EFFECT OF DYKURE A POLYHERBAL FORMULATION ON TYPE-I AND TYPE-II DIABETES

Transcription

1 EFFECT OF DYKURE A POLYHERBAL FORMULATION ON TYPE-I AND TYPE-II DIABETES Address for Correspondence: Harshul Parikh a, R. Balaraman b Prof. R. Balaraman Pharmacy Department Faculty of Tech. & Engg., The M. S. University of Baroda, Kalabhavan, Vadodara a harshul72@yahoo.com b rbalaraman2000@yahoo.com 1

2 INTRODUCTION Composition of Dykure capsules Name of Plant Azadirachta Indica Trigonella foenumgraceum Picrohiza kurroa Terminalia Chebula Saraca Indica Maduca Indica Coccinia Indica Asphaltum puniabiunm Abutilon Indicum Tinospora cordifolia Terminalia Balerica Aerva Lanata Per 100 gm of Dykure powder 10 gm 12 gm 05 gm 06 gm 08 gm 06 gm 06 gm 06 gm 08 gm 06 gm 07 gm 08 gm 2

3 MATERIALS AND METHODS ANIMALS: Healthy adult Wistar rats of either sex were used for the study. Rats were housed in small cages with free access to food and water ad libitum. During the experiments, all the animals were fed with standard laboratory animal diet. EXPERIMENTAL MODELS: 1. Streptozotocin(STZ)+ Nicotinamide(NIC) induced Type II diabetes in rats A) Parameter assessed in SERUM : Fasting serum glucose Glycosylated Hemoglobin (Hb1Ac) Total Cholesterol Triglyceride HDL Cholesterol B) Oral glucose tolerance test : 2. Streptozotocin(STZ) induced Type-I diabetes in rats. A) Parameter assessed in SERUM : Fasting serum glucose Glycosylated Hemoglobin (Hb1Ac) Total Cholesterol Triglyceride HDL Cholesterol 3

4 INDUCTION OF TYPE-II DIABETES Wistar rats were first administered with Nicotinamide (120mg/kg) in Normal saline intraperitoneally. After 15 min, they were administered with Streptozotocin (60 mg/kg) in 1M Citrate buffer (ph 4.5) intraperitoneally. After 7 days, rats with fasting blood glucose above 180 mg/dl were considered as diabetic and selected for further study. INDUCTION OF TYPE-I DIABETES Wistar rats were administered with Streptozotocin (60 mg/kg) in 1M Citrate buffer (ph 4.5) intraperitoneally. After 7 days, rats with fasting blood glucose above 180 mg/dl were considered as diabetic and selected for further study. EXPERIMENTAL DESIGN: (n=40 animals divided equally into following groups) Group 1: Control- Normal rats Group 2: Normal rats treated orally for 30 days with Dykure 1gm/kg/day divided in two equal doses. Group 3: Streptozotocin (60mg/kg) + Nicotinamide (120mg/kg) Group 4 : Streptozotocin (60mg/kg) + Nicotinamide (120mg/kg) induced diabetic rats treated orally for 30 days with Dykure 300mg/kg/day, p.o Group 5 : Streptozotocin (60mg/kg) + Nicotinamide (120mg/kg) induced diabetic rats treated orally for 30 days with Dykure 1gm/kg/day divided into two equal doses. Group 6 : Streptozotocin (60mg/kg) Group 7 : Streptozotocin (60mg/kg) induced diabetic rats treated orally for 30 days with Dykure 300mg/kg/day, p.o Group 8 : Streptozotocin (60mg/kg) induced diabetic rats treated orally for 30 days with Dykure 1gm/kg/day divided into two equal doses. 4

