Diabetic Mellitus (Hyperglycemia) is an endocrine disease and not a single disease which is a group of chronic metabolic or heterogeneous affliction due to the irregular secretions of insulin and action of insulin or both. Absence or reduced insulin in turn leads to abnormal high blood sugar level and glucose intolerance [1-2]. The Indian subcontinent has many natural remedies like Ayurveda,Yunani and Siddha. Based on these systems we can able to find new lead molecules upon further research may lead to complete drug. Positive results from clinical trials of these remedies require further investigations along with extensive clinical trials. Most of the plant compounds use as medicine in different diseases is secondary metabolites; they have no role in plant metabolism but has a significant role in defective mechanism of plant. Basic metabolic process of these compounds is almost similar in plants and animals[3-5]. Metformin is an oral antidiabetic drug in the biguanide class. It is the first-line drug of choice for the treatment of type 2 diabetes, in particular, in overweight and obese people and those with normal kidney function. Metformin works by suppressing glucose production by the liver. Metformin is the only anti-diabetic drug that has been conclusively shown to prevent the cardiovascular complications of diabetes. It helps reduce LDL cholesterol and triglyceride levels, and is not associated with weight gain. There is scope for the potential herb-interactions between Capsicum annuum and Metformin. This can cause few adverse reactions as a result, it precipitates potentially life-threatening effects. Hence, the study  need to be subjected to pharmacological studies in order to discover their effect on the patients who are taking the treatment with synthetic drugs.

MATERIALS AND METHODS

PLANT MATERIALS

Preparation, authentication and evaluation of Capsicum annuum Linn.

The dried fruits of Capsicum annuum Linn were procured from local market. The fruits were coarsely powdered and extracted in a Soxhlet extractor using ethanol. The extract was dried on a rotary evaporator to get a thick reddish brown oleoresin containing capsaicinoids. The oleoresin was analysed to quantitate the capsaicin content.

A stock solution of standard capsaicin (1mg/ml) was prepared and it was diluted to prepare a working standard of 100?g/ml. The calibration curve for capscaicin was prepared in the range of 20-100?g/ml. The linearity response for capsaicin was assessed in the range of 0.2 ?g/spot to 1ug/spot in terms of slope, intercept and correlation coefficient values. HPTLC was performed on precoated silica gel G plates in the solvent system Toluene: Ethylacetate (7:3) and was scanned at 280nm. A 1:50 dilution of the oleoresin was carried out and 10?l of the oleoresin was co chromatographed with standard capsaicin to determine the content of capsaicin in the oleoresin sample.[6]

DRUGS AND CHEMICALS

Adult male wistar-rats weight between 150±20grams were used in this Experimental study. These animals were acclimatized to standard laboratory’s conditions of suitable temperature (27oC + 1oC) and maintained on 12:12 hours light: dark cycle in animal’s house. They were maintained in elevated rat’s wire cages and provided with regular rat’s chow (Standard pellets contains diet), distilled water ad-libitum for 14 days. These experimental protocols were in conducted according with IAEC/ CPCSEA.

Bio analysis of Metformin in rat plasma

Chromatographic condition

A Shimadzu LC-6AT with SPD-10A detector was used for HPLC analysis. The reagents used for preparation of mobile phase were of HPLC grade. High performance liquid chromatographic method was developed using phenomenex C 18 ODS (5 ?) 250 x 4.60 mm column, mobile phase selected for this method contains acetonitrile, aqueous sodium di hydrogen phosphate buffer (40:60) pH adjusted to 6 with o-phosphoric acid which was filtered through 0.2 ? membrane filter. Flow rate employed was 1.0ml/ min. Detection of eluent was carried out at 234 nm and separation was carried out at ambient temperature

Extraction procedure:

Preparation of standard stock solution:

2 ug/ml solution of Metformin was prepared by dissolving in methanol-water 1:1 ratio.

Preparation of plasma stock solution:

Plasma stock solution was prepared by diluting 50 ul of the above standard stock solution with 950 ul of Plasma.

Extraction of Metformin from plasma:

To the plasma stock solution equal volume of acetonitrile was added for deproteinization and vortex for 10 mins followed by centrifugation at 10000nm for 10 mins. The supernatant was collected was filter and subjected to analysis at 234 nm by HPLC. A 20 microlitre sample was injected in HPLC for estimation of Metformin.

Retention Time of Metformin:

Retention time observed was at 3.8 min at flow rate of 1.0ml/min and variation in retention time was not significant at different injections.

