In-vitro alpha amylase inhibitory activity of the leaf extracts of Adenanthera pavonina

Air-dried, powdered leaves (200 g) of A. pavonina were extracted with methanol (MeOH) at room temperature for 2 × 24 h using 1500 ml of MeOH for each extraction. The combined MeOH extracts were concentrated under vacuum at 40 °C to 1 L and were partitioned into petroleum ether (2 × 500 ml). The combined petroleum ether fraction was filtered and dried over anhydrous Na₂SO₄ and was evaporated under vacuum to dryness. The dried petroleum ether extract was refrigerated in a tightly closed container until use. The remaining methanol fraction was evaporated under vacuum to dryness and residue was partitioned between ethyl acetate (EtOAc, 1 L) and water (1 L). The ethyl acetate extract was dried over anhydrous Na₂SO₄ and was evaporated under vacuum to dryness and refrigerated until further use. The aqueous fraction was frozen and freeze-dried and the residue was stored in the refrigerator until further use.

The α-amylase inhibition assay was performed using the 3,5-dinitrosalicylic acid (DNSA) method [8]. The leaf extract of A. pavonina was dissolved in minimum amount of 10% DMSO and was further dissolved in buffer ((Na₂HPO₄/NaH₂PO₄ (0.02 M), NaCl (0.006 M) at pH 6.9) to give concentrations ranging from 10 to 1000 μg/ml. A volume of 200 μl of α-amylase solution (2 units/ml) was mixed with 200 μl of the extract and was incubated for 10 min at 30 °C. Thereafter 200 μl of the starch solution (1% in water (w/v)) was added to each tube and incubated for 3 min. The reaction was terminated by the addition of 200 μl DNSA reagent (12 g of sodium potassium tartrate tetrahydrate in 8.0 mL of 2 M NaOH and 20 mL of 96 mM of 3,5-dinitrosalicylic acid solution) and was boiled for 10 min in a water bath at 85–90 °C. The mixture was cooled to ambient temperature and was diluted with 5 ml of distilled water, and the absorbance was measured at 540 nm using a UV-Visible spectrophotometer. The blank with 100% enzyme activity was prepared by replacing the plant extract with 200 μl of buffer. A blank reaction was similarly prepared using the plant extract at each concentration in the absence of the enzyme solution. A positive control sample was prepared using Acarbose (100 μg/ml–2 μg/ml) and the reaction was performed similarly to the reaction with plant extract as mentioned above. The α-amylase inhibitory activity was expressed as percent inhibition and was calculated using the equation given below: The % α-amylase inhibition was plotted against the extract concentration and the IC₅₀values were obtained from the graph.

The total phenolic content of A.pavonina was determined spectrophotometrically using Folin-Ciocalteu’s reagent. The extract (100 μl ;1 mg/ml) was mixed with 100 μl of Folin-Ciocalteu’s phenol reagent and kept for 5 min. A volume of 1.00 ml of a 7% Na₂CO₃ solution was added to the reaction mixture followed by addition of 1.30 ml of deionized (DI) water. The mixture was kept in the dark at ambient temperature for 90 min and thereafter the absorbance measured at 750 nm. A calibration graph was prepared using Gallic acid as the standard and the total phenolic content in the extract was expressed as milligrams of Gallic acid equivalents in one gram of extract [10, 11].

The plant extract was dissolved initially in 100 μl of DMSO and was further dissolved in artificial sea water to give concentrations ranging from 125 to 1000 μg/ml. Artificial sea water was prepared using the Cold Spring Harbor Laboratory protocol, 2011 [12]. A 26.29 g of NaCl (450 mM), 0.74 g KCl (10 mM), CaCl₂ 0.99 g (9 mM), MgCl₂•6H₂O 6.09 g (30 mM) and MgSO₄•7H₂O 3.94 g (16 mM) was dissolved in 1 L deionized water and was filtered by using Whatmann filter paper No. 42. The pH of the solution was adjusted to 7.8 and autoclaved and was store at 4 °C until use (maximum for 1 week). Two milliliters of the extract solution was added into separate test tubes and ten mature brine shrimps were added into each test tube. The test tubes were incubated at 30 °C for 24 h and counted the live shrimp under magnification. The number of deaths of shrimps was plotted against extract concentration and the LD₅₀ value was estimated [13]. Potassium dichromate dissolved in artificial seawater at concentrations ranging from 125 to 1000 μg/ml was used as positive controls of the experiment. Each experiment was performed in was performed in triplicates.

Evaluating the plot of % α-amylase inhibition as a function of extract concentrations (Fig. 1), the IC₅₀ values were calculated (Table 1). The crude MeOH extract exhibited the lowest IC₅₀ of 16.16 ± 2.23 μg/ml and the IC₅₀ values of EtOAc, petroleum ether, and the water extracts were 59.93 ± 0.25, 145.49 ± 4.86 and 214.85 ± 9.72 μg/mL respectively. The standard positive control Acarbose showed an IC₅₀ of 18.63 ± 1.21 μg/ml. In this study, significant differences were found between the IC₅₀ values of all extracts (P =2.23 × 10⁻¹¹, one-way ANOVA). However, the difference between the IC₅₀ of Acarbose and the methanol extract did not show a significant difference (P = 0.175, one-way ANOVA).

The DPPH radical scavenging activities of the A. pavonina extracts showed IC ₅₀ value of 751.66 ± 4.91, 423.62 ± 2.45 and 249.92 ± 3.35 μg/ml for the extracts of petroleum ether crude methanol and ethyl acetate respectively (Fig. 3, Table 1). The best activity was observed in the EtOAc fraction with an IC₅₀ value of 249.92 ± 3.35 μg/ml and IC₅₀ of the aqueous extract was not determined, as it was greater than 1 mg/ml. The % inhibition of the aqueous extract at 1 mg/ml was only 25.87 ± 1.75%. Significant differences were found between the IC₅₀ values of all extracts for the DPPH scavenging activity (P =0.00019, one-way ANOVA).

LD₅₀ values lower than 1000 μg/ml is considered to be cytotoxic in the toxicity evaluation of plant extracts using brine shrimp assay [13]. The LD₅₀ value of the crude methanol extract of A. pavonina leaves was estimated to be 1963.936 μg/ml indicating the absence of cytotoxic compounds in the extract.