 100 μl medium (RPMI 1640), and were let to adhere overnight. Different fractions of plant extract were then added to individual wells. Then incubated at 37°C for 24 hrs. after incubation each individual well was supplemented with 10 μl MTT reagent, and then incubated again for 4 hrs. For stabliztion of MTT crystals 100 μl of DMSO was incremented. Again the plates were incubated at 37°C for a night. Percentages inhibitions were determined and expressed in Results Percentage inhibition was calculated by the following formula Percentage inhibition= (B-A)-(C-

A) × 100 (B-A) A= Average absorbance of media B= Average absorbance of media with cells C= Average absorbance of extract sample 2.6.10 Statistical analysis All the tests were performed as individual triplicate experiment. Results obtained from crude extract activity were calculated for average % inhibition while data obtained from the results of activity of fractions was statistically analyzed by Graph pad prism 6.

2.7. Antioxidant Assays 2.7.1 DPPH free radicals scavenging activity For DPPH free radicals scavenging activity the method of (Brand-Williams et al., 1995) was followed with some modifications. DPPH (24 mg) was dissolved in 100 ml of methanol to prepare DPPH solution. The stock solutions of plant samples were prepared in methanol having concentrations of 1 mg/ml and then diluted to the concentrations 1000, 500, 250. 125 and 62.5 μg/ml. DPPH and sample solutions were mixed in a ratio of 1:1 and were incubated at 23°C for 30 min. Finally, absorbance was measured at 517 nm using UV spectrophotometer (Thermo electron corporation USA). Ascorbic acid was used as positive control. Percent radical scavenging activity was measured using the following equation; Scavenging effect % = control absorbance - sample absorbance/control absorbance x100

2.8 Heavy Metals Analysis Heavy metal analysis was done according to AOAC 1995 using flame atomic absorption spectroscopy. Using wet digestion method (Meena et al., 2010, Helaluddin et al., 2016). 2.8.1 Acid Digestion of Samples Wet digestion method was used for sample analysis. 0.5 g to 1.0 g of the each plants sample was weighed for analysis and digested with 15 ml of 20 % sulphuric acid solution in test tube. Now the test tube with sample in it was left for 24 hours so that the sample can be fully dissolved in the solution. For better digestion each test tube was heated for 10 to 15 minute. The solution of each test tube was filtered by using the whatman-42 filter paper and a filtrate was obtained of each plant sample. Distil water was added to each filtrate to make the final volume of the solution 30 ml. Flame Atomic Absorption Spectrophotometric techniques were used to measure the 39

concentration of Lead, Mercury, Arsenic, Cadmium and Zinc in the each extract of plant sample solution.

2.9 Proximate Analysis The proximate analysis (proteins, fats, carbohydrates, moisture and ash) of selected medicinal plants were resolved by using AOAC methods (Chemists, 1990, Feldsine et al., 2002). 2.9.1 Determination of Moisture

The sterilized (30 minute at 1050C in oven) and clean were weighed. Dry plant materials (3 gram) of individual samples were taken in Petri plate, and retained in oven (1050C for 6 hours). Selected samples were then exposed to cooling in desiccators and weighed. This procedure was implemented two times. For the calculation of moisture content the following formula was used. Moisture (%) = Weight of fresh sample – Weight of dry sample × 100 2.9.2 Determination of Ash

The crucible were washed and retained in oven (550 0C/30 minute), to dry out. The flasks/crucible were then placed in desiccators to cool out. The selected plants material was taken in quantity of 1.0±.01 gm in dried form in crucibles. After burning the sample at low flame the crucibles were placed in a Muffle furnace and treated with

5500C and for 3 hours till the white ash was attained. After getting the white ash, it was placed in desiccators for cooling. The crucible was weight. The percent ash was calculated as (Horwitz et al., 1970) Ash (%) = (%) weight of sample after ashing × 1 0 0 Weight of sample 2.9.3 Determination of Curd Fat Powder of selected anticancer medicinal plants (5gm) materials were processed in paper thimble and connected it to a goldfish of soxhlet extractor. 250 mL of n-hexane or petroleum ether solvent was poured on the top of the thimble, and run for six(06) hours on heating mantle. After extracting the crude fats, thimble was detached. In next step, the flasks were cooled in a desiccator and weighed. To determine the percentages of crude fats in sample the following formula was used (Howitz, 2000).

Crude Fat (%) = Weight of beaker with fat – Weight of empty beaker × 100 Weight of original sample

2.9.4 Determination of Protein Proteins were determined in the selected plant samples into three steps: i. Digestion. ii. Distillation. iii. Titration. i. Digestion To carry out digestion process dried material (1gm) of selected plants were taken in digestion flask and 2 gm of digestion mixture was added. The digestion mixture contain copper sulphate, Potassium sulphate, and ferrous sulphate, in the ratio of 1:18.5:0.25 (w/w/w) with further addition of 20 mL concentrated sulphuric acid. Until the clear appearance the heating process was continued. Then further boiled for two hours and cooled. To the digest (5ml), distil water (30 ml) mixed. Then in the volumetric flask the digest diluted to100 mL. ii. Distillation The parnas Wagner distillation assembly was set. Boric acid (4% ) 40 ml was taken with the addition of two droplets of methyl red indicator, was ,the acid become pink in color. In the next step the digest (5ml) was moved to the extraction assembly. Inside the assembly 10 ml of NaOH (40%) solution was supplemented to the digest. After 10 minutes when the distillation process completed, the Boric acid changed to Ammonium Borate indicated by yellow color iii. Titration The boric acid having trapped ammonia was titrated with 0.1N hydrochloric acid, the color of boric acid containing ammonia changed again to pink. The percent protein was calculated by the following formula. Protein (%) =1.4 × 6.25 × 0.1N HCl × Vol OF H2 SO4 (used) Weight of sample Where 1.4 = Weight of Nitrogen expressed in gram in the formula 6.25 = Protein factor for vegetable

2.9.5 Determination of Carbohydrate Carbohydrate was determined by using the following formula (Müller and Tobin, 1980). Carbohydrate (in grams) = 100-(%crude fat + %crude fiber + %Ash + %Protein)

2.10 Phytochemical Analysis of Plant Material Phytochemical screening of crude ethanolic extracts was carried out for chemical classes like alkaloids, glycosides, flavonoids and saponins (Sopowara. 1993; Trease & Evans, 1996:Iyengar.1995; Evan et al., 2002; Deshpande & Kadam. 2013). 2.10.1 Test for Alkaloids (Mayer’s reagent) In distilled water (60 ml) mercuric chloride (0.3555 g) was dissolved to make solution (A ) and to make another solution (B), distilled water (20 ml), was added with potassium iodide (5gm) and a solution was formed. The two solutions were mixed together and volume was enhanced to 1000 ml by the addition of distilled water. Dragendorff’s Reagent Two solutions were prepared Solution (A) and Solution (B). The contents and quantity of solutions (A) were basic Bismuth nitrate (1.7 g) and Tartaric acid (20g) dissolved in 80 ml of water. While Solution (B) contained Potassium iodide (16 g) dissolved 40 ml. After formation of Solution A and B, and mixing with each other in the ratio of 1:1. The plant extracts (0.5-0.6 g) each mixed with 1% HCl (8 ml) were heated and filtered. The filtrate of each selected medicinal plants (2ml) were treated with Mayer‘s Reagent and Dragendorff‘s reagent. The presence of Alkaloids were observed by precipitation or turbidity. 2.10.2 Test for Flavonoids For the removal of fatty Materials, the prepared extract (0.5 g) was shaken with pet ether. After defatting the selected plant extract residues was dissolved in 80% alcohol (20 ml) and then filtered. The filtrate was used for the following tests: a) The filtrate (3 ml ) was mixed with 1% AlCl3 (4 ml) in MeOH solvent in a test tube. Formation of yellow color was observed to indicate the presence of flavonols, flavones and/or chalcones was indicated by the presence of yellow color. b) The selected anticancer plant filtrate (3 ml) was mixed with 1% KOH (4 ml), which resulted in Dark yellow color shown the occurrence of flavonoids.

2.10.3 Test for Saponins The selected anticancer plants extracts (0.5 g) was mixed with distilled water and boiled, the presence of saponins were indicated. 2.10.4 Test for Glycosides Killer kiliani test –the extract [50mg] was dissolved in distilled water and then filtered. To 2 ml of filtrate 1ml of glacial acetic acid and a drop of ferric chloride and a drop of concentrated sulfuric acid was added. Green blue color to upper layer and reddish brown color at the junction of two layers indicates the presence of cardiac glycosides 2.10.5 Test for Phenols Each of the selected anticancer medicinal plant extracts (500mg) was dissolved in distilled (5 ml). after adding a few drops of neutral ferric chloride solution (5%) dark green color appeared which is the indication of phenolic compounds.

2.11 Isolation of Compounds After fractionation and on the basis of results of preliminary activities DCM (dichloromethane) fraction of Saxifraga flagellaris was used for compound isolation. Column chromatography was used for isolation. 2.11.1 Methodology of Column Chromatography Silica 60 (Sigma-Aldrich) with pore size 0.035-0.070mm was used for column Chromatography. First glass column was washed and dried. The column was adjusted in the stand and was fitted in it. Sufficient amount of n-hexane was added to the column. The tap of the column was closed. Then slowly sufficient quantity of silica was poured in the column very carefully up to 5-6cm. Then before the column to be loaded with the extract slurry, the column was allowed to drain by adding more n-haxen to the column in order to reduce chances of air bubbles in the silica slurry. Weighted extract, which was DCM fraction, was completely dissolved in small amount of distilled water. Dry silica was added and a fine slurry of extract was prepared. Sample was adsorbed on silica and kept to dry at room temperature to a fine powder. When sample was completely dried then it was carefully loaded over the silica slurry, preloaded in the glass column which form a uniform layer. Few grams of dry silica was added over the sample in the column to protect the uniformity of sample layer and keep its evenness during the further process. Elution of column was started 100% n-hexane. Column was eluted with mobile phase of organic solvents systems of polarity basis. Two solvent systems were used in the whole process. First was of n-haxane and

ethyl acetate system and the other second was of chloroform and methanol system. Firat of all 100% n-haxane was run through the column. And elution‘s were collected in glass vials. Then each glass vial was subjected to TLC and the elution‘s of similar Rf values were combined. The slowly the polarity of the system was changed to 90%n-haxane and 10% ethyl acetate. Elution‘s were collected in glass vials, subjected to TLC and vials of similar Rf values were combined. The process was repeated with slowly and gradually increasing the polarity of the solvent system as 80%n-haxane with 20% EA, 70% Hxn/30% EA, 60% Hxn/40% EA, 50% Hxn/50% EA, 40% Hxn/60% EA, 30 Hxn/70% EA, 20% Hxn/80% EA, 10% Hxn/90% EA and finally 100% EA were used. In all these elution‘s spots with similar Rf vales were combined. Same process was repeated with solvent system two (chloroform and methanol). Organic solvent system two was started with 100% chloroform, then slowly and gradually the polarity of the

system was changed (90% CHCl3 10%, Methanol, 80% / CHCl3 20% Methanol,70%/ CHCl3 30%,

Methanol, 60%, CHCl3 40%, Methanol, 50% /CHCl3 50% Methanol, 40% CHCl3 60%, Methanol,

30% CHCl3 70% Methanol, 20%/ CHCl3 80% Methanol, 10% CHCl3 90%, Methanol, and finally 100% Methanol,) and the elutions collected in each ratio were subjected to TLC and spots having similar Rf values, were combined. 2.11.2 Thin layer chromatography Thin layer Chromatography (TLC) was carried out on TLC Plates, silica 60 F254 with Fluorescent indicator, pre coated aluminum cards (0.2 mm thickness) from Merck (Germany). TLC was performed for all elutions of all ratios, collected from the column. Elutions having spots with similar Rf values were then combined together for further isolation. Following procedure was adopted for TLC. 2.11.3 Spotting and developing the TLC plate TLC plates of required size were cut by using sharp cutter. A pencil line drawn on one end of the cut TLC card. Cards were marked for application of elution spot. Using pointed capillary tube on the pencil line marked a spot of the sample was inject there, at one side of the card. Samples were applied at equal distance from each other. Different mobile phases (solvent systems) were prepared for different elutions depending on the mobile phase percentage during collecting elutions through the column. Then TLC cards were placed in the TLC tank and allowed to develop. After some time when mobile phase reached at upper end, the cards were removed. Solvent front was marked and cards were dried. TLC plates

were visualized using ultra violet (UV) light at 254nm and visualized spots were marked for the determination of Rf values. If the spots are not clear then H2SO4 spray was used and dry on hot plate, holding with forceps. Then see in the UV box and distances covered by mobile phases and compound, were measured and Rf value was calculated as, Rf = Distance travelled by visualized spot/Distance travelled by the solvent front Elutions with best separation and enough quantity were repeatedly, subjected to column chromatography using pencil column to get pure compound.

2.12 Identification and Spectral Analysis of Compounds Isolated compounds were subjected to identification and for spectral analysis. 2.12.1 Nuclear Magnetic Resonance Spectroscopy (NMR) Analysis Isolated compounds were dried, weighed (5-10mg) and dissolved in (2ml) deutrated solvent (Merck) for NMR. Compound isolated from DCM fraction of saxifrage flagilaris was dissolved in Deutrated chloroform as it was soluble in that and the compounds are soluble in DMSO. The solutions then pipetted in clean NMR tube by the help of Pasteur pipette to a height of 5.2cm.

Proton HNMR, Carbon C13NMR, DEPT 90 and 135 and correlation experiments were run on NMR. NMR spectra were recorded using a JEOL Eclips 400 NMR Spectrometer with JeoL Delta Version 7.2 control and processing software was used to perform different NMR experiments, where peak position were quoted on the scale relative to an internal standard.

2.13 Anticancer Activity of Pure Isolated Compounds 2.13.1 Chemicals and reagents 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma- Aldrich (Hamburg, Germany). Dichlorofluorescein diacetate (DCFHDA) was obtained from Molecular Probes (Eugene, USA). Annexin V-FITC Apoptosis Detection Kit (ab14085) and Caspase 3 Assay Kit (Colorimetric, ab39401) were obtained from abcam (330 Cambridge Science Park, Cambridge, UK). Antibodies (cleaved caspase-3, cleaved PARP, and β-actin) were purchased from Santa Cruz Biotechnology (Texas 75220, USA). All chemicals were used as prescribed by the manufacturers. 2.13.2 Cell culture HCT 116 (ATCC® CCL-247™), and PC-3 (ATCC® CRL-1435™) cell lines were cultured in DMEM medium. Medium was supplemented with 10% FBS, 1% penicillin streptomycin, and

cells were maintained at 37˚C in a humidified atmosphere of 5% CO2. 2.13.3 MTT assay Cell viability was measured by the MTT assay. Cells were seeded into 96-well plates at a density of 2 × 105 cells/well. Triplicate wells were treated with vehicle and different concentrations of Adlumidine for indicated times. After incubation at 37°C in 5% CO2, medium was removed, and 200μl fresh medium with 20 μl, 5 mg/ml MTT solution was added. After 4 hours, the medium with MTT was removed and 200 μl DMSO was added to each well. The plates were then gently agitated until the color reaction was uniform, and the OD540 (optical density at a wavelength of 540 nm) was determined using a 96-well plate reader. Microsoft® Excel 2007 was used for data analysis. Vehicle treated cells served as the indicator of 100% cell viability. 2.13.4 Measurement of ROS generation To detect ROS generation after treating cells with Adlumidine, 5 μM DCFHDA was added to the cell culture medium for 30 min in the dark. DCFHDA is a non- fluorescent dye that is converted to highly fluorescent DCF when oxidized by intracellular ROS and peroxides, and it has an excitation and emission wavelengths of 480 nm and 520 nm, respectively. The cells were washed with 1X PBS and then analyzed on a confocal laser scanning platform (DM/R-TCS, Leica) coupled to a microscope (Leitz DM REB). 2.13.5 Annexin-V-FITC apoptosis detection assay Apoptosis assay was performed as prescribed by the manufacturer (abcam: ab14085 protocol).

Briefly cells were seeded in 6 wells plate (2 × 105/well), and incubated with Adlumidine for indicated time. Next, cells were gently trypsinized, collected by centrifugation and re-suspended in 500 µl of 1X Binding Buffer. Finally, cells were incubated with 5 µl each of Annexin-V-FITC and Propidium Iodide (PI: 50mg/ml) at room temperature for 10 min in the dark, and analyzed by flow cytometry (BD FACSARIA III, BD Biosciences, USA). 2.13.6 Caspase-3 activity assay Cells were treated with Adlumidine for specified time and cell lysates were prepared using cell lysis buffer. Protein concentration was measured and adjusted to 50 – 200 µg protein per 50 µL cell lysis buffer for each assay (well). Further, assay was performed according to the manufacturer‘s protocol, and output (OD 400 - 405 nm) was measured on a microplate reader. 2.13.7 Western immunoblotting Cells were collected using a cell scraper and cell lysates were prepared by cell lysis buffer. The

protein concentration of the cell lysates was measured using the Bio-Rad Protein Assay, and subsequently, samples were prepared in SDS sample buffer containing 60 mM Tris–HCl (pH 6.8), 2% SDS, 10% glycerol, and 5% β-mercaptoethanol. Samples were then separated on a 10% SDS- polyacrylamide gel electrophoresis (PAGE) gel, and transferred onto a nitrocellulose membrane (Amersham). The blotted membranes were blocked using 3% albumin solution in distilled water (Gendepot, USA) for 1 h at room temperature. Next, the membranes were incubated with the primary and secondary antibody for the indicated time, and washed three times with washing buffer (20 mM Tris–HCl pH 7.5, 500 mM NaCl, and 0.1% Tween-20). Signals were detected using an ECL Prime Western Blotting Detection Reagent (Amersham) following the manufacturer's protocol.

CHAPTER 3 RESULTS

3.1 Antibacterial Activity In the present study, antibacterial study was performed using five different extracts of the respected plant and the results were observed by measuring the zone of inhibition. The results of the activity are shown in the tables 3.2-3.9. All the fractions obtained from the medicinal plants were tested for antibacterial potential against four bacteria namely, S. aureus, S. mutans, S. marcescens and MRSA. These species selected as an experimental organisms based on their pathogenic nature and mostly reported in different hospital of Khyber Pakhtunkhwa during screening. These species were mainly responsible for diseases in humans, therefore, we targeted these species to explore the strength of our extract against these pathogenic species to help our society and to provide them with cost effective treatment against these pathogenic species. A 6 mg/ml of the tested medicinal plant exudate were used against the experimental organisms. In the results, it was noticed that all the plant fractions were active against the pathogenic bacteria (Table 3.1). Crude methanolic extract inhibited S. aureus (10.0±0.45 mm zone and, 38.46%), S. mutans (12.0±0.55 mm zone and 42.86%), S. marcescens (09.0±0.67 mm zone and 42.86%) and MRSA (12.0±0.90 mm zone and 48%). The n- hexane fraction inhibited S. aureus (08.0±0.34 mm zone and 30.77%), S. mutans (10.0±0.68 mm zone and 35.71%), S. marcescens (10.0±0.87 mm zone and 47.62%) and MRSA (08.0±0.88 mm zone and 32%). Chloroform fraction inhibited S. aureus (09.0±0.33 mm zone and 34.62%), S. mutans (07.0±0.34 mm zone and 25%), S. marcescens (09.0±0.45 mm zone and 42.86%) and MRSA (11.0±0.56 mm zone and 44%). Ethyl acetate fraction inhibited S. aureus (12.0±0.22 mm zone and 46.15%), S. mutans (12.0±0.10 mm zone and 42.86%), S. marcescens (10.0±0.12 mm zone and 47.62%) and MRSA (07.0±0.14 mm zone and 28%). Aquous fraction inhibited S. aureus (05.0±0.45 mm zone and 19.23%), S. mutans (08.0±0.70 mm zone and 28.57%), S. marcescens (06.0±0.77 mm zone and 28.57%) and MRSA (09.0±0.55 mm zone and 36%).

