Antioxidant Activity and Lipoxygenase Enzyme Inhibition As- say with Total Flavonoid Assay of Garcinia porrecta Laness. Stem Bark Extracts

Introduction: The genus Garcinia which is rich of secondary metabolites, mainly flavonoids, have known to have antioxidant and anti-inflammatory activity through the inhibition of lipoxygenase. There isn’t found literature indicating research on inhibition of lipoxygenase activity been done in this plant. The purpose of this study is to obtain the data and determine the potential antioxidant activity, and inhibition of lipoxygenase activity of Garcinia porrecta Laness. stem bark extracts. Methods: This research is included FRAP (Ferric Reducing Antioxidant Power) method antioxidant assay, in vitro lipoxygenase inhibition assay, flavonoids qualitative analysis by thin layer chromatography, and total flavonoids assay in the most active extract. Results: The results showed the methanol, ethyl acetate and n-hexane extracts of G. porrecta Laness. stem bark using FRAP method, has antioxidant activity with EC50 values respectively 1.33; 4.97; and 19.96 μg/mL and lipoxygenase inhibition activity with IC50 values 0.23; 0.52; and 4.87 μg/mL. The most active extract in the both assay is methanol extract which has total flavonoids of 5.66 mg QE/g (quercetin equivalent). Conclusion: The results from the study show extracts of the stem bark of G. porrecta Laness. has antioxidant activity and potential for lipoxygenase inhibition.


INTRODUCTION
Free radicals are atoms or molecules that are unstable (one electron or more without a partner), so as to obtain an electron pair, free radicals tend to look for other molecules and atoms of lead compounds that are not normal and starts a chain reaction in the body. 1 One example of the kind of free radicals in the body is a reactive oxygen species (ROS) and reactive nitrogen species (RNS, such as nitric oxide, NO ) generated by the enzyme NO synthase (NOS) and NAD (P) H oxidase isoform.The beneficial effects of ROS / RNS (eg superoxide radicals and nitric oxide) occurs at concentrations of low/moderate and involves a physiological role in the cellular response to noxia, such as in the defense against infectious agents, in function of a number of cellular signaling pathways, and the induction of mitogenic response , Excess production of ROS (arising either from mitochondrial electron transport chain or excessive stimulation of NAD (P) H) produces oxidative stress, a process that became the mediator of the damage to cell structures, including lipids in cell membranes, proteins, and DNA.As a result of oxidative stress that accumulates can lead to degenerative diseases such as Alzheimer's, cancer and body triggers cell death (apoptosis) faster. 2 One way to deal with free radicals is with antioxidants.Antioxidants are compounds that can slow or prevent damage caused by free radicals by dampening the activity of free radicals or break the chain reaction of oxidation caused by free radicals. 3e of the effects of free radicals is the process of induction of cytokines, inflammatory mediators in the body, which causes an inflammatory response occurs. 4Inflammation is a protective response the body's normal when there is tissue injury which involves a variety of physiological processes in the body such as the activation of the enzyme inflammatory, inflammatory mediator release, the movement of white blood cells through the capillaries into areas of inflammation, cell migration, and the restoration of damaged tissue. 5Lipoxygenase (LOX) is one enzyme that has a role in inflammation, especially in the biochemical processes of leukotrienes.Leukotrienes are the main regulator of allergic reactions and inflammation.Currently, lipoxygenase inhibitors become a potentially important agent that shows significant anti-inflammatory activity. 6,7Flavonoids like baicalein and apigenin have been known to be useful as inhibitors of lipoxygenase in vitro. 8arcinia is the largest genus of the family Clusiaceae which has about 400 species is widespread in Asia, Africa, South America, and the Polynesian Islands.Garcinia contains many secondary metabolites, mainly triterpenes, flavonoids, xanthones, and phloroglucinol that have pharmacological activities such as anticancer, anti-inflammatory, antibacterial, antiviral, anti-HIV, antidepressants, and antioxidants. 9ne species that grows in Indonesia Garcinia, Garcinia porrecta Laness at which previous studies have shown that stem bark of the plant contains several kinds of compounds such as porsanton and dulsisan-Pharmacognosy Journal, Vol 9, Issue 2, Mar-Apr, 2017 Ts = Transmittan As = -log Ts As = Absorbance positive control/extract -The absorbance of the solution FRAP EC 50 of samples was calculated using the equation of nonlinear regression with aid analysis software GraphPad PRISM® version 7, the concentration of the sample is transformed into a logarithm as the x-axis and y-axis percent capacity.

Lipoxygenase Inhibition Activity Test
Optimization tests were done prior the sample test.Each procedure is described in Table 2 through 5. Optimization of Enzyme concentration was described in Table 11 .Inhibition of lipoxygenase activity by the extract of methanol, ethyl acetate, n-hexane Garcinia porrecta Laness.stem bark and baicalein (Table 6) can be known from the value of % inhibition calculated using the following equation.

Determination of Total Flavonoids with Colorimetric
Method of AlCl3 to Quercetin and G. porrecta Laness.

