Effect of Momordica charantia fruits on Diabetic Complications
Momordica Charantia (Cucurbitaceae) or Bitter Melon, is a Tropical vegetable, is a common food in Indian cusine and has been used extensively in folk medicine (Dasgupta, 2011). In Ayurveda, the fruit is considered as tonic, stomachic, stimulant, emetic, antibilous, laxative and alterative. Bitter melon has been used in various Asian traditional medicine systems for a long time. Like most bitter-tasting foods, bitter melon stimulates digestion. While this can be helpful in people with sluggish digestion, dyspepsia, and constipation, it can sometimes make heartburn and ulcers worse. The fact that bitter melon is also a demulcent and at least mild inflammation modulator, however, means that it rarely does have these negative effects, based on clinical experience and traditional reports (Kumar et al., 2010; Patel et al., 2010; Sharma et al.,2011).
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In the initial study, significant antihyperglycemic activiy of crude powder of M.charantia fruits was observed in STZ-induced diabetic rats. Among the various extracts aqueous extract showed significant effect on postprandial hyperglycemia in normal as well as STZ diabetic rats after oral administration of sucrose and also showed significant lowering of fasting blood glucose in STZ diabetic rats. Therefore the aqueous extract was subjected to the fractionation and two different fractions namely butanol and aqueous fractions were obtained. The fractions were again administered to the STZ diabetic rats in which both the fractions showed significant antihyperglycemic although the activity was greater in aqueous fraction treated group.
The present chapter mainly illustrates the long term multiple dose effect of aqueous fraction of M.charantia in high fructose diet fed low dose STZ-induced diabetic rats (HFD-STZ) and high dose STZ induced diabetic rats. Multiple dose study in the animal models provide the better understanding about the overall effect of the selected fraction on the various symptoms and associated compilcations of diabetes mellitus. Prior to the in vivo study, the aqueous fraction has also subjected to the in vitro study using L6 cells to find out its effect on the cellular glucose uptake and insulin signaling.
Design of study
Aqueous fraction of aqueous extract of T.belerica fruits were subjected to in vitro study using L6 cells to see the effect of fraction on glucose uptake, and insulin signaling. In vivo study was performed using high fructose diet fed low dose STZ-induced diabetic rats (HFD-STZ) and high dose STZ-induced diabetic rats. HFD-STZ rats were selected and grouped on the basis of elevated plasma triglycerides and cholesterol level while the high dose STZ-induced diabetic rats were grouped on the basis of their glycated haemoglobin (HbA1c) level. Multiple dose of aqueous fraction was carried out for one month and oral glucose tolerance test (OGTT) and biochemical analysis including plasma lipid profile analysis and hepatic and renal function tests were performed at regular intervals. Since chronic complications are more pronounced in high dose STZ rats, therefore at the end of the experiments the animals were sacrificed and the kidney of treated as well as untreated control groups were collected for the study of the effect of aqueous fraction on the molecular markers involved in oxidative stress induced diabetic nephropathy.
(1) Concentration dependent effect of aqueous fraction of aqueous extract of M.charantia fruits on glucose uptake in L6 cells:
Treatment of aqueous fractions led to increase of basal as well as insulin-stimulated glucose uptake in concentration dependent manner in L6 cells. Figure 1 shows significant increase of 1.41-fold (p<0.05) was observed in treated L6 myotubes at the minimum concentration of 5 ug/ml. Maximum increase of 1.66 fold (p<0.01) was observed at 10 ug/ml. Effect of aqueous fraction on insulin-induced increase in glucose uptake was also studied. When pre-incubated myotubes with various concentration of aqueous extract were provided with insulin for final 20 min, a dose-dependent increase of 1.88-fold (p<0.01), 1.91-fold (p<0.01) and 2.12-fold (p<0.01) respectively at 2.5 , 5.0 and 10.0 μg/ml concentration were observed.
(2) Effect of aqueous fraction of M.charantia on mRNA expression of insulin signaling gene in L6 cells:
Gene expression profile suggest that the expression of IRS-1 (Insulin receptor substrate, PI3K (Phosphatidylinositol 3-kinase), AKT2 (Protein kinase-B) and GLUT4 genes were upregulated by the treatment of aqueous fraction. Thus it is clear that the aqueous fraction of M.charantia stimulates the genes of insulin signaling pathway which may lead to the antihyperglycemic effect of the fraction.
