Antioxidant Activity and Lipoxygenase Enzyme Inhibition Assay with Total Flavonoid Content from Garcinia hombroniana Pierre Leaves

Objective: Garcinia hombroniana Pierre leaves extract have been known to contain flavonoid, but it has not been known yet for its antioxidant activity and inhibition of lipoxygenase activity. This study aims to determine antioxidant activity and inhibition of lipoxygenase activity of G. hombroniana leaves extract. Method: Antioxidant activity tested by using FRAP (Ferric Reducing Antioxidant Power) method and inhibition of lipoxygenase activity using baicalein as the positive control. Total flavonoid assay is also quantitatively done by AlCl3 colorimetric method on the most active extract using quercetin as the positive control. Results: The test result showed that the n-hexane, ethyl acetate and methanol extract of G. hombroniana Pierre leaves have antioxidant activity which showed by EC50 value consecutively are 36.260; 2.969; and 7.416 μg/mL, and also can inhibit lipoxygenase activity which showed by IC50 value consecutively are 2.052; 0.134; and 1.314 μg/mL. Ethyl acetate extract of G. hombroniana Pierre leaves has the most active antioxidant activity and inhibition of lipoxygenase activity. Total flavonoid content of ethyl acetate extract of G. hombroniana Pierre leaves is 42.004 mg QE/g sample. Conclusion: Garcinia hombroniana Pierre leaves extract has antioxidant activity and can inhibit lipoxygenase activity.


INTRODUCTION
Plants contain various chemical compounds such as phenolic, flavonoid, carotenoid, steroid, and thiol compound that may help protect cells from oxidative stress damage and reduce the risk of chronic disease.Flavonoid such as catechin and epicatechin can be used for the activity of antioxidant and anti-inflammatory. 1 Garcinia hombroniana Pierre are in Indonesia, one of them in the Bogor Botanical Garden, but has not been much research on this plant.People familiar with G. hombroniana Pierre as mangosteen family of plants that are ornamental plants, food crops, and as a traditional medicine for itchy. 2 Previous research has been conducted on the bark of G. hombroniana Pierre which showed that the ethyl acetate extract had the highest content of flavonoid and antioxidant activity EC 50 values obtained by FRAP method is 5579.8± 117.7 mol TE (Trolox Equivalent)/g. 2 Garcinia hombroniana Pierre leaves extract have been known to contain flavonoid, 3 but it has not been known yet for its antioxidant activity and inhibition of lipoxygenase activity.This study aims to determine antioxidant activity tested by using FRAP (Ferric Reducing Antioxidant Power) method and inhibition of lipoxygenase activity of G. hombroniana leaves extract.This research is expected to provide information on the strength of antioxidant activity and inhibition of lipoxygenase activity in the most active extract of n-hexane, ethyl acetate, and methanol leaves of G. hombroniana Pierre as a source of antioxidant and anti-inflammatory.

Antioxidant Activity Assay
For the antioxidant activity assay, 3.8 mL FRAP reagent solution reacted with 0.2 ml baicalein solution or sample, then incubated for 30 minutes at 37ºC.The solution absorbance was measured at 595 nm.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 percentage of capacity can be calculated using this equation: % Capacity = (1 -Ts) x 100% Ts = Transmittan As = -log Ts As = Absorbance of standard or sample solution -Absorbance of reference solution EC 50 value gained by using Microsoft Office Excel and GraphPad Prism 7.

Lipoxygenase Enzyme Inhibition Assay
For the lipoxygenase enzyme inhibition assay, first to be optimized pH borate buffer solution, stop solution, enzyme concentration, and substrate concentration.Test of inhibition of lipoxygenase activity carried out by reacting 10 mL of baicalein or sample solution (various concentration) with 1690 mL of 0.2 Pharmacognosy Journal, Vol 9, Issue 2, Mar-Apr, 2017

Antioxidant Activity Assay
Test of antioxidant activity using baicalein as the positive control.The test result obtained EC 50 value of baicalein is 1.165 µg/mL (Table 1).The test result of extract n-hexane, ethyl acetate and methanol leaves of G. hombroniana obtained EC50 values consecutively are 36.260;2.969; and 7.416 µg/mL (Table 2).

Lipoxygenase Enzyme Inhibition Assay
Lipoxygenase inhibition test is done by using baicalein as a positive control.The test result obtained IC 50 value of baicalein is 0.250 µg/mL (Table 3).The test result of extract n-hexane, ethyl acetate and methanol leaves of G. hombroniana obtained IC50 values consecutively are 2.052; 0.134; and 1.314 µg/mL (Table 4).

Thin Layer Chromatography (TLC) Analysis
Analysis with TLC (Thin Layer Chromatography) was conducted to determine the presence of flavonoid in the extract n-hexane, ethyl acetate and methanol leaves of G. hombroniana qualitatively.The TLC result showed that there aren't any flavonoid in the n-hexane and methanol extract, (Figure 1 and 3) but there is flavonoid in the ethyl acetate extract of the leaves of G. hombroniana (Figure 2).
Analysis by TLC (Thin Layer Chromatography) gave the result that ethyl acetate extract of the leaves of G. hombroniana has the highest content of flavonoid.Then the ethyl acetate extract of the leaves of G. hombroniana undergoes the determination of total flavonoid content by AlCl 3 colorimetric method.

