The Potential of Stem Bark of Kayu Sarampa (Xylocarpus moluccensis (Lam.) M. Roen)) as α-glucosidase Inhibitor

The prevalence of diabetes mellitus type 2 in the world is more than 230 million people, increases about 3% in a year.1 Hyperglycemia accelerates the formation of reactive oxygen species that increases lipid, DNA and protein modification in human tissue.2 Molecular modification in some tissues causes an imbalance between protective antioxidant and free radical production. That was the beginning of oxidative damage which is known as oxidative stress.3


INTRODUCTION
The prevalence of diabetes mellitus type 2 in the world is more than 230 million people, increases about 3% in a year. 1 Hyperglycemia accelerates the formation of reactive oxygen species that increases lipid, DNA and protein modification in human tissue. 2 Molecular modification in some tissues causes an imbalance between protective antioxidant and free radical production. That was the beginning of oxidative damage which is known as oxidative stress. 3 Antioxidant was used to inhibit or minimalize oxidative damage. One common method which is used to evaluate antioxidant activity is 1,1-diphenyl-2-pilcrylhidrazyl (DPPH). 4 This method measures synthetic radicals DPPH in a polar solute such as ethanol or methanol at room temperature which is scavenged by an antioxidant compound. 5 Native people in Ratahan, Sulawesi Utara use batu Nyirih (Xylocarpus moluccensis) to treat diabetic patients. This plant is known as Kayu sarampa. Xylocarpus genus spread from India beach, Ceylon, Burma, Malaysia and Indonesia. Many researchers study about active components in Kayu Sarampa. It contains antibacterial, antidiabetes, antioxidant, antifilarial, antidiarrhea, antidepressant and cytotoxic activity. 6 Kayu sarampa can be used to treat fever, joint pain, headache, and disorders such as cholerae, constipation, and diarrhea 7 .
Methanol extract of X. moluccensis was found to be significantly effective in scavenging DPPH method. 8 The fruits of X. moluccensis contain limonoid which can change enzyme activity to metabolize glucose and increase glucose absorption by muscle tissue. 9 In humans, alpha-glucosidase enzymes aid the digestion of dietary carbohydrates and starches to produce glucose for intestinal absorption, which in turn, leads to an increase in blood glucose levels. Elbakyan (1998) reported that a compound obtained from an isolate of ethyl acetic fraction was 2-methoxy-5-(1propenyl) phenol and 2-methoxy-4-(1-propenyl) phenol with m/z 180 and 164, respectively. 10 Extraction is an initial step to separate bioactive from plants. Solvent extraction is the most common technique to extract natural products from plants. 11 Secondary metabolites in plants have different polarity. Therefore, extraction with various solvent polarities was preferable. 12 The use of solvent-based on its polarity can be applied to dry powder of plant tissue using reflux. It was done at discontinued high temperature condition although soxhlet was done under continued high temperature. The advantages of reflux compared with soxhlet was the less use of solvent, and compared to creation, it only needs a short time of extraction. 13 Therefore, this research uses sequential reflux extraction in hexane, ethyl acetate, and methanol solvent. Kayu sarampa plant consists of bioactive components used to cure diabetic patients. However, there is no evidence of the use of stem bark of X. moluccensis to inhibit α-glucosidase. This research was done to sequentially extract bioactive component from stem bark of X. moluccensis and asses their α-glucosidase inhibitory activity.

Plant material
Sample used in this study was the stem bark of X. moluccensis which was obtained from Ratatotok district, North Sulawesi and was identified by Herbarium Bogoriensis, Biological research centre, Indonesian Institute of Science.

