Chemicals
All the chemicals used were of analytical grade, n-hexane, chloroform, acetone, and ethanol (Merck Lif. Sci. Pvt. Ltd., India) and Baker’s yeast α-glucosidase, P-nitrophenyl-α-D-glucopyranoside, and acarbose (Sigma-Aldrich Pvt. Ltd., India).
Collection and authentication of plant sample
The mature, wholesome, roughly equal size, and just ripened Manilkara zapota (L.) P. Royen fruits were purchased from the local market of Nanded, MS, India (latitude 19.13° N and longitude 77.32° E). The taxonomic identity, authentication (No.: -BSI/WRC/100-1/TECH./2019/68), and voucher specimen of fruit was deposited at the herbarium of Botanical Survey of India, Pune, India.
Sample preparation and extraction
The fruits were washed thoroughly with deionized water, shade dried, and peeled off with the help of a steel spoon. Peels were coarsely powdered in a pulverizer under room temperature (28 °C) at University’s Rashtriya Uchchatar Shiksha Abhiyan, Centre for Herbo Medicinal Studies and stored in an airtight container with wrapping in aluminum foil at room temperature in the dark.
After defatting by pet ether, the dried peel powder was individually extracted [12] using different organic solvents with increasing order of their polarity viz. n-hexane, chloroform, acetone, ethanol, and water. Five hundred milliliters of n-hexane was added to 50 g of dried peel powder and shaken in an orbital incubator shaker (Remi RIS 24 +) at 150 rpm, 28 °C for 2 h. Immediately after cooling down to room temperature using an ice bath, it was centrifuged (Remi Centrifuge, Mumbai, India) at 2500 rpm for 15 min, and Whatman filter paper No. 1 filtered filtrate in a petri dishes were evaporated for 3 days [13]. The same procedure was followed for extraction with chloroform, acetone, ethanol, and water. All procedures were carried out at room temperature and obtained extracts were stored in airtight dark bottles at 4 °C before further analysis. The extractive yield was calculated as
$$ \%\mathrm{Extractive}\ \mathrm{Yield}=\left(\mathrm{dry}\ \mathrm{extract}\ \mathrm{obtained}/\mathrm{weight}\ \mathrm{of}\ \mathrm{the}\ \mathrm{extraction}\ \mathrm{sample}\right)\times 100 $$
Evaluation of antioxidant activity
DPPH assay
DPPH assay was performed to assess the radical scavenging activity by n-hexane, chloroform, acetone, ethanol, water, and ethanol aqueous fraction extracts of MZFP. The MZFP extracts samples were dissolved in methanol. Ascorbic acid was used as standard and methanol (5 ml) as blank, while for control equal volume of DPPH in methanol was used. Three milliliters solution of DPPH in methanol (0.2 mM) was mixed with 1 ml of the extracts samples of various concentrations (0.1 to 1 mg/ml). After proper mixing in test tubes incubated in dark for 40 min at room temperature then absorbance was measured at 517 nm [14,15,16]. The mean values ± SEM of the assay carried out in triplicate were presented. The percentage inhibition was calculated by the formula
$$ \%\mathrm{inhibition}=\left[\left({\mathrm{A}}_{\mathrm{control}}-{\mathrm{A}}_{\mathrm{sample}}\right)/{\mathrm{A}}_{\mathrm{control}}\right]\ 100 $$
Where Acontrol is absorbance by DPPH solution and Asample absorbance by DPPH solution with the extracted sample.
H2O2-scavenging assay
The various concentrations (10 to 50 μg/ml) of each of n-hexane, chloroform, acetone, ethanol, water, and ethanol aqueous fraction extracts of MZFP was prepared by dissolving in 4 ml of 0.1 M phosphate buffer (pH 7.4) and added to 0.6 ml of H2O2 solution (43 mM). After 30 min., the absorbance of the reaction mixture was recorded at 230 nm, and then followed at every 10 min. Phosphate buffer (pH 7.4) was used as a blank sample for background subtraction [17, 18]. The % scavenging activity was compared against ascorbic acid as a standard and calculated as
$$ \%\mathrm{radical}\ \mathrm{scavenging}=\left[\left({\mathrm{A}}_{\mathrm{control}}-{\mathrm{A}}_{\mathrm{sample}}\right)/{\mathrm{A}}_{\mathrm{control}}\right]\ 100 $$
Where Acontrol is absorbance by H2O2 solution and Asample absorbance by H2O2 solution with the extracted sample.