Table 3. 1 Antibacterial potential of the crude extract of A. lebbeck

Cr. Met. Ext n- hexane CHCl3 EtOAc Aqueous Bacteria Standard Zone % Zone % Zone % Zone % Zone % S. aureus 26.0±0.76 10.0±0.45 38.46 08.0±0.34 30.77 09.0±0.33 34.62 12.0±0.22 46.15 05.0±0.45 19.23 S. mutans 28.0±0.60 12.0±0.55 42.86 10.0±0.68 35.71 07.0±0.34 25.00 12.0±0.10 42.86 08.0±0.70 28.57 S. marcescens 21.0±0.44 09.0±0.67 42.86 10.0±0.87 47.62 09.0±0.45 42.86 10.0±0.12 47.62 06.0±0.77 28.57 MRSA 25.0±0.34 12.0±0.90 48.00 08.0±0.88 32.00 11.0±0.56 44.00 07.0±0.14 28.00 09.0±0.55 36.00

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Figure 9 Antibacterial Activity of Albizia lebbeck

All the fractions obtained from the medicinal plants were tested for antibacterial potential against four bacteria namely, S. aureus, S. mutans , S. marcescens and MRSA. These species were selected as an experimental organisms based on their pathogenic nature and mostly reported in different hospital of Khyber Pakhtunkhwa during screening. These species were mainly responsible for diseases in humans, therefore, we targeted these species to explore the strength of our extract against these pathogenic species to help our society and to provide them with cost effective treatment against these pathogenic species. A 6 mg/ml of the tested medicinal plant exudate were used against the experimental organisms. In the results, it was noticed that all the plant fractions were active against the pathogenic bacteria table 3.2. Crude methanolic extract inhibited S. aureus (12.0±0.67 mm zone and, 46.15%), S. mutans (11.0±0.56 mm zone and 39.29%), S. marcescens (10.0±0.36 mm zone and 47.62%) and MRSA (13.0±0.35 mm zone and 52%). n- hexane fraction inhibited S. aureus (09.0±0.33 mm zone and 34.62%), S. mutans (10.0±0.55 mm zone and 35.71%), S. marcescens (11.0±0.45 mm zone and 52.38%) and MRSA (10.0±0.65 mm zone and 40%). Chloroform fraction inhibited S. aureus (10.0±0.99 mm zone and 38.46%), S. mutans (13.0±0.93 mm zone and 46.43%), S. marcescens (13.0±0.45 mm zone and 61.90%) and MRSA (11.0±0.65 mm zone and 44%). Ethyl acetate fraction inhibited S. aureus (12.0±0.55 mm zone and 46.15%), S. mutans (09.0±0.40 mm zone and 32.14%), S. marcescens (11.0±0.44 mm zone and 52.38%) and MRSA (11.0±0.64 mm zone and 44%). Aquous fraction inhibited S. aureus (08.0±0.56 mm zone and 30.77%), S. mutans (09.0±0.35 mm zone and 32.14%), S. marcescens (10.0±0.54 mm zone and 47.62%) and MRSA (10.0±0.66 mm zone and 40%).

Table 3. 2 Antibacterial Potential of the Crude Extract of M. azedarach

Cr. Met. Ext n- hexane CHCl3 EtOAc Aqueous Bacteria K2 Standard Zone % Zone % Zone % Zone % Zone % S. aureus 26.0±0.34 12.0±0.67 46.15 09.0±0.33 34.62 10.0±0.99 38.46 12.0±0.55 46.15 08.0±0.56 30.77 S. mutans 28.0±0.45 11.0±0.56 39.29 10.0±0.55 35.71 13.0±0.93 46.43 09.0±0.40 32.14 09.0±0.35 32.14 S. marcescens 21.0±0.22 10.0±0.36 47.62 11.0±0.45 52.38 13.0±0.45 61.90 11.0±0.44 52.38 10.0±0.54 47.62 MRSA 25.0±0.34 13.0±0.35 52.00 10.0±0.65 40.00 11.0±0.65 44.00 11.0±0.64 44.00 10.0±0.66 40.00

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Figure 10 Antibacterial Activity of Melia azedarach

All the fractions obtained from the medicinal plants were tested for antibacterial potential against four bacteria namely, S. aureus, S. mutans , S. marcescens and MRSA. These species were selected as an experimental organisms based on their pathogenic nature and mostly reported in different hospital of Khyber Pakhtunkhwa during screening. These species were mainly responsible for diseases in humans, therefore, we targeted these species to explore the strength of our extract against these pathogenic species to help our society and to provide them with cost effective treatment against these pathogenic species. A 6 mg/ml of the tested medicinal plant exudate were used against the experimental organisms. In the results, it was noticed that all the plant fractions were active against the pathogenic bacteria (Table 3.3). Crude methanolic extract inhibited S. aureus (13.0±0.67 mm zone and, 50.00%), S. mutans (13.0±0.87 mm zone and 46.43%), S. marcescens (12.0±0.60 mm zone and 57.14%) and MRSA (12.0±0.93 mm zone and 48%). n- hexane fraction inhibited S. aureus (10.0±0.60 mm zone and 38.46%), S. mutans (11.0±0.56 mm zone and 39.29%), S. marcescens (09.0±0.93 mm zone and 42.86%) and MRSA (10.0±0.98 mm zone and 40%). Chloroform fraction inhibited S. aureus ( 12.0±0.26 mm zone and 46.15%), S. mutans ( 12.0±0.87 mm zone and 42.86%), S. marcescens (10.0±0.67 mm zone and 47.62%) and MRSA (07.0±0.64 mm zone and 28%). Ethyl acetate fraction inhibited S. aureus (08.0±0.34 mm zone and 30.77%), S. mutans (10.0±0.55 mm zone and 35.71%), S. marcescens (07.0±0.55 mm zone and 33.33%) and MRSA (09.0±0.87 mm zone and 36%). Aquous fraction inhibited S. aureus (07.0±0.44 mm zone and 26.92%), S. mutans (06.0±0.56 mm zone and 21.43%), S. marcescens (07.0±0.60 mm zone and 33.33%) and MRSA (06.0±0.34 mm zone and 24

Table 3. 3 Antibacterial Potential of the Crude Extract of Hedera helix

Cr. Met. Ext n- hexane CHCl3 EtOAc Aqueous Bacteria Standard Zone % Zone % Zone % Zone % Zone % S. aureus 26.0±0.55 13.0±0.67 50.00 10.0±0.60 38.46 12.0±0.26 46.15 08.0±0.34 30.77 07.0±0.44 26.92 S. mutans 28.0±0.54 13.0±0.87 46.43 11.0±0.56 39.29 12.0±0.87 42.86 10.0±0.55 35.71 06.0±0.56 21.43 S. marcescens 21.0±0.64 12.0±0.60 57.14 09.0±0.93 42.86 10.0±0.67 47.62 07.0±0.55 33.33 07.0±0.60 33.33 MRSA 25.0±0.56 12.0±0.93 48.00 10.0±0.98 40.00 07.0±0.64 28.00 09.0±0.87 36.00 06.0±0.34 24.00

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Figure 11 Antibacterial Activity of Hedera helix

All the fractions obtained from the medicinal plants were tested for antibacterial potential against four bacteria namely, S. aureus, S. mutans , S. marcescens and MRSA. These species were selected as an experimental organisms based on their pathogenic nature and mostly reported in different hospital of Khyber Pakhtunkhwa during screening. These species were mainly responsible for diseases in humans, therefore, we targeted these species to explore the strength of our extract against these pathogenic species to help our society and to provide them with cost effective treatment against these pathogenic species. A 6 mg/ml of the tested medicinal plant exudate were used against the experimental organisms. In the results, it was noticed that all the plant fractions were active against the pathogenic bacteria (Table 3.4). Crude methanolic extract inhibited S. aureus (10.0±0.87 mm zone and, 38.46%), S. mutans (12.0±0.67 mm zone and 42.86%), S. marcescens (09.0±0.56 mm zone and 42.86%) and MRSA (11.0±0.93 mm zone and 44%). n- hexane fraction inhibited S. aureus (12.0±0.93 mm zone and 46.15%), S. mutans (13.0±0.66 mm zone and 46.43%), S. marcescens (10.0±0.88 mm zone and 47.62 %) and MRSA (12.0±0.90 mm zone and 48%). Chloroform fraction inhibited S. aureus ( 11.0±0.21mm zone and 42.31%), S. mutans 14.0±0.56 mm zone and 50%), S. marcescens (07.0±0.45 mm zone and 33.33%) and MRSA (10.0±0.57mm zone and 40%). Ethyl acetate fraction inhibited S. aureus (08.0±0.34 mm zone and 30.77%), S. mutans (10.0±0.34 mm zone and 35.71%), S. marcescens (08.0±0.44 mm zone and 38.10%) and MRSA (09.0±0.87 mm zone and 36%). Aquous fraction inhibited S. aureus (05.0±0.50 mm zone and 19.23 %), S. mutans (04.0±0.34 mm zone and 14.29%), S. marcescens (05.0±0.22 mm zone and 23.81%) and MRSA (08.0±0.23 mm zone and 32%).

Table 3. 4 Antibacterial Potential of the Crude Extract of S. flagellaris

Cr. Met. Ext n- hexane CHCl3 EtOAc Aqueous Bacteria Standard Zone % Zone % Zone % Zone % Zone % S. aureus 26.0±0.56 10.0±0.87 38.46 12.0±0.93 46.15 11.0±0.21 42.31 08.0±0.33 30.77 05.0±0.50 19.23 S. mutans 28.0±0.60 12.0±0.67 42.86 13.0±0.66 46.43 14.0±0.56 50.00 10.0±0.34 35.71 04.0±0.34 14.29 S. marcescens 21.0±0.87 09.0±0.56 42.86 10.0±0.88 47.62 07.0±0.45 33.33 08.0±0.44 38.10 05.0±0.22 23.81 MRSA 25.0±0.67 11.0±0.93 44.00 12.0±0.90 48.00 10.0±0.57 40.00 09.0±0.87 36.00 08.0±0.23 32.00

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Figure 12 Antibacterial Activity of Saxifraga flagellaris

All the fractions obtained from the medicinal plants were tested for antibacterial potential against four bacteria namely, S. aureus, S. mutans , S. marcescens and MRSA. These species were selected as an experimental organisms based on their pathogenic nature and mostly reported in different hospital of Khyber Pakhtunkhwa during screening. These species were mainly responsible for diseases in humans, therefore, we targeted these species to explore the strength of our extract against these pathogenic species to help our society and to provide them with cost effective treatment against these pathogenic species. A 6 mg/ml of the tested medicinal plant exudate were used against the experimental organisms. In the results, it was noticed that all the plant fractions were active against the pathogenic bacteria (Table 3.5). Crude methanolic extract inhibited S. aureus (12.0±0.34 mm zone and, 46.15%), S. mutans (13.0±0.45 mm zone and 46.43%), S. marcescens (11.0±0.97 mm zone and 52.38%) and MRSA (13.0±0.68 mm zone and 52%). n- hexane fraction inhibited S. aureus (10.0±0.33 mm zone and 38.46%), S. mutans (12.0±0.55 mm zone and 42.86%), S. marcescens (06.0±0.54 mm zone and 28.57%) and MRSA (11.0±0.56 mm zone and 44.00%). Chloroform fraction inhibited S. aureus ( 09.0±0.58 mm zone and 34.62%), S. mutans (14.0±0.98 mm zone and 50%), S. marcescens (0.0±0.00 mm zone and 0%) and MRSA (08.0±0.33mm zone and 32%). Ethyl acetate fraction inhibited S. aureus (10.0±0.46 mm zone and 38.46%), S. mutans (14.0±0.66 mm zone and 50%), S. marcescens (12.0±0.87 mm zone and 57.14%) and MRSA (12.0±0.98 mm zone and 48%). Aquous fraction inhibited S. aureus (06.0±0.44 mm zone and 23.08%), S. mutans (07.0±0.56 mm zone and 25%), S. marcescens (0.0±0.00 mm zone and 0%) and MRSA (08.0±0.44 mm zone and 32%).

Table 3. 5 Antibacterial Potential of the Crude Extract of V. jatamansi

Cr. Met. Ext n- hexane CHCl3 EtOAc Aqueous Bacteria K5 Standard Zone % Zone % Zone % Zone % Zone %

S. aureus 26.0±0.44 12.0±0.34 46.15 10.0±0.33 38.46 09.0±0.58 34.62 10.0±0.46 38.46 06.0±0.44 23.08

S. mutans 28.0±0.66 13.0±0.45 46.43 12.0±0.55 42.86 14.0±0.98 50.00 14.0±0.66 50.00 07.0±0.56 25.00

S. marcescens 21.0±0.34 11.0±0.97 52.38 06.0±0.54 28.57 0.0±0.00 0.00 12.0±0.87 57.14 0.0±0.00 0.00

MRSA 25.0±0.76 13.0±0.68 52.00 11.0±0.56 44.00 08.0±0.33 32.00 12.0±0.98 48.00 08.0±0.44 32.00

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Figure 13 Antibacterial Activity of Valeriana jatamansi

All the fractions obtained from the medicinal plants were tested for antibacterial potential against four bacteria namely, S. aureus, S. mutans , S. marcescens and MRSA. These species were selected as an experimental organisms based on their pathogenic nature and mostly reported in different hospital of Khyber Pakhtunkhwa during screening. These species were mainly responsible for diseases in humans, therefore, we targeted these species to explore the strength of our extract against these pathogenic species to help our society and to provide them with cost effective treatment against these pathogenic species. A 6 mg/ml of the tested medicinal plant exudate were used against the experimental organisms. In the results, it was noticed that all the plant fractions were active against the pathogenic bacteria (Table 3.6). Crude methanolic extract inhibited S. aureus (15.0±0.33 mm zone and, 57.69 %), S. mutans (13.0±0.34 mm zone and 46.43%), S. marcescens (11.0±0.66 mm zone and 52.38%) and MRSA (13.0±0.66 mm zone and 52%). n- hexane fraction inhibited S. aureus (12.0±0.44 mm zone and 46.15%), S. mutans (12.0±0.55 mm zone and 42.86%), S. marcescens (10.0±0.55 mm zone and 47.62%) and MRSA (11.0±0.77 mm zone and 44.00%). Chloroform fraction inhibited S. aureus (08.0±0.56 mm zone and 30.77%), S. mutans (11.0±0.34 mm zone and 39.29%), S. marcescens (11.0±0.68 mm zone and 52.38%) and MRSA (12.0±0.68 mm zone and 48%). Ethyl acetate fraction inhibited S. aureus (12.0±0.44 mm zone and 46.15%), S. mutans (14.0±0.45 mm zone and 50%), S. marcescens (06.0±0.66 mm zone and 28.57%) and MRSA (13.0±0.88 mm zone and 52%). Aqueous fraction inhibited S. aureus (09.0±0.34 mm zone and 34.62%), S. mutans (10.0±0.22 mm zone and 35.71%), S. marcescens (0.0±0.00 mm zone and 0%) and MRSA (07.0±0.66 mm zone and 28%).

Table 3. 6 Antibacterial Potential of the Crude Extract of F. cretica

Cr. Met. Ext n- hexane CHCl3 EtOAc Aqueous Bacteria K6 Standard Zone % Zone % Zone % Zone % Zone % S. aureus 26.0±0.44 15.0±0.33 57.69 12.0±0.44 46.15 08.0±0.56 30.77 12.0±0.44 46.15 09.0±0.34 34.62 S. mutans 28.0±0.66 13.0±0.34 46.43 10.0±0.55 35.71 11.0±0.34 39.29 14.0±0.45 50.00 10.0±0.22 35.71 S. marcescens 21.0±0.34 11.0±0.66 52.38 10.0±0.55 47.62 11.0±0.68 52.38 06.0±0.66 28.57 00.0±0.00 0.00 MRSA 25.0±0.76 13.0±0.66 52.00 11.0±0.77 44.00 12.0±0.68 48.00 13.0±0.88 52.00 07.0±0.66 28.00

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Figure 14 Antibacterial Activity of Fagonia cretica

All the fractions obtained from the medicinal plants were tested for antibacterial potential against four bacteria namely, S. aureus, S. mutans , S. marcescens and MRSA. These species were selected as an experimental organisms based on their pathogenic nature and mostly reported in different hospital of Khyber Pakhtunkhwa during screening. These species were mainly responsible for diseases in humans, therefore, we targeted these species to explore the strength of our extract against these pathogenic species to help our society and to provide them with cost effective treatment against these pathogenic species. A 6 mg/ml of the tested medicinal plant extracts were used against the experimental organisms. In the results, it was noticed that all the plant fractions were active against the pathogenic bacteria (Table 3.7). Crude methanolic extract inhibited S. aureus (12.0±0.56 mm zone and, 46.15%), S. mutans (13.0±0.78 mm zone and 46.43%), S. marcescens (10.0±0.88 mm zone and 47.62%) and MRSA (14.0±0.56 mm zone and 56.00%). n- hexane fraction inhibited S. aureus (13.0±0.67 mm zone and 50%), S. mutans (13.0±0.78 mm zone and 42.86%), S. marcescens (10.0±0.44 mm zone and 47.62%) and MRSA (12.0±0.34 mm zone and 48.00%). Chloroform fraction inhibited S. aureus (10.0±0.55 mm zone and 38.46%), S. mutans (14.0±0.56 mm zone and 50%), S. marcescens (11.0±0.55 mm zone and 52.38%) and MRSA (12.0±0.66 mm zone and 48%). Ethyl acetate fraction inhibited S. aureus (11.0±0.44 mm zone and 42.31%), S. mutans 16.0±0.33 mm zone and 57.14%), S. marcescens (09.0±0.56 mm zone and 42.86%) and MRSA (10.0±0.34 mm zone and 40%). Aquous fraction inhibited S. aureus (09.0±0.44 mm zone and 34.62%), S. mutans (09.0±0.56 mm zone and 32.14%), S. marcescens (06.0±0.88 mm zone and 28.57%) and MRSA (09.0±0.99 mm zone and 36%)

Table 3. 7 Antibacterial Potential of the Crude Extract of W. coagulans

Cr. Met. Ext n- hexane CHCl3 EtOAc Aqueous Bacteria K7 Standard Zone % Zone % Zone % Zone % Zone % S. aureus 26.0±0.44 12.0±0.56 46.15 13.0±0.67 50.00 10.0±0.55 38.46 11.0±0.44 42.31 09.0±0.44 34.62 S. mutans 28.0±0.66 13.0±0.78 46.43 12.0±0.78 42.86 14.0±0.56 50.00 16.0±0.33 57.14 09.0±0.56 32.14 S. marcescens 21.0±0.34 10.0±0.88 47.62 10.0±0.44 47.62 11.0±0.55 52.38 09.0±0.56 42.86 06.0±0.88 28.57 MRSA 25.0±0.76 14.0±0.56 56.00 12.0±0.34 48.00 12.0±0.66 48.00 10.0±0.34 40.00 09.0±0.99 36.00

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Figure 15 Antibacterial Activity of Withania coagulans

All the fractions obtained from the medicinal plants were tested for antibacterial potential against four bacteria namely, S. aureus, S. mutans , S. marcescens and MRSA. These species were selected as an experimental organisms based on their pathogenic nature and mostly reported in different hospital of Khyber Pakhtunkhwa during screening. These species were mainly responsible for diseases in humans, therefore, we targeted these species to explore the strength of our extract against these pathogenic species to help our society and to provide them with cost effective treatment against these pathogenic species. A 6 mg/ml of the tested medicinal plant extracts were used against the experimental organisms. In the results, it was noticed that all the plant fractions were active against the pathogenic bacteria (Table 3.8). Crude methanolic extract inhibited S. aureus (10.0±0.10mm zone and, 38.46%), S. mutans (12.0±0.20 mm zone and 42.86%), S. marcescens (09.0±0.32 mm zone and 42.86%) and MRSA (12.0±0.34mm zone and 48%). n- hexane fraction inhibited S. aureus (00.0±0.00 mm zone and 0%), S. mutans (10.0±0.44 mm zone and 35.71%), S. marcescens (07.0±0.98 mm zone and 33.33%) and MRSA (11.0±0.60 mm zone and 44%). Chloroform fraction inhibited S. aureus (12.0±0.34 mm zone and 46.15%), S. mutans (00.0±0.00 mm zone and 0%), S. marcescens (00.0±0.00 mm zone and 0%) and MRSA (10.0±0.65 mm zone and 40%). Ethyl acetate fraction inhibited S. aureus (00.0±0.00 mm zone and 0%), S. mutans (12.0±0.98 mm zone and 42.86%), S. marcescens (10.0±0.56 mm zone and 47.62%) and MRSA (00.0±0.00 mm zone and 0%). Aquous fraction inhibited S. aureus (04.0±0.76 mm zone and 15.38%), S. mutans (00.0±0.00mm zone and 0%), S. marcescens (00.0±0.00 mm zone and 0%) and MRSA (00.0±0.00 mm zone and 0%).

