Herbal formulation, DIA-2 and Rosiglitazone ameliorates hyperglycemia and hepatic steatosis in type 2 diabetic rats.

DIA-2 is a herbal mixture containing standardized extract of Allium sativum and Lagerstroemia speciosa. Recently we have reported the anti-diabetic effect of DIA-2 in high fat diet (HFD) and streptozotocin (STZ) induced type 2 diabetic (T2D) rats. The purpose of this study was to investigate and compare the effects of DIA-2 with Rosiglitazone (RG) on plasma biomarkers of hepatocellular injury, liver carbohydrate metabolizing enzymes, glycogen content, oxidant/antioxidant status and histopathological changes in T2D rats. ALT and ALP levels were significantly decreased after DIA-2 and RG treatment compared to T2D rats. Total protein and albumin remained unaltered in all the groups. Significant decrease in AST levels were observed after DIA-2 (125 mg/kg) and RG treatment. Hepatic hexokinase activity was significantly increased after RG and DIA-2 treatment and fructose-1, 6-bisphosphatase activity were inversely correlated with hexokinase activity. Hepatic gucose-6-phosphatase activity was significantly (p < 0.05) reduced after DIA-2 (62.5 mg/kg) and RG treatment. Lipid peroxides levels was significantly decreased in the liver of DIA-2 (62.5; p < 0.01 & 125 mg/kg; p < 0.05) treated animals. Hepatic glycogen content (p < 0.05) and antioxidant enzymes [SOD (p < 0.01; 62.5 mg/kg); GPx and GSH (125 mg/kg; p < 0.01)] were significantly increased after DIA-2 treatment. RG treatment on hepatic glycogen, GPx (p < 0.01) and SOD, GSH (p < 0.05) levels were significant when compared to T2D rats. These biochemical parameters were also correlated with histopathological evaluation. The above findings revealed that administration of DIA-2 could ameliorate the biochemical and histopathological changes in liver of T2D rats indicating the protective role of DIA-2 against HFD/STZ induced diabetes. In addition, DIA-2 and RG treatment resulted in amelioration of hepatic steatosis in T2D rats.

DIA-2, a polyherbal formulation ameliorates hyperglycemia and protein-oxidation without increasing the body weight in type II diabetic rats.

BACKGROUND: The dried bulbs of Allium sativum (Garlic) and leaves of Lagerstroemia speciosa (Banaba) are used as medicinal food for the treatment of diabetes and other ailments. AIM: The present study was undertaken to ascertain whether the combination of both garlic and banaba extract produces synergistic therapeutic effect in diabetic state. METHODS: In the in vitro studies, the effect of standardized aqueous extract of Allium sativum (ASE), methanolic extract of Lagerstroemia speciosa (LSE) and their mixture (1:1 ratio), DIA-2 on insulin stimulated glucose uptake in 3T3-L1 cells, erythrocyte sorbitol accumulation and protein glycation were evaluated. Impetus from the in vitro findings triggered to screen the anti-diabetic potential of DIA-2 in rat model of type II diabetes and associated oxidative stress. In the in vivo studies, acute oral toxicity of DIA-2 was determined following OECD-423 guidelines in female rats. Anti-diabetic activity of DIA-2 was investigated in high fat diet/low dose streptozotocin induced type II diabetes at four dose levels (62.5, 125, 250 and 500 mg/kg b.w) in rats. RESULTS: Combination of ASE and LSE produced synergistic and a dose dependent increase in glucose uptake in 3T3 adipocyte cell lines when compared to the individual extracts. A similar effect was observed in the inhibition of sorbitol accumulation and protein glycation tests. DIA-2 restored the glucose and lipid level near to normal level without gain in body weight which is the most commonly encountered side effect with the use of conventional antidiabetic agents, particularly insulin, insulin secretagogues, sulfonylureas and thiazolidinediones. DIA-2 also decreased hepatic protein carbonyl content levels significantly in the diabetic rats. CONCLUSIONS: The study concluded that DIA-2 posses potent anti-diabetic activity and anti-oxidant effects.  

In vitro cytotoxic, antioxidative and alpha-glucosidase inhibitory potential of a herbal mixture comprised of Allium sativum and Lagerstroemia speciosa.

