INTRODUCTION:

Diabetes mellitus is one of the most common chronic diseases in nearly all countries. There are an estimated 285 billion adults with diabetes in 2010; this number will continue to increase globally due to an aging population, growth of population size, urbanization and high prevalence of obesity and sedentary lifestyle¹. This disease is characterized by hyperglycemia resulting from reduced (or absent) secretion of insulin, often coupled with reduced sensitivity to its action (insulin resistance)². The increased plasma free radicals observed in diabetes mellitus may impair insulin action, thus contributing to the generation of hyperglycemia³. Defects in GLUT- 4 and GLUT-1 may explain the insulin-resistant glucose transport. Skeletal muscle is a major tissue for blood glucose utilization and a primary target tissue for insulin action. Insulin increases glucose uptake in skeletal muscle by increasing functional glucose transport molecules (GLUT-4) in the plasma membrane. Glucose transport in skeletal muscle can also be stimulated by contractile activity⁴. Free radical impairs insulin-stimulated GLUT-4 translocation and exerts an inhibitory effect on muscle contractility that is major pathological feature of diabetes^5,6,7.

The estimation of glucose content in rat hemi-diaphragm is a commonly employed and reliable method for in vitro study of peripheral uptake of glucose⁸.

Cayratia trifolia (L.) Domin is found in India from Jammu and Rajasthan to Assam, Tripura and West Bengal, extending into peninsular India, up to 600m⁹. In south-west region of Rajasthan, it is easily found in Sitamata wildlife sanctuary of Chittorgarh and Udaipur districts¹⁰. It is commonly known as amal-bel and ramchana in hindi and locally known as Khhata nimba and belongs to the family Vitaceae^9,11. It is a perennial climbing or spreading herb or under shrub with young and pubescent shoot. Leaves are 3-foliolate, long petioled; leaflets are ovate or ovate-lanceolate, with slender, leaf-opposed tendrils. Flowers are greenish-white in branched divaricate cymes. Fruits are turbinate, fleshy and 2-4 seeded^12,13,14. The stem, leaves and root contain hydrocyanic acid. Its leaves also contain delphinidin, cyanidin and yield yellow waxy oil and sterols⁹. The extract of tuber of this plant along with the infusion of its seeds is given orally to diabetic patients to check the sugar level of blood¹¹.

In view of alleged antidiabetic potential of C. trifolia, we have investigated the hypoglycaemic effect of extracts of root on isolated rat hemi-diaphragm by glucose uptake method.

MATERIALS AND METHODS:

Plant material

Cayratia trifolia (L.) Domin. was collected from Udaipur district of Rajasthan and identified by Dr. P.J. Parmar, Joint Director, Botanical Survey of India, Arid Zone Regional Centre, Jodhpur-342 008 and a voucher specimen was deposited in the college for future reference.

Preparation of extracts

The roots were cleaned and dried under shade at room temperature. After drying, roots were subjected to size reduction to a coarse powder by using dry grinder. This powder was packed into soxhlet apparatus and extracted successively with petroleum ether (60-80˚C), chloroform, ethyl acetate and ethanol. All the extracts were dried at 45˚C in hot air oven till solid mass was obtained and were stored in airtight containers in refrigerator below 10˚C.

Preliminary phytochemical screening

Preliminary phytochemical investigations were carried out that showed the presence of flavanoids in the ethanolic extract. Therefore, this extract was further used for the animal study.

Animals

Healthy adult albino rats of Wistar strain weighing 150-200 g were obtained from Animal House, Lachoo Memorial College of Science & Technology, Pharmacy Wing, Jodhpur. The animal house was well ventilated and animals had 12 ± 1 hour and day and night schedule with temperature between 15-20 ± 5˚C. The animals were housed in standard polypropylene hygienic cages (three animals per cage). The animals were fed with standard rat pellet feed. The current work was carried our after approval by Institutional Animal Ethical Committee.

Glucose uptake by isolated rat hemi-diaphragm

Glucose uptake by rat hemi-diaphragm was estimated by the methods described by Walaas¹⁵ with some modification. Albino rats of either sex weighing between 160-180 gm were selected. The animals were maintained on a standard pellet diet (water ad libitum) and fasted overnight. The animals were sacrificed by decapitation and diaphragms were dissected out quickly with minimal trauma and divided into two halves. The hemi diaphragms were then rinsed in cold Tyrode solution (without glucose) to remove any blood clots and were placed in small culture tubes containing 2 ml Tyrode solution with 2% glucose and incubated for 30 minutes at 37˚C in an atmosphere of 100 % O₂ with shaking. Four sets containing five graduated test tubes (n=5) each, were taken as follows:

Group 1: Served as control which contained Tyrode sol. with 2 % glucose.

Group 2: Contained Tyrode sol. with 2 % glucose + Insulin (0.25 IU/ml)

Group 3: Contained Tyrode sol. with 2 % glucose + Root extract (25 mg/ml)

Group 4: Contained Tyrode sol. with 2 % glucose + Root extract (25 mg/ml) + Insulin (0.25 IU/ml)

Two hemi-diaphragms from the same animal were not used for the same set of experiment. Following incubation, the hemi-diaphragms were taken out and weighed. The glucose content of the incubated medium was measured by GOD-POD method. The uptake of glucose was calculated in mg/g of moist tissue/ 30 min. Glucose uptake per gram of tissue was calculated as the difference between the initial and final glucose content in the incubated medium.

The data obtained were statically analyzed by one way ANOVA and expressed as mean ± S.E.M. followed by Dunnet’s t-test using computerized Graph Pad InStat version 3.06, Graph pad software, U.S.A.

RESULTS:

The estimation of glucose content in rat hemi-diaphragm was employed for in vitro study of peripheral uptake of glucose. The effect of ethanolic extract of C. trifolia on glucose uptake by isolated rat-hemi diaphragm is illustrated in Table 1 and Figure 1. Ethanolic extract enhanced the uptake of glucose by isolated rat hemi-diaphragm significantly (p<0.05). Administration of ethanolic extract and insulin together was found to be more effective than the ethanolic extract and insulin were given separately (p<0.05).

Table 1: Effect of root extract of C. trifolia on Glucose uptake by isolated rat hemi-diaphragm

S. No.	Incubation medium	Glucose uptake (mg/g/30 min.)
I	Tyrode sol. with 2 % glucose	03.46 ± 0.21
II	Tyrode sol. with 2 % glucose + Insulin (0.25 IU/ml)	11.27 ± 0.40*
III	Tyrode sol. with 2 % glucose + Root extract (25 mg/ml)	08.76 ± 0.26*
IV	Tyrode sol. with 2 % glucose + Root extract (25 mg/ml) + Insulin (0.25 IU/ml)	16.16 ± 0.19*

Each value represents the mean ± SEM (n = 5), *P < 0.05 vs Control (ANOVA followed by Dunnet’s t-test)

DISCUSSION:

Conventional antidiabetic agents can affect several pathways of glucose metabolism such as insulin secretion, glucose uptake by target organs as well as nutrient absorption. The experiment focused on exploring the competence of ethanol extract of C. trifolia for the correction of diabetes to substantiate folklore claim. Results of administration of ethanolic extract and insulin together showed synergistic effect between C. trifolia extract and insulin. Results indicated that C. trifolia possess significant hypoglycaemic potential and hence it could be used as a source of active principle for treating diabetes.

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Received on 07.04.2012 Modified on 26.04.2012

Research J. Pharm. and Tech. 5(5): May2012; Page 691-693