PHYTOCHEMICAL AND PHARMACOLOGICAL PROFILING OF Dysphania botrys L.
BY
MUHAMMAD NAEEM KHAN
A dissertation submitted to The University of Agriculture Peshawar, in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY AND GENETIC ENGINEERING
INSTITUTE OF BIOTECHNOLOGY & GENETIC ENGINEERING FACULTY OF CROP PRODUCTION SCIENCES THE UNIVERSITY OF AGRICULTURE PESHAWAR, PAKISTAN AUGUST, 2018 PHYTOCHEMICAL AND PHARMACOLOGICAL PROFILING OF Dysphania botrys L.
BY
MUHAMMAD NAEEM KHAN
A dissertation submitted to The University of Agriculture Peshawar, in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY AND GENETIC ENGINEERING
APPROVED BY:
Chairman Supervisory Committee Dr. Asad Jan Associate Professor
Co-Supervisor for Research Dr. Inamullah Khan Assistant Professor Pharmacy (UOP)
Member Major Field Dr. Safdar Hussain Shah
Member Minor Field Prof. Dr. Farhatullah
Chairman and Convener Board of Studies Prof. Dr. Iqbal Munir
Dean Faculty of Crop Production Sciences Prof. Dr. Zahir Shah
Director Advanced Studies and Research Dr. Shahid Sattar
INSTITUTE OF BIOTECHNOLOGY & GENETIC ENGINEERING FACULTY OF CROP PRODUCTION SCIENCES THE UNIVERSITY OF AGRICULTURE PESHAWAR, PAKISTAN AUGUST, 2018 TABLE OF CONTENTS
S. No. Title Page No.
List of Tables ...... i List of Figures ...... iii Abbreviations ...... iv Acknowledgments ...... vii Abstract ...... ix I INTRODUCTION ...... 1
II. REVIEW OF LITERATURE ...... 13
III. MATERIALS AND METHODS ...... 32
3.1 Plant collection and identification ...... 32 3.2 Extraction of plant material ...... 32 3.3 Fractionation procedure ...... 33 3.4 Phytochemical investigation ...... 35 3.4.1 Quantitative analysis of phytochmeicals ...... 35 3.4.1.1 Stock solution ...... 35 3.4.1.2 Test for crude alkaloids...... 35 3.4.1.3 Test for saponins ...... 35 3.4.1.4 Test for phenols ...... 35 3.4.1.5 Test for flavonoids ...... 35 3.4.1.6 Test for tannins ...... 36 3.4.1.7 Test for sterols ...... 36 3.4.2 Qualitative analysis of phytochemicals ...... 36 3.4.2.1 Determination of total phenol ...... 36 3.4.2.2 Determination of total saponins ...... 36 3.4.2.3 Determination of total flavonoids ...... 37 3.4.2.4 Determination of total alkaloids ...... 37 3.4.3 Proximate composition ...... 37 3.4.3.1 Moisture content ...... 37 3.4.3.2 Inorganic matter ...... 38 3.4.3.3 Crude lipid ...... 38 3.4.3.4 Dietary fiber ...... 38 3.4.3.5 Crude protein ...... 39 3.4.3.6 Nitrogen˗free extract ...... 39 3.4.4 Minerals analysis ...... 39 3.5 In-vitro studies ...... 41 3.5.1 Antimicrobial activity ...... 41 3.5.2 Strains and culture media ...... 41 3.5.3 Antibacterial activity...... 42 3.5.4 Antifungal activity ...... 42 3.5.5 Phytotoxic activity ...... 42 3.5.6 Antioxidant assay ...... 43 3.5.6.1 1, 1-diphenyl-2-picrylhidrazyl (DPPH)) radical scavenging activity ...... 43 3.5.6.2 ABTS (2, 2˗azinobis˗3-ethylbenzothiozoline-6-sulfonic acid) radical scavenging assay ...... 43 3.5.7 Lipoxygenase-inhibitory assay (LOX) ...... 44 3.6 In-vivo studies ...... 44 3.6.1 Acute toxicity study ...... 44 3.6.2 Anti-inflammatory effect ...... 45 3.6.2.1 Carrageenan induced paw edema model ...... 45 3.6.2.2 Xylene˗induced ear edema...... 45 3.6.3 Analgesic effect ...... 46 3.6.3.1 Formalin test ...... 46 3.6.3.2 Hot plate test ...... 46 3.6.4 Antipyretic effect ...... 47 3.6.5 Anti-diarrheal effect ...... 47 3.6.6 Anti-diabetic effect ...... 47 3.6.7 Hepatoprotective effect ...... 48
3.6.7.1 Carbon tetra chloride (CCl4) induced hepatotoxicity Model ...... 48 3.6.7.2 Biochemical investigations ...... 48 3.6.8 Sedative/hypnotic effect ...... 49 3.6.9 Anticonvulsant effect ...... 49 3.6.10 Antidepressant effect ...... 49 3.7 Statistical analysis ...... 50 IV RESULTS ...... 51 4.1 Phytochemical investigation ...... 51 4.1.1 Qualitative analysis of phytochemicals ...... 51 4.1.2 Quantitative analysis ...... 51 4.1.2.1 Total phenols...... 51 4.1.2.2 Total alkaloids...... 52 4.1.2.3 Total saponins ...... 53 4.1.2.4 Total flavonoids ...... 53 4.1.3 Proximate composition ...... 54 4.1.4 Mineral analysis ...... 55 4.2 In-vitro activities ...... 55 4.2.1 Antibacterial activity...... 55 4.2.2 Antifungal activity ...... 56 4.2.3 Phytotoxic activity ...... 57 4.2.4 Antioxidant activity ...... 58 4.2.4.1 DPPH radical scavenging activity ...... 58 4.2.4.2 ABTS radical scavenging activity ...... 59 4.2.5 Lipoxygenase-inhibitory assay ...... 60 4.3 In-vivo pharmacological activities ...... 60 4.3.1 Acute toxicity ...... 60 4.3.2 Anti-inflammatory activity ...... 61 4.3.2.1 Carrageenan˗induced paw edema model ...... 61 4.3.2.2 Xylene-induced ear edema...... 62 4.3.3 Analgesic activity ...... 63 4.3.3.1 Formalin test ...... 63 4.3.3.2 Hot plate test ...... 64 4.3.4 Antipyretic activity ...... 65 4.3.5 Antidiarrheal activity ...... 66 4.3.6 Anti-diabetic activity ...... 66 4.3.7 Hepativeprotective activity ...... 67 4.3.8 Sedative/hypnotic activity...... 68 4.3.9 Anti-convulsant activity ...... 69 4.3.10 Antidepressant activity ...... 69
V. DISCUSSION ...... 71 5.1 Phytochemical investigation ...... 71 5.1.1 Qualitative and quantitative analysis of phytochemicals ...... 71 5.1.2 Proximate composition: ...... 72 5.1.3 Mineral analysis ...... 73 5.2 In-vitro activities ...... 75 5.2.1 Antimicrobial activity ...... 75 5.2.2 Phytotoxic activity ...... 77 5.2.3 Antioxidant activities ...... 78 5.2.4 Lipoxygenase activity ...... 79 5.3 In-vivo pharmacological activities ...... 80 5.3.1 Acute toxicity ...... 80 5.3.2 Anti-inflammatory activity ...... 80 5.3.3 Analgesic activity ...... 82 5.3.4 Anti-pyretic activity ...... 83 5.3.5 Antidiarrheal activity ...... 85 5.3.6 Antidiabetic activity ...... 87 5.3.7 Hepativeprotective activity ...... 89 5.3.8 Sedative-hypnotic activity ...... 90 5.3.9 Anti-convulsant activity ...... 91 5.3.10 Antidepressant activity ...... 92 VI. SUMMARY ...... 94 VII. CONCLUSIONS AND RECOMMENDATIONS ...... 96 Conclusions ...... 96 Recommendations ...... 97 LITERATURE CITED ...... 98
LIST OF TABLES
Table No. Title Page No.
3. 1. Conditions for operation of micro and macro minerals ...... 40
3.2. Strains of bacteria for antibacterial activity ...... 41
3.3. Strains of fungi used for antifungal activity ...... 43
4.1. Phytochemical of methanolic crude extract and solvents fractions of D. botrys ...... 51
4.2. Proximate composition (%) of D. botrys whole plant ...... 54
4.3. Mineral composition of whole plant of D. botrys ...... 55
4.4. Antibacterial activity of methanolic crude extract and solvent fractions of D. botrys ...... 56
4.5. Antifungal activity of methanolic crude extract and solvent fractions of D. botrys ...... 57
4.6. Phytotoxic activity of methanolic crude extract and solvent fractions of D. botrys...... 58
4.7. DPPH radicals scavenging activity of methanolic crude extract and solvent fractions of D. botrys ...... 59
4.8. ABTS radicals scavenging activity of methanolic extract and solvent fractions of D. botrys ...... 59
4.9. Lipoxygenase-inhibitory assay of methanolic crude extract and solvent fraction of D. botrys ...... 60
4.10. In-vivo acute toxicity of methanolic crude extract of D. botrys ...... 60
4.11a. Anti˗inflammatory activity of methanolic crude extract of D. botrys on carrageenan provoked mice paw edema ...... 61
4.11b. Percent inhibition of carrageenan-induced paw edema by methanolic crude extract of D. botrys ...... 62
4.12a. Anti˗inflammatory effect of methanolic crude extract of D. botrys on xylene˗induced ear edema in mice ...... 62
i 4.12b. Percent inhibition of xylene-induced ear edema by methanolic crude extract of D. botrys ...... 63
4.13a. Analgesic effect of methanolic crude extract of D. botrys on formalin- induced pain in rats ...... 63
4.13b. Percent inhibition of formalin-induced pain by methanolic crude extract of D. botrys ...... 64
4.14. Analgesic effect of methanolic crude extract of D. botrys on pain induced by hot plate in mice ...... 64
4.15. Antipyretic effect of methanolic crude extract of D. botrys on brewer‟s yeast induced pyrexia in rats ...... 65
4.16. Antidiarrheal effect of crude extract of D. botrys on castor oil-induced diarrhea in rats ...... 66
4.17. Anti-diabetic activity of methanolic crude extract of D. botrys on alloxane induced diabetes in mice...... 67
4.18. Hepativeprotective activity of methanolic crude extract of D. botrys
extract on CCl4 stimulated toxicity in rats ...... 68
4.19. Sedative/hypnotic activity of methanolic crude extract of D. botrys on thiopental induced hypnosis ...... 68
4.20. Anticonvulsant Effect of methanolic crude extract of D. botrys on PTZ- induced convulsions in mice ...... 69
4.21. Antidepressant activity of crude extract D. botrys on the time of immobility in forced swim test model in rats ...... 70
ii LIST OF FIGURES
Fig.No. Title Page No.
3.1. Herbarium specimen of D. botrys ...... 32
3.2. Scheme of extraction and fractionation process ...... 34
4.1. Total phenols in methanolic crude extract and subsequent fractions ...... 52
4.2. Total alkaloids in methanolic crude extract and subsequent fractions ...... 52
4.3. Total saponins in methanolic crude extract and subsequent fractions ...... 53
4.4. Total flavonoids in methanolic crude extract and subsequent fractions ...... 54
4.5. Antibacterial activity of Ethyl acetate fraction and crude extract of D. botrys against ...... 56
4.6. Antifungal activity of crude extract and Ethyl acetate of D. botrys against ...... 57
iii ABBREVIATIONS
A. flavus Aspergillus flavus A. niger Aspergillus niger A.alternatum Acremonium alternatum ABTS 2, 2-azinobis-3-ethylbenzothiozoline-6-sulfonic acid scavenging assay ALP Alkaline phosphatase ANOVA Analysis of variance ATCC American type culture collection B. subtilis Bacillus subtilis C Centigrade C. michiganesis Clavibacter michiganesis Cm Centimeter
CuSO4 Copper Sulphate DCMF Dichloromethane fraction
DMSO Dimethyle sulphoxide DPPH 1, 1-diphenyl-2-picrylhydrazyl E. coli Escherichia coli EAF Ethyle acetate fraction Etc et cetra F. oxysporum Fusarium oxysporum F. solani Fusarium solani FST Forced swimming test HCl Hydrochloric acid
HNO3 Nitric acid hrs hours HxF n-Hexane fraction i.e that is i.p Intraperitoneal IC Inhibition concentration K. pneumonia Klebsiella pneumonia KOH Potassium Hydroxide L. minor Lemna minor LD Lethal dose
iv LOX Lipoxygenase assay M Molar M. pirimis Mucar pirimis MCE Mthanolic crude extract mg Milligram MIC Minimum inhibitory concentration min Minutes ml Milliliter mm Milimeter mM Millimolar n Number N Normal N/saline Normal saline NaCl Sodium chloride NaOH Sodium hydroxide NO Nitric oxide OD Optical density P. aeruginosa Peseudomonas aeruginosa P. vulgaris Proteus vulgaris PDA Potato dextrose agar
PDB Potato dextrose broth PEG Polyethylene Glycol PTZ Pentylene tetrazole R. communis Ricinus communis R. oryzae Rhizopus oryzae R.dominica Rhizopertha dominica S. aureus Staphylococcus aureus s.c Subcutinious sec Second SGOT Serum glutamic oxaloacetic transaminase SGPT Serum glutamic pyruvic transaminase TB Total bilirubin UV Ultraviolet µg Microgram
v µl Microliter v/v Volume by volume w/v Weight by volume X. campestris Xanthomonas campestris ZI Zone of inhibition
vi ACKNOWLEDGEMENTS
All praises are to Almighty ALLAH, the most Merciful and Compassionate, the only creator of the universe and source of all knowledge and wisdom, who blessed me with health, thoughts, loving parents, wife and children, talented teachers, helping friends and afforded me to complete this study. Countless salutations are upon the Prophet Muhammad (PBUH), the gleam of guidance, faith and knowledge for the humanity. I wish to express deepest gratitude and profound regards to my kind Supervisor Dr. Asad Jan, Associate Professor, IBGE, for his supervision, planning, execution and scholarly ideas that beautified the scientific nature of the research work carried out. He always encouraged me and kept my morale high by his suggestions, appreciation and motivation. Without his precious guidance and support I would never be able to complete my research. I feel great pleasure in expressing my ineffable thanks to my encouraging, inspirational and cool minded co-supervisor Dr. Inam Ullah Khan, Associate Professor, Department of Pharmacy, University of Peshawar for his sense of devotion, creativity, affectionate criticism and keen interest in my work; it was because of his inspiring guidance and dynamic cooperation during entire study program that I could complete this manuscript. I tender my thanks to Professor Dr. Iqbal Munir, Director IBGE for his administrative support and cooperation. Special thanks are extended to Professor Dr. Safdar Hussain Shah for his kind attitude, valuable suggestions and help. I am also thankful to Prof. Dr. Farhatullah, Dr. Aqib Iqbal, Dr. Shakoor and all the faculty members of IBGE and UAP for their moral help and support. Also thanks to the members of my supervisory committee for their cooperation and valuable suggestions during my research work. I would also like to acknowledge Department of Pharmacy, University of Peshawar and Department of Pharmacognocy, Faculty of Pharmacy, University of Karachi for providing me all technical and lab facilities. I convey my thanks from the deepest of my heart to my very caring seniors and friends Dr. Zahid Ullah (UOS), Dr. Zamarud Shah (USTB), Dr. Zafar Hashmi (COMSATS), Dr. Abdul Wajid (PCSIR), Muhammad Imran, Abid, Hezbullah,
vii Rameez and Medrar for their technical support and valuable suggestions during my research work. Words do not come out easy for me to mention the feelings of obligations towards my affectionate parents. My father proved the ocean of Love, care for me in which I saturated myself. Every aspect of my life is incomplete without him. I am most earnestly thankful to My Mother for the strenuous efforts done by her in enabling me to join the higher ideals of life. I am grateful to my parents, wife and sisters for their financial and moral support, patience and prayers they had made for my success. May ALLAH ALMIGHTY infuse me with the energy to fulfill their noble inspirations and expectations and further edify my competence.
Muhammad Naeem Khan
viii PHYTOCHEMICAL AND PHARMACOLOGICAL PROFILING OF Dysphania botrys L. Muhammad Naeem Khan and Asad Jan Institute of Biotechnology & Genetic Engineering Faculty of Crop Production Sciences The University of Agriculture Peshawar, Pakistan August, 2018
ABSTRACT
Dysphania botrys L. is an annual herbaceous plant belongs to family Amaranthaceae, native to Asia and Europe found in Pakistan, India and Iran. In the folk medicine D. botrys has been utilized for the treatment of different ailments like asthma, cold, influenza, head ach, liver and digestive problems and healing of wounds. The current work was designed to evaluate methanolic crude extract (MCE) of D. botrys for different in-vivo pharmacological activities and its various solvents fraction for phytochemical analysis and different in-vitro activities, in order to provide scientific authentication to its ethno-medicinal uses. Qualitative phytochemical study of MCE and solvent fractions of D. botrys confirmed the presence of phenols, alkaloids, flavonoids, sterols, tannins and saponins, however in n-hexane fraction (HxF) only flavonoids and saponins were detected. In quantitative analysis, amongst all the solvents, ethyl acetate fraction (EAF) had highest amount of phenol (27.4 mg/g), flavonoids (15.5 mg/g) and alkaloids (3.14 mg/g), while MCE displayed maximum amount of saponins (34.3 mg/g). In the proximate analysis, nitrogen-free extract were present in higher amount (38.45 ± 0.83%) followed by protein (30.26 ± 0.72%) while crude fibers were found least in amount (1.43 ± 0.53%). Among different minerals, reasonable amount of calcium (3268 ± 0.53 μg/g), potassium (2873 ± 0.71 μg/g), sodium (591 ± 0.23 μg/g) and iron (223 ± 0.46 μg/g) were found, while no cadmium and chromium was detected. MCE and EAF displayed considerable antibacterial activity against Xanthomonas campestris and Pseudomonas aeruginosa causing 12.6 ± 0.54 mm and 20.6 ± 0.53 mm zone of inhibition respectively which was analogous to that of cefixime, used as standard drug. In case of antifungal activity MCE hindered the growth of Fusarium oxysporum effectively, causing 19.3 ± 0.41 mm inhibition zone, while effect of other solvents was low to moderate. Highest phytotoxic effect was shown by MCE (1000 μg/ml) against the growth of Lemna minor, causing 70% reduction in its growth. EAF exhibited maximum scavenging activity against 1, 1- diphenyl-2-picrylhydrazyl (DPPH) and 2, 2-azino-bis-3-ethylbenzothiazoline-6- sulfonic acid (ABTS) radicals causing 57.17 ± 0.49% and 72.46 ± 0.59% scavenging activity respectively as compared to standard, ascorbic acid. The activity of lipoxygenase (LOX) enzyme was inhibited effectively by EAF (64 ± 0.16%), while HxF displayed least inhibiting effect (22 ± 0.21%). In the in-vivo pharmacological activities of crude extract of D. botrys, acute toxicity analysis showed no sign of mortality up to an amount of 2000 mg/kg. Crude extract (200 and 400 mg/kg) showed considerable (p<0.05) anti•inflammatory effect at early and late phase of carrageenan˗stimulated paw edema while in case of xylene˗induced ear edema dosage of 400 mg/kg was highly effective (p<0.01) in reducing ear inflammation (73.2%). Dose of 200 mg/kg of plant extract displayed considerable (p<0.05) peripheral
ix analgesic activity at both phases of analgesia, causing 60.71% and 67% reduction in severity of pain while in case of 400 mg/kg, its effect was highly significant (p<0.01) causing 78.57% and 82.14% pain inhibition. In the central analgesic activity (hot plate model) the effect of 400 mg/kg was highly significant (p<0.01) after 120 min of assessment time interval. In the antipyretic assay, effect of 400 mg/kg of plant extract was extremely significant (p<0.001) at all the assessment time intervals (1h-5h) and was comparable to that of standard drug paracetamol in reducing body temperature to normal, increased by brewer‟s yeast. In the antidiarrheal activity, plant extract of 400 mg/kg effectively (p<0.01) increased the latent period of diarrhea and caused a decline in the total wet fecal frequency and mean weight of fecal drops as compared to control. The elevated blood sugar induced by alloxane monohydrate in the anti-diabetic activity was significantly reduced by crude extract (400 mg/kg), however its effect was highly significant (p<0.01) at the 3rd and 4th hour of evaluation time. In the hepatoprotective assay, MCE of plant at dosage of 400 mg/kg markedly (p<0.05) declined high level of alkaline phosphatase (ALP) (179.22 ± 3.41 mg/dl) and total bilirubin (TB) (3.64 ± 0.13 mg/dl) while its effect was highly significant (p<0.01) in reducing the level of serum glutamic pyruvic transaminase (SGPT) (31.2 ± 1.28 U/ml) and serum glutamic oxaloacetic transaminase (SGOT) (48.31 ± 1.87 U/ml)) when compared to toxic control. Plant extract (100 and 200) showed a significant (p<0.05) synergetic effect on the thiopental induced hypnosis caused an early arrival of sleep and effectively (p<0.01) prolonged the duration of sleep from 88.80 ± 1.91 min to 145.20 ± 1.76 min. In the pentalyne tetrazol (PTZ) induced convulsion activity, plant extracts (200 and 400 mg/kg) effectively (p<0.05) delayed the onset of first clonus from 5.09 ± 0.22 min to 6.03 ± 0.28 min and 6.99 ± 0.07 min and prolonged the time of death from 9.72 ± 0.44 min to13.57 ± 0.6 min and 19.56 ± 0.15 min, respectively. The immobility time was significantly (p<0.05) decreased by MCE of plant from 193.98 ± 1.35 seconds to 96.78 ± 1.39 seconds, in the antidepressant activity.
Key words: Dysphania botrys, phytochemical analysis, antimicrobial activity, antioxidant assay, in-vivo pharmacological activities
x 1. INTRODUCTION
Plants not merely give food and raw material to humans but also the source of bioactive and medicinally important compounds. A plant is considered medicinal, when it contains active molecules or substances that can be utilized for healing purposes or act as precursor for making beneficial drugs. Phytotherapy also called phytomedicine, herbal medicine or botanical medicine refer to any medicine that is obtained from plants either in the crude or pure form having active ingredients or simply phytotherapy is the utilization of plants and its products for therapeutic and medicinal purposes against various ailments (Rates, 2001). The different parts of plants used in phythotherapy include leaves, branches, roots, flowers, seeds, bark, berries or whole plant. Medicinal plants are the back bone of folk medicinal system, which is based on the utilization of medicinal plants and their derivatives. The vast diversity of medicinal flora around the world provides a huge assets and infinite source for production of herbal medicine. More than three forth of world population uses herbal medicine for curing different diseases and relies upon it, because herbal medicine have no side effect and can easily obtained from nature (Farnsworth, 1985).
Human had been used plants for preventing and curing diseases throughout the history of mankind and analysis of fossils record shows, that the therapeutic use of plants is as old as 60,000 years (Solecki and Shanider, 1975). Extensive studies have indicated that phytomedicine represent the earliest form of medication. Documented record about herbal medicine is approximately 5,000 years, dated back to Sumerians, who explained different therapeutic uses of plants. However archeological investigations have revealed that the use of phytomedicine dates back sixty to eighty thousand years ago in Iraq and China respectively (Leroi Gourhan, 1975). Prehistoric analysis have showed that medicine extracted from medicinal plants, either in crude or pure form, represent the oldest method of medication. Archaeological investigations have revealed that peoples in the ancient time had knowledge about therapeutic properties of plants, since the practice of phytomedicine is as primal as human species (Halberstein, 2005).
Greek civilization has made a great role in the development of herbal- pharmaceutical science. Aristotle explained about five hundred crude drugs used against various diseases (Oktay et al., 2003). Hippocrates regarded as the founder of
1 modern allopathic medicine, described approximately four hundred substances of plant origin having therapeutic properties against various ailments (Sykiotis et al., 2006). Theophrastus cited about five hundred crude herbal drugs and their medicinal properties in his book. Claudius Galen Pergamum wrote three hundred books on medicinal plants. He made herbal drugs employing various extraction methods called Galenical and established the idea of pharmaceutical formulation to develop therapeutically secure and efficient medicine (Newman et al., 2000; Buerky and Higby, 2007).
Chinese traditional prescription is considered one of the earliest systems of medication based more than 85% on herbal products. Shen-Nong about five hundred years ago described certain plants in the Chinese herbal medicine and because of his efforts various plants were commonly used in the ancient China for health care (Dharmananda, 2013). Before the time of Shen-Nong, few earliest Chinese books, such as Shi-jing, Shang-Shu and Shing-Hai-Jing documented the use of plants as a remedy. Shi-Jing not only explained the medicinal uses of plants but also described their habitat and harvesting season. In this book eighty medicinal plants and ninety species of insects with their therapeutic uses were recorded. Shan-Hai-Jing, the ancient Chinese book documented nine plant species having nutritional value, forty five species having therapeutic properties, six species having toxic effect for pests and animals and two plants having noxious effect for human beings (Hu, 2008).
Ayurveda or Indian conventional medication is one of the ancient systems based on natural products and considered mother of all therapies. The documented proof of this system is available in the earliest literature for example, Atharva˗veda and Rig˗veda about 5000 years BC. These books had the names of medicinal plants in the form of poetry and transferred from generation to generation. (Mukherjee and Wahile, 2006).
Before the start of 19th nineteenth century, phytomedicine were utilized in crude form as infusion (herbal tea), tincture (alcoholic extract), syrup (sugar solution of extract), and decoction (stem, root, flower, leaves or bark boiled extract) or consumed externally as balms, ointments, essential oil and poultices (Newman et al., 2003). However later on, scientist started the separation, purification and recognition of therapeutically bioactive compounds from medicinal plants. This effort made it
2 possible to analyze and discover effective drugs which are even widely used in the modern pharmaceutical products (Kong et al., 2003; Gupta et al., 2005).
According to botanical survey there are approximately 250, 000 to 350, 000 species of plant present over the earth. Among these, very less number of plants species i-e 35, 000 to 70, 000 have been used for therapeutic purposes in different region of world which form 14% to 28% of total plants (Jin˗Ming et al., 2003). These curative plant derivatives are consumed generally in crude or partially processed shape, thus require scientific authentication to ascertain their medicinal uses (De Smet, 2002; Kinsel and Straus, 2003). Among all medicinal plants only 15% are phytochemically investigated and only 6% have biological screening data, while the remaining medicinal plants have no scientific data, thus have a great scope to search out for novel effective pharmacological agents (Newman et al., 2000; Harvey, 2000 and Verpoort, 2000).
Majority population of the world consumes a large diversity of plants for medicinal purposes, even though no scientific knowledge and information is present about the efficiency and effectiveness of these medicinal plants. Regarding the significance of drugs and bioactive compounds extracted from plants and lack of scientific knowledge, it is necessary conduct and research work an organized manner for herbal medicine and restorative plants (Schopene, 1983 and Awadeh et al., 2001). Over the earlier decades, a huge attention has been given for identification and extraction of bioactive compounds from plants, for pharmaceutical and dietary use (Ho et al., 1992; Oktay et al., 2003; Wangensteen et al., 2004). Different plant parts for example flowers, roots, stem and whole plant are used for obtaining bioactive compounds which are consumed in herbal medicines for the treatment of several ailments. Currently, because of development of advance scientific techniques and equipments, herbal experts take great interest to explore new therapeutic agents from bioactive metabolites of plants. These natural bioactive agents will serve infinite assets for herbal pharmaceutical industry (Yakubu et al., 2007).
Plants having medicinal properties played very important role and contribution in agriculture, pharmaceuticals and food industries. In spite of preeminence of synthetic chemistry and modern techniques to determine and synthesize drugs, the role and importance of phytochemicals for curing diseases and drug discovery is still remarkable (Raskin et al., 2002). Due to modern synthetic techniques, pharmaceutical industries
3 have developed a large number of synthetic drugs for curing various diseases, however the regular use of these drugs develop other chronic problems like microbe resistance and side effects, effecting other activities of body. Compared to herbal drugs, synthetics drugs are more expensive and ordinary peoples cannot afford it. The use of natural products for health care is steadily growing and there is a shift in general trend from synthetic to phytomedicine. More than four billion people are using phytomedicine as a remedy against various ailments (Fabricant and Farnsworth, 2001). Phytotherapy has been acknowledged by WHO as vital component for basic health care (Taylor, 2000).
In the modern scientific era the healing characteristics of medicinal plants have been investigated throughout the world, because of their strong therapeutic efficiency, antioxidant properties, no or less side effect and financial affordability. Medicinal plants provide raw material to pharmaceutical industry on large scale to extract drugs in pure form which are very efficient and economically viable. The significant therapeutic role of medicinal plants, in curing chronic diseases, have been reported and proved by scientific research (Martinez et al., 2008).
In case of cancer, researchers are expecting novel bioactive molecules having anticancer properties which can be more effective as compared to synthetic drugs. Flavonoids and other secondary metabolites extracted from various plants have revealed considerable inhibitory effect on malignant cells (Jiangrong and Jiang 2007; Zhao et al., 2007). Plants having hypoglycemic properties, offer a potential area for herbal research to control and cure diabetes, affecting a huge number of peoples all over the world (Renuka et al., 2009). Phytomedicine have been proved effective for treating various cardiovascular disorders, which are the frequent reasons of death of people all over the globe (Thippeswamy et al., 2009). Plants having hepativeprotective properties have the ability to lower down the elevated level of enzymes in liver, in case of viral infection and are the best alternative of synthetic drugs (Oshima et al., 1995; Bhawna and Kumar, 2009). Plants of different taxonomic groups and regions have shown antioxidant, antimicrobial, anti-inflammatory, antidiarrheal, antidepressant and analgesic property (Ibrahim et al., 2006; Ali et al., 2008). Due to widespread interest and application, medicines obtained from herb plays essential roles.
4 Economically medicinal plants play very important role because of their widespread industrial uses. These include folk medicine, herbal tea and health care products like neutraceuticals, phytopharmaceuticals, galenicals, semi-synthetic and synthetic pharmaceuticals. Up to now, 125 different compounds extracted from curative plants have been identified, screened and used in the pharmaceutical industry. Medicinal plants are the valuable resources making foreign exchange particularly for developing countries. Economically important phytochemicals and herbal medicine are vinblastine, vincristine, taxol, colchicines, podophyllotoxine, qeuuinine, tinctures, morphine, camptothecin, atropine, digitoxigenin, capsaicin, curcumin, capsaicin, aspirins, codeine, artimisinin and ephedrine. Therapeutic plants contribute significant role in the development of research in pharmaceutical industry which mainly focus on the separation and screening of bioactive compounds or synthesis of semi-synthetic products (Cordell, 2009).
The international market of plants derived natural products like phytochemicals, phytopharmaceuticals, flavors, fragrances, color ingredients, exceed annually several billion dollars. The trade of plants derived drugs and chemicals are increasing annually with a rate of 6.4%. In United state of America (USA), less the 5% people used plant derived products in 1991 but in 2004 this value increased to 50%. Net sale of herbal products is three fourth of total sale in the market. The worth of plant derived medicated confectioneries reached to 593 million US dollars (Patel, 2015; Tiwari, 2015). In 2014, there was an increase of 7%, in retail sale value of phytomedicine used for cough, cold and different allergies. In USA, Japan and other developed countries of world, the demand for neutraceuticals is more as compared to past and in 2010 its market sale value was more than 80-250 billion US dollars, with a similar value in Japan and Europe (Sapna, 2007).
