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ISSN: 2394 - 3726 Int. J. Phar. & Biomedi. Rese. 201 5, 2 (2): 34-40

Withania Coagulans (Stocks) Dunal – A Rare Ethnomedicinal of the Western Rajasthan Desert

Isha Pandey 1 and Krishnendra Singh Nama 2 1Dept. of Biochemistry, Jhalawar Medical College, Jhalawar, Rajasthan 2Dept. of Life Science, Career Point University, Kota (Rajasthan) *Corresponding Author E-mail: [email protected]

ABSTRACT The present paper deals with coagulans (Stocks) Dunal commonly known as “Indian cheese

maker” or “Vegetable rennet”, “Paneer doda, Paneer patta” is a ethnomedicinal plant distributed in

East of the Mediterranean region extending to South Asia. In Northern India, the fruits are being

used for the treatment of diabetic patients. The other uses of the plant are hypoglycemic,

hypolipidemic, cardiovascular, sedative, hepatoprotective, anti-inflammatory, wound healing, anti-

tumor, immune-suppressive, cytotoxic, antibacterial and antifungal properties. The aim of the present

paper to give correct identity against commonly occurring useful plant of this genus Withania

somnifera (L.) Dunal . Besides this, the paper also deals with correct botanical name, synonyms, local names, morphological characters, ecological notes, distribution, flowering and fruiting time, significant notes, uses, phytochemistry, causes of rarity, conservation, etc. Key words : Withania coagulans, Ethnomedicinal uses, phytochemistry, pharmacological properties, ex-situ multiplication.

INTRODUCTION The genus Withania Pauquy is having 10 species in the world, mostly in America and Africa; 2 species in India i.e. Withania coagulans (Stocks) Dunal and (L.) Dunal. The W. coagulans is having very limited distribution that is only in N. India, while W. somnifera is a common weed and also cultivated for its various uses and found throughout India. Both species are quite distinct and can easily be identified by morphological, anatomical and phytochemical characters. Now a day there is a growing focus on the importance of medicinal in the traditional health care system in India, because of various advantages over the synthetic drugs. The plants are used in medicine since antiquity. Much of medicinal plants are documented and are used successfully to treat different ailments. One of these plants which is used to treat various diseases is Withania coagulans (Stock) Dunal - a rare plant of the Western Rajasthan desert. The plant is also important for the property of coagulating milk, possessed by its fruits/berries; they are extensively used locally for this purpose in North-West India and adjoing countries. The milk coagulating properties is due to presence of an enzyme. The plant particularly fruits; leaves and roots are also locally used in the treatment of the various diseases. The present paper deals with correct botanical name, synonyms, local names, morphological characters, ecological notes, distribution, flowering and fruiting time, critical notes regarding identify of the plant, uses, phytochemistry, and causes of rarity, conservation and recommendation for the scientific multiplication in the nature. STUDY AREA The western sandy plain or Rajasthan desert covering a total area of about 1,96,150 sq. km spread over the districts of Sri Ganganagar, Bikaner, Jaisalmer, Barmer, Jodhpur, Churu and parts of Nagaur (96%), Jalore (88%) Jhunjhunu (69%), Sikar (65%), Pali (45%) and Ajmer (9%). Physiographically, it is the eastern extention of Saharo-Thar desert covered by shifting and stabilized sand dunes. Copyright © 2015; IJPBR 34

Pandey, I. & Nama, K.S. Int. J. Phar. & Biomedi. Rese. 2015, 2 (2): 34-40 ISSN: 2394 – 3726 The western 2/3 part extending over Bikaner, Jaisalmer, Churu, western parts of Nagaur and western 2/3 parts of Barmer and Jodhpur districts are covered by shifting and stabilized sand dunes of various types, magnitude and orientation like longitudinal, barkhan, transverse etc. This sandy plain tract is also known as “Marusthali” . The rocky outcrops are also common feathers, particularly in Barmer, Bikaner, Jaisalmer and Jodhpur districts, where elevated areas of Jurassic and Eocene rocks appears through sandy surface. The high temperature, low humidity, windstorms and less than 250 mm average rainfall are the common climatic feathers in the W. Rajasthan desert.