(3) Effect of aqueous fraction of aqueous extract of M.charantia on IRS-1, AKT and GLUT4 proteins in L6 cells:
Insulin signaling pathway can influence glucose uptake by the translocation of GLUT4 containing vesicles to the plasma membrane and thus facilitates in the transportation of glucose across the plasma membrane. Drugs affecting the insulin signaling may modulate the glucose uptake in this manner. In the present study, it was observed that similar to the gene expression profile, treatment of aqueous fraction increases the protein expression of of p-IRS-1, p-AKT and GLUT4. Thus it is clear that aqueous fraction of M.charantia do effect insulin signaling pathway in in vitro and as a consequence increase glucose uptake by cells.
(4) Effect of aqueous fraction of aqueous extract of M.charantia fruits on fasting blood glucose, oral glucose tolerance and plasma insulin level of High fructose diet fed low dose Streptozotocin-induced diabetic rats
Table 1 shows the remarkable effect of aqueous fraction on fasting blood glucose of treated animals. Highly significant improvement of 31.8% (p<0.01) and 56.0% (p<0.01) was observed in treated groups respectively on day 14 and 28 of treatment. The lowering observed on day 28 was even better than the metformin treated group i.e., 47.3% (p<0.01). It also had positive impact on oral glucose tolerance and significant improvement of 17.7% (p<0.05) and 29.1% (p<0.01) was registered on day 14 and 28 of the treatment. Plasma insulin level which get elevated in HFD-STZ rats was also brought down by 19.7% (p<0.05) as compared to untreated control group.
(5) Effect of aqueous fraction of aqueous extract of M.charantia fruits on lipid profile of High fructose diet fed low dose Streptozotocin-induced diabetic rats
High fructose diet generally cause disturbances in lipid profile leading to dyslipidemia which get further deified by the low dose treatment of STZ causing highly elevated plasma triglycerides, total cholesterol, LDL level and decreased plasma HDL level. Table 2 shows that the treatment with aqueous fraction for one month significantly improved plasma lipid profile and the significant declination of triglycerides, total cholesterol and LDL-cholesterol was found to the tune of 35.7% (p<0.01), 32.4% (p<0.01) and 33.8% (p<0.01) respectively on day 30 of the treatment. While at the same point of time, plasma HDL level was found increased by the significant extent of 41.5% (p<0.01). Thus the aqueous fraction of M.charantia was found sufficiently effective against diabetic dyslipidemia caused by high fructose and low dose STZ in animals.
(6) Effect of aqueous fraction of aqueous extract of M.charantia fruits on Hepatic and Renal parameters of High fructose diet fed low dose Streptozotocin-induced diabetic rats.
High fructose diet may cause dyslipidemia, hyperinsulinemia and insulin resistance which are the characteristics of diabetes type 2 and the extent of severity may get increased by the low dose of STZ which may further add the symptoms like hepatic and renal dysfunction in the experimental animals. Table 3 and 4 shows that in the present study treatment of aqueous fraction was found to improve the plasma level of hepatic and renal function markers. It is clear from Table 3 that there was significant decline of plasma AST and ALT level at ever time interval and the lowering of 31.2% (p<0.01) and 23.5% (p<0.05) respectively was observed on the final day of treatment. Accordingly Table 4 shows the significant decline of plasma level of urea, uric acid and creatinine to the tune of 38.4% (p<0.01), 27.5% (p<0.01) and 25.0% respectively was observed on day 30 of treatment.
(7) Effect of aqueous fraction of aqueous extract of M.charantia fruits on fasting blood glucose, oral glucose tolerance and plasma insulin of high dose streptozotocin-induced diabetic rats
Most of the beta cells get destroyed in high dose STZ-induced diabetic rats and therefore animals display high level of fasting blood glucose and intolerance towards external glucose administration. Table 5 shows that treatment of aqueous fraction for one month significantly improved the fasting blood glucose level to the tune of 15.4% (p<0.05) and 28.1% (p<0.01) as monitored on day 14 and 28 of the treatment. Improvement in oral glucose tolerance was not significant on day 14 while the significant improvement of 21.3% (p<0.05) was registered on day 28.