Certain reports have revealed that the trade of phytomedicine in United Kingdom (UK) has increased 43% in 1994-1998, having a worth of 50 million Euros, while in 2003, this value reached to 60%. In Germany, more than 1500 medicinal species belonging to 200 families have been therapeutically investigated and used in different pharmaceutical products on industrial scale. The worth of plant derived medicine in the market was 1.5 billion Euros. Currently, Poland, Germany and Bulgaria are the main exporters of herbal medicine (Mensah et al., 2008).
5 The trade of healing plants and its extracts in South Africa is common and more than 500 medicinal plant species are used on commercial scale. In Russia the sale of phytomedicine in the market reached to an amount of 35.8 billion Russian rubles in 2014 (Shikov et al., 2012). Brazil contain nearly 55,000 indigenous species among which 1200 are recognized as medicinal plants, while there are thousands of undocumented species, utilized by the native people for healing purposes (Fabricant and Farnsworth, 2001). The total sale of plant derived products in Brazil is 508 million Brazilian reals (BRL) in 2014. According to a survey carried out by WHO, the worth of herbal products in the current global market is 61 billion US dollar and it is estimated that this value will exceed 5 trillion US dollar by 2050 (Venessa, 2015).
It has been calculated that roughly 50% of medicines used in clinical practices, are originated from medicinal plants or its derivatives, because of having excellent potential of therapeutic activities and less side effects (Sofowora, 1984; Cowan, 1999). For pharmaceutical products plants are the primary source, due to certain features i-e firstly plants manufacture a number of primary and secondary bioactive metabolites, secondly they enable synthetic chemist to find out novel compounds or alter the existing compounds into more valuable drugs, thus provide a platform for development of a huge number of pharmaceuticals (Farnsworth, 1984). These bioactive compounds or phytochemicals defend plants against various pathogens, pests or any environmental stress. These compounds are organic in nature and can be categorized into primary and secondary metabolite. Primary metabolite, include protein, carbohydrates, nucleic acid and lipids which are fundamental bio molecules, necessary for development and growth. Secondary metabolites synthesized by plants are flavonoids, alkaloids, saponins, steroids, terpeniods, tannins, glycosides and volatile oils etc. These organic compounds are pharmacologically active and can be employed as a medicine or drug against different diseases. Among secondary metabolites, alkaloids have analgesic, diuretic, anti-malarial, antispasmodic properties, terpeniods have anthelmintic, anti- inflammatory, anti-malarial, anticancer, antiviral and antibacterial activities, flavonoids and phenols have antibacterial and anti-allergic and antioxidant properties, saponins have antiviral and anti-inflammatory activities and glycosides have antibacterial and antifungal properties (Chopra and Doiphode, 2002; Maurya et al., 2008).
6 Pakistan is among one of the developing country where the usage of herbal medication is extremely universal. The demand of phyto-medicine is rising with the passage of time due to easy accessibility and economic viability. Across the country, approximately 84% people use herbal products for their basic health care (Qureshi et al., 2007). Herbal medicines are prescribed by herbal practitioners locally called Tabib or Hakim (Saeed et al., 2011). Knowledge about therapeutic uses of plants is transmitted from one generation to next generation in the folkloric system of medicine. So these local practitioners have no scientific background, only have information about medicinal plants got from their predecessors or based on past experience and traditions. Therefore to provide logical background to herbal products, it is very necessary to study safety, quality and efficiency of medicinal plants on scientific bases. It is essential to evaluate pharmacological screening, invitro and invivo activities and finally the extraction and purification of bioactive molecules (Schensul et al., 2006).
The biodiversity of Pakistan is very unique due to diverse environmental and climatic conditions. Across the country approximately 6000 species of flowering plants are present belonging to 1572 genera. Among these species, 30% are commonly found everywhere throughout the country while 70% are endemic species found in particular regions and districts. Pakistan is alienated into 4 main phyto-geographic regions. Among these region, Irano-Turanian area contain 45% therapeutic plants, Himalayan and Sindian regions contain 10% and 9% medicinal plants whereas the smallest amount of therapeutic plants species, which is approximately 6%, are originated in the region near the Indian boarder (Ali and Qaiser, 2009).
In Pakistan scientific research on plants having medicinal potential is carried out mainly at academia or institutional levels. Such research activities include in-vivo and in-vitro assays of different extracts of medicinal plants, phytochemical screening and separation of therapeutically active compounds. These activities are carried out to ascertain their traditional uses on scientific bases and to search out therapeutic activities other than the folk uses of the plant. Such research activities have confirmed that most plants contained active ingredients which help in controlling many disorders (Shinwari, 2010).
7 In recent decades there is a great focus on obtaining chemicals from plants having significant antimicrobial and antioxidant activities. It is well established that certain chemicals, produced by plants, having antimicrobial properties and are lethal for bacteria and fungi (Harborne, 1988). The plants used these chemicals for their own defense and these compounds belong to various groups of phytochemicals for example phenols, flavonoids and iso-flavonoids etc. It is well documented that utilization of antioxidant obtained from plants, reduce chances of cardiac disease and cancer (Marchioli et al., 2001). These antioxidants decrease the chances of chronic diseases by activating the antioxidants naturally occur inside the living organisms or by giving it directly in diet (Stanner et al., 2004).
Dysphania botrys L. synonym Chenopodium botrys L. belongs to family Amaranthaceae, having the English names sticky goosefoot, Ambrosia, Jerusalem oak and feather geranium. Previously D. botrys was placed in the genus Chenopodium, but due to recent taxonomic and phylogentic investigations, it was placed in a separate genus, Dysphania. The transferring of fragrant species of Chenopodium to a separate genus took place nearly in 2000 (Clemants and Mosyakin, 2003). In all the contemporary phylogentic schemes, this updated circumscription was authenticated and as in Flora of North America North of Mexico (Mosyakin and Clemants, 2002; Mosyakin and Clemants, 2008) and Flora of China (Zhu et al., 2003), species were reorganized between Chenopodium and Dysphania, due to modern molecular studies (Kadereit et al., 2010; Fuentes-Bazan et al., 2012a).
D. botrys is an annual herb rising up to 0.6 m (2ft). It flowers from July to October which are minute, green, numerous, terminal, small, panicle, frequently reddening in fruiting phase, hermaphrodite and pollinated by wind. Its seeds are glossy and black in appearance, round in shape having 0.5-0.75 mm diameter. It is an aromatic herb having distinct odor and oak-like leaves (Kletter and Krichbaum, 2001). The immature leaves of D. botrys appear like tiny versions of those of the oak (Watts, 2007). Stem have many erect branches enclosed with stalked glandular hairs.
D. botrys is native to Asia and Europe found in Pakistan, Africa, India, Turkey, Australia, north Europe, Cyprus, and Southand North America (Seidemann, 2005). It grows in cultivated fields, road sides in cities and villages and found on distressed soil patch in grasslands and semi deserts, favoring sandy loose soil (Kletter and Krichbaum,
8 2001). It is cultivated sometime as garden plant, largely for its strongly scented foliage and flowers, used in dried flower arrangements (Small, 2006). It can tolerate high concentration of Cu and moderate amount of Zn, Fe and Mn and can grow in soil contaminated with heavy metals (Yousefi et al., 2011). It accumulates heavy metals in shoots and root (Nouri et al., 2009).
Species of Chenopodium have been utilized worldwide in herbal medicine for curing different ailments. It is well known that C. album acts as tonic, diuretic, laxative, and anthelmintic. From the immemorial time the C. ambrosioides is used against intestinal parasites in South America. For the catarrh and humoral asthema D. botrys has been utilized and is recognized as a good substitute for C. ambrosioides (Yadav et al., 2007).
D. botrys has been utilized worldwide in herbal medicine for curing different ailments. Extract of leaves and whole plant is used for the treatment of asthma, catarrh, influenza, headache and different digestive problems (Quattrocchi, 2012). In Iran D. botrys is used as a tonic and anticonvulsant agent for the cure of different nervous convulsions and as an expectorant for treating cough and asthma (Zargari, 1993). In Northern areas of Pakistan, an infusion is prepared from the whole plant of D. botrys, having analgesic, diuretic and laxative properties, used as a remedy for headache, liver, stomach and digestive ailments (Bano et al., 2014). In some areas of Kohistan, young and fresh leaves are used as an antiseptic and for wounds healing (Hazart et al., 2011). In Indian folk medicine D. botrys is well-known as diuretic, stimulant, carminative and antispasmodic, used as a medicine for urinary, digestive, respiratory, liver and stomach disorders (Khare, 2007). Its extract is utilized as a flavoring agent in soup of meat, barley and cheese in some areas of India (Maksimovic, 2005). In Himalaya region of Kashmir, extract is prepared from its seeds having vermicidal effect, used orally for removal of tapeworms, particularly in children, however its seeds are comparatively more toxic as compared to other parts (Kletter and Krichbaum, 2001).
In Ladakh it is used as vermicidal, laxative and diuretic agent in folk medicine. In province of Uttar Pardesh, juice is extracted from its leaf which is used to remove leeches from the nostrils of cattle and other domestic animals (Jain, 1984). In some areas of western Indian Himalaya, fresh and green leaves and branches are used as vegetable which also have analgesic effect and very effective to treat severe headache
9 (Sing, 2012). Inner bark of D. botrys is heated in boiled water and mix with sugar to make sugar coated tablets used for treatment of tuberculosis (Watts, 2007).
In Southern Europe it is used as a remedy for humoral asthma and catarrh and act as best alternative of C. ambrosioides (Yadav et al., 2007). In Germany, D. botrys was cultivated as medicinal plant and used to repel moths (Hanelt, 2001). D. botrys have characteristic odor and in the ancient time its leaves and branches were kept in cloths and garments to repel clothes moths (Artschwager, 1996). In Spain, special tea is prepared from its leaves and branches called „Te de valladolid‟, which is very effective to cure cough and digestive ailments (Pardo de Santayana, 2005). In Serbian conventional medicine, liquid extract is prepared from dried upper parts of plant having antidiarrheic, antispasmodic, diuretic and carminative properties. Its dried parts also used as a spice (Maksimovic, 2005).
D. botrys have characteristic odor due to presence of sesquiterpenes and monoterpenes (Kletter and Krichbaum, 2001). Monoterpenes include fenchone, camphor, linalool, δ˗3˗carene, nerol, menthone, pulegone, β˗pinene, thujone and terpinol˗4, while sesquiterpenes consist of β˗elemene, β˗eudesmol and elemol (Buchbauer et al., 1995). Other secondary metabolites present are alkaloids, flavonoids, phenols, terpenoids, ascaridole etc present in varying amount depending upon its origin and locality. Essential oil extracted from D. botrys is about 0.08-2%, however it has been shown by a number of studies that its composition and amount is not fixed, depending upon its origin and location it yield varying amount of oil (Yadav et al., 2010; Morteza-semnani and Babaezhad, 2007). Various studies have shown that ascaridole is the chief compound found in its essential oil (Rustembekova et al., 1975). Ascaridole consist of dicyclic monoterpene having peroxide functional group (Dembitsky et al., 2008). By heating ascaridole, isomerization occure forming isoascaridole (Tisser and Young, 2014). It has been reported that ascaridole has anthelmintic, anti-fungal, analgesic and sedative properties (Khare, 2007). It is a powerful inhibitor for Leishmania amazonesis, Trypanosome curzi and Plasmodium falciparum and tumor cell lines, inhibiting its in-vitro growth (Rai and Carpinella, 2006). Compounds like eudesmane, elemane, guaiane belong to sesquiterpenes and chenopodic acid belong to terpeniods was extracted as component of essential oil (Khare, 2007; De Pasual., 1980).
10 Various studies carried out on contents of flavonoids in D. botrys, led to the separation of flavonoids which include; quercetins, chrysoeriol, hispidulin, flavones, 7- methyleupatulin, 5-methylsalvigenin, salvigenin, jaceosidin and sinensetin (Kletter and Krichbaum, 2001). Among alkaloids, betaine is present in prominent amount in all parts of plant and has been isolated (Rustembekova et al., 1973). Investigation has been carried out about the presence of phytoecdysteriods, which are analogues of steroid hormones of invertebrates (Dinan et al., 1998).
D. botrys is natural growing wild plant, traditionally used by the rural and endemic inhabitants of different regions of Pakistan for the curing of asthma, cough, wounds, fever, pain, liver, respiratory, urinary and gastric complaints (Khare, 2007; Hazart et al., 2011, and Bano et al., 2014). Outstanding therapeutic properties of D. botrys has great potential and have fascinated the scientist to explore the active compounds having different pharmacological properties. Therapeutic utilization and health benefits of D. botrys are mainly based on legends and have no scientific authentication, making it a superior contender to congregate documentation including the phytochemical contents, in-vitro and in-vivo experiments using animal models available in the scientific studies. The current work was planned to explore the phytochemical composition and pharmacological potential of D. botrys in order to present scientific validation to its traditional uses.
11 Objectives of study
1) To perform phytochemical investigations of the crude extract of whole plant 2) To explore in-vitro and in-vivo toxicity plant crude extract 3) To evaluate certain in-vivo activities, in order to provide scientific validation to its folks use 4) To find out therapeutic uses other than folk uses for the whole plant by performing targeted pharmacological screening of crude extract
12 II. REVIEW OF LITERATURE
Al-sayed et al. (1989) examined chemical composition of essential oil of C. botrys found in Saudi Arabia. Rich amount of oil (2% v/w) were extracted from the plants. GC, GC-MS and spectroscopic study of gross terpenoids components were carried out. Among sesqui-terpenes α and β˗eudesmol were present in major amount. Antimicrobial potential of essential oil was also performed.
Bedrossian et al. (2001) analyzed the chemical composition of essential oil of C. botrys collected from California (central Sierra Nevada range). It was observed that 90 % compounds present in the essential oil belong to oxygenated sesquiterpeniods. Among the identified compounds present in the oil α and β˗chenopodiol, eudesma˗3, 2- dien˗6α˗ol, botrydiol, elemole, elemole acetate, γ˗eudesmol, α and β- eudesmol were present 36%, 9.4%, 9%, 6.5%, 5.5%, 5.4% and 3.7% respectively. Two novel alcoholic sesquiterpene, Guaia-3 9-dien-2-ol and eudesm-3-en 4α or 6α-diol were identified present in the oil and their structure were established from the analysis of their single crystal X-ray diffraction pattern.
Feizbakhsh et al. (2003) studied essential oil chemical composition of C. botrys by GC/MS, isolated from two different sites in Iran. Among all the components of the oil extracted from plants of different locations, thirty five and thirty components were identified representing eighty one and seventy one percent respectively. Among all the components of these two oils juniper camphor was (16 to 25%), elemol (13 to 14%) and α-cadinol (8 to 11%) respectively.
Maksimovic et al. (2005) examined that the upper aerial part of C. botrys contain essential oil, which after isolation (0.43%w/w) exhibited significant role against selected strains of fungi and bacteria, viz. Bacillus subtilis, Aspergillus niger, Klebsiella pneumonia, Candida albicans, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Sarcina lutea, Salmonella enteridis and Shigella flexneri.
Nahar and Sarkar (2005) isolated novel phenolic glycoside named as cheo- albuside from the methanolic extract of seeds of C. album. Its structure was determined by using ultra violet light, mass spectroscopy and Nuclear magnetic resonance
13 spectroscopy while antioxidant effect was estimated by using DPPH assay. The new compound showed significant radical scavenging activity as compared to standard.
Chalabian et al. (2006) analyzed essential oil of C. botrys extracted from its aerial parts by using n-hexane solvent extraction and hyro-distillation methods. By using n-hexane extraction method fourteen compounds were identified which forming 91% of total oil. Among these, major compounds were chenopodiol acetate and eudesma˗3, 12˗dien˗6-ol present in 35% and 11% respectively. Oil extracted by hydro- distillation method contained thirty four compounds of which twenty nine were identified. Among these eudesmol, epi-murolol and cubenol present in 15%, 11% and 10% respectively were present as the main components. The antibacterial activity of oil against Echerichia coli, Salmonella typhi and Shigella flexneri were studied.
Emamghoreishi and Heidari-Hamedani (2006) authenticated sedative/hypnotic effect of aqueous extract, hydro-alcoholic and essential oil of Coriandrum sativum in male abino rats, injected intraperitoneally, before 30 min of pentobarbital injection. It was observed that aqueous extract, hydro-alcoholic extract and essential oil prolonged sleeping time induced by pentobarbital, at different concentrations, but main active constituents causing sedative/hypnotic effect were found in aqueous plant extract.
Tzakou et al. (2007) extracted oil from upper branches of C. botrys isolated from different region of Greece and studied by using GC and GC/MS. In the extracted essential oil, 54 compounds were identified forming 94% of total oil. Majority of compounds belong to sesquiterpenes. Among these compounds elemol acetate, elemole, botrydiol, α˗chenopodiol, β˗eudesmol and selina˗3˗11-dien˗6α˗ol were present 16%, 14%, 11%, 9%, 7% and 6% respectively.
Ibironke and Ajiboye (2007) evaluated the effect of C. ambrosioides dried leaf methanolic extract for anti-inflammatory and analgesic. Folrmalin and hot-plate test were adopted for painkiller activities while for anti-inflammatory activity cotton pellet- induced granuloma and carrageenan-induced paw edema models were used. Dose of 300-700 mg/kg showed considerable anti-nociceptive and anti-inflammatory effect.
Jabbar et al. (2007) investigated the anthelmintic assay of seed kernal Caesalpinia crista and whole plant C. album against trichostrongylid (nematodes) of
14 sheep. Both the tested plants displayed time and dose dependent lethal effects against worms, causing an increase in the rate of mortality and decline in hatching of eggs. C. crista exhibited greater lethal effects as compared to C. album in eggs hatch test, having minimum lethal concentration LC50 value of 0.134 mg/mL than LC50 value of 0.449 mg/mL respectively. In in-vivo study, significant decline in egg per gram of feces was observed as 83.3% and 93.8% with C. album and C. crista aqueous methanolic extract at 3 g/kg after 5 and 13 days post-treatment.
Nsimba et al. (2008) studied antioxidant potential of C. quinoa seeds different extracts and their fractions by using FRAP (Ferric-reducing antioxidant power), DPPH and β-carotene bleaching assays. Satisfactory antioxidant activity was shown by different extracts of seeds and it was observed that the non-phenolic compounds, as compared to phenolic compounds, exhibited major radical scavenging activity.
Shen et al. (2009) evaluated antidepressant potential of methanolic extract of Bacopa monniera and its different fractions by using forced swimming test and tail suspension test models in mice. It was observed that methanol extract, ethylene acetate fraction and butyl alcohol fraction decreased significantly immobility time in both forced swimming and tail suspension tests after five days consecutive oral administration in mice, however the same doses did not show inhibitory effect in mice against locomotor activity.
Chekem et al. (2010) examined in-vitro antifungal activities of C. ambrosioides essential oil by broth micro dilution and well diffusion methods. The in-vitro assay of essential oil against fungi was concentration dependent and the values of least inhibitory concentrations ranged between 0.20 to 2 mg/ml. The antifungal effect was assessed on stimulated rodent‟s vaginal candidiasis and it was found independent of dose concentration.
Zapata-Sudo et al. (2010) evaluated effects of Dorstenia arifolia rhizome methanolic extract on the activities of nervous system. Plant methanolic extract was investigated for anticonvulsant, sedative and hypnotic effects by using pentylene tetrazole induced convulsion, locomotor activity assessment and pentobarbital stimulated sleeping time, respectively. Dosage of 50 mg/kg of methanolic extract significantly declined locomotor activity and increased significantly the period of
15 pentobarbital induced sleeping time. In case of PTZ-stimulated convulsions significant anticonvulsant activity was found in dosage dependent manner.
Dini et al. (2010) determined the amount of antioxidant compounds and their scavenging ability of bitter and sweet seeds of C. quinoa, before and after cooking, by using DPPH and FRAP assays. The bitter seeds exhibited greater antioxidant activity as compared to sweet seeds. It was also observed that cooking caused considerable loss in radical scavenging ability of bioactive compounds.
Gupta et al. (2010) examined anti-nociceptive and anti˗inflammatory effects of methanolic extract of Murraya koenigii dried leaves in albino rats. Analgesic activity of different doses of plant extract was determined by following formalin induced paw licking method and hot plate method, while anti-inflammatory activity was evaluated by following carrageenan-induced paw edema model. Plant extract displayed significant decline in paw edema stimulated by carrageenan and percent increase in pain and reaction time in formalin test and hot plate. The anti-nociceptive and anti- inflammatory effects depend upon the dose of methanolic extract of plant as compared to control and diclofenac sodium, as standard drug.
Hallal et al. (2010) investigated analgesic and antipyretic effect of C. ambrosioides fresh leaf water extract by using acetic acid, hot plate and yeast tempted pyrexia model in rats. Dose of 100 to 300 gm/kg of extract showed noticeable analgesic effect in both acetic acid and hot plate models. Similarly significant inhibitory effect was observed against pyrexia induced by yeast.
Pasko et al. (2010) determined the consequences of C. quinoa seeds in food on certain biochemical factors and vital elements present in the serum of rodents fed with elevated fructose. These include its effect on protein metabolism, glucose level, lipid metabolism and certain elements like Mg, Ca, K, Na level. Fructose caused a notable (p<0.05) decline in the level of low density lipo-protein (LDL) (42%) and alkaline phosphatase activity (p<0.05, 21%) while amplified level of triglcerides (p<0.01, 80%). The investigation of blood of rodents displayed that its seeds significantly declined blood level of cholesterol (p<0.05, 26%), LDL level (p<0.008, 57%) and triglycerides (p<0.05, 11%) as compared to untreated group. It also considerably declined blood cholesterol (p<0.01, 10%) and level of protein (p<0.001, 16%). It also caused an
16 effective decline in the level of HDL (p<0.05, 15%) but its seed after addation stopped its decreasing.
Amjid (2011) investigated pollens allergenicity in C. botrys and C. album. Pollen grains were congregated from vicinity of Kandovan, Karaj and Tehran and by mixing with phosphate-buffered saline (PH 7.4) extract were prepared. The pollen extract of these plants were give to male guinea pigs after which tests of skin were executed and measured based upon wheal diameter. The blood was taken directly from, pollen extract treated; guinea pig sera and heart obtained from test samples were kept for analysis at -20 oC. The allergenic sensitivity for C. botrys and C. album pollens observed during skin prick test was with average wheal thickness of 4 cm and 2.5 cm respectively. Blood analysis indicated an increase in the amount of IgE, in the number of eosinophils and neutrophils after treatment with pollen extract as compare to control group.
Foroghi et al. (2011) determined chemical composition of C. botrys by using gas chromatography and spectrometry. It‟s essential oil antibacterial activities were determined by employing agar well diffusion and agar disk methods. Macro broth tube test was carryout to indicate MIC. The common compound found in essential oil of C. botrys was α-eudesmol and it was studied that its oil having concentration 0.007 g/ml caused deterrence in growth of Staphylococcus aureus and Escherchia coli.
Song et al. (2011) investigated anti-diabetic activity of C. ambrosiodes in mice as test animal. Diabetes mellitus was induced by streptozocine (STZ) injection after feeding for two with high-fat diet. Different concentrations of crude extract (100, 200 and 300 mg/kg) exhibited considerable hypoglycemic role as contrast to control.
Mahboubi et al. (2011) studied chemical ingredient of hydro-distilled essential oil extracted from aerial branches of C. botrys by GC and GC-MS. By using micro broth dilution and disc diffusion methods antimicrobial assay of essential oil was analyzed against various kinds of microbes. Total of 43 components were identified which form 98% of essential oil. Among these, major compounds were 2, 3-dehydro-4- oxo-β-lonone and 7-epi-amiteol form 22.4% and 11.5% of total oil respectively. Strong antimicrobial activity was shown by essential oil against Klebsiella pneumoniae, Staphylococcus saprophyticus, Bacillus cerus, Streptococcus mutans, Staphylococcus
17 epidermidis, Salmonella typhimurium and Listeria monocytogenes. The growth of Aspergillus was inhibited while Candida albican was less affected by essential oil of plant.
Yousefei et al. (2011) inspected the consequences of different heavy metals on development of anthers and pollen grains of C. botrys at various developmental stages. As it is hyper accumulator for copper and fairly accumulator for manganese, iron, zinc reported by early studies, the effects of heavy metals individually were determined on the assembly growth and structure of anthers and pollen grains. This study was carried out in the nearby area of copper and iron mine where the amount of heavy metals was higher than the normal soil. C. botrys plants were grown on polluted and non-polluted soil and were observed for structural and developmental studies. The study of formation of anther in plants developed on polluted soil showed resemblance with that of plants grown on non-polluted soil, however developmental stages of anther and pollen were affected. The effects of heavy metals were stabilizing of tapetum layer, rising in tapetm layer number, thickening of callose wall in microspores mother cell stage, decreasing and varying size and shape of anther. Heavy metals also caused decline in number of pollen in plants grown on heavy metals contaminated soil.
Hazrat et al. (2011) performed ethno botanical investigation of some vital therapeutic plants in the area of Dir and Kohistan of Khyber Pakhtun Khwa (KPK), North West province of Pakistan. During the study total of forty species belonging to different families, including C. botrys, were found to be utilized by the local people as folkloric medicine for curing various ailments.
Akuodor et al. (2011) studied leaf methanolic extract of Bombax buonopozense for anti-diarrheal potential induced by castor oil, enteropooling and intestinal motility assay in rodents. The effect of methanolic extract was dose dependent and significantly decreased rate of feces, enteropooling and intestinal fluid motility. The LD50 value calculated in mice for methanolic extract was larger than 5000 mg/kg.
Guo et al. (2011) investigated anticonvulsant, antidepressant and potential bioactive components of Abelmoschus manihot ethanolic extract. It was observed that plant extract defended mice against pentylenetetrazole induced clonic, convulsion and mortality. Also caused decrease in immobility time in force swimming test in the tested
18 animals. The constituents detected in the ethanolic plant extract were hyperoside, isoquercitrin, hibifolin, quercetin˗3˗о˗glucoside and quercetin.
Gesinski and Nowak (2011) studied the yield and content of amino acid in protein obtained from the seeds of C. album and C. quinoa. It was observed that the proportion of amino acid in the protein obtained from the seeds of C. quinoa was greater than that of C. album. The biological value of C. quinoa protein, calculated with essential amino acid index, was significantly greater than that of C. album.
Pal et al. (2011) studied acetone and methanol extracts of dried plant of C. album against liver toxicity induced by paracetamol. Dosage of 200˗400 mg/kg exhibited noteworthy hepatoprotective effect as compared to standard drug silymarin and caused a remarkable decrease in the amount of enzymes like serum glutamate transaminase, serum glutamate oxaloacetate. Also caused decline in the amount of serum acid phosphatase, serum alkaline phosphatase and bilirubin. Plant methanol and acetone extracts inhibited prominent amount of lipid perioxide and restored the normal level of antioxidants. Hepativeprotective effect of acetone and methanol plants extracts were also confirmed from histopathological investegations.
Nigam and Paarakh (2012) studied the anti-diarrheal potential of hydro alcoholic extract of C. album aerial branches against diarrhea induced by castor oil using rats as model animal. It was observed that in comparison of standard reference drug i.e. loperamide (4 mg/kg), the potential of plant hydro alcoholic extract was dose dependent and amount of extract from 200 to 400 mg/kg showed considerable anti- diarrheal effect and caused notable decline in facial output and dropping frequency.
Amjid and Alizad (2012) studied the antibacterial effect of flower and leaf ethanolic and methanolic extracts of C. album against Staphylococcus aureus,Pseudomonas aeruginosa Escherichia coli and Bacillus cereus andby disc and well diffusion method. It was noted that both methanol and ethanol extracts of flowers and leaves did not show significant activity against the selected strains of bacteria.
Dwivedi and Singh (2012) evaluated antibacterial potential of Chenopodium murale leaves methanol and aqueous extracts against important five pathogenic bacteria i.e. Escherichia coli, Staphylococcus aureus, Pseudomonas aueruginosa, Salmonella
19 typhimurium and Proteus vulgaris by using disc diffusion method. Leaves methanol extract showed significant degree of inhibition against S.aureus while leaves aqueous extract ehibited strongest inhibition against P. aeruginosa.
Brend et al. (2012) determined the consequences of cooking and baking process on ferric reducing ability of plasma antioxidant activity and total phenol and flavonoids content of yellow and red C. quinoa plant seeds. Red quinoa seeds showed significant antioxidant activity and notable amount of total phenols and flavonoids, as compared to yellow quinoa plant seeds, thus might play significant role in the deterrence of chronic ailment linked with free radicals.
Ahmad et al. (2012) inquired analgesic and spasmolytic effect of ethanol extract of C. album and its, water, ethyl acetate, n-butanol and chloroform fractions. Crude extract of plant showed dose dependent increase of smooth muscles relaxation, while among the different fractions of C. album, n-butanol exhibited efficient relaxant activity. Analgesic potential of plant crude extract was evaluated by following tail flick method using mouse as model animal. Crude extract of 500 mg/kg dose exhibited considerable analgesic activity.
Nayak et al. (2012) studied a range of extracts of above ground parts of C. album for its hepativeprotective activity on liver toxicity tempted by CCl4 in rats. All the exracts (ethyl acetate, ether and methanolic extracts) of aerial plants parts exhibited significant hepativeprotective activity and caused considerable decrease in the elevated amount of serum glutamic pyruic transaminase, serum glutamic oxaloacetic transaminase, total cholesterol and bilirubin. It was observed from histopathological study that among all the extracts, methanolic extract showed significant activity against liver toxicity which was equivalent to silymarin used as standard drug.
Jain and Singhai (2012) studied radical inhibiting assay of leaves extract and different fractions of C. album and their effects on liver against toxicity tempted by carbon tetra chloride in model animals. It was observed that ethanol fraction was found effectual than the other tested extracts and fractions against DPPH and superoxide free radicals. The in-vivo investigation showed that ethanolic extract at concentration of 100, 200 and 400 mg/kg provide considerable protection against hazardous effects of carbon tetra chloride and displayed significant hepative-protective activity.
20 Sousa et al. (2012) extracted Kielmeyera neglecta Saddi and C. ambrosioides with diverse solvents like dichloromethane, hexane, ethyl acetate and ethanol. These different extracts were evaluated for their action against brine shrimp, antimicrobial potential and antifungal effect against Neurospora crassa cell wall. All the extracts of C. ambrosioides exhibited inhibiting potential against brine shrimps which might be due to cytotoxic effect against cancer cells, while that of K. neglecta only ethyl acetate and ethanol extracts were effective. Hexane and dichloromethane extracts of C. ambrosioides showed prominent antifungal activity against Candida krusei having minimum inhibition concentration value of 100 μg/mL.
Gawik-Dziki et al. (2013) assessed anticancer and antioxidant effect of leaves extract of C. quinoa through estimation of its phenolic constituents, analysis of its phenolic compounds effects on cancer cells properties and assessment of in-vitro antioxidant effects, bioavailability and bio accessibility. Significant amounts of rutin, isorhamentin, kampferol, gallic acid, sinapinic and ferulic were studied in the leaf extract. These compounds showed an inhibitory consequence on cellular ability for gap junctional communication and proliferation of prostate cancer cell.
Song et al. (2013) explored in-vitro hypoglycemic consequences of crude extract of C. ambrosioides, using mice as model animal. Mice were treated with streptozocine to induce diabetes. Treatments of 100 to 300 mg/kg of plant crude extract displayed considerable anti-diabetic effect as compared to control.
