GJRMI - Volume 4, Issue 9, September 2015 by Dr. Hari Venkatesh.K.Rajaraman

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INDEX – GJRMI - Volume 4, Issue 9, September 2015 MEDICINAL PLANTS RESEARCH Ethno-Botany ETHNO-MEDICINAL PLANTS USED BY THE GUJJAR-BAKERWAL TRIBE AND LOCAL INHABITANTS OF DISTRICT RAJOURI OF JAMMU AND KASHMIR STATE Toseef Riaz, Bhandari BS

182–192

Pharmacology & Therapeutics CO-ADMINISTRATION OF ETHANOLIC LEAF EXTRACT OF MORINGA OLEIFERA AND METFORMIN REVERSES POLYPHAGIA, POLYDIPSIA AND STABILIZES BODY WEIGHT IN ALLOXAN- INDUCED DIABETIC RATS IDAKWOJI Precious Adejoh, AKUBA Ojochegbe Barnabas, ELAH Onyeche Sarah

193–202

COVER PAGE PHOTOGRAPHY: DR. HARI VENKATESH K R, PLANT ID – FLOWER OF APARAJITA – CLITORIA TERNATEA L.* OF THE FAMILY LEGUMINOSAE PLACE – OFF KANAKAPURA ROAD, BANGALORE, KARNATAKA, INDIA *BOTANICAL NAME VALIDATED FROM www.theplantlist.org AS ON 05/10/2015

Research article ETHNO-MEDICINAL PLANTS USED BY THE GUJJAR-BAKERWAL TRIBE AND LOCAL INHABITANTS OF DISTRICT RAJOURI OF JAMMU AND KASHMIR STATE

Toseef Riaz1, Bhandari BS2* 1,2

Ecology Laboratory, Department of Botany and Microbiology, HNB Garhwal University, Srinagar Garhwal-246174, Uttarakhand, India. *Corresponding Author: email: bhandaribs11@gmail.com; Tel. +919412961154

Received: 22/06/2015; Revised: 20/07/2015; Accepted: 10/08/2015

ABSTRACT The Gujjar-Bakerwal is among the native societies of Jammu and Kashmir Himalaya with strong faith and belief in traditional health care systems based on indigenous ethno-botanical knowledge. Further, the living conditions are not strong enough to bear the expenditure of money based other health care methods. Therefore, the present study is aimed at providing information regarding ethnomedicinal uses of plants of Rajouri district of Jammu and Kashmir State by Gujjar-Bakerwal Tribe and other local inhabitants. Personal interviews were conducted with tribals, locals and traditional health practitioners including ‘hakeems’ and ‘vaids’. A total of 46 plant species belonging to 35 families were recorded from the study area which is being used by Gujjar-Bakerwal tribe and other local inhabitants. Most of the species are used in the treatment of cough, rheumatism or other joint pains, stomach-ear ache, diarrhoea, dysentery, diabetes, skin diseases, etc. Majority of plant species used in ethno-medicinal practices belong to herbaceous category (19 species) followed by trees (15 species), shrubs (10 species) and ferns (2 species). Leaves are mostly exploited for the preparation of traditional medicines (32.34%) followed by roots (26.08%), fruits (10.86%), seeds (8.69%), stems and flowers (4.34%), and rest remaining from other miscellaneous parts or exudes like latex, gums and resins. KEYWORDS: Ethno-medicines; Gujjar-Bakerwal Tribe; Nomadic; Traditional health care system; Treatment.

Cite this article: Toseef Riaz, Bhandari BS (2015), ETHNO-MEDICINAL PLANTS USED BY THE GUJJAR-BAKERWAL TRIBE AND LOCAL INHABITANTS OF DISTRICT RAJOURI OF JAMMU AND KASHMIR STATE, Global J Res. Med. Plants & Indigen. Med., Volume 4(9): 182–192

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Global J Res. Med. Plants & Indigen. Med. | Volume 4, Issue 9 | September 2015 | 182–192

INTRODUCTION The Himalaya is a vast mountain system and covers fully or partially eight countries of Asia i.e. Afghanistan, Bangladesh, Bhutan, China, India, Myanmar, Nepal and Pakistan. The Himalaya although cover only 18% of the geographical area of India, accounts for more than 50% of the India`s forest cover, and 40% of the species endemic to the Indian subcontinent (Maikhuri et al., 2000). In developing countries, rural population mainly comprised of herders, shepherds or other economically marginalized sections of the population use forests for grazing, fire wood and numerous other subsistence needs (Kothari et al., 1989; Van Schaik et al.,1997; Saberwal and Ranagarajan, 2003).

knowledge is also useful for policy makers for devising solution to local and regional problems (Anonymous, 1994). There are number of references pertaining to the ethnomedicinal studies of Jammu and Kashmir. Some concentrating on folk medicines in general while others have been devoted on the traditional knowledge of medicinal plants used by the tribal communities in particular (Azad and Shah, 2012; Bhat et al., 2012; Rashid, 2013 Jeelani et al., 2013; Mala and Sofi, 2014). The present study is aimed at providing traditional knowledge about ethno-medicinally plant used by the Gujjar-Bakerwal tribe and other local inhabitants of Rajouri District of Jammu and Kashmir State. MATERIAL AND METHODS

Plants have been unique sources of medicines and constitute the most common human uses of biodiversity (Hamilton, 2004). People depend on herbal remedies to treat abdominal pain, dysentery, dyspepsia, diarrhoea, indigestion, etc (Rashid, 2012). Medicinal plants are widely used by all section of the community, whether directly as folk remedies or as the medicaments of different indigenous system as well as in modern refined practices. The tribes and local communities have effectively developed their traditional knowledge system which incorporates the use of locally available plants and their products for the treatment of various ailments (Kala, 2005). Gujjar-Bakerwal is a nomadic tribe of Jammu and Kashmir State and keeps on moving from place to place, with their livestock in search of fodder and forage. In summer, the tribes migrate from the Rajouri and Poonch to the Pir-Panjal ranges of NorthWestern Himalaya while with the onset of winter, come back to Rajouri and Poonch (Rashid et al., 2008). Ethnobotanical studies bring out more useful information on unexplored, unexploited and/or overexploited natural resources and new uses of existing resources coupled with conservation of natural resources. This

