Chemical profiling and in-vitro α-amylase inhibitory activity of Sesbania sesban and Sesbania grandiflora seeds

ABSTRACT S. sesban is mainly used as economical purpose and S. grandiflora is known for medicinal as well as economical. Chemical characterization and antidiabetic activity of seed extracts were not explored yet. Seeds of S. sesban and S. grandiflora were separated in seed coat, germ, and endosperm by domestic grinder mixer. Successive soxhlet extraction of separated parts of Sesbania seed were carried out. Chemical profiling of methanolic extract was carried out by GC-MS, HPLC, and in vitro alpha amylase inhibitory assay. D-pinitol was found to be maximum (48.39%) in seed coat and minimum in seed (8.35%) of methanolic extracts of Sesbania seed. Alpha-amylase inhibitory activity of methanolic extracts of different parts were also studied in which DSSh, DSSp, & DSSe showed similar IC50 to acarbose, antidiabetic drugs. The result showed that S. sesban and S. grandiflora seed and endosperm is rich in health promoting mineral like Na (241 mg/100 gm and 238 mg/100 gm), K (103.1 mg/100 gm) and (60.2 mg/100 gm), Ca (525 mg/100 gm) and (500 mg/100 gm) and Fe (67.5 and 91.9 mg/100 gm). Hence, it is similar to some edible legumes which possess bioactive compound with therapeutic value and can be efficiently explored for the development of new functional or nutraceutically valued food products.


Introduction
The plants of Sesbania sp. (Fabaceae) belongs to leguminous; they are extensively found in many of the tropical countries of Asia (especially India, China, and Thailand) and in Africa. Although numerous subgenera and species have been reported, the most popular species include S. bispinosa (spiny Sesbania), S. drummondii (rattlebox), S. aculeata, S. grandiflora, and S. sesban, which are used for edible purpose by some ethnic groups. Leaves and young pods of S. grandiflora are believed to be nutritious, edible (eaten as string beans), and used as a vegetable. The dried leaves of S. grandiflora and S. sesban are known to be used in indigenous medicines and the preparation in the form of tea is believed to be effective to have antibiotic, antihelminthic, antitumor, and contraceptive properties. [1,2] Sesbania species commonly known as Dhaincha belongs to Fabaceae, are not food plants except in some cases where leaves and flower used as vegetables. [3] They are mainly used in agriculture as a green manure to improve soil fertility and also for animal fodder. The leaves and tender branches of Sesban are high in protein (20-25%) and have high digestibility when consumed by ruminants, such as goats and cattles and used as a potential seed include improved animal nutrition. Sesbania leaves, flowers, pods, and seeds are sources of animal feed and possibly also food for humans. The mature seeds of S. bispinosa are known to be cooked and eaten by Indian tribals like Katkharis and Ghonds [4] and important manure crop due to rapid growing behavior, succulent, easily decomposable along with low water requirement and produces maximum amount of organic matter enriched with nitrogen. [5,6] Seed and fibers have been already reported for cellulose, [7] lignins, [8] and galactomannans. [9] Seed, leaves, and flowers of S. bispinosa have been reported for good content of pinitol. [10] Phytochemicals were identified in S. bispinosa seed extracts through HPLC-ESI-MS/MS, that is, carboxylic acid (37%) and flavanoids (47%). Prosaikogenin and delphinidin −3-O-glucoside. MS fragment peak at 20.56 minute and hexagallolyl peak at 10.96 minute, which is consistent with six galloyl units. 18 compounds were identified with GC-MS in which sucrose and furanose were the major compounds. [11] Diabetes mellitus is one of the serious problems due to leading causes of death in all over the world and now nutraceuticals as the up and coming treatment of diabetes mellitus. When the metabolic regulation of carbohydrates and lipids is damaged, it induces an improper insulin function, and the blood sugar increases. Postprandial hyperglycemia can initiate multiple secondary complications, such as nervous lesions, retinopathy, and kidney disease. [12] S. sesban seed is also a source of many essential mineral especially sodium, potassium, phosphorus, iron which is responsible for nutrition. The leaves, flowers, seeds,and pods of the S. grandiflora are used in traditional Indian recipes. It is rich source of proteins, vitamins, antioxidant and minerals. It is used as human food and animal fodder. [13,14] Young pods are added to soups and vegetable curries. Moreover, different extracts of S. sesban and S. grandiflora showed various medicinal properties viz. antioxidation activity, anti-inflammatory, antidiabetic, and anticancer activity. 100, 200, and 400 mg/ kg of S. grandiflora leaves reported for antihyperglycemic activity in glucose overloaded hyperglycemic rats and hypoglycemic activity in overnight fasted normal rats. [15] Antidiabetic activity were studied in different doses of Sesbania sesban petroleum ether extracts of root, that is, 250 mg/kg, 250 mg/kg, and 1000 mg/kg body weight. Extract showed improvement in various body and serum parameters as well as regeneration β-cells of pancreas. [16] S. grandiflora extract showed maximum 81% alpha amylase inhibitory activity at 1000 µg/ml while Acarbose showed 93% at the same concentration. [17] Hence, it is important to investigate chemical composition of seed extracts by GC-Mass and reverse phase HPLC method and also evaluate the Anti-diabetic of methanolic extracts of separated parts of S. sesban and S. grandiflora seed. Similar to some of the edible legumes, even the wild and underexploited legume seeds might possess certain bioactive compounds with health promoting or with potential therapeutic value. This aspect can be efficiently explored for the development of new functional or nutraceutically valued food products.

