Hypoglycemic Effect of Kyllinga triceps in STZ Induced Diabetic Rats

Type 2 diabetes mellitus, also known as noninsulin dependent diabetes mellitus, develops in middle or later life and affects 2-6% of adults in most Western societies [1]. Diabetes mellitus has been recognized as a growing world-wide epidemic by many health’s advocacy groups including the World Health Organization (WHO) [2]. The WHO has estimated that diabetes will be one of the worlds leading causes of death and disability within the next quarter century. The statistics are alarming; 30 million people were diagnosed with diabetes world-wide in 1985, by 1995 the number had risen to 135 million, and at the current rate there will be 300 million by the year 2025 as predicted by the WHO [3].


Introduction
Type 2 diabetes mellitus, also known as noninsulin dependent diabetes mellitus, develops in middle or later life and affects 2-6% of adults in most Western societies [1]. Diabetes mellitus has been recognized as a growing world-wide epidemic by many health's advocacy groups including the World Health Organization (WHO) [2]. The WHO has estimated that diabetes will be one of the worlds leading causes of death and disability within the next quarter century. The statistics are alarming; 30 million people were diagnosed with diabetes world-wide in 1985, by 1995 the number had risen to 135 million, and at the current rate there will be 300 million by the year 2025 as predicted by the WHO [3].
The limitations of currently-available oral antidiabetic agents either in terms of efficacy/safety coupled with the emergence of the disease into a global epidemic have encouraged a concerted effort to discover drugs that can manage type 2 diabetes more efficiently [4]. Also, with increasing incidence of diabetes mellitus in rural population throughout the world and due to adverse effects of synthetic medicine, there is a clear need for development of indigenous, inexpensive botanical sources for anti-diabetic crude or purified drugs [5].
Medicinal plants use has turned out to be an alternative method for treatment of disease such as diabetes mellitus. Literature reports more than 800 plants have been utilized as empirical treatment for diabetes. One tenth of them have been characterized as hypoglycemic plants with active compounds such as mucilage gum, glycans, flavonoids, triterpenes and alkaloids [6].
Research conducted in last few decades on plants mentioned in ancient literature or used traditionally for diabetes has shown antidiabetic property.
The recommendation of the WHO committee on diabetes encouraging research on hypoglycemic agents of plant origin used in traditional medicine has greatly motivated research in this area [7]. Currently several hundred plants have been reported to have beneficial effect in treatment of diabetes [8][9][10].
Kyllinga triceps (Family: Cyperaceae) is common throughout India found specially in Northwestern India, Gujarat, Rajasthan and South India. It is also known as Nirvishaa, Nirbishi and Mustaa. Its root is used as diuretic (in polyuria), demulcent, antidermatosis and antidiabetic [11]. Decoction of the plant Kyllinga triceps showed antipyretic activity [12].
Different ethnic communities of Thoubal district of Manipur used to treat diabetes by 54 plant species. Boiled plant extract is used to treat diabetes by village priests cum doctors (Locally called Maida or Maidi) [13]. Jain et al reported that Kyllinga triceps is used to treat diabetes by local folklore and tribals of different districts of Madhya Pradesh and Chattisgarh [14].
In the present study we have selected Kyllinga triceps to examine its hypoglycemic activity because traditional documents claimed that to be an antidiabetic and used as folkloric medicine in different region of India.

Collection of plants
The plant was collected and authenticated by the comparison with the herbarium and voucher specimen (Voucher specimen No.-97767) was lodged in the department herbarium of National Botanical Research Institute, Lucknow.

Preparation of plant extract
Plants were shade dried, milled, and ground into coarse powder with the help of a mixer. The powdered material was subjected to cold maceration using sufficient quantity of ethanol and distilled water (1: 1) for 10 days with intermittent shaking in a round bottom flask. On 10 th day, it was strained and marcs were pressed. The pressed liquid was added to the strained liquid and the combined liquid was clarified by filtration and the filtrate was subjected to distillation at temperature 60°C for removing ethanol and water. After distillation, the semi solid obtained was kept for experiment.

Experimental animals
Healthy wistar albino rats were used for the study. They were maintained at standard laboratory condition and fed with commercial pellet diet and water ad libitum. The animals were acclimatized to laboratory condition for one week before commencement of experiment.