5 COLLECTION OF SERUM The blood samples were withdrawn from retro-orbital plexus under light ether anesthesia without any anticoagulant and allowed to clot for 10 minutes at room temperature. It was centrifuged at 2500 rpm for 20 minutes. The serum was kept at 4 c until used. Estimation of Serum Glucose By GOD / POD Method In vitro quantitative determination of serum glucose was done by using enzymatic kit (Beacon diagnostics) Principle: Enzymatic colorimetric determination of glucose according to the following reactions Glucose + O 2 Glucose oxidase Gluconic acid + H2O2 2H 2 O 2 + Phenol + 4-Aminoantipyrine Procedure: Peroxidase Red quinone + 4H 2 O Pipette out the solution in the test tubes as follows Reagents Blank Standard Sample Working reagent 2.0 ml 2.0 ml 2.0 ml Distilled water 10 μl Standard glucose μl Sample μl Mix and read the optical density (OD) after 10 minutes of incubation. The final colour is stable for at least 1 hour. Calculations: Fasting Glucose (mg/dl) = Absorbance of Sample /Absorbance of Standard X 100 5

6 Estimation of Glycosylated Hemoglobin By Ion exchange resin Method In vitro quantitative determination of Glycosylated hemoglobin Hb1Ac was done by using Ion exchange resin kit (Crest Biosystems) Principle: Glycosylated hemoglobin has been defined operationally as the fast fraction hemoglobin which elute first during column chromatography. A hemolysed preparation of whole blood is mixed continuously for 5 min. with a weakly binding cation-exchange resin. The labile fraction is eliminated during the hemolysate preparation. During the mixing, nonglycosylated hemoglobin binds to the ion exchange resin leaving glycosylated hemoglobin in the supernatant. After the mixing period, a filter separator is used to remove the resin from the supernatant. The percentage of Hb1Ac is determined by measuring absorbances of the glycosylated Hb and the total hemoglobin fraction. The ratio of the absorbances of the glycosylated hemoglobin (GHb) and the total hemoglobin fraction of the control and the test is used to calculate the percentage of Hb1Ac of the sample. Content of the Kit Ion exchange resin (Predispensed tubes) Resin separator Lysing reagent Reconstituted Control (10% GHb) Sample Material: Whole blood, preferably fresh was collected in EDTA tubes. Procedure: A. Hemolysate Preparation 1. Dispense 0.5 ml lysing reagent into tubes labeled as Control(C) and Test(T) 2. Add 0.1 ml of the reconstituted control and well mixed blood sample into the appropriately labeled tubes. Mix until complete lysis is evident. 3. Allow to stand for 5 min. 6

7 B. Glycosylated hemoglobin (GHb) Separation 1. The Ion exchange resin tubes were labeled as Control(C) and Test(T) ml of hemolysate from step-a was added into the appropriately labeled Ion exchange resin tubes. 3. A resin separator was inserted into each tube so that the rubber sleeve is approx. 1 cm above the liquid level of the resin suspension. 4. The contents were mixed on vortex mixer for 5 min. 5. Resin was allowed to settle down and resin separator was pushed into the tubes until the resin was firmly packed. 6. Supernatant was collected directly into the cuvette and absorbance was measured against distilled water. C. Total hemoglobin (THb) fraction ml of Distilled water was dispensed into tubes labeled as Control & Test ml of hemolysate from step A was added into the appropriately labeled tube and mixed well. 3. Absorbance was read against distilled water. Calculations: Ratio of Control (Rc) = Abs. Control GHb / Abs. Control THb Ratio of Test (Rt) = Abs. Test GHb / Abs. Test THb GHb in % = Ratio of Test (Rt) / Ratio of Control (Rc) X 10 (Value of Control) 7

8 Estimation of Serum Total Cholesterol By CHOD / POD Method In vitro quantitative determination of serum Cholesterol was done by using enzymatic kit (Recon diagnostics) Principle: The Cholesterol esters are hydrolyzed to free cholesterol by cholesterol esterase. The free cholesterol is then oxidized by cholesterol oxidase (CHOD) to cholesterol 4-en-3-one with the simultaneous production of hydrogen peroxide. The hydrogen peroxide reacts with 4-aminoantipyrine and phenolic compound in the presence of Peroxidase to yield a coloured complex which is read at 505nm. The intensity of colour produced is directly proportional to the concentration of total cholesterol in the sample. Procedure: Pipette out the solution in the test tubes as follows Read the absorbance of standard and test at 505nm against reagent blank Calculations: Reagents Blank Standard Sample Cholesterol reagent 1.0 ml 1.0 ml 1.0 ml Distilled water 10 μl Standard Cholesterol μl Sample μl Mix well and incubate for 10 min. at 37 c or 30 min. at room temp.(25±5 c). Distilled water 1.0 ml 1.0 ml 1.0 ml Total cholesterol (mg/dl) = Absorbance of Sample /Absorbance of Standard X 200 8