Aziz and Khan (2013) investigated sedative-hypnotic activities of Lycopus europaeus methanolic extract on central nervous system by using thiopental induced sleeping time and hole board methods. Diazepam was employed as standard reference drug to which the activities of plant extract were compared. Plant extracts of 800 to 1000 mg/kg significantly showed sedative-hypnotic activities as compared to standard drug.
Gqaza et al. (2013) analyzed and compared nutritional constituents of C. album young body parts and mature leaves. The contents of vitamin, carbohydrates, protein, fiber, amount of macro elements i.e. potassium, calcium, magnesium, amount of micro elements i.e. zinc, iron, copper and amount of rare elements i.e. arsenic, chromium, tin, in both young plant parts and mature leaves, were almost similar and no significant
21 difference was presents. It was inferred that both, young plant parts and mature leaves, served as important source of essential dietary nutrients.
Dziki et al. (2013) studied in vitro anticancer and antioxidant study of leaves extracts of C. quinoa. It was observed that notable amount of rutin, isorhamnetin, kaempferol, gallic acid, sinapinic and ferulic were present in the C. quinoa leaf extract having significant anti-carcinogenic, chemo-preventive and radical scavenging properties.
Panday and Gupta (2014a) estimated nutritional constituents of C. album in varying solvents (ether, methanol, petroleum, ethyl acetate, dichloromethane and water). Nutritional investigation revealed that C. album can act as vital resource of vigor, carbohydrate (glucose), proteins, beta˗carotene, ascorbic acid, micro amd macro minerals i.e. iron, zinc, magnecium, calcium, potassium and soudium.
Andov et al. (2014) in Macedonia analyzed essential oil extracted from upper branches of C. botrys isolated from five different sites and were studied using gas chromatography (GC) and mass spectroscopy (MS). Total compounds identified were seventy five, representing 90 to 91% of the oil. Its chemical composition has shown that sesquiterpene components were present in rich amount i-e. (83%-87%) containing seline-11-en-4α-ol (9.81%-13.5%), elemol acetat (9.88%-21.98%), elemol (5.57%- 9.49%) and selina˗4, 12˗dien-6α-ol (6.42%-9.71%) as major oxygen containing sesquiterpenes. The components of oil present in lower amount were α-chenopodiol (2.42%-5.43%), α-eudesmolacetat (3.24%-4.11%), α-chenopodiol-6-acetat (1.9%- 4.73%) and botrydiol (1.87-5.73%).
Karchegani et al. (2014) collected three ecotypes of C. botrys and analyzed its chemical components by using GC/MS. The plants collected from Fars, Mazandaran and Isfahan contained 19, 21, and 39 compounds respectively in upper parts which were identified and analyzed. The plants collected from Fars contained α-pinene (18.292%),Camphor (20.047%) and 1,8-Cineole (27.650%).The plants of Mazandaran were consist of Camphor (10.509%), β-Myrcene (11.246%) and 1,8-Cineole (39.873%) and the main chemical compounds present in plants of Isfahan were 1,8-Cineole (10.823%), β-Mycene (11.250%) and Camphene (24.785%).
22 Saleem et al. (2014) studied hepativeprotective activity of C. murale aqueous extract in mice used as model animal. It was observed that plant extract had a significant hepativeprotective effect and caused inhibition of the paracetamol stimulated high level of different enzymes in liver like aspartate transaminase, alanin transaminase, alkaline phosphatase and total bilirubin. The notable hepativeprotective activity of plant extract was confirmed from histopathalogical study and investigation of phytoconstiuents.
Kumar et al. (2014) studied chemical composition and anthelmintic effect of C. album leaves crude powder, methanol, diethyl ether and aqueous extract in different concentrations. Phytochemical study of its water extract had revealed the existence of triterpenes, sterols, resins, flavonoids, tannins, saponins and alkaloids. The anthelmintic effect, at 0.5%, 1% and 2% concentrations of all extracts, was almost 100%. The overall anthelmintic effects of crude and water extract was more effectual than methanol and diethyl ether extracts.
Parkash and Patel (2014) investigated the effect of different concentration of C. album leaves extracts against two gram positive bacteria B. subtilis and S aureus and two gram positive bacteria E. coli and P. aeruginosa. Significant antibacterial activity was shown by the leaves extract against all types of bacteria.
Panday and Gupta (2014b) evaluated antibacterial potential of C. album plant extracts in dissimilar solvents i-e. ether, petroleum, methanol, ethyl acetate, dichloromethane and water, against B. subtilis, E. coli, S. epidermidis and S. aureus. Among all extracts, methanolic plant extract exhibited significant antibacterial effect against all types of bacteria. The effect of extract of mixture of all solvents was found to be significant stopping the growth of S. aureus.
Panday and Gupta (2014c) evaluated antioxidant potential of C. album, using petroleum, dichloromethane, methanol, ether, ethyl acetate and water plant extracts in equal proportion. Among all extracts of C. album, aqueous, methanol and petroleum ether plant extracts exhibited significant antioxidant potential by using FRAP (ferric reducing antioxidant assay) and ABTS assays.
23 Nedialkova et al. (2014) investigated pharmacognostic properties of C. foliosum aerial parts and evaluated antioxidant properties of five flavonoids, isolated from plant methanolic extract, by using DPPH and ABTS radical scavenging assays. It was observed that plant extract had significant free radical scavenging properties and could be used as neutraceuticals with antioxidant properties.
Yao et al. (2014) determined four saponins fractions by HPLC-MS, extracted from the seeds of C. quinoa and investigated the anti-inflammatory effect of saponins fractions on RAW 264.7 macrophages cells. They observed a significant decrease in production of number of inflammatory mediators. The effect of fractions was dose dependent, caused the inhibition of secretion of inflammatory cytokines including tumor necrosis factor-α and interleukin-6.
Ullah and M. Ahmad (2014) determined hepativeprotective effect of ethanolic extract of C. mural whole plant in rodents intoxicated by carbon tetrachloride. The boosted level of serum markers like serum glutamic pyruvic transaminase, serum glutamic oxaloacetic transaminase, alkaline phosphatase and total bilirubin induced by carbon tetrachloride was effectively declined by plant extract, however the effect of 500 mg/kg of was highly significant (p<0.001). Histopathological investigation of liver tissue further validated its liver defensive effects.
Moilo et al. (2014) explored the in-vitro and in-vivo anti-schistosomal effect of extract of C. ambrosioides in mature worms. Different parts of plant (fruit, leaves, stem and roots) were soaked and extracted using different solvents. Leaves and fruits crude extract displayed remarkable effect (p<0.05) causing reduction in egg number. Among the different solvent fractions, hydrated fraction of leaf and methanol fraction of fruit caused 46% and 23% decline in the number of worms. The in-vitro observations depicted that fruit methanol extract executed greater number of worms (adult) as compared to leaf aqous extract. In case of fruit methanolic extract and leaf aqous extract effect on amature worms of S. mansoni, the fruit methanolic extract showed greater potential than leaf aqous extract. The mortality consequence of methanolic extract of fruit and aqueous extract of were leaf were statistically comparable to praziquantel.
24 Nowak et al. (2015) checked the antioxidant and cytotoxic abilities of bioactive molecules taken out from various parts of four different plants belong to genus Chenopodium. Greater amount of phenols was observed in seeds and herbaceous plants while highest level of free polyphenols were found in seeds and roots of C. urbicum (3.87 mg/g, 1.52 mg/g DW) respectively and extracts of C. album (3.36 mg/mg DW). Among the different extracts analyzed, C. urbicum and C. rubrum had the highest antioxidant activity. Significant anti-proliferative activity was shown, on the TOV-112 cell line, by the extract of seeds and herb of C. album and C. hybridum.
Parkash and Patel (2015) investigated the protective effect of extract of C. album leaves on liver against the toxicity induced by CCl4 in rodents. They studied SGPT, SGOT, alkaline phosphate, bilirubin and amount of protein in blood of various treatment groups. The leaves extract of C. album caused considerable decline in the level of mentioned serum enzymes and protein and its result was equivalent to that of silymarin. Its defensive effect was also confirmed by histopathalogical examination of cells of treated and control rodents.
Rahman et al. (2015) estimated the cytotoxic and anti-diarrheal activities of Maranta arundinacea leaves methanolic extract in brine shrimp and rats respectively. Cytotoxic activity was carried out by using lethality assay of brine shrimp while anti- diarrheal activity was studied by following enteropooling assay, castor oil induced assay and gastrointestinal motility assay in rodents, by using different concentration of plant extract. Methanolic plant extract between 200 to 400 mg/kg significantly inhibited diarrhea in all the three mentioned assays. It was also proved by cytotoxic assay that highest does of plant extract was not harmful to mice.
Bahekar and Ranjana (2015) examined effect of ethanolic extract of leaves of Manihot esculenta on diarrhea in rats used as model animal. The study was carried out by using castor oil stimulated accumulation of liquid in intestine and charcoal passage assay by using standard drug of Loperamide (5mg/kg) and atropine sulfate (5mg/kg) respectively. Leaves ethanolic extract significantly decreased accumulation of intestinal fluid and gastro-intestinal mobility in the test animals.
Socala et al. (2015) explored scientifically anticonvulsant, antidepressant and anxiolytic activities of cultured Ganoderma lucidium water extract in mice. For
25 evaluation of anticonvulsant activity, timed intravenous pentylenetetrazole infusion, maximal electroshock seizure (MES) and 6Hz psychomotor seizure models were used. For anxiolytic and antidepressant effects elevated plus maze assay (EPM) and forced swim test (FST) were evaluated. Plant water extract of 100 to 400 mg/kg increased appreciably threshold values for psychomotor seizure in 6 Hz seizure test, similarly the same amount of plant extract significantly decreased the time interval of immobility in the forced swim test.
Calado et al. (2015) examined anti-inflammatory and anti-nociceptive effects of hydrochloric crude extract (HCE) of C. ambrosioides in rodents by using osteoarthritis model. In order to compare ascaridole (monoterpene), which is found in hydrochloric crude extract, with NMDA receptors, molecular docking was carried out. After three days of treatment, HCE caused a decline in the knee edema. The HCE5 exhibited lower cellular infiltrate in synovium and cartilage and less intensity of allodynia from 3rd day and that of hyperalgesia from 7th day up to day of treatment. The HCE5 and HEC50 treated groups showed improvement in forced walking. HCE was useful in the curing of osteoarthritis because it reduced effectively synovial swelling and inflammation due to severe pain.
Tang et al. (2015) conducted antioxidant assay, and characterization of betanins and phenolics of three cultivar i-e black, white and red, of C. quinoa seeds. It was surveyed that the amount of phenols were different in the three cultivars each having radical scavenging properties, however the amount of phenols in the seeds of black colored cultivar were greater having significant antioxidant activity.
Nowak et al. (2016) appraised antioxidant and cytotoxic abilities of lipid soluble compounds of four Chenopodium species i.e. C. hybridium, C. album, C. urbicum and Chenopodium rubrum. Cytoxic assays of the four plants extracts were carried out against human lungs carcinoma A-549 and ovarian carcinoma TOV- 112D and human fibroblast cell lines and it was observed that seed extract of C. hybridum and C. album significant anti-proliferative activity on TOV-112 cell lines. The extract of C. urbicum and C. rubrum showed significant antioxidant activity among the extracts of other species.
26 Ajaib et al. (2016) evaluated the antibacterial potential of C. ambrosioides plant parts different extracts against various strains of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis by using agar well diffusion method. Bark petroleum ether extract displayed considerable inhibitory effect against B. subtilis while the hydrated extract of all parts did not exhibited any lethal effect against the selected strains.
Ozer et al. (2016) investigated the composition of phenol, enzymes inhibitory and antioxidant assays of water and ethanol extracts of C. botrys. Amount of saponins, tannins and flavonoids found in ethanolic were greater than present in water extract, however in case of phenol, its amount was higher in the water extract. Plant extracts were also screened for quantitative analysis of some selected compounds. Benzoic acid, among all compounds, was found to be the most plentiful compound in both extracts. Antioxidant effect of aqueous extract was greater as compared to ethanolic extract. Enzyme retarding effects of both extract were also studied on α-glucosidase, α-amylase, tyrosinase, butyryl cholin esterase (BChE) and Acetyl choline esterase (AChE). Its water extract displayed greater inhibitory on Acetyl choline esterase (AChE), tyrosinase, α-amylase, and α-glucosidase respectively. However the enzyme inhibitory activity of ethanolic extract on butyryl cholin esterase (BChE) was greater than water extract.
Abdollahnejad et al. (2016) investigated the sedative-hypnotic potential of Aloe vera hydrated extracts on rats. For the study of hypnotic effect of plant extract two tests i.e. open field and loss of righting reflex, were employed. Sedative-hypnotic activities of plant extract in the test animals were confirmed by investigation of electroencephalographic (EEG) recordings, according to which there was a simultaneous variation in rapid eye movement and non-rapid eye movement sleep in similar with total prolonged sleeping time. It was confirmed from the investigation that plant extract had sedative-hypnotic activities on electrical and functional actions of brain.
Santiago et al. (2016a) evaluated antibacterial potential of essential oil of C. ambrosioides against L. monocytogenes, S aureus, S. choleraesuis and E. coli by using agar cavity diffusion method. Essential oil exhibited significant inhibitory effect against all type of bacteria and rang of minimal inhibitory concentration was from 62.5-250 μl ml-1.
27 Santiago et al. (2016b) examined the antioxidant potential of C. ambrosioides essential oil extracted through hydro-distillation technique. The radical inhibiting consequence of essential oil was determined by checking the reduction of DPPH and by oxidation of β-carotene-linoleic acid assay. Synthetic butylated hydroxyl-toluene was for comparison in the same amount as that of essential oil. The antioxidant activity -1 exhibited by essential oil was with IC50 value equal to 455.7 μg ml .
Ajaib et al. (2016) investigated antioxidant activity of C. ambrosioides bark and fruit different extracts, by using ABTS, DPPH and metal chelating assays. It was inferred that aqueous extract of fruit and bark displayed significant antioxidant activity in ABTS and metal chelating assays while minimum activity in case of DPPH assay. Antioxidant activity of petroleum ether was observed highest in case of DPPH assay.
Teware (2017) investigated antibacterial effect of C. album extract against Proteus mirabilis and Streptococcus mutants. Ciprofloxacin was used as standard drug to which the activity of plant extract was compared. It was observed that antibacterial effect of plant extract was significant against both types of bacteria.
Zoufan et al. (2017) investigated heavy metal effects on the antibacterial activity of C. murale and other plants methanolic and ethanolic extracts, growing in polluted area near steel industry. The antibacterial activity was evaluated by using disc diffusion method against S. aureus, B. subtilis, E. coli and P. aeruginosa. Significant antibacterial potential was exhibited by methanolic and ethanolic extracts of C. murale and other plants against P. aeruginosa and E. coli.
Rauf et al. (2017) analyzed phytotoxic, cytotoxic and antibacterial activities of C. botrys, Teucrium stocksianum and Micromeria biflora found in Pakistan. Lemna acquinoctialis based phytotoxic activity, Brine shrimp cytotoxic activity, and agar well- diffusion method was used to carry out to evaluate phytotoxic, cytotoxic and antibacterial activities respectively. Baccilus subtilis and Klebsiella pneumonia showed noticeable venerability in response of aqueous and crude extract of the selected plants. Significant cytotoxic and phytotoxic activities were exhibited by its methanolic and aqueous extracts depending upon its concentration. Results obtained had shown that the abovementioned plants have greater cytotoxic, pytotoxic and antibacterial effects which
28 might contribute an imperative role in research and advancement of novel therapeutic agents.
Bojilov et al. (2017a) extracted volatile oil from the upper branches of C. botrys by hydro-distillation and studied by GC-MS and GC-FID. C. botrys was collected from six different location of Southern Bulgaria. Fifty three compounds were identified found in the oil extracted from C. botrys of different regions. There was only quantitative distinction in the chemical makeup of oil extracted from plants of different location. Majority of identified components were belonging to oxygenated sesquiterpenes (69% to 84%). The abundant components found in the oil were elemol acetate (14%to 26%), elemol (10%to 18%), α-eudesmol (7%to 17%), juniper camphor (3%to 11%), α-eudesmol acetate (5%to 6%), α-chenopodiol (4%to 6%). γ-costol was the new compound identified for the first time, present in the essential oil of C. botrys.
Bojilv et al. (2017b) analyzed composition of flavonoids in C. botrys by using different extraction and detection methods. For polyphenols primary extraction with two dissimilar solvents, HPLC/PDA fingerprint profiling and Orbitrap UHPLC-MS/MS detection were used. Study of fingerprint profile demonstrated that main components of polyphenols were jaceosidin, hispidulin, nepetin and methoxylated flavones while quercetin glycoside was present in least amount. For structural elucidation, ESI-MS analysis was used for examination of fragmentation of compounds. Novel information about methoxylated flavones fragmentation pathways was reported. Some components like quercetin-o3-O-galactoside, rutin, eupatilin, nobiletin and nepetin were identified for the first time in C. botrys polyphenols complex.
Ajayi et al. (2017) evaluated hydrated leaves extract of C. opulifolium for their anti˗inflammatory and analgesic effects in model animals. It was presumed that dosage of 100-400 mg/kg effectively reduced the intensity of pain both in the hot-plate and writhing models, however displayed no effect on the locomotory assay in the treated animals. The leaves extract notably (p<0.05) decrease (44.2%) paw edema stimulated by egg albumin after 12 minute assessment time interval.
Ahmad et al. (2017) studied chemical composition, separation and recognition of gallic acid and scopoletin in methanolic extract and subsequent fraction of n- butanol, ethyl acetate, chloroform and petroleum ether of C. murale growing in Iraq.
29 The various spectroscopic and chromatographic analyses revealed the presence of coumarin and galic acid. Gallic acid and scopoletin were found in the ethyl acetate solvent fraction of C. murale.
Wu et al. (2017) explored anti-inflammatory and antioxidant assay of leaves extract of C. quinoa by using water (cold), ethanol (95%), and methanol (50%). The crude extracts of methanol and ethanol at concentration of 50 mg/mL exhibited considerable scavenging effect against DPPH radicals causing 53% and 54% inhibition. Mthanol and aqueous extracts having concentration of 10 mg/mL showed elevated capacity of ferrous ion celating (26% and 29% respectively). Accumulation of nitric oxide is considerably suppressed by extract of ethanol (95%) and methanol (50%) in RAW (264.7) a cell, stimulated by LPS at concentration of 1 μg/mL and inhibitory effect of nitric oxide synthesis was represented in dose (concentration) dependent manner.
Oliveira et al. (2017) studied in-vitro effect of C. ambrosioides extract on cattle ticks (Rhipicephalus microplus). Total of 125 females animals were selected and divided into 5 groups on the basis of their weight, for the purpose to make sure that females employed for the experiment had a uniform weight. The treatments consist of C. ambrosioides (40 and 60%), ethanol, distilled water and amitraz (12%). It was observed that groups treated with ethanol and distilled water, 88% and 92% of female respectively sustained oviposition. While the effect of plant extract against the females cattle ticks was extremely significant (p<0.001) and the dosage of 40 and 60 plant extract, reduced the oviposition percentage up to 36 and 4 respectively.
Ren et al. (2017) studied lunasin content in C. quinoa and evaluated its radical inhibiting and anti-inflammatory properties. The contents of lunasin in fifteen different plant samples were found between 1.01× 10-30 g kg-10 to 4.89 × 10-30 g kg-1 plant dry seed. There was a considerable (p<0.05) difference in the contents of lunasin among various varieties of the similar region and the same variety of different regions. The purified isolated lunasin showed lower radical scavenging activity against DPPH free radicals, however exhibited strong activity against ABTS+ radicals. It also stopped the synthesis of nitric oxide, interleukin-6 on lipo-polysaccharide induced paw 374.6 macrophages and tumor inducing factor- α by up to 44.8%, 33.5% and 39.9% respectively.
30 Rios et al. (2017) determined the action of alcoholic aqueous crude extract of C. ambrosioides and its hexane fraction on bacterial growth, phagocytes activation and management of inflammatory responses by using model animals. It was noted that hexane fraction stopped growth of bacteria and inflammatory responses by the stimulation of phagocytes. However, alcoholic aqueous crude extract and hexane fraction treatment caused an elevation in ex-vivo secretion of nitric oxide and hydrogen peroxide by phagocytes. It also caused a decline in the serum level of pro-inflammatory cytokine, showing a systematic inhibitory effect on inflammation.
Sayyedrostami et al. (2018) explored the effect of essential oils of C. botrys leaves wound healing potential in rats. The animals were alienated randomly in to four groups, each group containing six animals, including control group, test groups and standard group. After treatment, the animals administrated with C. botrys caused a reasonable (p<0.01) decline in the area of wound as compared to untreated, basal cream and antibiotic (tetracycline) treated groups. Certain parameters like alignment of curing tissue and formation of epithelial layer in plant administrated animals displayed a notable increase in comparison with untreated animals. Its extract also narrowed the surface area of wound and count of neutrophills, lymphocytes and elevated effectively (p<0.01) the proportion of collagen and blood vessels number.
Kaur et al. (2018) determined the antibacterial consequences of diverse extracts of C. album extracted from three unlike solvents i.e. chloroform, acetone and methanol. Their detrimental effects were noted against B. subtilis, E. coli and L. bacillus, by employing well diffusion method. The 100% concentration of various extracts of plant exhibited highest potential against bacterial strains. It caused greatest inhibition activity against E. coli and L. bacillus caused 19 mm zone of inhibition, however exhibited no activity against B. subtilis.
31 III. MATERIAL AND METHODS 3.1 Plant collection and identification
The proposed study was conducted at Department of Pharmacy, University of Peshawar and Department of Pharmacognocy, Faculty of Pharmacy, University of Karachi, Pakistan. The specimen of D. botrys was collected from various parts of District Swat, Khyber˗Pakhtunkhwa (KPK), particularly from marginal areas of river Swat. The plant was identified by plant taxonomist Dr. Zahid Ullah, Center for Plant Sciences and Biodiversity, University of Swat. Specimen of plant having voucher number Swat000411 was deposited in herbarium of University of Swat for future reference.
Fig. 3.1. Herbarium specimen of D. botrys
3.2 Extraction of plant material
Plant material was cleaned with tap water and then dried out in shade at normal temperature for seventeen to twenty days time period. The dried plant material was minced in a Willy mills after which, 9.2 kg of powdered plant material was obtained. It was then mixed and extracted three times with 80% methanol for 72 hours at room temperature with occasional shaking using maceration method. The combined filtrates
32 were concentrated at 40-45 °C using rotary vacuum evaporator (Buchi, Switzerland) and the final residue formed was 790 g, which was the methanolic crude extract (MCE). The crude extract of whole plant was alienated into two parts, 300 g were used for in-vivo pharmacological activities while the remaining 490 g were added with water (distilled) for further fractionation in various solvents i.e. n-hexane fraction (HxF), dichloromethane fraction (DCMF) and ethyl acetate fraction (EAF) depending upon their elevating polarity.
3.3 Fractionation procedure
Plant methanolic extract (490 g) was mixed with distilled water (1 liter) and moved to separatory funnel for further fractionation. One liter solution of n-hexane was poured to separatory funnel and mixed strongly. After putting the solution on a stand, two layers were formed in which the outer layer was formed by hexane which was alienated from the lower layer. This procedure was performed three times and the pooled outer layers were condensed to 14 g n-hexane fraction, employing rotary evaporator at 40 °C temperature. After partition of n-hexane fraction, the similar method was pursued for DCM and EA solvents, yielded 31 g DCM and 34 g EA fraction (Wagenen et al., 1993). Crude plant extract and the three solvent fractions were utilized for phytochemical analysis and in-vitro activities.
33 Fig. 3.2. Scheme of extraction and fractionation process
34 3.4 Phytochemical investigation
3.4.1 Quantitative analysis of phytochmeicals
3.4.1.1 Stock solution
Stock solution was synthesized by dissolving 1 g of plant extract in 100 ml of individual solvents. The prepared stock solution was utilized for testing of secondary metabolites such as phenols, alkaloids, flavonoids, saponins, tannins and sterols by following the standard protocols. All the chemicals used for the detection of active biological molecules were of analytical grade.
3.4.1.2 Test for crude alkaloids
Alkaloids were detected in the plant extract by using Mayer‟s test. Dried plant extract of 50 mg and 10 ml dilute HCl was mixed through regular stirring and was then filtered. Two drops of Mayer‟s reagent were mixed to solution present in test tube. Formation of white color precipitates proved alkaloid presence (positive). Mayer‟s reagent employed in the test was of commercially grade (Tiwari et al, 2011).
3.4.1.3 Test for saponins
Saponins were detected by using Frothing test. Plant extract of 50 mg was diluted with distilled water up to 20 mL. Then the solution was poured in graduated cylinder and shake for 15 minutes. Formation of layer of foam indicated the presence of saponins (Kokate, 1999).
3.4.1.4 Test for phenols
Plant samples of 500 mg were mixed with 5 ml of distal water. Aqueous filtrate of each solvent was mixed with ferric chloride 2ml (5%) solution inside test tube. Formation of green color designated existence of phenols (Sofowora, 1993).
3.4.1.5 Test for flavonoids
Flavonoids were detected by using Alkaline‟s reagent test. Plant extract and fractions were mixed with solution of sodium hydroxide, formation of yellowish
35 (golden) color which becomes colorless by adding CH3COOH dilute solution, indicated the presence of flavonoids.
3.4.1.6 Test for tannins
Ferric chloride test was used for tannins detection. 50 mg of each sample was mixed with in 20 ml of deionized water. Then few drops of solution of ferric chloride (0.1%) were mixed to each sample. Appearance of blue or black color confirms tannins existence (Sofowora, 1993).
3.4.1.7 Test for sterols
Salkowski test was used for the confirmation of phytosterols. Chloroform having volume of 2 ml was mixed with plant extract of 3 mg in test tube. The 2 ml of pure H2SO4 was added to it. The formation of red color in the layer of chloroform after trembling the solution for 5 minutes designated the existence of phyto-sterols (Tiwarei et al., 2011).
3.4.2 Qualitative analysis of phytochemicals
3.4.2.1 Determination of total phenol
Total phenol contents in the methanolic extract and its consequent fractions were examined following the protocol of Khan et al. (2008). 10 mg extract was mixed with Folin˗Denis reagent (5 ml) and 20% sodium carbonate (10 ml). The solution was diluted by using distal water by a factor of hundred. Solution was filtered and kept at ambient temperature for 10 minutes. Spectronic 20 D (Milton Roy) was used for the calculation of extract absorbance at 770 nm against blank. The total phenol concentration in the plant crude extract and others fractions was examined by matching with tannic acid constructed standard curve.
3.4.2.2 Determination of total saponins
Total saponins constituent in the plant extract and derived fractions were determined by Khan et al. (2010) method. Test sample of 2 g was put in small beaker and then 50 ml of petroleum ether was mixed and warmed on water bath for 5 minutes
36 up to 40 oC with usual shaking. The solution was cleaned and twice repeated the process along with additional more ether (50 ml). It was further extracted on gentle heating with methanol (5×48 ml). Then on water bath layer of methanol was concentrated up to 25 ml after which 150 ml of dry acetone was mixed for saponins precipitation. It was filtered and dehydrated to a constant weight at 90-100 oC by using oven.
3.4.2.3 Determination of total flavonoids
To study the total amount of flavonoids, 10 g of CME and its fractions were mixed with 80% methanol (10 ml). It was then filtered through filter paper i.e. Whatman No. 42 and then the filtrate was put in crucible. Then using water bath it was evaporated and then weighed (Boham and Kocipai, 1994).
3.4.2.4 Determination of total alkaloids
To examine the total contents of alkaloids in the plant extract and its solvent fractions Khan et al. (2010) protocol was followed. 2 g of each sample was defatted by dissolving in ether and warmed up to 40 oC with regular shaking for 5 minutes on water bath. The solution was then acidified by treating with 100 ml acetic acid (20%) in
C2H5OH and kept for four hours. The final solution obtained was filtered through filter paper and treated with NH4OH in order to increase its pH value up to 9 followed by precipitation.
3.4.3 Proximate composition
Whole plant of D. botrys was utilized for proximate composition to investigate moisture, crude protein, inorganic˗content (ash), ether-extract oil (lipid), fibers and carbohydrates by employing protocol reported by AOAC (2005).
3.4.3.1 Moisture content
Grinded plant sample of 2 g was put in silica dish, formerly desiccated and weighed. It was then heated in an oven at 100 oC for 2-3 hours. After heating, the sample was cooled down in a desiccator and again noted its weight. The heating and weighing of the sample was sustained until it attained a stable weight.
37 Sample + dish Wt before drying sample after drying Moisture % = x 100 Wt of sample
3.4.3.2 Inorganic matter
Crucible dish was cleaned and heated in oven then 2 g of plant sample was put into crucible and weighed. By using burner flame, the sample was heated until charred completely. Whitish gray residue was formed when the dish containing the sample was placed approximately at 560 oC for 2 hours, in a muffle furnace.
3.4.3.3 Crude lipid
Plant sample of 3-5 g was taken in an extraction˗thimble employing Soxhlet‟s- apparatus. It was kept in small unit and linked with a flask (200 ml). Ether was used for extraction. After 4-5 minutes, siphoning would occur, when the process of extraction continued for 4-5 hours. At the temperature of 105 oC the flask was dried, chilled and again weighed at the end of process.
Wt of flask + lipid Wt of empty flask crude lipid % = x 100 Wt of sample
3.4.3.4 Dietary fiber
Plant sample of 2 g was put into a conical flask. Then mixed 200 ml (1.25%) sulphuric acid and boiled the solution for 30 minutes, keeping the amount of solution at steady level by pouring warm water. After boiling solution was removed, filtered and rinsed with warm water. Then added 200 ml (1.25%) sodium hydroxide and heated for further 30 minutes. After heating the sample was chilled and immediately filtered. The insoluble filtrate was moved to sintered crucible, rinsed three times with diethyl ether, and dried at 150 oC in an oven, till the time it gained a constant weight. Then for incineration, crucible was kept at 560 oC for 1 hour in a muffle furnace.
38
3.4.3.5 Crude protein
The reported procedure of Kjeldahl (1983) was followed for determination of crude protein. Crude protein in the plant sample was examined by following the reported method of Kjedahl, having digestion system and distillation unit with titration. The crude protein was determined by multiplying 6.25 factor with percent nitrogen of sample.
S B x N x 0.014 x D Nitrogen % = x 100 Wt of sample x V
Where N: Normality of acid, D: Dilution of sample, V: Volume after digestion of digest and percent crude protein = 6.24 x % N
3.4.3.6 Nitrogen˗free extract
The sum of nitrogen˗free extract (carbohydrates) was estimated by subtracting the amount of percentages of crude lipid, moisture, ash (inorganic matter), dietary fibers and crude protein from hundred (James, 1995).
Nitrogen-free extract = 100 – (% moisture + % crude lipid + % ash + % dietary fibers + % crude protein).
