Dr. Mahrukh Naseem

Title:

Effects of Ginkgo biloba and Panaz ginseng on metabolism of carbohydrate, lipids and insulin receptors genes in diabetic rats

Abstract

Diabetes is a major public health issue. As pharmaceutical agents carry adverse effects so the interest in the natural remedies is increased in the last few decades. Ginkgo biloba leaf extract (GBE) and Panax ginseng root extract (PGE) are ancient Chinese herbal drug, widely used for the treatment of diabetes and contain a prominent position in the list of the bestselling natural remedies. Anti-diabetic effects of GBE is attributed to the flavonoides and that of PGE are attributed to ginsenosides. GBE and PGE in combination shows significantly higher anti-diabetic effects than individual extracts in diabetic rats.
Adult Wistar rats were allowed to feed on high fat diet (HFD: 12.7% maize starch, 6.5% dextrose, 3.9% sunflower oil, 31.3% beef tallow and 28.6% casein by weight) for two weeks. The rats were divided into seven groups (08 rats in each group): Non-diabetic control group, Diabetic group, Diabetic + 100 mg/kg G. biloba leaf extract treated group (GBE), Diabetic + 300 mg/kg P. ginseng root extract treated group (PGE), mixed 1 group : Diabetic + combination of both GBE and PGE at dose of 200 mg/kg/day (50mg/kg/day of GBE and 150mg/kg/day of PGE), mixed 2 group : Diabetic + combination of both GBE and PGE at dose of 400mg/kg/day (100mg/kg/day of GBE and 300mg/kg/day of PGE), mixed 3 group : Diabetic + combination of both GBE and PGE at dose of 600mg/kg/day (150mg/kg/day of GBE and 450mg/kg/day of PGE). At the end of 14th day, the rats were kept in fasting condition overnight and then single intra-peritoneal injection of alloxan monohydrate (Sigma, USA) dissolved in 0.5 ml of saline solution at the dose of 120-130 mg/Kg body weight were injected to all rats except non-diabetic group which were injected with equal volume of normal saline. Body weight (BW) and blood glucose were measured at week 1 and week 14. At the end of the experimental period, blood samples in fasting/ basal state were collected from heart puncture for the biochemical parameters. Liver, muscles and adipose tissue were also collected for mRNA expression of genes involved in carbohydrate and fat metabolism.
Results were expressed as Means ± S.E.M. Statistical analyses were performed using Statview software (SAS Institute Inc., SAS Campus Drive, Cary, NC, USA). Two-ways repeated measure ANOVA followed by PLSD Fisher's test was performed for BW and blood glucose to assess the effects of time and herbal drugs. For the rest of the parameters, one-way ANOVA followed by PLSD Fisher's test was performed to assess the effect of herbal drugs. Differences were considered significant at P < 0.05.
A significant (P < 0.0001) reduction in the BW in diabetic group was recorded compared to non-diabetic and a significant reduction in the BW was occurred after treatment in all the five treated groups compared to diabetic group. Glycemia is significantly higher in diabetic rats (P < 0.0001) compared to non-diabetic and significant reduction in the blood glucose level was recorded in all the five treated groups (P < 0.0001) group in comparison to diabetic group. A significant reduction for FSG (P < 0.0001) for all groups was recorded for all the five treated groups compared to diabetic rats. We linked the glucose with the mRNA expressions of genes involved in the glucose metabolism and we studied gene expressions of GLUT-4, insulin receptor (IR), insulin receptor substrate-1 (IRS-1) and phosphoenolpyrovate carboxykinase (PEPCK) in liver, muscle and adipose tissue. A significant up-regulation for the mRNA expression of GLUT-4 was observed only in muscle in all the five treated groups, i.e. GBE (P < 0.001), PGE (P < 0.001), mixed 1 (P < 0.0001), mixed 2 (P < 0.0001) and mixed 3 (P < 0.0001). We found a significant down-regulation in the mRNA expression of IR in muscle (P < 0.0001) and adipose tissue (P < 0.05) in diabetic group compared to non-diabetic, however, a significant up-regulation was found in mixed 3 group in muscle (P < 0.001) and adipose tissue (P < 0.05). We found a significant down-regulation (P < 0.001) for IRS-1 in liver in diabetic state and a significant up-regulation was recorded in GBE (P < 0.05) and mixed 3 (P < 0.05) groups only. We found a significant down-regulation in IRS-1 in muscle (P < 0.0001) and adipose tissues (P < 0.0001) in diabetic group. None of the treated group showed significant results in muscles however, a significant up-regulation was found only in PGE (P < 0.001) and mixed 3 group (P < 0.0001) in adipose tissue. A significant up-regulation was recorded for PEPCK in GBE (P < 0.05), mixed 1 (P < 0.05), mixed 2 (P < 0.05) and mixed 3 (P < 0.05) groups in liver.