Materials
Seeds were procured from Lucknow, India through established indigenous supplier. For the authentication, seeds were sown in the month of May, 2016 in Barabanki and took four months to grow the crop. Specimen samples of S. sesban and S. grandiflora seeds were prepared, authenticated, and deposited in the NBRI, herbarium with LWG no. 107999 and 108456, respectively.

Separation
Seeds of S. sesban were too much smaller in comparison of S. grandiflora. It was quite difficult to separate seeds into seed coat, germ, and endosperm. So that wet method was selected to separate seeds of both species. Seeds were grinded by domestic mixer with optimize speed, cotyledons were separated by several times sieving and milling but separation of seed coat from endosperm was not easy, endosperm with seed coat soaked in distill water: ethanol (1:1) for 2 hour and grinded with domestic mixer at medium speed, yield of seed coat varied from (10.34-11.35%), germ (30.45-42.28%), and endosperm (26.67%). The standard pinitol purchased from Sigma Aldrich, Bengaluru.

Extraction of separated materials
Separated seed parts of S. sesban and S. grandiflora were powdered using mechanical grinder and passed through sieve ≠ 40 and successive soxhlet extraction from nonpolar to polar solvents were done. Separated seed powder was extracted with hexane, chloroform, acetone, ethanol, methanol, and water successively in a Soxhlet apparatus on water bath. The solvents were evaporated by rotavapor to obtain dry extracts and % yield was calculated as (dry extract weight/weight of sample)x100.

Mineral contents
Sample (100 mg) was taken in digestion tube, HNO 3 and H 2 O 2 (5:1) were added and heated upto 170-180°C until white residue obtained and volume made up to 50 ml milli q water and determined by ICP-MS.(7500cx) start at 40°C for 15 min; heating at 60°C for 15 min; stay at 60°C for 15 min; heating to 90°C for 20 min. ICP analysis of elements present in the digested sample was compared with standard sample viz. Ni, Cr, Cu, Na, Ca, K, Fe, Cd, Zn. Na, K, and Ca were estimated through flame photometer. [18]

GC-MS analysis of Methanolic extracts
GC-MS analysis of the methanolic seed extract was performed by using the equipment Thermo GC Trace Ultra Version: 5.0, Thermo MS DSQII. The equipment has a DB 35 -MS Capillary Standard nonpolar column with dimensions of 30 mm ×0.25 mm ID ×0.25 µm films with a split ratio 1:25. The carrier gas used is Helium with at flow of 1.0 ml/min and injector was operated at 250°C and the oven temperature was programmed as follows: 60°C for 15 min, then gradually increased to 280°C at 3 min. The identification of components was based on Willey and NIST libraries as well as comparison of their retention indices. [19]