Pretreatment:

Albino rats of either sex (150 to 180 g) were acclimatized in an air-conditioned room at20-220C for 2 weeks. They were housed in elevated wire cages and were provided with high fat diet (carbohydrates: proteins: fat in 42:18:40 ratios) and water ad libitum for a period of 15 days

Experimental Induction of Diabetes in Rats:

Hyperglycaemia was induced in overnight fasted adult wistar rats weighing 150-180g by a single intraperitoneal injection of freshly prepared Alloxan monohydrate in normal saline (150 mg/kg body weight) in a volume 1ml/kg body weight. Hyperglycaemia was confirmed by the elevated glucose level in plasma, determined at 48 h after injection. The Rats found hyperglycaemic were screened for the Antihyperglycaemic study[8].

Study design:

The diabetic rats are divided in to five groups 6 animals in each.

Group I:

Control group (0.5% Na.CMC suspension p.o)

Group II:

Capsicum annuum extract(50 mg/kg,p.o)

Group III:

Capsicum annuum extract (100 mg/kg.p.o)

Group IV:

Combination of Metformin (100 mg/kg.p.o) + Capsicum annuum extract (100 mg/kg).

GroupV:

Combination of Metformin (50 mg/kg.p.o) + Capsicum annuum extract (100 mg/kg).

Group VI: 

Metformin (100 mg/kg.p.o)

Pharmacokinetic study:

Single dose study

 The studies were carried out in diabetic albino rats (weighing about 180-230g).they were housed in elevated wire cages with free access to food and water ad libitum. The overnight fasted rats were divided in to six different groups (n=6);and the treat was given as mentioned above. Post- dosing the blood samples were collected at predetermined intervals of 1,2,4,8,12 and 24 hinto micro-centrifugal tubes containing sodium citrate from retro-orbital sinus under,mild ether anesthesia. The blood was subjected to centrifugation at 3000 rpm for 10 min and plasma was stored at -200C until analysis.

Multiple dose study:

The diabetic rats were divided into 6 different treatment groups same as mentioned above and the treatment was carried for 21 days(3 weeks).blood samples were collected from different groups on 0,7,14,21st day immediately after treatment. Blood samples were collected in to micro-centrifugal tubes containing sodium citrate from retro-orbital sinus under,mild ether anesthesia. The blood was subjected to centrifugation at 3000 rpm for 10 min and plasma was stored at -200 C until analysis.

Pharmacodynamic study in diabetic rats

Single dose study:

Determination of different biochemical parameters in Diabetic rats

Adults albino rats weighing 180-250g with fasting serum glucose >150 mg/dl are considered as diabetic. The treatment was given as mentioned above. Different biochemical parameters like serum glucose, cholesterol, urea concentrations are measured at different time intervals of 0, 1, 2, 4, 8, 12 and 24h by using semi auto analyzer. These values are considered as acute study values.

Multiple dose study:

The diabetic rats were divided into 6 different treatment groups same as mentioned above and the treatment was carried for 21 days(3 weeks).Different biochemical parameters like glucose, cholesterol, urea concentrations of the overnight fasted rats were  determined on 1,7,14,21 st day using semi auto analyzer.[9-13]

Statistical analysis:

• All data are expressed as mean+Sd. For comparison amongst different groups, One-way analysis of variance (ANOVA) followed by Dunnet test was performed. P value less than 5% (P <0>
• Pharmacokineticdata was calculated by using pk solver software and statistical analysis was done by INSTANT graph pad software.[14]

Histopathological studies

• After the last blood glucose estimation, the rats were sacrificed and pancreas were excised and subjected to histopathological studies to determine the inflammatory and necrotic changes. The tissues were stained using H&E stain and observed under 100 × magnifications. The volume of islet cells in pancreas was determined using eyepiece reticule following point count method using the formula.[15]

RESULTS

Values are given as mean± Standard deviation.

* *Statistical significance p < 0>

 *Statistical significance p < 0>

 n - number of animals used.

Values are given as mean± Standard deviation.

* *Statistical significance p < 0>

 *Statistical significance p < 0>

 n - number of animals used.

Table 3: Blood Urea levels at 0th,1st,2nd,4th,8th, 12th and 24thHour after oral administration of Capsicum annuum, Metformin and combination of Metformin + Capsicum annuum in diabetic rats (n=6)

Values are given as mean± Standard deviation.