3.4.4 Minerals composition
Sample of 1 g was taken in conical flask and solution of 10 ml (67%) HNO3 was mixed with it. The solution was kept at room temperature for 24 h after which 4 ml o (67%) of HClO4 was added to it. It was concentrated by heating on hot plate at 55 C until the formation of apparent solution having around 1 ml volume. After cooling the solution double deionized/distilled water was added and then filtered through filter paper (Whatman # 42). After that final solution of 100 ml was prepared by adding deionized/distilled water which can be used as stock solution (Saeed et al., 2010). The analysis of copper (Cu), chromium (Cr), cadmium (Cd), lead (Pb), zinc (Zn), iron (Fe) and nickel (Ni) was carried by employing atomic absorption spectrophotometer
39 (Polarized Zeiman-Hitachi 2000) while that of calcium (Ca), sodium (Na) and potassium (K) was performed by employing flame-photometer (Jinway PFP7, UK). The operational conditions maintained for each element on atomic absorption spectrophotometer and flame photometer are depicted in Table 3.1. Material of reference metals were purchased from Merck (Darmstadt, Germany). All the chemicals used were of analytical grade.
Table 3. 1. Conditions for operation of micro and macro minerals
Micro mineral Lamp‟s current Wavelength (nm) Silts width (nm) (mA) Cu 07.50 324.80 01.30 Cd 07.50 228.80 01.30 Cr 07.50 359.30 01.30 Fe 10.00 248.30 00.20 Zn 10.00 213.80 01.30 Ni 10.00 232.00 00.20 Pb 07.50 283.30 01.30 Macro˗minerals Filter-type Ca Ca filter 422.70 00.70 Na Na filter 589.00 02.00
K K filter 766.50 00.20
40 3.5 In-vitro studies
The detail procedure of different in-vitro activities of crude methanolic extract and solvent fractions are as follow.
3.5.1 Antimicrobial activity
3.5.2 Strains and culture media
Antibacterial activities of CME and various solvent fractions of D. botrys was studied against gram-negative bacteria such as Klebsiella pneumonia (clinical isolate), Peseudomonas aeruginosa (ATCC No. 9721), Escherichia coli (ATCCNo. 25922), Xanthomonas campestris (ATCC No. 33913), Proteus vulgaris (ATCC No.6380) and gram positive bacteria such as Staphylococcus aureus (ATCC No. 6538), Clavibacter michiganesis(ATCC No. 10202) and Bacillus subtilis (clinical isolate) followed by antifungal activities against Mucar pirimis (ATCC No. 52553), Aspergillus flavus (ATCC No. 9643), Aspergillus niger (ATCC No. 6275 ), Fusarium solani (ATCC No. 11712) and Fusarium oxysporum (ATCC No. 42355). All the bacterial and fungal strains were obtained from Department of Microbiology Quaid-e-Azam University Islamabad, Pakistan. Strains of bacteria were cultured, and kept at 38 °C (on agar slants) while the different colonies of fungi were inoculated and kept on PDA (potato dextrose agar) at temperature of 28-30 °C. Bacterial and fungal stock cultures were kept at 4 °C.
Table 3.2. Strains of bacteria for antibacterial activity
Bacterial species Gram-Type Detail Klebsiella pneumonia Nigative Clinicle-isolate, University of Peshawar Peseudomonas aeruginosa Nigative ATCC No. 9721 Escherichia coli Negative ATCC No. 25922 Xanthomonas campestris Negative ATCC No. 33913 Proteus vulgaris Negative ATCC No.6380 Staphylococcus aureus Positive ATCC No. 6538 Clavibacter michiganesis Positive ATCC No. 10202 Bacillus subtilis Positive Clinical isolate, University of Peshawar
41 Table 3.3. Strains of fungi used for antifungal activity
Fungal Species Detail Mucar pirimis ATCC No. 52553 Aspergillus flavus ATCC No. 9643 Aspergillus niger ATCC No. 6275 Fusarium solani ATCC No. 11712 Fusarium oxysporum ATCC No. 42355
3.5.3 Antibacterial activity
Antibacterial potential of plant extract was studied by using the protocol of Rios et al. (1988) with some amendment. In 1ml of dimethyl sulfoxide (DMSO), 10 mg of whole plant extract and other solvent fractions were dissolved. Inoculation of bacterial colonies was performed on sterile plates of agar by using sterilized cotton swabs in order to attain homogeneous growth. Disc (sterile) was soaked with 20 μl of MCE and other fractions, placed on the inoculated agar and incubated for 24 h at 37 oC. Cefixime was used as reference standard antibacterial drug and zone of inhibition (ZI) was noted in millimeters.
3.5.4 Antifungal activity Antifungal effect of plant extract was assessed by following Mbaveng et al. (2008) procedure. Sterilized potato dextrose (PDA) plates were inoculated with fungal cultures. Sterile disc was soaked with 20 μl of plant extract and placed on the media. At a temperature of 37 oC the plates were kept for 72 h and the same process was repeated three times. Clotrimazole was as standard antifungal drug and zone of inhibition was recorded in millimeter (mm). The process was repeated three times.
3.5.5 Phytotoxic activity
Pyhtotoxic effect of the plant MCE and various solvent fractions were investigated according to Atta˗ur˗Rahmman (1991) method, employing L. minor plant. Appropriate E-medium was prepared by dissolving different inorganic constituents in 100 ml distilled water having pH 5.5-6.5, which was adjusted by mixing solution of potassium hydroxide. It was then autoclaved at 121 °C for fifteen minutes. Sample of 20 mg/ml were mixed in methanol, serving as a stock. For each concentration three
42 separate medium sized flasks were used. These flasks were inoculated with 10, 100 and 1000 μL and kept overnight in sterile environment to evaporate the methanol. An amount of 20 ml of medium and L. minor plants (total plant used 10) each having three fresh fronds, were put in each medium flask and were reserved at ambient temperature in the growth room for seven days. Number of frond in each flask noted on the seventh day and the inhibition percentage was examined by applying the formula as under.
3.5.6 Antioxidant assay
3.5.6.1 1, 1-diphenyl-2-picrylhidrazyl (DPPH)) radical scavenging activity
The inhibiting effect of MCE and various fractions of D. botrys against DPPH free radicals were determined by employing Jain et al. (2008) procedure. Each fraction was mixed in ethanol (2˗3 ml, 20˗100 μg/ml) and then mixed with 1 ml (0.1 mM) solution of DPPH. Following an interval of 30 min, absorption was recorded by spectrophotometer at 517 nm. In case of ascorbic acid, utilized as standard, the similar method was pursued. The percent inhibiting potential was measured by applying the formula as under.
Where, Ao represents absorbance value of control while AI represents absorbance value of standard or samples.
3.5.6.2 ABTS (2,2˗azinobis˗3-ethylbenzothiozoline-6-sulfonic acid) radical scavenging assay Re et al. (1999) method was followed to explore the ABTS radical-scavenging activity of the plant extract. The cation radical i.e ABTS+ was formed by reacting 5 ml
4.9 mM potassium persulfate (K2S2O8) with 5 ml of 14 mM solution of ABTS and kept at normal temperature in dark for 16 hours. Prior to use, the concentration of solution was lowered by adding ethanol in order to acquire an absorbance value of 0.700 ± 0.020 at 734 nm. The extract of plant having different concentrations was homogenized with 1ml solution of ABTS and its absorbance was studied at wavelength of 734 nm. For every analysis ethanol blank was run and with a gap of 6 minutes, the entire
43 measurement was performed. By incorporating 950 μl solution of ABTS with 50 μl of Ascobic acid, standard group reaction mixture was obtained. Inhibition percentage of ABTS was calculated by a formula as under.
% ABTS inhibiting activity = (Ao – AI) / Ao x 100
Where Ao represents the value of control and AI represents absorbance value of standard or sample.
3.5.7 Lipoxygenase-inhibitory assay (LOX)
Yawer et al. (2007) spectrophotometric procedure was used for inhibition activity of lipoxygenase. Reaction mixture having lipoxygenase solution in 0.1 M phosphate buffer having pH 8 and inhibition solution (plant extract) was incubated at 25 oC for 10 minutes. By adding solution substrate, the reaction was started. Absorbance was measured after 6 minutes at 234 nm. The standard inhibitor, Baicalein was used in this study.
Lipoxygenase percent inhibition activity was measured as: Inhibition (%) = (1-A/B) x 100
Where, A represents enzyme activity in the absence of inhibitor, B represents enzyme activity along with inhibitors.
3.6 In-vivo studies 3.6.1 Acute toxicity study Acute toxicity test for plant methanolic extract was conducted by using mice in order to evaluate any probable toxicity. Animals were alienated into five different groups, each consist of six mice (n=6). Group I animals administrated with saline water (10 mg/kg, p.o), while animals of other test groups were given with increasing doses (100, 500, 1000, 2000 mg/kg) of plant extract. Before treatment body weight of each animal was determined. Animals of all groups were keenly monitored for any unpleasant consequence or mortality for a period of 24 hours (Bruce, 1985).
44 3.6.2 Anti-inflammatory effect 3.6.2.1 Carrageenan induced paw edema model
The anti-inflammatory effect of plant extract was investigated by using BALB/c mice having weight about 21-26 g. The procedure of Khan et al., (2009) was used according to which the animals were separated into five random groups, each consist of 6 animals. Group I animals were given 10 mg/kg N/saline and considered as control, whereas group II animals were given 5 mg/kg diclofenac sodium. The plant extract of 100 mg, 200 mg and 400 mg was injected intraperitonealy (i.p) to animals of test group (group III-V) with increasing doses. Carrageenan (1%; 0.05 ml) was injected subcutaneously after 30 minutes to the animals right hind paw in the sub plantar tissue. By using Plethysmometer (Plan-labe. S.L LE-7600). anti-edematous activity was determined for five hours (at 0, 1, 2, 3, 4, 5 hr) continuously.
Following equation was used to calculate edema % inhibition.
3.6.2.2 Xylene˗induced ear edema
Xylene˗tempted ear edema model was used by employing Okokon et al. (2010) procedure with some modification, by using BALB/c mice having weight 21-26 g. Animals were segregated into five random groups each consist of six animals. All the animals were fasted for 24 hours and then were treated with 0.5 mg/kg dexamethasone, 10 ml/kg N/saline and varying doses of plant extract. After 30 minutes, inflammation was motivated by applying xylene few drops on the interior right ear surface. Xylene was remained there for 60 minutes, after that the animals were given light anesthesia and then scarified. Both the ears were cut down and divided in spherical shape by a cork borer having 7 mm diameter. After weighing the cut section, the inhibition percentage of ear edema was calculated in reference with left ear which was without xylene.
45 3.6.3 Analgesic effect 3.6.3.1 Formalin test
Dubuisson and Dennis (1977) procedure was used according to Tjolsen et al. (1992) modified method. The selected animals (mice) were given diverse doses of plant extract i.e. 100, 200 and 400 mg/kg i.p. and after an interval of 30 min, 0.05 ml of 2.5% formaldehyde was injected into plantar surface of right hind paw of mice. The behavioral responses were observed as mice walking (running) or can stand on treated paw, paw partly elevated, treated paw overall elevations, biting or stinging of treated paw. Animals treated with formalin tempted behavioral responses distinguished by two different phases. Response of rats noted at the initial 0-10 min, was primary phase of pain, while response noted between 15 and 30 min, was last stage of analgesia. Diclofenac sodium (5 mg/kg subcutaneously) was utilized as standard reference drug.
3.6.3.2 Hot plate test Hot plate or thermal nociception test was used to examine the analgesic effect of plant extract. The animals were alienated into five random groups in which each consist of six animals (n=6). Two hours before to initiate experiment the animals were deprived of food. The rodents were pre-tested by placing them on hot plate (Havard apparatus) at temperature of 55 ± 0.10 °C. All those animals were discarded displaying latency time during pre-testing more than 15 seconds (Kang et al., 2008). Group I and II were given 10 ml/kg N/saline and 20 mg/kg tramadol, respectively, while group III to V were treated with different doses of plant extract. After thirty minutes the animals were put on hot plate one by one to record latency time of nociceptive responses (paw licking and flicking and jumping) in seconds. To avoid tissue damage 30 seconds cut- off time was opted for each animal. Latency period at 0, 30, 90 and 120 min for each group was noted.
The following equation was used to estimate percent analgesic potential:
46 3.6.4 Antipyretic effect
Antipyretic potential of plant extract was assessed by using yeast˗stimulated pyrexia test previously reported by Al˗Ghamdi (2001). Pyrexia was stimulated in rats by inserting subcutaneously 10 ml/kg 15% water solution of brewer‟s yeast (Sigma Aldrich, France). Clinical digital thermometer was used, by inserting about 3-4 cm in the rectum, to note the temperature of rectum of each rat before and after 24 h of yeast injection (Hartmann, Germany). All those animals were rejected that showed raise in body temperature lesser than 0.5 °C after twenty four hours of yeast administeration. The selected rodents were segregated into five groups, each consist of six animals (n=6). The group I treated with saline water (10 ml/kg), positive control group treated with 100 mg/kg paracetamol and test groups treated with varying amount (100, 200 and 400 mg/kg) of plant extract (Vimala et al., 1998; Khan et al., 2009). Temperature of rectum of each animal was noted at regular period of time (1h) after treatment. The magnitude of antipyretic activity was inferred that how much it reduce the induced pyrexia.
3.6.5 Anti-diarrheal effect
Anti-diarrheal effect of plant extract was evaluated in albino rats having 150- 200 g weight, by using Meite et al., (2009) protocol. Animals were deprived of food for eighteen hours before treatment and were separated into five groups, each consist of six animals (n=6). Group I was administrated with 10 ml/kg saline solution, while group II (toxic control) was treated with 2 mg/kg loperamide. Group III, IV and V (test group) animals were given diverse doses of plant extract i.e. 100, 200 and 400 mg/kg respectively. Animals of all groups were administrated by oral route. After an interval of 30 minutes of oral administration of plant extracts and standard drug, rats of all groups were administrated orally with 2 ml of castor oil in order to stimulate diarrhea. Different parameters like beginning of diarrhea, amount of wet feces and quantity of fecal output was studied in the next 4 hours time period.
3.6.6 Anti-diabetic effect For anti-diabetic activity of plant extract Kunnur et al., (2006) procedure was used in rats. By injecting 150 mg/kg alloxan monohydrate (i.p) in selected rats, diabetes was stimulated. The rats after treatment were maintained for seven days in an environment in which food and water was available to it. Then the animals were fasted
47 for 10-12 hours on the 8th day and sugar level of their blood were examined through one touch glucometer (Lifescan, Johnson & Johnson, California, USA). Animals having blood glucose level greater than 120 mg/dl were evaluated for further study and were indiscriminately alienated into 5 groups each having six animals (n=6). Group I, II and III was administrated with 100, 200 and 400 mg/kg of plant methanolic extract. Group IV was administrated with 150 mg/kg metformin, while group V animals were given 10 ml/kg normal saline. Samples of blood were taken from tail vein after fasting overnight with time intervals of 0, 1, 2, 3 and 4 hours. One touch glucometer (Lifescan, Johnson & Johnson, California, USA) was used for evaluation of blood sugar (glucose) level.
3.6.7 Hepatoprotective effect
3.6.7.1 Carbon tetra chloride (CCl4) induced hepatotoxicity model Hepatoprotective effect of plant extract was determined by carbon tetra chloride
(CCl4) provoked hepatotoxicity model in rats. Animals having weight 150-200 g were alienated into 5 groups each having 6 rats. Group I animals were served orally with 5% 1 ml/kg of body weight, gum acacia suspension daily with a single dose for five consecutive days along with 1 ml/kg liquid paraffin subcutaneously on the second and third day. Animals of group II (toxic control) were treated with 5% 1 ml/kg gum acacia suspension daily with a single dose for five consecutive days along with liquid paraffin and CCl4 having 1:1 ratio on the second and third day. Group III (standard) were served orally with 25 mg/kg silymarin. Group IV and V (test groups) animals were administrated with different doses of plant extract (200 mg/kg and 400 mg/kg) for five days. Group III-V animals were served with liquid paraffin and CCl4 with 1:1 ratio (2.5 ml/kg, s.c) on second and third day after silymarin and eupalitin glycoside. After 24 hours of last treatment rats were scarified. After collection and clotting of blood by centrifugating at 3000 rpm for 16-20 minutes serum was separated in order to perform other biochemical analysis.
3.6.7.2 Biochemical investigations Gornall et al., (1949) procedure was used for total level of protein while Mallory and Evenlyn. (1937) method was employed for total level of total bilirubin (TB). For other biochemical analysis like serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT) and alkaline phosphatase
48 (ALP), the method of Henry and Cannon (1974) and Bergemeyer (1974) was followed respectively.
3.6.8 Sedative/hypnotic effect Sedative/hypnotic effect of plant extract was examined by using Thiopental- provoked sleep model in rodents. Animals were alienated into 5 groups each consist of six animals (n=6) and diazepam was used as a reference standard drug. Group I animals were given distilled water, group II were administrated (i.p) with 3 mg/kg diazepam, while group III, IV and V animals were treated with different doses (50, 100 and 200 mg/kg) of methanolic plant extract. Thiopental (a sub-hypnotic dose) 60 mg/kg (i.p) was injected after 30 minutes in all the groups. The hypnotic effect was noted for onset of sleeping and duration of sleeping disappearance (latency) and reappearance (duration) of righting reflex. Hypnotic sleeping time was considered to be time interval between disappearance and reappearance of righting reflex (Williamson et al., 1996: Herrera-Ruiz et al., 2007).
3.6.9 Anticonvulsant effect Anticonvulsant effect of plant extract was determined by using Pentylene tetrazole (PTZ) induced convulsion model in mice (21-26 g). Animals were segregated into five groups, each consist of six animals (n=6). Group I animals were given saline solution 10 ml/kg, animals of group II administrated with 2 mg/kg clonazepam (i.p.) as a standard drug, while animals of test groups i.e. III, IV and V were administrated with different amount of plant extract (100, 200 and 400 mg/kg). After an interval of 30 minutes of treatment, convulsion was induced in all groups animals by injecting PTZ (90 mg/kg i.p.). After the administration of PTZ, animals belonging to all groups were thoroughly observed for the onset of first clonus. Animal‟s mortality rate was noted and then the obtained information were statistically evaluated and compared with standard (Nasir et al., 2008).
3.6.10 Antidepressant effect Antidepressant action of plant extract was carried out by using Forced swimming test (FST) following Porsolt et al., (1978) protocol. The animals were instigated to go ahead in a container having water up to 15 cm height and temperature of 25 oC. Rodents were separated into five groups in which six animals per group (n=6) were present. Group I animals (control) were treated 10 ml/kg sodium chloride
49 solution, animals of group II (standard control) treated with fluoxetine (30 mg/kg i.p.), while animals of groups III, IV and V (test groups) were given different doses (50, 100 and 200 mg/kg) of plant extract. In over all time interval of 10 minute, time period of immobility was observed for the final 6 minutes. Animals were assumed to be in state of immobility when they become stationary, stop struggling and motionless floating in water. The variation in time period of immobility was observed for each group.
3.7 Statistical analysis
Statistical significance was examined by ANOVA, followed by post˗hoc analysis.
50 IV. RESULTS 4.1 Phytochemical investigation 4.1.1 Qualitative analysis of phytochemicals D. botrys whole plant MCE and its three solvent fractions were studied for different important phytochemical as shown in the Table 4.1. MCE displayed positive results for alkaloids, phenols, flavonoids, saponins, tannins and sterols while in HxF only phenols and flavonoids were detected. DCMF and EAF gave negative results for tannins and sterols, respectively.
Table 4.1. Phytochemical of methanolic crude extractand solvents fractions of D. botrys Phytochemical Name of test MCE HxF DCMF EAF Alkaloids Mayer‟s test + - + + Phenols Ferric chloride test + + + + Flavonoids Alkaline reagent test + + + + Saponins Frothing test + - + + Tanins Ferric chloride test + - - + Sterols Salkowski test + - + - MCE: Methanolic crude extract, HxF: Hexane fraction, DCMF: Dichloromethane fraction and EAF: Ethyl acetate fraction. (+) = detected, (-) = not detected
4.1.2 Quantitative analysis 4.1.2.1 Total Phenols Reasonable amount of total phenolic compounds was observed in the crude extract and solvent fraction of D. botrys. Highest concentration of phenols was found in the EAF (27.4 mg/g) followed by MCE (21.4 mg/g), DCMF (19.2 mg/g) and HxF (13.5 mg/g) having the lowest phenols concentration (Figure 4.1).
51 35
30 27.4
25
21.4 19.2 20
15 13.5 mg/g of plant of mg/g 10
5
0 Crude extract Hexane fraction Dichloromethane fraction Ethyl acetate fraction
Figure 4.1. Total phenols in methanolic crude extract and subsequent fractions 4.1.2.2 Total Alkaloids Maximum amount of alkaloids were found in the EAF (3.14 mg/g) followed by DCM (2.6 mg/g) and crude extract (2.2 mg/g) while least amount of alkaloids were found in HxF (1.2 mg/g) as shown in Figure 4.2.
4
3.5 3.14 3 2.6 2.5
2.2
2
1.5
1.2 mg/g of mg/g plant 1
0.5
0 Crude extract Hexane fraction Dichloromethane fraction Ethyl acetate fraction
Figure 4.2. Total alkaloids in methanolic crude extract and subsequent fractions
52 4.1.2.3 Total saponins Regarding sponins content the plant was found very rich and MCE proved the richest source of saponins while hexane fraction contains least amounts of saponins. Highest amount of saponins were recorded in the MCE (34.3 mg/g) followed by EAF, DCMF and HxF having 28.6 mg/g, 14.3 mg/g and 11.8 mg/g respectively (Figure 4.3).
45
40 34.3 35
30 28.6
25
20 14.3 15
11.8 mg/g of mg/g plant 10
5
0 Crude extract Hexane fraction Dichloromethane fraction Ethyl acetate fraction
Figure 4.3. Total saponins in methanolic crude extract and subsequent fractions
4.1.2.4 Total Flavonoids EAF was found very rich regarding the total amount of flavonoids, having 15.5 mg/g flavonoids while the HxF had 4.7 mg/g flavonoids, proved weakest source. In MCE and EAF 12.4 mg/g and 11.2 mg/g flavonoids were found, respectively (Fig 4.4).
53 20
18 15.5 16
14 12.4
12 11.2
10
8 mg/g of plant of mg/g 6 4.7 4
2
0 Crude extract Hexane fraction Dichloromethane fraction Ethyl acetate fraction
Figure 4.4. Total flavonoids in methanolic crude extract and subsequent fractions
4.1.3 Proximate composition
The proximate composition analysis includes moisture, ash, protein, fiber, fat and carbohydrate of whole plant of D. botrys as presented in Table 4.2. It was observed that the plant contain reasonable amount of life basic essential nutrients like nitrogen- free extract (carbohydrates) (38.45%), protein (30.26%), fats (3.68%) while crude fibers (1.43%) were present in the least amount. The content of moisture was (7.45%) while the amount of ash which represents the inorganic matter was (18.73%).
Table 4.2. Proximate composition (%) of D. botrys whole plant
Content Percent composition Moisture 7.45±0.32 Ash 18.73±0.21 Fats 3.68±0.45 Protein 30.2 ±0.72 Fiber 1.43±0.53 Carbohydrates 38.4 ±0.83
All the values were taken as mean and standard error for each replicate (n=3)
54 4.1.4 Mineral analysis
Mineral composition analysis of D. botrys plant showed reasonable amount of macronutrients like Ca, K and Na, having 3268 μg/g, 2673 μg/g and 591 μg/g concentrations respectively. Our results also indicated that it is a good source of Fe and Zn having 223 μg/g and 46.7 μg/g respectively. All the tested metals were present within the permissible limit and no Cr and Cd were detected (Table 4.3).
Table 4.3. Mineral composition of whole plant of D. botrys Whole plant powder Concentration (μg/g) Ca 3268±0.53 K 2873±0.71 Na 591±0.23 Fe 223±0.46 Zn 46.7±0.32 Cu 8.3±0.48 Ni 1.2±0.16 Pb 0.4±0.13 Cr ND Cd ND
All the values were taken as mean and standard error for each replicate (n=3)
4.2 In-vitro activities 4.2.1 Antibacterial activity Antibacterial potential of D. botrys extract and its consequent fractions were evaluated as depicted in Table 4.4. Maximum antibacterial effect was shown by MCE and EAF followed by DCMF while HxF displayed least antibacterial activities. The range of inhibition was compared with standard drug cefixime which is used as broad spectrum antibiotic. CME of plant exhibited effective antibacterial activity against X. campestris and E. coli causing 12.6 ± 0.54 and 10.7 ± 0.43 mm zone of inhibition respectively. Among solvent fractions EAF proved the most effective with maximum antibacterial potential efficiently inhibited the growth of P. aerugonosa and P. vulgaris, causing 20.6 ± 0.53 mm and 9.8 ± 0.63 mm zone of inhibition respectively. HxF demonstrated least antibacterial ability against all the used strains of bacteria.
55 Table 4.4. Antibacterial activity of methanolic crude extract and solvent fractions of D. botrys Bacterial strain MCE HxF DCMF EAF *Standard (mm) (mm) (mm) (mm) (mm) C. michiganesis 9.7±0.15 6.4±0.61 8.5±0.32 8.7±0.42 13.8±0.04 B. subtilis 11.8±0.26 7.3±0.24 13.7±0.06 17.4±0.34 22.6±0.15 P. Aerugonosa 15.3±0.18 14.2±0.52 17.5±0.13 20.6±0.53 24.7±0.32 K. pneumonia 13.6±0.41 5.8±0.03 8.7±0.51 14.3±0.31 26.3±0.12 S. aureus 10.5±0.31 10.3±0.43 8.6±0.63 13.8±0.24 18.5±0.21 E. coli 10.7±0.43 5.3±0.51 9.7±0.32 7.5±0.41 13.8±0.13 P. vulgaris 6.3±0.14 7.2±0.31 8.5±0.52 9.8±0.63 12.5±0.01 X. campestris 12.6±0.54 6.8±0.42 10.4±0.61 7.4±0.71 14.6±0.13 MCE: Methanolic crude extract, HxF: Hexane fraction, DCMF: Dichloromethane fraction and EAF: Ethyl acetate fraction All the values were taken as mean and standard error for each replicate (n=3) *Standard: Cefixime
Figure 4.5. Antibacterial activity of Ethyl acetate fraction and crude extract of D. botrys against (a) P. aerugonosa (b) and X. campestris
4.2.2 Antifungal activity
Anti-fungal activity of plant extract and consequent fractions were assessed as represented in the Table 4.5. MCE exhibited highest antifungal activity against F. oxysporum followed by EAF, causing 19.3 ± 0.41 mm and 18.4 ± 0.33 mm zone of inhibition, respectively. EAF considerably stopped the growth of F. solani causing maximum zone of inhibition i.e. 12.5 ± 0.53 mm as compared to other solvents. Zone of inhibition was compared with reference drug clotrimazole, a standard antifungal
56 drug. The growth inhibiting effect of MCE and other fractions were low to moderate against other fungal strains, however, HxF showed no effect on the growth of A. flavus and A. niger.
Table 4.5. Antifungal activity of methanolic crude extract and solvent fractions of D. botrys Fungal strains MCE HxF (mm) DCMF EAF *Standard (mm) (mm) (mm) (mm) A.flavus 3.7±0.35 0.00±0.00 3.2±0.71 7.3±0.42 15.6±0.14 A. niger 4.2±0.13 0.00±0.00 3.5±0.63 6.8±0.35 18.3±0.41 M.piriformis 13.5±0.21 7.8±0.41 5.3±0.32 12.4±0.02 22.4±0.23 F. solani 9.5±0.51 6.2±0.18 6.4±0.12 12.5±0.53 14.7±0.36 F. oxysporum 19.3±0.41 10.8±0.13 13.6±0.71 18.4±0.33 24.8±0.15 MCE: Methanolic crude extract, HxF: Hexane fraction, DCMF: Dichloromethane fraction and EAF: Ethyl acetate fraction All the values were taken as mean and standard error for each replicate (n=3) *Standard: Clotrimazol
Figure 4.6. Antifungal activity of crude extract and Ethyl acetate of D. botrys against (a) F. oxysporum and (b) F. solani
4.2.3 Phytotoxic activity Plant methanolic crude extract and subsequent fractions were evaluated against Limna minor inorder to investigate its synergetic or antagonistic effect on its development as depicted in Table 4.6. The growth inhibiting effect of plant extract and various fractions were dose dependent and maximum inhibition of growth was
57 performed by high dose (1000 μg/ml) of plant sample as compared to low dose (10 μg/ml). MCE of D. botrys displayed highest inhibiting effect on the growth of L. minor at diverse concentrations i.e. 10 μg/ml, 100 μg/ml and 100 μg/ml caused 26%, 50% and 70% inhibition, respectively, HxF showed lowest retarding effect i.e. 10 μg/ml, 100 μg/ml and 1000 μg/ml caused 13%, 16% and 26%, respectively. Phytotoxic effect of DCMF and EAF was moderate.
Table 4.6. Phytotoxic activity of methanolic crude extract and solvent fractions of D. botrys. Sample % Total fronds Survived Died Sample concentration Phytotoxic No. fronds No. fronds No. (μg/ml) effect 30 10 22 8 26 MCE 30 100 15 15 50 30 1000 9 21 70 30 10 26 4 13 HxF 30 100 25 5 16 30 1000 22 8 26 30 10 23 7 23 DCMF 30 100 18 12 40 30 1000 15 15 50 30 10 24 6 20 EAF 30 100 22 8 26 30 1000 18 12 40 MCE: Methanolic crude extract, HxF: Hexane fraction, DCMF: Dichloromethane fraction and EAF: Ethyl acetate fraction
4.2.4 Antioxidant activity
4.2.4.1 DPPH radical scavenging activity
The DPPH radical scavenging activity of D. botrys crude extract and subsequent fractions were investigated as shown in the Table 4.7. The EAF at a concentration of 500 μg/ml showed highest scavenging activity (57.46 ± 0.49%) followed by MCE and HxF (49.83 ± 0.92% and 41.63 ± 0.71%) respectively. DCMF exhibited the least scavenging activity (37.52 ± 0.47) at all the tested concentration. The DPPH percent scavenging activity of all the tested samples was as EAF>MCE>HxF>DCMF.
58 Table 4.7. DPPH radicals scavenging activity of methanolic crude extract and solvent fractions of D. botrys Solution % Scavenging effect Standard Conc. MCE HxF DCMF EAF Ascorbic acid (μg/ml) 20 3.62±0.48 2.14±0.31 1.43±0.45 4.75±0.62 11.35±0.34 50 12.58±0.32 7.52±0.46 5.74±0.75 15.31±0.58 26.71±0.61 100 22.81±0.57 16.21±0.82 14.51±0.61 31.53±0.73 55.83±0.76 200 35.13±0.21 27.72±0.30 26.67±0.32 42.13±0.89 71.47±0.49 500 49.83±0.92 41.63±0.71 37.52±0.47 57.17±0.49 93.42±0.24 MCE: Methanolic crude extract, HxF: n˗Hexane fraction, DCMF: Dichloromethane fraction and EAF: Ethyl acetate fraction All the values were taken as mean and standard error for each replicate (n=3)
4.2.4.2 ABTS radical scavenging activity
The percent ABTS free radical scavenging activity of crude extract and solvent fraction were calculated as summarized in Table 4.8. Among the tested samples maximum scavenging activity was exhibited by EAF (72.17 ± 0.59%) while HxF showed least (53.76 ± 0.57%) activities against ABTS free radicals at concentration of 500 μg/ml respectively. The scavenging activity of MCE was 61.54 ± 0.34%while that of DCMF was 58.65 ± 0.47%. The ABTS radical scavenging activity of all the tested samples was as EAF>MCE>DCMF>HxF.