HPLC analysis of methanolic extracts
Pinitol was analyzed by using device prepared with PDA and Refractive index detector. HPLC XBridge Amide column (250mm x 4.6 mm, 3.5 µm: waters, USA) was used for estimation of pinitol. Standard stock solution was diluted separately in Acetonitrile and H 2 O 7:3, (v/v) at a concentration of 2 mg/ml. Sample study accomplished at 30°C using isocratic solvent system with flow rate 1 ml/min for 20 minute analysis, 20 µl volume of standard and extracts were injected for analysis. During study spectra were recorded in the range of 210-400 nm. [10] In vitro α-Amylase inhibitory assay The α-amylase inhibition assay was performed by 3, 5-dinitrosalicylic acid method. [20] The methanolic seed extract was dissolved in 100% DMSO solvent and further dissolved in sodium phosphate buffer (0.02 M), NaCl (0.006 M) at pH 6.9 to give concentration range from 6.25-200 μg/ml. 200 μl of αamylase solution (2unit/ml) was mixed 40 μl of plant extract was incubated for 10 minutes at 30°C and 200 μl of the starch solution (1% w/v in water) was added to each tube and incubated for 3 minutes. The reaction was stopped by the addition of 200 μl DNSA reagent (12 g of sodium potassium tartrate tetrahydrate in 8.0 ml of 2 M NaOH and 20 ml of 96 mM of 3, 5-dinitrosalicylic acid solution) and boiled for 10 min in a water bath at 85-90°C and 30 μl of reaction mixture was diluted with 270 μl of distill water on 96 well microtiter plate. Absorbance was measured at 540 nm wavelength using UV-Visible spectrophotometer and blank was prepared by using 200 μl buffer in place of plant extracts. A positive control was prepared using acarbose (6.25-200 μg/ml) and reaction was performed similar to the plant extract as given above. The α-amylase inhibitory activity was expressed as percent inhibition as given below % α-amylase inhibition = 100x Abs[(control) −Abs(Sample)/ Abs(Control)

Statistical analysis
Statistical analysis were performed using SPSS 16.0 software in triplicate. One way ANOVA with tukey's multiple comparison tests and results were expressed as a mean ± standard deviation accepted as significantly different at 95% confidence interval (p < .05).

Results and discussion
Successive extraction of seed material was done to fractionate nonpolar and polar metabolite. Nonpolar metabolites like fatty acid, sterols were present in hexane extract. Polar metabolites were present in ethanol, methanol and water extract. Maximum yield was obtained in water extract of the endosperm of S. grandiflora i. e. 14.57% while in methanol12.12% in the seed of S. sesban. The maximum yield was also obtained in aqueous extract of germ i.e. 16.2% while in methanol 5.8% in the seed coat.

GC-MS profile of sugars and polyols in different parts of Sesbania seed
A typical chromatogram was obtained after derivatization with pyridine and TMS of methanolic extract of different separated parts of Sesbania sesban and Sesbania grandiflora seed and peaks in chromatogram were correspond to TMS derivative of the corresponding sugar and polyalcohol shown by Table 1 [21] Low molecular weight carbohydrates were detected in edible legume extracts by GC-MS method. [22] Table 1 showed pinitol content varied from 8.35% to 48.39%, absent in germ of Sesbania sesban and endosperm of Sesbania grandiflora while D-fructose was present in seed coat and endosperm of Sesbania sesban 10.13% both and also in germ of Sesbania grandiflora. Myo-inositol content varied from 1.89% to 10.78% while absent in seed coat of Sesbania sesban, endosperm and seed coat of Sesbania grandiflora. TLC, GC, and GC-MS were used for the isolation and identification of sugar and cyclitols in carob powder. Major compounds identified were sucrose, fructose, glucose, D-pinitol, myo-inositol, and D-chiro-inositol along with traces of ononitol and sorbitol. [19] Carob and soyabean are considered to be richest pinitol source among Leguminosae family. [23] Arachidonic was found only in endosperm 43.60%. Mass fragmentation pattern was matched with standard pinitol in which pinitol showed RT at 22 Arachidonic acid is found only in endosperm of S. grandiflora (43.60%) and it is an important metabolite used in inflammation, starting material in the synthesis of two kinds of essential substances, the prostaglandins and leukotrienes. Arachidonic acid is a polyunsaturated omega-6-fatty acid (20:4), structurally related to the saturated arachidic acid found in cocoa butter. [24] Arachidonic acid used as nutritional supplement in new borns, neurological disorders, schistosomicidal action and tumoricidal potential. [25]