* *Statistical significance p < 0>

 *Statistical significance p < 0>

 n - number of animals used.

Values are given as mean± Standard deviation.

* *Statistical significance p < 0>

 *Statistical significance p < 0>

 n - number of animals used.

Table 5: Blood Cholesterol levels at 0th,7th, 14th and 21st day after oral administration of Capsicum annuum, Metformin and combination of Metformin + Capsicum annuum in diabetic rats (n=6)
       
           
       
    
    

Values are given as mean± Standard deviation.

* *Statistical significance p < 0>

 *Statistical significance p < 0>

 n - number of animals used.

* *Statistical significance p < 0>

 *Statistical significance p < 0>

 n - number of animals used.

Pharmacodynamic study:

The combination of high dose of Metformin ( 100 mg/kg) with 500mg/kg Capsicum annuum showed maximum hypoglycemic action, decrease in serum cholesterol and urea levels. The effect produced by combination of Metformin (50mg/kg) with Capsicum annuum was greater than the hypoglycaemic action produced by Capsicum annuum (100 mg/kg) alone and Metformin (50mg/kg).

Pharmacokinetic study:

The Single dose study shows that, 96?crease in AUC( 0 - ?)  in 100mg/kg of Capsicum annuum and 50mg/kg of Metformin,  97%   decrease AUC ( 0 - ?) in  100mg/kg  of Capsicum annuum and 50mg/kg of Metformin when compared with the 50mg/kg of Metformin group. C max was decreased by 91% in 100mg/kg of Capsicum annuum and 50mg/kg of Metformin, 99% in 100mg/kg   of Capsicum annuum and 50mg/kg of Metformin in single dose study when compared with the 5mg/kg of Metformin group. Significant decrease in absorption rate constant Ka by about 60% in Lower dose of 100mg/kg of Capsicum annuum and 50mg/kg of Metformin, 81% in 100mg/kg  of Capsicum annuum and 50mg/kg of Metformin when compared with the 5mg/kg of Metformin group. Significantly increase in clearance 2% in  100mg/kg of Capsicum annuum and 2.5mg/kg of Metformin. 5.45%  in 500mg/kg  of Capsicum annuum and 50mg/kg of Metformin compared  to 50mg/kg Metformin when compared with the 50mg/kg of Metformin group. The multiple dose study  shows that, 96?crease in AUC( 0 - ?)  in 100mg/kg of Capsicum annuum and 50mg/kg of Metformin.  98%  decrease AUC( 0 - ?) in  100mg/kg  of   Capsicum annuum and 50mg/kg of Metformin when compared with the 50mg/kg of Metformin group. C max was decreased by 88% in 500mg/kg of Capsicum annuum and 50mg/kg of Metformin, 96% in 100mg/kg of Capsicum annuum and 50mg/kg of Metformin in multiple dose study when compared with the 50mg/kg of Metformin group. Significant decrease in absorption rate constant Ka by about 58% in Lower dose of 100mg/kg of Capsicum annuum and 50mg/kg of Metformin, 76% in 100mg/kg  of Capsicum annuum and 50mg/kg of Metformin when compared with the 50mg/kg of Metformin group. Significantly increase in clearance  4% in  100mg/kg of Capsicum annuum and 50mg/kg of Metformin. 10% in 100mg/kg  of Capsicum annuum and 50mg/kg of Metformin compared  to 50mg/kg Metformin group. The exact reason behind the reduction in pharmacokinetic parameters was unknown but, it was understood that the combination of Metformin extract with  Metformin in fact reduces exposure of the synergic drugs without reducing the pharmacodynamic activity. The proposed combination allows a safe therapy with less adverse effects.  The histological study shows that the combination therapy  (Metformin + Capsicum annuum) involved in the increase the number of islets and recovered the partially damaged B cells in pancreas when compare to the Individual treatment.

Histological study:

The histological study shows that the combination therapy  (Metformin + Capsicum annuum) involved in the increase the number of islets and recovered the partially damaged B cells in pancreas when compare to the Individual treatment.

       
           
       
    H&S Staining of Pancreatic islets of Diabetic Control, Capsicum annuum alone Metformin alone and combination of Capsicum annuum & Metformin treated Diabetic Rats. 6A. diabetic control , 6B. 100mg of Capsicum annuum 6C. 100mg Capsicum annuum, 6D.50mg of  Metformin.6E.100mg of Capsicum annuum. +50mg of Metformin, 6F. 100mg of Capsicum annuum +50mg of Metformin.