Table 3. 8 Antibacterial Potential of the Crude Extract of M. oleifera

Cr. Met. Ext n- hexane CHCl3 EtOAc Aqueous Bacteria K8 Standard Zone % Zone % Zone % Zone % Zone %

S. aureus 26.0±0.44 10.0±0.10 38.46 00.0±0.00 0.00 12.0±0.34 46.15 00.0±0.00 0.00 04.0±0.76 15.38

S. mutans 28.0±0.66 12.0±0.20 42.86 10.0±0.44 35.71 00.0±0.00 0.00 12.0±0.98 42.86 00.0±0.00 0.00

S. marcescens 21.0±0.34 09.0±0.32 42.86 07.0±0.98 33.33 00.0±0.00 0.00 10.0±0.56 47.62 00.0±0.00 0.00

MRSA 25.0±0.76 12.0±0.34 48.00 11.0±0.60 44.00 10.0±0.65 40.00 00.0±0.00 0.00 00.0±0.00 0.00

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Figure 16 Antibacterial Activity of Moringa oleifera

3.2 ANTIFUNGAL ACTIVITY Medicinal plant species are very rich sources of antimicrobial compounds and due to this reason the WHO promoted that both developed and underdeveloped countries use the traditional medicine with a view of safe and effective remedies of various ailments. Plants have substantial medicinal activities and are usually used as a source of medicine worldwide. These plant species is chief source of various valuable and prevailing crude drugs. A huge number of medicinal plants have gained recognition due to their biological and antimicrobial potential (Walter et al., 2011; Shinwari et al., 2015). Raw drugs are generally extracted from various parts of medicinal plants due to their different medicinal properties. The diverse plant organs include leaves, root, stem, flower, fruit and various other modified parts of plant are used. These different parts of plants are gathered in small amount for local use and in bulk quantity they are collected for trading as well as for manufacturing of herbal products in herbal industries (Uniyal et al., 2006). In the present study antifungal study was performed using five different extracts of the selected plants and the results were observed by measuring the zone of inhibition. The results of the activity are shown in the Table.3.9-3-3.16. The results of the antifungal activity of the plant showed that the aqueous fraction showed high activity with minimum zone of inhibition of 20.0±0.52 by A.alternate, followed by 25.0 ±0.64 by P.pallidum, 25.0±0.53 by F.oxysporum and 30.0±0.56 by A.flavus. Cr.Met.Ext showed high zone of inhibition of 56.0±0.56 by A.alternate, and low of 40.0±0.46 by P.pallidum. N-hexane fraction showed zone of inhibition of 52.0±0.56, 50.0±0.78, 47.0±0.67 and 35.0±0.99 by F.oxysporum,

A.alternate, P.pallidum and A.falvus respectively. CHCl3 fraction showed zones of inhibitions of 60.0±0.33, 55.0±0.43, 50.0±0.54 and 45.0±0.22 by A.alternat, F.oxysporum, P.pallidum and A.flavus respectively. EtOAC fraction showed zones of inhibition of 55.0±0.77 by A.alternate, 45.0±0.63 by F.oxysporum, 40.0±0.74 by A.flavus and 30.0±0.66 by P.pallidum. From the results it is clear A.alternate is sensitive to aqueous extract of the plant.

Table 3. 9 Antifungal Potential of the Crude Extract of A. lebbeck

Cr. Met. Ext n- hexane CHCl3 EtOAc Aqueous Fungi Standard Zone % Zone % Zone % Zone % Zone % A.flavus 100.0±0.00 52.0±0.44 52.00 35.0±0.99 35.00 45.0±0.22 45.00 40.0±0.74 40.00 30.0±0.56 30.00 A. alternate 100.0±0.00 56.0±0.56 56.00 50.0±0.78 50.00 60.0±0.33 60.00 55.0±0.77 55.00 20.0±0.52 20.00 F. oxysporum 100.0±0.00 45.0±0.66 45.00 52.0±0.65 52.00 55.0±0.43 55.00 45.0±0.63 45.00 25.0±0.53 25.00 P. pallidum 100.0±0.00 40.0±0.46 40.00 47.0±0.67 47.00 50.0±0.54 50.00 30.0±0.66 30.00 25.0±0.64 25.00

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Figure 17 Antifungal Activity of Albizia lebbeck

The results of the antifungal activity of the plant showed that the aqueous fraction showed high activity with minimum zone of inhibition of 10.0±0.98 by P.pallidum, followed by 15.0 ±0.78 by F.oxysporum, 15.0±0.56 by A.alternate and 20.0±0.77 by A.flavus. Cr.Met.Ext showed high zones of inhibition of 30.0±0.67, 45.0±0.32, 55.0±0.66 and 65.0±0.45 by F.oxysporum, A.flavus, P.pallidum and A.alternate respectively. N-hexane fraction showed zones of inhibition of 40.0±0.44, 45.0±0.97, 50.0±0.99 and 60.0±0.50 by F.oxysporum, A.alternate, A.flavus and

P.pallidum respectively. CHCl3 fraction showed zones of inhibition of 40.0±0.21, 45.0±0.42, 55.0±0.22 and 60.0±0.32 by F.oxysporum, A.flavus, P.pallidum and A.alternate. EtOAC fraction showed zone of inhibition of 35.0±0.99 by F.oxysporum, 40.0±0.56 by F.pallidum, 40.0±0.45 by A.flavus and 55.0±0.50 by A.alternate. From the results it is clear that P.pallidum and F.oxysporum are sensitive to aqueous extracts of the respected plant.

Table 3. 10 Antifungal Potential of the Crude Extract of M. azedarach

Cr. Met. Ext n- hexane CHCl3 EtOAc Aqueous Fungi Standard Zone % Zone % Zone % Zone % Zone % A.flavus 100.0±0.00 45.0±0.32 45.00 50.0±0.99 50.00 45.0±0.42 45.00 40.0±0.45 40.00 20.0±0.77 20.00 A. alternate 100.0±0.00 65.0±0.45 65.00 45.0±0.97 45.00 60.0±0.32 60.00 55.0±0.50 55.00 15.0±0.56 15.00 F. oxysporum 100.0±0.00 30.0±0.67 30.00 40.0±0.44 40.00 40.0±0.21 40.00 35.0±0.99 35.00 15.0±0.78 15.00 P. pallidum 100.0±0.00 55.0±0.66 55.00 60.0±0.50 60.00 55.0±0.22 55.00 40.0±0.56 40.00 10.0±0.98 10.00

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Figure 18 Antifungal Activity of Melia azedarach

The highest antifungal activity was showed by n-hexane and chloroform fraction of the plant and they completely inhibited the growth of P.pallidum with 0.0 zone of inhibition. Aqueous fraction showed the zone of inhibitions of 13.0±0.12, 19.0±0.33, 22.0±0.44 and 35.0±0.33 by A.alternate, P.pallidum, F.oxusporum and A.flavus respectively. Cr.Met.Ext fraction showed zones of inhibition of 38.0±0.75, 43.0±0.62, 55.0±0.66 and 57.0±0.88 by F.oxysporum, A.alternate, A.flavus and P.pallidum respectively. EtOAC fraction showed zones of inhibition of 23.0±0.55, 34.0±0.46, 37.0±0.55 and 55.0±0.34 by P.pallidum, A.alternate, F.oxysporum and A.flavus respectively. From the results it is quite clear that P.pallidum is more sensitive to aqueous extract and can not grow in such medium while A.flavus was less sensitive to EtOAC extract of the plant.

Table 3. 11 Antifungal Potential of the Crude Extract of H. helix

Cr. Met. Ext n- hexane CHCl3 EtOAc Aqueous Fungi Standard Zone % Zone % Zone % Zone % Zone %

A.flavus 100.0±0.00 55.0±0.66 55.00 57.0±0.56 57.00 43.0±0.88 43.00 55.0±0.34 55.00 35.0±0.33 35.00

A. alternate 100.0±0.00 43.0±0.62 43.00 53.0±0.50 53.00 57.0±0.78 57.00 34.0±0.46 34.00 13.0±0.12 13.00

F. oxysporum 100.0±0.00 38.0±0.75 38.00 45.0±0.78 45.00 43.0±0.96 43.00 37.0±0.55 37.00 22.0±0.44 22.00

P. pallidum 100.0±0.00 57.0±0.88 57.00 00.0±0.00 0.00 00.0±0.00 0.00 23.0±0.55 23.00 19.0±0.33 19.00

o i

t 1 5 0

i S ta n d a rd

b i

h C r. M e t. E x t

n i

n - h e x a n e 1 0 0

e C H C l3

g a

t E tO A c n

e 5 0 A q u e o u s

e P

0 e s t m m u a u u v r n o d a r li fl e p l . lt s a y p A a x . o . A . P F F u n g a l s tra in s

Figure 19 Antifungal Activity of Hedera helix 69

The n-hexans fraction completely inhibited the growth of F.oxysporum, CHCl3 and EtOAC of P.pallidum while aqueous fraction showed a good antifungal activity against A.flavus and A.alternate with 0.0 zone of inhibition. Cr.Met.Ext fraction showed zones of inhibition of 35.0±0.44, 45.0±0.55, 57.0±0.23 and 65.0±0.54 by P.pallidum, A.flavus, F.pxysporum and A.alternate respectively. From the results it can be determined F.oxysporum is more sensitive to n-hexane extract of the plant, P.pallidum is sensitive to the CHCl3 and EtOAC extracts of the plant and A.flavus and A.alternate to the aqueous extract of the plant.

Table 3. 12 Antifungal Potential of the Crude Extract of S. flagellaris

Cr. Met. Ext n- hexane CHCl3 EtOAc Aqueous Fungi Standard Zone % Zone % Zone % Zone % Zone %

A.flavus 100.0±0.00 45.0±0.55 45.00 45.0±0.78 45.00 30.0±0.67 30.00 40.0±0.34 40.00 0.0±0.00 0.00

A. alternate 100.0±0.00 65.0±0.54 65.00 30.0±0.88 30.00 45.0±0.56 45.00 40.0±0.10 40.00 0.0±0.00 0.00

F. oxysporum 100.0±0.00 57.0±0.23 57.00 000.0±0.00 0.00 32.0±0.77 32.00 30.0±0.22 30.00 23.0±0.34 23.00

P. pallidum 100.0±0.00 35.0±0.44 35.00 29.0±0.67 29.00 00.0±0.00 0.00 00.0±0.00 0.00 30.0±0.21 30.00

i t

i 1 5 0

b S ta n d a rd i

h C r. M e t. E x t

n i

n - h e x a n e

1 0 0 e

g C H C l3 a

t E tO A c n

e 5 0 A q u e o u s

e P

0 e s t m m u u a r u v n d o i a r l l e p l f t s . l y a p A a x . o . A . P F F u n g a l s tra in s

Figure 20 Antifungal Activity of Saxifraga flagellaris

Aqueous fraction of the plant showed high antifungal activity and had completely inhibited the growth of A.flavus and A.alternate with 0.0 zone of inhibition. Cr.Met.Ext fraction showed zones of inhibition of 35.0±0.44, 45.0±0.55, 57.0±0.23 and 65.0±0.54 by P.pallidum, A.flavus, F.oxysporum and A.alternate respectively. N-hexane fraction showed high antifungal activity and completely inhibited the growth of F.oxysporum followed by the zone of inhibitions of 29.0±0.63,

30.0±0.88 and 45.0±0.78 by P.pallidum, A.alternate and A.flavus respectively. CHCl3 fraction showed high antifungal activity with 0.0 zone of inhibition aginst P.pallidum and low activity against A.alternate with zone of inhibition of 45.0±0.56. EtOAC fraction showed low antifungal activity with 0.0 zone of inhibition against P.pallidum and high activity against A.alternate with zone of inhibition of 40.0±0.10. From the results it is quite clear the test organisms are more sensitive to the plant extracts. A.flavus and A.alternate to the aqueous extract, and P.pallidum to the CHCl3 extract. While A.alternate was less sensitive to Cr.Met.Ext extract.

Table 3. 13 Antifungal Potential of the Crude Extract of V. jatamansi

Cr. Met. Ext n- hexane CHCl3 EtOAc Aqueous Fungi Standard Zone % Zone % Zone % Zone % Zone %

A.flavus 100.0±0.00 45.0±0.55 45.00 35.0±0.23 35.00 00.0±0.00 0.00 00.0±0.00 0.00 00.0±0.00 0.00

A. alternate 100.0±0.00 34.0±0.69 34.00 00.0±0.00 0.00 00.0±0.00 0.00 00.0±0.00 0.00 00.0±0.00 0.00

F. oxysporum 100.0±0.00 45.0±0.52 45.00 00.0±0.00 0.00 00.0±0.00 0.00 37.0±0.10 37.00 00.0±0.00 0.00

P. pallidum 100.0±0.00 48.0±0.56 48.00 00.0±0.00 0.00 00.0±0.00 0.00 34.0±0.34 34.00 00.0±0.00 0.00

o 1 5 0 i

t S ta n d a rd

i b

i C r. M e t. E x t h

n n - h e x a n e

i 1 0 0

e C H C l3 g

a E tO A c t

n A q u e o u s

e 5 0

e P

0 s te m m u a u u v n r d a r o li fl e p l . lt s a A y a x p . o . A . P F F u n g a l s tra in s

Figure 21 Antifungal Activity of Valeriana jatamansi

Aqueous fraction of the plant showed high antifungal activity and had completely inhibited the growth of P.pallidum with 0.0 zone of inhibition. While zone of inhibitions for F.oxysporum, A.flavus and A.alternate were 25.0±0.77, 30.0±0.67 and 35.0±0.56 respectively. Cr.Met.Ext fraction showed zones of inhibition of 39.0±0.48, 48.0±0.49, 50.0±0.33 and 60.0±0.44 by P.pallidum, F.oxysporum, A.flavus and A.alternate respectively. N-hexane fraction showed antifungal activity with zone of inhibition of 42.0±0.66, 43.0±0.54, 45.0±0.77 and 45.0±0.55 by

P.pallidum, A.flavus, F.oxysporum and A.alternate respectively. CHCl3 fraction showed high antifungal activity against A.alternate with zone of inhibition of 0.0±0.23 and low activity against F.oxysporum with 55.0±0.67 inhibition zone. EtOAC fraction showed high antifungal activity with 0.0 zone of inhibition against P.pallidum and low against A.alternate with 49.0±0.21 inhibition zone.

Table 3. 14 Antifungal Potential of the Crude Extract of F. cretica

Cr. Met. Ext n- hexane CHCl3 EtOAc Aqueous Fungi Standard Zone % Zone % Zone % Zone % Zone %

A.flavus 100.0±0.00 50.0±0.33 50.00 43.0±0.54 43.00 34.0±0.34 34.00 45.0±0.34 45.00 30.0±0.67 30.00

A. alternate 100.0±0.00 60.0±0.44 60.00 45.0±0.55 45.00 32.0±0.46 32.00 49.0±0.21 49.00 35.0±0.56 35.00

F. oxysporum 100.0±0.00 48.0±0.49 48.00 45.0±0.77 45.00 55.0±0.67 55.00 42.0±0.22 42.00 25.0±0.77 25.00

P. pallidum 100.0±0.00 39.0±0.48 39.00 42.0±0.66 42.00 45.0±0.77 45.00 00.0±0.00 0.00 00.0±0.00 0.00

n o

i 1 5 0 t

i S ta n d a rd b

i C r. M e t. E x t

h n

i n - h e x a n e

1 0 0

e C H C l3 g

a E tO A c t

n A q u e o u s

e 5 0

e P

0 e s t m m u a u u v n r d a r o i l e p ll .f t s l y a A a x p . o . A . P F F u n g a l s tra in s

Figure 22 Antifungal Activity of Fagonia cretica 75

Aqueous fraction of the plant showed high antifungal activity and had completely inhibited the growth of all the test organisms. Cr.Met.Ext fraction showed zones of inhibition of 25.0±0.55, 38.0±0.34, 45.0±0.33 and 53.0±0.66 by A.flavus, P.pallidum, A.alternate and F.oxysporum respectively. N-hexane fraction showed high antifungal activity against P.pallidum and

F.oxysporum with zone of inhibition of 0.0±0.0. CHCl3 fraction showed antifungal activity with zones of inhibition of 30.0, 32.0±0.88, 32.0±0.56 and 34 by P.pallidum, A.flavus, A.alternate and F.oxysporum respectively. EtOAC fraction showed high antifungal activity against P.pallidum and A.flavus with zones of inhibition of 0.0±0.0.

Table 3. 15 Antifungal Potential of the Crude Extract of W. coagulans

Cr. Met. Ext n- hexane CHCl3 EtOAc Aqueous Fungi Standard Zone % Zone % Zone % Zone % Zone % A.flavus 100.0±0.00 25.0±0.55 25.00 34.0±0.92 34.00 32.0±0.88 32.00 0.0±0.00 0.00 0.0±0.00 0.00 A. alternate 100.0±0.00 45.0±0.33 45.00 47.0±0.99 47.00 32.0±0.56 32.00 25.0±0.56 25.00 0.0±0.00 0.00 F. oxysporum 100.0±0.00 53.0±0.66 53.00 0.0±0.00 0.00 34.0±0.34 34.00 40.0±0.47 40.00 0.0±0.00 0.00 P. pallidum 100.0±0.00 38.0±0.34 38.00 0.0±0.00 0.00 30.0±0.54 30.00 0.0±0.00 0.00 0.0±0.00 0.00

o 1 5 0 i

t S ta n d a rd

i b

i C r. M e t. E x t h

n n - h e x a n e

i 1 0 0

e C H C l3 g

a E tO A c t

n A q u e o u s

e 5 0 c

r L e g e n d

e P

0 s te m m u a u u v n r d a r o li fl e p l . lt s a A y a x p . o . A . P F F u n g a l s tra in s

Figure 23 Antifungal Activity of Withania coagulans

Aqueous fraction of the plant showed high antifungal activity against P.pallidum with 0.0±0.0 inhibition zone and low against A.flavus with 26.0±0.32 inhibition zone. Cr.Met.Ext fraction showed zones of inhibition of 45.0±0.88, 45.0±0.76, 55.0±0.67 and 60.0±0.55 by F.oxysporum, P.pallidum, A.flavus and A.alternate respectively. N-hexane fraction showed high antifungal activity against P.pallidum with zone of inhibition of 0.0±0.0 and low activity against A.alternate with inhibition zone of 53.0±0.45. CHCl3 fraction showed antifungal activity with zones of inhibition of 34.0±0.44, 34.0±0.23, 45.0±0.34 and 60.0±0.22 by P.pallidum, A.flavus, F.oxysporum and A.alternate respectively. EtOAC fraction showed antifungal activity with zones of inhibition of 23.0±0.78, 28.0±0.88, 37.0±0.89 and 43.0±0.92 by P.pallidum, F.oxysporum, A.alternate and A.flavus respectively.