Table 4.8. ABTS radicals scavenging activity of methanolic extract and solvent fractions of D. botrys Solution % Scavenging effect Standard Conc MCE HxF DCMF EAF Ascorbic (μg/ml) acid 20 6.12±0.47 2.42±0.56 6.31±0.32 9.45±0.28 13.61±0.41 50 13.2±0.92 8.37±0.43 10.23±0.61 15.63±0.37 19.76±0.73 100 21.65±0.53 15.23±0.76 17.13±0.74 27.96±0.68 41.53±0.53 200 36.78±0.75 24.81±0.39 31.89±0.83 41.53±0.32 57.65±0.89 500 61.54±0.34 53.76±0.57 58.65±0.47 72.46±0.59 94.76±0.47 MCE: Methanolic crude extract, HxF: n˗Hexane fraction, DCMF: Dichloromethane fraction and EAF: Ethyl acetate fraction All the values were taken as mean and standard error for each replicate (n=3)
59 4.2.5 Lipoxygenase-inhibitory assay The lipoxygenase-antagonistic potential of crude extract and different fractions at a concentration of 100 mg/mL is summarized in the Table 4.8. The results indicated that maximum retarding effect was shown by EAF causing 64% inhibition followed by DCMF, inhibiting the activity of lipoxygenase enzymes by 58%. HxF displayed least inhibiting effect and decreased its activity only 22%. The effect of MCE was moderate reducing the activity of lipoxygenase enzymes by 43%.
Table 4.9. Lipoxygenase-inhibitory assay of methanolic crude extract and solvent fraction of D. botrys
Sample (100 mg/mL) % Inhibition
MCE 43±0.84 HxF 22±0.21 DCMF 58±0.48 EAF 64±0.16 Baicalein 87±0.31 MCE: Methanolic crude extract, HxF: n˗Hexane fraction, DCMF: Dichloromethane fraction and EAF: Ethyl acetate fraction All the values were taken as mean and standard error for each replicate (n=3)
4.3 In-vivo pharmacological activities 4.3.1 Acute toxicity Plant methanolic extracts of different concentrations were analyzed for acute toxicity using model animals. It was observed that plant extract up to amount of 2000 mg/kg showed no lethal consequences on tested animals and all the animals remained alive after 24 hrs of evaluation time (Table 4.10).
Table 4.10. In-vivo acute toxicity of methanolic crude extract of D. botrys No. of deceased Dose No. of survived Treatment animals Dead (24 (mg/kg) animals (24 hrs) hrs) 100 All Nil 500 All Nil Plant crude extract 1000 All Nil 2000 All Nil Normal saline 10 All Nil
60 4.3.2 Anti-inflammatory activity 4.3.2.1 Carrageenan˗induced paw edema model Our results indicated marked anti-inflammatory effect of methanolic extract of D. botrys at all the tested doses in different assessment time intervals, against paw edema induced by carrageenan in mice. The results of plant extract are illustrated and compared with standard drug and control in Table 4.11a,b. Injection of carrageenan in paw induced inflammation which enhanced slowly, getting highest size at 5th hour of injection. D. botrys extract at dose of 100 mg/kg exhibited anti-inflammatory activity that became significant (p<0.05) at the last phase (after 5h) of inflammation. Anti- inflammatory activity of 200 mg/kg and 400 mg/kg of plant extract was significant (p<0.05) in both early and last phase of inflammation, and was comparable to that of diclofenac sodium, used as standard drug. The overall anti-inflammatory activity of plant extract was in concentration dependent manner and was more efficient in the last phase as compared to early phase of inflammation.
Table 4.11a. Anti˗inflammatory activity of methanolic crude extract of D. botrys on carrageenan provoked mice paw edema
Increase in paw volume (mm) Dose Treatment (mg/kg) Early phase Late Phase (3 hours) (5 hours) Saline 10 5.73±1.09 5.81±1.21 100 4.89±1.27 4.63±1.89* Plant extract 200 4.27±0.92* 3.62±1.09* 400 3.01±0.86* 2.97±0.98* Diclofenac sodium 5 2.84±0.71** 2.41±0.67**
Values were taken as the mean and standard error from 6 replicates for each group. *p<0.05, **p<0.01. Standard and every test group were compared with toxic group. One-way ANOVA followed by Dunnet‟s test.
61 Table 4.11b. Percent inhibition of carrageenan-induced paw edema by methanolic crude extract of D. botrys
% inhibitions of paw edema Treatment Dose (mg/ kg) Early phase Late Phase (3 hours) (5 hours) Saline 10 - - 100 14.65 20.31 Plant extract 200 25.47 37.69 400 47.46 48.88 Diclofenac sodium 5 50.43 58.51
4.3.2.2 Xylene-induced ear edema All doses of D. botrys crude extract displayed considerable anti-edematous effect in comparison to control as depicted in the Table 4.12a,b. Topical application of plant crude extract (100, 200 and 400 mg/kg) inhibit significantly xylene-tempted ear inflammation (p<0.05 and p<0.01). Its effect was dose dependent and greater action was shown by amount of 400 mg/kg of plant extract. However, dexamethasone, used as reference drug exhibited marked activity against inflammation of ear.
Table 4.12a. Anti˗inflammatory effect of methanolic crude extract of D. botrys on xylene˗induced ear edema in mice
15 min 60 min Treatment Dose (mg/kg) Difference (mg) Difference (mg) Saline 10 35.25±1.42 36.65±1.72 100 20.16±1.78* 14.42±1.73*
Plant extract 200 17.31±1.37* 13.39±1.16**
400 13.54±2.51* 10.72±1.09**
Dexamethasone 5 9.89±1.83** 8.24±1.61**
Values were taken as the mean and standard error from 6 replicates for each group. *p<0.05, **p<0.01. Standard and every test group were compared with toxic group. One-way ANOVA followed by Dunnet‟s test.
62 Table 4.12b. Percent inhibition of xylene-induced ear edema by methanolic crude extract of D. botrys
% percent inhibition Treatment Dose (mg/kg) 15 min 60 min Saline 10 - - 100 42.4 59.3 Plant extract 200 50.7 63.7 400 61.5 73.2 Dexamethasone 5 71.2 77.3
4.3.3 Analgesic activity 4.3.3.1 Formalin test Plant extract of D. botrys displayed strong analgesic potential and the pain induced by formalin was strongly attenuated (p<0.05) by the various dosages of plant, however the analgesic effect of 400 mg/kg was extremely significant (p<0.01) and comparable to that of standard drug. There was no major difference in the analgesic potential of plant extract in the early and late phase of analgesia, however the pain relieving potential of the plant extract was highly pronounced in the late phase after formalin injection in comparison to early phase (Table 4.13a,b).
Table 4.13a. Analgesic effect of methanolic crude extract of D. botrys on formalin-induced pain in rats
Score of pain severity Treatment Dose (mg/kg) Early phase Late Phase (0-10) min (15-30) min Saline 10 2.8±0.2 2.8±0.3 100 1.6±0.1* 1.5±0.2* Plant extract 200 1.1±0.1* 0.9±0.1* 400 0.6±0.1** 0.5±0.3** Diclofenac sodium 5 0.2±0.1** 0.2±0.2**
Values were taken as the mean and standard error from 6 replicates for each group. *p<0.05, **p<0.01. Standard and every test group were compared with toxic group. One-way ANOVA followed by Dunnet‟s test.
63 Table 4.13b. Percent inhibition of formalin-induced pain by methanolic crude extract of D. botrys
% inhibition Treatment Dose (mg/kg) Early phase Late Phase (0-10) min (15-30) min Saline 10 - - 100 42.85 46 Plant extract 200 60.71 67 400 78.57 82.14 Diclofenac sodium 5 92.85 92.85
4.3.3.2 Hot plate test Central analgesic potential of D. botrys extract was assessed as an elevation in the latency time that was observed with a gap of time interval of 30 min starting from 0˗120 min, after the treatment of saline, different doses of plant extract and standard drug, tramadol (Table 4.14). Dose of 400 mg/kg of plant extract exhibited significant effect (p<0.05) after 30 min and 60 min (11.03 ± 0.56 and 11.98 ± 0.61, respectively) and become more pronounced (p<0.01) after 120 min (12.97 ± 0.49). The standard drug, tramadol, displayed highest analgesic potential (p<0.01) after 30 min of treatment which persist till 120 min.
Table 4.14. Analgesic effect of methanolic crude extract of D. botrys on pain induced by hot plate in mice
Dose Latency time of nociceptive responses (minutes) Traetment (mg/kg) 0 30 60 90 120 10 8.24±0.23 8.49±0.19 8.68±0.33 8.70±0.31 8.77±0.49 Saline 100 8.39±0.71 9.56±0.34 9.88±0.67 9.92±0.42 9.97±0.44
Plant 200 8.36±0.42 9.96±0.58 10.21±0.46 10.41±0.14 10.93±0.36 extract 400 8.35±0.36 11.03±0.56* 11.98±0.61* 12.05±0.13* 12.97±0.49**
Tramadol 20 8.52±0.57 12.62±0.38** 15.78±0.52** 15.75±0.13** 15.63±0.34**
Values were taken as the mean and standard error from 6 replicates for each group. *p<0.05, **p<0.01. Standard and every test group were compared with toxic group. One-way ANOVA followed by Dunnet‟s test.
64 4.3.4 Antipyretic activity
The elevated body temperature induced by brewer‟s yeast was extensively declined by all the tested doses of plant extract, however the effect of 200 and 400 mg/kg was highly significant (p<0.001) reducing the body temperature up to 37.02 oC and 36. 96 oC respectively, at the 5th hr of assessment time interval and was analogous to that of standard reference drug paracetamol (Table 4.15).
Table 4.15. Antipyretic effect of methanolic crude extract of D. botrys on brewer’s yeast induced pyrexia in rats
Temperature of rectum (oC) Dose Treatment Normal After 24 hrs After use of drug (mg/kg) Temperature 1 hour 2 hours 3 hours 4 hours 5 hours Saline 10 36.38±0.42 38.84±0.15 39.02±0.35 39.08±0.21 39.23±0.32 39.41±0.25 39.63±0.52
100 36.56±0.42 38.71±0.53 37.91±0.26* 37.51±0.41** 37.30±0.25** 37.27±0.61** 37.21±0.74**
Plant extract 200 36.87±0.63 38.80±0.83 37.62±0.41** 37.41±0.31** 37.19±0.37*** 37.14±0.42*** 37.02±0.74***
400 36.89±0.47 38.71±0.85 37.17±0.41*** 37.17±0.63*** 37.12±0.60*** 37.09±0.63*** 36.96±0.45***
Paractamol 150 36.62±0.28 38.95±0.27 37.18±0.14*** 37.03±0.37*** 36.72±0.36*** 36.45±0.43*** 36.18±0.45***
Values were taken as the mean and standard error from 6 replicates for each group. *p<0.05, **p<0.01, ***p<0.001. Standard and every test group were compared with toxic group. One-way ANOVA followed by Dunnet‟s test.
65 4.3.5 Antidiarrheal activity
Our observation regarding antidiarrheal activity showed that methanolic extract of plant have antidiarrheal effect in a dosage dependent manner and decreased the episodes of diarrhea in rats, tempted by castor oil. Plant extract of 200 mg/kg and 400 mg/kg showed an overall vital (p<0.05 and p<0.01) antidiarrheal effects as compared to control group. Both the doses of plant extract increased the latent period (118.25 ± 1.47 min and 241.5 ± 1.53 min) while caused a decline in total frequency of wet feces (4.13 ± 0.81 and 2.84 ± 0.63) and mean weight of fecal drops (0.29 ± 0.07 g and 0.16 ± 0.05 g) upon administration of castor oil (Table 4.16).
Table 4.16. Antidiarrheal effect of crude extract of D. botrys on castor oil- induced diarrhea in rats
Treatment Dose mg/kg Latent period (min) Total wet fecal Mean weight of frequency fecal drops Saline 10 64.27±1.29 7.83±0.74 1.3±0.12 100 72.41±1.79 5.89±1.03 0.43±0.09* Plant extract 200 118.25±1.47* 4.13±0.81* 0.29±0.07** 400 241.5±1.53** 2.84±0.63** 0.16±0.05** Lepromide 3 324.2±0.97** 2.19±0.94** 0.13±0.06**
Values were taken as the mean and standard error from 6 replicates for each group. *p<0.05, **p<0.01. Standard and every test group were compared with toxic group. One-way ANOVA followed by Dunnet‟s test.
4.3.6 Anti-diabetic activity
The effect of plant extract on the alloxane monohydrate induced diabetes in rats is summarized in the Table 4.17. It was noted that the dosage of 400 mg/kg plant extract effectively reduced elevated blood sugar level at all the assessment time intervals however its effect was extremely significant (p<0.01) at the 3rd and 4th hour of administration reducing blood sugar level up to 131.4 mg/dl and117.4 mg/dl respectively and was equivalent to that of standard drug metformin. The other dosages of 100 and 200 mg/kg showed considerable (p<0.05) effect in the last 2 hours of assessment time interval.
66 Table 4.17. Anti-diabetic activity of methanolic crude extract of D. botrys on alloxane induced diabetes in mice.
Dose Level of glucose in blood (mg/dl) mg/kg Treatment (i.p) 0 hour 1 hour 2 hour 3 hour 4 hour
10 168.7±0.73 169.0±0.54 169.6 ±0.62 169.2±0.98 169.5±0.59 Saline 100 169.5±0.45 166.6±0.58 162.7±0.32 154.6±0.45* 143.3±0.87*
Plant 200 170.6±0.43 163.2±0.56 157.6±0.62* 148.4±0.41* 145.7±0.42* extract 400 171.2±0.51 157.2± 0.76* 148.2±0.43* 131.4±0.76** 117.4±0.43**
150 169.9±0.41 141.4±0.53** 132.6±0.80*** 119.2±0.54*** 113.9±0.74*** Metformin Values were taken as the mean and standard error from 6 replicates for each group. *p<0.05, **p<0.01, ***p<0.001. Standard and every test group were compared with toxic group. One-way ANOVA followed by Dunnet‟s test.
4.3.7 Hepativeprotective activity Effect of methanolic extract of D. botrys on the elevated levels of serum hepative specific markers like SGPT, SGOT, ALP and TB in CCl4 treated rats was determined as shown in Table 4.18. Administration of CCl4 (2 mg/kg, s.c) considerably (p<0.001) elevated the serum level of SGPT, SGOT, ALP and TB (63.7 ± 1.62 U/ml, 84.3 ± 1.58 U/ml, 251.8 ± 3.41 U/ml and 5.56 ± 0.22 mg/dl, respectively) in group II as compared to control (group I). MCE of plant (400 mg/kg) caused a significant (p<0.01) decline in the elevated level of SGPT and SGOT (31.2 ± 1.28 U/ml and 48.31 ± 1.87 U/ml) and that of ALP and TB (179.31 ± 3.41 U/ml and 3.64 ± 0.13 mg/dl) effectively (p<0.05), however, its effect was less than silymarin, used as standard drug. While 200 mg/kg of methanolic plant extract have no prominent effect on the level of tested serum markers.
67 Table 4.18. Hepativeprotective activity of methanolic crude extract of D. botrys extract on CCl4 stimulated toxicity in rats
Dose SGPT (U/ml) SGOT ALP (U/L) TB (mg/dl) Treatment (mg/kg) (U/ml) Liquid 26.64±1.93 29.36±1.16 113±2.76 1.13±0.14 Saline parafin 2.5 63.7±1.62 84.3±1.58 251.8±3.41 5.56±0.22 CCl4 200 52.82±1.72 72.9±2.48 236.1±2.89 4.57±0.15 Plant extract + CCl4 400 31.2±1.28** 48.31±1.87** 179.31±3.41* 3.64±0.13*
Silymarin + 25 24.47±1.89** 33.17±2.41** 147.22±2.04** 2.45±0.78** CCl4 Values were taken as the mean and standard error from 6 replicates for each group. *p<0.05, **p<0.01. Standard and every test group were compared with toxic group. One-way ANOVA followed by Dunnet‟s test. TB: Total bilirubin, ALP: Alkaline phosphatase, SGOT: Serum glutamic oxaloacetic and SGPT: Serum glutamic pyruvic transaminase.
4.3.8 Sedative/hypnotic activity The synergetic effects of different doses of plant crude extracts on the thiopental induced hypnoses were evaluated as summarized in Table 4.19. Plant extract of 100 mg/kg and 200 mg/kg significantly (p<0.05) induced an early onset of sleeping reducing the time from 7.8 minutes to 6.41 and 5.93 minutes respectively. It also effectively prolonged the time of sleeping from 88.80 minutes to 130.40 and 177.60 minutes respectively. The overall efficiency of plant extract was lesser than the standard drug diazepam.
Table 4.19. Sedative/hypnotic activity of methanolic crude extract of D. botrys on thiopental induced hypnosis
Treatment Dose Onset of sleeping Sleeping duration mg/k (m) (m) Saline 10 7.81±0.47 88.80±1.91 50 7.18±0.23 107.60±1.08 Plant extract 100 6.41±0.37* 130.40±1.69* 200 5.93±0.27* 145.20±1.76** Diazepam 5 5.74±0.26* 177.60±1.44**
Values were taken as the mean and standard error from 6 replicates for each group. *p<0.05, **p<0.01. Standard and every test group were compared with toxic group. One-way ANOVA followed by Dunnet‟s test.
68 4.3.9 Anti-convulsant activity The effect of plant extract on the convulsion induced by pentylenetetrazole was studied as summarized in Table 4.20. Plant extract of 400 mg/kg dose notably (p<0.05) deferred the arrival of first clonus from 5.09 to 6.99 minutes (1.90 minutes). It also prolonged effectively (p<0.01) the duration of death from 9.72 to 19.56 minutes (9.84 minutes) and its effect was comparable to that of standard drug clonazepam. The other doses of plant extract (100 and 200) also showed significant effect on increasing the time of death.
Table 4.20. Anticonvulsant Effect of methanolic crude extract of D. botrys on PTZ-induced convulsions in mice
Treatment Dose mg/kg Onset of first clonus Time of death (min) (min) Saline 10 5.09±0.22 9.72±0.44 100 5.68±0.27 12.31±0.48* Plant extract 200 6.03±0.28* 13.57±0.61* 400 6.99±0.07* 19.56±0.15* Clonazepam 2 8.58±0.37** 23.84±0.62**
Values were taken as the mean and standard error from 6 replicates for each group. *p<0.05, **p<0.01. Standard and every test group were compared with toxic group. One-way ANOVA followed by Dunnet‟s test. 4.3.10 Antidepressant activity
The anti-depressant effects of different doses of plants extract using forced swim test are summarized in Table 4.21. The result showed that amongst the variuos tested doses of plant extract only 200 mg/kg of plant extract was effective and exhibited considerable (p<0.05) anti-depressant effect decreasing the time of immobility from 193.98 to 96.78 seconds. The standard drug flouxetine exhibited highest anti-depressant effect.
69 Table 4.21. Antidepressant activity of crude extract D. botrys on the time of immobility in forced swim test model in rats
Treatment Dose (mg/kg) Immobility time (seconds)
Saline 10 193.98±1.35
50 139.8±1.52
Plant extract 100 145.38±1.16
200 96.78±1.42*
Fluoxetine 30 76.5±1.39**
Values were taken as the mean and standard error from 6 replicates for each group. *p<0.05, **p<0.01. Standard and every test group were compared with toxic group. One-way ANOVA followed by Dunnet‟s test.
70 V. DISCUSSION 5.1 Phytochemical investigation 5.1.1 Qualitative and quantitative analysis of phytochemicals
In order to study the biological assays of herbal derivatives it is necessary to study their chemical composition. The active compounds that are naturally occurred in the plants play very important role in their biological activities. Screening of different secondary metabolites and their quantitative analysis in the crude plant extract and its fraction exposed the pharmacological importance of D. botrys (Table 4.1). Crude methanolic extract and its consequent fractions confirm presence of reasonable amount of phenols. Their quantitative analyses revealed that maximum amount of phenols were present in the EAF (27.4 mg/g) while HxF displayed least amount of phenol (13.5 mg/g) (Figure. 4.1). Our results regarding phenolics contents were slightly greater than the quantity reported by Ozer et al. (2016) in the same plant which may be due to its geographical location, soil and environmental condition. Phenolic compounds present in the plant provide defense mechanism against reactive oxygen species, herbivores, insects and microorganisms (Vaya et al., 1997).
The amount of alkaloids was maximum in the EAF (3.14 mg/g), however relatively low as compared to other secondary metabolites (Figure 4.2). Alkaloids are chemical compounds occurring naturally in nearly 20% of vascular plant species, most commonly in herbaceous dicot plants and comparatively little in monocots and gymnosperms. (Hegnauer et al., 1988). These active compounds have role in plant protection against pathogens and herbivores. Isolated pure alkaloids and their derivatives are employed all over the globe for medicinal purposes due to their bactericidal, antispasmodic and analgesic properties. (Hartmann, 1991; Harborne, 1988). Among all the phytochemicals in the tested solvents, MCE exhibited highest amount of saponins (34.3.41 mg/g) followed by EAF (28.6 mg/g) (Figure 4.3). Saponin help in improving immunity system of animals against pathogens, decreasing blood cholesterol level and decreasing the risk of intestinal cancer (Havsteen, 2002).
In case of total flavonoids, whole plant crude extract and its fraction had diverse amount of total flavonoids concentration. EAF displayed highest concentration (15.5 mg/g) (Figure 4.4), which was in agreement with the observation of Panday and Gupta
71 (2014) who reported 15.68 mg/g flavonoids contents in C. album plant, belongs to the same genus. Flavonoids have an imperative function in a broad range of biological activities such as induction of cell apoptosis, reticence of cell-proliferation, enzyme inhibition, antioxidant and antibacterial activities (Catoni et al., 2008; Cook and Samman, 1996).
5.1.2 Proximate analysis:
In proximate composition analysis, the content of moisture was 7.45%, which is very low from moisture content of C. album (84.8%) growing in Nigeria reported by Adedapo et al. (2011), but however our results are comparable with the moisture content of C. quinoa (11.2%) reported by Ogungbenle (2009). This low value of moisture in the whole plant extract is extremely useful in increasing shelf life of herbal drugs and decreases the chance of fungal and bacterial growth, which grows fast on substances having high moisture contents as compared to low moisture containing substances. The value of ash (18.73%) designates the existence of inorganic substances in the plant extract and could be an excellent source of minerals (Table 4.2). Fats which are also called triglycerides are the rich sources of energy are found in seeds and nuts of plants and adipose tissue of animals. The fats found in animals are usually saturated while in plants these are unsaturated and having low boiling and melting points and very useful for human health. The value of fats contents (3.68%) was in the range as reported by earlier literature in other members of this genus.
Reasonable amount of protein (30.26%) was observed which could contribute the daily protein requirement and are good source of different amino acids (NRC, 1975). Proteins are the fundamental biological macro molecules having structural and functional role inside living organisms. In our results 38.45% nitrogen free extract (carbohydrates) was observed in the whole plant. Plants are the efficient sources of carbohydrates and most of the carbohydrates are obtained from plant sources. Carbohydrates are the most plentiful organic compound on earth and are the good and efficient source of energy. Apart from energy source, carbohydrates perform vital significant functions inside living organisms. In D. botrys the amount of crude fibers was 1.43%, which is the main component of balance diet and help in the inhibition of different chronic disorders such as cancer, blood pressure and diabetes. Inside intestine fiber absorbs maximum amount of water, makes the stools softer and facilitate its flow
72 outside. Any one taking diet, having high amount of fiber, can alleviate hemorrhoids and constipation (Bowman and Russell, 2001).
5.1.3 Mineral analysis The amount of different metals analyzed in the whole plant of D. botrys may account in the ethno-pharmacological use of herbal extract for the cure of many diseases. The concentration range of different minerals in the plant may also helpful in employing its doses within the permissible limits. Among the micro-nutrients copper is involved in the ATP production, CO2 absorption, iron metabolism, connective tissue formation and neurotransmitters formation and metabolism. The concentration of copper found in the plant was 8.3 μg /g which is within the permissible limit of 10 μg/g for plants.
Prominent amount of iron (223 μg/g) was observed in the plant which further increased the nutritional value of the plant. Among trace elements, iron occurs most abundantly in human body. Optimal concentration of iron is necessary for survival of plants, animals and microbes (Arredando and Nunez, 2005). It has reported by world health organization that the world‟s 48% pregnant females and 46% kids are have problem of anemia due to scarcity of iron. The daily optimum intake of iron recommended by food and nutrition board is 8 mg, 18 mg and 27 mg per day for male, female and pregnant women, respectively (IOM, 2001). The amount of iron in the current plant was within the permissible limits (36-241 μg /g) and is therefore proved an excellent source of iron.
Cadmium is considered to be one of the most phytotoxic because in plants it disturbs various biochemical and physiological processes, leading to inhibition of growth and cell death (Sandalio et al., 2001; Guo et al., 2009; Xu et al., 2009). It is released mainly into the soil surface from industries and agricultural practices (Wagner, 1993) and has been rank seventh amongst top twenty toxins (Yang et al., 2004). The permissible limit of cadmium in plant recommended by WHO (2005) is 0.03 mg/kg. Our results revealed that no cadmium was detected in whole plant of D. botrys.
Lead is considered one of hazardous metals which enter human body through different sources such as contaminated water, fruits and vegetables. Lead has no metabolic function in the body and its intake in high amount may cause accumulation
73 inside body causing different noxious effects such as hepatic, cardiovascular and digestive disorders. The permissible maximum value of lead for plants is 10 μg/g. Our results displayed that whole plant powder of D. botrys had 0.4 μg/g of lead which fall within the recommended range, which further authenticated that the plant can be used safely for medicinal purposes.
Nickel occurred naturally in abundant amount in the earth crust and its scarcity rarely occurs in different types of dietary products. Intoxication of nickel may cause lung fibrosis, skin, heart and kidney problems. Nickel is stored inside pancreas and have important role in the secretion of insulin. Its deficiency leads to malfunctioning of liver (Denkhaues and Salnikow, 2002). For plant its acceptable concentration range is 1.5 μg/g. It is revealed by our results that the amount of nickle was 1.2 μg/g, which is within the allowable concentration range.
Zinc is the crucial metal performing very important functions in living organisms such as it act as antioxidant, anti-nociceptive, bone restorative agent and significant for discharge of hormones and cell signaling. There are more than three hundred proteins inside living organisms which are zinc dependent (Tapiero and Tew, 2003). The amount of zinc in our plant of study was 46.7 μg/g, slightly lower than the the permissible range which is 50 μg/g for plants (Srivastava et al., 2006).
In the current study reasonable amount of calcium (3268 μg/g) was observed which means that this plant can be used as an alternate source of calcium. Calcium have important role in metabolic reactions and along with phosphorous have role in the structure of teeth, bones and soft tissue (Shapiro and Heaney, 2003). Calcium played very prominent role in controlling of neurons and muscles activity, vasodilatation and vasoconstriction and glandular secretion. Deficiency of calcium causes weakness of bones and muscles present in skeleton and deformity in beating of heart. Intoxication of calcium causes kidney stone, constipation, nausea, loss of appetite, vomiting, convulsion, abdominal pain, and sometime coma (Gallagher et al., 2012).
Potassium is important element present in all living organisms and its normal concentration is important for regulating action potential and cell signaling in electrically active cells. The cellular regulatory mechanisms include balance of signal transduction, membrane potential, secretion of insulin and other hormones, regulation of cell volume, immune system and vascular tone (Curran, 1998). The concentration of
74 potassium found in the whole plant of D. botrys was 2873 μg/g which increased the nutritional value of the plant.
In our study, considerable amount of the amount of sodium was found 591μg/g in the whole plant. Sodium is very essential nutrient for all living organisms performs prominent role in different metabolic reactions. Normal amount of sodium is necessary for growth and any deficiency in its amount may disturb the whole metabolic reactions in all living organisms. Intracellular and extracellular movement of fluids also depends upon the normal amount of sodium and any variation in its amount may alter its balance and distribution (Morris et al., 2008).
It can be assumed on the basis of our observation that whole plant of D. botrys is an incredible resource of essential nutrients which are necessary for regular physio- chemical performance of healthy body. All of the tested metals were found within the permissible range while cadmium and chromium was not detected (Table 4.3). Therefore, the current observation further authenticated the folkloric use of the plant for different ailments.
5.2 In-vitro activities Plant methanolic crude extract and derived fractions were evaluated for diverse in-vitro activities.
5.2.1 Antimicrobial activity In recent decades, the irrational utilization of antimicrobial drugs has been increased which lead to the growth and propagation of multi˗drug challenging types of pathogenic microbes (WHO, 2001; Aibinue et al., 2003). Furthermore, there is an increase in the rate of morbidity and mortality due to high cost and un˗availability of latest antimicrobial drugs (Williams, 2000). So there is a great need to focus on obtaining compounds from plants having significant antimicrobial activities. It is well established that certain chemicals, produced by plants for their own defense, have antimicrobial properties and are lethal for bacteria and fungi (Harborne, 1988). Thus herbal crude extracts, various fractions and the extracted compounds offer efficient source for the synthesis of antimicrobial drugs (Maloo et al., 2014). In this connection, methanolic plant extract and its various fractions were screened for their antibacterial and anti-fungal potential against different strains of bacteria and fungi.
75 Our observations indicate that MCE and EAF were more effective than DCMF and HxF, in hampering the development of bacterial and fungal strains (Table 4.4 and 4.5). One of the probable reasons of highest antimicrobial potential exhibited by MCE and EAF may be the existence of active resulting metabolites like alkaloids, phenols, flavonoids, saponins, tannins and aromatic compounds (Bonjar et al., 2004). Such compounds have the potential to restrain the growth of many human pathogenic bacteria and fungi by attaching to proteins present at their surface, breaching peptide bonds and altering their biochemical composition or by inhibiting the intake of existing nutrient by microorganisms (Cowan, 1999).
In the present study, antibacterial and antifungal activities of crude extract and subsequent fractions of D. botrys against pathogenic bacteria like C. michiganesis, P. vulgaris and X. campestris and fungal strains such as A. flavus, A. niger, M. piriformis, F. solani and F. oxysporum are described for the first time. C. michiganesis causes a destructive disease of tomato called bacterial canker (Ark and Thompson, 1960). It survives and lives in seeds, soil and has many alternative hosts (Thyr et al., 1975; Moffet and Wood, 1984). Its growth was considerably hindered by MCE followed by EAF causing 9.7 ± 0.15 and 8.7 ± 0.42 mm zone of inhibition. P. vulgaris is widespread gram-negative bacteria commonly found in the environment and also present inside human body as normal flora of gut. It is motile, rod shaped, non-sporing and chemo-heterotrophic bacterium having diverse mode of propagation and transmission (Herter and Broeck, 1911). It is considered the third most important source of infections acquired from hospitals (Bahashwan and Shfey, 2013). Its growth was effectively restrained by EAF and DCMF causing 9.8 ± 0.63 and 8.5 ± 0.52 mm of zone of inhibition. X. campestris instigate citrus blight, rice blight and cabbage infection or black rot around the globe (Britto et al., 2011). MCE exhibited highest antibacterial activity against X. campestris followed by DCMF, causing 12.6 ± 0.54 mm and 10.4 ± 0.61 mm zone of inhibition respectively.
