Fractionation and α-glucosidase Inhibitory Activity of Fractions from Garcinia hombroniana Pierre Leaves Extracts

Background: Diabetes mellitus become one of the biggest global health problems of the 21st century. Type 2 diabetes play role for the majority of cases of diabetes worldwide which is characterized by the increase of postprandial blood glucose level. Maintaining postprandial glucose level through inhibition of α-glucosidase is one of the essential strategies in the treatment of diabetes. Inhibitory effect of α-glucosidase was commonly used to identify active compounds potentially to treat diabetes. Natural resources have potency as antidiabetic that can be used in diabetes treatment. Objective: The objective of the study is to separate active fraction in the crude extract of Garcinia hombroniana leaves to facilitate obtaining a pure biologically active compound as the α-glucosidase inhibitor. Methods: Fractionation to separate active fraction was performed using column and thin layer chromatography methods while α-glucosidase inhibitory activity assay was performed in vitro using spectrophotometric methods at λ 400 nm. Results: Ethyl acetate and methanol extract of G. hombroniana yielded 14 and 12 fractions, respectively. Two fractions with the higher percent inhibition compared to other factions are fraction 8 from ethyl acetate extract (FEA8) and fraction 3 from methanol extract (FM3). The IC50 values of FEA8, FM3 and acarbose are 16.370 μg/mL, 59.042 μg/mL, and 39.534 μg/mL respectively. Conclusion: Fraction 8 from ethyl acetate extract of G. hombroniana leaves (FEA8) was separated and known in this study as the most bioactive α-glucosidase inhibitor agent compared with another extract, fractions, and acarbose.


INTRODUCTION
Diabetes mellitus becomes one of the biggest global health problems of the 21st century.International Diabetes Federation (IDF) analyzed that, by 2040 the global figure will grow to 642 million if nothing is done to respond the growing diabetes epidemic. 1HO Global report on diabetes shows that since 1980 community is currently living with diabetes has risen from 180 to 422 million. 2Type 2 diabetes play role for the majority of cases of diabetes worldwide.Rising phenomenon in diabetes has become a critical concern that drives effort in searching for a new drug from natural resources to face this trend.Diabetes characterized by the increase of postprandial blood glucose level.The management of postprandial blood glucose level important in the treatment of diabetes.The α-glucosidase inhibitor is responsible for the degradation of carbohydrates, it slows down the degradation of carbohydrates by inhibiting the activity of α-glucosidase competitively, so postprandial blood glucose level can be decreased and the postprandial blood glucose level comes under control.α-glucosidase inhibitors can be an alternative first line agent and provide several benefits, 3 it has been recommended by American Association of Clinical Endocrinologists and Third Asia-Pacific Region Diabetes Treatment Guidelines as the first-line of treatment for lowering postprandial hyperglycemia.Their efficacy in lowering HbA1c, and reducing post-meal blood sugars, make them suitable for use in type 2 diabetes. 4,5-glucosidase inhibitors effective as monotherapy and polytherapy with other diabetes therapeutic agents. 6atural resources have provided a good source of a wide variety of bioactive compounds from which we can develop new therapeutic agents.Many plants used in the treatment of diabetes, and from the study on various parts of plant, leaves are the most promising part for obtaining active compound. 7,8ndonesia is a mega-biodiversity country, which has a lot of potential medicinal plants to be developed as an active herbal ingredient or lead compound from plant materials for drug development.α-glucosidase inhibitors that have been used clinically is voglibose, acarbose, and miglitol.Limited choice of α-glucosidase inhibitors has motivated research to get a new α-glucosidase inhibitors agents are expected to have better effectiveness.α-glucosidase inhibitory activity assay of plants was conducted on Garcinia fruticosa Lauterb, Garcinia rigida Miq, Garcinia daedalanthera Pierre, Garcinia hombroniana Pierre, Garcinia kydia Roxb, and Garcinia bancana Miq and resulting that these extracts

RESULTS
Extracts' test results of α-glucosidase inhibition, show that percent inhibition of n-hexane extract is the lowest, so fractionation performed only on ethyl acetate and methanol extract of G.hombroniana (Table 1).Fractionation of ethyl acetate and methanol extract of G.hombroniana yielded 14 and 12 fractions, respectively (Table 2).Two fractions with the higher percent inhibition compared to other factions are fraction 8 from ethyl acetate extract (FEA8) and fraction 3 from methanol extract (FM3), their TLC profile is shown in Figure 1 and their percent inhibition in various concentrations shown in Table 3. FEA8 became the most have α-glucosidase inhibitory activity, 9,10 but further studies to obtain the active fraction that has α-glucosidase inhibition activity has not been done.The aim of the study is to separate active fraction in the crude extract of G. hombroniana leaves to facilitate obtaining a pure biologically active compound as the α-glucosidase inhibitor.

Fractionation of G. hombroniana Leaves Extract
Fractionation extract of G. hombroniana (20 g) was performed by column chromatography (diameter 4 cm and height 50cm).Silica gel (70-230 mesh) as the stationary phase prepared by mixing 300 g of silica gel and n-hexane was entered into the column.Combinations of solvents with increasing polarity were used as the mobile phase.The wet packing method was used in preparing the silica gel column.Extracts were prepared in a ceramic mortar by silica gel (70-230 mesh) in acetone and left to dry and then gently layered on top of the column.Elution process was performed with solvent gradient systems in polarity using n-hexane, ethyl acetate, and methanol.Ratios of solvent combinations were sequentially used in the gradient elution; n-hexane: ethyl acetate 100:0, 90:10, 80:20, 70: 30, and so on until comparison 0: 100; and then ethyl acetate: methanol 100:0, 90:10, 80:20, 70: 30, and so on until comparison 0: 100.The eluted fractions were collected in aliquots of 100 ml in a bottle.Fractions are then evaporated and tested using thin layer chromatography.