MATERIAL AND METHODS Since last three years, many botanical collection tours have been undertaken by the authors in the different seasons of the years in the Western Rajasthan desert to collect the plant in the nature and also collect the ethnobotanical information by the local people of the area. The plant specimens collected have been made the voucher herbarium specimens as per the International herbarium techniques. The herbarium specimens have been identified by Scientist of Botanical Survey of India, Arid Zone Regional Centre, Jodhpur (Rajasthan) and consulted the herbarium of BSI Jodhpur (BSJO) and the herbarium, Botany Department, JNV, University, Jodhpur (JAC). The fruits of the plant have also been verified from the local markets and tradesmen. The fruits available in the local markets are mostly adulterated with the commonly occurring Withania somnifera , which is a common weed and extensively cultivated in the area and adjoing states, particularly Madhya Pradesh. The “Sindhi” community in particular extensively uses the plant in their daily life as “Paneer making plant” and against diabeties and in wound healing.

OBSERVATION AND RESULT The present study deals with a solitary species Withania coagulans belonging to the family , which is a rare vulnerable plant species in the Rajasthan desert and is commonly used as “Paneer making plant” and as medicines against diabeties, in would healing and other diseases by the local people in the area. Withania coagulans (Stock) Dunal (Synonym: Punneria coagulans Stocks) Family : Solanaceae. Hindi names: Paneer-bandh, Punir, Punirbandh, Akri, Binputakah, Paneer-doda, Paneer- dodi, Paneer- bed English: Indian Cheese maker, Indian Rennet. Plant character: Branched undershrubs upto 1 m tall; branches and leaves rigid, grayish-green-white, stellate-tomentose. Leaves leathery, elliptic-ovate to elliptic-lanceolate, obtuse, entire, petiolate. Flowers polygamous-dioecious, i.e. unisexual. Male flowers: calyx cupular-campanulate, lobes acute, stellate- tomentose; corolla exceeding calyx, stellate-tomentose outside, yellowish within; stamens reaching upto the top of corolla-tube, with glabrous filaments. Female flowers: stamens sterile, included in corolla tube; ovary ovoid, glabrous; style glabrous; stigma mushroom shaped, 2-lamilate. Fruits berry, 1.0-1.25 cm across, globose, red, smooth, surrounded by the enlarged fruiting calyx, which are rough, scurfy- pubescent outside. Seeds ca 2.5 mm across, ear-shaped, brownish, minutely rugose-reticulate. Fl. and Fr.: November - April. Found in dry, hot and stony/rocky habitats, rare, not common. Distribution: Distributed in East of the Mediterranean region extending to South Asia i.e. Iran, , (Sind and Baluchistan), Nepal and India, upto 1700 m. In India, it is found in (North-West India) Himachal Pradesh, Punjab, Uttarakhand and Rajasthan. In Rajasthan it is sporadically distributed in Barmer, Jaisalmer and Jodhpur districts of Western Rajasthan desert and it is not common, categorized as “vulnerable species” by Pandey, et.al., [1]. In Western Rajasthan desert the fruits are extensively used as emetic and diuretic; also have milk coagulating properties for making cheese, that’s why the plant locally known as “ Paneer-bandh or Paneer-Pata or Paneer-doda ”. Copyright © 2015; IJPBR 35