Fungal strains belonging to the genus of Aspergillus, have the ability to produce toxic substances called mycotoxins which degrade food quality and therapeutic potential of drugs extracted from plants (Gautam and Bhadauria, 2009). A. niger is filamentous saprophytic fungus found in organic debris, earth and food products causing stem and root rot of dracaena and sansevieria, cotton boll rot, discoloration of dates, cashew kernel, dried prune, vanilla pods and figs (Bugno et al., 2006; Gautam
76 and Bhadauria, 2008; Bobbarala et al., 2009). While A. flavus causes infection in maize, sorghum, wheat, rice, nuts producing a toxic substance called aflatoxins which are mutagenic, carcinogenic and fatal fungal secondary metabolites (Zain, 2011; Hua, 2013; Kiswii et al., 2014). The growth of both these fungal strains was moderately inhibited by EAF while HxF showed no effect on their growth. One of the probable reasons for its resistance is that HxF did not contain sufficient amount of active phtochemicals which retard its growth or might due to its complex structure.
F. solani is cosmopolitan in distribution and important plant pathogenic fungi which commonly infects 111 different plants species belonging to 87 genera (Kolattukudy and Gamble, 1995). Its growth was effectively retorted by EAF followed by MCE. F. oxysporum is found in soil and causing different human and plant diseases (Nelson et al., 1981). The antifungal activity of MCE and EAF against these two fungal strains was comparable with that of standard drug which can be very useful for the control and management of these pathogenic fungal strains. M. piriformis is a pathogenic fungus found in soil which causes severe post-harvest losses in different agriculture products. Fruits and vegetables such as tomatoes, pears, nectarines and peaches which have high water content are more susceptible to it and are easily attacked by this fungus (Moline and Kuti, 1984; Michailides and Spotts, 1990).
In conclusion, MCE and EAF exhibited considerable lethal effect against two fungal strains which can be further exploited for the extraction of active metabolites used for the control and management of these phyto-pathagenic fungi. However, the overall effect of all the tested solvent against other strains was low to moderate.
5.2.2 Phytotoxic activity In order to control weeds and to enhance crop production effectively, various herbicides and pesticides are used, however, the severe use of synthetic chemicals caused an increased risk of toxicity in the food chain and polluting the soil, water and air (Roger et al., 1994, Pell et al., 1998, Aktar et al., 2009 and, Heap, 2014). Therefore it is very necessary to search out alternative strategies for weed management, like natural herbicides which are eco-friendly, easily biodegradable and more economical as compared to synthetic herbicides. The phytotoxic effect of a plant extract or bioactive constituents derived from it is very useful in assessing their herbicidal ability. L. minor assay is an economical and simple method used for evaluating plants or their extracted
77 compounds for their inhibiting effects. L. minor is susceptible to majority of pollutants and toxic substances, making it helpful in toxicity measurements (Ateeq˗ur˗Rehman et al., 2009). Moreover, it has been noted that anti˗tumor substances reduce its development while there are some compounds which enhance its growth, thus it is very helpful for the screening of new plant development and growth inducers and fulfill the current demand of less toxic, biodegradable natural herbicide (Lewis, 1995; Wang, 2007; Cayuela et al., 2007).
The current study was executed in order to explore and inspect the inhibiting effect of plant extract and their various derived fractions against L. minor. Among all the tested samples maximum phytotoxic effect was shown by MCE, causing maximum inhibition of growth of L. minor followed by DCMF of the plant extract, showing that active allelopathic constituents are present in maximum amount in these solvents as compared to other fractions (Table 4.6). Some plants produce active secondary metabolites which may act as allelochemicals for other plants, effecting the growth of plants growing in its closed vicinity. These active compounds are released either by exudation or by some other epidermal sceration (Patil and Magdum, 2011). Hussain et al. (2010) studied phytotoxic effect of Roscoea nepalensis, Rumix dentatus, Rumix hastatus, Polygonum persicaria, polygonum plebejum and Rheum australe. Ali et al. (2009) evaluated growth inhibiting effect of Euphorbia wallichii root extract obtained from different solvent. All of the mentioned plant extracts and their solvent fractions showed considerable phytotoxic effect (55-100%) at higher concentration (1000 μg/ ml) as compared to low concentration (10 μg/ ml), displayed 25-60% inhibiting effect. Our findings are in line with these previous observations, which further authenticat the phytotoxic effect of D. botrys plant extract and solvent fractions.
5.2.3 Antioxidant activities Free radicals which are known to cause numerous health problems have been studied in order to avoid and control their detrimental effects. Antioxidants protect us from negative effect of these free radicals by either scavenging free radicals or shielding the antioxidant defense mechanisms (Umamaheswari and Chatterjee, 2008). But most of the currently available free radicals scavengers are manmade and have been alleged to cause harmful health effects (Barlow 1990; Sadiq et al. 2015). Due to the unwilling side effects of artificial antioxidants there is a tendency to replace them
78 with antioxidants which occur naturally, having maximum efficiency and less negative effects. Antioxidant based formulations of drugs are employed for the control and prevention of numerous chronic diseases. Plants are the chief source of naturally occurring antioxidants, produce large amount of secondary metabolites having anti- oxidative properties.
The antioxidant potential of plant extract and solvent fraction can be evaluated by employing various procedures but the frequently employed procedures are those to produce free radicals and then deactivate these free radicals by substances having antioxidant properties (Arnao et al., 2001). In the recent study, DPPH and ABTS models were used for the assessment of radical scavenging activity. These are the frequently and standard methods used for the evaluation of radical scavenging potentials of plant extracts and antioxidants substances (Sanchez-Moreno et al., 1998). In our observation EAF and MCE of the plant showed higher scavenging activities in both models and were comparable to that of ascorbic acid, used as standard antioxidant (Table 4.7 and 4.8). One of the probable reasons of higher scavenging activity is that these plants extracts may contain bioactive phytochemicals that donate hydrogen to free radicals reducing the potential damage (Jamshed et al., 2012). The phytochemical composition of EAF and MCE also revealed that it contain maximum amount of phenols and flavonoids which may be the other reason of their highest scavenging activity. The overall scavenging activity of all the tested samples of plant extract was in a dosage dependent manner and increased by increasing their concentration.
5.2.4 Lipoxygenase activity It is reported in the literature that lipoxygenase enzymes have the ability to alter linoleic, arachidonic and other various unsaturated fatty acid into bioactive metabolites that have role in immune and inflammatory responses (Catalano and Procopio, 2005). Lipoxygenases are the main enzymes causes the synthesis of leukotriens which have a vital role in many inflammation causing ailments like asthma, arthritis, allergic disorders and cancer (Rackova et al., 2007; Dobrian et al., 2011). The elevated levels of leukotriens could be noted in case of psoriasis, colitis ulcerosa, asthma, rheumatoid arthritis and allergic rhinitis (Schneide and Bucar, 2005). The synthesis of leukotriens can be hindered through stopping lipoxygenase pathway and targeting it with inhibitors, which can help in curing a number of human health disorders. It is suggested by
79 researchers that the inhibitors of lipoxygenase may help to plan pharmacologically and biologically targeted curative strategies stopping lipoxygenase isoforms and their bioactive metabolites which can be helpful in treatment of cancer (Pidgeon et al., 2007).
The current study demonstrated considerable anti-lipoxygenase potential of D. botrys crude extract and various solvent fractions (Table. 4.9). Among the crude extract and solvent fractions, EAF exhibited maximum lipoxygenase activity as compared to standard. Reactive oxygen species have a role in transmission of inflammation by inducing the secretion of cytokines and by stimulation of enzymes such as lipoxygenase from the inflamed tissues. Hence, plants having high antioxidant contents with high antioxidant activity are proved to be helpful to neutralize reactions causing inflammation. In our observation regarding the antagonistic potential against lipoxygenase, EAF also displayed maximum activity which suggests that this fraction must contained highest amount of active compounds, which could help in stopping the function of lipoxygenase enzymes. The elevated lipoxgenase inhibitory activity might also be due to considerable maximum phenolic, flavonoids, alkaloids, tannins and essential oils (Santha et al., 1991; Manosroi et al., 2013). The phytochemical analysis of crude extract and solvent fractions also displayed considerable amount of phenols, flavonoids, alkaloids and saponins which might exert a combine synergetic effect, inhibiting the function of lioxygenase enzyme.
5.3 In-vivo pharmacological activities 5.3.1 Acute toxicity Plant crude extract of different concentrations were analyzed for acute toxicity using model animal in order to investigate the minimum toxic limit of plant extract. It was observed that plant extract up to dosage of 2000 mg/kg showed no toxic effect on tested animals and all the animals remained alive after 24 hrs of evaluation time (Table 4.10). This observation confirm that the different doses of plant crude extract which are used for different in-vivo activities were within the tolerable range and did not cause lethal effect on the rodents used for the evaluation of these activities.
5.3.2 Anti-inflammatory activity The anti-inflammatory effect of MCE of D. botrys was evaluated by employing carrageenan induced paw edema model and xylene induced ear edema model in mice,
80 as model animals. Acute edema stimulated by carrageenan in mice paw is reputable model to evaluate the anti-inflammatory potential of active natural plant constituents. Injection of carrageenan at sub-plantar region stimulates local edema which increases slowly. It causes maximum inflammation and almost hundred percent larger volume of treated paw which steadily decreases with in 24 hour. The formation of carrageenan- induced edema in mice paw is biphasic process, consist of initial or early phase and second or late phase. The early phase (1-3 hr) is non˗phagocytic type of edema followed by late phase, with enlarged edema development that persisted up to an interval of 5 hrs (Rotelli et al., 2003 and Meckes et al., 2004). Different investigations have been explained the participation of various mediators in different phases of edema provoked by carrageenan. The initial phase of edema is attributed to the liberation of 5- hydroxy-tryptamine, histamine, platelet activating factors, bradykinin and serotonin. The late phase is ascribed by increased release of leukotriens and prostaglandins while the connection between early and late phases is provided by kinins in the inflammatory area (Nguemfo et al., 2007; Unnisa and Parveen, 2011).
It is inferred from our observations that crude extract of D. botrys displayed considerable (p<0.05) anti˗inflammatory effect, acting effectively in a dose dependent manner. Its effect was more pronounced and analogous to that of diclofenac sodium, in the late phase of carrageenan induced assay of paw edema (Table 4.11a). Our observations are in agreement with that of Vineger et al., (1969), Dirosa and Willoughby (1971) who reported that, clinically anti-inflammatory drugs are more efficient against the late phase of inflammation. Even though the exact mechanism of action is yet to be established, it is possible that crude extract of D. botrys have inhibitory effect on the synthesis of cyclo-oxigenase. Its effects seem similar to that of non-steroidal anti-inflammatory drugs (NSAIDs) like indomethacin and diclofenac, whose mechanism of action is stopping of cyclo-oxigenase enzyme, involved in the production of cyclic endo-peroxides which again have role in the synthesis of prostaglandins.
Xylene-stimulated model of ear edema is generally employed to assess the intensity of vessels dilatation and extravasations of neurogenic inflammation. This model is helpful in the assessment of steroidal and non-steroidal agents having anti- inflammatory properties and has accurate predictive values in the selection of such active substances (Kumawat et al., 2012; Sowemimo et al., 2013). Its topical
81 application on the surface of ear induces vasodilatation and enhances the permeability of vessels. It is associated with a neuro-modulator or neurotransmitter, substance P, which is widely distributed in the central or peripheral nervous system having role in a number of physiological processes (Junping et al., 2005). Release of this neurotransmitter from sensory neurons induces vasodilatation and membrane extravasations, signifying its role in neurogenic inflammation.
In our study, all the dosages of crude extract of D. botrys markedly decreased the severity of ear edema in a dose-dependent manner and the anti-edematous activity of 400 mg/kg was comparable to that of reference drug, dexamethasone (Table 4.12a). The inhibition of ear inflammation indicated that crude extract of D. botrys alleviated vasodilatation and plasma extravasations of neurogenic inflammation, which is necessary in managing the initial phase of acute inflammation. The results of the present study are in line with that of Ibironke and Ajiboye (2007) who evaluated the anti-inflammatory ability of methanolic extract of C. ambrosioides (300 mg/kg) which significantly inhibited the edematous effect, with increasing doses over time. The present study has proved that crude extract of D. botrys have active principles exhibiting anti-inflammatory properties, which authenticates the traditional use of this plant as a remedy of pain and inflammation.
5.3.3 Analgesic activity
Analgesic assay of methanolic extract of D. botrys was evaluated by employing chemical and thermal models of pain. The first test performed to investigate the analgesic property of plant extract was formalin test (Table 4.13a). Formalin is used as a chemical stimulant for inducing pain in animal behavioral studies, described by Dubuisson and Dennis (1978). Formalin test is responsive to non-steroidal and mild pain-relieving drugs. Pain induced by formalin consists of two different phases, probably due to dissimilar types of pain mechanisms. The early phase of pain starts immediately after formalin injection and remain for 5 minutes, due to direct stimulation of nociceptors causing bradykinin and substance P release which further stimulate afferent C fibers (Cui et al., 2004). The late phase initiates after 15-30 minutes of formalin injection and remains up to 40 minutes. In this phase pain is induced due to liberation of different pain mediators like histamine and prostaglandin (PG), augmentation of cyclooxigenase COX and liberation of nitric oxide (Bars et al., 2001;
82 Parada et al., 2001; Nakamoto et al., 2010). The biphasic mechanism of pain induction of formalin test can be employed to expose the probable pathway involved in reducing pain (Tjolsen et al., 1992). The action of various analgesic drugs differs in the early and late phase of pain irritated by formalin. Certain drugs like opioids which act centrally, exhibit good response and inhibit both phases of pain while other drugs like acetylsalicylic acid, which act peripherally, stop prostaglandin (PG) synthesis and COX activity, inhibit only late phase of pain (Hunskaar and Hole 1987; Shibata et al., 1989; Yuri et al., 2004; Paschapur et al., 2009).
The analgesic potential of crude extract of D. botrys was also evaluated by hot- plate test, a thermal˗nociception model which is considered one of the most frequent tests used for the evaluation of centrally acting analgesics. Hot-plate induced pain in experimental animals assesses the intricate response to acute nociceptive and non- inflammatory input used for examining central nociceptive activity (Sabina et al., 2009). Several drugs having peripheral analgesic activity, such as aspirin exhibits weak activity against hot plat-induced pain. But other analgesics like morphine and ibuprofen can reduce prostaglandin production through central inhibition of COX (cyclo- oxygenase) or attach to specific opioid receptors in CNS, showing both peripheral and central analgesic effects (Biorkman, 1995; Dolezal and Krsiak, 2002).
Regarding our observation of hot-plate assay, crude extract of D. botrys (400 mg/g) displayed significant analgesic result (p<0.01) after 60 min of treatment, while the standard drug tramadol, which is similar to morphine in action (opioid agonist), increased the threshold level of pain with in 30 min of treatment (Table 4.14). This dissimilarity in the peak analgesic position could be elucidated by the variation in the rate of metabolism or efficiency of active metabolites as the pain relieving potential of tramadol is greater than crude extract of D. botrys (400 mg/g).
5.3.4 Anti-pyretic activity Fever or pyrexia is a condition occurs due to abnormal elevation in body temperature. It is induced by several endogenous inflammatory mediators and pyrogens called cytokines like interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necroses factor-α, macrophage protein-1 and prostaglandins that are released by activated mononuclear peripheral phagocytes and immune cells (Roth, 2006). Brewer's yeast is commonly used exogenous pyrogen, which induces pyrexia in experimental
83 animals. It binds to a specific immunological protein called lipo-polysaccharide binding protein (LBP) (Ukwuani et al., 2012). Its binding causes the production and release of different cytokines mentioned above which trigger arachidonic acid pathway and finally the synthesis and liberation of prostaglandins E2 (PGE2) (Gege-adebayo et al., 2013). Pyrexia induced by yeast is known as pathogenic fever (Aman and Patrick, 2011).
According to classical view, pyrexia is stimulated by inflammation causing mediators (IL-1, IL-2, TNF-α etc), which are released by active peripheral mononuclear macrophages and other immune cells (Zeisberger, 1999). These fever-provoking cytokines are moved through definite carriers from blood to brain and enter the brain through circum-ventricular organs, where they interact with their receptors present on peri-vascular tissue or endothelial cells of brain (Banks et al., 1995; Schiltz and Sawchenko, 2003; Roth et al., 2004; Matsumura and Kobayashi, 2004). This assumed mechanism of induction of fever is commonly known as humoral hypothesis of fever induction. The mentioned inflammatory mediators act on anterior hypothalamus enhancing the release of PGE2 synthesized by cyclooxygenase (COX-2) causing an increase in body temperature (Saper and Breder, 1994). Certain effective antipyretic drugs like paracetamol works by inhibiting the effect of these pyrogenes on COX-2 and PGE2 formation, in temperature neurons present in the anterior hypothalamus of brain (Ashok et al., 2010).
In the current study, all the tested doses of plant crude extract showed a dose dependent moderate to extreme antipyretic effect. Doses of 200 and 400 mg/kg of MCE were highly significant in reducing the induced elevated body temperature of animals at all the assessment time intervals and was analogous to effect of standard drug paracetamol (Table 4.15). Substances having antipyretic potential have been reported to suppress fever by stopping prostaglandin synthetase production, causing an obstruction for the prostaglandin synthesis in brain or decreasing the synthesis of interleukins after the formation of interferons. The plant MCE possibly caused a decline in the body temperature by decreasing the amount of prostaglandins E2 in the temperature controlling centre of brain via its reaction on cyclooxygenase or by raising the synthesis of body’s individual substance having antipyretic properties i.e. arginine and vasopressin (Okokon and Nwafor, 2010). Its antipyretic activity might also be
84 attributed to active secondary metabolites like flavonoids, alkaloids and saponins which are present in considerable amount in the plant extract might have a synergetic effect on reducing the production of prostaglandin. These secondary metabolites suppress peroxidation of arachidonic acid which reduces the level of prostaglandin causing a decline in the intensity of pain and fever. Our results are in close concord with that of Hallal et al. (2010) investigations, according to which dose of 100 and 300 mg/kg of C. ambrosioides plant extract exhibited efficient antipyretic effect against the fever induced by Brewer's yeast.
In conclusion, our investigation scientifically validated antipyretic activity D. botrys, however additional exploration is required to segregate the active metabolites which are involved in its antipyretic action and to examine the exact mechanisms of their action.
5.3.5 Antidiarrheal activity Castor oil induced diarrheal model was employed to authenticate antidiarrheal potential of methanolic extract of D. botrys in selected animals. Diarrhea is a condition of frequent and abnormal defecation of feces caused as a result of distorted motility of water and electrolytes inside the intestinal tract. Castor oil has been commonly used for stimulation of diarrhea in experimental animals because it is metabolized into active molecule ricinoleic acid, which causes irritation and inflammation in the mucosal lining of intestine (Vieira et al., 2000; Afroz et al., 2006). Numerous mechanisms have been proposed which explain the diarrheal initiation potential of castor oil, including inhibition of Na+ K+ ATPase activity in intestinal track, reduction in normal water absorption (Capasso et al., 1994; Imam et al., 2012), motivation of mucosal cyclic adenosine monophosphate (cAMP) mediated active secretion or activation of adenylate cyclase (Pinto et al., 1992), stimulation of platelet activating factors and formation of prostaglandin. The production of prostaglandin induces vasodilatation, contraction of smooth muscle and secretion of mucus in small intestine. In both rodents and humans, E series prostaglandin are considered good diarrheal agent (Mascolo et al., 1994).
In the current analysis the methanolic extract of plant showed antidiarrheal potential in a dosage dependent manner and dose of 200 mg/kg and 400 mg/kg markedly reduce the severity of diarrhea i-e increased latent period and decreased frequency of total wet feces and mean weight of total wet feces, which is comparable to
85 the standard drug loperamide (Table 4.16). It efficiently antagonized diarrhea induced by castor oil and is one of most effective and common drug used for diarrheal treatment. The consequences advocate that methanol extract of plant have antidiarrheal potential, comparable to that of standard drug, but the exact active compound and its mechanisms of action is unknown. The antidiarrheal activity of plant extract can be anticipated from the mode of action castor oil that induces diarrhea i.e the plant extract show counter activity to that of castor oil and bring the condition to normal. The extract may increase electrolyte and water absorption or lower down the secretion of electrolytes and fluid which cause a marked decrease in the surplus intestinal fluid and frequency of diarrhea in a dosage dependent manner. The other possible mechanism of antidiarrheal consequence of plant extract may be the reticence of ricinoleic acid effect on prostaglandin E2 receptors. Blockage of prostaglandin receptors cause a significant decrease in the secretary and intestinal motility effect of prostaglandins that are produced due to the irritating activity of ricinolic acid derived from castor oil. The plant extract may inhibit rapid peristalsis in the large intestine which causes slow movement of feces through the intestine. The slow transit of feces also increases the chances of greater fluid absorption from the feces, causing the stool drier that further slows its passage. The antidiarrheal activity of plant extract may be due to its possible counter effect on the generation or action of cyclic nucleotides i.e. cyclic guanosine monophosphate and cyclic adenosine monophosphate. An elevation in either cyclic guanosine monophosphate (cAMP) stimulates chloride ion (Cl-) secretion and at the same time stops Na+/Cl- absorption (Murek et al., 2010).
Phytochemical investigation of plant extract have revealed that it consist of phenols, flavonoids, alkaloids, terpeniods, saponins, tanins and sterols which may play a role in its antidiarrheal properties. Previous investigations have revealed that plant derived active components like flavonoids, glycoside and tannins etc are the agents having anti-dysentery and antidiarrheal properties (Palombo, 2005). It is also reported that flavonoids have inhibitory effect on motility of fluid inside intestine (Mohammad et al., 2009). Various in-vitro and in-vivo investigation have revealed that flavonoids have the potential to restrain prostaglandin E2 stimulated intestinal secretion and spasmogens tempted contraction. It also hinders release of autocoids and prostaglandin (Dosso et al., 2011). Thus, flavonoids as the inhibitors of biosynthesis of prostaglandins are considered to reduce intensity of diarrhea induced by castor oil (Brijesh, 2009).
86 In conclusion, our investigation revealed that D. botrys extract contains pharmacologically active substances, which are effective for management of diarrhea. Further investigations are required to determine the active compounds and their mechanisms of action responsible for the observed antidiarrheal effect.
5.3.6 Anti-diabetic activity Diabetes has marked impact on human health, quality and life expectancy of patients and health care expenditures (Ngugi et al., 2012).It is considered among one of the chronic metabolic disorders and is allied with other disorders like hypertension and obesity (Akah et al., 2011). Alloxane monohydrate is the common diabeto-genic compound used for induction of diabetes in experimental animals, in order to estimate the anti-diabetic prospective of different plant extracts and active compounds (Viana, 2004; Etuk, 2010). Alloxane monohydrate is organic compound derived from urea, provokes diabetes by selective destruction of pancreatic beta-cells of islets of Langerhans (Iranloye et al., 2011), which causes a massive decline in the synthesis and release of insulin, making it biologically insufficient or unavailable and thus causes an increase in blood sugar level. It confers its toxic effect on pancreatic beta cells through blockage of glucokinase enzyme, formation of free radicals, oxidation of sulphydryl (- SH) group and an instability in calcium homeostasis within the cell (Dunn, 1983; Szkudelski 2001; Dhanesha, 2012). The basic mode of action involves selective absorption of the compound because of its structural resemblance with glucose and highly effective uptake mechanism of beta cells of pancreas (Lenzen, 2008).
In the present investigation, it was eminent that amount of 400 mg/kg considerably reduced blood sugar level at all the assessment time interval, however its effect was highly effective at the third and fourth hour of treatment and was analogous to that of standard drug metformin (Table 4.17). Our investigations are in line with that of Song et al., (2013) observations, in which it was reported that quantity of 300 mg/kg plant crude extract of C. ambrosioides considerably declined elevated blood sugar induced by alloxane monohydrate. Similarly, Kumar et al., (2015) analyzed that methanolic leaf extract of C. album exhibited higher anti-diabetic potential than EAF at various evaluation time intervals. It seemed from our study and the previous investigations that D. botrys and the other related plants belonging to this family contain some active metabolites, particularly in their crude extract which have anti- diabetic potential and decreased the elevated level of sugar in blood to normal. The
87 different apparent mechanisms through which MCE of plant brought the elevated sugar level to normal might be its action to restore the damaged cells of islets of Langerhans, increased the secretion of insulin (Bedoya et al., 1996; Shafighi and Amjad, 2013), increased its sensitivity for absorption of glucose (Yolanda and Adriana, 2006) and encouraged its conversion to glycogen. Its anti-diabetic potential might also be due to its effect to restore abnormal absorption of carbohydrate in the small intestine and facilitate uptake of glucose from blood by cells present in the peripheral regions arbitrated by a glucose transporter GLUT-4, which depend upon the level of insulin (Obatomi et al., 1994).
The restoring effect of plant extract on lowering blood sugar level might be due to presence of active phytochemicals such as flavonoids, saponins, alkaloids, tannins, sterols and terpeniods which have been allied with lowering elevated level of sugar in blood (Middleton et al., 2000). The effectiveness of the mentioned active metabolites against hyperglycemic condition has been reported by Venkatachalam et al., (2011) in Lantana camara fruit ethanolic extract. Glauce et al. (2004) reported that flavonoids caused a decline in the blood sugar level because of lipogenisis and elevated transportation of sugar in lipocytes. Saponin extracted from Momordica charantia caused an increase in the secretion of insulin and decrease in the high blood sugar level in alloxane stimulated mice (Han and Wang, 2008). It has been reported by researchers that alkaloids and its derivatives enhance pancreatic cells regeneration, reinstate insulin secretion and restore the maximum sugar level to normal (Middleton et al., 2000). Alkaloids extracted from Acanthus montanus leaves caused hypoglycemic effect at varying doses of 100, 200 and 300 mg/kg of body weight in alloxane-stimulated animals (Odoh and Ezugwu, 2012). Tannins isolated from plants exhibited anti-diabetic action due to their inhibitory effect on alpha-glucosidase and alpha-amylase enzymes (Kunyanga et al., 2011). It has been reported that terpenoids, extracted from plants, are commonly used by hyperglycemic and hyper blood pressure patients because they restore normal blood glucose level and decrease diastolic blood pressure (Piero et al., 2015). The leaves of Emblica officinalis, which contain high amount of terpenoids, are utilized for the curing of hyperglycemia (Treadway, 1994).
The current study had proved that D. botrys contains some active compounds which might act independently or synergistically in promoting the hypoglycemic potential of plant extract against the elevated sugar level induced by alloxane in rats.
88 However further study is required to isolate the active phytochemicals which are responsible for hypoglycemic activity and to investigate their mechanisms of action responsible for the observed anti-diabetic activity.
5.3.7 Hepativeprotective activity
The preventive action of methanolic extract of D. botrys against damage of liver provoked by CCl4 toxicity in rats was evaluated. CCl4 induced liver injury is one of the most common method employed for the evaluation of hepatoprotective effects of therapeutic plants and drugs in experimental model animals (Ahsan and Haque, 2009).
The effect of CCl4 on liver is similar to that of viral hepatitis (Rubinstein, 1962). It cause toxicity in liver mainly because of its active metabolite, trichloromethyl free radicals, which are formed due to specific type of cytochrome P-450 dependent mixed oxidase enzyme (Lesiuk et al., 1999). These stimulated radicals react again with oxygen and produce trichloro methyl peroxyl radicals. These radicals attack on lipids present in the lipo-protein membrane of endoplasmic reticulum, causing peroxidation, degradation and finally inducing cell death (Recknagel and Waller, 1989). Function of liver can be assisted by evaluating the concentration of SGPT, SGOT ALP and TB, which are normally present in elevated amount in cytoplasm. If there is some problem in the liver, these enzymes leak out into blood stream, however its amount depend upon the severity of liver damage (Nkosi et al., 2005).
In the present study when the animals treated with CCl4 alone for 24 h (group II), caused oxidative stress and significant liver damage as revealed by elevated blood level of SGPT, SGOT, ALP and TB as compared to animals of control group. It was noted that methanol extract of D. botrys (400 mg/kg) extensively declined the increased level of SGPT, SGOT, ALP and TB provoked by CCl4, indicating progress in the functional condition of liver (Table 4.18). The improvement towards normal histological architecture of liver and level of serum enzymes caused by D. botrys methanolic extract (400 mg/kg) is similar to the hepatoprotective effect of silymarin, a common drug having hepativeprotective properties. Similar notabl hepatoprotective properties were shown by the 300 mg/kg, efficiently reduced the high level of these enzymes in the blood (Parkash and Patel, 2005).
The hepatoprotective ability of plant extract might be due to the existence of active metabolites and biomolecules like phenol, monoterpenes, carotiniods,
89 glycosides, flavanoids, alkaloids and essential oil (Gupta and Misra, 2006). The active bio-molecules present in the plant extract restore the damaged liver condition towards normal by, inhibiting the activity of cytochrome P-450, stopping the process of lipid peroxidation, stabilizing the membrane of liver and promoting the biosynthesis of protein and glucoprotein. It is concluded from the result that the MCE of plant have significant hepative-protective potential, depending upon its dose, however, the active compounds which have hepativeprotective potential and the exact mechanism of its action is unknown. Further studies are required to isolate the active components and study its mode of action.
5.3.8 Sedative-hypnotic activity The sedative-hypnotic prospective of methanolic extract of D. botrys was estimated by using thiopental induced hypnosis and comparing with that of diazepam, which was used as standard reference drug (Table 4.19). This is a classical model used in behavioral pharmacology to investigate the sedative and hypnotic properties of substances in the model animals. A number of reports revealed that the central nervous system depressant barbiturates, such as thiopental, bind to barbiturates attaching sites on gamma amino butyric acid GABAA receptors and stimulate GABA-mediated increased polarization of post-synaptic neurons through allosteric alteration of GABAA receptors (Fernandez et al., 2004).
The sedative-hypnotic and anxiolytic prospective of benzodiazepines (BDZs) such as diazepam are typically credited to increase the potential of gamma amino butyric acid (GABAA) (Yemitan and Salahdeen 2005). It unites to gamma sub-unit of
GABAA receptor, causing structural alteration and elevation in GABAA receptors activity. Diazepam do not become an alternate for GABA, which attach at alpha (α) sub-unit, but elevate the rate of channel opening actions which causes a raise in chloride ion transmission and reticence of action-potential (Rang et al., 2003, Ali et al., 2008). According to some reports the sedative-hypnotic potential of diazepam may be because of direct instigation of glycine synapses inside brain (Snydar and Enna, 1975). This may also elucidate the possible way of action of investigated plant sample, as it is obvious from our findings, that sedative-hypnotic effect of plant samples was parallel to that of diazepam. Our results demonstrated that plant methanolic extract of 100 mg and 200 mg effectively induced early arrival of sleep and prolonged the duration of hypnosis caused by thiopental as depicted in Table 4.19. It is Obvious from the current
90 study that the MCE of plant contains some active substances which displayed synergetic effect on thiopental action on the central nervous system, induced early and prolonged hypnosis. It also showed a correlation between the sedative-hypnotic effect of plant extract and that of diazepam.
The initial phytochemical assessment of the plant confirmed the existence of phenols, flavonoids, alkaloids, tannins, sterols and saponins. There are many reports in the scientific literature which confirmed that phenol, flavonoids, alkaloids and saponins rich plants and their extracts possess effective sedative-hypnotic and anxiolytic potential mediated through similarity (in-vitro) with benzodiazepine site of GABAergic system (Trofimiuk et al., 2005; Awad and Ahmed 2009; Estrada-Reyes et al., 2010). Besides, tannins are also involved in non-specific central nervous system depression (Takahashi et al., 1986). It can be assumed that, along with other factors, the sedative- hypnotic effect of plant methanolic extract may be due to the existence of these active metabolites.
