Detection of Aspartic Proteinase Activities Using Gel Zymography.

Gel zymography is a two-stage process where the proteins from the test sample are first separated by electrophoresis followed by the detection of the activity of hydrolytic enzymes. Many zymography procedures use sodium dodecyl sulfate (SDS) polyacrylamide gels copolymerized with an appropriate substrate. The procedure described here uses native polyacrylamide gel electrophoresis (PAGE) in the absence of both SDS and substrate. In order to visualize aspartic proteinase activity, the gel is impregnated in bovine hemoglobin at pH 3.0 for 15 min after the electrophoresis procedure. Subsequently, the gel is incubated in a humid container in the absence of hemoglobin for 1 h at 37 °C. At the end, the gel is stained with amido black and destained. Clear areas against a dark background corresponding to aspartic proteinase activities can be detected.

Bioassay-guided fractionation and identification of α-amylase inhibitors from Syzygium cumini leaves.

CONTEXT: Pancreatic α-amylase and α-glucosidase inhibitors serve as important strategies in the management of blood glucose. Even though Syzygium cumini (L.) Skeels (Myrtaceae) (SC) is used extensively to treat diabetes; scientific evidence on antidiabetic effects of SC leaves is scarce. OBJECTIVE: SC leaf extract was investigated for α-amylase inhibitory effect and continued with isolation and identification of α-amylase inhibitors. MATERIALS AND METHODS: Bioassay-guided fractionation was conducted using in vitro α-amylase inhibitory assay (with 20-1000 µg/mL test material) to isolate the inhibitory compounds from ethyl acetate extract of SC leaves. Structures of the isolated inhibitory compounds were elucidated using 1H NMR and 13C NMR spectroscopic analysis and direct TLC and HPLC comparison with authentic samples. Study period was from October 2013 to October 2015. RESULTS: An active fraction obtained with chromatographic separation of the extract inhibited porcine pancreatic α-amylase with an IC50 of 39.9 µg/mL. Furthermore, it showed a strong inhibition on α-glucosidase with an IC50 of 28.2 µg/mL. The active fraction was determined to be a 3:1 mixture of ursolic acid and oleanolic acid. Pure ursolic acid and oleanolic acid showed IC50 values of 6.7 and 57.4 µg/mL, respectively, against α-amylase and 3.1 and 44.1 µg/mL respectively, against α-glucosidase. DISCUSSION AND CONCLUSIONS: The present study revealed strong α-amylase and α-glucosidase inhibitory effects of ursolic acid and oleanolic acid isolated from SC leaves for the first time validating the use of SC leaves in antidiabetic therapy.

α-glucosidase and glycation inhibitory effects of costus speciosus leaves.

BACKGROUND: Hyperglycaemia is a salient feature of poorly controlled diabetes mellitus. Rate of protein glycation is increased with hyperglycaemia leading to long term complications of diabetes. One approach of controlling blood glucose in diabetes targets at reducing the postprandial spikes of blood glucose. The objectives of this study were to assess the in vitro inhibitory effects of Costus speciosus (COS) leaves on α-amylase and α-glucosidase activities, fructosamine formation, protein glycation and glycation-induced protein cross-linking. METHODS: Methanol extracts of COS leaves were used. Inhibitory effects on enzyme activities were measured using porcine pancreatic α-amylase and α-glucosidase from Saccharomyces cerevisiae in the presence of COS extract. Percentage inhibition of the enzymes and the IC50 values were determined. In vitro protein glycation inhibitory effect of COS leaves on early and late glycation products were measured using bovine serum albumin or chicken egg lysozyme with fructose. Nitroblue tetrazolium was used to assess the relative concentration of fructosamine and polyacrylamide gel electrophoresis was used to assess the degree of glycation and protein cross-linking in the reaction mixtures. RESULTS: α-Glucosidase inhibitory activity was detected in COS leaves with a IC50 of 67.5 µg/ml which was significantly lower than the IC50 value of Acarbose (p < 0.01). Amylase inhibitory effects occurred at a comparatively higher concentration of extract with a IC50 of 5.88 mg/ml which was significantly higher than the IC50 value of Acarbose (p < 0.01). COS (250 µg/ml) demonstrated inhibitory effects on fructosamine formation and glycation induced protein cross-linking which were in par with 1 mg/ml aminoguanidine were detected. CONCLUSION: Methanol extracts of COS leaves demonstrated in vitro inhibitory activities on α-glucosidase, fructosamine formation, glycation and glycation induced protein cross-linking. These findings provide scientific evidence to support the use of COS leaves for hypoglycemic effects with an added advantage in slowing down protein glycation.

Analysis of glycation induced protein cross-linking inhibitory effects of some antidiabetic plants and spices.

BACKGROUND: Protein cross-linking which occurs towards the latter part of protein glycation is implicated in the development of chronic diabetic complications. Glycation induced protein cross-linking inhibitory effects of nine antidiabetic plants and three spices were evaluated in this study using a novel, simple, electrophoresis based method. METHODS: Methanol extracts of thirteen plants including nine antidiabetic plants and three spices were used. Lysozyme and fructose were incubated at 37 °C in the presence or absence of different concentrations of plant extracts up to 31 days. Standard glycation inhibitor aminoguanidine and other appropriate controls were included. A recently established sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) method was used to detect the products of protein cross-linking in the incubation mixtures. RESULTS: High molecular weight protein products representing the dimer, trimer and tetramer of lysozyme were detected in the presence of fructose. Among the nine antidiabetic plants, seven showed glycation induced protein cross-linking inhibitory effects namely Ficus racemosa (FR) stem bark, Gymnema sylvestre (GS) leaves, Musa paradisiaca (MP) yam, Phyllanthus debilis (PD) whole plant, Phyllanthus emblica (PE) fruit, Pterocarpus marsupium (PM) latex and Tinospora cordifolia (TC) leaves. Inhibition observed with Coccinia grandis (CG) leaves and Strychnos potatorum (SP) seeds were much low. Leaves of Gymnema lactiferum (GL), the plant without known antidiabetic effects showed the lowest inhibition. All three spices namely Coriandrum sativum (CS) seeds, Cinnamomum zeylanicum (CZ) bark and Syzygium aromaticum (SA) flower buds showed cross-link inhibitory effects with higher effects in CS and SA. PD, PE, PM, CS and SA showed almost complete inhibition on the formation of cross-linking with 25 µg/ml extracts. CONCLUSIONS: Methanol extracts of PD, PE, PM, CS and SA have shown promising inhibitory effects on glycation induced protein cross-linking.