Pandey, I. & Nama, K.S. Int. J. Phar. & Biomedi. Rese. 2015, 2 (2): 34-40 ISSN: 2394 – 3726 Significant notes: Withania coagulans (Stock) Dunal can be identified from Withania somnifera (L.) Dunal (is a common weed and cultivated for its roots, in some parts of Rajasthan (Nagaur, Pilani) and Madhya Pradesh (Manasa, near Indore) for its medicinal properties) by having stems and branches more or less rigid, grayish-green-white or ash in colour; fruits enclosed tightly within a coriaceous, pubescent fruiting calyx and plants polygamous-dioecious i.e. flowers unisexual; while in W. somnifera – stems and branches not rigid, green in colour; fruits loosely enclosed in a membranous fruiting calyx and plants bisexual i.e. flowers bisexual. The same may be summarized in key form also: 1a. Plants unisexual. Stems and branches more or less rigid, grayish or ashy-green-white in colour. Fruits tightly enclosed within rough, scurfy, coriaceous, pubescent fruiting calyx…………………………………………………………………………….. W. coagulans 1b. Plants bisexual. Stems and branches not rigid, green in colour. Fruits loosely enclosed in a membranous fruiting calyx………………………………………………………W. somnifera Besides taxonomic distinction between the above two species phytochemically and anatomically also both can be identified. The roots of Withania coagulans , characterized by the isolated groups of non-lignified cells in the xylem-tissues, microsphenoides of calcium oxalate in the phelloderm cells and triseriate medullary rays; while these characters are not found in the roots of Withania somnifera . Thus in the local markets the very commonly occurring species Withania somnifera fruits are being adulterated freely by the tradesmen with the fruits of W. coagulans , as there is less distinction in the fruits of both the species. The fruits of W. coagulans are locally used in “Paneer making” as well as in medicines; but in case Withania somnifera the roots are extensively used by body builders and medicines by the local people of the area. Specimens examined: Barmer: Bhandari 2169 (JAC); Jaisalmer: At the base City Fort, Aakal Fossil Park rocky area and Kuldhara rocks, Isha Pandey 1,2,3 (CPUK, BSJO); Bhandari 1025 (JAC); Jodhpur: Baleshar, BSI Botanical Garden, Balsamnd hillocks, Pal village, Chopasni Housing Board sector 15, and Near DPS School, Singhadiya and Maina 29692, 29693, 29694, 29695 (BSJO); Bhandari 502 (JAC); Isha Pandey 4,5 (CPUK, BSJO). USES The plant is famous for the property of coagulating milk, possessed by its fruits/berries. The fresh and dried fruits, are rubbed with a small quantity of milk and then added to the rest of milk, this property in the plant confined to only the pulp and husk of the fruits. The milk - coagulating property is due to the presence of an enzyme, which is a plant hormone that can easily be isolated by extracting the fruits with water and precipitating the enzyme either by Amonium sulphate or by adding two volumes of Acetone. The precipitate is dried at low temperature and the enzyme is so obtained a brownish-white powder. Under optimum condition, one part of concentrated enzyme will coagulate 90,000 parts of milk in half an hour. The concentrated enzyme can successfully be used in place of animal rennet to prepare soft cheese like Cheddar. The cheese thus obtained has a good texture and flavor. The active principle named “Withanin ” found in the seeds of the fruits is ferment like animal rennet. The plant is also extensively used by the local people (particularly fruits, seeds, leaves and roots) for the treatment of various diseases. The fruits in India commonly used for the treatment of Diabetes mellitus. For this, the local people soaked the fruits in water for whole night. After sieving the fruits the water so collected, drunk by the diabetic patient for about a month is very effective. It has also antimicrobial, carminative, flatulence, dyspepsia, anthelmintic, antifungal, hepatoprotective, hypoglycemic, hypolipidemic, cardiovascular, free radical scavenging, anti-inflammatory, antitumor, immunosuppressive, depresant properties, etc. The decoction of the plant taken orally for curing asthma, biliousness stranguary and fresh plant paste applied externally for healing wounds. The seeds are emmenagogue, diuretic, useful in lumbago, ophthalmic, lesion the inflammation of piles. The ripe fruits are sedative, in intestinal affections. Leaves are used as febrifuge and as blood purifier. The tender branches used as tooth brushes [2, 3, 4, 5, 6, 7]. Copyright © 2015; IJPBR 36