BACKGROUND: Hyperglycemia induced over production of free radicals in the mitochondrial electron transport chain is now considered as one of the central mechanisms in the pathogenesis of diabetic complications. Allium sativum and Lagerstroemia speciosa contains active principles possessing anti-diabetic and antioxidant properties. This study is aimed at evaluating the evidence that supports this traditional claim and investigates the possible synergistic effect on these herbs when given as a herbal mixture in vitro. AIM: The present study investigates the cytotoxic, antioxidant and a-glucosidase inhibitory potential of Allium sativum (ASE), Lagerstroemia speciosa (LSE) and their combinations using in vitro methods. MATERIALS AND METHODS: The total phenol, total flavonoid and total tannin content were determined in ASE and LSE. The cytotoxic effects of ASE, LSE and their combination in the ratio of 1:2, 1:1 w/w were evaluated using 3T3 L1 preadipocyte cells. Effect of ASE, LSE and its mixture on intracellular reactive oxygen species (ROS) production were determined by 2', 7'dichlorfluorescein diacetate (DCF DA) staining technique in 3T3-L1 adipocytes. The ability of the herbal extracts and their combination to scavenge super oxide radicals and to inhibit alpha-glucosidase enzyme (a carbohydrate metabolising enzyme) were measured using in vitro methods. RESULTS: The total phenols and tannins were expressed as microgram (microg) of gallic acid equivalents/mg of extract (GAE/mg), flavonoids as microg of quercetin equivalents/mg of extract (QE/mg). LSE had significant higher total phenol (300.11 +/- 1.99), flavonoid (53.12 +/- 0.48) and tannin content (118.90 +/- 0.15) compared to ASE which possessed total phenol (159.93 +/- 0.87); flavonoid (9.37 +/- 0.73) and tannin content (80.5 +/- 0.19). The IC50 value, the concentration of the extracts that cause 50% inhibition or cell death was measured as an index of cytotoxicity. The IC50 value was found to be in the following decreasing order: 1:2 mixture (98 microg/ml) > ASE (323.6 microg/ml) > 1:1 mixture (428.1 microg/ml) > LSE (2154 microg/ml). The 1:1 mixture was comparatively less cytotoxic under the tested concentration range (1 x 10(0) pg - 1 x 10(8) pg) than 1:2 combinations. The results observed with lactate dehydrogenase (LDH) release were similar to that of cell viability assay. The 1:1 mixture (DIA-2 hereafter) was considered for further investigations. DIA-2 inhibited the ROS levels, which is evidenced by the decreased DCF fluorescence. DIA-2 could also efficiently scavenge the super oxide radical generated from PMS/NADH-NBT system showing an IC50 value 69.99 microg/ml, the IC50 value of ASE (157.7 microg/ml), LSE (20.43 microg/ml), and ascorbic acid (49.64 microg/ml) used as positive control. The results of in vitro a-glucosidase inhibitory assay showed highest IC50 value with LSE (0.3 microg/ml) and DIA-2 (0.7 microg/ml) than ASE (136.3 microg/ml) and positive control miglitol (651.8 microg/ml). CONCLUSIONS: DIA-2 exerts synergistic effect in scavenging the ROS and inhibiting the enzyme alpha-glucosidase in vitro compared to its individual extracts. The possible synergistic therapeutic effects may be due the presence of the antioxidant rich flavonoids, phenols and tannins present in LSE and ASE.

Combinatorial chemopreventive effect of Azadirachta indica and Ocimum sanctum on oxidant-antioxidant status, cell proliferation, apoptosis and angiogenesis in a rat forestomach carcinogenesis model.

INTRODUCTION: We investigated the combinatorial chemopreventive efficacy of Azadirachta indica (AI) and Ocimum sanctum (OS) against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinogenesis, based on changes in oxidant-antioxidant status, cell proliferation, apoptosis and angiogenesis. METHODS: Male Wistar rats were assigned to four groups. Rats in groups 1 and 2 received MNNG (150 mg/kg body weight i.g.) three times with a gap of two weeks in between the treatment. Group 2 rats additionally received ethanolic AI (100 mg/kg body weight i.g.) and OS (150 mg/kg body weight i.g.) leaf extract three times per week for 26 weeks. Group 3 animals were given AI and OS leaf extract alone, whereas group 4 served as the control. RESULTS: Lipid and protein oxidation and status of the antioxidants, superoxide dismutases, catalase, reduced glutathione (GSH) and GSH-dependent enzymes together with markers of proliferation (proliferating cell nuclear antigen [PCNA], glutathione S-transferase-Pi [GST-P]), invasion (cytokeratin [CK]), angiogenesis (vascular endothelial growth factor [VEGF]) and apoptosis (Bcl-2, Bax, cytochrome C and caspase-3) were used to biomonitor chemoprevention. Rats administered MNNG developed forestomach carcinomas that displayed low lipid and protein oxidation coupled to enhanced antioxidant activities, and overexpression of PCNA, GST-P, CK, VEGF and Bcl-2 with downregulation of Bax, cytochrome C and caspase-3. Coadministration of AI and OS extract suppressed MNNG-induced gastric carcinomas accompanied by modulation of the oxidant-antioxidant status, inhibition of cell proliferation and angiogenesis, and induction of apoptosis. CONCLUSION: The results of the present study suggest that chemoprevention by AI and OS combination may be mediated by their antioxidant, antiangiogenic, antiproliferative and apoptosis inducing properties.

Proliferation, angiogenesis and apoptosis-associated proteins are molecular targets for chemoprevention of MNNG-induced gastric carcinogenesis by ethanolic Ocimum sanctum leaf extract.

INTRODUCTION: This study was designed to evaluate the chemopreventive effects of ethanolic Ocimum sanctum (OS) leaf extract on cell proliferation, apoptosis and angiogenesis during N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinogenesis. METHODS: The rats were divided into four groups of ten each. Rats in group one were given MNNG (150 mg/kg body weight) by intragastric intubation three times, with a two-week interval between treatments. Rats in group two were administered MNNG as in group one, and in addition, they received intragastric intubation of ethanolic OS extract (300 mg/kg body weight) three times per week, starting on the day following the first exposure to MNNG. The intubation of ethanolic OS extract continued until the end of the experimental period. Rats in group three were given ethanolic OS leaf extract only. Group four served as controls. All the rats were killed after an experimental period of 26 weeks. RESULTS: Intragastric administration of MNNG-induced well-differentiated squamous cell carcinomas that showed increased cell proliferation, and angiogenesis with evasion of apoptosis, as revealed by the upregulation of proliferating cell nuclear antigen (PCNA), glutathione S-transferase-pi (GST-pi), Bcl-2, cytokeratin (CK) and vascular endothelial growth factor (VEGF) and with downregulation of Bax, cytochrome C and caspase 3 protein expression. Administration of ethanolic OS leaf extract reduced the incidence of MNNG-induced gastric carcinomas. This was accompanied by decreased expression of PCNA, GST-pi, Bcl-2, CK and VEGF, and overexpression of Bax, cytochrome C, and caspase 3. CONCLUSION: This study provides evidence that, in MNNG-induced gastric carcinogenesis, the key proteins involved in the proliferation, invasion, angiogenesis and apoptosis, are viable molecular targets for chemoprevention using ethanolic OS leaf extract.