Phytochemical Screening , LC-MS Studies and Antidiabetic Potential of Methanol Extracts of Seed Shells of Archidendron bubalinum ( Jack )

Background: Some Malaysia and Indonesia people believed that root and seed shell of Archidendron bubalinum can treat diabetes. However, seed shell of Archidendron bubalinum has not yet to be scientifically proven and confirmed their ability to treat diabetes. The study of the potential of this seed shell was also scarcely available. Objective: The recent work was aimed to investigate the phytochemical screening of methanol extracts of seed shells of Archidendron bubalinum and to evaluate their chemical compositions and antidiabetic activities. Material and Methods: The methods of phytochemical screening were including alkaloids, flavonoids, tannins, polyphenols, saponins, and terpenoids. Their chemical compositions were determined by Liquid Chromatography-Mass Spectrometry (LC-MS) and antidiabetic activities were performed by α-glucosidase inhibitory method. Results: The phytochemical screening showed that methanol extracts of seed shells of Archidendron bubalinum contain flavonoids, tannins, polyphenols, and terpenoids. This extracts exhibited antidiabetic activity with IC50 7.77 μg/mL. This result was supported by LC-MS analysis which showed the presence of phlorizin and astilbin, in which these compounds had high inhibitory activity against α-glucosidase or diabetes. Conclusion: LC-MS analysis revealed the presence of polyphenol compounds namely phlorizin and astilbin in which had high α-glucosidase inhibitory activity, might largely contribute in the antidiabetic activity.

middle income countries, including China, India, Indonesia, Pakistan, Egypt and Mexico. 3Diabetes is a well-known metabolic disorder, which is characterized by an abnormal postprandial increase of blood glucose level.The control of postprandial hyperglycemia is believed to be important in the treatment of diabetes.α-glucosidase secreted from intestinal chorionic epithelium is responsible for the degradation of carbohydrates.α-glucosidase inhibitors slow down the process of digestion and absorption of carbohydrates by competitively blocking the activity of α-glucosidase.Consequently, the peak concentration of postprandial blood glucose is reduced and the blood sugar level comes under control. 4Some of α-glucosidase inhibitors, such as acarbose that obtained from natural sources, can effectively control blood glucose levels after food intake and have been used clinically in the treatment of diabetes. 5Only a few α-glucosidase inhibitors are commercially available.All of them contain sugar moieties and their synthesis involves tedious multistep procedures.Moreover, clinically they have been associated with serious gastrointestinal side effects.Therefore, it is necessary to search for alternatives that can display α-glucosidase inhibitory activity but without side reactions. 6-glucosidase inhibitors can be used as a new class of antidiabetic drug.411 compounds exhibiting α-glucosidase inhibitors activity were summarized and isolated them from medical plants.The compound classes isolated include: 61 terpenes, 37 alkaloids, 49 quinines, 103 flavonoids, 37 phenols, 73 phenylpropanoids, 8 steroids, and 43 other types of compounds.] The species Archidendron bubalinum belongs to the family Fabaceae or Leguminosae in the major groups of Angiosperms.It contains about 126 species and is a large and economically important family of flowering plants.Fabaceae is the most common family found in tropical rainforests and in dry forests. 9The species is indigenous to Sumatra in Indonesia, Peninsular Malaysia and Thailand.The vernacular name is kabau or julang jaling (Indonesia), kerdas (Malaysia) and nieng-nok (Thailand).The strong pungent smelling seeds have an odor like jering (Archidendron jiringa) and petai (Parkia speciosa).The bark of Archidendron bubalinum is used as febrifuge.Young seeds can be eaten in raw as traditional vegetable salad and can be used traditionally as a diuretic but excessive consumption can lead to poisonous and kidney problem. 10 Archidendron bubalinum seeds were found to have high moisture content, low fat content, high protein content but being limited in valine, methionine and tyrosine. 11Archidendron bubalinum contained of anti-nutrient: tannins, trypsin inhibitors and hemaglutinin.Studies in Thailand have been reported that crude extracts of Archidendron bubalinum seeds had superoxide scavenging activities. 124] The study of the potential of this seed is scarcely available and little attention is given due to limited resources.Archidendron bubalinum has not yet to be scientifically proven and confirmed their ability and chemical constituents to treat diabetes. 14he objective of this study is to identify the types of compounds found in the methanol extracts from seed shells of Archidendron bubalinum from Lampung Indonesia and to investigate its potential antidiabetic activity.