Phytogeographically, the state of Jammu and Kashmir is divisible into three distinct provinces: (i) the Jammu region with more of tropical, subtropical and temperate elements, (ii) Kashmir region with its characteristic temperate and alpine flora and (iii) the cold region of Ladakh with predominance of alpine mesophytes, cold desertic and oasitic elements, characteristic of Tibetian plateau and Siberia (Singh et al., 1999). Rajouri is one of the hilly districts of Jammu and Kashmir State bounded by Jammu in south, Poonch in the north, PoK (Mirpur) in the west and Udhampur district in the east (Figure 1). Geographically, Rajouri district lies between 30°50’ to 33°30’ North longitude and 74° to 74°10’ East Latitude covering an area of 2630 km2.In-depth ethno-botanical surveys were carried out in order to harvest traditional knowledge using semi-structural questionnaires. Priority was given to the ageold persons including ‘hakeems’ and ‘vaids’ in order to fetch maximum traditional knowledge prevailing in health care systems. Besides questionnaires, the documentation was done based on interview, informal discussion and participatory observations following standard methods (Martin, 1995; Reyes Garcia et al., 2007). Plants in use were brought to laboratory,

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pressed, dried and preserved by conventional methods. The specimens were identified with the help of relevant flora (Chopra et al., 1956; Naithani, 1984; Gaur, 1999; Sing et al., 2002). For the confirmation of identification, the specimens were systematically matched with the authentic specimens lodged at

internationally indexed Herbarium of Garhwal University, Srinagar (GUH). Plants have been properly labelled with botanical name(s), vernacular name(s), locality, family, date of collection and deposited in the Herbarium (GUH) obtaining collection number.

Figure 1: Location map of the study area in Jammu and Kashmir state (Source: Internet)

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RESULTS AND DISCUSSION A total of 46 ethno-medicinally plants belonging to 35 families were collected and identified, used in the treatment of various diseases by the Gujjar-Bakerwal tribe and other local inhabitants. Descriptions regarding botanical names, vernacular names, family, parts used and the diseases or ailments in which plants are used have been incorporated in Table1. These plant species belong to 35 different families including angiosperms, gymnosperms and pteridophytes. Out of the 46 species used in the traditional health care system, Fabaceae was found as the dominant family of angiosperms with 3 species in medicinal use. Apocynaceae, Euphorbiaceae, Lamiaceae, Liliaceae, Moraceae, Rutaceae and Scrophulariaceae each has 2 species used in curing various disorders. Pinaceae (Gymnosperms) and Adiantaceae (Pteridophytes) are also found in use with 2 species each. Further, herbaceous plants are in maximum use followed by trees, shrubs and ferns (Figure 2). It has also been observed that most of the plant species are used in the remedial measure for the treatment of cough, rheumatism and other joint pains, jaundice, syphilis, gonorrhoea, ear, stomach pain, skin diseases, etc. Leaves are maximum exploited (33%) for the medicinal use followed by roots (26%), fruits (11%), seeds (9%), flowers and stems (4% each), and rest remaining other miscellaneous parts including latex and gum (Figure 3). Earlier (Ahuja, 1965; Sharma and Singh (1989); Kiranjot et al., 2007; Shah et al., 2009; Bhellum and Singh, 2012) also discussed the ethno-botany of important medicinal plants used by different communities in different parts of India. Ethno-botany is a holistic approach involving the reciprocal and dynamic aspect of

interaction of indigenous people with plants (Schultes, 1962; Fords, 1980) and includes studies such as food, fibre, dyes, tannins, medicinal, useful as well as harmful plants, taboos and megico-religious belief, material use and the action of domestication, conservation, improvement or over-exploitation of plants (Ford, 1978; Jain, 1987, 1995). Simultaneously ethno-botany has evolved the relationship between the people-plant in a multidisciplinary manner linking botany, ecology, pharmacology and public health. These medicinal plants have been introduced in markets as a result of exploring traditional medicines and ethno-pharmacology (Balick, 1996; Bussmann, 2002). The local people have their own knowledge for the utilization and conservation of plants and this knowledge passes from generation to generation (Sharma et al., 2011). The Gujjar-Bakerwal tribe is among the native societies of Jammu and Kashmir Himalaya with strong faith and belief in traditional knowledge. Unfortunately this traditional knowledge is rapidly vanishing due to various logical and illogical reasons. Sometimes secrecy is being maintained for the conservation purposes of natural resources but on the other, due to misconception among the local practitioners of the traditional health care systems, that disclosing the secrecy affects the effectiveness of the folk medicines. This leads to overexploitation of natural resources due to lack of concrete information regarding the use of ethno-medicinal plants. Nevertheless, Gujjar-Bakerwal tribe of Rajouri district of Jammu and Kashmir State is rich in ethnobiological knowledge and this knowledge is being transmitted from one generation to another. Similar information related to manplant interaction of many tribal communities from different parts of the world has been reported by various workers from time to time (Farnsworth et al., 1985; Johnston and Colquhouns, 1996; Nautiyal et al., 2001b; Negi et al., 2002).

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Figure 2: Component wise representation of plants in traditional use 20 18 16 No. of species

14 12 10

6 10 4 2 2

0 Herb

Tree

shrub

Fern

Figure 3: Plant parts used to cure various ailments Others 9% Latex 4% Roots 26%

Seeds 9% flowers 4%

Stems 4%

Fruits 11%

Leaves 33%

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Table1. Detailed description of ethno-medicinally plants used by Gujjar-Bakerwal tribe and other local inhabitants of Rajouri District of Jammu & Kashmir State: No.

Botanical Name (Family)

Local name

Voucher no.

Part Used for Used*

01.

