Phytochemical, Biological and Safety Evaluation of Zaleya pentandra (Aizoaceae) MASTER OF PHILOSOPHY Pharmaceutical Chemistry SAHARISH KHALIQ Department of Pharmacy FACULTY OF PHARMACY & ALTERNATIVE MEDICINE The Islamia University of Bahawalpur Pakistan (Session: 2014-2016) Table of contents Sr. No. Contents Page Acknowledgements I List of tables III List of abbreviations VI List of figures VII Abstract VIII Chapter No. 1: INTRODUCTION 1 Introduction 1 1.1 Historical Sources Relevant for Study of Medicinal Plants 1 1.2 Phytochemicals 6 1.3 The family Aizoaceae 15 1.4 Botanical description of the family Aizoaceae 15 1.5 Botanical description of genus Zaleya 17 1.6 Botanical description of Zaleya pentandra 17 1.7 Medicinal Importance of Family Aizoaceae 18 1.8 Taxonomical classification 18 Chapter No. 2: LITERATURE SURVEY 2.1 Literature survey of family Aizoaceae 20 2.1.1 Literature survey on Pharmacological properties of family Aizoaceae 20 2.6 Literature survey on Phytochemical properties of family Aizoaceae 29 Chapter No. 3: MATERIAL AND METHODS 3 Materials and Methods 38 3.1 Collection of Plant material 38 3.2 Identification 38 3.3 Drying and Grinding 38 3.4 Maceration 38 3.5 Extraction 39 3.6 Equipment 39 3.7 Qualitative detection of secondary metabolites 40 3.7.1 Preparation of reagents 40 3.7.2 Preparation of solutions 40 3.7.3 Qualitative analysis of secondary metabolites 42 3.8 Quantitative analysis of Secondary metabolites 44 3.9 Procedure for Biological Evaluation 45 3.10 Toxicological studies/ Safety evaluation 46 3.10.1 Experimental birds and management 46 3.10.2 Experimental procedure 47 3.10.3 Parameters studied 47 3.10.3.1 Clinical signs and behavioral alterations 47 3.10.3.2 Haematological and biochemical parameters 47 3.10.3.3 Absolute and relative weight of body organs 57 Chapter No. 4: RESULTS 4 Results 59 4.1 Extraction 59 4.2 Phytochemical analysis of Zaleya pentandra 59 4.3 Quantitative estimation of phenolic and flavonoid contents 60 4.4 Biological screening 61 4.5 Toxicological screening 62 Chapter No. 5: DISCUSSION 5 Discussion 83 Chapter No.6: REFERENCES 6 References 86 ABSTRACT In present study Zaleya pentandra from family Aizoaceae was detail studied for evaluation of phytochemical, biological and toxicological parameters of plant. Zaleya pentandra is the only specie present in Pakistan and not much work is reported on this plant. Study was also carried out to observe the toxicology of drug on long term use. Traditionaly this plant has been used in various ailments like respiratory tract infection, skin diseases and as laxative and cathartic agent. Crude extract of dichloromethane and methanol were prepared using rotary evaporator under reduced pressure by maceration. Secondary metabolites were confirmed by phytochemical tests. Confirmatory tests were performed. Tannins, flavonoids, alkaloids, saponins and cardiac glycosides were reported. Dichloromethane and methanolic extract of whole plant of Zaleya pentandra were screened for total phenolic and total flavonoid content. Highest phenolic and flavonoid contenst were reported in Zaleya pentandra methanolic extract (ZPM) as compared to Zaleya pentandra dichloromethane extract (ZPD). Antioxidant activity of plant extract were calculated by DPPH (1,1 diphenyl pipryl hydrazyl), and ferrous reducing antioxidant power assay (FRAP). ZPM extract showed highest % inhibition in DPPH and FRAP when compared with ZPD extract. Dichloromethane and methanolic extract of Zaleya pentandra were administered in experimental chicks divided in 4 groups each with 5 individuals and 1 group considered as control. From lowest to highest different doses were given for 21 days. No drug was given to control group. Different physical, haematological and biochemical parameters were studied. Statistical analysis revealed that there was no toxic effect of plant even when given at higher dose. Plant is considered to be safe with many pharmacological and biological activities. Effort in doing more research on this plant is welcoming. viii 1. INTRODUCTION Curing the disease with medicinal plants is an oldest therapy. In ancient times people for the cure of their disease and ailments considered drugs in nature. It took many years and as a result awareness was developed regarding the use of medicinal plants against illness (Petrovska et al., 2012). Previously there was no information regarding the actual reason for the disease or illness and the part of a plant which can actually be used as a cure. For the treatment of specific disease medicinal plants were discovered and eventually their use increased (Kelly, 2009). 