MATERIALS AND METHODS
The seeds of Archidendron bubalinum were collected from uncultivated farm lands located at Lampung, Sumatera Island, one of province in Indonesia.The seeds were peeled to separate the seed shells and seed (Figure 1).The seed shells were collected, thoroughly washed and then sun dried for 4 h.The dried seed shells were soaked in methanol with ratio 1:10 (w/v) and extracted by maceration methods for 24 h.Then, the supernatant was filtered by filter paper.Soaking process was repeated once again in the same sample and the supernatant was filtered.All the supernatant were collected together and concentrated using rotary evaporator at 50 o C.

Alkaloids determination
About 100 mg of methanol extracts was mixed in 5 ml of 10% hydrochloric acid then pH was adjusted to 8 with dilute ammonia solution.The mixture was added with 20 ml chloroform and then dried.The extracts were tested with Dragendorff reagent and Mayer's reagent.The formation of precipitate was indicating the presence of alkaloids.

Flavonoids determination
About 100 mg of methanol extracts was boiled in 100 ml of distilled water for 5 min and filtered.5 ml of the filtrate was mixed with 0.1 mg magnesium powder, 1 ml concentrated hydrochloric acid and 1 ml amyl alcohol then shaked vigorously.The colour changed to yellow or red or orange, indicating the presence of flavonoid.

Terpenoids and steroids determination
About 100 mg of methanol extracts were mixed with 25 ml diethyl ether and shaked vigorously.The diethyl ether layer was separated then added 2-3 drops of Lieberman-Burchard reagents.A bluish colour of the interface was formed, indicating positive results for the presence of terpenoids while greenish colour, indicating positive results for the presence of steroids.

Saponins determination
About 100 mg of methanol extracts were added with 10 ml of boiled distilled water, cooled and shaked vigorously for 10 min.The foam formation was observed and will be stable if added with a few drops of hydrochloric acid, indicating the presence of saponins.

Tannins and polyphenols determination
About 100 mg of methanol extracts were extracted with 1 ml ethanol and 1 ml distilled water.The filtrate was mixed with a few drops of 1% FeCl 3 and into another filtrate added with gelatin salt.Then observe the colour changing, if a green or blue or purple colour was appeared, indicating the presence of tannins.

Liquid Chromatography-Mass Spectrometry (LC-MS) studies
The chemical constituents of the methanol extracts were determined using LC-MS.LC-MS analysis was performed using Mariner Bio spectrometry equipped with a binary pump.The HPLC was interfaced with a Q-TOF mass spectrometer fitted with an ESI source.Full-scan mode from m/z 100 to 1200 was performed with a source temperature of 140°C.HPLC column Phenomenex 5μ C8, (150 × 2 mm i.d.) was used for the analysis.Solvent was methanol with 0.3% formic acid.Solvents were delivered at a total flow rate of 0.1 mL/min.The solvent was run by isocratic elution.The MS spectra were acquired in the positive ion mode.The temperature of the drying gas (N 2 ) was 350 o C, at a gas flow rate of 6 mL/min, and a nebulizing pressure (N 2 ) of 25 psi.About 0.5 g of sample extracts was diluted with methanol and filtered with 0.22 µm nylon filter S79 prior to analysis.A 5 µl volume of the extracts were injected onto the analytical column for analysis.The mass fragmentations were identified by using spectrum database for organic compounds in SDBS application.
α-Glucosidase inhibition test 16,17 A 250 μL solution of p-nitrophenyl-α-D-glucopyranoside 5 mM and 495 μL phosphate buffer 0.1M pH 7 was added to the reaction tube containing 5 μL of the sample solution in DMSO with a concentrations variation of 100, 50, 25, and 10 μg/mL.After homogeneous, the solution was pre incubated for 5 min at 37°C, the reaction was initiated by the addition of 250 μL α-glucosidase solution (0.062 units), incubation was continued for 15 min.The reaction was stopped by the addition of 1 ml of Na 2 CO 3 0.2 M. The activity of the enzyme was measured, based on the reading of p-nitrophenol absorbance at λ 400 nm.Quersetin was used as a reference standard with concentration of 10, 7.5, 5, 2.5, and 1 µg/mL.% inhibition was measured by using the equation:

Phytochemical screening
The phytochemical screening of methanol extracts of Archidendron bubalinum seed shells revealed the presence of flavonoids, tannins, total phenols, and terpenoids, while alkaloids and were not detected (Table 1).These results are the same as in the husks sample analysis.However, this extract provides a different bioactivity, in which this extract gives positive results against antidiabetes (Table 3).This is interesting so it proceeds with the analysis of phytoconstituents using LC-MS

Liquid Chromatography-Mass Spectrometry (LC-MS) studies
LC-MS analysis of methanol extracts of Archidendron bubalinum seed shells had detected five peaks with the retention time 0.41, 1.28, 1.90, 2,63, and 2.80 minutes (Figure 2).Then each peak was fragmented, resulting 5 fragmentation spectrum with candidates mass (m/z) 365, 436, 436, 247 and 450.(Figure 3 and Table 2) The LC-MS spectrums interpretation was performed using a spectrum database for organic compounds in SDBS application.The results of spectrum interpretation on methanol extracts of Archidendron bubalinum seed shells, indicating that there are substance of phlorizin and astilbin with the retention time of 1.90 and 2.80, respectively.These results were confirmed by each of the fragmentation pattern as figure-4 and figure-5
Based on the results of antidiabetic test in Table 3, Figure 6, it can be seen that the methanol extracts of Archidendron bubalinum seed shells had high antidiabetic activity with IC 50 of 7.77+0.11µg/mL.This value was almost same as IC 50 of quercetin that was 6.04+0.14µg/mL.] Phlorizin is a natural product and dietary constituent found in a number of fruit trees.It has been used as a pharmaceutical and tool for physiology research for over 150 years. 21Both the phlorizin which was alkaloid compound, as well as astilbin have many phenolic groups in their molecules.Polyphenols were often present in polar glycosides and were easily soluble in polar solvents.The antioxidant and antidiabetic properties of these compounds were related to the presence of phenolic groups that can donate hydrogen atoms to a free radical so that the radicals become less reactive.The antidiabetic mechanism of methanol extracts of Archidendron bubalinum seed shells depended on the content of the compounds therein.Methanol extracts of Archidendron bubalinum seed shells was useful as an antidiabetic through several mechanisms: 1. Methanol extracts of Archidendron bubalinum seed shells were rich in flavonoids and phenolic compounds such as phlorozin and astilbin.Phenolic compounds can donate their hydrogen atoms and function as free radical inhibitors, where phenolic compounds will protect organs such as the pancreas from free radicals attack.(Figure 7) 2. Another mechanism was by competitively inhibiting α-glycosidase activity, these inhibitors help to prevent the fast breakdown of sugars and thereby control the blood sugar level.

CONCLUSION
Phytochemical screening showed that methanol extracts from Archidendron bubalinum seed shells containing many compounds either flavonoids or phenolic compounds.Based on the LC-MS analysis which was interpreted by SDBS application, it can be identified that some of the compounds in the methanol extracts were phlorizin and astilbin.From the results of antidiabetic test, it was proved that methanol extracts of Archidendron bubalinum seed shells had high antidiabetic activity with IC 50 of 7.77+0.11µg/mL which was almost same with IC 50 of quercetin standard, 6.04+0.11µg/mL.High antidiabetic activity of methanol extracts of Archidendron bubalinum seed shells were coming from the compounds of phlorizin and astilbin.From these results, it can be concluded that methanol extracts of Archidendron bubalinum seed shells had great potential as biomedicine for diabetes diseases.

mentioned. 18
The spectrum of fragmentation mass of phlorizin was written by m/z 436 of molecular ion of C 21 H 24 O 10 resulted from phlorizin substance when it was exposed with 70 eV of energy.Molecular ion of C 21 H 24 O 10 was experiencing fragmentation and McLafferty reorganization by releasing C 6 H 10 O 5 and producing fragments by m/z 274 originating from C 15 H 14 O 5 .(Figure 4) The mass spectrum of astilbin was written by m/z 450 originating from the molecular ion of C 21 H 22 O 11 which was formed when it was exposed by 70 eV of electron.Molecular ion of C 21 H 22 O 11 was experiencing fragmentation and McLafferty reorganization by releasing C 6 H 10 O 5 and producing fragments by m/z 288 originating from C 15 H 12 O 6 .The pattern of fragmentation of astilbin can be seen in Figure 5.

Figure 6 :
Figure 6: Graph of the relationship between concentration and % inhibition for IC 50 determination.A) Quercetin B) Methanol extracts.

Figure 7 :
Figure 7: Reaction of phenolic compounds with free radicals.