Sample preparation
The stem bark of X. moluccensis. from Ratatotok, North Sulawesi, was freshly picked, collected, sorted, and dried. The Stem bark were then crushed until they became smaller.
Microscopic observations by scanning electron microscope (SEM) and light microscope

Extraction and Fractionation
Four kilograms of stem bark powder of dried X. moluccensis was extracted using hexane. Extraction was initially done using reflux apparatus to obtain hexane extract. Hexane extract then was evaporated using a rotary evaporator to obtain a crude hexane extract (HE). It was stored under room temperature. The same way was done for ethyl acetate solvent to obtain ethyl acetate extract (EAE). Then, methanol was used to extract the residue to obtain methanolic extract (ME).
Determination of free radical 1.1-diphenyl-2picrylhidrazyl (DPPH) scavenging activity DPPH radical scavenging activity was done according to the procedure declared by Burda & Oleszek (2001) with slight modification. 14 Five hundred microliter extracts were added into 1.5 mL DPPH solution, mixed for 2 minutes, and incubated in dark room for 30 minutes. The absorbance of the solution was measured for 5 minutes before incubation time ends. Color change from purple to yellow means free radical scavenging efficiency. Free radical scavenging activity was calculated as the percentages of color decreasing of DPPH solution using the following equation: Free radical scavenging activity (%) = × 100% After the inhibition percentage of each concentration was obtained, linear regression was made so that the equation y = a + bx was obtained, where x is the concentration (μg/mL) and y is the percentage of inhibition (%). Antioxidant activity is expressed by 50% Inhibitory Concentration or IC 50 , which is the concentration of the sample that can reduce DPPH radicals by 50% from the initial concentration.

FRAP assay test
This tes was conducted using the method described by Bhagat

Standard and sample examination of Alpha-glucosidase Inhibitor
Acarbose as a standard and sample was weighed and dissolved in phosphate buffer solution pH 6.8. Sample with low solubility in phosphate buffer was dissolved in maximum 10% DMSO. Then standard and sample were diluted into some concentrations.
Thirty microliter standard and sample solution were added to 17 μL PNPG substrate. The solution was incubated for 5 minutes at 37 o C, and added to 17 μL alpha-glucosidase solution. Solution was incubated again at 37 o C for 15 minutes. After that, 100 μL sodium carbonate 267 mM was added. Solution absorbance was measured using a microplate reader at 405 nm wavelength.
Alpha-glucosidase inhibitory activity was calculated by the following equation 16 :

Statistical analysis
All the experimental data do triplicates and the results are expressed as mean ± SD. IC 50 was analyzed using Probit. Antioxidant power and alpha-glucosidase inhibitory activity were analyzed using One way ANOVA analysis followed by Duncan Multiple Range Test. Analysis was performed using SPSS software version 20.

RESULTS AND DISCUSSIONS
Extraction X. moluccensis was extracted sequentially using reflux with hexane, ethyl acetate, and methanol as the solvent. Relux is an extraction using heat. Miller (1975) stated that heating under reflux process can increase the possibility to damage tissue membrane of plant material, so that solvent can penetrate into tissue void which contains active components. 7 Active components will dissolve in because of its different concentration between active component inside and outside the tissue, so concentrated solution is pulled outside. The yield of reflux sequential extraction was displayed in the following table 1.

Free Radicals DPPH Scavenging Activity
The evaluated X. moluccensis concentration were 25, 50, 75, 100, and 125 mg/L, respectively. Free radical DPPH commonly used as substrate to evaluate antioxidant activity. Antioxidant properties of X. moluccensis extract reacted with DPPH, the solution color changed from purple to yellow. The change in solution color affected to DPPH absorbance. The higher concentration of antioxidant component in solution, the lower the absorbance of DPPH.
The change in solution color is caused by a component that donates hydrogen atom to DPPH radical. Antioxidant reducted DPPH radical to more stable form namely DPPH-H (2,2-diphenyl-1-picrylhidrazine). 4 Evaluation of antioxidant activity using DPPH method is presented in Figure 4. Figure 4 showed that antioxidant activity of X. moluccensis through DPPH method is increased in a concentration-dependent manner. Methanol extract exhibited the highest free radical DPPH scavenging activity 82,93%, followed with ethyl acetate extract 74,75%, and Hexane extract 27,83%, respectively. Ascorbic acid as antioxidant standard exhibited 88,09% activity. Lai et al. (2001) stated that antioxidant activity using radical DPPH mostly increase in a concentrationdependent manner. 17 IC 50 value represents the ability of X. moluccensis extract to scavenge 50% of DPPH free radical. The lowest IC 50 value of an extract represents high antioxidant activity. 18 IC 50 was obtained from a regression linear equation in Figure 1 which plotted x axis (concentration) with y axis (free radical DPPH scavenging activity), so a regression equation and IC 50 obtained is presented in Table 3. Table 3 showed that ME IC 50 of X. moluccensis exhibits the highest activity compared with EAE, and HE. IC 50 value of an extract represents its potential to inhibit free radicals. A strong level IC 50 categorized with IC 50 value 50-100 mg/L, medium range category 100-250 mg/L, and low level category was 250-500 mg/L. 19 High antioxidant activity of methanolic extract of kayu sarampa is caused by polar secondary metabolite such as flavonoid. Interaction of flavonoid with nitric oxide synthases (NOS) activity may modulate the NO production. Xanthine oxidase (XO) is considered as a key source of free radicals, and some flavonoids such as quercetin, silibin, and luteolin have been shown to inhibit such activity. Flavonoids may also reduce the activity of peroxidase and may inhibit the release of free radicals by neutrophils and activation of these cells by α1-antitrypsin. 20 Ascorbic acid used as a control in this study is a strong antioxidant. IC 50 of ascorbic acid exhibits IC 50 value 10,49 mg/L. It was caused possibly by 2 groups of hydroxy (-OH) on its double bond which is easily oxidized by free radicals. In addition, vitamin C used in this study is a pure substance so it exhibits strong IC 50 or high antioxidant activity. 21

