Materials
The fruits of M. charantia var. charantia (MCC) and M. charantia var. muricata (MCM) were bought from the local market in Chengalpet, Tamil Nadu, India. They were taxonomically identified by a botanist and verified by DNA barcoding. Porcine α-amylase and yeast α-glucosidase were bought from Sigma Aldrich, and Acarbose from Bayer AG (Germany).
Protein extraction
Proteins were extracted from the fruit pulp of the two varieties of M. charantia as described before [24] with minor modifications. Fresh pulp was ground with ice-cold acid-ethanol, filtered through a muslin cloth, and centrifuged at 8000 × g for 10 min. The pH of the supernatant was adjusted to 3.0 using ammonia solution. Four volumes of acetone was added, mixed gently, and incubated at 4 °C for 24 h. The mixture was centrifuged at 6000 × g for 10 min. The pellet was washed with 80 % acetone, air dried, and dissolved in 10 mM Tris–HCl, pH 8.0.
α-amylase inhibition assay
Stock solutions of protein extracts and Acarbose were prepared in water. Inhibition of porcine α-amylase activity was determined using dinitrosalicylic acid as described before [25]. Protein extract or Acarbose (100 μl of 2 to 20 mg/ml) was added to 100 μl of α-amylase (1 U/ml) and 200 μl of sodium phosphate buffer (20 mM, pH 6.9) to get 0.5 to 5.0 mg/ml final concentration. The samples were pre-incubated at 25 °C for 10 min, and 200 μl of 1 % starch prepared in 20 mM sodium phosphate buffer (pH 6.9) was added. The reaction mixtures were incubated at 25 °C for 10 min. The reactions were stopped by incubating the mixture in a boiling water bath for 5 min after adding 1 ml of dinitrosalicylic acid. The reaction mixtures were cooled to room temperature, diluted to 1:5 ratio with water, and absorbance was measured in a spectrophotometer (Amersham Biosciences, USA) at 540 nm. Percentage of inhibition of enzyme activity was calculated as
$$ \%\ \mathrm{Inhibition} = \left[\left({{\mathrm{A}}_{540}}^{\mathrm{Control}}\hbox{--}\ {{\mathrm{A}}_{540}}^{\mathrm{Treatment}}\right)/{{\mathrm{A}}_{540}}^{\mathrm{Control}}\right]\ \mathrm{x}\ 100 $$
wherein A540
Control is absorbance at 540 nm in control sample without protein extract and A540
Treatment is absorbance at 540 nm in treatment with protein extract
α-glucosidase inhibition assay
Inhibition of α-glucosidase activity was determined using yeast α-glucosidase and p-nitrophenyl-α-D-glucopyranoside (pNPG) as described before [26]. Protein extract or Acarbose (100 μl of 2 to 20 mg/ml) was added to 50 μl of α-glucosidase (1 U/ml) prepared in 0.1 M phosphate buffer (pH 6.9), and 250 μl of 0.1 M phosphate buffer to get 0.5 to 5.0 mg/ml final concentration. The mixture was pre-incubated at 37 °C for 20 min. After pre-incubation, 10 μl of 10 mM pNPG prepared in 0.1 M phosphate buffer (pH 6.9) was added, and incubated at 37 °C for 30 min. The reactions were stopped by adding 650 μl of 1 M sodium carbonate, and the absorbance was measured in a spectrophotometer (Amersham Biosciences, USA) at 405 nm. Percentage of inhibition of enzyme activity was calculated as
$$ \%\ \mathrm{Inhibition} = \left[\left({{\mathrm{A}}_{405}}^{\mathrm{Control}}\hbox{--}\ {{\mathrm{A}}_{405}}^{\mathrm{Treatment}}\right)/{{\mathrm{A}}_{405}}^{\mathrm{Control}}\right]\ \mathrm{x}\ 100 $$
wherein A405
Control is absorbance at 405 nm in control sample without protein extract and A405
Treatment is absorbance at 405 nm in treatment with protein extract
Analysis of proteolytic activity
Proteolytic activity of the plant extracts was tested against α-amylase (2 U/ml), α-glucosidase (0.05 U/ml) and a mixture of six unrelated proteins (β-lactalbumin, lysozyme, soybean trypsin inhibitor, ovalbumin, bovine serum albumin, and phosphorylase-b, 5 μg). These samples were treated with 20 μg of the protein extracts from MCC and MCM for 10 min at 25 °C (α-amylase and mixture of six proteins) or 37 °C (α-glucosidase). Treatment with Proteinase K (55 °C for 1 h), and heat denatured protein extracts were used as positive and negative controls, respectively. The reactions were stopped by heating the samples at 100 °C for 5 min after adding protein loading dye to a final concentration of 1X. The samples were analyzed by 12 % SDS-PAGE.