Determination of the total flavonoid content (TFC)
The standard solution with the various concentration of quercetin used to create a calibration curve 6 (Table 5).The absorbance of ethyl acetate extract of leaves of G. hombroniana plotted against the quercetin calibration curve and then calculated its total flavonoid content.The content of flavonoid in the sample expressed in QE (quercetin Equivalent).QE is the equality number of milligram quercetin in 1 gram sample.The result obtained total flavonoid content of ethyl acetate extract of the leaves of G. hombroniana is 42.004 mg QE/g sample.
M borate buffer pH 8.5 and 1000 mL of linoleic acid solution (900 μM).The mixture solution was then incubated for 10 minutes at 25ºC.300 mL of lipoxygenase solution (5000 units/mL) was added and incubated another 15 minutes at 25ºC.1000 mL of stop solution is added and measured its absorbance using UV-Vis spectrophotometer at 235 nm.
Inhibition of the activity of lipoxygenase can be known from the value of the inhibition percentage that was calculated using the following equation:

Thin Layer Chromatography (TLC) Analysis
Analysis by thin layer chromatography (TLC) was conducted to determine the leaves extract of Garcinia hombroniana that have the highest content of flavonoid qualitatively.Eluent used after optimization of the mobile phase is ethyl acetate-formic acid (40:1) for methanol extract, toluene-ethyl acetate-formic acid (61:30:9) for ethyl acetate extract and n-hexane-ethyl acetate (6:4) for n-hexane extract.The plate of silica gel 60 F 245 is used as the stationary phase.
In this TLC analysis, quercetin is used as a standard solution that is treated similarly to the extract.The distance covered by the standard solution (Rf standard) is then compared to the distance traveled by each extract (Rf sample).

Determination of the total flavonoid content (TFC)
For total flavonoid content, 0.5 mL standard or sample solution was reacted with 1.5 mL methanol pro analysis; 0.1 mL AlCl 3 10%; 0.1 mL 1M sodium acetate solution; and 2.8 mL distilled water.The mixture solution was incubated for 30 min at room temperature.Absorption was measured by using UV-VIS spectrophotometer at 435 nm.The calibration curve of quercetin was required to obtain the linear regression equation so that the level of flavonoid in the sample can be calculated.presence of flavonoid, but the result showed that the ethyl acetate extract of leaves of G. hombroniana has the low level of flavonoid.In the previous study, xanton was found at the twigs of G.hombroniana. 7The antioxidant activity and inhibition of lipoxygenase activity in leaves extract of G. hombroniana may be caused by xanton and other compounds that have antioxidant activity and may inhibit lipoxygenase activity.

CONCLUSION
Based on the research that has been done, it can be concluded that the ethyl acetate extract of leaves of Garcinia hombroniana Pierre has the most active antioxidant activity and also the most active in inhibiting lipoxygenase activity.Total flavonoid content of ethyl acetate extract of the leaves of Garcinia hombroniana Pierre is 42.004 mg QE/g sample.

Antioxidant Activity Assay
Test of antioxidant activity using baicalein as the positive control to ensure that the testing method performed properly and can be used.Baicalein works by binding to the iron element and undergoes oxidation to slow or prevent the oxidation of other molecules. 4he capacity percentage of baicalein is better compared to the sample leaves extract of G. hombroniana.This may cause by baicalein is a purely positive control (there are no impurities or other compounds), whereas the leaves extract of G. hombroniana contains various compounds that attracted during the extraction process and not all of the compounds are reducing agents to FRAP.Ethyl acetate extract of leaves of G. hombroniana has the best antioxidant activity presumably because the ethyl acetate extract contains higher flavonoid than another extract.

Lipoxygenase Enzyme Inhibition Assay
Lipoxygenase inhibition test is done by using baicalein as a positive control to ensure that the testing method performed properly and can be used.Baicalein acts as an inhibitor which will inhibit the reaction between lipoxygenase and linoleic acid.Inhibition of lipoxygenase caused by the presence of phenolic compounds such as flavonoid that work as a reductive inhibitor. 4Ethyl acetate extract of leaves of G. hombroniana has the best inhibition activity presumably because the ethyl acetate extract contains flavonoid higher than another extract.

Thin Layer Chromatography (TLC) Analysis
Analysis of TLC using stationary phase silica gel 60 F 254 and the mobile phase is different for each extract because the extract obtained by maceration process so that each has different polarity.Mobile phase nhexane and ethyl acetate (6: 4) is used for n-hexane extract; mobile phase toluene, ethyl acetate and formic acid (61: 30: 9) is used for ethyl acetate extract; and mobile phase ethyl acetate and formic acid (40: 1) is used for methanol extract.
The TLC result showed that there aren't any flavonoid in the n-hexane and methanol extract, but there is flavonoid in the ethyl acetate extract of the leaves of G. hombroniana.The absence of flavonoid in the methanol extract may cause by the abundance of chlorophyll which causes most of the spot produced red color at 366 nm. 5 Determination of the total flavonoid content (TFC) The antioxidant activity and inhibition of lipoxygenase activity contained in the leaves extract of G. hombroniana presumably caused by the ABOUT AUTHORS 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 IC 50 value gained by using Microsoft Office Excel and GraphPad Prism 7.

Figure 1 :
Figure 1: TLC result of n-hexane extract of the leaves of G. hombroniana at 254 nm (a) and 366 nm (b).

Figure 2 :
Figure 2: TLC result of ethyl acetate extract of the leaves of G. hombroniana at 254 nm (a) and 366 nm (b).

Figure 3 :
Figure 3: TLC result of methanol extract of the leaves of G. hombroniana at 254 nm (a) and 366 nm (b).

Shinta Marlin:
Undergraduate Student from Faculty of Pharmacy, University of Indonesia Enrolling Apothecary Program in Faculty of Pharmacy University of Indonesia Elya: Lecturer, Researcher, and Laboratory of Phytochemistry and Pharmacognosy, Faculty of Pharmacy, Universitas.