Pandey, I. & Nama, K.S. Int. J. Phar. & Biomedi. Rese. 2015, 2 (2): 34-40 ISSN: 2394 – 3726 PHYTOCHEMISTRY The aqueous and methanol extracts of fruits of the plant contains different phytoconstituents viz. steroids, esterases, alkaloids, phenolic compounds, tannins, saponins, carbohydrates, protein, free amino acids, fatty oils, essential oils and organic acids. Pharmacological investigations have elucidated association of these activities with the specific steroidal lactones known as “ Withanolides ” present in Withania coagulans [6, 4, 8, 7, 3, 9, 10, and 11]. The above different phytochemicals present in W. coagulans , which makes it remarkable for its use by traditional practioners. A variety of Whithanolides (Steroidal lactones) such as coagulin-F (27-hydroxy-14, 20-epoxy-1-oxo (22R)-witha-3, 5, 24-trienolide); coagulin-G (17-beta, 27-dihydroxy-14, 20-epoxy-1-oxo-(22R)-witha-2, 5, 24-trienolicle), coagulanolide (175, 205, 22R) – (14-alpha-15-alpha, 17-beta, 20-beta-tetrahydroxy-1- dxowitha-2,5,24-trienolide), Withacoagulin have been isolated from the whole plant of Withania coagulans . The photochemical studies suggest that alkaloids and steroids isolated from the plant are responsible for antidiabetic (hypoglycemic) activity of the plant [8, 12]. The pharmacognostic effect has been studied in details by Gupta [8], which are summarized as follow: 1. Antihyperglycaemic and antihyperlipidemic (antidiabetic) effect : The aqueous and chloroform extract of Withania coagulans fruits showed pharmacological effect on blood glucose, lipid profile. And it is having significant decrease in blood levels of glucose, triglyceride, total cholesterol, LDL and VLDL and on increase in HDL [4, 6]. 2. Wound healing activity : The hydroalcoholic fraction of the methanolic extract of this plant has wound healing activity in streptozotocin induced diabetic rats. 3. Cardiovascular effects : An isolated new withanolide from the fruits with a special chemical structure that was similar to the aglycones of the cardiac glycosides shows drop of blood pressure in dogs (34+/2.1 mm Hg) which was blocked by atropine and not by mepyramine or propranolol [13]. 4. Hepatorotective effect : Liver protective effect of 3-beta hydroxyl-2, 3- didydro withanolide-F isolated from W. coagulans is having high protective effect [13]. 5. Immuno – suppressive effect : Six new withanolides, withacoagulins A to F (1 to 6 resp.), together with ten known withanolides, 7 to 16, isolated from the aerial parts of the plant. The crude extracts of this plant, exhibited strong inhibitory activities on the T- and B – cells proliferation. 6. Diuretie effect : The aqueous extract of the fruits indicated significant increase in urine volume by 79.12% and 71.02% at 500 mg/kg and 750 mg/kg body weight doses respectively compared to controls. 7. Antimutagenic and anticarcinogenic effect : The genotoxic nature of any herbal drug is determined based on presence of phytoconstituents. W. coagulans contains withanolides - a plant hormone, which are reported for antitumor activity and flavonoides, which have been shown to possess antimutagenic and anticarcinogenic activities by the fruits extract of the plant [14]. 8. Anti-inflammatory activity : The fruit extracts having anti-inflammatory properties [15]. 9. Anti- fungal activity : The ethanolic extract of whole plant show two new withanolides 14, 15 – beta- epoxywithanolide 1[(20S, 22R) 178, 208 - dihydroxy- 148, 158- epoxy- 1-oxo-witha-3, 5, 24- trienolide] and 17 beta- hydroxylwithanolide K [(20S, 22R) 14-alpha, 178, 208 trihydroxy 1-oxo- witha-2, 5, 24-trienolide]. These two new withanolides have very active against a number of potentially pathogenic fungi [16, 17]. 10. Antibacterial and antihelmintic activities : The alcoholic extract of the fruits of this plant yield a volatile oil having anti-bacterial activities against Streptozotocin aureus and Vibrio chloera and it is also found to have antihelmintic activity [3, 8]. CAUSES OF RARITY The flora of Rajasthan comprising 2090 indigenous and naturalized species belonging to 819 genera under 159 families, which also includes a solitary species of Gymnosperm [1]. As per IUCN categories of “Threat”, the present flora of Rajasthan also having 65 Rare, endangered and threatened taxa (RET) [1]. Copyright © 2015; IJPBR 37