Optimization of extraction conditions
In accordance with high extractive yield and antioxidant activity, the optimization of extraction conditions with the ethanol solvent was carried out according to the method of Woo et al. 2013 [12] with some modifications. The first step was initiated by using three different concentrations of ethanol (40%, 70%, and 100%) for extraction. Five hundred milliliters of each above ethanol concentration solvent was added to 50 g of each dried MZFP powders. The mixtures were shaken in an orbital incubator shaker at 150 rpm, 28 °C for 2 h. Then the extracts were rapidly cooled to room temperature using an ice bath, after that extracts were centrifuged at 2500 rpm, for 15 min using a centrifuge, and the supernatants were filtered by Whatman filter paper No. 1. The filtrates were then evaporated in a rotary evaporator at 40 °C to remove the solvent.
The optimum concentration of ethanol solvent to extract MZFP was identified by comparing the radical scavenging (DPPH, H2O2) assays. In the second step, with optimum concentration of ethanol, by following the same procedure as above, the extraction was done with three different extraction times viz. 6, 9, and 12 h. While in the third step, with optimum concentration of ethanol and the optimum extraction time, the extraction procedure was done at 3 different temperatures for extraction viz. 40 °C, 50 °C, and 60 °C. The whole extraction process was carried out in a dark environment and the extracts obtained were kept in dark bottles in the freezer (4 °C) before further analysis.
Evaluation of in vitro antidiabetic potential
In vitro evaluation for α-glucosidase enzyme inhibition
Assay was performed as per the method described by Wan et al. (2013) and K. Savikin et al. (2018) [19, 20] with slight modifications. An optimum (about 2 ml) volume of 0.1 M phosphate buffer (pH 6.8) was prepared. Each of 200 μl Baker’s yeast α-glucosidase enzyme (0.5 U/ml) extract, MZFP extract samples in the concentration ranging from 25 to 150 μg/ml and phosphate buffer were mixed, kept for pre-incubation at 40 °C for 3 min. To start the reaction, a 200-μl of substrate p-Nitrophenyl-α-D-glucopyranoside (15 mg/10 ml) was mixed into the above mixture and again incubated at 40 °C for 25 min. An 800 μl of 0.2 M sodium hydroxide was added to this mixture to terminate the reaction. For measuring the α-glucosidase enzyme inhibition activity by sample extract, the release of p-nitrophenol from p-nitrophenyl-α-D-glucopyranoside was recorded at 405 nm. Acarbose was used as a reference α-glucosidase inhibitor. The % inhibition was calculated by the formula
$$ \%\mathrm{Inhibition}={\mathrm{Abs}}_{405}\ \left(\mathrm{control}\right)-{\mathrm{Abs}}_{405}\ \left(\mathrm{extract}\right)/{\mathrm{Abs}}_{405}\ \left(\mathrm{control}\right)\times 100 $$
Statistical analysis
The antioxidant activity study results were expressed as mean ± SEM of three determinations, while IC50 values were calculated by linear regression analysis by program Excel 2010. The α-glucosidase inhibition assay procedures were carried out with three determinations and mean ± SD values were determined. The plots of percent inhibition versus log inhibitor were calculated by linear regression analysis from the mean inhibitory values to estimate IC50 values.
The presence of significant differences between inhibition by acarbose and extract samples was calculated by one-way ANOVA followed by Tukey’s multiple comparisons test at 95% confidence interval by GraphPad Prism version 9.1 (221) software. While the p values < 0.05 were considered statistically significant.