5.3.9 Anti-convulsant activity Anti-convulsion potential of crude extract of D. botrys was examined in experimental animals against PTZ-induced clonic seizures. PTZ is the common drug used to induce convulsion in experimental animals due its interaction with ion channel of GABAA receptors. GABA is the main inhibitory neurotransmitter present inside brain and any reticence in its neurotransmission is considered to be the primary cause of epilepsy (Silambujanaki et al., 2010). Increase in the GABAergic neurotransmission is considered to inhibit seizers, while decrease in its transmission enhances seizers (Amabeokua et al., 2007). The standard common antiepileptic and anticonvulsant drug like phenobarbetone, diazepam and clonazepam are considered to enhance GABA- induced opening of chloride ion channels on GABAA receptors causing more chloride ions to enter the neurons which ultimately decreases the activity of neurons inside brain (Mcddonald and Kelly, 1993; De-Sarro et al., 1999). Administration of moderate doses of PTZ (90 mg/kg) provokes clonic seizures with severe negative neuro-chemical effects such as decline in the level of GABA and subsequent reduction of inhibitory responses, causing a condition which aggravates excitation (Walsh et al., 1999; Eloqayli et al., 2003). Racine (1972) reported that there are five stages of epileptic seizure or convulsion also called Racine-seizures score, according to which the first
91 phase is ear facial trembling of model animal, second phase is spastic signal across the body, third phase is cyclonic jerk, fourth phase is clonic-tonic seizures (it proceed into the side position) and the final fifth phase is comprehensive clonic-tonic convulsions (it proceed into the backward position).
In the current investigation, the consequence of 400 mg/kg of MCE was comparable to that of standard drug clonazepam, radically delayed the onset of first clonus and prolonged the duration of death effectively (Table 4.20). The resemblance of plant extract with that of standard drug suggests that it might activate GABAA receptors and enhance its inhibitory neurotransmission. Phytochemical investigation of MCE of D. botrys showed the presence of considerable amount of active metabolites such as saponins, flavonoids, steroids, tannins and alkaloids, among which various phytochemicals have been observed to have activities effecting central nervous system. Previously, flavonoids (Asl et al., 2007), alkaloids (Taesotikul et al., 1998), essential oil (Dallmeier and Carlini, 1981), saponins, sterols are reported to have anticonvulsant potential in experimental convulsion models such PTZ and MES (Chaohan et al., 1988 and Kastur et al., 2002). Different flavonoids extracted from plants have properties like benzodiazepine, work in the nervous system and alter GABA-produced chloride flow in different convulsion, sedation and anxiety models using experimental animals (Asl et al., 2007).
In our observation the anti-convulsant activity shown by the plant extract might be due to collective effect of its all active metabolites present in it, which need to be isolated and investigate their proper mechanism of action. However our observation confirmed the folkloric use for the treatment of convulsion and neurological disorders.
5.3.10 Antidepressant activity
The forced swim test (FST) is commonly employed for the evaluation of anti- depressant prospective of drugs and herbal extracts in experimental animals. According to this model, the extension in duration of mobility reflects anti-depressant effect, while decrease in duration of mobility indicates neurological depression in the central nervous system (Subarnas et al., 1993). The animal found motionless for longer interval of time shows that it is in situation of exhaustion, sadness and fatigue are the common indications of depression as also found in human (Aladeokin and Umukoro, 2011). There is close relationship between clinical efficiency of antidepressant drugs
92 and their influence in FST, which is not found in any other behavioral model (Steru et al., 1985; Porsolt, 1981).
The data obtained from our results showed that only higher dose (200 mg/kg) of plant extract extensively condensed the immobility time interval and was analogous to that of fluoxetine, used as standard antidepressant drug (Table 4.21). Fluoxetine reduce the neurological depression by stopping nor-epinephrine (NE) re-uptake. Its effect in the FST might be due to improved accessibility of neurotransmitters NE and serotonin at post-synaptic site following reuptake inhibition (Pal and Dandiya, 1993). According to primary hypothesis of depression projected 40 years ago, the common cause of depression is the functional insufficiency of cerebral mono-aminergic transmitters like dopamine (DA), serotonin (5-hydroxytryptamine) 5HT and NE which are situated at synapses (Roiser et al., 2012). Certain studies have reported stabilizing effect of plant extract through regularization of mono-aminergic level and stress parameters, which provide evidence that the anti-depressant ability of plant extract might be due to restoration of mono-aminergic neurotransmitters (Rai et al., 2003).
93 VI. SUMMARY
Dysphania botrys is an annual herbaceous plant belongs to family Amaranthaceae, native to Asia and Europe found in Pakistan, India and Iran. Previously this plant belonged to genus Chenopodium but due some variation in anatomic and taxonomic characteristics it was placed in a separate genus of Dysphania. In conventional medicine, D. botrys has been utilized for the cure of diverse diseases like cold and influenza, asthma, head ach, liver and digestive problems and healing of wounds. The current work was designed to explore MCE and its various solvent fractions for phytochemical analysis, various in-vitro and in-vivo pharmacological activities (crude extract only) in order to furnish scientific authentication to its ethno- medicinal uses. Qualitative phytochemical assessment of D. botrys proved the existence of alkaloids, phenols, flavonoids, tannins, saponins, and sterols in the MCE while in HxF only flavonoids and saponins were detected. Highest amount of phenol, flavonoids, alkaloids were found in EAF while crude extract contained maximum amount of saponins. In the proximate analysis, nitrogen-free extract were present in higher amount followed by protein, inorganic matter, moisture and fats while crude fibers were found least in amount. Among different minerals calcium was found in maximum amount, followed by potassium, sodium, iron, zinc, copper, nickel and lead while no cadmium and chromium were detected. MCE and EAF displayed considerable antibacterial activity against X. Campestris and P. Aerugonosa respectively, while exhibited moderate bactericidal action against other strains of bacteria. In case of antifungal activity MCE hindered the growth of F. oxysporum effectively, while HxF showed no effect on the growth of A. flavus and A. niger. Maximum phytotoxic effect was shown by MCE, inhibiting the growth of L. minor, while HxF displayed least effect on its growth. EAF exhibited maximum DPPH and ABTS scavenging activity followed by MCE. EAF showed considerable antagonistic effect while HxF displayed least inhibiting effect against lipoxygenase activity. In the in-vivo pharmacological activities of MCE of D. botrys, toxicity test showed no sign of severe abnormality and mortality up to a dose of 2000 mg/kg. Crude extract (200 and 400 mg/kg) showed substantial (p<0.05) anti˗inflammatory action, both at the early and late phase of carrageenan-induced paw edema while in case of xylene induced ear edema dose of 400 mg/kg was highly effective (p<0.01) in reducing ear inflammation. Dosage of 200 mg/kg of plant extract depicted peripheral analgesic activity at both phases of
94 analgesia, causing considerable (p<0.05) diminution in severity of pain while dose of 400 mg/kg was highly significant (p<0.01) causing 78.57% and 82.14% pain inhibition. In the central analgesic activity (hot plate test) the action of 400 mg/kg was highly efficient (p<0.01) after 120 min of assessment time interval. In the brewer‟s yeast-provoked pyrexia model the effect of 400 mg/kg of plant extract was extremely significant (p<0.001) and was parallel to that of standard drug paracetamol in reducing body temperature to normal, at all the assessment time intervals (1h-5h). Plant extract (400 mg/kg) displayed substantial (p<0.01) antidiarrheal effect, increased the latent period of diarrhea and caused a decline in the total wet fecal frequency and mean weight of fecal drops as compared to control. The elevated blood sugar induced by alloxane monohydrate in the anti-diabetic activity was significantly (p<0.05) reduced by crude extract (400 mg/kg), however its effect was highly significant (p<0.01) after 4 h of evaluation time. In the hepatoprotective assay, plant extract at dosage of 400 mg/kg notably (p<0.05) declined elevated level of ALP and TB while its effect was highly significant (p<0.01) reducing the level of SGPT and SGOT, when compared to toxic control. Plant extract (100 and 200 mg/kg) demonstrated a remarkable (p<0.05) synergetic effect on the thiopental induced hypnosis caused an early arrival of sleep and effectively (p<0.01) prolonged the duration of sleep as compared to standard drug diazepam. In the PTZ-induced convulsion activity, plant extracts (200 and 400 mg/kg) effectively (p<0.05) postponed the initiation of first clonus and prolonged the time of death as compared to control. In the antidepressant assay, amount of 200 mg/kg extensively (p<0.05) decrease the immobility time as compared to control while the other doses showed no significant effect.
95 VII. CONCLUSIONS AND RECOMMENDATIONS Conclusions 1. Phytochemical investigation of the whole plant of D. botrys showed that it contain considerable amount of phenol, flavonoids, alkaloids, saponins, nitrogen free extract, protein, calcium, magnesium, sodium, iron and zinc while all the tested metals were found within the permissible limit and no cadmium and chromium was detected. 2. MCE and EAF showed maximum antibacterial, antifungal, DPPH and ABTS radical scavenging and lipoxygenase activities while the other extracts showed moderate effect. 3. MCE up to 2000 mg/kg was found safe and had no toxic effect after 24 h of evaluation time. 4. MCE of plant showed significant pharmacological effects in model animals and all the ethno-medicinal uses of the plant were scientifically authenticated. 5. MCE of 400 mg/kg of extract of plant displayed significant anti-analgesic, anti˗inflammatory, antipyretic, anti-diabetic, antidiarrheal, anticonvulsant, hypnotic and hepativeprotective effects in all the tested animals‟ models. 6. In-vitro activities like antifungal, lipoxygenase, phytotoxic activities and all the in-vivo pharmacological activities of D. botrys were described for the first time.
96 Recommendations
1. The plant of D. botrys is an excellent source of a variety of nutrients and noxious metals were found within tolerable range, so this plant may possibly be utilized as a foodstuff supplement and also can be added in silage and feed for domestic animals. 2. Further research is suggested to study phytochemical and pharmacological consequence of crude extract and derived solvent fractions of leaf, stem and roots individually. 3. Further investigation is necessary to separate the active compounds having pharmacological properties and study its mechanism of action. Once its mechanism of action is recognized then its effectiveness may be enhanced by varying its configuration synthetically.
97 LITERATURE CITED
Abdollahnejad, F., M. Mosaddegh, S. Nasoohi, J.M. Zadeh, M. Kamalinejad and M. Faizi. 2016. Study of sedative-hypnotic effects of Aloe vera aqueous extract through behavioral evaluations and EEG recording in rats. Iran. J. Pharm. Res. 15(1): 293-300.
Aibinu, I. E., V. C. Ohaegbulam, E. A. Adenipekun, F. T. Ogunsola, T. O. Odugbemi, and B. J. Mee. 2003. Extended-spectrum β-lactamase enzymes in clinical isolates of Enterobacter species from Lagos, Nigeria. J. Clin. Microbiol. 41(5): 2197-2200.
Adedapo., A., F. Jimoh and A. Afolayan. 2011. Comparison of the nutritive value and biological activities of the acetone, methanol and water extracts of the leaves of Bidens pilosa and Chenopodium album. Acta Pol. Pharm. 68(1): 83-92.
Afroz, S., M. Alamgir, M.T. Khan, S. Jabbar, N. Nahar and M.S. Choudhuri. 2006. Antidiarrhoeal activity of the ethanol extract of Paederia foetida Linn. (Rubiaceae). J. Ethnopharmacol. 105(1-2): 125-130.
Ahmad, M., O.A. Mohiuddin, Mehjabeen, N. Jahan, M. Anwar, S. Habib, S.M. Alam and I.A. Baig. 2012. Evaluation of spasmolytic and analgesic activity of ethanolic extract of Chenopodium album L. and its fractions. J. Med. Plants. 6(31): 4691-4697.
Ajaib, M., T. Hussain, S. Farooq and M. Ashiq. 2016. Analysis of antimicrobial and antioxidant activities of Chenopodium ambrosioides: an ethnomedicinal plant. J. Chem. 4: 1-11.
Akah, P. A., S.U. Uzodinma and C.E. Okolo. 2011. Antidiabetic activity of aqueous and methanol extract and fractions of Gongronema latifolium (Asclepidaceae) leaves in alloxan diabetic rats. J. Appl. Pharmaceut. Sci. 1(9): 99-103.
Aktar, M.W., D. Sengupta, and A. Chowdhury. 2009. Impact of pesticides use in agriculture: their benefits and hazards. Interdiscip.Toxico. 2: 1-12.
98 Akuodor, G.C., I. Muazzam, M.U. Idrees, U.A. Megwas, J.L. Akpan, K.C. Chilaka, D.O. Okoroafor and U.A. Osunkwo. 2011. Evaluation of the antidiarrheal activity of methanol leaf extract of Bombax buonopozense in rats. Ibnosina J. Med. B.S. 3(1): 15-20.
Al-Ghamdi, M.S. 2001.The anti-inflammatory, analgesic and antipyretic activity of Nigella sativa. J. Ethnopharmcol. 76: 45-48.
Ali, A and M.S. Memon. 2008. Incorporation of Enteromorpha procera Ahlner as nutrition supplement in chick's feed. Int. J. Biotechnol. 5(3): 211-214.
Ali, N., N.K. Rubina, G.R. Wahib, M.I. Choudhary. 2009. Biological screening of different root extracts of Euphorbia wallichii. Pak. J. Bot. 41(4): 1737-1741
Alnamer, R., K. Alaoui, E.H. Bouidida, A. Benjouad and Y. Cherrah. 2011. Sedative and hypnotic activities of the methanolic and aqueous extracts of Lavandulaofficinalis from Morocco. Adv. Pharmacol. Sci. 20: 12.
Al-sayed, A.M., M.A. Al-Yahya and M.A. Hassan. 1989. Chemical Composition and Antimicrobial Activity of the Essential Oil of Chenopodium botrys growing in Saudi Arabia. Int. J. Crude Drug. Res. 27(4): 185-188.
Aly, M., M.A. El-Sayeda, M.A. Al-Yahyaa and M. Hassanb. 1989. Chemical composition and antimicrobial activity of the essential oil of Chenopodiumbotrys growing in saudiarabia. Pharm. Biol. 27(4): 185-194.
Amabeokua, G.J, I. Green, J. Kabatende. 2007. Anticonvulsant activity of Cotyledon orbiculata L. (Crassulaceae) leaf extract in mice. J. Ethnopharmacol. 112: 101- 107.
Amjad, L. and Z. Alizad. 2012. Antibacterial Activity of the Chennopodium album Leaves and Flowers Extract. Int. J. Med. Health Res. 6(1): 14-17.
Amjad, L., 2011. Comparative study of pollen extracts allergenicity of Chenopodium album L. and ChenopodiumbotrysL. an in vivo study. International Conference
99 on Biosciences, Biochemistry and Bioinformatics IACSIT. Press, Singapore. 338-352.
Andov, L. A., M. Karapandzova, I. Cvetkovikj, G. Stefkov and S. Kulevanova. 2014. Maced. Pharm. Bull. 60(1): 45-58.
Arnao.M.B., A. Cano, J.F Alcolea and M. Acosta. 2001. Estimation of free radical- quenching activity of leaf pigment extracts. Phtochem.Analysis. 12 (2): 138- 143.
Artschwager, M. 1996. Healing with plants in the American and Mexican West.1st ed. University of Arizona Press. 133.
Asl, M.A, S.S. Rad and F. Zamansoltani. 2007. Anticonvulsant effects of aerial parts of Passiflora incarnata extract in mice: involvement of benzodiazepine and opioid receptors. BMC Complement Altern. Med. 7: 1-6.
Ateeq-ur-Rehman, A., S. Mannan, M. Z. Inayatullah, M. Akhtar, Qayyum and B. Mirza. 2009. Biological evaluation of wild thyme (Thymus serpyllum). Pharm. Biol. 47: 628-633.
Awad R., F. Ahmad and N. Bourbonnais-Spear. 2009. Ethnopharmacology of Qeqchi Maya antiepileptic and anxiolytic plants: effects on the GABAergic system. J. Ethnopharmacol. 125(2): 257-264.
Awadh, A.N.A., W.D. Juelich, C. Kusnick and U. Lindequist. 2001. Screening of Yemeni medicinal plants for antibacterial and cytotoxic activities. J. Ethnopharmacol. 74: 173-179.
Aziz, A. and I.A. Khan. 2013. Pharmacological evaluation of sedative and hypnotic activities of methanolic extract of Lycopus europaeus in mice. J. Phytopharm. 2(4): 8-12.
Bahashwan, S.A. and H.M. El-Shafey. 2013. Antimicrobial resistance patterns of Proteus isolates from clinical specimens. Eur. Sci. J. 9: 188-202.
100 Bahekar, S.E. and S.K. Ranjana. 2015. Antidiarrheal activity of ethanolic extract of Manihot esculenta Crantz leaves in Wistar rats. J. Ayurveda Integr. Med. 3: 9475-9476.
Bano, A., M. Ahmad, T. Be- Hadda, A. Saboor, S.Sultana, M.Zafar, M.P. Khan, M. Arshad and M.A. Ashraf. 2014. Quantitative ethnomedicinal study of plants used in the Skardu valley at high altitude of Karakoram-Himalayan range, Pakistan. J. Ethnobiol. Ethnomed. 10: 43-44.
Bedoya, F.J., F. Solano and M. Lucas. 1996. N-monomethyl-arginine and nicotinamide prevent streptozotocin-induced double strand DNA break formation in pancreatic rat islets. Experientia. 52: 344-347.
Bedrossian, G., P.S. Beauchamp, B. Bernichi, V. Dev, K.Z. Kitaw and H. Rechtshaffen. 2001. Analysis of North American Chenopodium botrys Essential Oil Isolation and Structure of Two New Sesquiterpene Alcohols. J. Essent. Oil Res. 3(6): 393-400.
Benzie, I.F.F and J.J. Strain. 1996. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant powe: The FRAP assay. Anal.Biochem. 239(1): 70-76.
Bergemeyer, H.U. 1974. Editor in: Method of enzymatic analysis. New York: Academic Press Inc. 2: 856-864.
Bhawana, S. and S.U. Kumar. 2009. Hepatoprotective activity of some indigenous plants. Int. J. Pharmtech. Res. 4: 1330-1334.
Bojilov, D., S. Dagnon and I. Ivanov. 2017. Constituent composition of Chenopodium botrys essential oil. Bulg. Chem. Commun. 49: 124-129.
Bojilov, D., S. Dagnon and I. Ivanov. 2017.New insight into the flavonoids composition of Chenopodium botrys. Phytochem.Lett. 20: 316-321.
Bonjar, G.H. and A. Nik, S. Aghighi. 2004. Antibacterial and antifungal survey in plants used in indigenous herbal-medicine of south east regions of Iran. J. Biol. Sci. 4: 405-12.
101 Brend, Y., L. Galili, H. Badani, R. Hovav and S. Galili. 2012. Total phenolic content and antioxidant activity of red and yellow quinoa (Chenopodium quinoa wild.) seeds as affected by baking and cooking conditions. Food Nutr. Sci. 3: 1150- 1155.
Buchbauer, G., L. Jirovetz, M. Wasicky, J. Walter and A. Nikiforov. 1995. Head space volatiles of Chenopodium botrys (Chenopodiaceae). J. Essent. Oil Res. 7: 305- 308.
Buerki, R.A. and G.J. Higby. 2007. Dosage forms and basic preparations: History. Pine Hurst, North Carolina, USA. Encyclopedia of pharmaceutical technology. 2: 125-129.
Catoni, C., H. Schaefer and A. Peters. 2008. Fruit for health: The effect of flavonoids on humoral immune response and food selection in a frugivorous bird. Funct. Ecol. 22(4): 649-654.
Cayuela, M. L., P. Millner, J. Slovin and A. Roig. 2007. Duckweed (Lemna gibba) growth inhibition bioassay for evaluating the toxicity of olive mill wastes before and during composting. Chemosphere. 68(10): 1985-1991.
Chalabian F., M. Azam. L. kambiz and S. Sadouzi. 2006. Comparison of the essential oils of Chenopodium botrys, Ferulago subvelutina, Rosa gallica and antimicrobial activity of the oils against some microbes. Iran. J. Med. Aroma. Plants. 22(2): 146-154.
Chattopadhyay, D., G. Arunachalam, A.B. Mandal and S.C. Shwan. 2002. Mandal. Evaluation of antipyretic activity of leaf extracts of Mallotuspeltatus(Geist) Muell. Arg. varacuminatus: A folk medicine. Phytomedicine. 9:727-730.
Chauhan, A.K, M.P. Dobhal and B.C. Joshi. 1988. A review of medicinal plants showing anticonvulsant activity. J. Ethnopharmacol. 22: 111-123.
Chekem, M.S., P. K. Lunga, J.D.D Tamokou, J.R. Kuiate, P. Tane, G. Vilarem and M. Cerny. 2010. Antifungal Properties of Chenopodiumambrosioides Essential Oil AgainstCandida Species. The P. J. 3(9): 2900-2909.
102 Chopra, A. 2002.Doiphode Ayurvedic medicine: Core concept, therapeutic principles and current relevance. Med. Clin. of North Am. 86:75-89.
Clemants, S.E. and S.L. Mosyakin. 2003. Dysphania R. Brown; Chenopodium Linnaeus. Flora of North America Editorial Committee (ed.), Flora of North America north of Mexico. Oxford Univ. Press, New York and Oxford. 4: 267- 299.
Cook, N.C and S. Samman. 1996. Flavonoids-chemistry, metabolism, cardioprotective effects, and dietary sources. J. Nutr. Biochem 7: 66-76.
Cordell, G. 2009. Sustainable drugs and global health care. Quim.Nova. 32(5): 1356- 1364.
Cowan, M.M. 1999. Plant products as antimicrobial agents.Clin. Microbiol. Rev. 12: 564-582.
Cui, M., S. Khanijou, J. Rubino, and K.R. Aoki. 2004. Subcutaneous administration of botulinum toxin A reduces formalin-induced pain. Pain. 107(1-2):125-133.
Dallmeier, K. and E.A. Carlini. 1981. Anesthetic, hypothermic, myorelaxant and anticonvulsant effects of synthetic eugenol derivatives and natural analogue. Pharmacol. 22: 113-127.
Dembitsky, V., I. Shkrob and L.O. Hanus. 2008. Ascaridole and related peroxides from the genus Chenopodium. Biomed. 152: 209-215.
De-Pascual, J., I.S. Bellido and M.S. González. 1980. Chenopodiaceae components: polyoxigenated sesquiterpenes from C. botrys. Tetrahedron. 36: 371-376.
De-Pascual, J., J.B. Sanchez and M.G. Sanchez.1978.Chenopodiaceae components I. sesquiterpenoids from Chenopodium botrys L. An. Quim. 74: 91-96.
De-Smet, P. 2002. Herbal remedies. N. Engl. J. Med. 3: 2042-2046.
103 Dinan, L., P. Whiting and A.J. Scott 1998..Taxonomic Distribution of phytoecdysteroids in seeds of members of the Chenopodiaceae. Biochem. Syst. Ecol. 26: 553-576.
Dini, I., G.C. Tenore and A. Dini. 2010. Antioxidant compound contents and antioxidant activity before and after cooking in sweet and bitter Chenopodium quinoa seeds. J. Food Sci. Technol. 43(3): 447-451.
Dubuisson, D. and S.G. Dennis. 1977. The Formalin test: quantitative study of the analgesic effects of morphine, meperidine and brain stem stimulation in rats and cats. Pain. 4: 161-174.
Dwivedi and Singh. 2012. Antibacterial potential of Chenopodium murale l. against resistant human pathogenic bacteria. Int. J. Inst. Pharm. Life. Sci. 2(3): 115-20.
Dziki, U.G., M. Swieca, M. Sulkowski, D. Dziki, B. Baraniak and J. Czyz. 2013. Antioxidant and anticancer activities of Chenopodium quinoa leaves extracts. In vitro study.Food and Chem. Taxicol. 57: 154-160.
El-Faky, F.K., O. Attif, A. Ela and M. N. Gaanem. 1995. Antimicrobial evaluation of extracts from some Yemeni plants. Alexanderian J. Pharma. Sci. 9: 35-37.
Elliot, M., K. Chithan and C.T. Нeohdris. 2000. The effect of plant flavonoids on mammalian cells: Implications for inflammation, heart disease and cancer. Pharmacol.Rev. 52: 673-751.
Emamghoreishi, M. and G. Heidari-Hamedani. 2006. Sedative-hypnotic activity of extracts and essential oil of Coriander seeds. Iran J. Med Sci. 31(1): 22-27.
Estrada-Reyes R., C. López-Rubalcava, L. Rocha, G. Heinze, A.R. González Esquinca and M. Martinez-Vazquez. 2010. Anxiolytic-like and sedative actions of Rollinia mucosa: possible involvement of the GABA/benzodiazepine receptor complex. Pharm. Biol. 48(1): 70-75.
Fabricant, D. and N.R. Farnsworth. 2001. The value of plants used in traditional medicine for drug discovery. Environ. Health Perspect. 109: 69-75.
104 Fabricant, D.S and N.R. Farnsworth. 2001. The value of plants used in traditionalmedicine for drug discovery. Environ. Health.Perspect. 1: 69-75.
Farnsworth, N.R. 1984. . The role of medicinal plants in drug development.In Natural products and Drug Development. Tindall and Cox, London.
Farnsworth, N.R., O. Akerele, A.S. Bingel, D.D. Soejarto and Z. Guo. 1985. Medicinal plants in therapy. WHO. 63: 965-981.
Farnsworth, N.Ra and D. Soejarto. 1991. Global importance of medicinal plants. In: Conservation of Medicinal Plants. Cambridge University Press, Cambridge.
Feizbakhsh, A., S. Sedaghat and M.S. Tehrani. 2003. Chemical composition of the essential oils of ChenopodiumbotrysL. from two different locations in Iran. J. Essent. oil Res.15(3): 193-194.
Fernandez S., C. Wasowski, A.C. Paladini, M. Marder. 2004. Sedative and sleep- enhancing properties of linarin, a flavonoid-isolated from Valeriana officinalis. Pharmacol. Biochem.and Behav. 77 (22): 399-404.
Folch, J., M. Lees and G.H.Sloane-Stanely. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226: 497-509.
Foroughi, A., P. Pouya, F. Najafi, M.Z. Zangeneh, A. Zangeneh and R. Moradi. 2016. Chemical Composition and Antibacterial Properties of Chenopodiumbotrys L. Essential Oil. Int. J. Pharmacognosy. Phytochem. Res. 8(11): 1881-1885.
Fuentes-Bazan, S., G. Mansion, and T. Borsch. 2012. Towards a species level tree of the globally diverse genus Chenopodium (Chenopodiaceae). Mol. Phylogenetics. Evol. 62: 359-374.
Fuentes-Bazan, S., P. Uotila, and T. Borsch. 2012. A novel phylogeny-based generic classification for Chenopodium sensu lato, and a tribal rearrangement of Chenopodioideae (Chenopodiaceae). Willdenowia. 42: 5-24.
105 Gallego, F., A.Swiatopolk-Mirski and E. Vallejo. 1965. Contribution to the study of Chenopodium botrys L. in relation to its essential oil. Rev. Bras. Farmacogn. 25: 69-87.
Gallagher, J.C., V. Yalamanchili and L.M. Smith. (2012). The effect of vitamin D on calcium absorption in older women. J. Clin. Endocrinol. Metab. 97(10): 3550- 3556.
Gawlik-Dziki, U., M. Swieca, M. Sulkowski, D. Dziki, B. Baraniak, J. Czy. 2013. Antioxidant and anticancer activities of Chenopodium quinoa leaves extracts in- vitro study. Food and Chem. Toxicol. 57: 154-60.
Gesinski, K. and K. Nowak. 2011. Comparative analysis of the biological value of protein of Chenopodium quinoa (wild) and Chenopodium album l. Part i. amino acid composition of the seed protein. Acta Sci. Pol. Agricultura. 10(3): 47-56.
Glauce, S.B, C,M., Ana-Carolina, R.L. Ana-Michelle, A.M. Kalyne and G.V. Tiago. 2004. Hypoglycemic and anti-lipemic effects of the aqueous extract from Cissus sicyoides. Biomed.Central Pharmacol. 4: 9-12.
Gornall, A., J. Bardawil and M.M. david. 1949. Determination of protein by biuret modified method. Boil. Chem. 177: 751.
Gqaza, B.M., C. Njume, N.I. Goduka and G. George. 2013. Nutritional assessment of Chenopodium album l. (imbikicane) young shoots and mature plant-leaves consumed in the Eastern Cape province of South Africa. International Conference on Nutration and food Science. 53(19): 97-102.
Guo, J., C. Xue, J.A. Duan, D. Qian, Y. Tang and Y. You. 2011. Anticonvulsant, antidepressant-like activity of Abelmoschus manihot ethanol extract and its potential active components in vivo. Phytomedicine. 18(14): 1250-1254.
Gupta, S., M. George, M. Singhal, G.N. Sharma and V. Garg. 2010. Leaves extract of Murraya koenigii L. for anti-inflammatory and analgesic activity in animal models. J. Adv. Pharm. Technol. Res. 1(1): 68-77.
106 Halberstein, R. 2005. Medicinal plants: Historical and cross-cultural usage patterns. Ann. Epidemiol. 15: 686-699.
Hallala, A., S. Benalia, M. Markouk, K. Bekkouchea, M. Larhsinia, A. Chaitb, A. Romanec, A. Abbada and M. K. El-Abdounid. 2010. Evaluation of the Analgesic and Antipyretic Activities of Chenopodiumambrosioides L. Asian J. Exp. Biol. Sci. 1(1): 189-192.
Han, C., Q. Hui and Y. Wang. 2008. Hypoglycaemic activity of saponin fraction extracted from Momordica charantia in PEG/salt aqueous two-phase systems. Nat. Prod. Res. 22: 1112-1119.
Hanelt, P. 2001. Mansfeld's Encyclopedia of Agricultural and Horticultural Crops.1st ed. Springer Verlag.Publ. Berlin. 248.
Harborne, J.B. 1988. Introduction to ecological biochemistry, Third edition.Academic press, New York.
Hartmann, T. 1991. Alkaloids.In herbivores; their interaction with secondary plant metabolites. Rosenthal and Berenbaum, eds. Academic press, San Diego. 2(1): 33-85.
Harvey, A. 2000.Strategies for discovering drugs from previously unexplored natural products.Drug Discov. Today 5: 294-300.
Havsteen, B.H. 2002.The biochemistry and medical significance of the flavonoids. Pharmacol.and Ther. 96: 67-202.
Hazrat, A., M. Nisar, J. Shah, S. Ahmad. 2011. Ethnobotanical study of some elite plants belonging to dir, Kohistan valley, Khyber Pukhtunkhwa, Pakistan. Pak. J. Bot. 43(2): 787-797.
Hegnauer, R. 1988. Biochemistry, distribution and taxonomic relevance of higher plant alkaloids.Phytochem. 27: 2423-2427.
Herborne J.B. 1973. Phytochemical methods. Chapman and Hall, London. 110-113.
107 Herter, C.A. and C.T. Broeck (1911) A biochemical study of Proteus vulgaris Hauser. J Biol. Chem. 9: 491-511
Ho, C.T., C.Y. Lee and M.T. Huang. 1992. Phenolic compounds in food and their effects on health, analysis, occurrence, and chemistry. ACS Symposium Series 506, American Chemical Society, New York.