RP-HPLC-PDA-RI analysis
Pinitol (%) was calculated in respect of peak area of standard solution ( Table 2). Chemical finger printing profile of all methanolic extracts in different parts of S. sesban and S. grandiflora seed were developed. Maximum pinitol was found in germ and endosperm of S. grandiflora, that is, 6.85% and 6.11%, respectively, while in the endosperm of S. sesban, that is, 14.5%. Minimum Pinitol was found in seed coat of S. sesban, that is, 2.95%. Pinitol was absent in the seed coat of S. grandiflora and seed powder of S. sesban. It is renowned natural antidiuretic, anti-inflammatory, hypoglycemic and considered as an effective phytotherapeutic. Therefore, RP-HPLC-PDA-RI protocol is a useful tool in fingerprint profile of S. sesban and S. grandiflora methanol extracts. Hence, there is prospects of isolation of D-pinitol from germ of S. grandiflora and endosperm of S. sesban. Pinitol and other sugars were estimated by HPLC showed maximum concentration of pinitol at 84.59 mg/gm in wild type carob pods. Correlation between sugars profile (glucose, sucrose, and fructose) and D-pinitol content of different types of carob pods (wild and cultivated) was also reported. [26]

Mineral content analysis
Trace elements are randomly distributed and speciated throughout the cereal grain. The germ and the outer layers of the grain have the highest concentrations of trace elements. A large fraction of the trace elements is therefore lost during the milling process which was analyzed by ICP-MS (Table 3)

Alpha-amylase inhibitory activity
A graph has been plotted against % alpha-amylase inhibition and concentration ( A sufficient amount of D-pinitol reported to significantly reduce the blood glucose level during 0.5-2 h after administration was estimated to 10 mg/Kg body weight. Thus, an average person weighing Ca 0 ± 0 525 ± 0.003 500 ± 0.04 0 ± 0 60 kg should consume at least 600 mg of D-Pinitol in order to achieve any beneficial health effect. [27] These α-amylase inhibitors are starch blockers as they stop the hydrolysis of slows the absorption of starch into the body mainly by blocking the hydrolysis of 1,4-glycosidic linkages of starch glucose level in blood plasma [28] into body and decrease the and other oligosaccharides into maltose, maltriose and other simple sugars. The α-amylase inhibitory activity in methanol extract is most likely to be due to polar compounds and is worth investigating further and isolating pure active compounds. Alpha amylase inhibitory activity was observed in D. villosa from 10.71% to 71.88% with concentration 1.5-1000 µg/ml and IC 50 value was also calculated as 71.44 µg/ml in comparison to acarbose (83.23 µg/ml). [29] Alpha-amylase inhibitory activity of hexane and ethanol extract of P. amarus was also studied. It was found that both showed appreciable amount of inhibitory activity in comparison of acarbose. [30] Alpha amylase inhibitory activity of S. grandiflora extract was studied and it was found that maximum inhibition shown at 1000 µg/ml concentration, that is, 81% while acarbose 93% at same concentration. 17

Conclusion
Different metabolites were identified in different parts of Sesbania seeds viz. D-Pinitol, myo-inositol, D-fructose and sucrose and also have appreciable alpha-amylase activity. Arachidonic acid was found as major marker in endosperm of S. grandiflora. Hence, further studies on isolation of such metabolites from Sesbania seed as a reference marker and in therapeutics serve as potential source in drug development as health supplement.