Phytochemical, Biological and Safety Evaluation of Zaleya pentandra ()

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 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 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.

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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).

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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

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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. In pharmacotherapy, various plants were introduced by Arabs, most of them from India a country with whom they had trade relations. Many of the plans that were of extreme medicinal importances were enlisted in all pharmacopoeias in the world are present till date. Deadly nightshade, aloe, coffee, pepper, cinnamon, senna and many others were used by Arabs. Drugs that were severe in action were interchanged by the drugs mild in action.

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Arabs work “De Re Medica” was consulted by physicians from Europe John Mesue (850 ADA) “Canon Medicinae” by Avicenna (980-1037), and “Liber Magnae Collectionis Simplicum Alimentorum Et Medicamentorum” by Ibn Baitar (1197-1248), almost 1000 medicinal plants were described in it (Tucakov, 1964). In (1254-1324) Marco Polo’s travelling in tropical Asia, China, and Persia, (1942) discovery of America and (1948) journey of Vasco De Gama to India resulted in identification and discovery of many natural medicinal plants brought into Europe. Materia medica was self-sufficient and was enriched with many new medicinal plants after the discovery of America like cinchona, cacao, jalap, Vanilla, Podophylum, Tobacco, etc. Cinchona succirubra was used in European medicines. Though there were many opponents for the use of quinine but its use spread rapidly among England, France and Germany. Paracelsus (1493-1541) claimed that drugs prepared from raw plants and minerals should be determined astrologically. Emphasizing his strong belief he supported “Signatura Doctorinae” _ The Signature Doctorine. According to his belief, God created his own sign on all healing substances that indicates their use in certain diseases. For instance haselwort resembles liver in shape so it must be used for liver diseases. Marigold and dandelion and many other plants with flowers yellow in color, used to treat jaundice, while pansies, were utilized for heart problems because their petals are similar in shape with heart (Brown et al., 1995). Ancient people used medicinal plants as pharmaceutical aid in the form of infusion, maceration and decoction. In 16th and 18th compound drug demand was raised. Compound drugs were formulated by the combination of medicinal plants along with drugs from plant and animal origin. These compound drugs prepared from medicinal plants, animal and minerals were very valuable and were sold very expensively (Bojadzievski 1992; Toplak 2005). Plantarium (1753) Linnaeus (1707-1788) in 18th century briefly described and classified all the species of that time. All the present species were named and classified without considering that some of the species were previously named somewhere else. For the purpose of naming a specie polynomial system was developed in which 1st word described the genus and 2nd the specie. Linnaeus

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changed the naming system into binominal one. In this system specie consisted of genus name with capital letter and specie name with initial small letter (Jancic, 2002). 19th century was a revolutionary period for the use of medicinal plants. Alkaloids (1806), ipecacuanha (1817), strychnos (1817), and quinine (1820) were isolated. Glycosides were also identified and isolated. Active substances from medicinal plants using chemical methods were discovered like tannins, saponins, oils, vitamins and hormones (Dervendzi, 1992). There was a high risk in elimination of medicinal plants from the therapy in 19th and 20th century. Various authors claimed that drugs obtained from medicinal plants had many short falls because of the destruction of enzymes during drying of medicinal plants i.e. healing action of medicinal plants depends on process of drying. In 19th century alkaloids and glycosides obtained from medicinal plants assured that they are in pure form and had long lasting action. In 20th century stability studies were designed for fresh medicinal plants and for the plants that consist of labile active components. Besides that great effort was done in the manufacturing and cultivation of medicinal plants. Presently all around the world all the pharmacopoeias prescribe medicinally important drugs. Few countries have separate herbal pharmacopoeia like Germany and Russia. Number of drugs are unofficial that are in use. Their use is based on experience of famous medicine, latest research or experimental results. Medicinal Plants used are self-administered or applied on the recommendation of doctor or pharmacist. These drugs are used individually or in combination with synthetic drug. New law regarding drug and medical devices dated September 2007 per motes that dry or fresh part of plants can be used in manufacturing of herbal products. Herbal preparations are dispensed without a prescription as OTC preparation (Over the Counter). Pakistan is among one of those countries where belief on phytomedicine is more, under the name of Unani system of medicines or 'Hikmat'. But there is more to it because plant extracts are used in other forms of treatments like homeopathic and

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allopathic. In Pakistan the use of herbs is not carried out the way they deserve because of less research and interest in this field (Petrovska et al., 2012).