Elevated fasting blood glucose and reduced glucose tolerance are characteristic of STZ-induced diabetic rats. Table 5 shows that the treatment with Aqueous fraction for 30 consective days improved glucose tolerance of the treated animals to the tune of 18.0% (p<0.05) and 29.9% (p<0.01) at 14th and 28th day of treatment respectively. Fasting blood glucose was declined to 15.0% (p<0.05) and 27.5% (p<0.05) as compared to untreated control on 14th day and 28th day respectively. Plasma insulin level in treated group was found elevated to 42.3% (p<0.01).
(8) Effect of Aqueous fraction of Aqueous extract of M.charantia fruits on glycated haemoglobin level of high dose streptozotocin-induced diabetic rats.
Glycated haemoglobin or HbA1c reflects the average concentration of glucose in blood for a prolonged period of time and HbA1c level are generally found elevated above normal in the untreated or late diagnosed diabetes. Hence the reduction in HbA1c level reflects the effect control of blood glucose level. In the present study animals showing HbA1c level 10 and above were selected for study. Figure 10 shows that the oral administration of Aqueous fraction of M.charantia declined the HbA1c level to the tune of 24.1% (p<0.01) which was close to the 40.8% (p<0.01) reduction showed by metformin treated group on 30th day.
(9) Effect of Aqueous fraction of Aqueous extract of M.charantia fruits on lipid profile of high dose streptozotocin-induced diabetic rats.
High dose STZ-induced diabetic animals which develop high HbA1c level and other complications in long duration are generally lean animals with disturbed lipid profile also but the level of triglycerides and cholesterol are not as much elevated as in diet induced model. In the present model medium elevation of triglycerides, total cholesterol and LDL was noticed and slight declination of HDL level. Table 6 shows that the plasma triglyceride level was found reduced by 25.4% (p<0.01) while total cholesterol and LDL-cholestrol were dropped down by 18.2% (p<0.05) and 38.8% (p<0.01) on day 30 of treatment. HDL-cholesterol level was found raised by the level of 13.7% which is not significant on the above said day of treatment. There was no considerable improvement in metformin treated group except the plasma LDL-cholesterol which was found reduced by the significant level of 18.3% (p<0.05) on final day of treatment.
(10) Effect of Aqueous fraction Aqueous extract of M.charantia fruits on Hepatic and Renal parameters of Streptozotocin-induced diabetic rats
It is evident from Table 7 and 8 that Aqueous fraction treated group showed marked decline in plasma AST and ALT as well as urea, uric acid and creatinine level which clearly reflects improvement in hepatic and renal performance as compared to the untreated control group.
Effect of aqueous fraction of M.charantia fruits on activated stress signaling pathway in the kidney of STZ-induced diabetic rats:
To determine the effect of hyperglycemia in the kidney of STZ-induced diabetic rats treated with aqueous fraction of M.charantia, the expression of p-PKCδ was studied and figure 11 shows no significant change of expression in M.charantia treated rats in comparison to STZ-untreated rats.
Effect of aqueous fraction of M.charantia fruits on expression of pp-38(MAPK) ,ERK1/2 and JNK1/2 in the kidney of STZ-induced diabetic rats
The effect of M.charantia treatment on glucose-induced changes in MAPK family members was also studied. MAPKs have been identified as transducers linking high glucose to biochemical deficits in diabetes. p38 mediates responses to osmotic stress including the regulation of genes such as aldose reductase together with JNK which is activated by oxidative stress (208). Aldose reductase-sensitive phosphorylation and nuclear migration of p38 MAPK has been demonstrated in DRG sensory neurons of diabetic rodents and specific inhibition of p38 prevents Na+ channel phosphorylation (346). Figure 12 demonstrates a clear increase in pp38 MAPK in response to increasing glucose. (Yuan et al 2009).But on the contrary no change was observed in pp38 and its associated members (JNK1/2 and ERK1/2 )
Effect of aqueous fraction of M.charantia fruits on the expression of IKKβ, NFκβ and antiinflamatory AKT in the kidney of STZ-induced diabetic rats
In order to assess the effect of aqueous fraction of M.charantia on IKKβ expression in the STZ induced kidney, the immunoblotting of IKKβ and NFκβ were performed and its effect on anti-apoptotic molecules such as AKT was also analysed. As it is clear from figure 13, that the fraction did not showed any significant effect in expression level of IKKβ, NFκβ and phosphorylated AKT .
