1. INTRODUCTION:

Diabetes mellitus is a chronic metabolic disorder affecting millions of individual worldwide characterized by hyperglycemia due to inherited or acquired deficiency in the production of insulin in turn resulting in complications associated with neurological, cardiovascular, retinal and renal disorders. There are an estimated 2.85 billion adults with diabetes in 2010 and this number will continue to increase globally due to an aging population, growth of population size, urbanization, high prevalence of obesity and sedentary lifestyle.^1,2 The treatment with modern medicines such as insulin therapy, sulfonylureas, thiazolidinediones, biguanides, meglitimides, and etc., mostly associated with the side effects. To overcome the toxic effects caused by these drugs an alternative system of treatment is required. Therefore, it is crucial to search the necessary potential in herbal medicines.³Moreover, medicinal plant extracts have been used by indigenous people to treat various ailments, including infectious diseases⁴. In view of this, we investigated a medicinal plant which is claimed to possess anti-diabetic activity.

Crotolaria verrucosa commonly called as “Blueflower Rattlepod” belongs to the family Fabaceae is an erect much branched herb. This plant is widely distributed in South Asia, Australia.⁵

In India it is used ethno medically in the treatment of diarrhea, diabetes, leprosy, and migraine. The literature survey reveals that no reports were found on the anthelmintic activity of crude extract of Crotolaria verrucosa. ⁶Therefore the current study was performed to evaluate the in vitro anthelmintic activity of crude extract of Crotolaria verrucosa against alloxan induced diabetic albino rats.

2. MATERIALS AND METHODS:

2.1 Chemicals

Alloxan was purchased from Crescent Trading Company, Hyderabad. Glibenclamide was obtained as a gift sample from Arandy Laboratories Ltd, Hyderabad. All other chemical and reagents used were of analytical grade.

2.2 Preparation of plant extract

The dried powder of C. verrucosa whole plant was extracted with Ethanol (80%) by soxhlation process. The filtrates were concentrated at reduced pressure by Rotary Falsh Vaccum Evaporator.⁷

2.3 Qualitative Phytochemical Studies

In order to determine the presence of alkaloids, glycosides, flavones, tannins, terpenes, sterols, saponins, and sugars, a preliminary phytochemical study (color reactions) with various plant extracts was performed.^8,9

2.4 Quantitative screening

2.4.1 Determination of Alkaloid content^[10]

Alkaloids were quantitatively determined according to the method described by Harbone. 200 mL of 10% acetic acid in ethanol was added to 5 g of powdered sample and allowed to stand for about 4 hours. The filtrate was then concentrated to 1/4^th of its original volume. Further, concentrated ammonium hydroxide was added in drop by drop manner to the extract until the precipitation was complete. Then, the whole solution was allowed to settle and the obtained precipitates were washed with dilute ammonium hydroxide and filtered. Finally, the alkaloid residue was dried, weighed and expressed quantitatively.

2.4.2 Determination of Flavonoid content^[11,12]

The total flavonoid content was quantitatively determined according to the Dowd method. 5mL of 2% aluminium trichloride (AlCl₃) in methanol was mixed with equal volume of the extract solution (0.3mg/mL). The same solution was placed in a cuvette and absorption was read at 415 nm using Shimoda UV-Visible spectrophotometer. Prior, blank solution absorption was measured consisting of 5mL of the extract solution and 5mL of methanol without AlCl₃. The total flavonoid content was determined using a standard curve plotted with quercetin (1-100mg/L) as the standard. Total flavonoid content is expressed as quercetin equivalents (QE)/g of the extract.

2.4.3 Determination of Total Phenolic content^[13]

The total phenolic content was quantitatively determined according to the Folin-Ciocalteu method described by Cheung et al. and Singleton et al. Briefly, 0.02mL of extract of different concentrations (4, 8, 12, 16 and 20mg/ml) and control (methanol) were mixed with 1.58 ml of distilled water. Then, 0.1 ml of Folin-Ciocalteu’s phenol reagent was added to each test tube. After 3 min, 3ml of Na₂CO₃(2%) was added and the mixture was shaken on a shaker for about 2 hours at room temperature. Finally, absorbance was determined at 765 nm with a Shimoda UV-Visible spectrophotometer. All assays were conducted in triplicate and the results were averaged. A dose response linear regression was generated by using the pyrocatachol standard absorbance and the levels in the samples were expressed as pyrocatachol equivalents (mg of PAEs/g of extract).

2.4 Experimental Animals

Healthy albino Wistar rats (200–225 g) of either sex were housed in polypropylene cages lined with husk in standard environmental conditions. The rats were fed on a standard pellet diet ad libitum and had free access to water (Turner .A, 2009). The experiments were performed after approval of the protocol by the Institutional Animal Ethics Committee (IAEC) and were carried out in accordance with the current guidelines for the care of laboratory animals.

2.5 Experimental Design

Antidiabetic activity of C. verrucosa ethanolic extract was assessed in normal, and alloxan induced diabetic rats (Thirupathi Reddy et al., 2006). In all studies, the animals were fasted overnight for 16hrs with free access to water throughout the duration of experiment.