Table 3. 16 Antifungal potential of the crude extract of M. oleifera

Cr. Met. Ext n- hexane CHCl3 EtOAc Aqueous Fungi K8 Standard Zone % Zone % Zone % Zone % Zone %

A.flavus 100.0±0.00 55.0±0.67 55.00 45.0±0.34 45.00 34.0±0.23 34.00 43.0±0.92 43.00 26.0±0.32 26.00

A. alternate 100.0±0.00 60.0±0.55 60.00 53.0±0.45 53.00 60.0±0.22 60.00 37.0±0.89 37.00 30.0±0.31 30.00

F. oxysporum 100.0±0.00 45.0±0.88 45.00 35.0±0.33 35.00 45.0±0.34 45.00 28.0±0.88 28.00 20.0±0.28 20.00

P. pallidum 100.0±0.00 45.0±0.76 45.00 00.0±0.00 0.00 34.0±0.44 34.00 23.0±0.78 23.00 00.0±0.00 0.00

n o

i 1 5 0 t

i S ta n d a rd

b i

C r. M e t. E x t

h n

i n - h e x a n e 1 0 0

e C H C l3

g a

t E tO A c

n A q u e o u s

e 5 0

e P

0 e s t m m u a u u v n r d a r o i l e p ll .f t s l y a A a x p . o . A . P F F u n g a l s tra in s

Figure 24 Antifungal Activity of Moringa oleifera

3.3 Phytotoxicity of Selected Medicinal Plants Allelopathy is a biological term in which one organism produce such biochemical that effect the growth, existence, improvement, and reproduction of other individual and these biochemical are known as allelochemicals.. Plant allelopathy is the types of relations concerning receptor (the plant which is effected) and donor plants (which effect) and may exert either positive effects (weed control) negative effects (e.g., , soil sickness, biological invasion or auto toxicity) (Cheng and Cheng, 2015). Germination of seeds and plant development is altered by allelopathy and therefore germination is important way for the learning of allelopathy. Allelopathy has a significant part in agriculture and disturbs the progress and magnitude of the crops by the exchange of chemicals among crops, weeds and trees. Allelochemicals secreted by unknown plants significantly affect the intrinsic plants regardless of native species secreted allelochemicals or not (Sertkaya et al., 2010, Hussain et al., 2010). Allelochemicals can alter the contents of plant growth hormones or make inequalities in many phyto-hormones, which hinders plant growth and development, for instance, with respect to germination of seed and sapling growth. Allelochemicals of phenolic nature can motivate IAA oxidase activity and obstruct the reaction of POD with IAA, unavoidable GA or IAA to effect endogenous hormone stages (Rauf et al., 2012, Cheng and Cheng, 2015). For allopathic study sandwich method of Fuji et al., (2003 & 2004) was followed, in which Lactuca sativa seeds were used in agar gel bedding with different amount of plants parts pieces. The results of the phytotoxicity of selected medicinal given in Table (3.5.1) indicated that seeds of Lactuca sativa respond differentially to different medicinal plants. The L. sativa seedlings were very susceptible to the 10 mg powder of S. flagellaris. The radicle length of L. sativa seedlings germinated in the presence of S. flagellaris residues was 0.49 cm, followed by V. jatamansi (0.54 cm) and A. lebbeck (0.57 cm). The recorded length of the L. sativa radicle in the presence of F. cretica, H. helix, M. oleifera, W. coagulans, and M. azedarach was 0.69, cm 1.01 cm, 2.91 cm, 3.04 cm, and 3.92 cm, respectively. Moreover, L. sativa showed different responses regarding the length of plumule. S. flagellaris restricted plumule length 0.52 cm. Followed by V. jatamansi and A. lebbeck powder with plumule length of 0.50 cm and 0.55 cm. The effect F. cretica came up with plumule length of 1.14 cm. H. helix, M. oleifera, W. coagulans, and M. azedarach restricted the length of plumule of L.sativa up to 1.50 cm, 2.91 cm, 2.64 cm, and 3.91 cm respectively 80

The effect of 20 mg powdered sample of the selected medicinal plants on the seedlings growth of L. sativa showed more quantitative effects. As expected, L. sativa seedlings were very susceptible to S. flagellaris that attained mean radicle length of 0.32 cm, followed by V. jatamansi (0.47 cm) and A. lebbeck (0.49 cm). The effect F. cretica showed with radicle length of 1.13 cm. While H. helix, M. oleifera, W. coagulans and M. azedarach restricted the length of radicle of L.sativa up to 1.51 cm, 2.93 cm, 3.64 cm and 1.99 cm respectively. The inhibitory and stimulatory responses of the plumule to 20 mg quantities were as follows. S. flagellaris restricted Plumule length 0.31 cm, followed by V. jatamansi and A. lebbeck powder with plumule length of 0.44 cm and 0.48 cm, the effect F. cretica came up with plumule length of 1.05 cm. While, H. helix, M. oleifera, W. coagulans, and M. azedarach restricted the length of plumule of L.sativa up to 0 .50 cm, 2.44 cm, 2.65 cm and 2.49 cm respectively. Considering the effects of 40 mg quantity of powder of selected plants on seeds of Lactuca sativa showed more quantifiable properties. L. sativa seedlings were very susceptible to S. flagellaris with radicle length of 0.32 cm. Followed by V. jatamansi and A. lebbeck powder with radical length of 0.47 cm and 0.49 cm. The effect F. cretica showed with radicle length of 1.13 cm. While H. helix, M. oleifera, W. coagulans, and M. azedarach restricted the length of radicle of L.sativa up to, 1.51 cm, 2.93 cm, 3.64 cm and 1.99 cm respectively. While to 40 mg quantity of powder of plants, L. sativa showed different responses regarding length of Plumule. S. flagellaris restricted Plumule length 0.52 cm. Followed by V. jatamansi and A. lebbeck powder with plumule length of 0.41 cm and 0.55 cm. The effect F. cretica came up with plumule length of 1.14 cm. While H. helix, M. oleifera, W. coagulans, and M. azedarach restricted the length of plumule of L.sativa up to 0.55 cm, 1.84 cm, 1.11 cm, and 1.89 cm respectively.

Table 3. 17 Phytotoxicity of Selected Medicinal Plants 10mg 20mg 40mg S. No. Plants names Radicle Plumule Radicle Plumule Radicle Plumule

1 A. lebbeck 0.57±0.15 0.55±0.14 0.49±0.05 0.48±0.07 0.46±0.07 0.41±0.04

2 M. azedarach 3.92±0.23 3.91±0.24 1.99±0.16 2.49±0.17 1.86±0.11 1.89±0.13

3 H. helix 1.01±0.07 1.50±0.06 0.51±0.02 0.29±0.02 0.50±0.07 0.55±0.10

4 S. flagellaris 0.49±0.09 0.52±0.13 0.32±0.01 0.31±0.02 0.40±0.01 0.31±0.02

5 V. jatamansi 0.54±0.14 0.50±0.12 0.47±0.04 0.44±0.07 0.42±0.07 0.37±0.04

6 F. cretica 0.69±0.06 1.14±0.091 1.13±0.19 1.05±0.16 1.04±0.16 1.06±0.11

7 W. coagulans 3.04±0.15 2.64±0.15 3.64±0.17 2.65±0.16 1.78±0.11 1.11±0.09

8 M. oleifera 2.91±0.21 2.91±0.22 2.93±0.14 2.44±0.15 1.82±0.11 1.84±0.11

5 P lu m u le 4 0 m g

4 R a d ic le 4 0 m g

m P lu m u le 2 0 m g m 3

n R a d ic le 2 0 m g

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e L 1

0 k s i s h ix i s a ra c c l r n ic n e a e la t a fe b r h l a e l i b a e m r u le e d ra g a c g o l e a t a z e fl a ia o a ia d j c z a e a n g i a o a n b ia H g n g i ri l l a a a n o A e fr ri a i F M M x le th a a i S V W

Figure 25 Phytotoxicity of 10/20/40 mg against Radicle of L. sativa

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l a

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t 2 h

g 1 n s i i s s e h r n a k x n a r c i a c a L 0 c a l ll a i l e r t if e e e m u b a h e g e g a r l b d a t c a o e a l le r f ja o z a c a e i a a d a a n g i g a z a e n o i in i li a a g n r b H r i l e if r a a o A x e F h M l it M a a S V W

Figure 26 Phytotoxicity of 10 mg against Radicle of L. sativa

m 5

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h 1

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e 0 L i h ix is s a s a k c l r c n r c a e a n ti fe e r h ll a la i b a e u e a e m r l b d r g a c g o le e e a t a z fl a ia o a a a d j n c g i e a z a a o a in i li H g n g i r lb e ra ia a n o A if r F a M M x le th a a i S V W

Figure 27 Phytotoxicity of 10 mg against Plumule of L. sativa

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Figure 28 Phytotoxicity of 20 mg against Radicle of L. sativa

n 3

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l u

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h 1

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Figure 29 Phytotoxicity of 20 mg against Plumule of L. sativa

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t h

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Figure 30 Phytotoxicity of 40 mg against Radicle of L. sativa

2 .5

2 .0

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L 1 .0

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0 .5

0 .0 k s i s h ix i s a ra c c l r n ic n e a e la t a fe b r h l a e l i b a e m r u le e d ra g a c g o l e a t a z e fl a ia o a ia d j c z a e a n g i a o a n b ia H g n g i i l l a n r e r ia a o A if r F a M x le th M a a i S V W

Figure 31 Phytotoxicity of 40 mg against Plumule of L.sativa

3.4 Heavy Metal Analysis Plants hold together toxic and non-toxic metals in wide-ranging concentration. It is an established fact that plants absorb heavy metals from soil and store it in their parts from environments. The concentration of, Pb, As, Cd, Hg, and Zn in selected medicinal plants are given in Table.3.18 Table 3. 18 Heavy Metal Concentration of Selected Medicinal Plants

Lead Arsenic Cadmium Mercury Zinc S.No Plants Names (Pb) µg/g (As) µg/g (Cd) µg/g (Hg) µg/g (Zn) µg/g

1 A. lebbeck 0.204±0.11 Not detected 0.021±0.002 0.041±0.01 0.13±0.002

2 M. azedarach 3.72±0.25 3.74±0.24 1.84±0.13 2.39±0.18 1.76±0.15

3 H. helix 1.25±0.07 1.30±0.06 0.41±0.02 0.26±0.02 0.49±0.08

4 S. flagellaris Not found 0.195±0.135 Not found 0.051±0.031 0.108±0.007

5 V. jatamansi 0.048±0.022 0.552±0.14 Not found Not found 0.203±0.15

6 F. cretica Not found 0.375±0.171 0.041±0.001 Not found 0.395±0.15

7 W. coagulans 0.044±0.026 0.542±0.17 Not found Not found 0.287±0.16

8 M. oleifera Not found 0.208±0.151 0.060±0.021 Not found 0.360±0.18

) 4 g

/ Z in c (Z n ) µ g /g g

 M ercu ry (H g ) µ g /g (

s C a d m iu m (C d ) µ g /g

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k h x s i a s a li i s c n r c c e r n i e e a a a t la f b r h ll e i b a a e m r u le le d r g a c g o e e t a a a z d la a i o a i a e f j n c g z a i a H a o a in lb li g n g i r e a ia a n o A r r F a M if e h M x l it a a S V W

Figure 32 Heavy Metal Concentration in Selected Medicinal Plants, Lead Concentration The concentration of lead was different in the selected species, but almost all were in nontoxic level in S. flagellaris, M. oleifera, and F. cretica no lead were detected while in W. coagulans, had 0.044±0.026 and V. jatamansi, 0.048±0.022 were having minor

concentration A. lebbeck, 0.204±0.11 and H. helix moderate 1.25±0.07 and highest was (

found in M. azedarach 3.72±0.25

) n

g 4

t 

a 3

r t

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e c

1 n i s h s o i s k c a n a r n r c a x c a C 0 i a l e r l l a i e l t u f b a e i e m e g b d h r e g a a l e e . t c l a o z l a o H j . . . a f c . F A . . . M S V M W

Figure 33 Lead Concentration in Selected Medicinal Plants, Using Wet Digestion Method

Arsenic concentration Arsenic concentration in S. flagellaris, 0.195±0.135, M. oleifera, 0.208±0.151, F. cretica, 0.375±0.171 and W. coagulans, 0.542±0.17 while V. jatamansi 0.552±0.14 and H. helix

1.30±0.06, there were no trace found in A. lebbeck, 0 and highest accumulation were found in M. azedarach 3.74±0.24 (Fig.34).

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g /

g 4



(

n 3

t a

r 2

e c

n 1

o C 0 i s k h x is s a a c i r n c n r e c l i a e a e la a t l f b r h l e u i b a e m r e a a c g l le d r g t a o e e la a ia o a z f j c a i a d n g iz e a a o a a g n i in lb li H a g n r e a i a A fr r F a o M i e h M x l it a a S V W

Figure 34 Arsenic Concentration in Selected Medicinal Plants, Using Wet Digestion Method I. Cadmium Concentration Cadmium traces was not found in S. flagellaris, 0 W. coagulans, 0 and V. jatamansi, 0 while in others M. oleifera, 0.060±0.021 F. cretica, 0.041±0.001 A. lebbeck, 0.021±0.002 and H. helix 0.41±0.02 among all was at the top in having the heavy metal M. azedarach 1.84±0.13 (Fig. 35).

(

) 2 .0

o 

i 1 .5

r t

1 .0

e c

n 0 .5 i

o s s i s n k h r n a a

C a r c c x a a c l i l i e 0 .0 e a l l t u f r e m i b e e g a a r e b h g t a l d a c e a o o l e a l j r f a c z i a a e a i a a n a g d n i z g o n i a e a n i i a i g b l r r a r l H f a e i e h o A l F t M x i M a a S V W

Figure 35 Cadmium Concentration in Selected Medicinal Plants, Using Wet Digestion Method II. Mercury concentration The highest Mercury concentration was confirmed in M. azedarach 2.39±0.18 followed by H. helix 0.26±0.02 S. flagellaris, 0.051±0.031 and A. lebbeck, 0.041±0.01 and. F. cretica, 0 V.

jatamansi, 0 W. coagulans, 0 M. oleifera,0 indicated no results (Fig. 36).

(

)

 a

r 2

e c

1 n

o s i s i s r n a h n a

C k c a a r c x l a c l a i l i e e l t u f r e m i b e e g a g a e 0 b h t r a l d a c e l a o o l e a f j z a c r i a a a e a a i n a g z d g n i a o n i i e a a n i b l i g a r l r r e H f a h o i e t A x l F M i M a a S V W

Figure 36 Mercury Concentration in Selected Medicinal Plants, Using Wet Digestion Method

III. Zinc concentration M. azedarach 1.76±0.15 was having highest Concentration of zinc followed by H. helix 0.49±0.08 F. cretica, 0.395±0.15 M. oleifera, 0.360±0.18 and, W. coagulans 0.287±0.16 V. jatamansi 0.203±0.15, A. lebbeck, 0.13±0.002 the least concentration was found in S.

flagellaris, 0.108±0.007 (Fig.37).

(

)

g 2 . 0

 o

i 1 . 5

a r

t 1 . 0

n e

c 0 . 5 n

o s i i s s h n a k x r n a r C 0 . 0 c i a c a c l l a i l e e a l t f b r e m e u i e g e b a h g a r l e d t c a o l e a a a o r l j a z f i c a a a e i d a a n a g z i n i a e g n o i b i a a g n l l H i r e r r a a o A f h i e F t M x l i M a a S V W

Figure 37 Zinc Concentration in Selected Medicinal Plants, Using Wet Digestion Method 3.5 Antioxidant Activity Right after the fractionation of selected medicinal plants, which are A. lebbeck, M. azedarach, H. helix, S. flagellaris, V. jatamansi, F. cretica, W. coagulans and M. oleifera. All four (04) frictions were tested for their antioxidant activity. For the anti-oxidant activity, we used DPPH assay. Human body produce free radicals, these free radicals are the main cause of cancer. Antioxidants are those substances, which scavenges those free radicals. In our study we used DPPH (1, 1-diphenyl-2-picrylhydrazyl) assay. DPPH is used extensively to know the antioxidant potential of plant extracts. These plant extract donate protons to free radical like DPPH and thus scavenge it. In our results we used four fractions i.e. Ethyl acetate, chloroform, Methanol extract and n-haxane extract. Aqueous extract were avoided because studies shows that DPPH scavenging is not good with Hydrophilic extracts. The method used in DPPH assay was by (Brand-Williams et al., 1995). Ascorbic acid was used as standard.

Inhibition values and IC50 were calculated. Overall results were concentration dependent. The results exhibited that all species were active in scavenging DPPH but mainly dependent upon

the concentrations of fractions. In our antioxidant activity, the results are shown in table3.6. In DCM extract, the best among all was S. flagellaris 8.52±0.121, followed by V. jatamansi 13.12±0.153, A. lebbeck 14.7±0.572, H. helix 16.14±0.259, W. coagulans 16.49±0.350, M. azedarach 17.78±0.251, F. cretica 20.79±0.112 and M. oleifera 21.55±0.158. While Ethyl acetate extracts scavenged DPPH in the following ascending order S. flagellaris 19.81±0.311, V. jatamansi 20.12±0.137, H. helix 20.14±0.261, F. cretica 24.79±0.232, A. lebbeck 25.6±0.756, M. azedarach 28.88±0.246, W. coagulans 30.49±0.403 and M. oleifera

34.55±0.158. the Cr. Methanol extract showed the IC50 value in (μg/ml) are, S. flagellaris 9.32±0.102, V. jatamansi 15.12±0.144, M. azedarach 18.40±0.157, H. helix 19.61±0.978, W. coagulans 18.82±0.092, F. cretica 21.65±0.348, A. lebbeck 25.93±0.075 and M. oleifera 26.65±0.149. The n-Haxan Extract showed S. flagellaris 17.11±0.321, A. lebbeck 20.79±0.112, M. oleifera 24.43±0.339 F. cretica 25.57±0.156, H. helix 25.45±0.152, V. jatamansi 29.13±0.155, M. azedarach 30.49±0.403 and W. coagulans 44.12±0.184. Table 3. 19 In vitro Antioxidant Activity of Different Extracts of Selected Medicinal Plants Cr.Eth Ex DCM IC50 Meth Ex IC50 n-hexane IC50 Scientific name IC50 (μg/ml) (μg/ml) (μg/ml) (μg/ml) Ascorbic acid 10.158±0.475 10.158±0.475 10.158±0.475 10.158±0.475 A. lebbeck 14.7±0.572 25.6±0.756 25.93±0.075 20.79±0.112 M. azedarach 17.78±0.251 28.88±0.246 18.40±0.157 30.49±0.403 H. helix 16.14±0.259 20.14±0.261 19.61±0.978 25.45±0.152 S. flagellaris 8..52±0.121 19.81±0.311 9.32±0.102 17.11±0.321 V. jatamansi 13.12±0.153 20.12±0.137 15.12±0.144 29.13±0.155 F. cretica 20.79±0.112 24.79±0.232 21.65±0.348 25.57±0.156 W. coagulans 16.49±0.350 30.49±0.403 18.82±0.092 44.12±0.184 M. oleifera 21.55±0.158 34.55±0.158 26.65±0.149 24.43±0.339

)

l m

/ 5 0 g

 D C M IC 5 0 (µ g /m l)

( 4 0 C r.E th E x IC 5 0 (µ g /m l)

0 M e th E x IC 5 0 (µ g /m l) 5

C 3 0

I n -h e x a n e IC 5 0 (µ g /m l)

n 2 0

a r

t 1 0

n e

c 0

n o

C d x i k h li s i a s a c c c e i s ic n r e a r n t a fe a b r h la a e l i c b a a l r u e i e d r e m c g l b l e e g ta a a o r a z d la a i o a o zi a e f j n c g c i a a o n b i H a g ia i s l l g n a r A A e a a n o r ri F a M if e h M x l it a a S V W

Figure 38 Antioxidant Activity of Selected Medicinal Plants, Using DPPH Assay

2 5

o i

t 2 0

r t

n 1 5

e c

n 1 0 o

C 5

s i s i s a k h r a n id x n a r 0 c c i a c l e c e l l a i f a l t u i a b r e e m e b a r g e h g a l ic e d t c a o l a a a o b e r l j a r a z f i c a i e g o a a a n a z d i n c i a e g n o i s b i a a g n r l l H i a e r r a o A A f h i e F t M x l i M a a W S V

Figure 39 Antioxidant Activity of DCM Fractions of Selected Medicinal Plants, Using DPPH Assay

4 0 A s c o r b ic a c id

A lb iz ia le b b e c k 3 0

n M e lia a ze d a r a c h

i t

a H e d e ra h e lix

r t

n 2 0 S a x ifr a g a fla g e lla r is

e c

n V a le r ia n a ja ta m a n s i o

C 1 0 F e g o n ia c re tic a

W ith a n ia c o a g u la n s

d i a i k h ix is s a s r c c c l r n c n e a e a ti a fe a b r h l a e l i b a l r u e ic e d a e m c g l l e r g ta a o b z e a a a r ia a d l j i o a o z e f n c g c i ia a a o a in s lb l H g n g i r e a ia e n o A A r r F a M if e h M x l it a a S V W

Figure 40 Antioxidant Activity of Ethyl Acetate Fractions of Selected Medicinal Plants, Using DPPH Assay

3 0 A s c o r b ic a c id

n A lb iz ia le b b e c k

o i t M e lia a z e d a r a c h

a 2 0 r

t H e d e r a h e lix n

e S a x ifr a g a fla g e lla r is c

n 1 0 V a le r ia n a ja ta m a n s i o

C F a g o n ia c r e tic a W ith a n ia c o a g u la n s

0 M o r in g a o le ife r a s i s id k h x i s a n a c c c li r n c r e a a a ti a e a b r e l l f h l m e u i c b a e r g le i e d a g ta c a b l e r o r a z e la ja ia o o i a d f c a z a n g c i e a o a n s ia n i i lb l H g a g n r A e a i a a o A r r F M if e th x l i M a a S V W

Figure 41 Antioxidant Activity of Cr. Methanol Fractions of Selected Medicinal plants, Using DPPH Assay

5 0

A s c o r b ic a c id n

o 4 0 A lb iz ia le b b e c k

i t

a M e lia a z e d a r a c h r

t 3 0 H e d e r a h e lix n

e S a x if r a g a f la g e lla r is c 2 0

n V a le r ia n a ja ta m a n s i o

C 1 0 F a g o n ia c r e tic a W ith a n ia c o a g u la n s

0 s i s d k h i s a n a M o r in g a o le if e r a i c x r n c r c c li i a e e a la a t l f a b r e l e u i b h e m r e c a a g l i le d a g t c a o b e r a a a o r a z e l j i c a i f o z a d a n g c i e a o ia n a g n i s lb i g n r l H a ia a A A e r r a o if e F h M l it M x a a W S V

Figure 42 Antioxidant Activity of n-Hexane Fractions of Selected Medicinal Plants, Using DPPH Assay

3.6 PROXIMATE ANALYSIS Proximate composition of plants be responsible for a valuable data about its therapeutic and dietetic quality. The values of moisture, ash, fats, protein and carbohydrate of selected medicinal plants are shown in Table 3. 20. (Fig 43).