1.1 Historical Sources Relevant for Study of Medicinal Plants Use of synthetic drugs and their decreasing efficiency opened a new horizon for the use of natural drugs. Herbs were 1st agent used to cure pharmacological diseases. Treatment with herbs was the natural thing because herbs were the 1st thing man interacts with. Herbalism was practiced all over the world. Sumerian clay tablet from Nagpur was the oldest evidence of plants used medicinally for preparing drugs. It was almost 5000 years old clay slab that consist of 12 recipes for preparing drugs from almost 250 different plants (Kelly, 2009). Emperor Shen Nung Circa 2500 BC wrote a Chinese book on roots and grasses named as “Pen T’ Sao” comprising 365 drugs. Many of the drugs are even now commonly used named as gentian, ginseng, ephedra and camphor. Holy books from India like Vedas specified treatment using plants native to that country. Various spices that are now used basically originate from India like clove, pepper, nutmeg etc. In 1500 BC, The Ebers Papyrus, consist of 800 prescriptions from almost 700 different plants and drugs like aloe, senna, garlic, onion, fig, coriander were used in therapy (Glesinger 1954; Tucakov 1964). Bible and the holy Jewish book Talmud referred that aromatic plants used for treatment like myrtle and incense (Dimitrova, 1999).Various plants belonging from the genus Artemisia were commonly referred as a sign of strength and good health. Their name was basically derived from Artemis Greek word that means “healthy” (Toplak, 2005). 1 About 300 different medicinal plants were categorized according to their physiological action by Hippocrates (459---370 BC). For fever, wormwood and common centaury were used. Garlic was used against intestinal parasites. Deadly nightshade, henbane, and mandrake were commonly used as narcotics. Parsley and garlic act as diuretic. Pomegranate and oak were used as astringent (Bojadzievski, 1992). Threophrast (372---287 BC) classified almost 500 plants of that time in his books De Causis Plantarium— Plant Etiology and De Historia Plantarium—Plant History (Katic 1958; Pelagic 1970). Threophrast was awarded with a title “Father of botany “ as he had the knowledge for describing and classifying different medicinal plants (Bazala, 1995). De-Re-Medica (25BC---50AD) famous medical writer constituted almost 250 medicinally important plants in his work like poppy, pepper, aloe, henbane, cardamom etc (Tucakov, 1948). Dioscorides, “Father of Pharmacognosy” in ancient history was 1 of the famous writer on plant drugs. He worked as a military physician and pharmacognosist of Nero’s Army. During his travelling with Roman army he studied almost every kind of plant. In 77 AD he wrote the work “De Materia Medica” in which he presented sufficient data regarding medicinal plants (Thorwald, 1991). Total 944 drugs were described out of which 657 were of plant origin. Their appearance, method of collection, locality and their therapeutic action were described. Along with the description of plants their names in other language coupled with the areas and localities where they grow and originate from were referred as folk names. Plants with mild and strong effects were described (Nikolovski 1961; Katic 1980). Dioscorides listed domestic plants as willow, nettle, sage, common centaury, sea onion, etc. Chamaemelon used as anti-phlogistic to cure wounds, stings, burns, ulcer and as a cleanser for eyes nose and mouth. It was healthy to use in children for its mild carminative action. Dioscorides claimed that it had abortive action and according to him menstruation can be increased by flower, roots and entire plant. Discharge of urine, stone and release of embryo can also happen. Both the Romans and Arabs accepted this myth, Matricaria the Latin name was derived 2 from mater means “mother” i.e. matrix denoting uterus. From the genus Mentha number of species were derived, many of them were used as aid in stomachache and headache. Historia Naturalis a book written by Pliny the Elder (23 AD-79), who travelled across Germany and Spain, in his book he mentioned almost 1000 medicinal plants. Dioscorides and Pliny’s work comprised of the complete knowledge of all the medicinal plants of that time (Toplak, 2005). Galen a Roman physician (Pharmacist) presented a list of drugs with similar action (parallel drugs). Galen also described some of the drugs of that time which were not enlisted by Dioscorides like Uvae ursi folium, used as uroantiseptic and a mild diuretic even now a days. Slavic people in 7th century AD used Rosmarinus officinalis, Mentha viridis, Iris germanica in cosmetics and Artemisia maritime L., Lavandula officinalis, Sambuci flos against several insects like fleas, moths, mosquito and spiders. Aconitum napellus was used as a poison in hunting (Bojadzievski, 1992). The art of healing, cultivating medicinal plants and preparation of drugs moved to monasteries. About 16 medicinal plants were used in therapy that commonly grew within the monasteries like anise, mint, sage, tanvy, etc. Founder of a well reputed medical school Charles the Great (742AD—814) in Salerno ordered in “Capitularies” nature of medicinal plants that must be cultivated on state owned lands. Almost 100 various plants were enlisted, many of them are used commonly now a days like sage, common centaury, poppy, marsh mallow etc. Sage (Salvia officinalis) was greatly appreciated by the emperor. Sage a Latin word termed as salvare meaning “to save or cure” even now a day’s grown in all catholic monasteries.