Centratherum anthelminticum minimizes the risk of insulin resistance in fructose-induced type 2 diabetes

Article history: Received on: 20/02/2015 Revised on: 23/03/2015 Accepted on: 18/04/2015 Available online: 15/05/2015 The study was focused on investigating the effect of Centratherum anthelminticum ethanolic seed extract in fructose-induced type 2 insulin resistance diabetic rabbits after determining its qualitative analysis, acute toxicity, and effect on glucose tolerance. The phytochemical analysis revealed the presence of alkaloids, flavonoids, tanins, gallotannins, phlabotanins, phenols resins, saponins, and steroids. The same extract was found completely harmless up to 3000 mg/kg by showing no sign of acute toxicity in experimental rabbits. In oral glucose tolerance test, all doses (200-600mg/kg) of seed extract effectively produced percent reduction in blood glucose levels at 60 and 120 min. However, its high doses (400 and 600mg/kg) efficiently induced percent reduction (-10 to 11.9%) in post-prandial blood glucose level after 30 min as compared to diabetic control group. Similarly, the oral administration of same three doses (200-600 mg/kg) of extract for 14 days consecutively were found effective in decreasing blood glucose and serum total cholesterol, triglycerides, low& very low-density lipoproteins while increasing high-density lipoprotein in fructose-induced type 2 diabetic test rabbits. In addition, fasting insulin resistance index (FIRI) was also decreased by normalizing insulin levels in these test groups as compared to fructose-induced type 2 diabetic control group (P<0.05). Therefore it is concluded that ESEt of C. anthelminticum would be effective in improving hyperglycaemia, hypertriglyceridemia, hypercholesterolemia, hyperinsulinemia in fructose-induced type 2 diabetic rabbits by either decreasing insulin resistance or inhibiting fructose absorption in intestine.


INTRODUCTION
Fructose is an essential sweet component found in honey, fruits and corn syrup (Kumar et al., 2014).Over the past 30 years, increase in worldwide consumption of artificial sweeteners like corn syrup also increases the risk of various noncommunicable diseases including obesity, diabetes, hypertension and kidney diseases (Johnson et al., 2007, Segal et al., 2007).Literature described that high consumption of dietary fructose induced insulin resistance, postprandial hyperglycaemia and blood pressure in humans more than glucose (Brown et al., 2008, Stanhope et al., 2008).Insulin resistance is one of the major characteristics of type 2 diabetes/non-insulin dependent diabetes which usually associated with obesity that affects 90-95% of cases (Tamrakar et al., 2011).The impaired carbohydrate utilization in diabetes accelerates lipolysis causing hypertri-.glyceridemia that further increases the risk of premature arteriosclerosis (Gracheva et al., 2014).The estimated rate of deaths in people with diabetes is about 70-80% and is mainly due to vascular diseases (Segal et al., 2007).Therefore, for the treatment of diabetes, the potential drug would not only effective in lowering blood glucose but also to prevent other complications (Upendra et al., 2010).The available synthetic antidiabetic medicines produce various side effects including hypoglycaemic coma, hepatorenal disturbances and are unsafe during pregnancy (Ouyang et al., 2008).
Beside conventional medicines of diabetes, the high percentage of utilization of medicinal plants as hypoglycaemic and antihyperlipidemic agents was reported in rural areas of developing countries just to avoid insulin injections or unavailability of basic medical facilities (Paydar et al., 2013).Medicinal plants are famous in the treatment of many ailments including diabetes as these are rich in phytoconstituents viz., glycosides, alkaloids, terpenoids, flavonoids, carotenoids, etc, and have few or no side effects (Singh, 2011;Galani and Panchal, 2014).
Centratherum anthelminticum (L.) Kuntze or Vernonia anthelminticum belongs to the family Asteraceae.The seeds of this plant called as black cumin/wild cumin and in Urdu it is called as kali zeeri.
The seeds of this plant have been reported for many pharmacological activities like anti-inflammatory, antibacterial, larvicidal, antiviral, antifungal, anticancer, anthelmintic, antioxidant, analgesic, antipyretic, anti-inflammatory, diuretic, wound healing and antidiabetic effects (Pitchai et al., 2010).On the basis of its significant antidiabetic effects reported in alloxanand streptozotocin-induced diabetic rat models (Aguilara et al., 1998;Fatima et al., 2010), this study was aimed to investigate the effect of ethanolic seed extract of C. anthelminticum in fructoseinduced type 2 insulin resistance diabetic rabbits.In addition, the phytochemical screening, acute toxicity and effect of ESEt of C. anthelminticum on oral glucose tolerance were investigated.