2.5.1 Acute Toxicity Studies

Acute toxicity of C. verrucosa ethanolic extract was performed on albino rats, according to OECD Guidelines 425. The first group was treated after fasting overnight with a oral dose of 1000 mg/ kg of body weight with the ethanolic extract of the plant “C. verrucosa”, suspended in 0.6 % Na CMC. And extracts were given in two different groups. And the animals were observed continuously for 2-3 hrs for general behavioral, and finally death after 24 hrs. There was no mortality and no signs of toxicity and the extracts were found to be safe at this dose level. A higher dose of 2000 mg/ kg body weight of the ethanolic extract of whole plant. For the assessment of all the biological activities, three dose levels (200 mg/ kg, 100 mg/ kg, 400 mg/ kg) were chosen.

2.5.2 Evaluation of extract in Alloxan- induced diabetic rats

Diabetes was induced in rats by injecting 120 mg/kg of Alloxan monohydrate intraperitoneally in 0.6% w/v CMC to overnight-fasted rats. After 72 h of injection, fasting blood glucose level was measured. The animals that did not develop more than 200 mg/dl glucose levels were rejected. The selected diabetic animals were divided into five groups (n = 6) and one more group of normal non-alloxanised animals was also added to the study. Group 1 was kept as normal control (non-alloxanised rats), which received a single dose of 0.5 ml/100 g of the vehicle; Group 2 was kept as negative control, alloxan induced and received a single dose of 0.5 ml/100 g of the vehicle; group 3, 4, and 5, diabetic induced were treated with ethanolic extract that exhibited antidiabetic activity at three dose levels (100mg/kg, 200mg/kg, 400mg/kg); Group 2 was kept as negative control, alloxan induced and received a single dose of 0.5 ml/100 g of the vehicle. The treatment was continued for 7 consecutive days (p.o). at the end of 7th day, the rats were fasted for 16h and blood parameters were determined.¹⁴The determination of blood glucose levels is done by tail tipping method using Accuchek-sensor glucometer. After 7 days blood is collected by Tail – Cut method to measure blood glucose levels.¹⁵

3. RESULTS AND DISCUSSIONS:

The percentage yields of ethanol extract were found to be 10% w/w respectively. Preliminary phytochemical analysis showed the presence of alkaloids, terpenoids, flavonoids, carbohydrates and glycosides. Quantitative determination was performed for alkaloids, flavonoids and phenols. Alkaloids content was determined according to Harborne method and the amount of total alkaloids content was found to be 8.26% (w/w). Flavonoids are large class of benzo-pyrone derivatives and their total content was estimated by Dowd method. The total flavonoid content was found to be 35.1mg quercetin equivalent. Total phenolic content assay was performed as described by Slinkard- Singleton method and found to be 17.8mg pyrocatachol equivalent.

Table 1: Antidiabetic Activity of ethanolic extract of C.verrucosa

	1^st Day	3^rd Day	7^th Day
Normal Control	106.33 + 3.84	109.83 + 4.56	112.7 + 2.16
Diabetic Control	252.08 + 3.51	249.002 + 4.21	248.67 + 1.065
Ethanolic Extract (100 mg/Kg)	251.83 + 3.15	231.50 + 4.023*	212.17 + 4.70 **
Ethanolic Extract (200 mg/Kg)	243.02 + 2.68 *	212.17 + 3.416 **	182.50 + 1.035 **
Ethanolic Extract (400 mg/Kg)	248.50 + 1.256**	198.004 + 2.64***	139.64 + 2.49 ***
Standard(Glibenclamide 5 mg/kg)	211+ 3.114**	151.17+ 4.325***	117.864 + 3.169***

*P <0.05, **P< 0.01, ***P <0.001 was considered significant comparing to the Diabetic controlled group.

The present study was undertaken to evaluate the antidiabetic activity of ethanolic extract of C. verrucosa. Alloxan induced hyperglycemia in rodents is considered to be a good preliminary screening model and is widely used. Alloxan causes diabetes through its ability to destroy the insulin-producing beta cells of the pancreas. In vitro studies have shown that alloxan is selectively toxic to pancreatic beta cells, leading to the induction of cell necrosis. The cytotoxic action of alloxan is mediated by reactive oxygen species, with a simultaneous massive increase in cytosolic calcium concentration, leading to a rapid destruction of beta cells.

Experimental studies revels that the ethanolic extract from C. verrucosa whole plant (400 mg/kg) orally administered for 7 days produced a significant decrease in the blood glucose level in the model of alloxan-induced diabetes in rats. It also proves the traditional claim with regard to C. verrucosa for its anti-diabetic activity. Thus, the significant antidiabetic effect of C. verrucosa ethanolic extract could be due to the presence of various phytoconstituents detected in the phytochemical screening which alone or in synergism can impart therapeutic effect. Majorly, due to the presence of alkaloids in the crude extract might be the reason for the effective hypoglycemic activity of the crude extract of C. verrucosa.

4. CONCLUSION:

The present investigation clearly reveals the indigenous system of medicine claim of the importance of C. verrucosa as an economical antidiabetic agent. The plant bears a potential for further research to isolate anti-diabetic principle. Further extended research may include isolating and characterizing the active constituent present in the crude extract and also to investigate its mechanism of action.

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Received on 10.10.2013 Modified on 25.10.2013

Research J. Pharm. and Tech. 7(1): Jan. 2014; Page 20-22