Mode of inhibition assay
Mode of inhibition of α-amylase and α-glucosidase by the protein extracts was determined as described before [27]. For α-amylase, the enzyme solution (1 U/ml) was pre-incubated with protein extracts (10 mg/ml), Acarbose (10 mg/ml) or phosphate buffer (pH 6.9) at 25 °C for 10 min. The reactions were started by adding 5 to 25 mg/ml starch, and continued at 25 °C for 10 min. The reactions were stopped by adding 0.5 ml of dinitrosalicylic acid followed by boiling for 5 min. For α-glucosidase, the enzyme solution (1 U/ml) was pre-incubated with protein extracts (10 mg/ml), Acarbose (10 mg/ml) or phosphate buffer (pH 6.9) at 25 °C for 10 min. The reactions were started by adding 50 to 250 mg/ml pNPG, and continued at 25 °C for 10 min. The reactions were stopped by adding 0.05 ml of 1 M sodium carbonate. Release of reducing sugars was quantified using maltose and paranitrophenol standard curve. Double reciprocal plot (1/v versus 1/[S]) where v is reaction velocity and [S] is substrate concentration was plotted. Mode of inhibition was determined by analysing Lineweaver-Burk plot using Michaelis-Menten kinetics.
Induction of diabetes in Wistar rats
Forty male Wistar rats (3-months old) were purchased from King’s Institute, Chennai, and kept under 12:12 h light and dark cycle at 25 ± 2 °C. The diabetic animals were fed with high fat diet and water ad libitum throughout the treatment period of 30 days. The experimental protocols were conducted in accordance with internationally accepted principles for laboratory animal use and were approved by the Institutional Animal Ethical Committee (087/835/IAEC-2014). Diabetes was induced in 18 h fasted animals by intraperitoneal injection of 110 mg/kg nicotinamide followed by 45 mg/kg Streptozotocin (freshly dissolved in 0.1 M citrate buffer, pH 4.5). Tail bleeds were performed 7 days after injecting Streptozotocin, and animals with blood glucose concentration above 250 mg/dL were considered diabetic.
Oral starch and sucrose tolerance tests
Oral starch and sucrose tolerance tests were performed as described before [28]. Twenty fasted diabetic and non-diabetic rats were divided into four groups of five each, and orally treated with 10 mg/kg body weight of the protein from MCC, MCM, Acarbose (positive control) or distilled water (negative control). Ten minutes after the treatment, blood glucose level was estimated (0 min), and the rats were orally administered with 3.0 g/kg starch or 4.0 g/kg sucrose. Blood glucose level (BG) was estimated 30, 60 and 120 min after the administration. Peak blood glucose (PBG) was determined by observing the blood glucose level during the above mentioned time intervals and area under the curve (AUC) was calculated using the formula given below
$$ \mathrm{A}\mathrm{U}\mathrm{C}\ \left(\mathrm{mg}/\mathrm{dL}.\ \mathrm{H}\right) = \left({\mathrm{BG}}_0 + {\mathrm{BG}}_{30}\mathrm{x}\ 0.5\right)/2 + \left({\mathrm{BG}}_{30} + {\mathrm{BG}}_{60}\mathrm{x}\ 0.5\right)/2 + \left({\mathrm{BG}}_{60} + {\mathrm{BG}}_{120}\mathrm{x}\ 1.0\right)/2 $$
wherein BG0 is the blood glucose level before oral administration of starch and glucose, and BG30, BG60, and BG120 are the blood glucose level 30, 60 and 120 min after the administration.
Statistical analysis
All experiments were performed in triplicate. Means, standard errors, and standard deviations were calculated from replicates within the experiments. Statistical analysis was done by one way analysis of variance (ANOVA). Statistical significance was accepted at P < 0.05. IC50 was calculated using graph pad prism software.
