Antioxidant Activity and Lipoxygenase Enzyme Inhibitory Assay with Total Flavonoids Content from Garcinia hombroniana Pierre Stem Bark Extract

Introduction: Garcinia has been known as a rich source of xanthones, flavonoids, and phenols. The aim of this research is to obtain data of antioxidant activity and to observe potential inhibition of lipoxygenase activity that most active from methanolic, ethyl acetate and n-hexane extracts with total flavonoids content from most active extracts from the bark of Garcinia hombroniana Pierre. Methods: The antioxidant activity was measured using the ferric reducing antioxidant power (FRAP), the anti-inflammatory assay was measured using inhibition of lipoxygenase activity test, qualitative analysis of flavonoids using thin layer chromatography, and total flavonoids content was measured using AlCl3 colorimetric method. Results: The results showed that the ethyl acetate extract from G. hombroniana Pierre stem bark as the most active extract for antioxidant and lipoxygenase inhibition activity with EC50 and IC50 value consecutively 15.34 μg /ml; 0.26 μg /ml. Total flavonoids content of ethyl acetate is 7.430 mg QE/g extract. The results of this study showed bark extract Garcinia hombroniana Pierre has antioxidant activity and potent to inhibit lipoxygenase activity. Conclusion: Based on the research for methanolic, ethyl acetate and n-hexane extract, it can be concluded that the ethyl acetate extract of G. hombroniana Pierre as the most active extract for antioxidant and lipoxygenase inhibition activity.


INTRODUCTION
Garcinia is a plant that rich source of secondary metabolites, especially xanthones, flavonoids, and polyphenols. 1A wide variety of biological and pharmacological activity have been reported such as anti-HIV, anti-cancer, antioxidant, anti-tuberculosis, anti-fungal, antibacterial, anti-inflammatory, and anti-Alzheimer 2 Anti-inflammatory activities showed in the presence of 12-lipoxygenase inhibition by α-mangostin from Garcinia mangostana with IC 50 was 0.58 µM. 3 Garcinia hombroniana Pierre is a native plant from Sumatra.At this time, G. hombroniana Pierre has been cultivated in Bogor Botanical Garden, West Java.Some constituents were identified as lupeol acetate, β-sitosterol, xanthones, leucodin, betulin, betulinic acid, and stearic acid 4 that may be used for treatments.The previous study provides that G. hombroniana Pierre stem bark extract has antioxidant activity (ethyl acetate extract 5579.8 ± 117.77; methanol extract 4709.6 ± 88.0; n-hexane extract <2000 mol TE /g), anti-cholinesterase, and anti-bacterial.Traditionally, this plant also used as anti-inflammatory.Total flavonoids content of G. hombroniana Pierre was 3317.6 ± 131.0 QE (quercetin equivalent)/g extract. 2 However, few studies that discuss anti-inflammatory and antioxidant activity using Ferric reducing antioxidant power (FRAP) method of G. hombroniana Pierre.Therefore, further research to determine the inhibi-tion of lipoxygenase activity and antioxidant activity using FRAP.Furthermore, the phytochemical screening using thin layer chromatography (TLC) and total flavonoids contents at the most active extracts also discussed in this study.

Antioxidant Activity Assay using FRAP Method
FRAP assay was done according to the method described by Benzie and Strain (1996) 5 with some modifications.Working solution of FRAP was prepared by mixing 300 mM acetate buffer (pH 3.6), 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) (Sigma-Aldrich) in 40 mM HCl and 2 20 mM FeCl 3. 6H 2 O (10:1:1).3.8 ml FRAP reagent solution reacted with 0.2 ml baicalein solution or sample, then incubated for 30 minutes at 37ºC.For sample or standard control, 3.8 ml FRAP reagent solution reacted with 0.2 ml ethanol pro analysis, then treated the same as well as the sample or standard.The absorbance of the product was then measured at 595 nm.The percentage of capacity can be calculated using % capacity calculation.The analysis was done in triplicate.The result was expressed as EC 50 (µg/ml), and obtained by using Microsoft Office Excel or GraphPad Prism 7.
electrons, thereby increasing the redox potential. 9FRAP methods using reagents tripyridyl triazine (TPTZ) as an iron-binding ligand.FRAP test is a method that is simple, fast, and cheap enough and does not require special equipment. 10Antioxidant capacity was expressed as EC 50.EC 50 is a concentration of sample or standard that can exhibit 50 % of FRAP capacity.From the experiments, data showed the EC 50 of baicalein (positive control), ethyl acetate extract, methanolic extract, and n-hexane extract respectively 1.16; 15.34; 27.21, and 110.9 µg/ ml.Ethyl acetate as the most active extract because has the lowest EC 50 value but less potent than baicalein as a positive control.Sequentially, that has the highest antioxidant activity then was baicalein > ethyl acetate extracts > methanolic extract > n-hexane extract.

