Investigation of Phytochemical and Biological Properties on Traditional Medicinal Plants for Hypoglycemic Activity

The herbal medicines are the major remedy in traditional medicinal system and are being used in medicinal practice for thousands of years. These have made a great contribution in maintaining human health. Diabetes mellitus is a metabolic disorder of hyperglycemia with disturbances of carbohydrate, lipid and protein metabolism resulting from defects of insulin secretion, insulin action and both. Management of diabetes mellitus without any side effects is still a challenge to the medical system. This is the increasing demand by patients to use natural products with hypoglycemic activity, because insulin and oral hypoglycemic drugs have undesirable side effects. Medicinal plants are good sources of natural antioxidants believed to exert their effects. In order to identify the herbal based plant drug, the present study was focused on pharmacognostical, phytochemical and pharmacological studies of Barleria noctiflora. Barleria noctiflora was collected during the winter season in and around Erode District, Tamilnadu, India. It was identified and authenticated by Prof. P. Jayaraman, Director, National Institute of Herbal Science, Chennai-45, Tamilnadu, India. This is the first report of pharmacognostical studies on the leaf and stem parts of Barleria noctiflora. Pharmacognostical studies that microscopical, macroscopical, physicochemical constituents and fluorescent analysis were carried out; these are valuable tools for selected plant and identification of powdered drugs. The dried plant materials were subjected to extraction by Soxhlet process separately. All the extracts were concentrated to dryness under reduced pressure and controlled temperature. The ethanol extract was further fractionated with ethyl acetate and n-butanol. The extract was used to in vitro antioxidant and the fractions were used in vitro and in vivo pharmacological studies. The phytochemical studies of extracts showed the presence of flavonoides, alkaloids, saponins, steroids, glycosides, tannins, phenols, protein and carbohydrates in various extracts. The crude drugs of powder presence of vitamin C, vitamin D and inorganic elements like sodium, iodine, phosphate, and chloride. The total phenolic and flavonoids content of Barleria noctiflora of crude ethanolic and aqueous extract was obtained using the regression calibration curve and is expressed as tannic acid and rutin equivalents. The total phenolic and flavonoids content was higher in the ethanolic extract compared to the aqueous extract. It shows the ethanolic extract of Barleria noctiflora posse’s high antioxidant ability. The IC50 values were found to be more than 100 μg/ml for all the extracts indicating the nontoxic nature of the extracts. Among, the five extracts of Barleria noctiflora tested for in vitro antioxidant activity, the ethanol extract of Barleria noctiflora exhibited potent antioxidant activity when compared to other extracts. Hence, the study of in vitro antioxidant activity shows potent in ethanol extract selected and fractioned with different solvents from the ethyl acetate and nbutanol fractions were tested for in vitro antidiabetic activity by inhibition of α-amylase and inhibition of α-glucosidase. The results were expressed as IC50, which is the concentration of the fraction to inhibit enzyme by 50%. The in vitro α-amylase and α- glucosidase inhibitory studies demonstrated EAFBN had inhibitory activity of intestinal digestive enzyme. Hence, ethyl acetate fraction of Barleria noctiflora (EAFBN) was selected for in vivo evaluat of acute toxicity, sub acute toxicity, anti diabetic activity and wound healing activities. In acute toxicity studies, the LD50 was determined as per OECD guidelines for fixing the dose for biological evaluation. The LD50 of the ethyl acetate fractions of Barleria noctiflora (EAFBN) falls under category 4 values with no death and no signs of acute toxicity at doses of 2000 mg/kg. The subacute studies, the treatment of EAFBN 100, 250 and 500mg/kg body weight doses was no changes of body and organ weight was observed at all doses. The significant (p<0.01) reduction of serum glucose level, total cholesterol, triglyceride and LDL-Cholesterol, hence HDL-Cholesterol level was significant (p<0.01) increases. There might be due to the presence of hypoglycemic components in the ethyl acetate fraction of Barleria noctiflora. The liver parameter of AST was showed no significant changes and the ALT level was significant (p<0.01) changes, but the values are in between of the normal values. There is no significant change in hematological parameter like RBC, WBC, Hb and PCV. This observation gives credence to the use of the herbal product as a hypoglycemic agent and showed reduced the risk factors of CHD. So the result reveals, there was no pathological indication in toxicity study. Histopathological studies of EAFBN treated 100, 250 and 500 mg/kg body weight doses of animal no degenerative changes and exhibited normal architecture with glomerular and tubular structures in the kidney. The histology of liver exhibited normal liver parenchyma with no distortion of architecture, with normal lobules, acini, and no bile stasis kuffer cell prominence. Overall, these findings support the hypothesis of EAFBN reduce the risk of toxicity in the long duration of drug administration and reduce the risk of diabetes and cardiovascular patients. The treatment with the EAFBN at the dose of 500 mg/kg body weight showed the normal structure of kidney and liver tissue. In an OGTT, the EAFBN doses of 100, 200 & 400 mg/kg have shown increase the tolerance for glucose and blood glucose levels were significantly reduced in the dose dependent after oral administration (2g/kg). The EAFBN tested drug showed significant activity, according to the dose dependent manner. In hypoglycemic activity, the decrease body weight might be due to the low utilization of uptake blood sugar in the cell. While there was a significant (p < 0.01) gain of body weight in EAFBN treated diabetic rats when compared to diabetic control. The administration of EAFBN restores body weight seems to be a result of its facility to reduce hyperglycemia. The treatment with the EAFBN significant (p < 0.01) decrease in blood glucose level was observed in HFD-STZ induced diabetic rats, when compared to diabetic rats. EAFBN at 100, 200 and 400 mg/kg caused a significant (p < 0.01) reduction in serum lipid profile of total cholesterol, triglycerides, LDL- Cholesterol and significant (p < 0.01) increase in HDL-Cholesterol level was observed when compared to diabetic rats. The liver parameters were ALT, AST and ALP significant decrease in EAFBN treated rats when compared to diabetic rats. A significant (P < 0.01) increase the level of insulin and significantly decreases the level of HbA1c was observed in EAFBN (100, 200 and 400mg/kg) body weight dose when compared to diabetic control. The renal parameters like serum urea and creatinine level shows significant (p < 0.01) decrease was observed in for EAFBN (100, 200 and 400mg/kg) body weight dose when compared to diabetic control. There was a marked significant (p < 0.01) reduction in the liver glycogen levels of diabetic rats and EAFBN (100, 200 and 400mg/kg) treatment showed significant (p < 0.01) increase in liver glycogen levels when compared with the diabetic rats. The glucose uptake by hemidiaphragms showed a significant reduction of diabetic rats when compared to control group. The glucose uptake were significant (p < 0.01) enhancement of rats treated with EAFBN (100, 200 and 400mg/kg), when compared to diabetic control. The glucose transport showed a significant (p < 0.01) enhancement in diabetic rats as compared to normal rats. Also, both EAFBN (100, 200 and 400 mg/kg) treated rats showed a significant (p<0.01) inhibitory effect on glucose transport in liver tissue. In histopathology of the pancreas, β-cells of pancreatic islets were damage in diabetic animals. There was the regeneration of β-cells in EAFBN treatment groups since the cells recovered from the initial injury. The treatment with the EAFBN at the dose of 400 mg/kg body weight showed slightly normal structure of pancreas tissue. These results suggest the potent hypoglycemic nature of the EAFBN and support the CHD risk and other complications in type 2 diabetes. The EAFBN was assessed for the wound healing activity in rats using excision and incision wound models. The percentage closure of the excision wound area for the control rats was found to be 15.35±0.07 to 90.1±0.27 between 4 to 16 days of the experiment. The treatment with the EAFBN at 5% w/w and 10% w/w showed a percentage closure, 19.5 ± 0.42 to 95.8 ± 0.27 and 19.5 ± 0.18 to 96.6 ± 0.10 between 4 to 16 days of the treatment. All the values were found in significant (p < 0.01), when compared to diabetic control animals. The complete epithelization was observed in the same day of EAFBN (10% w/w) and standard drug, hence diabetic control rats (23.66 ± 0.55) were 5 days increase the period of epithelization compare to normal control (18.83 ± 0.16). In the incision wound model, the animal treated with the topical application of EAFBN 10% w/w treated animals skin breaking strength and standard drug have shown more or less same skin breaking strength. All the values were found in significant (p < 0.01), when compared to diabetic control animals. In the present investigation the Barleria noctiflora fraction promotes significant wound healing activity by increasing proliferation, formation of granulation tissue, synthesis of collagen and increase the rate of wound contraction compared with diabetic control. Based on the results, we can conclude the plant of Barleria noctiflora could be used for diabetes and to prevent the complications in type 2 diabetes. The preliminary phytochemical tests of the extract indicated the presence of flavonoides, alkaloids, saponins, steroids, glycosides, tannins. Many of these are known to possess antioxidant, hypoglycemic and wound healing activities. These activities observed in the fraction may be due the presence of flavonoids, tannins and phenolic in the EAFBN fraction. The ethyl acetate fraction of Barleria noctiflora was subjected to column chromatography and isolation of active constituents. The compound was isolated and their purity was confirmed by TLC using different solvent systems. Based on the spectral analysis viz, IR, NMR and UV spectra, they were identified as Acyclic a, b unsaturated ketone. In conclusion, the present study provides the phytochemical and biological investigation of the Barleria noctiflora plant. The plant extracts were shown to possess in vitro antioxidant properties. Among them, the ethanolic extract Barleria noctiflora was found to be most potent. The in vivo studies carried out ethyl acetate fraction of Barleria noctiflora rats proved the potent hypoglycemic activity. Detailed biochemical and histological evidence was provided to prove the activity. The ethyl acetate fraction of Barleria noctiflora was also found to possess in vivo wound healing activity in excision and incision models. In the phytochemical analysis, Acyclic a, b unsaturated ketone compound was isolated