Phytochemical composition and in-vitro Anti-diabetic potential of Swietenia mahagoni seed pod

Naveen Y P. Swietenia mahagoni seed pod was screened for its anti-hyperglycemic potential by using In-vitro assays. Dried powder of seed pod was used in glucose adsorption and glucose diffusion retardation index assay, where as the aqueous and methanol extract of the seed were used to study the inhibition of enteric enzymes (α-amylase and sucrase). The bark powder showed dose dependent glucose adsorption and glucose diffusion retardation index. Aqueous extract was more potent in inhibiting the activity of αamylase than methanol extract. There was no notable activity observed in case of sucrase assay. Overall, assays partly indicate the anti-hyperglycemic potency of the seed pod which may be mediated through the inhibition of enteric enzymes. Further investigations are needed to validate this observation Copy Right, IJAR, 2016,. All rights reserved.


Methods:-Preparation and extraction of sample:-Collection and preparation of samples:-
The seed pod of the sweitenia mahagoni fruits were collected from University of Mysore campus in the month of Fed 2016.

Sample preparation:-
The collected seed pod was thoroughly washed under running water to remove adhering dirt and other foreign particles, dried for a day at 50°c.The dried sample was powdered and passed through 60 mesh sieve. Thus obtained powder was stored in air tight container at 4°c till further use.

Preparation of aqueous extract of S.mahagoni seed pod :-
The aqueous extract of the sample was prepared by extracting powder material with distilled water in a mechanical shaker for 24 h at ambient temperature. The solution obtained was filtered through Whatman filter paper (No.4) to separate insoluble component and thus obtained filtrate was freeze dried. A extract with brown colored and fine powdered texture was obtained after freeze drying.

Preparation of methanol extract of S.mahagoni:-
The methanol extract of the sample was prepared by extracting powder material with methanol in a mechanical shaker for 24 h. Filtered to separate insoluble component and the filtrate was flash evaporated . The brown colored methanol extract obtained after flash evaporation was used in all in-vitro assays.
Phytochemical composition:-Estimation of total phenolics:-Total phenolics: Total phenolic content of the leaf was assayed by the method of Folin Ciocalteau (Slinkard and Singleton., 1977). In brief, various aliquots of aqueous methanol extract of the seed pod (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 seed pod 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 the Concentration as gallic acid equivalents (μg/ml)

Saponins:-
The Saponin content was analyzed according to a gravimetric method previously described (Miliauskas et.al., 2004). 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 filtrate 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, whereas 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 of the seed pod powder.

Tannins:-
Tannins were estimated according to spectrophotometric method described by Trease & Evans (Hudson and El-Difrawi, 1979). 5 g of the seed pod 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.

Estimation of total alkaloids:-
Alkaloids were estimated by the gravimetric method (Naveen Y P and Asna Urooj, 2015). The sample (0.5 gm) 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. 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 until the precipitation was complete. The above solution was allowed to settle and the precipitate was collected and washed with dilute ammonium hydroxide and filtered. Thus resulting alkaloid precipitate was dried, weighed and expressed in alkaloids per gm of sample taken.

Estimation of Flavonoids:-
The content of flavonoids was determined by a pharmacopeia method (Bickel H and Schultz G, 1976) using rutin as a reference compound. For brief, One ml of aqueous extract in methanol (10 mg/ml) was mixed with 1 ml aluminiumtrichloride in ethanol (20 g/l) and diluted with ethanol to 25 ml. The absorption at 415 nm was read after 40 min at 20 C. Blank samples were prepared from 1 ml plant extract and 1 drop acetic acid, and 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.

Anti-diabetic assays:α-amylase Inhibition Assay:-
The αamylase enzyme assay was carried out according to the procedure defined by Ou et al . Effect of seed pod aqueous and methanol extract on activity of enzymes was assayed using enzymes starch system. Various concentrations of aqueous and methanol extract and 100 mg of α amylase enzyme powder added to 25ml 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 2ml of 0.1 M NaOH was added and heated on a boiling water bath for 10min 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. % = − × 100 Glucose adsorption assay:-Glucose adsorption assay was carried out according to method of a previous study (Ou et.al., 2001). Seed pod 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 original solution and G2 is glucose concentration of the same solution after incubation for 6h.

