Isolation and Identification of Chemical Compounds from Garcinia fruticosa Lauterb Stem Bark Extract

Garcinia is a plant that contains many phenolic compounds and their derivatives, such as xanthon, flavonoids, benzophenone, lactone, and phenolic acids.5 Research conducted by Shen et al. shows that Garcinia contains various flavonoid compounds, such as kaempferol, quercetin, and flavanone and flavonoids-O-glycosides.1 In addition, according to Shen and Yang, Garcinia also contain steroid compounds, such as β-sitosterol and stigmasterol.1


INTRODUCTION
Garcinia is a tropical plant that grows in Indonesia. Garcinia is known to have many health benefits for the body. From several studies that have been conducted on various types of Garcinia plants, it is known that Garcinia is a source of secondary metabolite compounds that can be medically useful for the treatment of certain diseases, such as antidiabetic and antioxidant. 1,2 Garcinia fruticosa Lauterb. comes from the family Clusiaceae. 3 In Indonesia, G. fruticosa Lauterb grows in Irian Jaya. Garcinia itself is widely found in tropical regions, such as Africa and Asia. 4 (Panasonic), capillary pipes, drip pipettes, rotary evaporators (IKA, Germany), infrared spectrometers (Shimadzu), magnetic resonance spectrometers core (Jeol, Japan), analytical scales (Accu-Lab), analytical scales, UPLC-LC-MS / MS (Waters), 10 ml vials, Chemdraw (software).

Extraction and fractination
Ethyl acetate extract of Garcinia fruticosa bark was obtained from the Phytochemical Laboratory of the Faculty of Pharmacy, University of Indonesia. Extraction uses the maceration method in sequence with n-hexane, ethyl acetate, and methanol as solvents. A total of 30 grams of ethyl acetate extract was fractionated using column chromatography with polarity gradient n-hexane -ethyl acetate -methanol as the mobile phase and silica gel G60 as the stationary phase. The elution process starts from n-hexane 100; followed by n-hexane: ethyl acetate 95: 5; 90:10; 85:15; 80:20; and so on at intervals 5 to obtain the ethyl acetate fraction 100, then continued with ethyl acetate: methanol 95: 5; 90:10; 85:15; 80:20; and so on at intervals of 5 to get the methanol fraction 100 so that eluents with different polarity gradients are obtained. Each fraction is collected every 100 ml and evaporated and then combined into several fractions based on the thin layer chromatography profile.

Isolation and purification of subfractions D
Subfraction D has brownish yellow deposits. Subfraction D is then refined by recrystallization with n-hexane and ethyl acetate as solvent to obtain pure compounds. From the recrystallization results obtained by a chromatogram profile which is then accommodated in vials. Subfraction was carried out by thin layer chromatography using 3 different eluents (chloroform: aceton: formic acid, 4: 1: 0.25) with a single spot yield. The compound obtained was weighed, and put in a glass vial with a well-closed container. The compounds obtained are identified their compound structure.

Isolation and purification of subfraction I
Subfraction I shows white sediment. Subfraction I was then refined using preparative TLC with the mobile phase of n-hexane: ethyl acetate (9: 1). From the preparative TLC results obtained by chromatogram profiles which are then separated and accommodated in vials, and the dominant peak is in the chromatogram pattern number 1 (I-1) in the form of white needle crystals. I-1 subfraction was carried out thin layer chromatography using eluent (n-hexane: ethyl acetate: formic acid, 9: 1: 0.3) with a single spot result, the compound I-1 obtained was weighed, and put in a vial of glass with a well-closed container. The compound I-1 obtained was identified by its compound structure.
Thin layer chromatography (TLC) 1 mg of isolate was dissolved in ethyl acetate and filtered before spotting on the TLC plate. Chamber Saturation time is 15 minuts.

Compound identification
The compounds were identified by analyzing spectroscopic data from IR, MS spectrophotometry, Nuclear Magnetic Resonance proton ( 1 H-NMR) and carbon ( 13 C-NMR) spectroscopy, and NMR-2D techniques which included HMQC and HMBC.
a. Infrared Spectrum Check (IR) 1 mg of isolate are crushed with 100 mg KBr until homogene. The mixture is pressed with a strength of 10 tons/cm 3 to form a thin pellet, then the infrared absorption is measured. b. Nuclear Magnetic Resonance (NMR) Examination 10 mg of isolate was dissolved in CD3OD, put into a glass tube placed in the middle of a magnetic field tank containing helium gas and sealed with liquid nitrogen. From the results of data processing will be obtained the proton core magnetic resonance spectrum ( 1 H-NMR), carbon ( 13 C-NMR), and 2D-NMR techniques which include HMQC and HMBC.
c. Mass Spectrum Examination by HPLC-MS 1 mg of pure isolate was dissolved in methanol. 10 µL sample was taken and injected into LC-MS through the Heliflex AT-5 ms column, 30 m x 0.25 mm x 0.25 µm flow velocity of 0.5 mL/min.