Hosseinzadeh, H and V. Khosravan. 2002. Anticonvulsant effects of aqueous and ethanolic extracts of Crocus sativus stigmas in mice. Arch. Intern. Med. 5: 44- 47.
Hu, L. 2008. Textual research of medicinal and edible plants in Shanhaijing.Chinese Materia Medica. 33(10): 1226-1230.
Hua, S. 2013. Biocontrol of Aspergillus flavus.Pich.Anoml. 2: 34-37.
Hussain, F, I. Hameed, G. Dastagir, S. Nisa, I. Khan and B. Ahmad. 2010. Cytotoxicity and phytotoxicity of some selected medicinal plants of the family Polygonaceae. Afr. J. Biotechnol. 9(5): 770-774.
Ibironke, G.F. and K.I. Ajiboye. 2007. Studies on the anti-inflammatory and analgesic properties of Chenopodium ambrosioides leaf extract in rats. Inter. J. Pharm. 3(1): 111-115.
Ibrahim, M., M.L. Rude, N.J. Stagg, H.P. Mata and T.W. Vanderah. 2006. Cb2 cannabinoid receptor mediation of anti-nociception. Pain. J. Ethnopharmacol. 12: 36-42.
Jain, S. and H.S. Puri. 1984. Ethnomedicinal plants of Jaunsar-Bawar hills, Uttar Pradesh, India. J. Ethnopharmacol. 12: 213-22.
Jamshed, I., S. Zaib, U. Farooq, A. Khan, I. Bibi and S. Suleman. 2012. Antioxidant, Antimicrobial, and Free Radical Scavenging potential of aerial parts of Periploca aphylla and Ricinus communis. Inter. Schol. Res. Network, 10: 5402- 5408.
108 Janovska, D., K. Kubikova and L. Kokoska. 2003. Screening for antimicrobial activity of some medicinal plants species of traditional Chinese medicine. Czech J. Food Sci.21: 107-110.
Jiangrong, L. and Y. Jiang. 2007. Litchi flavonoids: Isolation, identification and biological activity. Molecules. 12: 745-758.
Jin-Ming, K., G. Ngoh-Khang, C. Lian-Sai and C. Tet-Fatt. 2003. Recent advances in traditional plant drugs and orchids. Acta Pharm.Sin. 24: 7-21.
Junping, K., N. Yun, N. Wang, L. Liang and H. Zhi-Hong. 2005. Analgesic and anti- inflammatory activities of total extract and individual fractions of Chinese medicinal plants, Polyrhachislamellidens. Biol. Pharm. Bull. 28: 176-180.
Kabat, E.A and M.M. Mayer. 1961. Experiment of Immunochemistry. In: Thomas, Springfield. 2: 123-30.
Kadereit, G., D. Gotzek, S. Jacobs, and H. Freitag. 2005. Origin and age of Australian Chenopodiaceae. Org. Divers.Evol. 5: 59-80.
Kadereit, G., E.V. Mavrodiev, E.H. Zacharias, and A.P. Sukhorukov. 2010. Molecular phylogeny of Atripliceae (Chenopodioideae, Chenopodiaceae): implications for
systematics, biogeography, flower and fruit evolution, and the origin of C4 photosynthesis. Amer. J. Bot. 97: 1664-1687.
Kadereit, G., T. Borsch, K. Weising, and H. Freitag. 2003. Phylogeny of Amaranthaceae and Chenopodiaceae and the evolution of C4-photosynthesis. Intl. J. Pl. Sci. 164: 959-986.
Kang, J. Y., M.N. A. Khan, N.H. Park, J.Y. Cho, M.C. Lee, H. Fujii and Y.K. Hong. 2008. Antipyretic, analgesic, and anti-inflammatory activities of the seaweed Sargassum fulvellum and Sargassum thunbergii in mice. J. Ethnopharmcol. 116(1): 187-190.
Kannur D. M., V.I. Mukkeri and P. Akki. 2006. Antidiabetic activity of Caesalpinia bonducella seed extracts in rats. Fitoterapia. 77: 546-554.
109 Karchegani H.M., S.Z. Hosseini and S.A. Kazemeini. 2014. Allelopathic effects of sorghum on milk thistle (Silybum marianum L.) seed germination and growth.Res. Ecophysiol. 9: 115-123.
Karchegani, M, L. Amjad, M. Ranjbar. 2014. Comparative Analysis of Chemical Composition of three Ecotypes of Chenopodium botrys L. in Iran. Adv. Environ. Biol. 8(24): 43-47.
Kasture, V.S., S.B. Kasture and C.T. Chopde. 2002. Anticonvulsive activity of Butea monosperma flowers in laboratory animals. Pharmacol. Biochem. Behav. 72: 965-972.
Khan, I., M. Nisar, F. Ebad, S. Nadeem, M. Saeed, H. Khan and Z. Ahmad. 2009. Anti- inflammatory activities of Sieboldogenin from Smilax china Linn: experimental and computational studies. J. Ethnopharmcol. 121(1): 175-177.
Khanberg, P., E. Lager and C. Rosen. 2002. Refinement and evaluation of a pharmacophore model for flavone derivatives binding to the benzodiazepine site
of the GABAA receptor. J. Med. Chem. 45(19): 4188-4201.
Khare, C.P. 2007. Indian Medicinal Plants: An illustrated dictionary. Spring. 142.
Khyalibova, B. 1968. Pharmacy of Jerusalem oak (Chenopodium botrys) Alkaloids. Tr Alma-At Zootekh-Vet Ins. 15:21-23.
Kinsel, J. and S. Straus. 2003. Complementary and alternative therapeutics: Rigorous research is needed to Support Claims. Annu Rev. of Pharmacol. Toxicol. 43: 463-484.
Kletter, C and M. Krichbaum. 2001. Tibetan Medicinal Plants. Med. Pharm. Scient.Pub.241-250.
Koelz, W. 1979. Notes on the Ethno-botany of Lahul, a province of the Punjab.Quart. J. Crude Drug Res. 17: 1-56.
Kosem, N., K. Ichikawa, H. Utsumi and P. Moongkarndi. 2013. In vivo toxicity and antitumor activity of mangosteen extract. J. Nat. Med. 67: 255-263.
110 Kumar, .R., S. Vatsya and C.L Yadav CL. 2016. Anthelmintic Activity and Phytochemical analysis of Chenopodium album against Haemonchus contortus. J. Vet. Sci. 2: 144-56.
Kumar, P., S. Kumar, S. Kumar and R. Kumar. 2015. In vitro study of plant extract from Chenopodium album that inhibits a key enzyme in diabetes and its role in diabetic oxidative stress. Der. Pharm. Sin. 6(12): 48-61.
Kunyanga, C.N, J.K. Imungi, M. Okoth, C. Momanyi and H.K. Biesalski. 2011. Antioxidant and antidiabetic properties of condensed tannins in acetonic extract of selected raw and processed indigenous food ingredients from Kenya. J. Food Sci. 76: 560-567.
Leonard, T.B. and J.G. Dent. 1984. Hepatotoxicity induced by carbon tetrachloride, paracetamol and Dgalactosamine in rats. Res. Commun. Pathol.Pharmacol. 44 (3): 387-388.
Leroi Gourhan, A. 1975. The flowers found with Shanidar IV, a Neanderthal burial in Iraq. Sci. 190(4): 562-564.
Lewis, M. A. 1995. Use of freshwater plants for phytotoxicity testing: A review. Environ. Pollut. 87: 319-336.
Lowry, O. H., N.J. Rosebrough, A.L. Farrand and R.J. Randall. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265-275.
Macsimovic, Z.A., S. Dordevic and M. Mraovic. 2005. Antimicribial activity of Chenopodiumbotrysessential oil. Fitoterapia. 76: 112-114.
Mahboubi, M., F.G. Bidgoli and N. Farzin. 2011. Chemical Composition and Antimicrobial Activity of Chenopodium botrys L. Essential Oil. J. Esset. Oil. Bear. Pl. 14(4): 498-503.
Maksimovic, Z., S. Dordevic and M. Mraovic. 2005. Antimicrobial activity of Chenopodium botrys wssential oil. Fitoterapia. 76:112-114.
111 Mallory, H.T and E.A. Evenlyn. 1937. The determination of bilirubin with photoelectric colorimeter. J. Biol Chem. 119: 481-485.
Maloo, A., S. Borade, R. Dhawde, S.N. Gajbhiye and S.G. Dastager. 2014. Occurrence and distribution of multiple antibiotic-resistant bacteria of Enterobacteriaceae family in waters of Veraval coast, India. Environ. Experimen. Biol. 12: 43-50.
Marchioli., R.C., G. Schweiger, L. Levantesi, Tavazzi, and F. Valagussa. 2001. Antioxidant vitamins and prevention of cardiovascular disease: epidemiological and clinical trial data. Lipids. 36: 53-63.
Martinez, M., R.M, Lazaro,L.B. Del-Olmo and P.B. Benito. 2008. Anti-infectious activity in the anthemideae tribe. In: Atta-ur- (Ed.) Studies. J. Nat. Prod. Chem. 35: 45-516.
Maurya R, G. Singh and P. Yadav. 2008. Anti-osteoporotic agents from Natural sources. In: Atta-ur-Rahman (Ed.) Studies. Nat. Prod. Chem. 35: 517-545.
Mbaveng, A.T., B.Ngameni, V.Kuete. 2008. Antimicrobial activity of the crude extracts and five flavonoids from the twigs of Dorsteniabarteri (Moraceae),” J. Ethnopharmacol.116 (3): 483-489.
Mcdonald, R.L. and K.M. Kelly. 1993. Antiepileptic drugs: Mechanisms of action. Epilepsia. 34:1-8.
McDonald, S., P.D. Prenzler, M. Antolovich and K. Robards. 2001. Phenolic content and antioxidant activity of olive extract. Food Chem. 73: 73-84.
Meite, S., J.D. N‟guessan, C. Bahi, H.F. Yapi, A.J. Djaman and F.G. Guina, F. G. 2009. Antidiarrheal activity of the ethyl acetate extract of Morindamorindoides in rats. Trop. J. Pharm. Res. 8 (3).
Middleton, E. and C. Kandaswami. 1992. Effects of flavonoids on immune and inflammatory cell function. Biochem.Pharmacol. 43: 1167-1179.
112 Middleton, E., C. Kandaswami and T.C. Theoharides. 2000. The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol. Rev 52: 673-751.
Moffet, M.L and B.A. Wood. 1984. Survival of Corynebacterium michiganense subsp. michiganense within host debris in soil. Australas Plant Path. 13: 1-3
Mohlenbrock, R.H. 2001. The Illustrated Flora of Illinois: Flowering Plants. Southern Illinois Uni. Pres. 53.
Morteza-Semnani, K and E.Babanezhad. 2007. Essential oil composition of Chenopodium botrys L. from Iran. J. essen oil bear. Pl. 10: 314-317.
Mosyakin, S.L. and S.E. Clemants. 2002. New nomenclatural combinations in Dysphania R. Br. (Chenopodiaceae): Taxa occurring in North America. Ukrayins‟k. Bot. Zhurn. 59(4): 380-385.
Mosyakin, S.L. and S.E. Clemants. 2008. Further transfers of glandular-pubescent species from Chenopodium subg. Ambrosia to Dysphania (Chenopodiaceae). J. Bot. Res. Inst. Texas. 2: 425-431.
Nahar, L. and S.D. Sarkar. 2005. Chenoalbuside: an antioxidant phenolic glycoside from the seeds of Chenopodium album L. (Chenopodiaceae). Braz. J. Pharmcog. 15(4): 279-282.
Nasrin, M.K., A. Leila and R. Monireh. 2014. Comparative Analysis of Chemical Composition of three Ecotypes of Chenopodium botrys L. in Iran. Adv. Environ. Biol. 1: 278-292.
Nayak, D.P., S.C. Dinda, P.K. Swani, B. Kar, and V.J. Patro. 2012. Hepatoprotective activity against CCL4-induced hepatotoxicity in rats of Chenopodium album aerial parts. J. Phytother. Pharm. 1(2): 33-41.
Nedialkova, Z.K., P.T. Nedialkov and S.D. Nikolov. 2014. Pharmacognostic investigations of the aerial parts of Chenopodium foliosum Asch. and radical-
113 scavenging activities of five flavonoids isolated from methanol extract of the plant. J. Phcog. 6(4): 43-48.
Newman, D., G. Cragg and K. Snader. 2000. The influence of natural products upon drug discovery. Nat. Prod. Rep. 17:215-234.
Nigam, V., and P.M. Paarakh. 2012. Evaluation of anti-diarrhoeal activity of hydro alcoholic extract of Chenopodium album L. Ind. J. Nat. Prod. Resour. 4(1): 61- 66.
Nouri, J., N. Khorasani, B. Lorestani, M. Karami, A.H. Hassani and N. Yousefi. 2009. Accumulation of heavy metals in soil and uptake by plant species with phytoremediation potential. Environ. Earth Sci. 59: 315-23.
Nowak, R., K. Szewczvk, U.G. Dziki, J. Rzymowska and L. Komsta. 2015. Antioxidative and cytotoxic potential of some Chenopodium L. species growing in Poland. Saudi J. Biol. Sci. 23(1): 15-23.
Nsimba, Y.R., H. Kikuzaki and Y. Konishi. 2008. Antioxidant activity of various extracts and fractions of Chenopodium quinoa and Amaranthus spp. Seeds. Anal. Nutr. Clin. Methods. 106(2): 760-766.
Obadoni, B.O and P.O. Ochuko. 2001. Phytochemical studies and comparative efficacy of the crude extract of some homeostatic plants in Edo and Delta, States of Nigeria. Global .J. Pure and Appl. Sci. 2001. 8: 203-208.
Obatomi, D.K., E.O. Bikomo and V.J. Temple. 1994. Antidiabetic properties of the African mistletoe in streptozotocin-induced diabetic rats. J. Ethnopharmacol. 43: 13-17.
Odoh, U.E and C.O. Ezugwu. 2012. Anti-Diabetic and Toxicological Studies of The Alkaloids of Acanthus montanus (Acanthaceae) Leaf. Planta Medica. 78.
Ogungbenle, H.N. 2003.Nutritional evaluation and functional properties of quinoa (Chenopodium quinoa) flour.Int. J. Food Sci. Nutr. 54(2): 153-158.
114 Okokon, J.E and P. Nwafor. 2010. Anti-inflammatory, analgesic and antipyretic activities of ethanol root extract of Croton zambesicus. Pak. J. Pharm. Sci. 23(4): 385-392.
Okokon, J.E. and P.A. Nwafor. 2010. Antiinflammatory, analgesic and antipyretic activities of ethanolic root extract of Croton zambesicus. Pak J. Pharm. Sci. 23(4): 385-392.
Oktay, M., I. Gulçin and O.I. Kufrevioglu. 2003. Determination of in vitro antioxidant activity of fennel (Foeniculum vulgare) seed extracts. Lebensmittel- Wissenschaft Technologie. 36: 263-271.
Oshima, M., M. Takahashi, H. Oshima, M. Tsutsumi, K. Yazawa and Y. Sugimura. 1995. Effects of docosahexaenoic acid (DHA) on intestinal polyp development in Apc delta 716 knockout mice. Carcinogenesis. 16(11): 2605-2607.
Ozer, S., C. Sarikurkcu and B. Tepe. 2016. Phenolic composition, antioxidant and enzyme inhibitory activities of ethanol and water extracts of Chenopodium botrys. Roy. Soci. Chem. 6: 64986-64992.
Pal, A., B. Banekjee, T. Banerjee, M. Masih and K. Pal. 2011.Hepatoprotective activity of Chenopodium album linn.plant against Paracetamolinduced hepatic injury in rats. Int. J. Pharm. Sci. 3(3): 55-57.
Panday, S. and R.K. Gupta. 2014. Screening of nutritional, phytochemical, antioxidant and antibacterial activity of Chenopodium album (Bathua). J. Pharmacogn. Phytochem. 3(3): 1-9.
Pardo, M, E. Blanco, R. Morales. 2005. Plants known as te in Spain: an ethno- pharmaco-botanical review. J. Ethnopharmacol. 98: 1-19.
Parkash, J. and K.R. Patel. 2014. Evaluation of antibacterial activity of different concentrations of Chenopodium album leaves extract. J. D. D T. 4(1): 123-126.
115 Patil, S. B and C. S. Magdum. 2011. Determination of LC50 values of extracts of Euphorbia hirta linn. and Euphorbia neriifolia (linn) using brine shrimp lethality assay. AJP. Sci. 1 (2): 42-43.
Patoprotective effect of Chenopodium murale in mice.Bangladesh J. of Pharmacol. 9(1): 124-128.
Piero, N.M., N.S. Kimuni, J. Ngeranwa, G.O. Orinda and J.M. Njagi. 2015. Antidiabetic and Safety of Lantana rhodesiensis in Alloxan Induced Diabetic Rats. J. Develop. Drugs. 4: 129-132.
Porsolt, R.D., G. Anton, N. Blavet, M. Jalfre. 1978. Behaviouraldespairin rats: a new model sensitive to antidepressive treatments. Eur. J. Pharmacol. 47: 379-391.
Porsolt, R.D., M. Le-Pichon, and M. Jalfre. 1977. Depression: a new animal model sensitive to antidepressant treatments. Nat. 266-730.
Quattrocchi, U. 2012. World Dictionary of Medicinal and Poisonous Plants.CRC Pre. 1504.
Qureshi, R. M.A. Ghufran, S.Gilani, K. Sultana and M.Ashraf. 2007. Medicinal plant. Pak. J. Bot. 39: 2271-2275.
Raghuramulu, N.,N.K., Madhavan and S. Kalyanasundaram. 2003. National Institute of Nutrition. Indian Council of Medical Research, Hyderabad, India.Manu. Lab. Tech. 56-58.
Rahman, M.K., M.A. Chowdhury, M.T. Islam, M.A. Chowdhury, M. Erfanuddin and C.D. Sumj. 2015. Evaluation of antidiarrheal activity of methanolic extract of Maranta arundinacea L. Leaves. Adv. Pharmacol. Sci. 3: 1-6.
Rai, M. and M.C. Carpinella. 2006. Pharmacological characterization. Adv. in Phytomed. 3(1): 337-8.
Raskin, I., D.M, Ribnicky, S. Komarnytsky, S. Ilic and N. Pouley. 2002. Plants and human health in the twenty-first century. Trends Biotechnol. 20(12): 522-531.
116 Rauf, A., S. Uysal, T. Hadda, B.S. Siddiqui, H. Khan , M.A. Khan, M. Ijaz, M. S. Mubarak, S. Bawazeer, T. Abu-Izneid, A. Khan and U. Farooq. 2017. Antibacterial, cytotoxicity, and phytotoxicity profiles of three medicinal plants collected from Pakistan. Curr.Top. Med. Chem. 1: 122-134.
Re, R., N. Pellegrini, A. Proteggente, A. Pannala, M. Yang and C. Rice-evans. 1999. Antioxidant activity applying an improved ABTS radical decolorization assay. Free Radic. Biol. Med. 26: 1231-1237.
Renuka, B, F. Hibin and H. Jain. 2009. Fructooligosaccharide fortification of selected fruit juice beverages: Effect on the quality characteristics LWT. J. Food Sci. Technol. 42: 29-37.
Rios, J.L., M.C. Recio and A. Villar. 1988. Screening methods for natural products with antimicrobial activity: A review of the literature. J. Ethnopharmacol. 23: 127-49.
Roger, P. A., I. Simpson, R. Oficial, S. Ardales, and R. Jimenez. 1994. Effects of pesticides on soil and water microflora and mesofauna in wetland rice fields: a summary of current knowledge and extrapolation to temperate environments. Aust. J. Exp. Agr. 34: 1057-1068.
Roiser, J.P., R. Elliott and B.J. Sahakian. 2012. Cognitive mechanisms of treatment in depression. Neuropsychopharmacology. 37(1): 117-119.
Rustembekova, B., M.I Goryaev and P.P Gladyshev.1973. Isolation of betaine from Chenopodium botrys. Chem. Nat. Compd. 9: 543-547.
Rustembekova, B., M.I. Goryaev, G.I. Krotova and A.D. Dembitski. 1975. Substances added to the composition of essential oils, 60 oxygen containing compounds of Chenopodium botrys essential oil. Izv Akad Nauk kaz SSR Ser Khim. 25: 32- 34.
Saleem, M., B. Ahmed, M.I. Qadir, Mahrukh, M. Rafiq, M. Ahmad and B. Ahmad. 2014. Hepatoprotective effect of Chenopodiummurale in mice Bangladesh. J. Pharmacol. 9: 124-128.
117 Sanchez-Moreno, C., J.A. Larrauri, and F. Saura-Calixto. 1998. New parameter for evaluation of free radical scavenging capacity of polyphenols, 2nd International Electronic Conference on Synthetic Organic Chemistry (ESCOC-2).
Santiago, J., M. Cardso, L.R. Batista, E.D. Castro, M.L. Teixeira and M.F. Pires. 2016. Essential oil from Chenopodium ambrosioides L.: secretory structures, antibacterial and antioxidant activities. Acta Scientiarum. Biol. Sci. 38(2): 139- 147.
Sayyah, M., J. Valizadeh and M. Kamalinejad. 2002. Anticonvulsant activity of the leaf essential oil of Laurusnobilisagainst pentylenetetrazole and maximal electro shock-induced seizures. Phytomedecine. 9: 212-216.
Schopen, A. 1983.TraditionelleHeilmittel in Jemen.Franz Steiner Verlag GmbH. Berlin.
Seidemann, J. 2005 World Spice Plants: Economic Usage, Botany, Taxonomy. Berlin Heidelberg: Springer. 8: 592-593.
Shafighi, M. and L. Amjad. 2013. Evaluation of Antioxidant Activity, Phenolic and Flavonoid Content in Punica granatum var. Isfahan Malas Flowers. Int. J. Agri. Crop Sci. 5: 1133.
Shamkuwar, P.B and D.P. Pawar. 2013. Antidiarrhoeal and antispasmodic effect of Berberisaristata. Int. j. Pharmacognosy.and Phytochem. 5(1): 24-26.
Shen, Y.H., Y. Zhou, C. Zhang, R. H. Liu, J. Su, X.H. Liu and W.D. Zhang. 2009. Antidepressant effects of methanol extract and fractions of Bacopa monnieri. Pharm. Boil. 47(4): 340-343.
Silambujanaki, P., V. Chitra, S. Kumari, M. Sankari, D. Raju and C.H. Chandra. 2010. Anti-convulsant activity of methanolic extract of Butea monosperma leaves. RJPBCS. 1: 431-435.
118 Singh, K. 2012. Traditional knowledge on ethnobotanical uses of plant biodiversity: a detailed study from the Indian Western Himalaya. Biodiv. Res. Conserv. 28: 63- 77.
Small, E. Culinary Herbs. 2006. Canadian Science Publishing NRC Res. Pre. 2: 298- 309.
Socala, K., D. Nieoczym, K. Grzywnowicz, D. Stefaniuk and P. Wlaz. 2015. Evaluation of anticonvulsant, antidepressant, and anxiolytic-like effects of an aqueous extract from cultured mycelia of the Lingzhi or Reishi medicinal mushroom Ganoderma lucidum (Higher Basidiomycetes) in mice. Int. J. Med. Mushrooms. 17(3): 209-218.
Solecki, R. and Shanidar. 1975. A Neanderthal flower burial in northern Iraq. Science. 190: 880-881.
Song, M.J., S.H. Lee and D.K. Kim. 2011. Antidiabetic effect of Chenopodium ambrosioides. Phytopharm. 1(2): 12-15.
Stanner, S., A.J. Hughes, C.N.M. Kelly and J. Buttriss. 2004. A review of the epidemiological evidence for the „antioxidant hypothesis‟. Public Health Nutr. 7: 407-422.
Steru.L. and R. Chemat. 1985. The tail suspension test: A novel method for screening antidepressants in mice. Psychopharmacology. 85: 367-370.
Swinyard, A. and H.J. Kupferberg. 1985. Antiepileptic drugs: detection, quantification and evaluation. Federal proceedings. 44: 2629-2633.
Sykiotis, G., G. Kalliolias and A. Papavassiliou. 2006. Hippocrates and genomic medicine. Arch. Med. Res. 37: 181-183.
Kiswii, T.M., E.O. Monda, P.O. Okemo, C. Bii and A. E. Alakonya. 2014. Efficacy of selected medicinal plants from eastern kenya against Aspergillus flavus,” J. Plant Sci. 2(5). 226-231.
119 Taesotikul, T., A. Panthong, D. Kanjanapothi, R. Verpoorte, J.C. Scheffer. 1998. Neuropharmacological activites of the crude alkaloidal fraction from stems of Tabernaemontana pandacaqui poir. J. Ethnopharmacol. 62: 229-234.
Takahashi, R. N., T.C. De Lima and G.S. Morato. 1986. Pharmacological actions of tannic acid; II.Evaluation of CNS activity in animals. Planta Medica. 4: 272- 275.
Tang, Y., X. Li, B. Zhang, P.X. Chen, R. Liu and R. Tsao. 2015. Characterization of phenolics, betanins and antioxidant activities in seeds of three Chenopodium quinoa-Wild genotypes. Food Chem. 166: 380-388.
Teware, T. 2017. Antibacterial activity of Chenopodium album extract. World J. Pharm. Sci. 6(11): 917-923.
Thamarai, P. Selvi, M. SenthilKumar, R. Rajesh, T. Kathiravan. 2012. Antidepressant activity of ethanolic extract of leaves of Centellaasiatica.Linnby In vivo methods. Asian J. Res. Pharm: 2(2): 76-79.
Thippeswamy, B.S., S.P. Thakker, S. Tubachi and G.A. Kalyani, 2009.Cardioprotective Effect of Cucumis trigonus Roxb on isoproterenol-induced myocardial infarction in rat. Am. J. Pharmacol. Toxicol. 4: 29-37.
Thyr, B.D., M.J. Samuel and P.G. Brown, 1975. New solanaceous host records for Corynebacterium michiganense. Plant Disease Reporter. 59(7): 595-598.
Tisser and, R. and R. Young. 2014. Essential Oil Safety. 2nd ed. Churchill Livingstone Elsevier. 5: 471-494.
Tjolsen, A., O.G. Berge, S. Hunskaar, J.H. Rosland and K. Hole. 1992. The formalin test: an evaluation of the method. Pain 51: 5-17.
Treadway, L. 1994. Amla, traditional food and medicine. J. Amer. Bot. Counc. 31: 26.
Trofimiuk, E., A. Walesiuk, J. Braszko and J. John's wort. 2005. Hypericum perforatum diminishes cognitive impairment caused by the chronic restraint stress in rats. Pharmacol. Res. 51(3): 239-246.
120 Tzakou, A., F. Pizziment, V. Sdrafkakis and E.M. Galati. 2007. Composition and Antimicrobial Activity of Chenopodium botrys L. Essential Oil from Greece. J. Essent. Oil. Res. 19(3): 292-294.
Vaya, J., P.A. Belinky and M. Avira. 1997. Antioxidant constituents from licorice roots: Isolation, structure elucidation and antioxidative capacity towards LDL Oxidation. Free radic. Biol. Med. 23: 302-313.
Venkatachalam, T., V.K, Kumar, P.K, Selvi, A.O. Maske and V. Anbarasan. 2011. Antidiabetic activity of Lantana camara Linn.hemoglobin fruits in normal and streptozotocin-induced diabetic rats. J. Pharm. Res. 4: 1550-1552.
Verpoorte, R., 2000. Pharmacognosy in the New Millennium: Lead finding and Biotechnology. J. Pharm. Pharmacol. 52: 253-262.
Wang, L., Y. Zhao, L. Zhou and J. Zhou. 2007. Chemical Constituents of Houttuynia cordata. Chinese traditional and herbal drugs. 38: 1788-1790.
Wangensteen, H., A.B. Samuelsen and K.E. Malterud. 2004. Antioxidant activity in extracts from coriander. Food Chem. 88: 293-297.
Watts, D.C. 2007. Elsevier's Dictionary of Plant Lore.Elsevier Inc. 214.
WHO. 2005. Global Atlas of Traditional, Complementary and Alternative Medicine. World Health Organization, Geneva. Herbal Gram. 2(1): 28-37.
Winter, C.A., E.A. Risley and G.W. Nuss. 1962. Carrageenin-induced edema in hind paw of the rat as an assay for anti-inflammatory drugs. Proc. Soc. Exp. Biol. Med. 111: 544-547.
Woolfe, G and A.D. MacDonald. 1944. The evaluation of the analgesic action of pethidine hydrochloride (DEMEROL). J. Pharmacol. Exp. Ther. 80: 300-307.
Xu, Y., E. A. Hubal and J. C. Little. 2009. Predicting residential exposure to phthalate plasticizer emitted from vinyl flooring: sensitivity, uncertainty and implications for biomonitoring. Environ. Health Perspect. 118(2): 253-258.
121 Yadav, N., N. Vasudeva and S. Singh and S. Sharma. 2007. Medicinal properties of genus Chenopodium Linn. Nat. Prod. Rad. 6: 131-134.
Yakubu M.T., M.A. Akanji and A.T. Oladiji. 2007. Haematological evaluation in male albino.Rats following chronic administration of aqueous extract of Fadogia agrestis stem. Pharmacol.Manag. 3: 34-38.
Yao, Y., X. Yang, Z. Shi and G. Ren. 2014. Anti-Inflammatory activity of saponins from Quinoa (Chenopodium quinoa Wild.) seeds in lipopolysaccharide- stimulated RAW 264.7 macrophages cells. J. Food Sci. 79(5): 1018-1023.
Yawer, A., E. Ahmed, A. Malik, M. Ashraf, M.A. Rasool and N. Afza. 2007. New lipoxygenase-inhibiting constituents from Calligonum polygonoids. Chem. Biodiver. 4: 1578-1585.
Yolanda, B.L. and G.C. Adriana. 2006. Effects of dietary polyunsaturated n-3 fatty acids on dyslipidemia and insulin resistance in rodents and humans. J. Nut. Biochem. 17: 1-13.
Yousefi, N., A. Chehregani, B. Malayeri, B. Lorestani and M. Cheraghi. 2011. Investigating the effect of heavy metals on developmental stages of anther and pollen in Chenopodium botrys L. (Chenopodiaceae). Biol. Trace Elem. Res. 140: 368-76.
Zain, M. E. 2011. Impact of mycotoxins on humans and animals,” J. Saudi Chem. Soc. 15(2): 129-144.
Zapata-Sudo, G., T.C Mendes, M.A. Kartnaller, T.O Fortes, N.F. Freitas, M.A. Kaplan and R.T. Sudo. 2010. Sedative and anticonvulsant activities of methanol extract of Dorstenia arifolia in mice. J. Ethnopharmacol. 130(1): 9-12.
Zargari, A. 1993.Medicinal Plants.Tehran University Publications, Tehran, Iran. 4: 218- 219.
122 Zhao, Y., C. Tong and J. Jiang. 2007. Hedgehog regulates smoothened activity by inducing a conformational switch. Nature. 450(71): 252-258.
Zoufan, P., R. Jalali, A. Karimiafshar and H. Motamedi. 2017. Assessment of heavy metal accumulation and antibacterial activity of some medicinal herbs grown in an industrial area of steel production, Ahvaz. Iran J. Pharm. Sci. 13(1): 73-86.
123