Materials and methods
2.4 In vitro studies
2.4.1 Estimation of total polyphenol content
The total polyphenol content of each extracts and/or fraction was determined (as gallic acid equivalent) according to the method described by McDonald et al. (2001) with slight modifications.
Briefly, a 200 µL of the extract (240 µg/mL) was incubated with 1 mL of 10 times diluted Folin Ciocalteau reagent and 800 µL of 0.7 M Na2CO3 for 30 minutes at room temperature. The absorbance values were then determined at 765 nm in a Shimadzu UV mini 1240 spectrophotometer (Shimadzu Corporation, Kyoto, Japan). All measurements were done in triplicate.
Quantification was done on the basis of a standard curve of gallic acid. Results were expressed mg/g (GAE) and calculated using following formula,
Total polyphenol content (mg/g GAE) = [GAE (mg/mL) × V (mL)]/W (g) Where GAE (mg/mL) = (y-c)/m
GAE: Gallic acid equivalent (mg/mL), V: Total volume of sample (mL), W: Sample weight (g), y: absorbance of the sample, c: intercept at the y-axis, m: slope of the standard curve
2.4.2 Determination of total flavonoid content
The total flavonoid content of the plant extracts and/or fractions was determined using a method reported by Chang et al. (2002) with slight modification. Briefly, a 500 µL (240 µg/mL) of each sample was mixed with 500 µL methanol, 50 µL of 10% AlCl3, 50 µL of 1 mol/L potassium acetate and 1.4 mL water, and allowed to incubate at room temperature for 30 minutes. The absorbance of the reaction mixture was subsequently measured at 415 nm using the spectrophotometer mentioned above. The total flavonoid content was calculated as quercetin equivalent (QE) in mg per g of dry extract.
Quantification was done on the basis of a standard curve of quercetin. Results were expressed mg/g (QE) and calculated using following formula,
Total polyphenol content (mg/g QE) = [QE (mg/mL) × V (mL)]/W (g) Where QE (mg/mL) = (y-c)/m
QE: quercetin equivalent (mg/mL), V: Total volume of sample (mL), W: Sample weight (g), y:
absorbance of the sample, c: intercept at the y-axis, m: slope of the standard curve.
2.4.3 DPPH radical scavenging activity
The total free radical scavenging activity of the extracts and/or fractions was determined and compared to that of ascorbic and gallic acids by using a slightly modified method described by Tuba and Gulcin (2008). An aliquot of 500 µL of a 0.3 mM solution of 1,1-diphenyl-2-picryl-hydrazyl (DPPH) in methanol was added to 1 mL of the extracts and/or fractions at different concentrations (30, 60, 120 and 240 µg/mL). These solutions were mixed and incubated in the dark for 30 minutes at room temperature. The absorbance was measured at 517 nm against blank samples lacking the free radical scavengers.
The results of the DPPH were expressed as a percentage of the control (blank) according to the following formula:
% Inhibition = [(Abs of control-Abs of sample)/Abs of control] x 100
2.4.4 Ferric (Fe
3+) reducing anti-oxidant power assay
The ferric reducing anti-oxidant power method of Oyaizu (1986) was used with slight modifications to measure the reducing capacity of the samples. To perform this assay, 1 mL of each extract and/or fractions (30, 60, 120 and 240 µg/mL) were incubated with 1 mL of 0.2 M sodium phosphate buffer (pH 6.6) and 1% potassium ferricyanide at 50°C for 30 minutes. After 30 minutes incubation, the reaction mixtures were acidified with 1 mL of 10% trichloroacetic acid. Thereafter, 1 mL of the acidified sample of this solution was mixed with 1 mL of distilled water and 200 μL of FeCl3
(0.1%). The absorbance of the resulting solution was measured at 700 nm in a spectrophotometer.
Increased absorbance of the reaction mixture indicated the greater reductive capability of the extracts (Gulcin et al. 2004).
The results of the ferric reducing anti-oxidant power assay was expressed as a gallic acid equivalent according to the following formula:
% Inhibition = (Abs of sample/Abs of Gallic acid) x 100
2.4.5 Inhibition of hemoglobin glycation
Inhibition of non-enzymatic glycosylation of hemoglobin by various extracts and/or fractions was measured by the modified method of Pal and Dutta (2006). Glucose (2%), hemoglobin (0.06%) and gentamycin (0.02%) solutions were prepared in phosphate buffer 0.01 M, pH 7.4. An aliquot of 1 mL of each of the solution above was mixed with 1 mL of extracts with different concentrations (30, 60, 120 and 240 µg/mL) in dimethyl sulfoxide (DMSO). These mixtures were incubated in the dark at room temperature for 72 hour. The percentage inhibition of glycosylation of hemoglobin was calculated from the absorbance measured at 520 nm. Gallic acid was used as a standard.