Abrus precatorius (Fabaceae) Abutilon indicum (Malvaceae) Aconitum heterophyllum (Ranunculaceae) Adhatoda vasica (Acanthaceae) Adiantum capillus (Adiantaceae) Adiantum venustum (Adiantaceae) Aegle marmelos (Rutaceae)

Ratie

TR-17365(GUH)

Peeli booti Patees

TR-17406(GUH)

TR-17380(GUH)

Basooti, Adusa Hans raj Kali kakes Bill, Vilva

TR-174141(GUH) LE

02. 03.

04. 05. 06. 07.

08. 09. 10.

11.

12. 13.

14. 15.

Albizia lebbeck (Fabaceae) Amaranthustricolor (Amaranthaceae) Asparagus adscendens (Liliaceae) Asparagus racemosus (Liliaceae) Azadirachta indica (Meliaceae) Bauhinia variegata (Caesalpiniaceae) Berberis lycium (Berberidaceae) Bergenia ciliata (Saxifragaceae)

To cure ulcer, skin disease and rheumatic pains. To cure piles, ulcer and paralysis. Effective against fever, cough and vomiting. In chest disease, wound, fever and swellings. Paste applied in demulcent and as expectorant tonic. To check vomiting.

TR-17393(GUH)

TR-17366(GUH)

TR-17390(GUH)

Dhrienk TR-17413(GUH)

Ghanar

TR-17370(GUH)

Safedmusli

TR-17411(GUH)

Satmooli

TR-17368(GUH)

As aphrodisiac.

Neem

TR-17386(GUH)

Kachna r

TR-17400(GUH)

LE & BA LE & BU

Simblu

TR-17367(GUH)

Zakhme TR-17384(GUH) -eHayat Jalneem TR-17404(GUH)

Leaves diuretic, Bark as bitter tonic used tooth paste. Leaves in skin diseases and wound, buds in dysentery, blood purifier. As gastric tonic, mild laxative and eye tonic. Powder to cure fever, wound, diarrhoea and eye diseases.

In stomach ache and liver disorder. In dysentery, stomach-ache and diabetes

16.

Bacopa monnieri (Scrophulariaceae)

17.

Bombax ceiba (Bombacaceae)

Simbal

TR-17369(GUH)

18.

Cannabis sativa (Cannabinaceae)

Bhang

TR-17412(GUH)

To recover diarrhoea and dysentery, juice as coolant in summer. Boiled extract against stomach infection. To cure dysentery and diarrhoea. Tubers used in diabetes and dysentery.

Paste in cuts antioxidant.

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and

Global J Res. Med. Plants & Indigen. Med. | Volume 4, Issue 9 | September 2015 | 182–192

Carissa opaca (Apocynaceae) Cedrus deodara (Pinaceae)

Garand a Devdar

TR-17373(GUH)

TR-17389(GUH)

21.

Phyllanthus emblica (Euphorbiaceae)

Aamla

TR-17405(GUH)

22.

Euphorbia hirta (Euphorbiaceae)

Dodal

TR-17375(GUH)

23.

Ficus palmata (Moraceae)

Kamri

TR-17399(GUH)

FR& LA

24.

Fragaria vesca (Rosaceae) Hypericum perforatum (Hypericaceae) Ipomoea (Convolvulaceae) Mentha longifolia (Lamiacea) Mimosa pudica (Mimosaceae) Morus alba (Moraceae)

Kunchi

TR-17371(GUH)

Fruits to cure lungs and bladder disease. Latex is applied externally on wounds. Digestive disorders.

Basanti phool

TR-17403(GUH)

Curing dysentery.

Khrisha TR-17383(GUH) -beej Pudina TR-17372(GUH)

As galactogogue and fever.

Lajwant TR-17398(GUH) -i Toot TR-17377(GUH)

LE& R FR

In indigestion and controlling vomiting. In kidney troubles.

Jajooli

TR-17397(GUH)

Tulsi

TR-17374(GUH)

TR-17401(GUH)

TR-17376(GUH)

TR-17394(GUH)

Powder in dyspepsia.

To control high blood pressure, antidotes in snake bite. Paste used in fungal infection.

19. 20.

25.

26. 27. 28. 29. 30. 31.

32. 33. 34. 35. 36. 37. 38.

Mucuna pruriens (Fabaceae) Ocimumsanctum(La miaceae)

Olea ferruginea Kaow (Oleaceae) Pinus roxburghii Chir (Pinaceae) Pistacia integerrima Kangar (Anacardiaceae) Rauvolfia serpentina (Apocynaceae) Rumex hastatus (Polygonaceae) Syzygium cumini (Myrtaceae) Tagetes minuta (Asteraceae)

SapTR-17382(GUH) gandhi Khattim TR-17395(GUH) -al Jamun TR-17378(GUH) Guta

TR-17392(GUH)

LE SE& BA LE

Extract to overcome poor digestion. Oil applied to treat piles, arthritis and skin diseases of goats and sheeps known as ‘Makku’ disease. Treatment of piles, liver, stomach, anaemia and urinary troubles. Latex is used in ear-ache and internal wound.

Against dyspepsia and good for digestive system. Infusion in nerve disorder, impotency and paralysis. Against bronchitis, respiratory and urinary troubles and also mixed with tea known as ‘Tulsi Tea’. Whooping cough and throat sore. As crack cream. dysentery

and

Diabetes. Juice useful in ear-ache and as insect repellent.

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Taxus wallichiana (Taxaceae) Tinospora cordifolia (Menispermaceae)

Barmi

TR-17402(GUH)

Guloh

TR-17379(GUH)

41.

Vervascum thapsus (Scarphulariaceae)

Gider tamaku

TR-17388(GUH)

42.

Vitex negundo (Verbenaceae)

Bana

TR-17381(GUH)

R& LE

43.

Viola odorata (Violaceae)

Banafsh TR-17396(GUH) -a

44.

Withania somnifera Ashwag (Solanaceae) -anda

TR-17385(GUH)

To overcome general as well as sexual weakness.

45.

Zanthoxylum armatum (Rutaceae) Ziziphus mauritiana (Rhamnaceae)

Timar

TR-17391(GUH)

To relieve tooth-ache.