Table 3. 20 Proximate Analysis of Different Parts of Selected Medicinal Plants all Values are Mean ± Sem of Three Values. %age %age %age S. No. Plant Name %age of Ash %age Fat Moisture Protein carbohydrate 1 A. lebbeck 23± 0.07 16.36± 0.43 2.85± 0.19 45.79± 0.42 12± 0.23 2 M. azedarach 23.34± 0.12 12.41± 0.56 3.36± 0.09 56.69± 0.34 4.2± 0.17 3 H. helix 25.08± 0.21 12.3± 0.43 6.01± 0.13 49.31± 0.72 7.3± 0.08 4 S. flagellaris 9.92± 0.13 15.5± 0.54 4.37± 0.27 62.79± 0.54 7.42± 0.11 5 V. jatamansi 17.67± 0.22 10.27± 0.09 3.39± 0.25 64.6± 0.71 4.07± 0.24 6 F. cretica 15.01± 0.08 12.66± 0.11 2.31± 0.04 64.32± 0.63 5.7± 0.13 7 W. coagulans 27.43± 0.15 10.27± 0.26 3.39± 0.16 52.62± 0.34 6.29± 0.18 8 M. oleifera 8.01± 0.06 9.17± 0.14 3.88± 0.09 72.63± 0.06 6.31± 0.05

8 0 % a g e F a t % a g e c a rb o h y d ra te 6 0

% a g e P r o te in s

e % a g e M o is tu r e

g 4 0

a % a g e o f A s h %

2 0

0 i s k s s a a c h ix i c n r e c l r n i a e b a e a a t l if r h ll e u b a e m r le e a a c g l d r g t a o e a a a o ia z e l j i c a a d f n g iz e a a a a o i in lb i H g n g r l a a a n A e r i a o f r F h M i le it M x a a W S V

Figure 43 Proximate Composition of Selected Medicinal Plant

Determination of Percentage of Ash Percentage of ash was determined by with help of furnace by taking temperature up to 600 Co (fig.44). The results revealed that W. coagulans had the highest ash contents (27.43± 0.15), followed by H. helix (25.08± 0.21), M. azedarach (23.34± 0.12), A. lebbeck (23± 0.07), V. jatamansi (17.67± 0.22). Minimum quantities of ash was found in F. cretica (15.01± 0.08), S. flagellaris (9.92± 0.13), and M. oleifera (8.01± 0.06), respectively.

s e

g 4

a %

0 i s k is s a a c h x n r c li r n ic a e e a a a t l f b r e ll e u i b a h e m r g le le d a g ta c a o e r a a a o a z e l j i c a i f g z a d a n i e a o ia n b ia g n i l l H a g n r a i a o A e r r a f e F h M i l it M x a a W S V

Figure 44 Ash Composition of Selected Medicinal Plants Determination of Percentage Moisture The moisture percentage is shown in (fig.45). The highest value of moisture was confirmed in A. lebbeck it showed the value of 16.36± 0.43, followed by S. flagellaris 15.5± 0.54 and M. azedarach with percentage of 12.41± 0.56. F. cretica 12.66± 0.11, and H. helix moisture percentage was 12.3± 0.43 and V. jatamansi 10.27± 0.09 while W. coagulans 10.27± 0.26. M. oleifera 9.17± 0.14, were with least moisture values respectively.

1 8

1 6 s

e 1 4

g a

% 1 2

1 0

8 i s k is s a a c h x r n r c i n ic a e e a l a a t l f b r e ll e u i b a h e m r g le e d g a c a l a t o e r la a a o a z e f j i c a i a d n g iz e a a a a n o i in lb i H g g n r l a ia a A e r r a o if e F h M l it M x a a W S V

Figure 45 Moisture Composition of Selected Medicinal Plants Determination of Percentage Protein The protein content was prominent in H. helix with the value of 6.01± 0.13, followed by S. flagellaris 4.37± 0.27, and M. oleifera 3.88± 0.09 as it is clear from (fig.46). W. coagulans 3.39± 0.16, and V. jatamansi proteins percentage was 3.39± 0.25, the next three species with least protein contents were M. azedarach 3.36± 0.09, A. lebbeck 2.85± 0.19, and F. cretica 2.31± 0.04, respectively.

8 0

7 0

s e

g 6 0

a %

5 0

4 0 k s i a s a c h ix i s n r e c l r n ic a e a e a a t l f b r h l e u i b a l m r e e a e c g l l d g ta a o e r a a o ia z e l ja i a z a d f n c g i e a a b a a o i in l li H g n g n r A e a ia a a o fr r F h M i le it M x a a W S V

Figure 46 Protein Composition of Selected Medicinal Plants Determination of Percentage Carbohydrate The maximum Carbohydrates contents were found in M. oleifera with the value of 72.63± 0.06, followed by V. jatamansi 64.6± 0.71 and F. cretica 64.32± 0.63, as it is clear from (fig.47). S. flagellaris 62.79± 0.54, and M. azedarach Carbohydrates percentage was 56.69± 0.34, the next three species with least Carbohydrates contents were W. coagulans 52.62± 0.34, H. helix 49.31± 0.72, and A. lebbeck 45.79± 0.42, respectively.

8 0 A lb iz ia le b b e c k M e lia a z e d a r a c h 7 0

H e d e r a h e lix s

e S a x if r a g a f la g e lla r is

g 6 0

a V a le r ia n a ja ta m a n s i % F a g o n ia c r e tic a 5 0 W ith a n ia c o a g u la n s M o r in g a o le if e r a 4 0

k x s i a s a c h i i s c n r e c l r n i a e b a e a a t l if b r h ll e u e a e m r g l le d a a c o e r g t a a z e a a ia o a i d fl j c g iz a e n b a a a o ia in l li H g n g n r A e a ia a o r r F a f h M M i le it x a a W S V

Figure 47 Carbohydrate Composition of Selected Medicinal Plants

Determination of Percentage Fat Among the selected medicinal plants the fats contents was highest in A. lebbeck with the value of 12± 0.23, followed by S. flagellaris 7.42± 0.11, and H. helix 7.3± 0.08, as it is clear from (fig.48). M. oleifera 6.31± 0.05, and W. coagulans fat percentage was 6.29± 0.18, the next three species with least fat contents were F. cretica 5.7± 0.13, M. azedarach 4.2± 0.17, and V. jatamansi 4.07± 0.24, respectively.

100

1 5

1 0

a % 5

0 i s k is s a a c h ix r c n r e c l n i a e a e a a t l f b r ll e u i b a h e m r g le e d a g a c a o l r t e la a a o ia z e f j i c a z a d n g i e a a o a n b a n i i l li H g g n r a ia a o A e r r a f e F h M i l it M x a a W S V

Figure 48 Fat Composition of Selected Medicinal Plants 3.7 Phytochemical Analysis of Selected Medicinal Plants Phytochemical analysis showed that all the medicinal plants possessed the considered phyto- constituents, but at varying degree (Table.3.21). A. lebbeck had higher amounts of alkaloids and Phenols, moderate amounts of glycosides and lower amounts of Saponins and Flavonoids. M. azedarach contained higher quantities of phenolics, glycosides, moderate quantities of Alkaloids, and lower quantities of saponins and flavonoids. glycosides were found low H. helix, where saponins were present in higher amounts. The presence of alkaloids, flavonoids and phenols were at moderate levels. S. flagellaris exhibited higher contents of saponins and flavonoids, but the other three phyto-chemicals, i.e. phenols, glycosides and alkaloids were found in moderate concentrations. V. jatamansi contained low quantity of saponins and flavonoids. glycosides were recorded in high points while alkaloids and phenols in moderate Levels. F. cretica displayed high content of saponins, and phenols in moderate points but the other three phyto-chemicals i.e. flavonoids, glycosides and alkaloids were found in low concentrations. In W. coagulans the concentrations of glycosides, and flavonoids were high, phenols, and alkaloids were moderate while saponins were low. During the tests saponins were found high, glycosides in low levels while phenols, flavonoids and alkaloids were found in moderate concentration in M. oleifera.

101

Table 3. 21 Phytochemical Analysis of Selected Medicinal Plants Pants names Alkaloids Saponins Glycosides Flavonoids Phenols

A. lebbeck ++- +-- +++ +-- ++-

M. azedarach ++- +-- +++ +-- +++

H. helix ++- +++ +-- ++- ++-

S. flagellaris ++- +++ ++- +++ ++-

V. jatamansi ++- +-- +++ +-- ++-

F. cretica +-- +++ +-- +-- ++-

W. coagulans ++- +-- +++ +++ ++-

M. oleifera ++- +++ +-- ++- ++-

Key: - : Not found, +:Low, ++: Moderate, +++: High.

3.8 Anticancer Activity The present study was undertaken to find out the anticancer activity of selected medicinal plants using MTT bioassay against available cancer cell line (Colon cancer cells--- HT-29).

120 100 80 60 40 20

Control

K5 25K5uM K1 25K1 uM 50K1 uM 25K2 uM 50K2 uM 25K3 uM 50K3 uM 25K4 uM 50K4 uM 50K5 uM 25K6 uM 50K6 uM 25K7 uM 50K7 uM 25K8 uM 50K8 uM

K2 100K2 uM 100K3 uM 100K4 uM 100K5 uM 100K6 uM 100K7 uM 100K8 uM K1 100K1 uM

102

Figure 49 Anticancer Activity of Different Fraction of Selected Medicinal Plants Extracts at All Concentrations against (Colon cancer cells--- HT-29). Cell Line Using MTT- Assay . Table 3. 22 Anticancer Activity of Different Fractions of Selected Medicinal Plants At 25 µm

Plants plant code n- hexane %age CHCl3 %age EtOAc %age

Control 0.517 100 0.51 100 0.5135 100 A. lebbeck K1 25 µM 0.576 111.41 0.467 91.57 0.5215 101.56 M. azedarach K2 25 µM 0.594 114.89 0.515 100.98 0.5545 107.98 H. helix K3 25 µM 0.465 89.94 0.486 95.29 0.4755 92.60 S. flagellaris K4 25 µM 0.053 10.25 0.052 10.20 0.0525 10.22 V. jatamansi K5 25 µM 0.407 78.72 0.363 71.18 0.385 74.98 F. cretica K6 25 µM 0.357 69.05 0.451 88.43 0.404 78.68 W. coagulans K7 25 µM 0.437 84.53 0.527 103.33 0.482 93.87 M. oleifera K8 25 µM 0.4 77.37 0.351 68.82 0.3755 73.13

0 .8 E tO A c

C H C l3

l 0 .6

a n - h e x a n e

v r

u 0 .4

l e

C 0 .2

0 .0

l k x s i a s a o c h i i s c n r r e c l r n i a e t b a e a a t l if n b r h ll e u e o a e m r g l le d a a c o C e r g t a a ia z e la a i o a z a d f j n c g i e a b a a o ia in l li H g n g n r A e a ia a o r r F a f h M M i le it x a a W S V

103

Figure 50 Anticancer Activity of Different Fraction of Selected Medicinal Plants Extracts at 25 μM Concentrations against (Colon cancer cells--- HT-29). Cell Line Using MTT- Assay The result of 25 μM fractionations of selected medicinal plants showed different anticancer activities, three (03) fractions were tested for their anticancer potential against cancer cell line using MTT bioassay against available cancer cell line (Colon cancer cells--- HT-29. We used six (25) μM dose concentration of each fraction, found that all the some fractions showed good anticancer activities. Using n- hexane fraction the S. flagellaris showed the most encouraging result by restricting cell growth to 10.25% followed by F. cretica 69.05% M. oleifera 77.37% V. jatamansi 78.72% W. coagulans 84.53% H. helix 89.94% A. lebbeck 111.41% and M. azedarach% 114.89 while the an exception of F. cretica the same trend was followed by chloroform fractions of selected medicinal plants, which are S. flagellaris showed the most encouraging result by restricting cell growth to 10.20% followed by M. oleifera 68.82 % V. jatamansi 71.18% F. cretica 88.43%W. coagulans 103.33% H. helix 95.29% A. lebbeck 91.57% and M. azedarach 100.98%. The ethyl acetate fractions of selected medicinal plants showed the following effects S. flagellaris showed the most promising result by restricting cell growth to 10.22% followed by M. oleifera 73.13 % V. jatamansi 74.98% F. cretica 78.68 %W. coagulans 93.87% H. helix 92.60% A. lebbeck 101.56% and M. azedarach 107.98% Cell viability. Table 3. 23 Anticancer Activity of Different Fractions of Selected Medicinal Plants at 50 μM Concentrations

Plants plant code n- hexane %age CHCl %age 3 EtOAc %age Control 0.517 100 0.51 100 0.5135 100 Albezia lebbeck K1 50 µM 0.536 103.68 0.486 95.29 0.511 99.51 M. azedarach K2 50 µM 0.48 92.84 0.631 123.73 0.5555 108.18 H. helix K3 50 µM 0.451 87.23 0.453 88.82 0.452 88.02 S. flagellaris K4 50 µM 0.053 10.25 0.05 9.80 0.0515 10.03 V. jatamansi K5 50 µM 0.273 52.80 0.337 66.08 0.305 59.40 F. cretica K6 50 µM 0.413 79.88 0.439 86.08 0.426 82.96 W. coagulans K7 50 µM 0.488 94.39 0.442 86.67 0.465 90.56 M. oleifera K8 50 µM 0.37 71.57 0.363 71.18 0.3665 71.37

104

0 .8

E tO A c l

a C H C l3

v 0 .6 i

v n - h e x a n e

r u

s 0 .4

l e

C 0 .2

s i s k i s a n a 0 .0 h x r n r l c c i a c a e o e l l a ti l f a l u i r b r e m e t b a h e r g e g a l n e d t c a o o l a a o e r l ja a a z e f i c a C i a g z d a a n a i e n o i n ia g i lb l a a g n r H i a a A e r r o if e F h M l it M x a a W S V

Figure 51 Anticancer Activity of Different Fraction of Selected Medicinal Plants Extracts at 50 μM Concentrations against (Colon Cancer Cells--- ht-29). Cell Line Using MTT- Assay The result of 50 μM fractionations of selected medicinal plants showed different anticancer activities, three (03) fractions were tested for their anticancer potential against cancer cell line using MTT bioassay against available cancer cell line (Colon cancer cells--- HT-29. We used fifty-(50) μM dose concentration of each fraction, found that all the some fractions showed good anticancer activities. Using n- hexane fraction the S. flagellaris showed the most encouraging result by restricting cell growth to 10.25%, followed by V. jatamansi 52.80 %, M. oleifera 71.57 %, F. cretica 79.88 %, H. helix 87.23%, M. azedarach 92.84 %, W. coagulans 94.39 % and A. lebbeck 103.68%. The chloroform fractions of selected medicinal plants showed the following cell vitality percentage. The S. flagellaris indicated the most promising result by restricting cell growth to 9.80%, followed by V. jatamansi 66.08%, M. oleifera 71.18%, F. cretica 86.08%, W. coagulans 86.67%, H. helix 88.82%, A. lebbeck 95.29% and M. azedarach 123.73%. While the ethyl acetate fractions of selected medicinal plants showed the following cell viability percentage. The S. flagellaris indicated the most promising result by restricting cell growth to 10.03%, followed by V. jatamansi 59.40%, M. oleifera 71.37%, F. cretica 82.96%, W. coagulans 88.02%, H. helix 108.18%, A. lebbeck 90.56% and M. azedarach 99.51%.

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Table 3. 24 Anticancer Activity of Different Fractions of Selected Medicinal Plants at 100 μM Concentrations

Plants plant code n- hexane %age CHCl3 %age EtOAc %age

Control 0.517 100 0.51 100 0.5135 100 A. lebbeck K1 100 µM 0.532 102.90 0.496 97.25 0.514 100.10 M. azedarach K2 100 µM 0.489 94.58 0.512 100.39 0.5005 97.47 H. helix K3 100 µM 0.353 68.28 0.411 80.59 0.382 74.39 S. flagellaris K4 100 µM 0.057 11.03 0.052 10.20 0.0545 10.61 V. jatamansi K5 100 µM 0.359 69.44 0.343 67.25 0.351 68.35 F. cretica K6 100 µM 0.458 88.59 0.376 73.73 0.417 81.21 W. coagulans K7 100 µM 0.541 104.64 0.43 84.31 0.4855 94.55 M. oleifera K8 100 µM 0.383 74.08 0.419 82.16 0.401 78.09

0 .6 E tO A c

C H C l3 l

a 0 .4 n - h e x a n e

l l

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0 .0 l o k h x s i a s a r c li i s c n r t e c e r n ti a e b a a a e l if n b r h ll r u e o e a a e m c g l C l d r g ta a o a e e a a o i z d l ja i c a iz a e f n g b a a a o ia in l li H g n g r A e a a a n o r i F a f r h M M i le it x a a W S V

Figure 52 Anticancer Activity of Different Fraction of Selected Medicinal Plants Extracts at 100 μM Concentrations against (Colon cancer cells--- HT-29). Cell Line Using MTT- Assay The result of 100 μM fractionations of selected medicinal plants showed different anticancer activities, three (03) fractions were tested for their anticancer potential against cancer cell line 106

using MTT bioassay against available cancer cell line (Colon cancer cells--- HT-29). We used hundred-(100) μM dose concentration of each fraction, found that all the some fractions showed good anticancer activities. Using n- hexane fraction the S. flagellaris showed the most encouraging result by restricting cell growth to 11.03%, followed by H. helix 68.28%, V. jatamansi 69.44%, M. oleifera 74.08%, F. cretica 88.59%, M. azedarach 94.58%, A. lebbeck 102.90% and W. coagulans 104.64%. The chloroform fractions of selected medicinal plants showed the following cell vitality percentage. The S. flagellaris indicated the most promising result by restricting cell growth to 10.20%, followed by V. jatamansi 67.25%, F. cretica 73.73%, H. helix 80.59%, M. oleifera 82.16%, W. coagulans 84.31% A. lebbeck 97.25% and M. azedarach 100.39%. While the ethyl acetate fractions of selected medicinal plants showed the following cell viability percentage. The S. flagellaris indicated the most promising result by restricting cell growth to 10.61%, followed V. jatamansi 68.35%, H. helix 74.39%, M. oleifera 78.09%, F. cretica 81.21%, W. coagulans 94.55%., M. azedarach 97.47% and A. lebbeck 100.10% .

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3.9 Results of Isolated Compound 1H NMR spectrum (CDCl3): 2.49 ddd, 1 H, J = 15.5, 3.1, 3.0 (H-5a); 2.55 dd, 1 H, J = 11.0, 10.9, 3.0 (H-6a); 2.61 s, 3 H (NMe); 2.82 m, 1 H, (H-5b); 3.08 m, 1 H, (H-6b); 3.99 s, 2 H (O- CH2-O); 5.5 s, 2 H (O-CH2-O); 4.18 d, 1 H, J = 3.4 (H-14); 5.88 dd, 1 H, J = 3.4, 0.9 (H-13); 6.09 s, 2 H (–OCH2O–); 6.34 s, 1 H (H-4); 6.52 s, 1 H (H-1); 6.88 d, 1 H, J = 7.9 (H-11); 7.13 dd, 1 H, J = 7.9, 0.9 (H-12).

The ‗HNMR spectrum of compound 2 (CDCI3,) showed the presence of one N-methyl (2.49) and two methylene dioxy group protons (3.99 and 6.09), two aromatic ring protons, which gives two singlets (6.42 and 6.34, H-1 .H-4), one aromatic proton doublet at 6.88 (H-11) and another aromatic proton doublet at 7.13 (H-12). The two multiplets at 2.82 and 2.55 were due to H-5 and H-6.

The assignment of the 13C NMR signals of 2 was assisted by the use of DEPT and the 2D techniques. It shows twenty resonances, one methyl twelve aromatic carbons, five saturated carbons along with one ester type carbonyl.

The down field signals at 26.9 and 49.1 are C-5 and C-6 respectively. The methyl attached to N appears at 45.1 where as two methylene dioxy O-CH2-O shows signals at 101.1and 103.4. The signals at 67.2 and 85.1 were assigned to C-14 and C-13, whereas the aromatic carbons appeared at 110.2, 146.0, 146.9, 110.2, 130.7 and 124.5 are for C-1, C-2, C-3, C-4, C-15 and C-16. The signals at 108.7, 144.7, 149.3, 113.4, 115.6 and 140.5 were assigned to C-7, C-8, C-9, C-10, C-11 and C-12 respectively. The ester carbony;l appeared at 167.7. From all the data the compound was Adlumidine, compared with the reported data in the literature.

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Figure 53 1H NMR of Compound

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Figure 54 13C NMR of Compound

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Figure 55 DEPT 90 of Compound

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Figure 56 DEPT 135 of Compound

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Structure of Compound isolated from DCM fraction of Saxifraga flagellaris

Figure 57 Structure of Adlumidine Compound

Adlumidine (2) (C20H17NO6). Obtained as a crystalline substance (4.0 mg), m.p. 239-241 °C, UV spectrum (MeOH), ʎmax, nm (log e): 285 (3.94), 330 (3.90). FAB-MS, m/z 367.105 (M + H)+. EI-MS, m/z (%): 206 (100), 190 (10), 177 (3).

