This chapter deals with the evaluation of alpha amylase inhibitory activity of different extracts isolated from leaves of Aloe vera L. and leaves of Azadiracta indica A Juss. collected from Bharatpur and Jaipur districts of Rajasthan using different biochemical methods. MATERIAL AND METHODS Plant collection and authentication Leaves of Aloe vera Linn. and leaves of Azadiracta indica A Juss. were collected from different localities of Jaipur and Bharatpur districts. Plant collection and authentication is same as discussed in chapter 1. Extractions: Extraction of plants parts in different polar and non polar solvents (Water, Methanol, Ethanol, Acetone, Pet ether and Toluene) and for their secondary metabolites (Flavonoids and Alkaloids) was carried out as discussed in chapter 1by well established methods. Salivary alpha amylase assay: 1. Starch Iodine color assay (Xiao et al., 2006): Reagents 63
1% Starch solution, Phosphate buffer of ph 6.9 and of 0.02 molarity, Iodine reagent, Salivary alpha amylase enzyme. Procedure: Screening of plant extracts for α-amylase inhibitors were carried out in test tubes according to Xiao et al. with slight modifications based on the starch iodine test. Total assay mixture composed of 120 µl 0.02M sodium phosphate buffer (ph 6.9 containing 6 mm sodium chloride), 1.5 ml of salivary amylase and plant extracts of concentration range from 0.3-1.5 mgml -1 (w/v) were incubated at 37 C for 10 min. Soluble starch (1%, w/v) was then added to each reaction mixture and were incubated at 37 C for 15 min. Thereafter 1 M HCl (60 μl) was added to stop the enzymatic reaction, followed by the addition of 300 μl of iodine reagent (5 mm I 2 and 5 mm KI). Colour change was observed and the absorbance was recorded at 620 nm. Control reaction tubes representing 100% enzyme activity did not contain any plant extract. To eliminate the absorbance produced by plant extract, appropriate extract controls without the enzyme were also examined. Appearance of dark-blue colour indicates the presence of starch; a yellow colour indicates the absence of starch while a brownish colour indicates partially degraded starch in the reaction mixture. In the presence of inhibitors from the extracts the starch added to the enzyme assay mixture was not degraded and gave dark-blue colour complex 64
whereas no coloured complex was developed in the absence of the inhibitor, indicating that starch was completely hydrolysed by α-amylase. 2. Glucose-DNSA color assay (Miller, 1959): Reagents: DNSA reagent, Phosphate buffer of ph 6.9 and of 0.02 molarity, 1% Starch solution, Salivary alpha amylase enzyme. Procedure: Inhibition assay was performed using chromogenic DNSA method. Total assay mixture composed of 500 μl of 0.02 M sodium phosphate buffer (ph 6.9 containing 6 mm sodium chloride), 1ml of salivary amylase and 400 μl extracts of concentration ranging from 0.3-1.5 mgml -1 (w/v) were incubated at 37 C for 10 min. After pre-incubation, 580 μl of 1% (w/v) starch solution was added to each tube and were subjected to incubation at 37 C for 15 min. Reaction was then terminated by adding1.0 ml DNSA reagent, placed in boiling water bath for 5 min, cooled to room temperature and the absorbance were measured at 540 nm. Control containing no plant extracts showed 100% enzyme activity. To eliminate the absorbance produced by plant extract, appropriate extract controls with the extract in the reaction mixture except for the enzyme were also included (negative control). 65
Percent inhibition of alpha amylase was calculated as follows: Percent Relative enzyme activity= (enzyme activity in test sample with extract/enzyme activity in control)*100. % Inhibition in the α-amylase activity= (100 % Relative enzyme activity). Statistical Data Analysis All experiments were performed in three different sets each in triplicates. The data are expressed as mean ± SEM (standard error of the mean). Statistical difference, ANOVA and linear regression analysis were performed using Graph pad prism 5 statistical software. The IC 50 values were determined from plots of percent inhibition versus log inhibitor concentration and calculated by logarithmic regression analysis from the mean inhibitory values. The IC 50 values were defined as the concentration of the extract, containing the α- amylase inhibitor that inhibited 50% of the alpha amylase activity. Results: Aloe vera L. Results revealed that various extracts of leaves of Aloe vera L. exhibit alpha amylase inhibitory activity of different level. 66