1.2 Phytochemicals Secondary metabolites are biologically active plant compounds but are not nutritious. These chemicals impart various functions to system. Secondary plant substances mean “Plant chemicals”. Latest research showed that numbers of photochemical are present in vegetables, fruits, nuts, beans, whole grains and seeds. Cardiovascular and chronic diseases like cancer can be treated with taking fruits and vegetables enriched with photochemical. Some photochemical are toxic enough that may cause death even. Phytochemical from plant extract material are classified in different classes. Various photochemical present in plant kingdom are discussed as follow:

Alkaloids Alkaloids are compounds that are alkali like or basic in nature. They are present in living organisms exhibiting many activities significant for life. Single or multiple heterocyclic rings containing nitrogen atom are present in these compounds. Alkaloids containing oxygen, hydrogen, carbon and nitrogen are derivatives of quinoline, isoquinoline, indole, pyrole and pyrolidine. Alkaloids derived from various parts of plants like from leaves datura, in seeds physostigma and nuxvomica, in stem withania, cinchona in bark and in whole aerial parts of lobelia and ephedra. Various families are enriched with alkaloids that are capable to exhibit pharmacological activities. Alkaloids common in nature isolated from plant parts are caffeine (1), strychnine (2), brucine (3), piperine (4), quinine (5) those are given below (Ali, 1998).

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1

2

7

3

4

8

5

Glycosides Those compounds which on hydrolysis break down into sugar and non-sugar parts are known as glycosides. Glycosides are composed of 2 parts. Glycone refers to sugar part and Aglycone or genin for non-sugar portion. Sugar residue in glycosidic compound is linked to C-1 through oxygen (O-glucoside), nitrogen (N- glucoside) or sulphur (S-glucoside) moiety. B-D-Glucose is commonly found in glycosides. Rhamnose, digitoxose and cymarose (deoxy sugar) are also detected along. Majority of the glycosides are crystalline and colorless. Flavone glycosides are yellowish in color, while anthracene glycosides are orange or red colored glycosides. Glycosides are soluble in water and alcohol but insoluble in organic solvents like chloroform, carbon tetrachloride, petroleum ether and solvent ether. Salicin (6) a glycoside when treated with water produces salicyl alcohol and sugar part. Various glycosides are very important as they are used in treating various diseases like cardiac, anthraquinone and many other glycosides (Trease and Evans, 2009).

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6 Saponins Word “saponin” is a Latin word derived from sapo meaning soap. Saponins are identified from their property of forming foam in aqueous solution. They are derived from squalene consisting of 6 isoprene units. When dissolved in water they are highly soluble, have high molecular weight and neutral in action. On hydrolysis saponin yield a sugar and non-sugar part (Trease and Evans, 2009).

Tannins Tannins are derivatives of polyhydroxy benzoic acid. They are complex non nitrogenous compounds extensively present in plant kingdom. They are important active constituents of material like oak bark which are used in tanning of skin (Ali, 1998). Tannins on hydrolysis yield Gallic acid and sugar part so termed as glycosides. In 1796 Sequin purposed the theory that the substance present in the plants having the ability to combine with animal hide protein, inhibiting putrefaction and converting them into leather are tannins. Tannins are basically of two types Hydrolysable tannin includes hexa hydroxyl diphenic acid and ellagic acid (7), whereas condensed tannins comprised of catechin (8), epicatechin (9) (Trease and Evans, 2009).