Analytical Thin Layer Chromatography (TLC) and Pooling of Fractions
Analytical TLC used a plate of silica gel 60 F 254 (Merck).A plate of silica gel was cut out.With capillary pipe, a spot of the sample was applied on a plate about 1.0 cm from the edge.The plate was entered into a small chamber containing the solvent system and then viewed using UV lamp (Camag) to identify chromatogram pattern.The fraction that has the same chromatogram pattern then merged and tested its activity in inhibition enzyme α-glucosidase.Potential fraction as the α-glucosidase inhibitor was identified with analytical TLC (silica gel 60 F 254 ) (Merck), using spray reagent (1% ethanolic solution of aluminum chloride and 10% methanolic solution of sulphuric acid ) and then be observed under UV light.

Inhibition of α-glucosidase Assay
The inhibition of α-glucosidase was assessed using adopted method. 11 volume of 30 μl of sample, 36 μL of 0.1 M phosphate buffer (pH 6.8) and 17 µL substrate p-nitrophenyl-α-D-glucopyranoside at concentration of 4 mM were put in 96 well microplate and then preincubated at 37 ºC for 5 min.After preincubating at 37 o C for 5 minutes, 17 μL (0.08 units/ml) α-glucosidase was added and incubated at 37 o C for 15 minutes to get the complete reaction.The reaction was stopped by adding 100 μL Na 2 CO 3 200 mM into each well, and absorbance was measured at 400 nm by a microplate reader (Versamax ELISA Microplate Reader, USA).The IC 50 value which defined as the concentration of the sample needed to inhibit 50% of α-glucosidase activity in assay conditions, analyzed using Graph-Pad Prism.bioactive fraction in α-glucosidase inhibition activity.The IC 50 values of FEA8, FM3, and acarbose as positive control are presented in Table 4.

DISCUSSION
This study was conducted in order to evaluate α-glucosidase inhibitor potentials of fractions separated from extract of G.hombroniana leaves.Antidiabetic properties were determined in terms of the activity to inhibit α-glucosidase.Inhibition of α-glucosidase was exhibited to delay the degradation of carbohydrates.Therefore, α-glucosidase inhibitors have efficacy as therapeutic agents for the management of type 2 diabetes. 12tracts' test results of α-glucosidase inhibition show that percent inhibition of n-hexane extract is the lowest, so fractionation performed only on methanol and ethyl acetate extract of G.hombroniana (Table 1).Methanol and ethyl acetate extract of G.hombroniana leaves yielded 14 and 12 fractions, respectively using column chromatography.Each fraction produced was evaluated in an in vitro α-glucosidase inhibitory studies using microplate reader to facilitate selection of the bioactive fraction.
In vitro α-glucosidase inhibitory studies of fractions demonstrated that all samples had inhibitory activity (Table Fraction 8 from ethyl acetate extract (FEA8) and fraction 3 from methanol extract (FM3) exhibited potency as better α-glucosidase inhibitor compared to other fractions with percent inhibition 71.057 % ± 2.040 and 30.263 % ± 1.716, respectively.Percent inhibition of FEA8 and FM3 in various concentrations shown in Table 3.The highest inhibitory activity of samples was obtained from FEA8 fraction with IC 50 value 16.370 ug/mL.The IC 50 value of FEA8 fraction was much smaller than the IC 50 of acarbose as a positive control (IC 50 acarbose: 39.534 μg/mL) (Table 4).It shows that FEA8 fraction inhibits the activity of α-glucosidase better than acarbose in a smaller concentration.Assay of α-glucosidase inhibition activity shows that fractions obtained have the higher potency than extract origin, it may because the increase in activity after fractionation may be as a result of separation the active ingredients from the nonactive fractions.The active fraction would usually yield desired biological activity which in this case, is inhibition of α-glucosidase in vitro assay.Active fractions, FEA8 was identified by thin layer chromatography using eluent chloroform : ethyl acetate: acetic acid (1:1:0.1),then sprayed with 1% ethanolic solution of aluminium chloride, observed under UV light (366nm), while FM3 was identified by thin layer chromatography using eluent ethyl acetate: methanol (9:1), sprayed with 10% methanolic solution of sulphuric acid and then be heated.Profile TLC of FEA8 show yellow and green fluorescence under UV light (366nm) and FM3 show violet spot (Figure 1).Flavonoids resulting blue, green or yellow fIuorescence, which depends on the use of different spray reagents, 13 while terpenoid compounds will form pink to purple or violet after being sprayed with 10% methanolic solution of sulphuric acid and be heated. 14his result does not rule out the possibility that the active fractions contain another phytoconstituents.
][21] This study can be confirmed by further experiments such as flavonoid, phenol, tannin, glycoside, terpenoid, alkaloid isolation from FEA8 and FM3 through preparative HPLC, preparative TLC, column chromatography or recrystallization and α-glucosidase inhibitory activity assay of isolate that have been separated from other compounds.

CONCLUSION
Fraction 8 from the crude ethyl acetate extract of G.hombroniana leaves (FEA8) with eluent H/E 5:5 was separated as the most bioactive α-glucosidase inhibitor agent with the highest percentage inhibition compared with another fractions and extracts.The IC 50 values of FEA8 fraction (16.370 μg/mL) were smaller than the IC 50 of acarbose as a positive control (39.534 μg/mL), it exhibited higher potency of FEA8 compared to acarbose as positive control.However, further study is needed to isolate bioactive compound in this plant which is responsible for this activity.

Figure 1 :
Figure 1: A.TLC Profile of FEA8 B. TLC Profile of FM3