3.10 Anticancer Activity of Isolated Compound

3.10.1 Adlumidine Reduce the Cell Viability of HCT 116 And PC3 Cells Cell viability assays are often conducted for screening test compound to determine if these are having some positive or negative regulatory effects on cell proliferation, or show direct cytotoxicity that eventually lead to cell death. In order to determine the effects of Adlumidine on cell viability of HCT 116 and PC3 cells, we conducted MTT assay. Cells were treated with different concentrations (5-100 µg/ml) of Adlumidine for the 24 h. We found that 20 µg/ml of Adlumidine killed 25.96% HCT 116 cells (Figure 58a). In case of PC3 cells, Adlumidine caused 21.72% cell death at 10 µg/ml concentration (Figure 58b). This compound proved toxic for HCT 116 and PC3 cell lines at ≥ 20 µg/ml concentrations. These findings suggested that Adlumidine reduced the cell viability of HCT 116 and PC3 cells.

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Figure 58 Adlumidine reduce the cell viability of HCT 116 and PC3 cells Triplicate wells were treated with vehicle and different concentrations of Adlumidine for the indicated times. Control cells served as the indicator of 100% cell viability. 10-20 µg/ml concentration of these compound proved to be largely nontoxic for HCT 116 and PC3 cells. Data were presented as means ± SD.

From the cell viability results, we selected 20 µg/ml concentration of Adlumidine for HCT 116 cells, and 10 µg/ml concentration for PC3 cell lines. In order to examine whether Adlumidine specifically induce apoptosis in HCT 116 cells, we conducted Annexin-V/PI apoptosis assay through flow cytometer and found that, compared to untreated cells, Adlumidine increased early apoptosis to 19.5%, while boosted late apoptosis to 10.4% (Figure 59). These findings elucidated that, Adlumidine specifically induced apoptosis in HCT 116 cell lines.

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Figure 59 Adlumidine Induce Apoptosis in HCT 116 Cells HCT 116 and PC3 were treated with Adlumidine for indicated times. Cells were washed with 1X PBS and collected by centrifugation. 2 x 105 cells were incubated with 5 µl each of Annexin-V-FITC and Propidium Iodide (50mg/ml) at 25°C for 10 min in the dark. Cells were then analyzed by flow cytometry. From Annexin-V chart: (1) Lower left= negative Annexin- V and negative PI, indicating viable cells. (2) Lower right= positive Annexin-V and negative PI, indicating early apoptosis. (3) Upper right= positive Annexin-V and positive PI, indicating late apoptosis. (4) Upper left negative Annexin-V and positive PI, indicating necrotic cells. Upper right plus Lower right, indicate the summation of apoptosis. Experiment was repeated at least three times and data were presented as means ± SD.

ROS generation is considered the most important tool used by anticancer drugs for killing cancer cells. ROS damage the DNA, proteins, lipids and other biological molecules which subsequently lead to cell death. Additionally, ROS are also involved in caspase-dependent cell death (Cadenas, 2004, Simon et al., 2000). In order to investigate the involvement of ROS pathway in Adlumidine-induced apoptosis, we treated HCT 116 with Adlumidine for 24 h and incubated with 5 µM

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DCFHDA for 30 minutes in the dark. Compared to control cells, a significant increase was noticed in oxidant-induced 2′, 7′-dichlorofluorescein fluorescence in treated HCT 116 cells within 30 min of DCFHDA addition (Figure 60). Hydrogen peroxide (H2O2) was used as positive control (Figure 60). These result suggested that Adlumidine induced ROS generation in HCT 116, which may contribute in apoptosis induction.

Control H2O2 Adlumidine

Figure 60 Adlumidine Induce ROS Generation in HCT 116 cells Cells were exposed to Adlumine and Adlumidine and then incubated with DCFHDA for 30 min. Images of cells were obtained under a fluorescence microscope (λ min = 480 nm, λ max = 520 nm). H2O2 was used as positive control for ROS generation.

Caspases are a set of cysteine proteases that upon activation by some stimuli, induce cell death pathways, which eventually lead to cell death (Elmore, 2007). Among them, caspase-3 is a frequently activated death protease, which catalyzes the specific cleavage of many key cellular proteins (Porter and Janicke, 1999). Poly (ADP-ribose) polymerase (PARP) cleavage is linked with caspase-3 activation (Boulares et al., 1999). In order to investigate the activation of caspase-3 pathway, we conducted caspase-3 activity assay using HCT 116 and PC3 cell lines, which was based on spectrophotometric detection of the chromophore p-nitroaniline (p-NA) after cleavage from the labeled substrate DEVD-pNA [manufacturer protocol]. In case of HCT 116, compared to untreated cells, we found a significant increase of 10.50 fold in caspase-3 activity in Adlumidine treated cells, (Figure 61). Similarly, this compound elevated caspase-3 activity 7.63, in PC3 cell lines (Figure. 61). Next, we conducted western immunoblotting and found an increase of the p11 subunit of caspase-3, as well as of the 116

subsequently cleaved PARP in both HCT 116 and PC3 cells upon treatment with Adlumidine (Figure 62). Actin was used as internal control. These results imply that Adlumidine induced caspase-3- dependent apoptosis in HCT 116 and PC3 cell lines.

Figure 61 Caspase-3 Activity in Adlumidine Based on Spectrophotometric Detection

Figure 62 Adlumidine Lead to Caspase-3 Dependent Apoptosis In HCT 116 And PC3 Cells Cells were treated with Adlumidine for 24 h. Cellular proteins were extracted by cell lysis buffer, and western blot was performed using antibodies specific for cleaved caspase-3, cleaved PARP and β-actin. Actin was used as a loading control in all experiments.

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CHAPTER 4 DISCUSSION

4.1 Antifungal Activity

The use of medicinal plants against fungi is not new to human kind. It produces eminent beneficial compounds. These compounds have a vital role in the development of antimicrobial drugs. Fungal pathogens are one of the key infectious agents in plants and animals causing loss of quality, limited shelf life, and damages to the crops. There are increased demands to develop active biomolecules to cope with such problems (Redo et al., 1989). The opportunistic fungi cause diseases in humans and can be controlled by using drugs that have no side effects (Bansod and Rai, 2008). Medicinal plants are the source of new drug discovery (Silva and Fernandes Júnior, 2010). There are many drugs, which are isolated from natural plants and are very effective against various diseases. Today, the importance of medicinal plants based drugs are more than ever before. In the developing countries, the quality and quantity of modern synthetic drugs are insufficient, so plants can be used as cheap natural sources to combat diseases safely. The old traditions of using medicinal plants as cure for many diseases has still a key role in discovery of modern drugs and chemotherapeutic agents (Ullah et al., 2013, Shinwari et al., 2013). In recent years, the interest in antimicrobial drugs has increased and new methodologies in research are developing to cope with increasing microbial resistance. Therefore, the urge to develop new antimicrobial activities and screening mythologies has increased (Balouiri et al., 2016). The present antifungal activity specified that these particular medicinal plants have worthy outcomes contrary to microbes‘ i.e. The results of the antifungal activity of selected medicinal plant showed much significance. Among all fractions, Cr. Met. Ext. Fraction showed high activity with maximum zone of inhibition 65.0±0.54 mm (65%), 65.0±0.54 mm (65%) by S. flagellaris and V. jatamansi against Alternaria alternate, a similar result was given by Hadizadeh et al., 2009 against A.Alternate which confirm our result that our selected plant has also remarkable antifungal activity (Hadizadeh et al., 2009). Potillo et al., (2001) reported that that over all the cr. Met. Ext showed maximum antifungal activities which is similar to our research work (Portillo et al., 2001). The lowest was shown by W. coagulans 25.0 ± 0.55 (25%) mm of zone inhibition .

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in the selected medicinal plants H. helix , A. lebbeck, F. cretica, W. coagulans, M. oleifera and M. azedarach showed remarkable antifungal activities against A.flavus Similar reported by (Rios and Recio, 2005). It is clear from the results that the selected medicinal plants possess very good antifungal properties and specially the crude Methanolic and Ethanolic extracts, which also clarify that the polar solvent dissolves the polar drugs. these polar solvent fractions are good antifungal and antibacterial drugs. Same results were reported by (Duraipandiyan et al., 2006). The n-Hexane Fraction of A. lebbeck, M. azedarach, H. helix and S. flagellaris in the selected medicinal plants provided significant anti-fungal activities against P. pallidum and F. oxysporum which is in accordance with the findings of (Silva and Fernandes Júnior, 2010). Similarly V. jatamansi, F. cretica, W. coagulans and M. oleifera showed mild anti fungal activity, related results were stated by (Kordali et al., 2009). While against A. alternate the all selected plants were mild to high antifungal which is in accordance to (Cowan, 1999). The results of the antifungal activity of the selected medicinal plants showed that the Chloroform fraction has high activity with maximum (mm) zone of inhibition 60.0 ± 0.22 (60%) by M. oleifera against P. pallidum and the lowest was shown by V. jatamansi 0.0±0.00 (0%) mm of zone inhibition our result coincide with the findings of (Nikkon et al., 2003, Muanza et al., 1994). According to the results of (Ali et al., 2001) A. lebbeck , M. azedarach, H. helix , S. flagellaris, F. cretica, W. coagulans and M. oleifera contain good antifungal activities against fungal strains A.flavus, F. oxysporum, A. alternate and P. pallidum. while the Chloroform fraction of V. jatamansi contain least antifungal activities against the selected strains similar findings are given by (Dua et al., 2008) and (Charu and Kaushik, 2003). According to (Srinivasan et al., 2001, Buwa and Van Staden, 2006) Ethyl acetate fraction A. lebbeck contain exceptional anti-fungal activity in our results Ethyl acetate fraction of A. lebbeck showed with maximum (mm) zone of inhibition 55.0 ± 0.77 (55%). However, V. jatamansi came up with the least antifungal activity, which shows the fact that it has least antifungal agents against the selected strains in the fractions which is in accordance with the findings of (Lin et al., 2009). Apart from the above two plants, other selected medicinal plants which are H. helix , S. flagellaris, F. cretica, W. coagulans, M. oleifera and M. azedarach hold high antifungal activity against all fungal strains, except P. pallidum which was less vulnerable in most of cases. Our result are supported by the work of (Carpinella et al., 1999, Carpinella et al., 2003) and (Dorman and Deans, 2000). Agreeing to (Freile et al., 119

2003, Qasem and Abu‐ Blan, 1995) the antifungal activity of aqueous fractions has least value among all fractions, but still F. cretica , A. lebbeck, H. helix and M. oleifera fraction showed mild to moderate activity while S. flagellaris, V. jatamansi, H. helix and M. azedarach were least effective against fungal strain P. pallidum which is similar to the findings of (Satish et al., 2007).

4.2 Anti-Bacterial Activities All over the ages plants were used for remedial purposes along with other uses. They are used in almost any type of disease from minor wound to chronic heart disease. Still at large, people of the world particularly the developing countries rely continuously on traditional herbal system to deal with various diseases. A layman in rural may have no knowledge of the different phytochemicals present in these medicinal plants, but they have a belief that these medicinal plants that were used by their forefathers possesses astringent power to heal the diseases. Several hundred genera of plants are utilized for medicinal purposes, which act as key sources for effective and powerful drugs (Perry and Metzger, 1980, Sofowora, 1982, Ross, 2007). Botany has a key role in health of mass ―the medicine man‖ (Pinsari) called as the first professional botanist known to human race. As for as the western medicine is concerned it is taught in most of the medical faculties all over the world and has largely dependent on manufactured drugs (Hebbar et al., 2004, Bauer and Brönstrup, 2014). However, from the last decade, the trend is shifting from modern medicinal to natural medicinal system, which is totally dependent on herbal drugs and therapies. As per European Union, 1400 herbal preparations are currently in wide use. In Europe and Germany, herbal preparations got great importance in healthcare needs. The popularity of herbal preparations can be judged by their increasing importance in cosmetic industry, such as skin tissue regenerators, anti-wrinkling agents and anti-age creams (Hoareau and DaSilva, 1999). In the Eastern Europe, the herbal products are either unmodified or slightly altered before use (Bahadur et al., 2007, Health, 1997).

In the present study, five different fractions were taken and their anti-bacterial activities were carried out. These five fractions were cr. Methanol. extract, n-Hexane, Chloroform, cr. Ethanol extract and aqueous extracts. The bacterial strains Streptococcus mutans, Methicillin- Resistant Staphylococcus aureus (MRSA), Staphylococcus aureus, (Gram-positive), and

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Serratia marcescens,, (Gram-negative) were used in the anti-bacterial activity. Jigna et al. (2005) reported that F. cretica contained significant antibacterial activity. Similar results have been found in our research, where Cr. Methanolic extract from F. cretica showed maximum inhibition zones (57.69 %) against all bacterial strains. Among other plants, H. helix and V. jatamansi showed very high anti-bacterial properties (50-52%) against S. aureus and S. marcescens, which is in accordance with the results of Bonjar (2004). As for as the antibacterial activity of A. lebbeck, M. azedarach, S. flagellaris, W. coagulans and M. oleifera concerned, they exhibited potent activity, but comparatively lower than that of earlier mentioned species. Similar results were reported about A. lebbeck, M. azedarach, S. flagellaris, W. coagulans and M. oleifera by (Nikkon et al., 2003, Kala, 2006). Who worked on M. oleifera and other different plants against different bacterial strains. Among all selected plants W. coagulans (56%) showed exceptional results against MRSA, which is in agreement with Maurya et al. (2010). In our results of n-Hexane fractions, we found that M. azedarach (52%) and W. coagulans (50%) contained significant antibacterial activity against S. marcescens and S. aureus, respectively. Hirasawa et al., (1999), who also reported that S. flagellaris contain good antifungal activity, have recorded similar observations. In our result, we also found that S. flagellaris (46-47%) showed antibacterial activities against all four selected bacterial strains. W. coagulans, with exception of S. aureus, showed moderate results against S. mutans, S. marcescens and MRSA, which is an agreement with findings of Basile et al. (2000). Similarly, with exception of S. mutans (35%), F. cretica inhibited the bacterial growth to moderate level (40-47%). Dastagir et al. (2012) reported the same about F. cretica. While in our selected plants A. lebbeck, M. azedarach, H. helix , V. jatamansi, and M. oleifera, inhibited all the four selected bacterial stains from low to moderate level (30-47%). Jan et al. (2012) worked on different plants belonging to 26 families and found moderate antibacterial activities in n-hexane fractions, which coincide with our findings. Using chloroform fractions we established that M. azedarach, (61.90%), F. cretica, ( 52.38%), and W. coagulans,( 52.38%), extracts was the most potent plant species to inhibit S. marcescens. After studding different research readings of other scientists (Aziz et al., 1998, Khan et al., 2010) we found that antimicrobial activity increases or decreases due to the occurrence of active constituents (phenolic compounds). The concentration of phenolic compounds varied with varying environmental conditions. According to different researchers, anti-bacterial 121

activities linked with phenolic contents in medicinal plants (Vaquero et al., 2007, Alberto et al., 2006, Caceres et al., 1991). In our results, we found that the Chloroform extract of S. flagellaris (33.33%),, V. jatamansi,( 0.00%), and M. oleifera( 0.00%) contain least significance against S. marcescens that has been confirmed by Lima-Filho et al. (2002) got the same trend in result while he used chloroform extract of other species against bacteria S. marcescens. In the study of plants against S. mutans (Hirasawa et al., 1999) found that S. mutans is susceptible to many plants extract, we confirm the findings of (Asad et al., 2018) that S. flagellaris (50%), V. jatamansi, (50%),and W. coagulans(, 50%), indicated significant results in inhibiting the growth of the bacteria. Apart from M. oleifera ( 0.00%) the other plants A. lebbeck, 25.00%), and F. cretica, 39.29%), low inhibition while M. azedarach, (46.43%), and H. helix , (42.86 %) inhibited the S. mutans on moderate level. (Fahey, 2005) reported that M. oleifera (46.15%), and H. helix , (46.15%), chloroform extracts are effective in inhibition of S. aureus . according to (Alade and Irobi, 1993) and (Ghaima et al., 2013) the bacteria is moderately inhibited by the chloroform extracts of (Ghaima et al., 2013) plant. We found similar results that S. aureus is moderately inhibited by A. lebbeck, 34.62%), M. azedarach, (38.46%), S. flagellaris (42.31%),, V. jatamansi, 34.62%), F. cretica, 30.77%) and W. coagulans, 38.46%). Johnson et al. (2001) reported that MRSA has great resistance against most of the drugs; therefore, there is a need to develop new antimicrobial drugs with high efficacy against MRSA. In our findings, we noticed that except H. helix, 28.00%), all the selected medicinal plants showed higher activity against MRSA. (Sato et al., 2000) used Scutellaria barbata D. Don (Lamiaceae) to discover antibacterial compounds with strong inhibitory effect on MRSA, reported that S. aureus, and MRSA are inhibited by the used plant extracts. The antibacterial trend of our selected plants also showed significant results. The antibacterial trends in ascending orders are as follows Albizia, (46.15%), W. coagulans, (42.31%), M. azedarach, (46.15%), and F. cretica, (46.15%), showed high level of inhibition while H. helix , (30.77%), S. flagellaris (30.77%), and V. jatamansi, 38.46%), also inhibited S. aureus (Ali et al., 2001) used Ethanolic extracts of 20 selected plant species for antibacterial activity. Fourteen of which were significant against MRSA in conformity to that our selected medicinal plants showed promising results against the bacteria in which, F. cretica, (52.00%), V. jatamansi, (48.00%), M. azedarach, (44.00%), and W. coagulans, (40.00%), were highly significant, while H. helix , 36.00%), S. flagellaris (36.00%), and A. 122

lebbeck, 28.00, %), showed moderate inhibition against MRSA. (Basile et al., 2000) reported that Ethanolic extracts have significant antibacterial properties that is in accordance to our findings. (Sakanaka et al., 1989, Kapoor et al., 2015) reported that W. somnifera contain certain antibacterial compounds and the ethyl acetate extracts are best among all, we found results that agree with these scientists findings. Our results showed that Ethyl acetate fraction has highest inhibitory potential against bacteria S. mutans. The highest inhibition was shown by W. coagulans (57.14%), followed by F. cretica,( 50.00%), and V. jatamansi, (50.00%) against S. mutans. While A. lebbeck, (42.86%), and M. oleifera (42.86%) good antibacterial properties. The other three species M. azedarach, (32.14%), H. helix, 35.71%) and S. flagellaris (35.71%), among selected medicinal plants indicated anti-bacterial potential. (Erdogrul, 2002) examined that some Indian medicinal plants via agar disc diffusion technique against bacteria S. marcescens, in which the Ethyl Acetate extract proved to be substantial inhibitor for the pathogenic bacteria, in our results the ethyl acetate extract exhibited the outstanding inhibition by V. jatamansi, (57.14%),and M. azedarach, (52.38%). While three of the selected plants A. lebbeck, (47.62%), M. oleifera (47.62%) and W. coagulans, 42.86%), extracts were high in the inhibition. The other three plants H. helix, 33.33%), S. flagellaris (38.10%), and, F. cretica, (28.57%) also provided inhibition zones in mild level, which are in accordance to (Sukanya et al., 2009). (Gnan and Demello, 1999) reported that aqueous extracts of selected plants contain Mild to moderate level of inhibition. In our findings we agree to the findings, as the aqueous extracts are best when use fresh. In our results, we found M. azedarach, (40-47.62%), very effective in inhibition of S. marcescens and MRSA bacterial strains. While all other seven plants, which are A. lebbeck, 19.23%), H. helix, 26.92%), S. flagellaris (19.23%), V. jatamansi, 23.08%), F. cretica, 34.62%), W. coagulans, 34.62%), and M. oleifera 15.38%), showed moderate inhibition of the selected bacterial strains similar findings are given by (Voravuthikunchai and Kitpipit, 2005) In conclusion, we say that the Cr. Methanolic and Ethyl acetate fraction as whole were more effective which is in agreement with (Ghaima et al., 2013). By large it can be easily concluded that, these difference in antimicrobial activity of the fraction is due the presence of different phyto- constituents in different extract. The main factor of presence or absence of a constituent is related to polarity of fractions. 123