9 Estimation of Serum Triglycerides By GPO Method In vitro quantitative determination of serum Triglyceride was done by using enzymatic kit (Reckon diagnostics) Principle: Lipase hydrolyses triglycerides sequentially to Di & Monoglycerides and finally to glycerol. Glycerol kinase (GK) by using ATP as a phosphate source, converts glycerol to glycerol-3-phosphate and ADP is formed. Enzyme glycerol-3-phosphate oxidase oxidizes glycerol-3-phosphate into dihydroxyacetone phosphate and hydrogen peroxide is formed. Enzyme Peroxidase utilizes this hydrogen peroxide to oxidize 4-aminoantipyrine and ADPS which results in formation of purple coloured complex. The absorbance of coloured complex is measured at 546nm which is proportional to Triglyceride concentration present in the sample. Procedure: Pipette out the solution in the test tubes as follows Read the absorbance of standard and test at 546nm against reagent blank Calculations: Reagents Blank Standard Sample Working Reagent 1.0 ml 1.0 ml 1.0 ml Distilled water 20 μl Standard Cholesterol μl Sample μl Mix well and incubate for 15 min. at 37 c or 30 min. at room temp. (25±5 c). Distilled water 1.5 ml 1.5 ml 1.5 ml Triglycerides (mg/dl) = Absorbance of Sample /Absorbance of Standard X 200 9

10 Estimation of Serum HDL Cholesterol By/ PTA Method In vitro quantitative determination of serum HDL Cholesterol was done by using enzymatic kit (Recon diagnostics) Principle: High density lipoproteins (HDL) are separated from other lipoprotein fractions by treating serum with phosphotungstic acid and magnesium chloride. HDL remains in solution while all other lipoprotein fractions are precipitated; cholesterol content of which is estimated by enzymatic method described earlier in total cholesterol estimation. Procedure: Separation of HDL fraction Pipette out the solution in the ependroff tube as follows Serum sample 3-HDL-Cholesterol solution 0.2 ml 0.2 ml Mix well and centrifuge at rpm for ten minutes. Separate the clear supernatant immediately and determine cholesterol content as follows. Estimation of HDL Cholesterol Pipette out the solution in the test tubes as follows Mix well and read the absorbance of standard and test at 505nm against reagent blank Calculations: Reagents Blank Standard Sample Cholesterol reagent 1.0 ml 1.0 ml 1.0 ml Distilled water 0.1 ml Standard HDL Cholesterol ml Supernatant of separated fraction ml Mix well and incubate for 20 min. at 37 c or 30 min. at room temp.(25±5 c). Distilled water 2.0 ml 2.0 ml 2.0 ml HDL cholesterol (mg/dl) = Abs. of Sample /Abs. of Standard X Conc. of std. X D.F HDL cholesterol (mg/dl) = Abs. of Sample /Abs. of Standard X 50 X2 10

11 RESULTS Treatment Serum Glucose Glycosylated Hemoglobin HbA1c Total Cholesterol Triglyceride HDL Cholesterol Control 72.04± ± ± ± ±1.64 PF 1gm/kg 65.58± ± ± ± ±1.26 STZ+NIC 199.5± ± ± ± ±1.36 STZ+NIC+PF 300mg/kg 146.4± ± ± ± ±1.02 STZ+NIC+PF 1gm/kg 98.80± ± ± ± ± STZ 302.0± ± ± ± ±1.35 STZ+PF 300mg/kg 194.9±2.65 ### 6.56±0.34 ### 100.3±1.50 ## 90.37±1.65 ### 37.82±1.82 STZ+PF 1gm/kg 96.60±2.26 ### 5.13±0.20 ### 80.23±2.50 ### 73.71±2.84 ### 46.91±2.10 ## PF = Polyherbal formulation * Compare to Control + Compare to STZ+NIC # Compare to STZ Or +++ Or ### indicates p<0.001 ** Or ++ Or ## indicates p<0.01 * Or + Or # indicates p<

