Plant materials and preparation of the samples
Leaves of Orthosiphon stamineus were obtained from Kepala Batas, Pulau Pinang Malaysia. The plant was identified by the School of Biological Sciences, Universiti Sains Malaysia, and a voucher specimen (10810) was deposited at the Herbarium of the School of Biological Sciences, Universiti Sains Malaysia. The dried leaves were powdered using a milling machine and were extracted with 50% (v/v) ethanol using the maceration method (200 g dried leaves in 2 l of 50% ethanol at 55°C for 24 h; two cycles) for 7 days. The extracts were filtered and concentrated at 60°C using a rotary evaporator (Buchi Labortechnik, Flawil, Switzerland). Finally, the concentrated extract was freeze-dried (Labconco Corporation, Kansas City, MO, USA) to yield 10.3% dry powder.
HPLC study of the 50% ethanolic extract of O. stamineus
High-performance liquid chromatography (HPLC) analysis was performed using a Shimadzu-LC system (Shimadzu, Japan) equipped with a CBM-20A controller, LC-20AT pump, DGU-20A5 degasser, SIL-20A auto-sampler, SPD-20AV detector and a CTO-10ASvp column oven.
Chromatographic separations were achieved using an Agilent Eclipse Plus C18 (250 × 4.6 mm i.d.; 5 μm). A Zorbax guard fittings kit packed with a replaceable Eclipse Plus C18 Guard column (12.5 × 4.6 mm i.d.; 5 μm) was used to protect the analytical column. A reverse-phase HPLC assay was carried out using an isocratic system at a flow rate of 1 ml/min, a column temperature of 25°C and a mobile phase of acetonitrile, isopropyl alcohol and 0.02 M phosphate buffer (NaH2PO4) (30:15:55 v/v) with the pH adjusted to pH 3.5 using 85% phosphoric acid. The UV detection was set at 340 nm. The injection volume was 20 μl of solution. The total run time was less than 20 min for each injection. Data were acquired and processed using LC-Solution Software. The peaks were detected at 340 nm and identified using standard substances, namely, sinensetin, eupatorin and 3′-hydroxy-5,6,7,4′-tetramethoxyflavone. The 50% ethanol extract of O. stamineus at 10 mg/ml was used as a stock solution. The stock solution was then diluted with the mobile phase to 1 mg/ml as the sample solution. The total amounts of 3′hydroxy-5,6,7,4′-tetramethoxyflavone, sinensetin and eupatorin (purchase from Indofine Chemical Company, NJ, USA) in the 50% ethanol extract of O. stamineus were quantified using developed HPLC method (n = 3). The contents of these three compounds were expressed as percentages of the dried extract [18].
Isolation of the compounds
The 50% ethanolic extract of O. Stamineus was separated into ethyl acetate, butanol and water-soluble fractions. The ethyl acetate fraction with antihyperglycaemic activity was separated via silica-gel column chromatography to give two sub-fractions: namely ESF-1 (non-active) and ESF-2 (active) (ethyl acetate fraction and sub-fraction ESF-2 were active in antihyperglycaemic effect, unpublished result). The ESF-2 was fraction was then subjected to silica gel chromatography for bioactive compound isolation [19]. Isolated compound was subjected NMR study [19]. All the spectroscopic data in good agreement with Yam et al, 2010, it can be concluded that the isolated compound was sinensetin [19].
In vitro α-glucosidase inhibition study
The α-glucosidase was assayed using a method modified by Apostolidis et al[20], where a 50% ethanolic extract of O. stameinus (62.5, 31.25, 15.6, 7.8, 3.9, and 1.95 mg/ml in dimethylsulphoxide:distilled water (1:1) solution) and sinensetin (2.5, 1.25, 0.625, and 0.3125 mg/ml in methanol) were prepared. Then, 50 μl of 50% ethanolic extract of O. stameinus or sinensetin solution will mixed well with 100 μl of 0.1 M phosphate buffer (pH 6.9) containing α-glucosidase (Sigma, St. Louis, USA) solution (1.0 U/ml) and the mixture were then incubated in 96-well plates at 25°C for 10 min. After pre-incubation, 50 μl of 5 mM p-nitrophenyl-α-D-glucopyranoside (Sigma, St. Louis, USA) solution in 0.1 M phosphate buffer (pH 6.9) was added to each well at timed intervals. The reaction mixtures were incubated at 25°C for 5 min. Before and after incubation absorbance readings were recorded at 405 nm using a micro-plate reader (Thermomax, Molecular device Co., Virginia, USA) and compared to a control which contained 50 μl of the buffer solution instead of the extract. The experiments were performed in triplicate and the α-glucosidase inhibitory activity was expressed as percentage inhibition. Acarbose (Bayer Pharmaceuticals, Leverkusen, Germany) was prepared in distilled water at 10, 5, 2.5 and 1.25 mg/ml were made and used as positive controls.
Alpha-amylase inhibition assay
The inhibition of α-amylase was determined using an assay modified from the Worthington Enzyme Manual [21], where a 50% ethanolic extract of O. stameinus (62.5, 31.25, 15.6, 7.8, 3.9, and 1.95 mg/ml in dimethylsulphoxide:distilled water (1:1) solution) and sinensetin (2.5, 1.25, 0.625, and 0.3125 mg/ml in methanol) were prepared. A total of 500 μl of each concentration of extract and sinensetin mixed with and 500 μl of 0.02 M sodium phosphate buffer (pH 6.9) containing α-amylase (Sigma, St. Louis, USA) solution (0.5 mg/ml) and incubated at 25°C for 10 min. After pre-incubation, 500 μl of a 1% starch solution in 0.02 M sodium phosphate buffer (pH 6.9) was added to each tube at timed intervals. The reaction mixtures were then incubated at 25°C for 10 min. The reaction was stopped with 1.0 ml of dinitrosalicylic acid (Sigma, St. Louis, USA) colour reagent. The test tubes were then incubated in a boiling water bath for 5 min and cooled to room temperature. The reaction mixture was then diluted after adding 15 ml of distilled water, and the absorbance was measured at 540 nm using a micro-plate reader (Thermomax, Molecular device Co., Virginia, USA). The experiments were performed in duplicate and the absorbance of sample blanks (buffer instead of enzyme solution) and a control (buffer in place of sample extract) were also recorded. The absorbance of the final extract was obtained by subtracting its corresponding sample blank reading. Sinensitin was dissolved in methanol and serial dilutions of 2.5, 1.25, 0.62 and 0.31 mg/ml were prepared. Acarbose was prepared in distilled water at 10, 5, 2.5 and 1.25 mg/ml were made and used as positive controls.
Statistical analysis
All the data were showed as mean ± SD and statistical analysis were performed using one-way analysis of variance (ANOVA) followed by Tukey multiple comparison test with P < 0.05 taken as significant (GraphPad Prism 5.0).