Pandey, I. & Nama, K.S. Int. J. Phar. & Biomedi. Rese. 2015, 2 (2): 34-40 ISSN: 2394 – 3726 The Withania coagulans is rare in the Western Rajasthan desert only countable collections have been made from the area like Barmer, Jaisalmer and Jodhpur districts. The probable reasons for the scarcity of this plant is due to over exploitation for various economic uses like medicinal “Paneer making” properties and over population both human being and livestock. The other probable reasons for the rarity of the plant due to badly disturbed ecosystem, lower range of adaptability, grazing, rapid urbanization, climatic factors and polygamous – dioecious, unisexual flowers. The natural regeneration by the seeds of the plant, which are only available in female plant. The fruits are commonly used by the local people as “Cheese making” ( Paneer doda , Paneer band ). All these above factors put this plant in “ vulnerable ” state. Hence, the present authors recommend that “ Ex-situ ” conservation and multiplication of germplasm by Biotechnological methods may give desirable results. Emphasis should be given for the establishment of National parks and Sanctuaries in the state. The recent establishment of “Thar Biosphere Reserve” in Jaisalmer and Barmer districts in Rajasthan (3,100 sq. Km area) may play a better role in the conservation and multiplication of this plant and other RET taxa. Thus, there is urgent need of “Desert Botanic Garden” (DGB) in the heart of W. Rajasthan like Botanical Survey of India, Jodhpur campus, where preliminary affords have already been made in this direction. The Jodhpur is now-days-a “educational hub”, where top many scientific and educational institutes have been working. This will be very useful for the future generation studying in these institutes. The proposed DBG may also serve as an intrinsic laboratory at nature for study of phenotypes and genotypes of all 65 species including this species, which show some outstanding properties and so also develop a “National Cactarium” [1]. For immediate conservation of this plant, we should create awareness among local people and particularly students of the area by educate them for identity and more scientific methods for the use of this plant. In local markets the tradesman freely adulterated by mixing the fruits of commonly cultivated and available another species Withania somnifera (L.) Dunal (Asvagandha). There is little difference in the fruits of both species and due to lack of knowledge for the correct identity of both plants; the people are purchasing the adulterated fruits of both species (Paneer Band, Paneer Doda ) - W. coagulans . The body builders ( Nagauri-Asvagandha ) extensively use the roots powder of Asvagandha. However, the fruits of Paneer band are used in diabetes and as cheese/ paneer making. Thus from the above discussion both species morphologically and phytochemically also quite distinct and also having different uses as medicinal plants. So considering above facts the plant should immediately conserved.

Fig.1. Withania coagulans seedling Fig.2. Withania coagulans rootstock

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Pandey, I. & Nama, K.S. Int. J. Phar. & Biomedi. Rese. 2015, 2 (2): 34-40 ISSN: 2394 – 3726 CONCLUSION The medicinal plants are important part and parcel of human population to combat diseases from Withania coagulans i.e. fruits/berries, seeds, leaves, roots, etc. have been used as a treatment in many disorders. This plant has been found to possess a vast variety of biologically active compounds viz. esterases, free amino acids, fatty oils, essential oils, tannins, saponins, carbohydrates, proteins, organic acids and withnolides. Withanolides are plant hormones steroidal lactones and several of them posses’ significant pharmacological and therapeutic potentials. Very little work has been done on the biological activity and plausible medicinal uses of the compounds and has extensive investigations and researches are required by the pharmaceutical companies to explore their therapeutic utility to combat diseases of future human society. Although crude extracts from various parts of W. coagulants , particularly fruits have medicinal and cheese making applications, modern drugs can be synthesized only after extensive investigations of their bioactivity, mechanism of action, pharmacotherapeutic, toxicity and other proper standardization and clinical trials. The plant’s fruits ( Paneer Doda ) are a very good drug to treat diabetic mellitus, it not only lowers the blood sugar level, but it also minimizes the future complications of diabetic patients like neuropathy, retinopathy, nephropathy and cardiovascular pathologies by its very potent antioxidant action. It also has a very good hepatoprotective, anti-tumor, anti-angiogenic, chemo- preventive and anti-inflammatory activities. Besides ethnomedicinal uses of this plant, the causes of rarity conservation and multiplication have also been dealt in the present study. The correct identity of this plant from commonly occurring and cultivated plant species Withania somnifera has also been in dealt. So one can purchase the fruit of pure Withania coagulans from the market instead of adulterated fruits. The specimens collected during the botanical collection tours have been deposited in the Department, Life science, Carrier Point University, Kota (CPUK), Rajasthan.

ACKNOWLEDGEMENTS We are very much thankful to Prof. Dr. V.D. Bohra, Head and Dr Ajay Bhargave, Associate Professor, Department of Biochemistry, Jhalawar Medical College, Rajasthan for ceaseless encouragement and facilities providing during the course of present study. We are also thankful to all teaching staff of this medical college for suggestions and inspiration during the study. Thanks are also extended to Dr. R.P. Pandey (Ex-Scientist ‘D’ & HoO of Botanical Survey of India, Jodhpur) for their support during conducting the study and identifying the plant specimens.

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