12 Oral glucose tolerance test The oral glucose tolerance test was performed on over night (18 hr) fasted rats. Rats were divided into four groups as shown in table. Glucose (2gm/kg) was orally administered to the rats. In case of treatment groups it was administered 30 min after the administration of Dykure. Blood was withdrawn form the retro orbital plexus under light ether anesthesia at 0, 30, 60, 120 min of glucose administration. The blood glucose level were estimated by glucose reactive strips on glucometer (Accuchek, Roche diagnostics) Minutes Normal Diabetic Diabetic + PF Diabetic + PF 300mg/kg 1g/kg ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

13 mg / dl Serum Glucose control NS NS PF 1gm/kg STZ+NIC STZ+NIC+PF 300mg/kg STZ+NIC+PF 1g/kg Groups STZ STZ+PF 300mg/kg STZ+PF 1g/kg control PF 1gm/kg STZ+NIC STZ+NIC+PF 300mg/kg STZ+NIC+PF 1g/kg STZ STZ+PF 300mg/kg STZ+PF 1g/kg Figure 1 : Effect of Dykure (300 mg/kg and 1gm/kg) on Serum Glucose in normal and Streptozotocin (STZ) +Nicotinamide (NIC) and Streptozotocin(STZ) induced diabetic rats (* p<0.05, **p<0.01, p<0.001, NS p>0.05) 13

14 Percentage of Hb1Ac Glycosylated Hemoglobin Hb1AC NS * Control PF 1gm/kg STZ+NIC STZ+NIC+PF 300mg/kg STZ+NIC+PF 1gm/kg Groups STZ STZ+PF 300mg/kg STZ+PF 1g/kg Control PF 1gm/kg STZ+NIC STZ+NIC+PF 300mg/kg STZ+NIC+PF 1gm/kg STZ STZ+PF 300mg/kg STZ+PF 1g/kg Figure 2 : Effect of Dykure (300 mg/kg and 1gm/kg) on Glycosylated Hemoglobin (Hb1Ac) in normal and Streptozotocin (STZ) +Nicotinamide (NIC) and Streptozotocin(STZ) induced diabetic rats (* p<0.05, **p<0.01, p<0.001, NS p>0.05) 14

15 mg / dl Serum Cholesterol CONTROL NS ** PF 1gm/kg STZ+NIC STZ+NIC+PF 300mg/kg STZ+NIC+PF 1gm/kg STZ Groups STZ+PF 300mg/kg STZ+PF 1g/kg CONTROL PF 1gm/kg STZ+NIC STZ+NIC+PF 300mg/kg STZ+NIC+PF 1gm/kg STZ STZ+PF 300mg/kg STZ+PF 1g/kg Figure 3 : Effect of Dykure (300 mg/kg and 1gm/kg) on Serum Cholesterol in normal and Streptozotocin (STZ) +Nicotinamide (NIC) and Streptozotocin(STZ) induced diabetic rats (* p<0.05, **p<0.01, p<0.001, NS p>0.05) 15

16 mg / dl Serum Triglyceride CONTROL * PF 1gm/kg STZ+NIC STZ+NIC+PF 300mg/kg STZ+NIC+PF 1gm/kg STZ STZ+PF 300mg/kg STZ+PF 1g/kg CONTROL PF 1gm/kg STZ+NIC STZ+NIC+PF 300mg/kg STZ+NIC+PF 1gm/kg STZ STZ+PF 300mg/kg STZ+PF 1g/kg Groups Figure 4 : Effect of Dykure (300 mg/kg and 1gm/kg) on Serum Triglyceride in normal and Streptozotocin (STZ) +Nicotinamide (NIC) and Streptozotocin(STZ) induced diabetic rats (* p<0.05, **p<0.01, p<0.001, NS p>0.05) 16