Beer

TR-17387(GUH)

In indigestion, diarrhoea and stomach-ache.

39. 40.

46.

Head-ache and to overcome indigestion. Against malarial fever, stomach trouble and coolant for animals. Extract used in bronchitis, asthma and stomach pains. Used as diuretic and in joint pain. Useful in liver disorder.

*R (Roots), ST (Stem), LE (Leaf), FL (Flower), BU (Bud), FR (Fruit), SE (Seed),BA (Bark), WO (Wood), LA (Latex), PO (Pods), RE (Resin), GA (Galls).

The dwindling down of traditional ethnobotanical knowledge is due to lack of sustainable development policy towards promoting knowledge based programmes on one hand and accessibility of allopathic medicines on the other. Pharmaceutical researches acknowledge that screening plants on the basis of information derived from traditional knowledge saves billions of dollars in time and resources (Hafeel and Shanker, 1999). Thus, there is urgent need to conserve the traditional knowledge existing among tribal communities in different parts of India including Gujjar- Bakerwal tribe through implementing some incentive programmes.

nomadic shifting seasonally in different parts of the State on one hand and largely due to costly modern health care systems. Nevertheless, tribals are well aware about availability of medicinal plants and parts used in curing various ailments. Cough, cold, fever, rheumatism and other joint pains, stomachache, weakness are among the common ailments treated traditionally using ethnomedicinal plants. But unfortunately, due to various reasons this traditional knowledge is dwindling down in the modern generations. Need is to conserve this traditional heritage through the implementation of some incentive programmes for the tribal communities.

CONCLUSION

ACKNOWLEDGEMENT

The study reveals the utilization pattern of some ethno-medicinal plants by the GujjarBakerwal Tribe and other inhabitants of Rajouri District of Jammu and Kashmir State. They have developed their utilization pattern based on traditional knowledge existing from centuries. Further, they have strong faith in the traditional health care systems partially due to accessibility of natural sources as these are

Authors are thankful to the University Grants Commission (UGC) New Delhi for financial support to one of the authors (Toseef Riaz) as Ph.D. Fellowship. Gujjar-Bakerwal Tribe and other local inhabitants deserve special thanks that have been kind enough in providing ethno-medicinal knowledge and other relevant information.

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Sharma PK, Singh V (1989). Ethno-botanical studies in north-west and trans HimalayaV. Ethno-veterinary medicinal plants used in Jammu and Kashmir, India. Journal of Ethnopharmacology; 27: 63–70. Singh DK, Uniyal BP, Mathur R (1999). Jammu & Kashmir. In: Mudgal, V & P.K.(eds.). Floristic Diversity and Conservation Strategies in India 2. Botanical Survey of India. Kolkata, pp. 905–974.

Source of Support: NIL

Singh NP, Singh DK, Uniyal BP (2002). Flora of Jammu and Kashmir. Botanical Survey of India, Calcutta. Van Schaik CP, Terborgh J, Dugelby B (1997). The silent crisis: the state of rain forest nature preserves. In: R. Kramer, C. van Schaik and J. Jonson, (eds.), Last stand: protected area and defence of tropical biodiversity, pp. 64–89. Oxford University Press, New York, USA.

Conflict of Interest: None Declared

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Research article CO-ADMINISTRATION OF ETHANOLIC LEAF EXTRACT OF MORINGA OLEIFERA AND METFORMIN REVERSES POLYPHAGIA, POLYDIPSIA AND STABILIZES BODY WEIGHT IN ALLOXAN- INDUCED DIABETIC RATS IDAKWOJI Precious Adejoh1*, AKUBA Ojochegbe Barnabas2, ELAH Onyeche Sarah3 1

Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria 2 Department of Science Laboratory Technology, Kogi State Polytechnic, Lokoja 3 Department of Chemical Sciences, Faculty of Natural and Applied Sciences, University of Mkar, MkarNigeria *Corresponding Author: E-Mail: sirprecious@yahoo.com; Phone: (+234) 8077039243

Received: 10/07/2015; Revised: 20/08/2015; Accepted: 10/09/2015

ABSTRACT This study examined the effect of Ethanol extract of Moringa oleifera (MOE) leaves and metformin co-administration on the glucose levels, bodyweight, food and water intake of diabetic Wistar rats. Diabetes was induced in rats by administration of 150 mg alloxan/kg body weight intraperitoneally. Six groups of diabetic rats and one group of normoglycaemic rats (n = 6) were used for the study. Groups I and II served as non-diabetic and diabetic controls respectively. Groups III and IV received 400 and 800 mg MOE /kg respectively, groups V and VI received the same doses of MOE respectively but co-administered with metformin (150 mg/kg) and group VII received metformin (150 mg/kg) alone. Daily food and water intake, weekly body weight change and fasting blood glucose levels were estimated for 28 days. MOE (400 mg/kg)/ metformin and MOE (800 mg/kg)/ metformin administration showed significant reduction in FBS (p< 0.001) on days 7, 14, 21 and 28, food intake and water intake (p< 0.001) in weeks 2, 3 and 4 but no significant difference was observed in the body weight of treated rats when compared to diabetic control. It was concluded that the extract and metformin co-use may be useful in ameliorating symptoms of diabetes. KEY WORDS: Diabetes mellitus, Metformin, Moringa oleifera, Polydipsia, Polyphagia

Cite this article: IDAKWOJI Precious Adejoh, AKUBA Ojochegbe Barnabas, ELAH Onyeche Sarah (2015), CO-ADMINISTRATION OF ETHANOLIC LEAF EXTRACT OF MORINGA OLEIFERA AND METFORMIN REVERSES POLYPHAGIA, POLYDIPSIA AND STABILIZES BODY WEIGHT IN ALLOXAN- INDUCED DIABETIC RATS, Global J Res. Med. Plants & Indigen. Med., Volume 4(9): 182–192