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7

8

11

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Resins A compound when exposed to heat gets soft and then eventually melts. Resins are hydrophobic, not soluble in water but highly soluble in organic solvents. Resins are complex mixtures of resin phenol, resin alcohol, resenes and esters (Trease and Evans, 2009) .

Flavonoids Flavonoids consist of phenyl propane and 3 acetate units. Almost 2000 flavonoids occur. They are yellow in color. Pharmacologically they are anti-inflammatory, anti-allergic and antioxidant in nature. Quercetin (10) and luteolin (11) are commonly occurring flavonoids (Trease and Evans, 2009).

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10

11

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Table 1.1 Types of Phytochemicals

Phytochemi Functions Sources cals Flavonoids May impede inflammation Apples, citrus fruits, onions, (anthocyani and tumor growth; may soybeans and soy products, coffee ns, help immunity and and tea quercetin) increase production of detoxifying enzymes in the body

Polyphenos May inhibit cancer Green tea, grapes, wine, berries, (ellagic formation, stop citrus fruits, apples, whole grains acid, inflammation and act as and peanuts resveratrol) antioxidants

Indoles and May persuade Cruciferous vegetables (cabbage, Glucosinol detoxification of collard greens, kale, cauliflower) ates carcinogens, decrease (sulforapha formation of ne) cancer‐related hormones, inhibit carcinogens and tumor growth

Carotenoid Inhibit cancer cell growth, Red, orange and green fruits and s function as antioxidants vegetables including carrots, cooked (beta‐carote and change immune tomatoes, leafy greens, sweet ne, reaction Potatoes, winter squash, apricots, lycopene, oranges and watermelon lutein)

Isoflavones May prevent tumor Soybeans and soy products (daidzein, growth, stop formation of genistein) cancer‐related hormones and commonly function as antioxidant

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Inositol May hinder cell Bran from corn, oats, rice rye and (phytic development and function wheat, nuts, soybeans and soy acid) as antioxidant products

Isothiocyan May encourage Cruciferous vegetables (cabbage, ates detoxification of collard greens, kale, cauliflower) carcinogens, stop tumor development and function as antioxidants

Terpenes May inhibit cancer Cherries, citrus fruit peel, rosemary (perillyl formation, stop alcohol, inflammation and function limonene, as antioxidants carnosol)

1.3 The Family Aizoaceae Aizoaceae, a small family consist of 128 genera and 1170 specie, out of all these many of the genus and specie are cultivated and distributed in South America,tropical , West indies, Mediterranean and tropical Asia (Willis and Shaw 1973; Mabberley 1987). Aizoaceae are included in . In Pakistan this family is divided into 8 genera (Perveen and Qaiser, 2000). Aizoaceae is considered to be the family comprised of leaf succulent plants (Klak et al., 2003). Aizoaceae family is recognized and differentiated by certain characteristics that other families do not possess. This family consist of parygynous flowers, long funicles, absence of wood in xylem, epidermal bladder cells, expanding tissues and hygrochastic base (Hartmann, 2002).

1.4 Botanical Description of Family Aizoaceae Botanically aizoaceae is divided into 5 subfamilies. It is described as following

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Subdivision of Aizoaceae Aizoaceae consist of 5 sub families named as Aizooideae, Mesembryanthemoideae, Ruschoideae, Sesuvioideae, and Tetragonioideae. Out of all five subfamilies, four subfamilies including Ruschioideae, Sesivioideae, Mesembryanthemoideae, Tetragonioideae are considered to be monophyletic and Aizooideae is paraphyletic (Klak et al., 2003).

Sesuvioideae One of the small subfamilies consists of 4 genera and almost 36 species. This family is found almost in whole world in tropics and subtropics. In past this family was considered as monophyletic. Plants are perennials or annuals. Leaves are succulent, stipulate, petiolate or sometimes cylindrical. Flowers are perigynous, arilate seeds and fruit is circumscissile.

