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Materials and Methods

Collection and processing of sample

Plant samples were collected from University of Mysore campus, Mysore and subsequently identified by Dr G.R Janardhana, Department of studies in Botany, University of Mysore, Mysore. The dark brown bark pieces were cut into smaller ones and dried in hot airoven at 45°C until completely dried. The dried bark was ground to fine powder and passed through 60 size mesh. Then the prepared sample was stored at 4°C until further use.

Preparation of water extract of bark

50 g bark powder was taken in 500 mL conical flask, 250 mL of distilled water was added and the conical flask was kept on shaker for 6 h at ambient temperature. The brown colored solution was centrifuged at 100 ×g to sediment water insoluble components and the supernatant obtained was filtered through filter paper. The solution obtained was freeze dried to get dark brown colored waterextracted powder. Then the extract was stored at 5°C for further use.

Preparation of crude enzymes from rat small intestine

In the preparation of crude enzymes, procedure of Dahlquist [8] was followed. Male rats of wistar strain weighing 140-160 g were obtained from central animal house, Department of Zoology, University of Mysore. The rats were sacrificed by cervical dislocation,the intestine was immediately excised. The excised intestine was washed with maleate buffer of pH 6 (0.1 M). The brush border was carefully scraped and homogenized in maleate buffer (5:1 W/V) in cold condition. Resulting homogenate was centrifuged at 1000 ×g for 15 min at 4°C. The supernatant was pipetted and stored at -20°C forfurther use as crude enzyme source of α-glycosidase and sucrase.lled the inclusion criteria were included for this study.

Phytochemical estimation

Total phenolics: Total phenolic content of the plant leaf was assayed by the method of Folin Ciocalteau [9]. In brief, various aliquots of aqueous methanolic extract of the bark (10 mg/mL) were mixed with 5 mL Folin-Ciocaleu reagent and 4 mL of sodium carbonate (75 g/L). The resulting solution was vortexed and incubated at 40°C for 30 min. The absorption was read at 765 nm. A calibration curve was prepared by using gallic acid as standard. All determinations were performed in triplicate. Total content of phenolic compounds in bark methanol extracts in gallic acid equivalents (GAE) was calculated by the following formula:

A = 0.980C + 9.925×10.3

Where, A is the absorbance of the sample and C is theConcentration as gallic acid equivalents (μg/ml).

Flavonoids: The flavonoid content was determined by apharmacopeia method using rutin as a reference compound [10]. In brief, one mL of aqueous methanolic extract in methanol (10 mg/ mL) was mixed with 1 mL aluminium trichloride in ethanol (20 g/L) and diluted with ethanol to 25 mL. The absorption at 415 nm was read after 40 min. Blank samples were prepared from 1 mL plant extract and 1 drop acetic acid, diluted to 25 mL. A standard graph was constructed using rurtin as the reference standard using the above method. All determinations were carried out in triplicate. The amount of flavonoids in plant extracts in rutin equivalents (RE) was calculated by the following formula.

     

Where, X- Flavanoids content (mg/g) plant extract in rutin equivalents

A-Absorbance of the Sample

Ao- Absorbance of the standard

m- Weight of the sample in mg

mo- Weight of rutin in the solution

Estimation of Total Alkaloids: The sample (0.5 g) was taken in 250 mL beaker and 200 mL of acetic acid (10%) in ethanol was added and incubated at room temperature for 10 h [11]. The resulting solution was filtered and the extract was concentrated on a water bath to one quarter of the original volume. To the concentrate, concentrated ammonium hydroxide was added drop wise to the extract until the precipitation was complete. Then, the solution was allowed to settle and the precipitate was collected, washed with dilute ammonium hydroxide and filtered. Thus resulting alkaloid precipitate was dried, weighed and expressed in alkaloids per g of the sample taken.

Saponins: The Saponin content was analyzed according to a gravimetric method previously described [12]. In brief, 250 mL of 20% ethanol was added to 10 g of the pulverized bark powder and stirred at 55°C using a magnetic stirrer for 12 h. The resulting solution was filtered using filter paper (Whatman No. 1) and the filterate volume was reduced to 40 mL under vacuum, to this 20 mL diethyl ether was added in a separating funnel and shaken vigorously. The pH of the aqueous layer was adjusted to 4.5 by adding NaOH, where as the ether layer was discarded. The pH adjusted aqueous part was extracted with 60 mL of n-butanol. The butanol extract was washed twice with 10 mL of 5% NaCl and evaporated to dryness to give a crude saponin extract which was weighed and expressed in g/g to the sample.

