Chemicals
Human recombinant aldose reductase, DL-glyceraldehyde, glucose, lithium sulfate, 2-mercaptoethanol, NADPH, quercetin, dimethylsulfoxide, sorbitol, sorbitol dehydrogenase, and NAD+ were obtained from Sigma-Aldrich (St. Louis, MO). All other chemicals and solvents were of analytical grade and were obtained from local companies.
Sample collection and preparation
Fresh samples of selected fruits, apple (Malus domestica Borkh.), banana (Musa paradisiaca Linn.), pawpaw (Carica papaya Linn.), and watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai), were obtained from designated fruits store in Ojo Area (6.4579° N, 3.1580° E) of Lagos, Nigeria. Proper identification and authentication of the fruits were done at the Department of Botany, Lagos State University, Lagos, by Dr. O. J. Sharaibi with the reference numbers LSH/20/651, LSH/20/652, LSH/20/653, and LSH/20/654 for apple, banana, pawpaw, and watermelon, respectively. The fruits were washed under running tap water to remove sand. Pawpaw, watermelon, and apple were peeled and sliced, and their seeds were removed, while banana was only peeled. This is followed by extraction of the juice from all the fruits using household juice extractor. The juice was centrifuged, filtered, and the supernatant freeze-dried in a lyophilizer. The percentage yield of the extracts of apple, banana, pawpaw, and watermelon is 10.50%, 6.71%, 23.24%, and 45.27%, respectively. Extracts were dissolved in distilled water to give stock solutions of 1.0 mg/mL and different concentrations (12.5, 25, 50, 100, and 200 μg/mL) of the extracts were prepared using serial dilution method with distilled water. All extracts were stored at 4 °C prior to analysis.
Aldose reductase inhibition assay
The aldose reductase inhibition assay was performed according to the method of [10] with minor modifications. The reaction mixture contained 0.15 mM NADPH, 10 mM DL-glyceraldehyde, 5 μL of aldose reductase, and 100 μL of fruit extract (12.5–200 μg/mL) or distilled water in a total volume of 1.0 mL of 100 mM sodium phosphate buffer (pH 6.2). After the reaction mixtures were incubated at 25 °C for 5 min in advance, the reaction was initiated by addition of the enzyme, and then the change in absorbance was measured at 340 nm for 10 min using a Cary50 Bio UV-VIS spectrophotometer. The aldose reductase inhibition activity was calculated as percentage inhibition, thus
% Inhibition = [(∆Abscontrol − ∆Absextract)/∆Abscontrol] × 100
The inhibition assay of the standard (quercetin) was performed using the same procedure but replacing the extract with quercetin (12.5–200 μg/mL). The concentration of extract or standard (quercetin) that inhibited 50% of aldose reductase activities (IC50) were evaluated using Microsoft Excel (2010).
Kinetics of inhibition of aldose reductase by C. papaya
The kinetics of inhibition of aldose reductase by aqueous extract of pawpaw (Carica papaya) was performed according to the method described by [11]. In one set of tubes, the reaction mixture contained 0.15 mM NADPH, DL-glyceraldehyde (10–200 mM), 5 μL of aldose reductase, and 100 μL of pawpaw extract (50 μg/mL) in a total volume of 1.0 mL of 100 mM sodium phosphate buffer (pH 6.2). In another set of tubes, 100 μL distilled water replaced the pawpaw extract and this serves as control. The change in absorbance was measured at 340 nm and converted to reaction velocities. Lineweaver-Burk plot was plotted to determine the mode of inhibition.
Sorbitol dehydrogenase inhibition assay
Sorbitol dehydrogenase inhibition activity was determined in accordance with the method of [12]. The assay mixture contained 100 mM tris-HCl buffer (pH 9.0), 0.5 mM NAD+, 50 μL sorbitol dehydrogenase, 100 mM sorbitol (substrate), and fruit extract (12.5–200 μg/mL). The reaction was initiated by the addition of NAD+. The rate of change in the absorbance of the mixture was measured at 340 nm using a Cary50 Bio UV-VIS spectrophotometer. The sorbitol dehydrogenase inhibition activity was calculated as percentage inhibition, thus
% Inhibition = [(∆Abscontrol − ∆Absextract)/∆Abscontrol] × 100
The inhibition assay of the standard (quercetin) was performed using the same procedure but replacing the extract with quercetin (12.5–200 μg/mL). The concentration of extract or standard (quercetin) that inhibited 50% of aldose reductase activities (IC50) were evaluated using Microsoft Excel (2010).
Kinetics of inhibition of sorbitol dehydrogenase by C. papaya
The kinetics of inhibition of sorbitol dehydrogenase by aqueous extract of pawpaw (Carica papaya) was performed according to the method described by [13]. In one set of tubes, the reaction mixture contained 100 mM tris-HCl buffer (pH 9.0), 0.5 mM NAD+, 50 μL sorbitol dehydrogenase, sorbitol (100–500 mM), and 50 μg/mL pawpaw extract. In another set of tubes, distilled water replaced the pawpaw extract and this serves as control. The change in absorbance was measured at 340 nm and converted to reaction velocities. Lineweaver-Burk plot was plotted to determine the mode of inhibition.
Phytochemical analysis
Due to the low IC50 values of pawpaw extract for the inhibition of aldose reductase and sorbitol dehydrogenase, the qualitative and quantitative phytochemical composition of the pawpaw extract was determined using the modified spectrophotometric methods of [14, 15], respectively.
Statistical analysis
All analyses were performed in triplicates unless otherwise stated. Data were expressed as mean ± SEM and statistical significance was considered at p < 0.05. IC50 values were obtained from percentage inhibitions using Microsoft Excel software (Microsoft, 2010). Analysis of variance (ANOVA) was used to assess differences in the percentage inhibitions and IC50 values of the extracts as well as standard. Kinetics of inhibition of enzymes were determined by linear regression using GraphPad Prism statistical package (GraphPad Software, USA).