Plant material
The dried and matured plant parts of thirty Korean medicinal herbs were obtained from “Korean Collection of Herbal Extracts” a Biotech company in Korea. A collection of voucher specimen is available with the company (Korea Collection of Herbal Extracts, 2000).
Extraction
The dried plant parts individually were chopped into small pieces and pulverized into a fine powder. The powdered plant materials (100 g, dry weight) were kept for extensive decoction in 80% methanol for 3 days at room temperature. The extracts were then concentrated using rotary vacuum evaporator at 20-30°C to obtain the dried crude extracts.
Reagents
α-Glucosidase (from Saccharomyces cerevisiae type I) and 4-nitrophenyl α-D-glucopyranoside were purchased from Sigma-Aldrich (St. Louis, MO, USA). Other commercially available reagents and solvents were used as received.
α-Glucosidase assay
The enzyme inhibition activity for α-glucosidase was evaluated according to the method previously reported by Shibano et al. (7 (link)) with minor modifications. The reaction mixture consisted of 50 μL of 0.1 M phosphate buffer (with pH of 7.0), 25 μL of 0.5 mM 4-nitrophenyl α-D-glucopyranoside (dissolved in 0.1 M phosphate buffer, with pH of 7.0), 10 μL of test sample and 25 μL of α-glucosidase solution (a stock solution of 1 mg/mL in 0.01 M phosphate buffer, with pH of 7.0 was diluted to 0.1 Unit/mL with the same buffer, with pH of 7.0 just before assay). This reaction mixture was then incubated at 37°C for 30 min. Then, the reaction was terminated by the addition of 100 μL of 0.2 M sodium carbonate solution. The enzymatic hydrolysis of substrate was monitored by the amount of p-nitrophenol released in the reaction mixture at 410 nm using microplate reader. Individual blanks were prepared for correcting the background absorbance, where the enzymes were replaced with buffer. Controls were conducted in an identical manner replacing the plant extracts with methanol. 1, 2, 3, 4, 6-penta-O-galloyl-β-D-glucose was used as positive control. All experiments were carried out in triplicates. The inhibition percentage of α-glucosidase was assessed by the following formula:
I α-glucosidase% = 100 X (ΔAControl - ΔASample) / ΔAControlΔAControl = ΔATest - ΔABlankΔASample = ΔATest - ΔABlankStatistical analyses
All assays were performed at least three times with triplicate samples. All results are expressed as mean ± SD. IC50 values were only determined for the plant extracts with inhibition ≥ 50% at 5 mg/mL by plotting a percent inhibition versus concentration curve, in which the concentration of sample required for 50% inhibition was determined and expressed as IC50 value.
The dried and matured plant parts of thirty Korean medicinal herbs were obtained from “Korean Collection of Herbal Extracts” a Biotech company in Korea. A collection of voucher specimen is available with the company (Korea Collection of Herbal Extracts, 2000).
Extraction
The dried plant parts individually were chopped into small pieces and pulverized into a fine powder. The powdered plant materials (100 g, dry weight) were kept for extensive decoction in 80% methanol for 3 days at room temperature. The extracts were then concentrated using rotary vacuum evaporator at 20-30°C to obtain the dried crude extracts.
Reagents
α-Glucosidase (from Saccharomyces cerevisiae type I) and 4-nitrophenyl α-D-glucopyranoside were purchased from Sigma-Aldrich (St. Louis, MO, USA). Other commercially available reagents and solvents were used as received.
α-Glucosidase assay
The enzyme inhibition activity for α-glucosidase was evaluated according to the method previously reported by Shibano et al. (7 (link)) with minor modifications. The reaction mixture consisted of 50 μL of 0.1 M phosphate buffer (with pH of 7.0), 25 μL of 0.5 mM 4-nitrophenyl α-D-glucopyranoside (dissolved in 0.1 M phosphate buffer, with pH of 7.0), 10 μL of test sample and 25 μL of α-glucosidase solution (a stock solution of 1 mg/mL in 0.01 M phosphate buffer, with pH of 7.0 was diluted to 0.1 Unit/mL with the same buffer, with pH of 7.0 just before assay). This reaction mixture was then incubated at 37°C for 30 min. Then, the reaction was terminated by the addition of 100 μL of 0.2 M sodium carbonate solution. The enzymatic hydrolysis of substrate was monitored by the amount of p-nitrophenol released in the reaction mixture at 410 nm using microplate reader. Individual blanks were prepared for correcting the background absorbance, where the enzymes were replaced with buffer. Controls were conducted in an identical manner replacing the plant extracts with methanol. 1, 2, 3, 4, 6-penta-O-galloyl-β-D-glucose was used as positive control. All experiments were carried out in triplicates. The inhibition percentage of α-glucosidase was assessed by the following formula:
I α-glucosidase% = 100 X (ΔAControl - ΔASample) / ΔAControlΔAControl = ΔATest - ΔABlankΔASample = ΔATest - ΔABlankStatistical analyses
All assays were performed at least three times with triplicate samples. All results are expressed as mean ± SD. IC50 values were only determined for the plant extracts with inhibition ≥ 50% at 5 mg/mL by plotting a percent inhibition versus concentration curve, in which the concentration of sample required for 50% inhibition was determined and expressed as IC50 value.
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