Preparation of ethanolic seed extract
Seeds of C. anthelminticum were purchased, authenticated and kept (KU/BCH/SAQ/02) in Department of Biochemistry, University of Karachi, Karachi-75270, Pakistan.The dried seeds were grinded and 40 g of seed powder was soaked in ethanol (1L; 95%) for overnight at room temperature.Then filtered through Whatmann No 42 (125mm) filter paper twice and concentrated at 40⁰C till dryness in a rotary vacuum evaporator.Finally obtained brown residue termed as ethanolic seed extract (ESEt) that was stored in refrigerator below 10ºC until used (Qureshi et al., 2009).

Induction of fructose-induced insulin resistance type 2 diabetes
It was done by giving oral administration of 30 ml of 35% fructose solution once in a day in overnight fasted rabbits for 14 connective days (Neeharika et al., 2012).

Positive control
Commercially available Pioglitazone (Zolid, 15mg/kg) of Getz Pharma, Pakistan Ltd. was used as positive control in the present study.

Acute toxicity study
Acute toxicity was measured by oral administration of ESEt from 100-3000mg/kg.For this, overnight fasting rabbits were randomly divided into different groups (4/group).Doses of ESEt was individually administered orally to the rabbits of their respective test groups, whereas distilled water (1ml/kg) were given orally to rabbits in control group.The rabbits were then allowed free access to food and water.Acute toxicity was observed over a period of 12-24 hours in terms of behavioural change (sedative or not), mortality rate and other side effects such as itching, ruffled hair, clumping together, etc.

Oral glucose tolerance test (OGTT)
The experimental rabbits in overnight fasted state were randomly divided into 5 groups (4 / group), as follows  Group I: Control group: treated with distilled water 1 ml/kg  Group II: Negative control treated with 0.05% DMSO 1 ml/kg  Group III: Test group: treated with ESEt 200mg/kg  Group IV: Test group: treated with ESEt 400mg/kg  Group V: Test group: treated with ESEt 600mg/kg Each group, after receiving its respective treatment orally, immediately administrated with glucose load 2 g/kg from same route.Glucometer (Optimum Xceed, Diabetes Monitoring System by Abbot) was used to monitor blood glucose levels at 0, 30, 60, and 120 minutes in rabbits of each group by pricking the ear vein.Finally, percent glycemic change between control and test groups was calculated by using the following formula x 100 Where G o = mean blood glucose level of control group at different time intervals G x = mean blood glucose levels of each test group at different time intervals respective to control

Effect of ESEt in fructose-induced insulin resistance type 2 diabetic rabbits
Albino rabbits weighing from 1-1.2 kg were purchased from the local supplier of Karachi University and kept in animal house of university according to the international guidelines of animal handling.The standard laboratory diet was given to these rabbits along with free access to water ad libitum and divided them in control and diabetic groups on the basis of treatments (Fig. 1).All treatments were given orally to experimental rabbits in overnight fasted state for consecutive 14 days (once in a day).
Rabbits were sacrificed on 15 day after completing 14 doses to collect blood, serum and liver tissues for analysing the biochemical parameters.Prior to this, fasting blood glucose (FBG) levels were also determined on initial (0) and final ( 14) day with the help of glucometer from their ear vein.

Determination of biochemical parameters
Commercially available enzymatic kits (Randox, United

Statistical analysis
The results are expressed as mean ± SEM (Standard Error Mean) and considered significant at p<0.05 when data were examined by one-way ANOVA followed by LSD (least significant difference) test (SPSS version 18).

Acute toxicity and OGTT
No acute toxicity was found by ESEt up to 3000 mg/kg in the form of sedation, itching and mortality in experimental rabbits.
In OGTT, ESEt 200mg/kg after 30 min produced significant (P<0.05)hypoglycaemia as compared to control.A significant reduction in the concentration of blood glucose was found in all doses of ESEt from 200-600 mg/kg (P<0.05) after 120 min.The values of percent glycaemic change after 60 and 120 min of ESEt 200-600 mg/kg administration were -12.35, −24.58, −18.53% and −39.88, −43.25, −30.89% respectively as compared to control group.ESEt 200 and 400mg/kg decreased blood glucose level after 0 min as −2.79 and −1.95% whereas the same extract in doses of 400 and 600 mg/kg showed percent glycemic change about −11.04 and −10.9% respectively after 30 min as compared to control group (Table 1).

Effect of ESEt on FBG and biochemical parameters in fructose-induced insulin resistance diabetic rabbits
ESEt in doses of 200, 400 and 600 mg showed significant decrease up to 115, 103 and 102 mg/dl respectively (P<0.05) in FBG values as compared to diabetic and positive control groups (Fig. 2).2).A significant decrease (P<0.05) in FIRI was also observed in test groups treated with ESEt (200-600mg/kg) by normalizing the insulin level as compared to diabetic control group that indicated increased insulin resistance (Fig. 3).