Lipoxygenase Enzyme Inhibition Assay
From the preliminary test obtained data for optimum conditions for lipoxygenase enzyme inhibition assay, there was 8.5 for optimum pH, 1 ml of methanol for stop solution, 5000 U/ml for enzyme concentration, and 900 µM for substrate concentration.The result showed that IC 50 of baicalein, methanolic extract, ethyl acetate extract, n-hexane extract respectively 0.24; 0,26; 0.95; 5.09 µg/ml.Ethyl acetate as the most active extract because has the lowest IC 50 value but less potent than baicalein as a positive control.The previous study reported that Garcinia contains xanthones and benzophenone derivative, and rich in flavonoids that have anti-inflammatory activity. 11ytochemical Screening using Thin Layer Chromatography Analysis using thin layer chromatography was used for screening the presence of flavonoids.The results showed that ethyl acetate and methanolic extract contains flavonoid that proved by yellow color in silica gel plates at 366 nm while n-hexane doesn't.The yellow color in ethyl acetate extract is more and intensive than methanolic extract.The result corresponded in antioxidant activity and lipoxygenase inhibition assay that state the ethyl acetate as the most active extracts.The result can be seen in Figure 1, 2 and 3.

Phytochemical Screening using Thin Layer Chromatography
Thin Layer Chromatography analysis for methanolic, ethyl acetate, and n-hexane extract using mobile phase that has been optimized.Stationary phase using silica gel 60 F 254 plates.Mobile phase for methanolic extract is ethyl acetate-formic acid (4:0.2),toluene-ethyl acetate-formic acid (61: 30: 9) for extract ethyl acetate and n-hexane-ethyl acetate (6:4) for extract n-hexane.each extract solution was spotted on a TLC plate.TLC plates which had been eluted dried and sprayed with AlCl 3 5% to analyze the presence of flavonoids compounds.The plates were examined under UV light at a wavelength of 254 nm and 366 nm.Quercetin is used as the reference standard that treated similarly to extract.Determined the Rf between quercetin and sample.

Determination of Total Flavonoids Content (TFC)
Total flavonoid content was measured using the modified method adapted from Chang et al. 6 The most active extract in antioxidant activity and inhibition of lipoxygenase highest measured the total flavonoids content.Prepared samples (0.5 ml) by reacting 1.5 ml of methanol 95% (v/v), 0.1 ml of 10% aluminum chloride (w/v), 0.1 ml of 1 M sodium acetate, and 2.8 ml of distilled water.The mixture samples were incubated at a temperature of ± 25 °C for 30 min.The absorbance was read at wavelength 435 nm.The analysis was done in triplicate.Quercetin is used as a standard with the same treatment as the samples for the calibration curve.The total flavonoid content was reported as total quercetin equivalent per g extract (mg QE/ g extract).

RESULT Antioxidant Activity Assay using FRAP Method
FRAP method is based on the ability of antioxidants to reduce Fe 3+ to Fe 2+ in the presence of 2,4,6-tri (2-pyridyl) -s triazine (TPTZ), forming intense blue color of the complex Fe 2 + -TPTZ that measured in maximum absorbance at 595 nm. 7This reaction depends on pH (optimum pH was 3.6). 8FRAP methods performed at acidic pH conditions (3.6) for an iron solubility and the most important is to facilitate the transfer of

Determination of Total Flavonoids Content (TFC)
The total flavonoids contents were measured by using AlCl 3 colorimetric method for ethyl acetate extract.The principle of determination of the flavonoid content consists in the fact that aluminum chloride forms stable acid complexes with the carbonyl group at C4 and hydroxyls at C3 (flavonols) and C5 in flavonols and flavones, besides forming labile acid complexes with hydroxyls in the ortho position in A or B rings of flavonoids. 6Obtained linear regression 0,0431x + y = 0.1394 from calibration curve of quercetin, with R 2 =0.9957According to the formula 12 total flavonoids content of ethyl acetate extract is of 7.430 mg QE (quercetin equivalents / g extract).

CONCLUSION
Based on the research for methanolic, ethyl acetate and n-hexane extract, it can be concluded that the ethyl acetate extract of G. hombroniana Pierre as the most active extract for antioxidant and lipoxygenase inhibition activity with EC 50 and IC 50 value consecutively 15.34 µg /ml; 0.26 µg/ ml.Total flavonoids content of ethyl acetate is 7.430 mg QE/g extract.However, there should be more research for ethyl acetate extract as the most active extract include isolation and characterization of compounds.

A
= Absorbance of reference solution with enzyme B = Absorbance of reference solution without enzyme C = Absorbance of standard or sample solution with enzyme D = Absorbance of standard or sample solution without enzyme The result was expressed as IC 50 (µg/mL), and obtained by using Microsoft Office Excel or GraphPad Prism 7.