Effect of aqueous fraction of M.charantia fruits on apoptotic markers (caspase-3, pp53 and cleaved PARP-1)
High glucose concentrations found in diabetic patients trigger cellular apoptosis. As given such an elaborated knowledge of effect of pro-apoptotic molecules the effect of the fraction on these apoptosis triggered protein expression that is activated–caspase-3 was studied and the results in figure 14 and 15 indicates no change in expression of active-caspase-3 and cleaved PARP-1 in treated group when compared to untreated STZ induced rats.
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Momordica charantia is a native to tropics (Hamissou, 2013) and its medicinal properties are mentioned in ancient literatures (Kumar, 2010). It also forms the part of many polyherbal antidiabetic formulations and also known for anticancer, antibacterial, antiulcer, antifertility, antihelminthic, antimalerial, antipsoriasis and immunomodulatory activities (Dasgupta, 2011; Sharma, 2011). The previous study confirmed the significant antihyperglycemic effect of aqueous extract in various animal models and therefore it was further fractionated to obtain butanol and aqueous fraction. Both these fractions showed significant antihyperglycemic activity in STZ-induced diabetic rats although higher activity was observed in aqueous fraction and therefore the same was selected for the study of secondary complications of diabetes.
Aqueous fraction treatment in L6 cells enhanced basal as well as insulin-stimulated glucose uptake in concentration dependent manner. GLUT4 translocation and distribution is vital in the glucose upatake by cells (Leney and Tavare, 2009). Effect of aqueous fraction on GLUT4 expression was studied by treating L6 myotubes with aqueous fraction and as a result expression of GLUT4 significantly increased at both mRNA and protein level. Hence the increase in glucose uptake was due to the upregulation of the GLUT4 expression by L6 myotubes. Present study also suggests that the aqueous fraction increased tyrosine phosphorylation of IRS-1 in L6 myotubes and also increased the mRNA level of the same. PI3K xpression was also found increased in treated L6 myotubes. Beside this the aqueous fraction also increased mRNA level of AKT in L6 myotubes and also stimulated the phosphorylation of AKT at Ser-473 suggesting that the stimulatory effect of aqueous fraction of A.indica on glucose uptake is mediated via PI-3-K/AKT pathway.
Further the aqueous fraction was subjected to the multiple dosing in HFD-STZ rat model which shares some characteristics with human type 2 diabetes (Salama et al., 2013). Fasting blood glucose level and oral glucose tolerance was markedly improved in aqueous fraction treated group which supports the outcome of in vitro study showing increased glucose uptake by treated cells. Dyslipidemia is the characteristic feature of HFD-STZ rats (Panchal and Brown, 2011) and aqueous fraction effectively restored the lipid profile of treated rats by the significant lowering of triglycerides, total cholesterol, LDL and enhancing the plasma HDL level. Declined level of hepatic transaminases and plasma level of urea, uric acid and creatinine indicates towards hepato and reno protective action of aqueous fraction.
Since the diabetic complications are more severe in low dose STZ-induced diabetic rats with untreated hyperglycemia of several weeks reflected in elevated level of HbA1c. Therefore the aqueous fraction was also studied in low dose STZ-induced diabetic rats showing abnormally high level of HbA1c. Such animals were treated with aqueous fraction for one month and there was significant improvement in fasting blood glucose level and oral glucose tolerance of treated animals. The improvement in fasting blood glucose was well reflected in the declination of HbA1c level of the treated animals by the significant extent. Plasma triglycerides, total cholesterol and LDL were significantly reduced and HDL-level was raised significantly which confirms the antidyslipidemic effect of aqueous fraction in diet induced model. There was also marked lowering of hepatic transaminases and plasma level of urea, uric acid and creatinine indicating towards the hepato and reno protective activity of aqueous fraction of M.charantia. Further study was carried on to see the effect of M.charantia on kidney of STZ-induced rats .The study revealed that M.charantia does not showed any significant effect on any of pro-apoptotic or stress-inducing pathway. Thus indicating that this fraction does not exerts significant effect on oxidative-stress induced nephropathy, therefore it was not relevant to move forward towards compounds elucidation and study.
Therefore it may be concluded that aqueous fraction of aqueous extract of M.charantia fruits is moderately effective in control of diabetic hyperglycemia and dyslipidemia and also improves hepatic and renal function but less effective against secondary complications like nephropathy.
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