Ferric reduction antioxidant power
The FRAP method is a method of testing antioxidant activity through the ability of antioxidant compounds to reduce Fe 3+ ions to Fe 2+ in the presence of 2.4,6-tri (2-pyridyl)-s-triazine (TPTZ) in an acidic atmosphere which produces intensive blue colour from the Fe 2+-TPTZ complex and causes an increase in absorbance when it is measured using a microplate reader at a maximum absorption of 593 nm. In the test using the FRAP method, ferrous sulfate heptahydrate (FSH) is used as a standard. First, the standard FSH calibration curve is made. The antioxidant activity is measured based on the sample equality with the AFS standard. The linear regression equation of FSH obtained y = 0.00257 x + 0.04715. Antioxidant power of sarampa extract is shown in following table 4.  Table 4, samples extracted with methanol and ethyl acetic show higher antioxidant power compared to n-hexane extract. This is due to the high polarity of methanol and ethyl acetic, whilst hexane is non polar. These results are influenced by the polarity of solvent because methanol is the most polar solvent used in this experiment, while ethyl acetate and hexane in descending order were less polar than methanol. The higher total antioxidant content in extract means more compounds can reduce Fe 3 + to Fe 2 + (blue), and compounds that reduce Fe 3 + is an

Alpha-glucosidase inhibitory activity
Concentration of stem bark kayu sarampa extract used in this test is 100 ppm. Alpha-glucosidase inhibitory activity of stem bark X. moluccensis extract was presented in Table 5.
The result shows that kayu sarampa stem bark exhibits antidiabetic activity due to its high inhibition compared with control. Methanolic extract shows inhibition 53,11% followed with ethyl acetate extract 49,7%, Hexane extract 44,53%, and acarbose as control 29,32%.  Srivastava et al reported that antihyperglycemia and anti dyslipidemia activity of ethyl acetate fraction of X. moluccensis (EAXm) effectively increase glucose recovery, decrease blood glucose level and fructosamin serum dose in diabetic mice induced by streptozotocin. EAXm also decreases cholesterol serum, triglyceride, LDL cholesterol, but increases HDL cholesterol, liver function, and kidney fuction in HFD/ HSD-STZ mice for 10 consecutive days. Moreover, EAXm increases glucose absorption by muscle tissue L-6 and inhibites alpha glucosidase enzyme in vitro with IC 50 28,4 μg/ml. Although there is no research about secondary metabolite in the stem bark of X. moluccensis as α-glucosidase inhibitor. 8 The secondary metabolite which possibly exhibites antidiabetic activity through α-glucosidase inhibition in kayu sarampa is limonoid. Das et   al reported that xyloccensin-L contains interaction and can bond when it is close to the target then confirm the possibility antidiabetic action mode by isolated compound from X. granatum. xyloccensin-L is a novel limonoid from X. granatum. 23