Preparation of plant extracts and fractions
Fruits of Phaleria macrocarpa were collected from Kepala Batas, Seberang Perai, Pulau Pinang, Malaysia in August, 2010. They were identified by Mr. V. Shunmugam a/l Vellosamy and a voucher specimen of the plant (voucher number 11259) was deposited in the herbarium unit, School of Biological Sciences, Universiti Sains Malaysia. The pericarps of the fruits were sliced, dried, and powdered using a milling machine. About 2,400 g were successively extracted with petroleum ether and methanol using Soxhlet apparatus (40°C) for 48 h each. Thereafter, the residue from the methanol extraction after complete drying was extracted with water by maceration at 60°C for 24 hours. Extraction with each solvent was done in triplicate and the extracts obtained were filtered with Whatman No. 1 filter paper and concentrated in vacuo by rotary evaporation (Buchi Labortechnik, Flawil, Switzerland). The concentrated extracts were finally lyophilized to obtain 73.6 g (3.06%), 445.36 g (18.55%) and 146 g (6.08%) each of dried petroleum ether extract (PEE), methanol extract (ME) and water extract (WE), respectively. Earlier results from hypoglycaemic and anti-hyperglycaemic tests with these extracts showed that the methanol extract was the most effective in lowering blood glucose , and thus this alone was used in the present study.
Successive liquid-liquid fractionation of the methanol extract
The methanol extract of Phaleria macrocarpa was fractionated with polarity graded solvents in separating funnels. In brief, 110 g of the methanol extract was first extracted with 3 × 360 ml of chloroform-water (6:5). The combined chloroform fractions were dried with anhydrous sodium sulphate and further concentrated in a rotary evaporator. The aqueous layer was extracted with 3 × 250 ml ethyl acetate and the combined ethyl acetate fractions were concentrated as above. Finally, the aqueous layer was extracted with n-butanol 5 × 250 ml and the combined n-butanol fraction was concentrated as well as the remaining aqueous fraction. The concentrated fractions were thereafter freeze-dried to obtain 18 g (5.45%), 24.3 g (7.36%), 110.1 g (33.3%) and 89.1 g (27%) of chloroform (CF), ethyl acetate (EAF), n-butanol (NBF) and aqueous (AF) fractions, respectively. Previous results from the hypoglycaemic and anti-hyperglycaemic tests with these fractions revealed that the n-butanol fraction was the most effective , and hence this alone was selected for the present investigation.
Fractionation of the active n-butanol fraction by dry-column flash chromatography
A chromatographic glass column (27 × 5 cm) used in the separation was gently loaded with 100 g of silica gel (Merck, 7730) in 300 ml petroleum ether. The silica was carefully packed by applying vacuum suction, and a levelled and well-compacted bed yield was ensured. The n-butanol fraction (12 g) was pre-adsorbed onto the adsorbent (silica gel, 200-400 mesh) by first dissolving in methanol (100 ml), followed by addition of the silica gel (24 g). The mixture was evaporated to dryness using a rotary evaporator, and the resultant dried extract-adsorbent mixture was then loaded onto the top of the already packed column evenly by applying suction. The column was first eluted with 2 × 300 ml 100% chloroform, followed serially by 2 × 300 ml chloroform-methanol in graded ratios: (9:1), (8:2), (7:3), (6:4), (5:5), (4:6), (3:7), (2:8), (1:9), (0:10) and finally with chloroform-methanol-water (7:13:2). Fractions were collected in a fixed volume and examined with thin layer chromatography using n-butanol-acetic acid-water (4:1:5) as the mobile phase. Fractions with similar profiles were pooled together offering two sub-fractions namely SFI and SFII which were freeze-dried to obtain 20 g (40%) and 8 g (16%) respectively. When these sub-fractions were subjected to hypoglycaemic and anti-hyperglycaemic screening, sub-fraction I was found to be the most active , therefore it was used for the current α-glucosidase and α-amylase inhibition tests.
Healthy male Sprague Dawley (SD) rats weighing 200-250 g obtained from the Animal Research and Service Centre, Universiti Sains Malaysia (USM) were used for this study. These were housed in the Animal Transit Room, School of Pharmaceutical Sciences, USM. They were allowed free access to food (standard laboratory chow, Gold Coin Sdn. Bhd., Malaysia) and tap water. The animals were maintained according to accepted international and national guidelines and the procedure for this experiment approved by the Animal Ethics Committee of Universiti Sains Malaysia, Penang, Malaysia (AECUSM). Diabetes was induced in the rats by intra-peritoneal injection of 65 mg/kg b.w. of streptozotocin (Sigma, St Louis, MO, USA), after an overnight fast . Seventy-two (72) hours after, their blood glucose levels were measured using the Accu-check Advantage II Glucose meter (Roche Diagnostics Co., USA) and rats with fasting blood glucose ≥ 15 mmol/L were considered diabetic and included in the study. The effects of P. macrocarpa extract, fraction and sub-fraction on oral carbohydrate tolerance (starch, sucrose and glucose), an indirect measure of α-glucosidase and α-amylase activities, were evaluated in non-diabetic rats (NDRs) and streptozotocin diabetic rats (SDRs) categorized into groups as shown below. Acarbose, a conventional α-glucosidase inhibitor, was used as a positive control in the two sets of experiments.