4.3. Heavy Metals in Medicinal Plants The medicines, which are acquired from plants, are considered less toxic than allopathic drugs, However, if these plants contain heavy metals then in spite of biological activities, they are unfit to be used as medicines. Metal noxiousness has extraordinary influence and significance concerning plants and ultimately the entire ecosystem is affected with it. Apart from the accumulation, these toxic heavy metals effects metabolism and retard growth and development of the host plants, which then effect the biophysical characters of the plants and its consumers (Nagajyoti et al., 2010). There are plants, which are ―hyper accumulator‖ regarding their ability to adopt to metalliferous soils. The ―hyper accumulator‖ have elevated tendency for heavy metal uptake, faster translocation and ability to detoxify heavy metals (Rascio and Navari-Izzo, 2011). The damage by heavy metal to human beings in special and other living being in general is increasing day by day. Among the 92-elements, 30-elements are toxic to human health, when consumed at higher doses. The toxic elements, include Al, As, B, Be, Co, Cr, Cu, Li, Mn, Ni, Se, Ti, V, Sb, Te, Cs, Au, Hg, Pb, Sr, Mo, Pd, Bi, Ba, W, Pt, Sn, Ag and Cd. The metallic elements are regarded as heavy metals, when their atomic weights get higher than 40 amu (Morais et al., 2012, Yu and Tsunoda, 2004, Otte, 2006). Among the metals, arsenic (As), lead (Pb), mercury (Hg), and cadmium (Cd), are present in every environment, which are harmful to humans even at lower concentrations (Vieira et al., 2011) Lead is brought to the surface of earth by humans and is more toxic,in oceans the concentration of lead is about, 0.01-0.02 μg/L but near surface the ocean water contain as much as 0.3 μg/L (Castro-González and Méndez-Armenta, 2008). Plants having high lead concentrations may trigger food chain with toxic effects and ultimately influences human health (Carlin et al., 2015). Humans when inhale leads, 50% of that becomes the part of the body, 90% of which accumulates in bones and teeth (Yu and Tsunoda, 2004). The effect of lead on children growth is very severe and it has very bad effect on their mental health (Castro-González and Méndez-Armenta, 2008). Children are particularly sensitive to this metal because of their more rapid growth rate and metabolism, with critical effects on their developing nervous system (ATSDR, 2007; Castro-González & Méndez-Armenta, 2008). The Joint FAO/ World Health Organization Expert Committee on Food Additives (JECFA) 124

established a provisional tolerable weekly intake (PTWI) for lead, i.e 0.025 mg/kg body weight (JECFA, 2004). The WHO provisional guideline of 0.01 mg/L of led has been adopted as a standard for drinking water (Organization, 2004). Plants hold together toxic and non-toxic metals in wide-ranging concentration. It is an established fact that plants absorb heavy metals from soil and store it in their parts from environments. The concentration of, Pb, As, Cd, Hg, and Zn in selected medicinal plants are given in Table.3.18. Another heavy metal that is very common to human is cadmium. Human uses cadmium from earlier times and with the advancement in technology cadmium use have been increased over the years. However, in recent years cadmium is considered as serious pollutant. It is present naturally in water, air and soil. The era of industrialization led to increase in cadmium concentration in environment than ever (Fytianos et al., 2001). Tobacco contain highest amount of cadmium and its absorption via lungs is greater than gastrointestinal tract. It is present in the form of inorganic salts, and the organic compounds, which contain cadmium, are very unsteady. Plants easily absorb it in the form of ions, and these ions are present in all parts, such as roots, seeds and leaves. A researcher has found that the kernel of wheat and rice contain more cadmium than other parts (Figueroa et al., 2008). The safe limit of cadmium set by FAO/WHO is 0.007 mg/kg (JEFCA, 2004). The EPA maximum pollutant level for cadmium in drinking water is 0.005 mg/L, while the WHO accepted the conditional recommendation of 0.003 mg/L (Organization, 1980). According to our results, the selected medicinal plants were having safe concentration for cadmium. We agree with the findings of other researchers like Küpper and Leitenmaier, (2013), who also found that M. azedarach contain a bit high but safe amount of heavy metals and that that a medicinal plants should have least concentration of heavy metals (Küpper and Leitenmaier, 2013, Leitenmaier and Küpper, 2013). In our findings we found that M. azedarach contained highest concentration among all plants 2.39 ±0.18 µg/g followed by H. helix 0.26 ±0.02 µg/g S. flagellaris, 0.051 ±0.031 µg/g and A. lebbeck, 0.041 ±0.01 µg/g and. Fagonia cretica, Valeriana jatamansi, W. coagulans, and Moringa oleifera indicated no results. One of the most toxic heavy metals in environment is mercury, operate through farming industry (fungicides, seed stabilizers), drugs industry, paper industry, and batteries (Zhang and Wong, 2007) adds it into environment. Exposure to mercury can damage central nervous system and causes other health hazards like renal failure and different types of cancers in 125

humans. In our study, the highest mercury concentration was found in M. azedarach (2.39±0.18) µg/g, followed by H. helix (0.26±0.02 µg/g), S. flagellaris (0.051 ±0.031 µg/g) and A. lebbeck (0.041 ±0.01 µg/g). F. cretica, V. jatamansi, W. coagulans, and M. oleifera, have shown no heavy metal accumulation, which is in agreement with Beauford et al. (1977). Majority of food contain up to 50 μg/kg of heavy metals (Jaishankar et al., 2014). The revised JECFA standards for toxic level of mercury is 3.3 g/kg body weight/week and PTWI of 1.6 g/kg body weight (JECFA, 2004). As, mercury is extremely toxic to human health, the current standards for drinkable fresh water by EPA and WHO are 0.002 mg/L and 0.001 mg/L, respectively (JECFA, 2004). According to Koeppe (1977), the concentration of lead in plant is mostly localized to the arears where lead is emitted as industrial effluent, in our results we found that, S. flagellaris, M. oleifera, and F. cretica no lead were detected while in W. coagulans, had 0.044 ±0.026 µg/g, and V. jatamansi, 0.048 ±0.022 µg/g, were having minor concentration A. lebbeck, 0.204 ±0.11 µg/g. while H. helix contain moderate 1.25 ±0.07 µg/g and highest was found in M. azedarach 3.72 ±0.25 µg/g. Arsenic is rarely found in usual environment, but present as arsenides in the ore of sulphur. it is more abundant in oxidized states in the aquatic environment (Zhao et al., 2009). Also, arsenic may be abundant in the areas where arsenical insecticides are frequently used (Verbruggen et al., 2009). Inorganic arsenic is cancer causing and specially cause cancer in lungs and skin (Smith et al., 2003). The JECFA set the standards for arsenic consumptions as 0.05 mg/kg body weight/day and PTWI as 0.015 mg/kg body weight (JECFA, 2004). The results of the current study revealed variable arsenic concentration in the tested medicinal plants. The concentration of arsenic in S. flagellaris M. oleifera, F. cretica, W. coagulans, V. jatamansi and H. helix was 0.195±0.135 µg/g, 0.208 ±0.151 µg/g, 0.375 ±0.171 µg/g, 0.552±0.14 µg/g, 0.542 ±0.17 µg/g and 1.30±0.06 µg/g. The highest concentration of arsenic was found in M. azedarach 3.74±0.24 µg/g, whereas A. lebbeck did not have any traces of arsenic Zinc is vital element for fitness and growth specially plants, but in excess amount may cause severe health problems and toxicity. If the quantity of zinc exceeds from 225 µg/g then it shows toxicity. Zinc is an important co-factor for many nucleic acid synthesizing enzymes, but consumption in higher amounts may cause pyrexia, nausea and laziness The consumption of zinc should not exceed 10,000 and 20,000 µg/day (Bhutta et al., 1999). M. azedarach 126

1.76±0.15 µg/g was having highest Concentration of zinc followed by H. helix 0.49±0.08 µg/g F. cretica, 0.395±0.15 µg/g M. oleifera, 0.360±0.18 µg/g and, W. coagulans 0.287±0.16 µg/g,V. jatamansi 0.203±0.15 µg/g, A. lebbeck, 0.13±0.002 µg/g, the least concentration was found in S. flagellaris, 0.108±0.007 µg/g. Medicinal plants are sources of active ingredients for the treatment of degenerative diseases, ranging from common cold to toxic ailments, like cancer. Pakistan has rich medicinal plants flora, which are used as medication as well as food supplements. Besides, the presence of heavy metals in these medicinal plants make them unfit for human consumption. In the current tested plants, the level of studied heavy metals was very low in general and well under the set limits by JECFA and WHO, which make them the potential source of natural remedies.

4.5 Proximate Analysis of Selected Medicinal Plants Medicinal plants usage runs parallel with the creation of life on this planet Earth. Man knows plants uses for remedial purposes since human civilization. For the long period, plants were the sole and valuable resource of natural products for maintaining human health. These plants were used to treat severe diseases all over the world before the advent of modern drugs as well as nourishing Humans (Van Wyk and Wink, 2017). The world most primitive manuscript relating to medicinal plants dates back to 60,000 BC and was found at the oldest archaeological remains at Shanidar IV, an archeological site in now Iraq (Tomlinson and Akerele, 2015). This record shows that the medicinal plant are serving as curing agents from Neanderthal man 60,000 BC to the modern man (Wangchuk and Tobgay, 2015). The life is impossible without the basic nutrients, like proteins, carbohydrates, lipids, minerals and vitamins. All these nutrients are required for dietary needs, such as growth and developments (Underwood et al., 1994). Due to the over population, imbalance diet or irregular distribution of food resources, most of the countries around the world are facing malnutrition . Protein calories malnutrition is one of the most important and frequent deficiencies all over the world that need to be solved on prior basis (Shah and Khalil, 1988). According to an estimate, medicinal plants fulfill the healthcare/nutritional requirements of one third of the world population at the moment, the production and processing of these plants is encouraged all over the world, including developed countries. Similarly, in emerging nations, like Pakistan and India, most of the people need good and healthy food (Begum et al., 2015). In the present study, we determined the proximate composition (moisture, ash, fats, protein and 127

carbohydrate) of the selected medicinal plants species. The protein content was higher in H. helix with the mean value of 6.01± 0.13%, followed by S. flagellaris 4.37± 0.27%. The next with highest in percentage was M. oleifera 3.88± 0.09% as it is clear from W. coagulans 3.39± 0.16%, and V. jatamansi proteins percentage was 3.39± 0.25%, the next three species with least protein contents were M. azedarach 3.36± 0.09%, A. lebbeck 2.85± 0.19%, and F. cretica 2.31± 0.04%, respectively. According to Kochhar et al. (2006) the plants having less carbohydrates and more fiber are best for diabetics. In this study, we have found that there are plants like W. coagulans, H. helix which may have potential to cure the disease. Percentage of ash was determined by with help of furnace by taking temperature up to 600 Co (fig. 44). After analyzing the results data we found that the highest value of percentage of ash was found in W. coagulans seeds it showed the value of 27.43± 0.15%, followed by H. helix 25.08± 0.21%, and M. azedarach with percentage of 23.34± 0.1%. Hussain et al., (2009) also selected eight medicinal plants from different areas and examined their proximate value but in our results, we found more ash percentages of Ash. Among the selected plants. A. lebbeck 23± 0.07%, and V. jatamansi 17.67± 0.22%. The three minimum percentages of eight (08) selected medicinal plants were F. cretica 15.01± 0.08, S. flagellaris 9.92± 0.13, and M. oleifera 8.01± 0.06 respectively. Plants are triumphed for centuries to feed humans and treat different diseases and still they reasonably guarantee their great medicinal value, efficiency and safety and have nutritional values. (Rahman et al., 2018). The moisture percentage shown in (fig. 45). The highest value of moisture was confirmed in A. lebbeck it showed the value of 16.36± 0.43, followed by S. flagellaris 15.5± 0.54 and M. azedarach with percentage of 12.41± 0.56. F. cretica 12.66± 0.11, and H. helix moisture percentage was 12.3± 0.43 and V. jatamansi 10.27± 0.09 while W. coagulans 10.27± 0.26. M. oleifera 9.17± 0.14, were with least moisture values respectively. Herbal medicines have usually been used in the form of fruit or vegetable, flowers or leaves, root or stem, plants parts or their essences for the cure of infections and maintaining health (Hamayun et al., 2006b). The carbohydrates contents in our selected medicinal plants were as follows. The maximum Carbohydrates contents were found in M. oleifera with the value of 72.63± 0.06, followed by V. jatamansi 64.6± 0.71 and F. cretica 64.32± 0.63, as it is clear from (fig. 47). S. flagellaris 62.79± 0.54, and M. azedarach Carbohydrates percentage was 56.69± 0.34, the next three species with least Carbohydrates contents were Withania coagulans 52.62± 0.34, H. helix 49.31± 0.72, and A. lebbeck 45.79± 128

0.42, respectively. It is very clear that good health requires good food and man will continuously search sources, which will lead to good food and health. In our quest, we found that among our selected medicinal plants, the fats contents was highest in A. lebbeck with the value of 12± 0.23,followed by S. flagellaris 7.42± 0.11, and H. helix 7.3± 0.08, as it is clear from (fig. 48). M. oleifera 6.31± 0.05,, and W. coagulans fat percentage was 6.29± 0.18, the next three species with least fat contents were F. cretica 5.7± 0.13, M. azedarach 4.2± 0.17,, and V. jatamansi 4.07± 0.24, respectively. Almighty Allah has endowed Pakistan with the natural wealth of flora including chirruping mosses, thriving fields, lush green woodlands, delightful, striking and scented flowers, pleasant fruits and the natural economy of medicinal plants with matchless biodiversity. It is a God gifted country with over six thousand species of higher plants. There are thirteen plant hotspots from alpine prairies to mangrove forest (Hamayun et al., 2006a, Shinwari, 2010, Baquar, 1989). From Conclusion, It is settled after the current research work that these anticancer therapeutic plants are foundations of remedy for some very significant ailments as well as have potential for the looking after of healthy life and normal body functioning by given that vitality of full nutrients for healthy life.

4.6 Phytotoxicity of Medicinal Plants Allelopathy is a biological term in which one organism produce such biochemical that effect the growth, existence, improvement, and reproduction of other individual and these biochemical are known as allelochemicals. Plant allelopathy is the types of relations concerning receptor (the plant which is effected) and donor plants (which effect) and may exert either positive effects (weed control) negative effects (e.g., , soil sickness, biological invasion or auto toxicity) (Cheng and Cheng, 2015). By the use of 10 mg quantity of powder of selected medicinal plants in our experiment, seeds of Lactuca sativa showed different responses. L. sativa seedlings were very susceptible to S. flagellaris with radicle length of 0.49 cm, followed by V. jatamansi and Albezia lebbeck powder with radical length of 0.54 cm and 0.57 cm. The effect F. cretica showed with radicle length of 0.69 cm and H. helix , M. oleifera, W. coagulans, and M. azedarach restricted the length of radicle of L.sativa up to 1.01 cm, 2.91 cm, 3.04 cm, and 3.92 cm respectively. Which confirms that S. flagellaris is the most efficient at 10 mg concentration against radicle length. Germination of seeds and plant development is altered by allelopathy and therefore germination is important way for the learning of allelopathy. Allelopathy has a significant part in agriculture and disturbs the 129

progress and magnitude of the crops by the exchange of chemicals among crops, weeds and trees. Allelochemicals secreted by unknown plants significantly affect the intrinsic plants regardless of native species secreted allelochemicals or not (Sertkaya et al., 2010, Hussain et al., 2010). The results of 10 mg quantity of powder of selected medicinal plants against the Plumule growth L. sativa showed different responses regarding length of Plumule. S. flagellaris restricted Plumule length 0.52 cm. Followed by V. jatamansi and Albezia lebbeck powder with plumule length of 0.50 cm and 0.55 cm. The effect F. cretica came up with plumule length of 1.14 cm. While H. helix , M. oleifera, W. coagulans, and M. azedarach restricted the length of plumule of L.sativa up to 1.50 cm, 2.91 cm, 2.64 cm, and 3.91 cm respectively. It is clear from the results that two plants i.e. H. helix and S. flagellaris is showing more phytotoxicity. Allelochemicals can alter the contents of plant growth hormones or make inequalities in many phyto-hormones, which hinders plant growth and development, for instance, with respect to germination of seed and sapling growth. Allelochemicals of phenolic nature can motivate IAA oxidase activity and obstruct the reaction of POD with IAA, unavoidable GA or IAA to effect endogenous hormone stages (Rauf et al., 2012, Cheng and Cheng, 2015). In the category of 20 mg, quantity of powder of selected plants on the growth of L.sativa showed more quantitative effects. L. sativa seedlings were very susceptible to S. flagellaris with radicle length of 0.32 cm, followed by V. jatamansi and Albezia lebbeck powder with radical length of 0.47 cm and 0.49 cm. The effect F. cretica showed with radicle length of 1.13 cm. While H. helix , M. oleifera, W. coagulans and M. azedarach restricted the length of radicle of L. sativa up to 1.51 cm, 2.93 cm, 3.64 cm and 1.99 cm respectively. In which is very clear that the S. flagellaris has inhibitory effects while W. coagulans has stimulatory effects. The inhibitory and stimulatory responses of the plumule to 20 mg quantities were as follows. S. flagellaris restricted Plumule length 0.31 cm, followed by V. jatamansi and Albezia lebbeck powder with plumule length of 0.44 cm and 0.48 cm, the effect F. cretica came up with plumule length of 1.05 cm. While, H. helix , M. oleifera, W. coagulans, and M. azedarach restricted the length of plumule of L.sativa up to 0 .50 cm, 2.44 cm, 2.65 cm and 2.49 cm respectively. After evaluation the allelopathic activities of 239 medicinal species using the sandwich method and 223 species of them were found to inhibit the seeds germination, while 17 species were found to stimulate lettuce radicle growth. The allelopathic influence after leaf 130

litter leachates on L.sativa seed germination and found inhibitory activity carried out by the sandwich method (Fujii et al., 2003). After screening 81 Pakistani medicinal plants and found that plants with allelopathic potentials also have stimulatory effects side by side with inhibition (Gilani et al., 2000). Considering the effects of 40 mg quantity of powder of selected plants on seeds of Lactuca sativa showed more quantifiable properties. L. sativa seedlings were very susceptible to S. flagellaris with radicle length of 0.32 cm. Followed by V. jatamansi and Albezia lebbeck powder with radical length of 0.47 cm and 0.49 cm. The effect F. cretica showed with radicle length of 1.13 cm. While H. helix , M. oleifera, W. coagulans, and M. azedarach restricted the length of radicle of L.sativa up to, 1.51 cm, 2.93 cm, 3.64 cm and 1.99 cm respectively. Four different species belonging to Anthemis, Inulaand Lactuca genera were collected from Hindukush ranges of Pakistan. These plants were studied with the help of two different methods Sandwich and Homogenated Sandwich. It was determined by application of different concentration leaf leachates has very much substantial value (Khan et al., 2009). The results of 40 mg quantity of powder of plants, L. sativa showed different responses regarding length of Plumule. S. flagellaris restricted Plumule length 0.52 cm. Followed by V. jatamansi and Albezia lebbeck powder with plumule length of 0.41 cm and 0.55 cm. The effect F. cretica came up with plumule length of 1.14 cm. While H. helix , M. oleifera, W. coagulans, and M. azedarach restricted the length of plumule of L.sativa up to 0.55 cm, 1.84 cm, 1.11 cm, and 1.89 cm respectively. We agree to the above- cited researchers from results, and affirm that our selected plants have very substantial value regarding phytotoxicity.