A significant increase was found for cholesterol in diabetic state (P < 0.0001) and significant reduction was found only in mixed 3 (P < 0.001) treated group. A significant decreased was found for VLDL-C in mixed 1 (P < 0.05), mixed 2 (P < 0.0001) and mixed 3 (P < 0.0001) groups. A significant decreased was observed for LDL-C in mixed 1, mixed 2 and mixed 3 (P < 0.0001) groups which previously found to be enhanced in diabetic condition. In case of HDL-c a significant decreased was found for GBE (P < 0.001), PGE (P < 0.05), mixed 1 (P < 0.001), mixed 2 (P < 0.0001) and mixed 3 (P < 0.0001) which previously found to be increased in diabetic group (P < 0.0001). Whereas, a significant increase was seem for TG (P < 0.0001) in diabetic state and significant reduction was found in all the five treated groups (P < 0.0001). We further studied a genes involved in lipid metabolism. A significant up-regulation was found for SREBP-1c in diabetic group (P < 0.0001) and a significant down-regulation was found to be occur in mixed 2 (P < 0.05) and mixed 3 (P < 0.001) treated groups compared to diabetic rats. In liver a significant up-regulation in the mRNA expression of FAS was found only in mixed 2 (P < 0.05) and mixed 3 (P < 0.05) treated groups which found to be down regulated in diabetic group (P < 0.001). A significant down-regulation in the mRNA expression of PPAR-α was found in diabetic rats skeletal muscle (P < 0.05), however, a significant up-regulation was found in GBE (P < 0.001), PGE (P < 0.05) mixed 1 (P < 0.001), mixed 2 (P < 0.001) and mixed 3 (P < 0.001) groups in comparison to diabetic rats. We studied PPAR-γ in adipose tissue and found a significant up-regulation in PGE (P < 0.05), mixed 1 (P < 0.001), mixed 2 (P < 0.001) and mixed 3 (P < 0.0001) groups which previously found to be down regulated (P < 0.001) in diabetic rats compared to non-diabetic rats.
We found that the body of the diabetic rats suffer with oxidative stress and measured a significant decrease for CAT (P < 0.0001) in diabetic group and significant increase was found in GBE (P < 0.05), PGE (P < 0.05), mixed 1 (P < 0.05), mixed 2 (P < 0.05), mixed 3(P < 0.05) groups compared to diabetic rats. Whereas, a significant decreased was recorded for MDA in GBE (P < 0.05), PGE (P < 0.05), mixed 1 (P < 0.001), mixed 2 (P < 0.001) and mixed 3 (P < 0.0001) groups, which previously showed a significant increased (P < 0.001) in diabetic group compared to non-diabetic. We linked oxidative stress with TNF- α and found a significant up-regulation (P < 0.0001) for all the three studied organs in diabetic groups compared to non-diabetic group. In case of liver a significant down-regulation was found for GBE (P < 0.0001), PGE (P < 0.0001) and mixed 3 (P < 0.0001) groups compared to diabetic rats. A significant down-regulation in the expression of TNF- α in muscle was recorded only in mixed 2 (P < 0.001) and mixed 3 (P < 0.0001) groups compared to diabetic rats. However, a significant down-regulation in the expression of TNF- α in the adipose tissue was observe for all the treated groups (P < 0.0001 for all groups) in comparison to diabetic group.
For serum creatinine a significant enhancement was observed for PGE (P < 0.05), mixed 1 (P < 0.05) and mixed 3 (P < 0.05) groups which previously found to be reduced in diabetic rats. A significant increased for AST was found in diabetes (P < 0.0001) compared to non-diabetic, while a significant reduction was found to be occur only for PGE (P < 0.05), mixed 2 (P < 0.05) and mixed 3 (P < 0.001) treated groups in comparison to diabetic group. Like AST a significant reduction was recorded for ALT in diabetic group (P < 0.001) and only GBE (P < 0.001), PGE (P < 0.05) and mixed 3 (P < 0.05) showed a significant decreased in ALT level compared to diabetic rats.
In conclusion, we found that both these natural remedies have strong individual anti-hyperglycemic, anti-hypertriglyceridemic and anti-oxidative effects. It also shows strong influence on the activation on the expression of genes involved in the metabolic pathways of glucose and lipid which previously became dysfunction in diabetic rats. When both these natural remedies were given in combination, synergistic effects were recorded in dose dependent manner.