Tannins: Tannins were estimated according to spectrophotometric method described by Trease & Evans [13]. 5 g of the bark powder was extracted with 50 mL of boiling water and filtered. 0.5 mL of the filtrate was added to 0.5 mL of ferric solution (0.5M) in an alkaline medium and allowed to stand for 30 min for color development. The absorbance was read at 760 nm and the amount of tannin was extrapolated from a standard calibration curve for tannic acid.

HPLC analysis

The HPLC system (Waters) consisting of photodiode array detector (W2998), dual pump system (515 waters), temperature control module II (TC2 waters), pump control module (PC2 waters), system controller (EMOAA01712), and a reverse phase HPLC analytical waters Symmetry column (C18, 5 μm particle size, 4.6 × 250 mm). The mobile phase was 1% acetic acid in water (solvent A)and acetonitrile: methanol (solvent B, ratio 1:1) and the separation was performed by the following linear gradient: 70:30 (Solvent A:B) for 10 min, 60:40 (solvent A:B) for another 10 min and the flow rate was adjusted to 0.6 mL/min. Injection volume was 20 μL. The chromatogram of water extract was compared with the individualchromatogram of standards bergenin, caffiec acid, cinnamic acid and kaempferol (Sigma Chemical Co, St. Louis, USA). Data analysis was done using Empower software.

Sucrase assay

The sucrase assay was carried out according to the procedure described by Honda [14]. In brief, 10 μL of crude enzyme solution (prepared as described in section 2.3), 10 μL of sample (water extract of the bark, 10 mg/mL) and 100 μL of maleate buffer were mixed in a test tube and incubated at 37°C for 10 min. The reaction was started by adding 100 μL of substrate (60 mmol of sucrose) and incubated for 30 min at 37°C in a water bath. The reaction was stopped by heating the contents on a boiling water bath for 10 min. The liberated glucose was then estimated by GOD-POD method. A control sample was also run by following the exact procedure but without adding sample. The activity, as percent inhibition of sucrase was calculated using the formulae below.

     

Alpha amylase assay

The alpha amylase enzyme assay was carried out according to the procedure defined by Ou et al [15]. Effect of Bark powder on activity of enzyme was assayed using enzyme starch system. Bark powder (1-4% w/v) and 100 mg of alpha amylase enzyme powder were added to 25 mL of potato starch solution in 50 mL plastic tubes and mixed well using vortex. The tubes were incubated at 37°C for 60 min in a shaking water bath. After incubation 2 mL of 0.1 M NaOH was added and heated on a boiling water bath for 10 min to stop the enzymatic reaction. The tubes were cooled and liberated glucose was measured using GOD-POD method. A control was also run without adding any test sample. The amylase inhibitory activity in percentage was calculated using the below formula.

     

Alpha-glucosidase assay

The glycosidase assay was carried out according to the procedure described by Honda [14]. Crude enzyme (solution prepared as described in section materials and methods), sample (water extract of bark powder), and maleate buffer were mixed in a test tube andincubated at 37°C for 10 min. The enzyme reaction was started by adding 200μL of substrate (p-nitrophenyl-a-D-glucopyranoside 2 m mol) and incubated at 37°C. After 30 min, the reaction wasterminated by treating the mixture with boiling water for 5 min. After the addition of 1.0 mL of disodium hydrogenphosphate (0.1 M), the absorbance of the liberated p- nitrophenol was read at 400 nm. An untreated enzyme solution was used as the control.

     

Glucose adsorption assay

Glucose adsorption assay was carried out according to method of Ou et.al [15]. Bark powder at various concentrations (1, 2, 3 and 4%) were added to 25 mL of glucose solution at various concentrations (5, 10, 15, 20 and 25 m mol) in 50 mL volume centrifuge tubes. The tubes were incubated on a shaking water bath for 6 h at 37°C.After incubation, tubes were centrifuged at 2500 ×g for 5 min. The remaining glucose in the supernatant was measured using GODPOD method. A control was also run without adding any test samples. Glucose adsorbed by the sample was calculated by the following relation mentioned below.

     

Where, G1 glucose concentration of original solution and G2 is glucose concentration of the same solution after incubation for 6h.

Statistical analysis

All the values are mean ± SD of triplicates determinations of three experiments. The data was analyzed by ANOVA followed by Tukey’s multiple comparisons test for significant differences using SPSS 14.0 software and the graphs were plotted using Origin 6.1 software. Thevalues were considered significant at p ≤ 0.05. IC50 value for all the enzyme assays were calculated by slope equation from the scatter graph in Microsoft excel.