Fractination of Ethyl Acetat Extract from G. fruticosa Lauterb Stem Bark.
Column chromatography results obtained 401 reservoirs. Each storage result was seen by chromatography profile by thin layer chromatography. The beds with the same chromatogram pattern are combined into one so that we get 11 final fractions of ethyl acetate extract, namely A, B, C, D, E, F, G, H, I, J, and K fractions. The weight of each fraction can be seen in Table 1 and 2.

Isolation and Purification of Compounds D
Compound D was obtained by column chromatography which was subsequently recrystallized with n-hexane and ethyl acetate as much as 19.5 mg in the form of a brownish-yellow solid. Chromatogram pattern of compound D with silica gel stationary phase and chloroform: acetone: ac. format (4: 1: 0.25), Rf = 0.625; can be seen in Figure 1.

IR Spectrophotometry Measurement Results Data of Compound D
The IR spectrum shows the presence of a hydroxy group (-OH) from a carboxylic group (-COOH) and phenol appears in the region of wave number 3483 cm -1 , aromatic presence at 1609 cm -1 , can be seen in Figure 2.    The above assumption is reinforced by the results of the C-NMR measurement, namely the presence of a carboxylic-COOH group appearing at 166.57 ppm, the presence of 2 x C-OH appearing at 147.18 and 151.18.
And by predicting H and C-NMR values, the correlation of H and C can be determined and also supported by the 2D-NMR spectrum (HSQC).
To determine the position of methoxy (-OCH 3 ) whether in C-3 or C-4, it can be ascertained by measurements of 2D-NMR -HMBC, long distance correlation (2-3 bonds).
On the HMBC spectrum, it is shown that the -OCH 3 position is located at C-3 due to the correlation between signal 3.79 (s) and signal C at the chemical friction of 147.18 and not with the chemical friction at 166.57 (-COOH). The signals at 6.92 (d) and 7.60 (dd) are close to -COOH with the HMBC correlation of 166.57 (Figures 11,12).       These results confirm that this compound is 4-hydroxy-3-methoxy benzoic acid, known as Vanillic acid (Fig. 12). This compound has never been isolated from Garcinia but has been isolated from the Bistorta manshuriensis plant 7 and Delonix regia (Boj.) Raf roots (Family Fabaceae). 8 Determination of Molecular Structure of Compounds I-1 Compound I-1 was obtained by preparative TLC by solvent n-hexane and ethyl acetate (9: 1), as much as 4 mg in the form of needle crystals. Chromatogram pattern of compound I-1 with stationary silica gel and eluent as mobile phase n-hexane: ethyl acetate: formic acid (9: 1: 0.3), Rf = 0.95; can be seen in Figures 13-15.
The H-NMR spectrum shows the specific signal of a β-sitosterol as the main component and stigmasterol as a minor component (3: 1), seen in the integrase ratio of 1: 0.73, of which 0.73 should be 2 H, so the ratio of the two compounds is 3: 1.
The specific signal at 5.34 (1H, d, 5 Hz) is a double bond signal at C-5, while the specific signal at 5.16 (dd, 15.5; 8.5 Hz) and 5.00 (dd; 8.5; 15.5 Hz) which is double bond H-C = C-H of C22-C23 but the integral value is only 0.73, so this is considered a minor component.
The signal at 3.52 (m) is an H-C-OH signal at C-3. For other characteristics that this compound is β-sitosterol, it can be seen in more detail the signal in the region of 1 ppm, where there are several methyl groups (s, d and t). Signals at 0.68 (3H, s), 0.81 (t, 6.5 Hz), 0.82 (d, 6.5 Hz), 0.84 (d, 6.5 Hz), 1.01 (s) and 0.88 (d, 6.5 Hz).
Based on these results and compared with literature (Saputra, Handayani, & Wartono, 2016), that compound I-1 has the same chemical shear value with β-Sitosterol and Stigmasetrol, which can be seen in Table 3. The structure of β-Sitosterol and Stigmasterol can be seen in Figure 16.