4.7 Antioxidant Activity Free radicals also known as oxidants, are chemicals with free or unpaired electrons. These are very common in nature, play very important roles in nature both harmful and beneficial. In medicines these are very important. There are two very important Reactive species i.e. reactive Oxygen species (ROS) and reactive Nitrogen species (RNS). It is proved that ROS/RNS induce and maintain oncogenic stimulation. The production of free radicals may be due to exogenous or endogenous sources. These free radicals may damage DNA of mitochondria and Nucleus as well as damage lipids and proteins by producing oxidative stress (Valko et al., 2006). 131

The body produce free radicles through food breakdown or can be acquired from polluted environment e.g. cigarette smoke etc. The processes that produce free radicals in body are Fenton-Haber-Weiss reaction Due to cellular metabolism or confronting xenobiotic or radiation may produce free radicals. These reactive oxygen species (ROS) or free radical is equivalent to 5% of the total oxygen taken daily (Pryor, 1982, Cowan et al., 1986, Yendala et al., 2017). Human body antioxidant system can control the level of oxidants to a certain level, if it exceed that level, then oxidation reaction starts which is harmful for the body. Excessive oxidants start Cell damage by inducing cellular oxidative stress, which triggers several diseases including cancer (Poprac et al., 2017). It is believed that cancer and other chronic disease are contributed by the accumulation of free radicals, which bind to Cell membrane, proteins and Nucleic acids, promoting cell damage and lipid oxidation (Höhn et al., 2017, Poprac et al., 2017, Kagan, 2018)

The substances, which provide protection against free radicles and oxidative damage, are known as antioxidants. Antioxidants are scavengers of free radicles being an important part of our diets; these prevent oxidation and repair cell damage. These are natural scavengers of free radicals and protect our body from oxidative harm against free radicals, biological antioxidants are Like α-tocopherol, ascorbic corrosive, carotenoids, and retinoids are extensively studied against natural cancer prevention (Rehman et al., 2018, Khan et al., 2018, Ravi et al., 2018). Techniques like chemical and cell cultures, have shown that antioxidant slow down and even neutralize cancer, but it is still to be investigated further (Aghajanpour et al., 2017) It is believed that cancer metastasis and invasion depends on angiogenesis. Angiogenesis can also be prevented by applying plant natural products as current therapeutic have more problems associated with it. Several anticancer compounds of plant origin have been identified (Bahmani et al., 2017). In our study we used DPPH (1, 1-diphenyl-2- picrylhydrazyl) assay. DPPH is used extensively to know the antioxidant potential of plant extracts. These plant extract donate protons to free radical like DPPH and thus scavenge it. In our results we used four (04) fractions i.e. Ethyl acetate, chloroform, Methanol extract and n- haxane extract. Aqueous extract were avoided because studies shows that DPPH scavenging is 132

not good with Hydrophilic extracts. The method used in DPPH assay was by (Brand-Williams et al., 1995). Ascorbic acid was used as standard. Inhibition values and IC50 were calculated. Overall results were concentration dependent. In our results, we found all species were active in scavenging DPPH but mainly dependent upon the concentrations of fractions. In our antioxidant activity the results are shown in table3.6. In Ethyl acetate extract the best among all was S. flagellaris 8.52±0.121, which is in accordance to the results of (Chevalley et al., 2000) who isolated some new compounds from Saxifraga cuneifolia. Next high scavenging properties were present in followed by V. jatamansi 13.12±0.153, A. lebbeck 14.7±0.572, H. helix 16.14±0.259, W. coagulans 16.49±0.350 and M. azedarach 17.78±0.251, while F. cretica 20.79±0.112 and M. oleifera 21.55±0.158 showed moderate antioxidant acivity with relative high concentrations. The chloroform extracts scavenged DPPH in the following ascending order S. flagellaris 19.81±0.311, V. jatamansi 20.12±0.137, H. helix 20.14±0.261, F. cretica 24.79±0.232, A. lebbeck 25.6±0.756, M. azedarach 28.88±0.246, W. coagulans 30.49±0.403 and M. oleifera 34.55±0.158. it is clear from the results that chloform and n- hexane extract are less effective than Ethyl acetate and Cr. Methanol extract (Sohn et al., 2008). Cr. Methanol extract was the next effective fraction and it showed the second lowest

IC50 value in (μg/ml). The values of plants were S. flagellaris 9.32±0.102, V. jatamansi 15.12±0.144, M. azedarach 18.40±0.157, H. helix 19.61±0.978, W. coagulans 18.82±0.092, F. cretica 21.65±0.348, A. lebbeck 25.93±0.075 and M. oleifera 26.65±0.149. Similar results were given by (Ganesan et al., 2008) who reported that Methanol extracts are very effective. The n-Haxan Extract were least effective, in comparison with ethyl acetate. The values of the extracts were S. flagellaris 17.11±0.321, A. lebbeck 20.79±0.112, M. oleifera 24.43±0.339 F. cretica 25.57±0.156, H. helix 25.45±0.152, V. jatamansi 29.13±0.155, M. azedarach 30.49±0.403 and W. coagulans 44.12±0.184 our findings about selected medicinal plants n- hexane fractions is in accordance with (Senevirathne et al., 2006)

4.8 Phytochemical Analysis of Selected Medicinal Plants Nature offer limitless possibilities, there are 12,000 secondary metabolites isolated from plants till now, it is estimated that these are only 10% and a lot more to be discovered. Secondary metabolites are produced for plant protection against insect and micro-organisms. Different properties of plants are shaped by these metabolites like; flavor given by capsanin, odor by terpenoids and pigmentation by tannins and quinines. Most important is the medicinal 133

properties are due to secondary metabolites (Peteros and Uy, 2010, Wallace, 2004). Even elements can also be important phytochemicals, relative to complex organic molecules e.g. selenium is an important component of many vegetables and fruits, involved in many metabolic pathways (Brown and Arthur, 2001). In particular it is a cofactor for enzymes involved in the production of glutathione, a very important antioxidants (Papp et al., 2007). Phytochemicals are plant chemicals; contain a heterogeneous set of constituents. These phytochemical compounds are also recognized as secondary plant metabolites and have biological properties such as antimicrobial effect, antioxidant activity, platelet aggregation reduction, enzymes modulation and detoxification, modulation of hormone metabolism, immune system stimulation, and anticancer property (Sen et al., 2010). Phytochemicals are not crucial nutrients as these are not required by human for nourishing life, but have key characteristics to stop or to combat most of common diseases. The results of phytochemical analysis (Table.3.21) showed that all medicinal plants possess the considered phyto-constituents to varying degree. According to (Hansen et al., 1995, Willaman and Schubert, 1961). Alkaloids are more common in dicots than monocots. Alkaloid rich plants are present in families all over plant kingdom but some family are very rich in secondary metabolites, we agree with these scientist and found the same pattern in our results A. lebbeck showed the presence of Alkaloids and Phenols to moderate level and Glycosides to high level, while Saponins and Flavonoids to low level. M. azedarach contained high level of phenols, glycosides and Alkaloids to moderate amounts, while the levels of Saponins and Flavonoids were found low. H. helix was found low in Glycosides and high in Saponins. The presence of Alkaloids, Flavonoids and Phenols were moderate. According to (Liu et al., 2008) genus Saxifraga contain very diverse nature of phytochemicals, we agree with that from our results as S. flagellaris exhibited high content of Saponins and Flavonoids, but the other three Phyto-chemicals i.e. Phenols, Glycosides and Alkaloids were found in moderate Concentrations. V. jatamansi contained low quantity of Saponins and Flavonoids. Glycosides were recorded in high points while Alkaloids and Phenols in moderate Levels. Phytochemical are bioactive compounds produced by medicinal plant in some quantities, are used as originators to prepare modern drugs (Surh, 2003). F. cretica displayed high content of Saponins, and phenols in Moderate points but the other three Phyto-chemicals i.e. Flavonoids, Glycosides and Alkaloids were found in low Concentrations. In W. coagulans the 134

concentrations of Glycosides, and Flavonoids were high, phenols, and Alkaloids were moderate while Saponins were low. During the tests Saponins were found high, Glycosides in low levels while Phenols, Flavonoids and Alkaloids were found in moderate concentration in M. oleifera. Acording (Kumar and Pandey, 2013) Flavonoids possess three-ring structure. These contain to phenolic group benzo-γ-pyrone, while the oligomers of these flavonoids are Proanthocyanidins. These universally exist in plants. From our results we conclude that our plants contain good potential against different diseases, because these have different phytochemicals from moderate to high level.

4.9 Anticancer Activity When penicillin was discovered, it opened a new way to different drug discovery. The medicinal plants are sources and The perception of these medicinal plants as a source of remedies and important instrument for modern drugs, as these are the springs of maximum novel combinations and compounds related to modern drug industries (Ginsberg, 2005). It is well known now that 80% of beneficial dug compounds and pharmaceutical product are derived from medicinal plants (Diallo et al., 1999). Cancer is life-threatening disease and in some stages even certain death that ruins the complete family economy and structure. The disease is present all over world and increasing day by day in Pakistan in particular. There are 26 types of cancers known according to the GLOBOCAN project of IARC. In twenty large from all over the world new cases were almost 10.9 million and deaths caused by cancer were 6.7 million, people those who were suffering from cancer with three years from diagnosis their number were 24.6 million. The cancers which caused most of the deaths are, half a million (1.15 million) from breast cancer, one million (1 million) from colorectal cancer lung. While the cancer which deaths toll were higher are; liver cancer (598,000 deaths), more than a million (1.18 million deaths) from lung cancer and the notorious stomach cancer killed 0.7 million people (700,000 deaths). The breast cancer caused highest deaths of all was 4.4 million. Though there are variations in threats of dissimilar cancers by geographical zones. The key factors are lifestyle and environment (Parkin et al., 2005). Cancer is prevailing day by day and cause of ill health and death worldwide, according to an estimation by GLOGOCAN in 2012 there are 14 million fresh victims were registered with 8 million expiries, it is estimated that by the end of 2030 there will be 70% increase. Although there is no direct data input in 135

most of the developing countries. According to (WHO) 34 of 194 countries of UN reported national mortality data of high quality,while68 other countries provided data of high-quality incidence for the latest work of Cancer incidence in five continents. GLOBOCAN, a project of the(IARC) provides estimates by cancer site using the best available data in each country (Antoni et al., 2016). According to GLOBOCAN the common types of cancers found in Pakistan are of bladder, breast, cervix uteri, colon rectum, lip and oral cavity. There are 27 types of cancers responsible for deaths in the world. In Pakistani population both genders contain high ratio of different cancers; of the total 148,041 cancer cases includes; breast (n = 34038, 23%), colorectal (n = 5335, 3.6%), lip and oral cavity cancer killed near thirteen thousand (n = 12761, 8.6%), people while lung cancer death toll were near seven thousand (n = 6800, 4.6%) and the cancer of the WBC also known as NHL or non-hodgkin lymphoma killed near six thousand (n = 5964, 4%). The death toll of cancer in Pakistani population is 101,113. (Sarwar and Saqib, 2017, Begum, 2018, Maucort‐ Boulch et al., 2018). The quest for plant extract and therapeutics is not new to man; due the experience based on many millennia such constituents are now known that form important remedies as well as have developed basic and modern pharmaceutics. Names such as Belladonna, Cinchona Colchicum,, Digitalis and opium etc. are very recent. Ancient remedies are the basis of discovery of natural substances having major biological and therapeutic interest. These also include new anticancer drugs. The NCI screened approximately thirty five thousand plants extracts and tested it for anticancer activity in 1960. Which resulted in approximately 2619 compounds isolation (Sarkar et al., 1996a). The present study was undertaken to find out the anticancer activity of selected medicinal plants using MTT bioassay against available cancer cell line (Colon cancer cells--- HT-29). The result of 25 μM fractionations of selected medicinal plants showed different anticancer activities, three (03) fractions were tested for their anticancer potential against cancer cell line using MTT bioassay against available cancer cell line (Colon cancer cells--- HT-29. We used six (25) μM dose concentration of each fraction, found that all the some fractions showed good anticancer activities. Using n- hexane fraction the S. flagellaris, showed the most encouraging result by restricting cell growth to 10.25% our result is supported by (ARISAWA et al., 1992) who isolated novel anticancer compounds from Chrysosplenium grayanum (Saxifragaceae). Next moderate to low activities were shown by F. cretica 69.05%, M. oleifera 77.37%, V. jatamansi 78.72%, W. coagulans 136

84.53%, H. helix 89.94% Albezia lebbeck 111.41% and M. azedarach 114.89%. While with an exception of F. cretica the same trends were followed by chloroform fractions of selected medicinal plants, which are S. flagellaris showed the most encouraging result by restricting cell growth to 10.20% followed by M. oleifera 68.82 %, V. jatamansi 71.18%, F. cretica 88.43%, W. coagulans 103.33%, H. helix 95.29%, Albezia lebbeck 91.57%, and M. azedarach 100.98%. The ethyl acetate fractions of selected medicinal plants showed the following effects S. flagellaris showed the most promising result by restricting cell growth to 10.22% followed by M. oleifera 73.13%, V. jatamansi 74.98%, F. cretica 78.68 %W. coagulans 93.87% H. helix 92.60% Albezia lebbeck 101.56% and M. azedarach 107.98% Cell viability. The result of 50 μM fractionations of selected medicinal plants showed different anticancer activities, three (03) fractions were tested for their anticancer potential against cancer cell line using MTT bioassay against available cancer cell line (Colon cancer cells--- HT-29. We used fifty-(50) μM dose concentration of each fraction, found that all the some fractions showed good anticancer activities. Using n- hexane fraction the S. flagellaris showed the most encouraging result by restricting cell growth to 10.25%, followed by V. jatamansi 52.80 %, M. oleifera 71.57 %, F. cretica 79.88 %, H. helix 87.23%, M. azedarach 92.84 %, W. coagulans 94.39 % and A. lebbeck 103.68%. The chloroform fractions of selected medicinal plants showed the following cell vitality percentage. The S. flagellaris indicated the most promising result by restricting cell growth to 9.80%, followed by V. jatamansi 66.08%, M. oleifera 71.18%, F. cretica 86.08%, W. coagulans 86.67%, H. helix 88.82%, A. lebbeck 95.29% and M. azedarach 123.73%. While the ethyl acetate fractions of selected medicinal plants showed the following cell viability percentage. The S. flagellaris indicated the most promising result by restricting cell growth to 10.03%, followed by V. jatamansi 59.40%, M. oleifera 71.37%, F. cretica 82.96%, W. coagulans 88.02%, H. helix 108.18%, A. lebbeck 90.56% and M. azedarach 99.51%. Plant polyphenols are very effective chemo-preventive and anticancer substances. Flavones has anticancer and antioxidant activities. Seventeen derivatives of flavones were tested out using MTT assay, against human cancer lines: brain epithelial (U87), breast (MCF-7), lung (A549), thirteen tri hydroxyl flavones influenced anticancer activity against at least one tested cancer cell line (Grigalius and Petrikaite, 2017). Polyphenolic compounds like anthocyanidins, flavonoids, phenolic acids, and tannins, are 137

secondary metabolites produced by plants. These compounds possess notable antioxidants and anticancer properties (Prasad et al., 2009, Cai et al., 2004). Ethno-medicinal plants have inclusive and marvelous involvement in the discovery of new medications against diverse ailments as well as cancer. Recent surveys with plant products have recovered fame for their natural implication and utility of their fundamental variety. So, citations of usually active anticancer plants and assessment of their essential mechanisms could be supportive in searching of novel anticancer drugs(Fritz and Kaina, 2006). The US National Cancer institute (NCI) introduced the quest for novelty of anticancer substitutes from plants in 1957. Since then 35,000 plant species had probed and contain more or less anticancer drugs, such as Camptothecin, Etoposide analogs, , Indicine–N-oxide, Taxol, Vinblastine, and Vincristine and their derivatives (Suffness and Douros, 1982). Cancer is surrounded by many causes of infections and loss commonly. Plants-derived compounds like; alkaloids, Taxol, and Vinca, upsets microtubule gathering (Ren et al., 2006, Clarke et al., 2006). The result of 100 μM fractionations of selected medicinal plants showed different anticancer activities, three (03) fractions were tested for their anticancer potential against cancer cell line using MTT bioassay against available cancer cell line (Colon cancer cells--- HT-29. We used hundred-(100) μM dose concentration of each fraction, found that all the some fractions showed good anticancer activities. Using n- hexane fraction the S. flagellaris showed the most encouraging result by restricting cell growth to 11.03%, our result is favored by (Uniyal and Tewari, 1991) who also reported another species Bergenia ligulata (Wall). Engl. (Saxifragaceae) for anticancer activity, pointing to the fact that the family has a lot of anticancer potential. Other plants showed moderate anticancer activities which are, H. helix 68.28%, V. jatamansi 69.44%, M. oleifera 74.08%, F. cretica 88.59%, M. azedarach 94.58%, A. lebbeck 102.90% and W. coagulans 104.64%. The chloroform fractions of selected medicinal plants showed the following cell vitality percentage. The S. flagellaris indicated the most promising result by restricting cell growth to 10.20%, followed by V. jatamansi 67.25%, F. cretica 73.73%, H. helix 80.59%, M. oleifera 82.16%, W. coagulans 84.31% A. lebbeck 97.25% and M. azedarach 100.39%. While the ethyl acetate fractions of selected medicinal plants showed the following cell viability percentage. The S. flagellaris indicated the most promising result by restricting cell growth to 10.61%, which is supported by (Balunas and Kinghorn, 2005), that plants are important and untiring source of drug 138

discovery. The second significant anticancer activity was shown by V. jatamansi 68.35%, followed by H. helix 74.39%, M. oleifera 78.09%, F. cretica 81.21%, W. coagulans 94.55%., M. azedarach 97.47% and A. lebbeck 100.10% . Our findings are in agreement with (Chen et al., 2008) who reported that Saxifraga stolonifera, which is another species of the genus contain strong anticancer activity. So in present context we expect to find our selected plant S. flagellaris as the novel source of anticancer compounds.

4.10 Isolation of Compound In human body, cells maintain homeostasis by using an active dedicated molecular program of cell death (apoptosis). Apoptosis or programmed cell death is as important as cell division and cell migration. It controls the cells number and tissue size and protects the organism from rogue cells that threaten homeostasis. Apoptosis is a complicated process. It is controlled by a number of proteins. Most of the morphological changes are caused by caspases. Caspases are a set of cysteine proteases that are activated in apoptotic cells. Under normal conditions, caspases are inactive proteins called procaspases. Once triggered by some stimuli, they get activated, inducing cell death pathways, which eventually lead to cell death (Hengartner, 2000). Various means of detecting apoptotic cells are available. These include flow cytometry, Immunohistochemistry, Annexin-V assay, M30 neoantigen assay, In situ3 -end labelling method, TUNEL assay and many more (Archana et al., 2013). Cells undergoing apoptosis exhibit several morphological modifications such as protein cross-linking, protein cleavage, DNA breakdown, and phagocytic recognition that together result in the distinctive structural pathology (Hengartner, 2000). Many anticancer drugs induce apoptosis in cancer cells by multiple pathways and inhibit the growth and metastasis of certain cancers. In the current study we analyzed the apoptosis inducing effect of Adlumidine against human colon and prostate cancer cell lines. As depicted from figure 59, Adlumidine specifically induced apoptosis in the said cell lines, which was detected by flow cytometer through Annexin-V- FITC assay. Adlumidine treatment significantly increased the levels of early and late apoptosis in HCT 116 and PC3 cells (Fig 59). As discussed earlier about the role of ROS generation in killing the cancer cells, we detected a significant increase in ROS levels through DCFHDA staining in Adlumidine treated cells compared to untreated cells (Fig 60). Further, we analyzed the activation of caspase 3 upon Adlumidine treatment. Once caspase 3 is 139

activated, there seems to be an irreversible commitment towards cell death (Elmore, 2007). We found that Adlumidine caused a significant increase of 10.50 fold in HCT 116 cells, while 7.63 fold increase in PC3 cell lines (Fig 62). In order to analyze the impact of caspase 3 activation on its substrates like PARP, we conducted western immunoblotting by using total cell lysates. In parental cells, caspase 3 cleavage was minimal, and in line with this, PARP cleavage was also negligible. Adlumidine treatment increased the expression of cleaved caspase 3 in both HCT 116 and PC3 cell lines, which was evident from the PARP cleavage. These findings clearly show that Adlumidine induce apoptosis in human colon and prostate cancer. Although we confirmed the induction of apoptosis by Adlumidine in HCT 116 and PC3 cells, still many things are unclear. The way of entry of the drug into the cell is the most important factor. Regarding Adlumidine, we have no idea whether these compounds interact with some cell-surface receptors or directly diffuse into the cell membrane. We are at very early stage about the mechanism of action of Adlumidine. We lack the knowledge about the cross-linking of various other pathways with the general apoptotic pathway triggered by Adlumidine. What is the fate of Adlumidine, we do not know yet. All these questions can be answered after further investigation. Currently we are sure that these compounds possess potent anticancer activities, and we hope that further investigation upon these compounds may insight into new cell signaling pathways and provide a therapeutic basis to inhibit growth and metastasis in human colon and prostate cancer.

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CONCLUSION

From the present study it is very easy to conclude that medicinal plants are not only important source of valuable chemicals that can cure cancer, but also contain other constituents, which are beneficial in treating different ailments. The present study might also help in the quest of anticancer drugs and drugs for other diseases, with minimum side effects. We selected eight medicinal plants and tested their biological activities. of these biological activities our selected plants showed remarkable antibacterial and antifungal properties. Which may open a gate to discover new broad-spectrum antibiotics. Our selected medicinal plants were first time thoroughly persuaded for anticancer drugs. Starting from heavy metal analysis of these plants was to ensure their safe use for human beings. Some of our selected medicinal plants were put to cancer cell line for the first time. The most important thing about is about S. flagellaris plant, this unique plant was not evaluated for anticancer properties, till now by any researcher except the supervisor and team as we know to the best of our knowledge. Built on the several bioassays and particularly on anticancer activity, S. flagellaris was carefully chosen for compound isolation, and compound adlumindine was isolated for the first time from DCM fraction of the plant. Which is very effective anticancer compound. In the last but not the least we suggest that our selected medicinal plants are significant sources of treatment for different human ailments.

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FUTURE PROSPECTS

i. The extracts of these selected medicinal plants should checked against other cancer cell line, which will help to develop a broad-spectrum drug. ii. These plant extracts can be tested against other viral diseases like herpes Flue etc. iii. Further studies are needed to isolate broad spectrum antibiotics from tested plants. iv. It is more likely that the active fractions of the selected plants contain more compound, so there is a need of further studies. v. For the isolated compound mechanism of action should be studied. vi. The isolated compounds should be checked against other pathogens. vii. Nature is full of valuable medicinal plants which should be evaluated for anticancer and Biological activities.

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