463-40-1 br where AB is the recorded absorbance of the blank
where AB is the recorded absorbance of the blank solution and Ats is the recorded absorbance of the tested sample solution.
2.6. α-amylase inhibition assay
The α-amylase inhibitory activity of each solvent fraction was as-sessed according to the standard method of Nyambe-Silavwe et al. with minor modifications . Briefly, each extract fraction was dissolved in a few milliliters of 10% DMSO (Riedel-de-haen, Germany) and then European Journal of Integrative Medicine 30 (2019) 100933
further dissolved in a buﬀer ((Na2HPO4/NaH2PO4 (0.02 M), NaCl (0.006 M) at pH 6.9) to a concentration of 1000 μg/mL. A dilution series was prepared (10, 50, 70, 100, 500 μg/mL). A volume of 0.2 mL of porcine pancreatic α-amylase enzyme (Sigma-Aldrich, USA) solution with a concentration of 2 units/mL was mixed with 0.2 mL of the plant fraction, then incubated for 10 min at 30 °C. Thereafter, 0.2 mL a freshly prepared starch solution (1%) was added and the mixture was in-cubated for at least 3 min. The reaction was stopped by the addition of 0.2 mL dinitrosalicylic 463-40-1 (DNSA) (Alf۔aAesar, UK), then the mixture was diluted with 5 mL of distilled water and heated for 10 min in a water bath at 90 °C. The mixture was left to cool down to room tem-perature, and then the absorbance was measured at 540 nm. A blank was prepared following the same procedure by replacing the plant fraction with 0.2 mL of the buﬀer.
Acarbose (Sigma-Aldrich, USA) was used as a positive control and prepared in the same procedure. The α-amylase inhibitory activity was calculated using the following equation:
where Ab is the absorbance of the blank and AS is the absorbance of the tested sample or control.
2.7. α-glucosidase inhibitory activity assay
The α-glucosidase inhibitory activity of each plant fraction was carried out according to a standard protocol with some modification . In each test tube, a reaction mixture was prepared containing 50 μL of phosphate buﬀer (100 mM, pH 6.8), 10 μL α-glucosidase (1 U/ mL) (Sigma-Aldrich, USA) and 20 μL of varying concentrations of ex-tract fractions (100, 200, 300, 400 and 500 mg/ml), which was then incubated at 37 °C for 15 min. Then, 20 μL of pre-incubated 5 mM PNPG (Sigma-Aldrich, USA) was added as the substrate of the reaction and the samples were incubated at 37 °C for a further 20 min. The reaction was terminated by adding 50 μL of Na2CO3 (0.1 M). The absorbance of the released p-nitrophenol was measured by a UV/Vis spectrophotometer at 405 nm. Acarbose at similar concentrations as the plant fractions was used as a positive control.
The inhibition percentage was calculated using the following equation:
Where; Ab is the absorbance of the blank and AS is the absorbance of the tested sample or control.
2.8. Cell culture and cytotoxicity assay
HeLa (cervical adenocarcinoma) and Colo-205 (colon) cancer cells were cultured in RPMI-1640 media supplemented with 10% fetal bo-vine serum, 1% penicillin/streptomycin and 1% L-glutamine. Cells were grown in a humidified atmosphere with 5% CO2 at 37 °C. Cells were seeded at 2.6 × 104 cells/well in a 96-well plate. After 48 h, cells were incubated with various concentrations of the tested compounds for 24 h. Cell viability was assessed by the CellTilter 96® Aqueous One Solution Cell Proliferation (MTS) assay according to the manufacturer’s instructions (Promega Corporation, Madison, WI). Briefly, at the end of the treatment, 20 μL of MTS solution per 100 μL of media was added to each well and incubated at 37 °C for 2 h. Absorbance was measured at 490 nm .
2.9. Statistical analysis
The determination of TPC, TFC and TTC and the inhibitory eﬀects of the plant fractions on α-amylase, α-glucosidase and porcine pancreatic enzymes were conducted in triplicate for each sample. The cytotoxic test was carried out in duplicate for each sample. The results are
Phytochemical screening assessment of four solvent fractions of C. curetum.
Phytochemical Aqueous Methanol Acetone Hexane Classes fraction fraction fraction fraction
Cardiac glycosides + – + + Saponin glycoside + – – – Alkaloids + + + + Protein + – – – Starch + – – – Phenols + + – – Terpenoids – + + + Tannins + + – – Steroids – – + + Reducing sugar + + – – Monosaccharide – – – – Flavonoids + + – –
presented as means ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism software version 6.01. Comparisons between three or more groups were performed by one-way analysis of variance (ANOVA), followed by the Bonferroni post hoc test.