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INTRODUCTION Diabetes mellitus (DM) is a multi-factorial disease which is characterized by hyperglycemia (Ugochukwu et al., 2003), lipoprotein abnormalities (Scoppla et al., 2001), raised basal metabolic rate (Okwu et al., 2006), defect in reactive oxygen species scavenging enzymes and altered intermediary metabolism of major food substances (Unwin et al., 2001). Diabetes results from an absolute deficiency in insulin secretion (type 1 DM), insulin action (type 2 DM) or both (AndradeCetto et al., 2007) leading to hyperglycaemia. Diabetes mellitus is a life threatening disorder and it is estimated that its annual incidence rate will continue to increase in the future worldwide (Vasavda et al., 2014). Hyperglycaemia which is the hallmark of diabetes mellitus is responsible for its cardinal symptoms namely: weight loss, polyphagia, polydypsia and polyuria (Robert, 2001). In hyperglycaemic state, the renal threshold for glucose is exceeded and it is thus excreted in the urine. The excreted glucose then acts as osmotic solute in urine, producing an excessive loss of water (polyuria). This excessive loss of water in urine also leads to another cardinal symptom of diabetes mellitus which is polydypsia. Polydipsia arises when there is a depletion of the Extra Cellular Fluid volume and dehydration following excessive loss of water in urine. This activates the thirst centre hence, the excessive thirst for water (polydypsia). Polyphagia in diabetes mellitus could be explained by the “Glucostat Theory of Feeding Regulation” which suggests that differences in the arteriovenous glucose content of the satiety and feeding centers in the hypothalamus regulates the feeding response (Guyton and Hall, 2006). It explains that if the difference is high due to high glucose utilization by the satiety center, then the satiety center is activated. However, if the difference is low, then feeding center is activated. In diabetes mellitus, the inability of glucose to permeate the cells of the satiety center leads to a low arteriovenous difference and the feeding center is chronically activated thus leading to

polyphagia (Guyton and Hall, 2006; Gannong, 2006). In diabetes mellitus, the gluconeogenic pathway is activated as a result of the inability of the cells to utilize glucose for energy production. Thus the weight loss in diabetes mellitus is linked to the utilization of muscle protein and excessive mobilization of fats from the adipose tissues for energy production in the gluconeogenic (Champe et al., 2005). Moringa Oleifera belongs to the family Moringaceae. Various parts (leaves, flower and seed) of Moringa Oleifera have been reported to be potent in ameliorating symptoms of diabetes mellitus including hyperglycaemia (Chinwe and Isitua, 2010). Metformin is a firstline antidiabetic agent for the management of type 2 diabetes (Esposito et al., 2011). Metformin is suitable irrespective of age, body weight, severity of hyperglycemia and provides a convenient pharmacological base for combined therapy with other antidiabetic agents (Scarpello and Howlett, 2008). The ameliorative effects of ethanol extract of Moringa oleifera leaves and Metformin coadministration on the symptoms of diabetes (weight loss, polyphagia and polydypsia) in alloxan-induced diabetic rats is thus investigated in this study. MATERIALS AND METHODS Collection and identification of plant material The fresh leaves of Moringa oleifera were obtained from Area A. Staff Quarters of Ahmadu Bello University, Zaria in June, 2014 and were identified by Mallam U. Mohammed of the herbarium unit of the Department of Biological Sciences, Ahmadu Bello University, Zaria where a voucher specimen (Voucher No. 571) was deposited for future reference. Preparation of ethanol leaf extract of Moringa oleifera The fresh leaves were rinsed with water to remove sand and debris and then shade dried to a constant weight and pulverized with a manual

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blender into a fine powder to give a gram weight of 820g. This was cold macerated with ethanol for 24 hours and filtered with Whatman filter paper (Size No1) to obtain ethanol extract of M. oleifera leaves (MOE). The final filtrate was evaporated to dryness over a water bath maintained at 40oC and refrigerated at 4℃ until required for use. Chemicals and Materials Metformin Hydrochloride (Glucophage®) was purchased locally, Alloxan monohydrate and absolute ethanol were purchased from the country representative of Sigma Chemical, St. Loius USA while a digital glucometer and corresponding test strips (Fine Test®, Infopia Co., Ltd. USA) was purchased from a pharmacy store in Samaru, Sabon Gari Local Government Area of Zaria, Kaduna State. All other chemicals used were of analar grade and obtained commercially. Animals Adult Wistar rats of either sex weighing 100–150g were purchased from the Animal House of the Department of Pharmacology and Therapeutics, Ahmadu Bello University, Zaria, Nigeria. They were kept in stainless steel cages under standard laboratory conditions of temperature 22 ± 1oC, relative humidity 14 ± 1% and 12h light /dark cycle. They were maintained on clean water and standard rodent chow. All experiments were performed according to the “Principles of Laboratory Animal Care” (NIH Publication No. 85; rev. 1985). Experimental Design Acute toxicity study The oral median lethal dose (LD50) of the extract was determined in rats according to the method of Lorke (1983) with slight modifications using 18 rats weighing between 100–150g. The study was carried out in two phases. In the first phase, 9 rats were divided into 3 groups of 3 rats each and were treated with the ethanol leaf extract of Moringa

oleifera at doses of 10, 100 and 1000 mg/kg body weight respectively. They were observed for 24 hours for signs of toxicity and/ or mortality. Based on the results of the first phase, 9 rats were again divided into 3 groups of 3 rats each and were also treated with ethanol extract of Moringa oleifera at doses of 1600, 2900 and 5000 mg/kg body weight respectively in the second phase. The rats were also monitored closely for the first 4 h after treatment and subsequently daily for 14 days for signs of toxicity and/or mortality. The median lethal dose (LD50) was estimated based on the observations in the second phase. Induction of diabetes mellitus The method described by Dunn and McLetchie (1943) was adopted. Diabetes mellitus was induced by a single intraperitoneal injection of alloxan at a dose of 150 mg/kg body weight of overnight fasted rats. The animals were thereafter allowed food and water ad libitum. The state of diabetes was observed after 72 hours using a Fine Test® digital glucometer and the corresponding test strip to determine the blood glucose levels following an overnight fast. Rats having fasting blood glucose (FBS) levels ≥ 200 mg/ dl were considered diabetic and selected for the study. Experimental protocol Seven (7) groups (6 groups of hyperglycaemic rats and 1 group of normoglycaemic rats) of 6 animals each were used for the study. Group I and II served as the normoglycaemic and diabetic controls respectively and received 1ml distilled water/kg body weight. Groups III and IV rats received 400 and 800 mg MOE/kg respectively. Groups V and VI rats were treated with these same doses (400 and 800 mg/kg respectively) but coadministered with a fixed dose of metformin (150 mg/kg body weight). Rats in group VII were treated with 150 mg metformin/ kg body weight only. Fasting blood sugar determination Blood samples were obtained from the tail vein of the rats and glucose level was estimated