Levels of percent inhibition of salivary alpha amylase by different extracts of Aloe vera L. and Azadiracta indica A Juss. are shown in Table 2.1. Percent inhibition of alpha amylase enzyme by different extracts of Aloe vera L. and their IC 50 values are shown in Table 2.2 and 2.3 respectively in plants of Jaipur and Bharatpur districts. Percent inhibition in plants of Jaipur and Bharatpur districts is graphically represented in Figure 2.1 and Figure 2.2 respectively. Water extracts (at concentration ranging from 0.3 mg/ml to 1.5 mg/ml) showed 41.73±0.14% to 43.64±0.13% and 41.10±0.13% to 43.05±0.10 % inhibition of salivary alpha amylase activity in plants of Jaipur and Bharatpur districts respectively. IC 50 values of the extracts as calculated were 0.575 g/ml and 0.063 g/ml in plants of Jaipur and Bharatpur districts respectively. Percent inhibition of alpha amylase by methanol extracts (at concentration ranging from 0.3mg/ml to 1.5 mg/ml) was found to be 43.77±0.14% to 45.66±0.14% with an IC 50 Value of 0.091 g/ml (in plants of Jaipur district) and 43.61±0.10% to 45.65±0.07% with an IC 50 value of 0.091 g/ml (in plants of Bharatpur district). Ethanol extracts (at concentration ranging from 0.3 mg/ml to 1.5 mg/ml) showed 50.13±0.90 % to 52.38±0.17 % inhibition (IC 50 value 0.00031 g/ml) in 67
plants of Jaipur district and 50.12±0.10 % to 51.83±0.13 % inhibition (IC 50 value 0.00032 g/ml) in plants of Bharatpur district. Acetone extracts (at concentration ranging from 0.3 mg/ml to 1.5 mg/ml) showed 39.94±0.11 % to 42.03±0.22 % and 39.12±0.10 % to 41.53±0.11 % inhibition of salivary alpha amylase in plants of Jaipur and Bharatpur districts respectively. IC 50 values of extracts were 0.471 g/ml and 0.199 g/ml in plants of Jaipur and Bharatpur district respectively. Percent inhibition of alpha amylase by pet ether extracts (at concentration ranging from 0.3 mg/ml to 1.5 mg/ml) was found to be 16.12±0.14 % to 18.54±0.08 % with IC 50 value of 2290.86 g/ml (in plants of Jaipur district) and 16.12±0.18 % to 18.27±0.09 % with IC 50 value of 4879.77 g/ml (in plants of Bharatpur district). Very high value of IC 50 showed insignificant inhibition of salivary alpha amylase by the extract of plants collected from both districts. Toluene extracts (at concentration ranging from 0.3 mg/ml to 1.5 mg/ml) showed 16.55±0.13 % to 20.10±0.16 % inhibition (IC 50 value 4.74 g/ml) in plants of Jaipur district and 16.82±0.10 % to 20.01±0.12 % inhibition (IC 50 value 807.23 g/ml) in plants of Bharatpur district. Very high value of IC 50 68
showed insignificant inhibition of salivary alpha amylase by the extract of plants collected from both districts. Alkaloid extracts (at concentration ranging from 0.3 mg/ml to 1.5 mg/ml) were found to exhibit 7.36±0.10 % to 17.34±0.10 % inhibition of alpha amylase with IC 50 value of 0.032 g/ml in plants of Jaipur district and 7.24±0.10 % to 17.20±0.12 % inhibition of the enzyme with IC 50 value of 0.043 g/ml in plants of Bharatpur district. Percent inhibition of alpha amylase by flavonoid extracts (at concentration ranging from 0.3 mg/ml to 1.5 mg/ml) was found to be 55.83±0.12 % to 57.70±0.09 % with IC 50 value of 0.0002 g/ml in plants of Jaipur district and 54.75±0.12 % to 57.11±0.15 % inhibition with IC 50 value of 0.000003 g/ml in plants of Bharatpur district. All experiments were performed in triplicates. One way analysis of variance (ANOVA) was used to show significance of difference with respect to control. In all experiments ρ value was found to be lower than 0.05 which indicate that differences were significant. Percent inhibition and IC 50 values are significantly different among different extracts but there is no significant difference in % inhibition and IC 50 values of same extracts of plants at same concentration in two districts of Rajasthan. 69