The results of the inhibition of hemoglobin glycation were expressed as a percentage of the control (blank) according to the following formula:
% Inhibition = [(Abs of control-Abs of sample)/Abs of control] x 100
2.4.6 α-Amylase (E.C. 3.2.1.1) inhibitory effect
The α-amylase inhibitory effect of the extracts and/or fractions was carried out using a modified method of McCue and Shetty (2004). Briefly, a 250 µL aliquot of extracts, fractions and/or compounds at different concentrations (30, 60, 120 and 240 µg/mL) was placed in a tube and 250 µL of 0.02 M sodium phosphate buffer (pH 6.9) containing α-amylase (2.0 U/mL) solution was added. This solution was pre-incubated at 25°C for 10 minutes, after which 250 µL of 1% starch solution in 0.02 M sodium phosphate buffer (pH 6.9) was added at a time interval of 10 seconds and then further incubated at 25°C for 10 minutes. The reaction was terminated after incubation by adding 1 mL of dinitrosalicylic acid (DNS) reagent. The tube was then boiled for 10 minutes and cooled to room temperature. The reaction mixture was diluted with 5 mL distilled water and the absorbance was measured at 540 nm using a Shimadzu UV mini 1240 spectrophotometer. A control was prepared using the same procedure, replacing the extract with distilled water.
The results of the α-amylase assay were expressed as a percentage of the control (blank) according to the following formula:
% Inhibition = [(Abs of control-Abs of sample)/Abs of control] x 100
2.4.7 α-Glucosidase (E.C. 3.2.1.20) inhibitory effect
The inhibitory effect of the plant extracts and/or fractions on α-glucosidase activity was determined according to the method described by Kim et al. (2005) using α-glucosidase from Saccharomyces cerevisiae. The substrate solution 5.0 mM p-nitrophenylglucopyranoside (pNPG) was prepared in 20 mM phosphate buffer, pH 6.9. An aliquot of 500 µL of α-glucosidase (1.0 U/mL) was then pre-incubated with 250 µL of the different concentrations of the extracts, fractions and/or compounds (30, 60, 120 and 240 µg/mL) for 10 minutes. Thereafter, a 250 µL of 5.0 mM pNPG was dissolved in 20 mM phosphate buffer (pH 6.9) as a substrate to start the reaction. The reaction mixture was incubated at 37°C for 30 minutes. The α-glucosidase activity was determined by measuring the yellow coloured p-nitrophenol released from pNPG at 405 nm.
The results of the α-glucosidase assay were expressed as a percentage of the control (blank) according to the following formula:
% Inhibition = [(Abs of control-Abs of sample)/Abs of control] x 100
2.4.8 Calculation of IC
50values
Concentrations of extracts,fractions or compounds resulting in 50% inhibition of radical or enzyme activities (IC50) were calculated from the plot of percentage inhibition against log(concentration of the samples).
IC50= 10x
Where x (mg/mL) = (y-c)/m
y: 50%, c: intercept at the y-axis, m: slope of the graph of log(concentration of sample used) against the percentage inhibitions.
2.4.9 Mechanism of α-glucosidase and α-amylase inhibitions
The most active fractions or compounds were subjected to kinetic experiments to determine the type of inhibition exerted on α-glucosidase and α-amylase. The experiment was conducted according to the protocols as described above at a constant concentration of the fractions or compounds (30 µg/mL) with a variable concentration of substrate. For the α-glucosidase inhibition assay, 0.313-5.0 mmol/L of pNPG was used and 0.063-1.0% of starch was used for the α-amylase inhibition assay. The initial rates of reactions were determined from calibration curves constructed using varying concentrations of p-nitrophenol (0.313-5.0 mmol/L) and maltose (0.063-1.0%) for the α-glucosidase and α-amylase inhibition assays, respectively. The initial velocity data obtained were used to construct Lineweaver-Burke’s plot to determine the KM (Michaelis constant) and vmax (maximum velocity) of the enzyme as well as the Ki (inhibition binding constant as a measure of affinity of the inhibitor to the enzyme) and the type of inhibition for both enzymes.
2.4.10 Gas Chromatography-Mass Spectroscopic (GC-MS) analysis
Based on the results of in vitro anti-oxidative and anti-diabetic studies, the most active extracts and/or fractions were subjected to GC-MS analysis. The GC-MS analysis was conducted with an Agilent technology 6890 GC coupled with an Agilent 5973 Mass Selective Detector and driven by Agilent Chemstation software. Compounds were identified by direct comparison of the retention times and mass spectral data with those in the National Institute of Standards and Technology (NIST) library.