Acetylation of γ -mangostin Isolated from the Mangosteen Pericarp ( Garcinia mangostana Linn.) and Their Antidiabetic Activity

—Mangosteen (Garcinia mangostana Linn) is one of the most well-known plants in Indonesia. Mangosteen contains many derivatives of oxygenated and prenylated xanthones from phenolics which exhibit diverse biological activities such as anticancer, antidiabetic. Modification of xanthone compounds is known to increase antidiabetic activity and it is known that α - and β -mangostin acetylated can be produced from the modification of α - and β -mangostin using acetic anhydride. In this study, as many as 1.43 grams (1.59%) of the γ -mangostin compound were successfully isolated from the ethyl acetate extract of mangosteen pericarp. Modification of γ mangostin through the acetylation reaction produces acetylated γ -mangostin in the form of 3,6,7-tri-methylester- γ -mangostin as much a s 32.9 mg (63%). Antidiabetic test results showed γ mangostin had an IC50 value of 8.55 μM, while the IC50 value of 3.6.7-tri-methylester- γ - mangostin was 1.82 μM. Acarbose as a positive control has an IC50 value of 4.48 μM. This shows that modification can increase antidiabetic activity.


I. INTRODUCTION
ARCINIA is a genus of plants from the Clusiaceae tribe where no less than 200 species of Garcinia grow spread all over the world [1], [2]. Garcinia specimens stored in the Bogoriense Herbarium and literature studies show that there are 64 species of Garcinia through out Indonesia with the most distribution in the island of Borneo. Garcinia species in Kalimantan are more numerous compared to other islands. As many as 25 species of Garcinia are on the island of Borneo, while in Sumatra and Sulawesi only 22 species each, Maluku and Irian Jaya (Papua) 17 species each, eight Java species, and Nusa Tenggara only five species of Garcinia [3].
Mangosteen (Garcinia mangostana Linn) is reported as a major source and is rich in oxygenated and prenylated xanthones. A total of 50 xanthones compounds that have been successfully isolated from mangosteen pericarp [4]. Some xanthones compounds that have been found in large numbers in mangosteen are α-mangostin, β-mangostin, γ-mangostin, 3-isomangostin [5], [6]. One of the xanthones compounds such as γ-mangostin are xanthones which are reported to have pharmacological effects such as, antioxidants, anticancer, anti-inflammatory, antifungal and antibacterial [4]. In addition, γ-mangostin has also been reported to have quite good antidiabetic activity with an IC50 value of 4.2 μM [7].
Diabetic is a disease caused by the pancreas not being able to produce enough insulin for the body or when the body cannot effectively use the insulin it produces. Insulin is a hormone that regulates blood sugar or glucose in the body [8]. Decreasing the action of insulin will result in changes in carbohydrate, protein and lipid metabolism resulting in an increase in blood sugar levels or called hyperglycemia. Hyperglycemia causes damage to the body's systems, especially blood vessels and nervous system [9], [10]. Diabetes mellitus (DM) is a disease that contributes to the high mortality rate in the world. These complex metabolic diseases are classified into type I and type II DM. Type I DM occurs because the pancreas fails to produce insulin, whereas type II DM refers to the condition of the body's inability to effectively use insulin produced by the body. This causes an increase in glucose levels in the blood or often called hyperglycemia.
Treatment for diabetic patients is taking oral medications (Oral Antidiabetic Drugs -OADs) or herbal medicines derived from nature. Oral medications commonly used for the treatment of diabetes are Metformin, Sulfonylurea and the αglucosidase inhibitor group [11]. Metformin decreases gluconeogenesis in the liver due to insulin sensitivity in the liver [12]. While sulfonylurea works by stimulating insulin secretion [13]. α-glucosidase inhibitors work by inhibiting the enzyme α-glucosidase which catalyzes the division of disaccharides and oligosaccharides [14]. Oral use of the drug has several side effects in its use. Some side effects include diarrhea, vitamin B12 deficiency, weight gain, hypoglycemia, and nausea in diabetics [14]- [17]. The use of inhibitors as antidiabetic drugs sourced from natural ingredients can be an alternative approach for the treatment of diabetics. These inhibitors have advantages in addition to cheap and affordable prices and also do not have side effects when compared with various types of commercial drugs that have been circulating today [18]. Research on isolation of xanthone compounds has been done, but not many researchers have modified xanthone compounds to get new xanthones derivatives. One technique for modification of xanthone compounds is acetylation. Acetylation is the process of adding an acetyl group to the structure of the desired compound. Alcohol and phenol protection is one of the most commonly used synthetic strategies to mask hydroxyl functions during multistep synthetic procedures. In addition, O-acetylation procedure is widely used to obtain derivatives of new compounds that can increase biological activity [19]. Modification of the αmangostin compound to 3,6-di-O-acetyl-α-mangostin using anhydrous acetate as an acetylation agent has been successfully carried out [20]. The addition of the acetyl group in the xanthone structure will certainly affect the results of the 13 C-NMR test. The increase in number (δc) naturally depends on the number of acetyl substituents substituted in xanthones.
The acetylation of 1,3-dihydroxycantone to 1,3diacetoxysantone compounds can increase the inhibitory activity of the α-glucosidase enzyme from 160.8 μM to 31.9 μM [21]. α-glucosidase is an enzyme in digestion which breaks down carbohydrates from oligo-or poly-saccharide forms into monosaccharides including glucose, galactose and fructose [18]. Inhibition of the α-glucosidase enzyme will inhibit glucose absorption so that glucose levels in the blood do not increase. Thus, α-glucosidase inhibitors can be used as antidiabetic [22].