Acute oral toxicity study
In present study observation of acute toxicity study carried according to OECD guideline 423, none of the rats showed observable signs of toxicity upon single administration of kyllinga extract (2 g/ kg, p.o.) on day one. Observations twice daily for 14 days also did not reveal any drug related observable changes. The study was repeated with another set of animals for 14 days and no signs of toxicity were observed.

Induction of diabetes
Rats were fasted overnight before inducing diabetes with streptozotocin. The rats were given an intraperitoneal injection of streptozotocin (50 mg\kg) freshly prepared in 0.1M sodium citrate buffer. The diabetic state was confirmed 48 h after streptozotocin injection. Threshold value of fasting blood glucose was taken as > 200 mg\dl.
Control and diabetic rats were weighed matched for body weight and divided into following group consisting five animals each. The acute study involved estimation of blood glucose levels at 0, 2, 4, 6 and 24 hrs after administration of Glibenclamide and KTE.
The sub acute study involved daily administration of Glibenclamide and KTE for 28 days and blood glucose levels were estimated on 0, 7,14,21 and 28 days.

Blood glucose estimation
Blood samples were obtained through puncture tail vein and collected blood samples were analyzed by Accu-Check Glucometer. Blood glucose levels were expressed in terms of mg\dl.

Body weight determination
Weight of rats was recorded during the study period of 28 days. Percentage (%) change in body weight was calculated and tabulated.

Results
In acute toxicity studies of Kyllinga extract given by oral route did not show any mortality and toxic effects up to the dose of 2000 mg\kg body weight. So 1\20 th and 1\10 th of safe dose is used for the experiments.
STZ-induced diabetic rats exhibiting persistent hyperglycemia (Blood Glucose>200 mg\dl) were selected for assessing the effect of KTE. Table 1 shows the blood glucose level at various time intervals to observe acute effect of two different doses of KTE and Glibenclamide. Data showed significant (p < 0.05) difference in blood glucose of all the treatments compared to diabetic control. Glibenclamide showed highest % decrease (45.7%) followed by KTE (200 mg/kg) (40.0%). KTE (100 mg/kg) showed lowest % decrease (36.4%) in blood glucose level. It is clear from the data that KTE (200 mg/kg) showed more hypoglycemic activity than KTE (100 mg/kg) in acute study.
To observe long term effect, sub acute study of 28 days was done. Table 2 shows the results of this study. Glibenclamide and KTE (200 mg/ kg) showed highly significant (p < 0.01) hypoglycemic effect compared to diabetic control while KTE (100 mg/kg) also showed significant (P < 0.05) hypoglycemic activity. KTE (200 mg/kg) showed less % reduction (47.2%) than Glibenclamide (55.4%). In sub acute study also KTE (200mg/kg) is more effective than KTE (100 mg/kg). All values are expressed as mean ± S.E.M (n=5). *P<0.05 as compared to diabetic control. One-way ANOVA followed by Dunnet multiple comparison test    Table 3 shows the body weight of rats at different time intervals during the study. The % change in body weight of all the treatments was calculated. Non diabetic control showed 3.4% increase in body weight while diabetic control showed 10.1% weight decrease .Glibenclamide, KTE (200 mg/kg) and KTE (100 mg/kg) showed 6.4, 5.0 and 5.5% increase in body weight respectively. All the treatments showed more % increase in body weight than non diabetic control. KTE (200 mg/ kg) and KTE (100 mg/kg) showed nearly similar effect on body weight. So KTE not only showed significant hypoglycemic activity but also effective in preventing the weight loss due to diabetes.

Discussion
Streptozotocin is well known for its selective pancreatic islets β cell cytotoxicity and has extensively used to induce diabetes in experimental rat model. It interferes with cellular metabolic oxidative mechanism. Glibenclamide is often used as a standard antidiabetic drug in STZ induced moderate diabetes to compare the efficacy of variety of hypoglycemic compounds or plant extracts. The present study was conducted to assess the hypoglycemic activity of Kyllinga extract in STZ induced diabetic rats. Results shows both the doses of Kyllinga extract significantly improving the body weight and controlling the blood glucose level in diabetic rats. 200 mg\kg dose is more effective than 100 mg\kg and comparable to Glibenclamide. Result of the study scientifically validates the folkloric use of Kyllinga triceps in different ethnic communities of Manipur, Madhya Pradesh and Chhattisgarh.
It may be concluded that Kyllinga extract possess hypoglycemic activities and the plant extract may be used as hypoglycemic agent. The plants should be considered as an excellent candidature for further studies on diabetes mellitus.