Azadiracta indica A Juss. Results revealed that various extracts of leaves of Azadiracta indica A Juss exhibit alpha amylase inhibitory activity of different level. Percent inhibition of alpha amylase enzyme by different extracts of Azadiracta indica A Juss. and their IC 50 values are shown in Table 2.4 and 2.5 respectively in plants of Jaipur and Bharatpur districts. Percent inhibition by extracts of plants of Jaipur and Bharatpur districts is graphically represented in Figure 2.3 and Figure 2.4 respectively. Water extracts of leaves of the plant (at concentrations ranging from 0.3 mg/ml to 1.5 mg/ml) exhibited 16.43±0.12 % to 19.47±0.20 % inhibition with an IC 50 value of 21.38 mg/ml in plants of Jaipur district and 16.63±0.10 % to 18.52±0.16 % inhibition with an IC 50 value of 4786.30 g/ml in plants of Bharatpur district. Very high value of IC 50 showed insignificant inhibition of salivary alpha amylase by the extract of the plant collected from Bharatpur district. 70
Percent inhibition by methanol extracts (at concentrations ranging from 0.3 mg/ml to 1.5 mg/ml) was found to be 13.35±0.13 % to 15.16±0.17 % with an IC 50 value of 426.57 g/ml and 12.53±0.13 % to 14.44±0.14 % with an IC 50 value of 380.19 g/ml in plants of Jaipur and Bharatpur districts respectively. Very high value of IC 50 showed insignificant inhibition of salivary alpha amylase by the extract of plants collected from both districts. Ethanol extracts (at concentrations ranging from 0.3 mg/ml to 1.5 mg/ml) were found to exhibit 16.53±0.12 % to 18.53±0.14% inhibition with an IC 50 value of 2290.86 g/ml in plants of Jaipur district and 15.95±0.17 % to 18.07±0.16 % inhibition with an IC 50 value of 177827.91 g/ml in plants of Bharatpur district. Very high value of IC 50 showed insignificant inhibition of salivary alpha amylase by the extract of plants collected from both districts. Acetone extracts (at concentrations ranging from 0.3 mg/ml to 1.5 mg/ml) showed 15.12±0.11 % to 17.52±0.06 % inhibition (IC 50 value 1174.89 g/ml) in plants of Jaipur district and 15.25±0.12 % to 17.12±0.23 % inhibition (IC 50 value 1174.89 g/ml) in plants of Bharatpur district. Very high value of IC 50 showed insignificant inhibition of salivary alpha amylase by the extracts of plants collected from both districts. 71
Percent inhibition by Pet ether extracts (at concentrations ranging from 0.3 mg/ml to 1.5 mg/ml) was found to be 20.36±0.13 % to 24.57±0.19 % with an IC 50 value 11.74 g/ml (In plants of Jaipur district) and 21.57±0.10 to 23.94±0.17 % with IC 50 value of 295.12 g/ml (In plants of Bharatpur district). Very high value of IC 50 showed insignificant inhibition of salivary alpha amylase by the extract of plants collected from Bharatpur district. Toluene extracts (at concentrations ranging from 0.3 mg/ml to 1.5 mg/ml) were found to exhibit 16.35±0.07 % to 18.53±0.10 % inhibition (IC 50 value 2290.86 g/ml) in plants of Jaipur district and 16.22±0.10 % to 18.55±0.18 % inhibition (IC 50 value 2290.86 g/ml) in plants of Bharatpur district. Very high value of IC 50 showed insignificant inhibition of salivary alpha amylase by the extract of plants collected from both districts. Percent inhibition by alkaloid extract (at concentrations ranging from 0.3 mg/ml to 1.5 mg/ml) was found to be 15.60±0.12 % to 19.69±0.0.09 % and 16.23±0.11 % to 18.99±0.12 % in plants of Jaipur and Bharatpur districts respectively. IC 50 values were 16.66 g/ml and 2333.45 g/ml in plants of Jaipur and Bharatpur districts respectively. Very high value of IC 50 showed insignificant inhibition of salivary alpha amylase by the extract of plants collected from Bharatpur district. 72
Flavonoid extracts (at concentrations ranging from 0.3 mg/ml to 1.5 mg/ml) showed 42.74±0.1.03 % to 46.85±0.13 % inhibition (IC 50 value 0.009 g/ml) in plants of Jaipur district and 45.11±0.25 % to 47.40±0.19 % inhibition (IC 50 value 0.006 g/ml) in plants of Bharatpur district. All experiments were performed each in triplicates. One way analysis of variance (ANOVA) was used to show significance of difference with respect to control. In all experiments ρ value was found to be lower than 0.05 which show that differences were significant. Percent inhibition and IC 50 values were significantly different in different extracts of same plant but there was no significant difference in % inhibition and IC 50 values of same extracts of same plants in two districts. However, IC 50 values of water, pet ether and alkaloid extracts of plants showed significant difference in two districts of Rajasthan. 73