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on 0, 7, 14, 21 and 28th day of the study by FineTest® glucometer Determination of water intake Water intake was measured using calibrated feeding bottle with stainless steel nozzles. The daily water intake was obtained by subtracting the volume of water remaining at the end of 24 hours of feeding from the initial amount in the water bottle at start of the day. The difference was the amount consumed for the day. Determination of food intake The food intake was measured by weighing the amount of food left in the container after 24 hours and subtracting it from the initial amount of food at start of the day’s feeding. The food containers were medium sized stainless steel plates to avoid spillage of food. Weight measurement All the rats in the different experimental groups were weighed at the start of the experiment. This was taken to be the initial weight. Weekly weight measurements were taken in all the groups. The final weight measurement was conducted on the last day of the experiment. And the differences in the weight in the various groups recorded accordingly. Statistical analysis All results are presented as mean ± standard error of mean and statistical differences between means were determined by one-way ANOVA followed by Dunnett’s post–hoc test for multiple comparison tests using GraphPad version 5.0. P-values less than 0.05 were considered significant. RESULTS Acute toxicity studies:

The oral median lethal dose (LD50) of MOE was therefore, estimated to be greater than 5000 mg/kg body weight in rats. Effect of Ethanol Extract of Moringa oleifera Leaves (MOE), Metformin and MOE/Metformin Co-administration on FBS of Rats When compared to the diabetic control, MOE (400 mg/kg) treated group showed a significant (p< 0.05) reduction in FBS only on day 28, MOE (800 mg/kg) treated group showed a statistically significant (p<0.01) reduction in FBS On days 21, MOE (400 mg/kg)/ metformin co-administration showed significant reduction in FBS on day 7 (p<0.05), days 14 and 21(p< 0.01) and day 28 (p< 0.001) respectively, MOE (800 mg/kg)/ metformin co-administration showed significant (p< 0.001) reduction on days 7, 14, 21 and 28 while Metformin (150 mg/kg) showed significant reduction on day 14 (p<0.05) and days 21 and 28 (p<0.01). Effect of MOE, Metformin and MOE/Metformin Co-administration on Weekly Food consumption of AlloxanInduced Diabetic Rats When compared to the diabetic control, MOE (400 mg/kg) treated group showed statistically significant (p<0.05) reduction in food consumption in week 4. MOE (800 mg/kg) treated group showed significant reduction in food consumption in week 2 (p< 0.05) and weeks 3 and 4 (p< 0.01) when compared to the control group. MOE (400 mg/kg)/Metformin and MOE (800 mg/kg) /Metformin treated groups showed significant reduction in week 1 (p< 0.05) and weeks 2, 3 and 4 (p< 0.001). The metformin-treated group showed significant reduction in food consumption in weeks 2 and 3 (p<0.05) and week 4 (p<0.01) when compared to the diabetic control (Table 2).

MOE did not produce any sign of toxicity or mortality at all the doses administered orally.

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Table 1. Effect of MOE, Metformin and MOE/Metformin Co-administration on Fasting Blood Sugar (FBS) (mg/dl) of Alloxan- Induced Diabetic Rats Post- Treatment Time in days (d) Treatment 0 7 14 21 28 Mg/kg 74.3 ± 5.60 76.3 ± 4.62 79.8 ± 2.41 78.9 ± 3.66 75.4 ± 2.56 NDC 481.4 ± 43.13 487.5 ± 31.87 509.8 ± 11.08 497.3 ± 15.83 477.3 ± 17.11 DC 473.0 ± 27.91 469.5 ± 39.15 457.5 ± 36.00 398.8 ± 27.18 306.8 ± 30.18* MOE 400 465.0 ± 32.13 423.5 ± 31.18 419.6 ± 26.00 261.2 ± 32.15** 252.0 ± 41.33** MOE 800 253.0 ± 35.13* 238.5 ± 27.33** 180.4 ± 27.83*** 100.6 ± 15.14*** MOE 400+Met 458.7 ± 27.52 124.8 ± 22.15*** 99.3 ± 6.34*** 80.8 ± 5.43*** MOE 800+Met 438.2 ± 59.27 168.5 ± 21.19*** 445.3 ± 38.31 438.8 ± 43.19 288.6 ± 24.39* 265.2 ± 23.21** 249.0 ± 37.21** Met 150 Data represented as mean ± S.E.M of FBS. Data was analyzed by one- way ANOVA followed by Dunnett’s post – hoc test for multiple comparisons, (n=6).Statistically significant *p < 0.05, **p < 0.05, 0.01, ***p < 0.001 compared with the diabetic control (DC). NDC= Non Diabetic Control

Table 2. Effect of MOE, Metformin and MOE/Metformin Co-administration on Weekly Food Consumption (g) in Alloxan- induced Diabetic Rats Post- treatment time in weeks (wk) Treatment (mg/kg) NDC