A. Instrument
Equipment used in this study includes glassware, a set of chromatography tools, analytical balance, Cimarec hot plate SP131320-33 (Cimarec, China), 60 GF254 silica gel TLC plates (Merck kGaA 64271

C. Isolation of γ-mangostin
The γ-mangostin isolate was obtained from the maceration process of dried mangosteen pericarp using ethyl acetate solvent. The extract obtained was then evaporated using a rotary evaporator at low pressure so that the solvent was extracted. Concentrated ethyl acetate extract obtained was fractionated using silica gel chromatography column. The profiles of pure compounds in a fraction are marked by a single stain on the TLC plate. The compound is confirmed to be pure and through the process of structural identification using UV, IR, MS and NMR instruments.

D. Acetylation
Acetylation of γ-mangostin is carried out through the acetylation reaction with the sodium acetate catalyst [21]. γmangostin (39.6 mg, 1 mmol) was dissolved in anhydrous acetate (2.5 mL). Subsequently, sodium acetate (103 mg, 0.125 mmol) was added to the solution and the reaction mixture was stirred at 60 o C (TLC monitoring). After the reaction is complete, the solvent is evaporated using a rotary evaporator and the residue obtained is partitioned using distilled water: chloroform (1:1). The organic layer was separated, the solvent was evaporated at low pressure, and TLC was tested with eluent proteleum ether: acetone (7: 3). Acetylation products obtained were identified by the 13 C NMR spectroscopic method.

E. Antidiabetic test
Antidiabetic activity tests were carried out using the αglucosidase enzyme inhibition method [23]. Samples of 10 μL, 0.1 M phosphate buffer (pH 6.9) 30 μL, 100 μM sucrose 20 μL, glucose kit 80 μL, and 20 μL enzyme supernatant were incubated at 37°C for 10 minutes. Akarbosa is used as a positive control. Absorbance was recorded at a wavelength of 490 nm on a UV-Vis spectrophotometer. Inhibitory activity is determined from the following equation: The α-glucosidase enzyme inhibition test was performed on various dilution samples to look for logarithmic regression equations so that IC50 values of the samples could be determined.

A. Isolation γ-mangostin
The isolation process begins with the maceration process of dried mangosteen pericarp powder using ethyl acetate. Maserat obtained from maceration results as much as 15 L, then concentrated with a rotary evaporator. The first maceration process gave 374.14 ethyl acetate extract, while the second and third maceration produced 121.05 extracts, and 39.82 grams. Ethyl acetate extract (90 g) was fractionated using a Vacuum Liquid Chromatography (VLC) column with 60 G (250 g) silica gel and n-hexane-EtOAc (Increasing Polarity) eluent gradient until a single stain profile was obtained. After that the solvent fraction is evaporated solvent at low pressure. The solids formed were then recrystallized, tested for purity, and the identification of the structure obtained compounds (1) γ-mangostin as in Figure 1 totaling 1.43 g.

B. Acetylation
The γ-mangostin compound that was successfully obtained from the ethyl acetate extract of G. mangostana pericarp was modified by the acetylation method. 0.1 mmol of γ-mangostin compound was dissolved in 2.5 ml of anhydrous acetate. Next, add sodium acetate catalyst 0.25 mmol.
The reaction mixture is stirred at 60 o C and the running reaction is monitored by TLC. Monitoring of the acetylation reaction using TLC showed that the reaction in the 60 th minute had gone perfectly marked by the absence of a reactant stain or γ-mangostin (the reactor had finished reacting). Based on this, the acetylation reaction is then stopped. Then the reaction mixture is partitioned using aquades-chloroform (1:1). The organic phase obtained is then evaporated at a low pressure temperature.
The formed crystals are washed using aquades to remove impurities. The crystal was tested for purity using TLC with three different eluents. The purity test results show that the reaction crystals are pure compounds that are characterized by the presence of a single stain on each plate and there are no impurities at the top or at the bottom of the stain. The pure compound resulting from the acetylation reaction is referred to as compound (2). The compound (2) produced from the acetylation reaction was 32.9 mg (63%).
Structural identification was carried out using 13 C-NMR spectroscopy. The obtained spectrum results were compared with the 13 C-NMR γ-mangostin spectrum. The entry of the acetyl group will certainly affect the number of carbon peaks in the 13C NMR spectrum of compound (3). Acetylation of xanthones at 60 o C can substitute free hydroxy groups in xanthones [21]. γ-mangostin contains three free hydroxyl groups in xanthone structure, then it is possible that the three hydroxyl groups are acetylated.
Based on the 13 C-NMR spectrum of compound (2), the addition of 3 methyl signals and 3 carbonyl signals indicates that 3 acetyl groups have successfully entered the xanthone structure by substituting three free hydroxy groups. The following is a comparison table of 13 C-NMR γ-mangostin and compound (2).
Based on the results of the 13 C-NMR analysis of compound (2) and its comparison with the 13 C-NMR spectrum of γmangostin, it can be concluded that the compound formed from the acetylation reaction is 3,6,7-tri-methylester-γmangostin ( Figure 3).

C. Antidiabetic Test
Antidiabetic test through the mechanism of α-glucosidase inhibition in various dilution samples is done to find the logarithmic regression equation so that the IC 50 value of the sample can be determined. IC50 is the concentration of a compound needed to inhibit the activity of the α-glucosidase enzyme by 50%. α-glucosidase is an enzyme in digestion which breaks down carbohydrates from oligo-or polysaccharide forms into monosaccharides including glucose, galactose and fructose [18]. Inhibition of the α-glucosidase enzyme will inhibit glucose absorption so that glucose levels in the blood will not continue to increase. Thus, α-glucosidase inhibitors can be used as antidiabetic [22].