In vitro α-glucosidase (EC 184.108.40.206) inhibition study
The assay was performed using our earlier procedure . In brief, 50 μl of 4 graded concentrations (100 μg/ml, 50 μg/ml, 25 μg/ml, 12.5 μg/ml) each of sample (extract/fraction/sub-fraction) and acarbose, the positive control, were suspended in 100 μl of 0.1 M phosphate buffer (pH 6.9) containing yeast α-glucosidase (Sigma Aldrich Chemical Co, USA) solution (1.0 U/L) and pre-incubated in a 96-well microplate at 25°C for 10 min. After pre-incubation, 50 μl of 5 mM p-nitrophenyl-α-D-glucopyranoside solution (the enzyme substrate), in 0.1 M phosphate buffer (pH 6.9) was added to each well. An equivalent volume (50 μl) of buffer solution was added to the blank or control in place of the extract. The reaction mixtures were incubated at 25°C for 5 min. The absorbance of the reaction mixtures before and after substrate incubation was measured at 405 nm on a micro-plate reader (Power Wave Biotek Instrument Inc, USA). The α-glucosidase inhibitory activity was calculated from the difference in the two absorbances and expressed as % inhibition as follows:
The experiment was performed in triplicate. The IC50, i.e. the concentration of the extract/fraction/acarbose resulting in 50% inhibition of the enzyme was calculated by regression analysis.
In vitro α-amylase (EC 220.127.116.11) inhibition study
A total of 500 μl of each sample and 500 μl of 0.02 M sodium phosphate buffer (pH 6.9) containing porcine α-amylase solution (0.5 mg/ml) were incubated at 25°C for 10 min. After pre-incubation, 500 μl of 1% starch solution in 20 mM sodium phosphate buffer (pH 6.9) was added to each test tube. The reaction mixtures were then incubated at 25°C for 10 min and thereafter stopped by addition of 1 mL of 3,5-dinitrosalicylic acid (DNS) colour reagent. The test tubes were then incubated in boiling water for 5 min and then cooled to room temperature. After dilution of the reaction mixtures with 10 ml of distilled water, the absorbance was measured at 540 nm. Acarbose was used as the positive control. The inhibition activity was calculated as follows:
Control incubations representing 100% enzyme activity were carried out in a similar fashion by replacing the plant extract/fraction with vehicle (500 μl DMSO and distilled water). For the blank incubation, the enzyme solution was replaced with distilled water and the same procedure was followed as above. Separate incubations conducted for the reaction of t = 0 min was performed by adding samples to the DNS solution immediately after addition of the enzyme. The experiment was also performed in triplicate and the IC50, i.e. the concentration of the extract/fraction/acarbose resulting in 50% inhibition of the enzyme was calculated by regression analysis.
Confirmatory in vivo studies in non-diabetic rats (NDRs)
Starch tolerance test
In this test, 30 overnight-fasted non-diabetic rats divided into five groups of six each were respectively treated (p.o.) with ME (1 g/kg), NBF (1 g/kg), SFI (1 g/kg), acarbose (positive control, 10 mg/kg), and distilled water (negative control). Ten minutes after, the rats were administered starch (3 g/kg body weight) orally and blood was collected via tail puncture for blood glucose estimation before (0 min) and at 30, 60 and 120 minutes after starch treatment . The recorded blood glucose concentrations peak blood glucose (PBG) and area under curve (AUC) were determined. Whereas the maximum blood glucose concentration for each group was taken as PBG for the group, AUC was calculated using the relationship:
Where BG represents the blood glucose concentration measured at time intervals 0, 30, 60 and 120 minutes.
Sucrose tolerance test
The sucrose tolerance test was carried out using the same procedure as for the determination of starch tolerance. However, in this test, sucrose at a dose of 4 g/kg body weight was used instead of starch.
Glucose tolerance test
The oral glucose tolerance test was also carried out using the same procedure as for the determination of starch tolerance, but glucose at a dose of 2 g/kg body weight was used instead of starch.
Confirmatory in vivo tests using streptozotocin-induced diabetic rats (SDRs)
This second set of tests also evaluated the effects of the active methanol extract (ME), fraction (NBF) and sub-fraction (SFI) on the tolerance of diabetic rats to orally administered starch, sucrose or glucose. In each test, 5 groups of rats (n = 6) were treated as follows. Groups 1-3 were treated with 1 g/kg each of ME, NBF and SFI, respectively and groups 4 (positive control) and 5 (negative control) were treated with acarbose (10 mg/kg) and an equivalent volume of distilled water (p.o.), respectively. As in earlier tests, 10 minutes after oral starch (3 g/kg)/sucrose (4 g/kg)/glucose (2 g/kg) treatment, blood glucose was measured at 0, 30, 60 and 120 min and used for PBG and AUC determinations similar as described above.
Data are expressed as mean ± SEM. Analysis of variance (ANOVA) followed by post hoc analysis (Dunnett’s test) were used for data analysis using the SPSS statistical package, version 17.0. Differences at P < 0.05 were considered significant.