DISCUSSION
Nature has provided effective balancers in the form of medicinal plants that regulate important functions and provide various nutrients that body fails to receive due to poor diet.The herbal medicines are considered safe than conventional medications and providing a natural alternative treatment for many health problems including type 2 diabetes (Medagama et al., 2014).
ESEt was found non-toxic as there was no sedation, itching and mortality seen in experimental rabbits by using doses of same extract up to 3000 mg/kg.Therefore, according to our pervious study, three doses of ESEt viz., 200, 400 and 600 mg/kg which showed significant antihyperlipidemic activity in high-fat induced hyperlipidemic rabbits (Lateef and Qureshi, 2013) were selected for further study.
Oral glucose tolerance test is used to check whether the individual is diabetic, insulin resistant and suffering from the disorders of carbohydrate metabolism (Li et al., 2014).The current study showed that ESEt of C. anthelminticum (200-600mg/kg) was effective in decreasing blood glucose level after 60 and 120 min of glucose load (2g/kg) in their respective test groups.However, the high doses (400 and 600mg/kg) of extract found more efficient in reducing postprandial blood glucose level at 30 min after glucose load.The hypoglycaemic activity of ESEt observed in OGTT might be due to its extra-pancreatic effect by producing delay in glucose absorption at intestinal level or enhancing glucose uptake in muscle and liver tissues in order to increase glucose tolerance and in that way stimulating glycolysis and glycogenesis (Qureshi et al., 2009).Several phytoconstituents including flavonoids, alkaloids, phenols, saponins, steroids which were detected in ESEt could also be responsible for the hypoglycaemic activity of ESEt as it has been reported earlier that phytochemicals have protective or disease preventive properties (Hashim et al., 2013).High intake of fructose induced weight gain that gradually induced insulin resistance and contributed in the progress of type 2 diabetes.
Fructose is a lipogenic agent as it is reported to involve in accelerating the synthesis of triglycerides (TGs) in liver after entering in hepatocytes through insulin-independent glucose transporters (GLUT-5) thereby inducing hypertriglyceridemia (Armato et al., 2015).This increased level of TGs could be the reason of insulin resistance by masking the insulin receptors on target cells especially muscles, adipocytes and hepatocytes, thus impaired carbohydrate utilization, induced hyperglycaemia and hyperinsulinemia.This situation is accompanied with high LDL-c, VLDL-c levels and low HDL-c levels (Hsiehet al., 2013).The same was observed in fructose-induced diabetic control group in the present study that showed hyperglycaemia, hypertriglyceridemia, hypercholesterolemia, and hyperinsulinemia.On contrary, three of the doses of ESEt (200, 400, & 600 mg/kg) found effective (P<0.05) in decreasing in serum TC, TG, LDL-c, VLDL-c levels and increasing HDL-c levels in their respective test groups.Interestingly, all doses of extract were also found to normalize insulin levels in their respective test groups thereby showing decrease in insulin resistance which also confirmed by observing low values of FIRI in test groups whereas diabetic control group showed high values of same index.Pioglitazone (15mg/kg) was also found effective in reducing insulin resistance in positive control group (P<0.05).Previously, methanolic seed extract of C.anthelminticum has been reported to have insulin secretary effect in type 2 diabetic rats (Arya et al., 2012).Thus, our study would add new concept in the antidiabetic action of C.anthelminticum that it could reduce insulin resistance by normalizing the levels of serum insulin and inhibiting triglyceride synthesis thus enhance insulin-receptor sensitivity and improve carbohydrate utilization in body cells.

CONCLUSION
The present findings conclude that hypoglycaemic effect of ESEt of C.anthelminticum in fructose-induced type 2 diabetic rabbits may be due to correcting insulin resistance by inhibiting either fructose absorption in intestine or triglycerides synthesis in liver.Thereby enhancing receptor sensitivity for insulin and correcting carbohydrate utilization in body cells.

Fig. 2 :
Fig. 2: Effect of ESEt on FBG (mg/dl) levels in fructose induced diabetic rabbits.Each bar represents the mean ± SEM (n=4).*p <0.05, when compared with Diabetic and Positive control groups.All doses of ESEt (200-600 mg/kg) showed significant decrease (P<0.05) in the values of serum TC, TG, LDL-c, VLDLc, and significant increase (P<0.05) in the values of HDL-c in test groups as compared to diabetic and positive control groups (Table2).A significant decrease (P<0.05) in FIRI was also observed in test groups treated with ESEt (200-600mg/kg) by normalizing the insulin level as compared to diabetic control group that indicated increased insulin resistance (Fig.3).

Fig. 3 :
Fig. 3: Effect of ESEt on FIRI in fructose induced type 2 diabetic rabbits.Each bar represents the mean ± SEM (n=4).*p <0.05, when compared with Diabetic and Positive control groups.

Table 1 :
Effect of ESEt on Glucose Tolerance in Rabbits.p<0.05, when compared with control group, b p<0.05, when compared with Negative control group Values in parenthesis show percent blood glucose decrease (-) /increase (+). a

Table 2 :
Effect of ESEt on Biochemical Parameters (mg/dl).p<0.05, when compared with Diabetic and Positive control group, + p<0.05, when compared between test groups. *