Tetragonioideae Tetragonioideae is a large family consisting of 2 genera Tetragonia and Tribulocarpus and 85 species. These species occur most abundantly in tropical and temperate regions of southern hemispheres mainly covering southern Africa. Plants of this family differ from others by possessing characteristics of winged or hanged fruits, short funicles, complete or semi inferior ovary.

Aizooaideae This subfamily consists of six genera. Plants are annual to perennial herbs, shrubs or sub shrubs with woody or herbaceous branches. Leaves are succulent. Almost all genera of Aizooaideae are monophyletic. This subfamily is present mainly in southern Africa, , western Arabia, East Africa and Mediterranean.

Mesembryanthemoideae This subfamily consists of 11 genera and almost 100 species. Plants are annual herbs or shrubs. Leaves are succulent, flat to almost cylindrical in shape. Ovary half inferior while fruit is hygrochastic capsule, bladder cells are large and conspicuous.

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Ruschioideae This subfamily consists of 111 genera and almost 1585 species .Plant is a perennial shrub sometimes annual. Leaves are succulent trigonous or cylindrical, epidermis xenomorphic with conspicuous bladder cells. Ovary is inferior and flowers along with petals are of staminodial origin while fruit is a hygrochastic capsule (Klak et al., 2003).

1.5 Botanical Description of Genus Zaleya Zaleya, a genus belongs to ice plant family called Aizoaceae. Members of the genus are annuals or perennials specified by fleshy, opposite, unequal, smooth- margined leaves, a prostrate growth form, flowers with five perianth segments subtended by a pair of bracts, and a fruit with a winged lid (Geethalakshmi et al., 2010). Almost 6 different species of this genus are found in Asia, Africa and Australia and only 1 specie from this genus is available in Pakistan. Zaleya has diverse medicinal applications. Zaleya and Trianthema both are genera of family Aizoaceae belonging to sub family Sesuviodeae. Close relationship is found between both these genera. Earlier Zaleya was merged and used within genera Trianthema but in few studies minute differences were notified in both genera (Hsaan et al., 2005). In some studies word Trianthema is used as a synonym for Genus Zaleya (Hemadri et al., 1987).

1.6 Botanical Description of Zaleya pentandra Zaleya pentandra from the genus Zaleya is the only specie found in Pakistan. Z.pentandra more or less is a prostate herb. Stem succulent mostly 60cm long. Leaves are opposite in a pair in different sizes up to 4.5cm long oblanceolate to more or less elliptic. Flowers 5-20 flowered auxiliary sessile clusters pale green to purple in color. Fruit splitting 1/3 from the base along a thickened rim. Trianthema pentandra is used as a synonym for Zaleya pentandra. It’s English or common name is African pursulane. Pentandra acquired its name for having the specification of five stamens. It’s mostly found in grassland, bush land, near

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riversides and cultivated areas (Mapaura and Timberlake, 2004). Its common name is salathi (Mahmood et al., 2011).

1.7 Medicinal Importance of Family Aizoaceae Family Aizoaceae has broad medicinal uses. All the plants from this family are used widely in several ailments like diseases of skin, tooth aches, fever, wound healing (Upadhyay et al., 1998). Several plants are used in treatment of opthalmics, corneal ulcers, night blindness, itching, and dimness of sight. Black quarter is mostly treated by leave extract. Plants from this family also act as a cathartic and in stomach ailments in treatment of edema, rheumatisms and also as an antidote in alcohol poisoning (Ahluwalia, 1968).

1.8 Taxonomical Classification

Table 1.2 Taxonomical Classification of Zayleya pentandra

Kingdom Plantae

Phylum Angiosperm

Class Dicotyledoneae

Subclass Sesuvideae

Order Caryophyllales

Family Aizoaceae

Genus Zaleya

Specie pentandra

Synonym = Trianthema pentandra

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Figure 1.1 Flower of Zaleya pentandra

Figure 1.2 Whole plant of Zaleya pentandra

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