131.5 ± 13.63

115.3 ± 02.88

119.5 ± 02.75

118.7 ± 02.22

160.3 ± 09.12

192.5 ± 04.61

200.7 ± 04.79

215.7 ± 03.38

MOE 400 MOE 800

163.0 ± 05.62 165.0 ± 02.52

186.8 ± 04.29 177.8 ± 02.86*

199.3 ± 09.84 178.3 ± 06.00*

200.0 ± 06.60* 183.3 ± 16.67**

MOE 400 +Met

126.7 ± 04.76*

125.0 ± 01.59***

137.5 ± 05.79***

123.2 ± 02.01***

MOE 800 +Met Met 150

123.8 ± 10.57* 161.2 ± 06.35

115.8 ± 02.10*** 176.8 ± 02.04*

119.2 ± 05.21*** 178.8 ± 05.79*

120.3 ± 02.32*** 181.3 ± 04.07**

Data represented as mean ± S.E.M of quantity of feed in (g), analysed by one-way ANOVA followed by Dunnett’s post – hoc test for multiple comparisons, (n=6). Statistically significant *p < 0.05, **p < 0.05, 0.01, ***p < 0.001 compared with the diabetic control (DC). NDC= Non Diabetic Control

Effect of MOE, Metformin and MOE/Metformin Co-administration on Weekly Water intake of Rats Compared to the diabetic control, MOE (400 mg/kg) treated group showed statistically significant reduction in water intake in weeks 3 and 4 (p<0.05). MOE (800 mg/kg) treated group showed significant reduction in water intake in week 2 (p< 0.05) and weeks 3 and 4 (p< 0.01) when compared to the control group. MOE (400 mg/kg)/Metformin treated groups showed significant reduction in week 1 (p< 0.01) and weeks 2, 3 and 4 (p< 0.001). MOE (800 mg/kg) /Metformin showed significant reduction in water intake in weeks 1, 2, 3 and 4

(p< 0.001). The metformin-treated group showed significant reduction in water intake in weeks 2 and 3 (p<0.05) and week 4 (p<0.01) when compared to the diabetic control (Table 3). Effect of MOE, Metformin and MOE/Metformin Co-administration on Body Weight of Alloxan- Induced Diabetic Rats There was no statistically significant difference in the mean body weight of rats in the hyperglycaemic rats (Table 4).

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Table 3. Effect of MOE, Metformin and MOE/Metformin Co-administration on Weekly Water Intake in Alloxan-induced Diabetic Rats Post- treatment time in weeks (wk) Treatment (mg/kg)

NDC

168.3 ± 10.14

165.7 ± 06.12

164.3 ± 05.71

158.6 ± 05.95

DC MOE 400 MOE 800

328.3 ± 28.22 256.7 ± 15.63 255.0 ± 15.44

394.3 ± 09.67 392.6 ± 05.95 361.4 ± 09.11*

424.3 ± 06.85 365.7 ± 10.20* 360.6 ± 09.11*

404.3 ± 03.69 368.6 ± 04.59* 322.9 ± 05.65**

218.3 ± 12.76** 198.3 ± 14.47*** 303.0 ± 22.61

214.3 ± 07.19*** 184.3 ± 02.02*** 390.7 ± 09.48

201.4 ± 05.53*** 175.7 ± 05.71*** 367.9 ± 05.65*

172.9 ± 05.22*** 140.0 ± 03.78*** 320.0 ± 05.35**

MOE 400 + Met MOE 800 + Met Met 150

Data represented as mean ± S.E.M of volume of water in (ml). Data was analysed by one- way ANOVA followed by Dunnett’s post–hoc test for multiple comparisons. (n=6), *p < 0.05, **p < 0.01, ***p < 0.001 statistically significant reduction compared with the diabetic control (DC). NDC= Non Diabetic Control

Table 4. Effect of MOE, Metformin (Met) and MOE/Metformin Co-administration on Average Body Weight (g) of Alloxan-Induced Diabetic Rats Post-treatment time in days (d) Treatment (mg/kg)

NDC

133.5 ± 14.26

149.0 ± 17.18

151.2 ± 15.55

152.6 ± 02.67

155.4 ± 16.91

121.2 ± 13.75

110.0 ± 13.50

108.4 ± 12.74

108.0 ± 12.17

103.4 ± 09.70

137.3 ± 14.24 132.2 ± 13.76 134.2 ± 14.10 138.2 ± 13.11 147.5 ± 12.13 MOE 400 125.5 ± 12.91 116.8 ± 13.45 120.3 ± 13.49 124.8 ± 14.45 131.2 ± 13.13 MOE 800 125.8 ± 12.67 114.8 ± 14.75 120.0 ± 16.10 121.2 ± 16.15 126.8 ± 15.92 MOE 400 +Met 138.7 ± 12.99 127.3 ± 14.11 132.7 ± 13.14 136.5 ± 13.56 144.8 ± 13.18 MOE 800 +Met 130.0 ± 57.81 119.0 ± 5.62 121.3 ± 5.03 126.5 ± 4.92 131.8 ± 5.45 Met 150 No statistically significant difference between treated groups and diabetic control

DISCUSSION The primary aim of diabetes management is to reduce blood glucose levels to within the normal range. Various hypoglycemic medications are available for this purpose but most of them are not without severe adverse effects. Consequently, some diabetics often resort to medicinal plants and foods with antidiabetic activity but with fewer or no adverse effects while some co-use anti-diabetic herbs with conventional anti-diabetic agents for better management. This study investigated the ameliorative effect of ethanol extract of Moringa oleifera leaves co-administered with

metformin on the cardinal symptoms of diabetes mellitus. In this study alloxan was used to induce experimental diabetes. Alloxan induces diabetes by damaging the insulin secreting pancreatic beta-cells of the islets of Langerhans resulting in reduced synthesis and release of endogenous insulin (Ghosh, 2005). The cytotoxic action of alloxan is mediated by reactive oxygen species with a simultaneous massive increase in cytosolic calcium concentration leading to a rapid total destruction of beta-cells producing a type 1-like diabetes mellitus (Szkudelski, 2001).

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Polyphagia, polydypsia, polyuria and weight loss are established classical symptoms and signs of diabetes mellitus. These were clearly demonstrated in this study, with increased food and water intake in the diabetic groups and also marked weight loss in these groups when compared to the non-diabetic control.

food and water intake are controlled by satiety and thirst centers respectively in the hypothalamus, it could also be that apart from controlling glycaemia, the extract/ metformin synergistically stimulated the satiety centre and inhibited the thirst centre as well as hunger (Okon et al., 2012).