17 mg / dl Serum HDL Cholesterol CONTROL ** NS NS PF 1gm/kg STZ+NIC STZ+PF 300mg/kg STZ+NIC+PF 300mg/kg STZ+NIC+1gm/kg STZ Groups NS ** STZ+PF 1g/kg CONTROL PF 1gm/kg STZ+NIC STZ+NIC+PF 300mg/kg STZ+NIC+1gm/kg STZ STZ+PF 300mg/kg STZ+PF 1g/kg Figure 5 : Effect of Dykure (300 mg/kg and 1gm/kg) on Serum HDL Cholesterol in normal and Streptozotocin (STZ) +Nicotinamide (NIC) and Streptozotocin(STZ) induced diabetic rats (* p<0.05, **p<0.01, p<0.001, NS p>0.05) 17

18 Plasma Glucose Level mg/dl ** NS * OGTT Normal Diabetic Diabetic + PF300 mg Diabetic + PF1 gm/k Minutes Figure 6 : Effect of Dykure (300 mg/kg and 1gm/kg) on Oral glucose tolerance test in normal and Streptozotocin (STZ) +Nicotinamide (NIC) induced diabetic rats (* p<0.05, **p<0.01, p<0.001, NS p>0.05) 18

19 DISCUSSION Induction of diabetes in both STZ+NIC and STZ groups was confirmed by significant elevated level of fasting serum glucose (Fig1. p<0.001). Oral administration of Dykure for 30 days has significantly reduced the blood glucose level both in STZ + NIC and STZ group (p<0.001). Significant elevation in blood level of Total cholesterol (Fig.3 p<0.001) and Triglyceride (Fig.4 p<0.001) was found in both diabetic groups as compared to normal animals and significant reduction in blood level of these parameters was observed after the treatment with Dykure. Glycosylated hemoglobin is formed continuously by the adduction of glucose by co-valent bonding to the amino-terminal valine of the hemoglobin beta chain progressively and irreversibly over a period of time. It is stable till the life of the RBC. This process is slow, non enzymatic and is dependent on the average blood glucose concentration over a period of time. A single glucose determination reflects the glucose level at the time while glycosylated hemoglobin reflects the mean glucose level over an extended period of time. Thus Glycosylated hemoglobin is now widely recognized as an important test for the diagnosis of Diabetes mellitus and is a reliable indicator of the efficacy of therapy. In present study, level of glycosylated hemoglobin HbA1c was found to be increased significantly in diabetic groups (Fig.2 p<0.001). Treatment with Dykure has reduced the elevated level of HbA1c significantly (p<0.001) as compared to diabetic groups. HDL cholesterol was found to be reduced in diabetic groups (Fig.5 p<0.001). Dykure 300mg/kg wasn t found to improve the HDL cholesterol level as compared to diabetic groups but Dykure 1g/kg has improved the HDL cholesterol to a significant (Fig.5 p<0.01) level In Oral glucose tolerance test, Dykure was found to be effective in improving glucose tolerance indicated by significant reduction in plasma glucose level (Fig.6 p<0.001) in treatment and normal animals as compared to diabetic animals. 19

20 The anti-diabetic and lipid lowering activity was fount to be dose dependent as there was significant difference in blood glucose, Hb1Ac and lipid profile between 300mg/kg and 1g/kg treated groups. There was no death reported in Normal animals treated with Dykure 1g/kg indicating its non-toxic nature. The difference in estimated parameters between normal animals and normal animals treated with Dykure was found to be statistically non-significant. The fundamental mechanism of occurrence of hyperglycemia in diabetes is hepatic overproduction of glucose by glycogenolysis & gluconeogenesis and reduction of utilization of glucose by the tissues. Under normal circumstances, insulin activates enzyme lipoprotein lipase and hydrolyses the triglyceride. Deficient action of Insulin in diabetes results in hypertriglyceridemia. Deficiency of insulin results in lipolysis and elevated serum level of cholesterol. The possible mechanism of hypoglycemic action of Dykure may be due to release of insulin from existing beta cells or due to reduction in insulin resistance. Improvement in lipid profile is indication of lipid lowering property of Dykure. Dykure may be useful in treatment of diabetes and its associated metabolic abnormalities. Study on Chronic model and mechanism based research work is further required to confirm its anti-diabetic and lipid lowering activity. 20