Glucose uptake in the yeast cells:-
Yeast cells were prepared according to the method ou et al. Briefy, commercial barker's yeast was washed by repeating centrifugation (3,000 RPM for 5min) in distilled water until the supernatant fluids were clear and a 10%(v\v) suspension was prepared in distilled water . various concentration of aqueous and methanol extracts (1-5mg) were added to 1ml of glucose solution (5.10 and 25Mm) and incubated for 10min at 37°c reaction was started by adding 100µl of yeast suspension, vortex and further incubated at 37°c for 60min. After 60min the tubes were centrifuged (2,500×g for 5min) and glucose was estimated in the supernatant.

Glucose diffusion retardation Index:-
Potato starch solution (1%) was prepared in phosphate buffer (0.05 M, pH 6.5). 25mL of starch solution was added with α-amylase (100 mg) and seed pod powder at various concentrations in dialysis bags of 12 KD cutoff, and dialyzed against 200mL of deionized water at 37°C in a shaker water bath. The glucose diffused into the dialysate was determined at 20,40,60 and 120 min using GOD /POD diagnostic kit. The values are indices of glucose diffusion retardation index (GDRI). A control, without samples, was also run in the same conditions.

Results:-Phytochemical composition:-
The phytochemical composition of the Swietenia mahagoni seed pod extract is presented in the Table. From the  table it can be observed the extract has a significant amount of total polyphenols, tannins and saponins.

Glucose uptake in yeast cells:-
Both the extracts showed moderate inhibitory activity on the glucose uptake by the yeast cells.

Glucose diffusion retardation index:-
Effect of the seed pod powder on glucose diffusion retardation index is given the Table 3. The glucose retardation potential increased with the increase in sample concentration.  (Nijveldt et.al., 2001). The saponins which are surface-active glycosides, have been shown immensely investigated for their immunostimulant, hypocholesterolaemic and anticarcinogenic potency (Francis et.al., 2002).
Mahagoni seed pod posses all the phytochemical groups in a considerable amount signifying its pharmacological value.
Carbohydrates are major energy source for the individuals, which are present in the diet in the form of polysaccharides and simple monosaccharide. Monosaccharide sugars such as glucose, fructose, galactose are directly taken up by intestinal epithelial cells . In case of complex polysaccharides, they have to be digested into simpler monosaccharides prior absorption. Digestion of these complex polysaccharides takes place with the aid of enzymes (Levin, 1994). Enzymes such as α-amylase, α-glycosidase, sucrase and lactase play a crucial role in complete digestion of polysaccharides into simpler monosaccharide units. The liberated mono-saccharides diffuse to intestinal epithelial cells where they are taken by the passive diffusion, facilitated diffusion through transporters named Glut transporters and by co-transport with other ions, mainly sodium ion . The process of glucose absorption is hindered if any of the above process is hindered. Phytochemicals/synthetic components which retard any of the processes can be considered as anti hyperglycemic agents and act by inhibiting the entry of glucose into blood stream.
Glucose adsorption onto adsorbents is a physical phenomenon, in the intestine the adsorption of glucose onto food matriz can result in decreased free glucose in the solution, thus can reduce the available glucose for uptake by the intestinal epithelial cells (Gallagher et.al., 2003). The seed pod powder has shown good glucose adsorbing and glucose diffusion retardation potential thus indicating its ability to decrease the glucose availability to diffusion into blood stream. Fiber is one of the main components in the bark and seed coat of the most plants and the insoluble fiber has shown to have potent glucose adsorbing potency (Aditomre et.al., 1990), thus fiber content in the seed pod powder may be component responsible for glucose adsorption and glucose retardation index. Inhibition of αamylase decreases the starch breakdown and thus breakdown of complex carbohydrate into simple absorbable sugars (William and Schenker, 1976).

Conclusion:-
Preliminary assays in the present investigation indicated the anti-diabetic potential of Swietenia mahagoni seed pod. The observed activity may be attributed to the presence of potential bio actives in the seed pod. Further research support is needed to validate the observed potential.