Twenty eight (28) days of treatment with ethanol extract of Moringa oleifera leaves and its co-administration with metformin ameliorated significantly these classical symptoms of diabetes mellitus as remarkable reductions in the fasting blood sugar, quantity of food and volume of water intake were observed in the treated groups throughout the duration of the study when compared to the diabetic control.

The body weights of diabetic rats decreased following alloxan treatment. This is in agreement with the symptoms of diabetes as stated by American Diabetic Association (ADA) (2000) to include unexplained weight loss. This observation could be attributed to the increased conversion of storage fat and proteins to glucose (gluconeogenesis) (Champe et al., 2005). However, after the first week of treatment, probably with improvement in glucose uptake by cells and subsequent reversal of gluconeogenesis, the body weights of the treated diabetic groups showed a steady increase, though with no significant difference throughout the course of the experiment. This study did not reveal any significant difference in the weights of the rats in the groups that received graded doses of extract coadministered with metformin suggesting that the use of metformin and Moringa oleifera extract by diabetics is not likely to bring about an increase in weight. This observation is good for diabetes management as a number of oral hypoglycaemic drugs in use such as the sulphonylureas are associated with weight gain (Campbell and Howlett, 1995).

The therapeutic actions of Moringa oleifera medication has been attributed to the relatively high antioxidant activity of its leaves, flowers, and seeds (Atawodi et al., 2010). The leaves contain antioxidants particularly quercetin and kaemferol (Fuglie, 1999) which are reported to possess hypoglycaemic activity (Desokky and Youssef (1997). The hypoglycaemic activity of these antioxidants is due to their ability to scavenge the free radicals generated by alloxan hence, regenerating the destroyed beta-cells and subsequently, release of insulin. The ameliorative effect of ethanol extract of Moringa oleifera leaves co-administered with metformin on the symptoms of diabetes mellitus could be attributed to the synergistic reduction in hyperglycaemia as seen in table 1. Hyperglycemia gives rise to glycosuria which is accompanied by osmotic diuresis (Polyuria). Polyuria which causes water depletion, signals the thirst centre in the hypothalamus for increased water intake. This cycle of polyuria and polydypsia continues depending on the level of hyperglycemia (Okon et al., 2012). The ethanol extract of Moringa oleifera leaves coadministered with metformin synergistically reduced the magnitude of food and water intake though out the duration of this study when compared to the diabetic group. Given that

CONCLUSION This study revealed that the anti-diabetic activity of the extract/metformin coadministration was far greater than that of the extract or metformin administered alone. This suggests that the extract and metformin showed synergistic anti-diabetic activities. The implication of this is that, the co-use of Moringa oleifera and metformin can play a significant role in the management of the symptoms associated with diabetes.

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REFERENCES American Diabetic Association. (2000). Standards of medical care in diabetes. Diabetes Care. 33 (1), S11–S61

Dunn, J. S and McLetchie, N. G. B (1943) Experimental alloxan diabetes in the rat. Lancet.11 (245): 384–387.

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Fuglie, L. J., (1999). The Miracle Tree: Moringa oleifera: Natural nutrition of the tropics .Church world service, Dakar, 68. Gannong, W.F. (2006). Review of Medical Physiology, 21st Edition. California: Lange Medical Publications Ghosh, M. N. (2005). Fundamentals of Experimental Pharmacology. 3rd Edition. Calcutta: Hilton and Company. Pp 184. Guyton, A. C. & hall, J.E. (2006). Textbook of Medical Physiology. Philadelphia: W.B. Saunders Lorke, D. (1983). "A new Approach to Practical Acute Toxicity Testing." Archives of Toxicology .54(4): Pp 275– 287 NIH, author. National Institutes of Health Guide for the Care and Use of Laboratory Animals. 1985 Publication No. 85-23, revised 1985 Okon, U. A., Owo, D. U., Udokang, N. E., Udobang, J. A. and Ekpeyong, C. E.,(2012) Oral Administeration of Aqueous Leaf Extract of Ocimum gratisimum Ameliorates Polyphagia, Polydipsia and Weight- Loss in Streptozotocin- Induced Diabetic Rats. American Journal of Medicine and Medical Science. 2(3): 45–49

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Okwu, D. U., Antai, A.B., Udofia, K. H., Obembe, A. O., Obasi, K. O., Eteng, M. U. (2006). Vitamin C improves basal metabolic rate and lipid profile in alloxan-induced diabetes mellitus in rats. Journal of Bioscience. 31(5): 575– 570. Robert, W.C. (2001), Ultrasound imaging: Principles and applications in rodent research. International Laboratory Animal Research.42 (23), 233–247. Scarpello, J.H.B. and H.C. Howlett, 2008. Metformintherapy and clinical uses. Diab. Vasc. Dis. Res., 5:157–167. PMID: 18777488 Scoppola, A., Montecchi, F. R., Mezinger and G., Lala, A. (2001). Urinary mevalonate excreation rate in type 2 diabetes: Role of metabolic control. Antherosclerosis. 156 (2); 357–361.

Source of Support: NIL

Szkudelski, T. (2001). The mechanism of alloxan and streptozotocin action in beta cells of rat pancrease. Physiology Resource. 50(2): 536–546. Ugochukwu, N. H., Babady, N. E., Cobourne, M., Gasset, S. R. (2003). The Effect of Gangronema latifolium extracts on serum lipid profile and oxidative stress in hepatocytes of diabetic rats. Journal of Bioscience. 28 (1): 1–5. Unwin, N., Sobngwi, E., Alberti, K. G. M. M. (2001). Type 2 diabetes: The challenge of preventing a global epidemic. Diabetes International. 11(9); 4–8. Vasavda Krup, Joshi Vilaxana, Hegde Prakash L., Harini A. (2014), Ant-diabetic Herbs in Ayurveda: An Update, Global J Res. Med. Plants and Indigen. Med., Volume 3(12): 489–496.

Conflict of Interest: None Declared

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