Stem Bark Extract
The test procedure involves reacting methanol extract at a concentration of 6000 mg / mL 0.5 ml, then added 1.5 mL of methanol, 0.1 ml of a solution of AlCl 3, a 10% 0.1 ml, and 2.8 ml aquadest.The mixture was incubated for 30 min at room temperature.Uptake is measured using UV-VIS spectrophotometry at a wavelength of 435 nm.Quercetin was used as a positive control and treated similarly to extract and quercetin calibration curves made with varying concentrations.The linear regression equation quercetin produced can be used to calculate the concentration of flavonoids contained in the sample.Controls for quercetin and extracts made by replacing the solution of AlCl 3 10% with distilled water and treated the same as quercetin and samples.If needed all the prepared solution was filtered with filter paper before measuring absorbance.Levels of flavonoids total calculated in quercetin equivalent (QE), with the formula:

Test Antioxidant Activity Method FRAP
Based on testing FRAP, extracts methanol, ethyl acetate, and n-hexane G. porrecta Laness.stem bark shows the same thing with baicalein of increased extract concentrations proportional to the increase in antioxidant capacity.Data Values of EC 50 tests of antioxidant activity methods FRAP was described in Table 7.Meanwhile, data values of EC 50 nhexane, ethyl acetate and methanol extract tests of antioxidant activity methods FRAP was described in Table 8-10.When compared with the antioxidant capacity baicalein reaching 50% at a concentration of 1.16 ton which is a compound class and compounds porlanosterol xanthones.Testing the antioxidant activity in extracts of G. porrecta Laness.already been done by the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenger which showed significant antioxidant activity (IC 50 <50 μg mL -1) . 10ntil now, this has never been tested inhibition of lipoxygenase enzyme activity and antioxidant activity test, especially with methods FRAP (Ferric Reducing Antioxidant Power) of the plant G. porrecta Laness.It encourages the study of the antioxidant activity of G. porrecta Laness.stem bark extracts in vitro with FRAP method and on the inhibition of lipoxygenase enzyme activity which is one of the inflammatory factors.Extracts with antioxidant activity and inhibition of lipoxygenase and analyzed the content of flavonoids qualitatively by thin layer chromatography.Extract the most active in the antioxidant activity and inhibition of lipoxygenase assay will be set on total flavonoids assay with AlCl 3 quantitative method.

Material Comparative
Baicalein (Sigma-Aldrich) as a positive control of the test the antioxidant activity with FRAP and test methods lipoxygenase inhibition activity.Quercetin (Sigma-Aldrich) as a positive control on a thin layer chromatography and the assay methods flavonoids AlCl 3.

Work Stages
Stages of the work done in this study started with the preparation of the methanol extract, ethyl acetate and n-hexane bark of Garcinia porrecta Laness., the antioxidant activity FRAP method and the inhibitory activity of lipoxygenase done on the third step in order to obtain an extract that has the highest activity.The extract containing flavonoids will be assayed quantitatively by the method of AlCl 3 .

Antioxidant Activity Test FRAP Ferric Reducing Antioxidant Power) Method
FRAP method procedure of antioxidant activity assay is in Table 1.Having obtained the data, calculated the percentage of capacity reduction of Fe ions 3+ by the positive control baicalein/extract the FRAP solution.The percentage of capacity can be calculated using the formula % Capacity = (1 -Ts) x 100% ies because in this study the optimization tests were used to generate maximum data.

Inhibition of Lipoxygenase Activity Assay of Methanol, Ethyl Acetate, and N-Hexane G. porrecta Laness. Stem Bark Extract
Based on inhibition of lipoxygenase activity assay that has been made to extract methanol, ethyl acetate, and n-hexane bark G. porrecta Laness.IC 50 values obtained successively by 0.23 ug / ml; 0.52 ug / ml; and 4.87 ug / ml, can be seen in Table 13 to 15.The test results showed that the extract has the most active inhibition of lipoxygenase activity is methanol extract.When compared with the positive control baicalein which reaches 50% inhibition at a concentration of 0.25 mg / mL, then either extract ethyl acetate and n-hexane from the bark of G. porrecta Laness.showed inhibition of lipoxygenase activity is lower than baicalein.The methanol extract stem G. porrecta Laness.have a better activity than the positive control baicalein in inhibiting lipoxygenase can be seen from the IC 50 values at lower concentrations.The inhibition of lipoxygenase activity is proportional to the concentration of the extract used in the test.The higher the concentration of the extract is used, the higher the lipoxygenase inhibitory activity as well, so that the product of a reaction between the substrate and lipoxygenase in the form of HpODE will decrease.It is characterized by decreasing absorption of the sample minus the control samples to the increased concentration of the extract.

Determination of Total Flavonoids Level of Methanol
Extract from G. porrecta Laness.Stem Bark.
Based on testing by the FRAP method antioxidant activity and inhibition of lipoxygenase activity, it is obtained methanol extract as the most active extract.Towards the methanol extract of the stem bark of G. porrecta Laness.then performed the assay method flavonoid AlCl 3 using UV-Vis spectrophotometer.The results of the optimization maximum wavelength show that the greatest uptake is at a wavelength of 435 nm.Therefore, for the assay positive control quercetin and methanol extract of the mg / mL, then either extract methanol, ethyl acetate and n-hexane from the G. porrecta Laness.stem bark showed lower antioxidant activity with FRAP method.Based on the classification, 11 the methanol extract, ethyl acetate and n-hexane is classified as a very powerful antioxidant.
The antioxidant activity could have been influenced by the presence of hydroxyl groups, such as those found in the phenolic compounds, flavonoids, and tannins.The antioxidant activity depends on the number and position of hydroxyl contained in the compound. 12The antioxidant activity of the extracts of methanol and ethyl acetate allegedly influenced by the presence of flavonoids compounds in both the extract.The content of flavonoids in the extract will be analyzed further using thin layer chromatography.Flavonoids in the extract are expected to be a reductant/electron donor from the -OH group at his disposal to reduce the Feions 3+ to Fe2+.
In the n-hexane extract, antioxidant activity is also quite active although it is lower than both the other extracts.The antioxidant activity of nhexane extracts suspected because of the non-polar compounds such as terpenoids interested in the n-hexane extract of G. porrecta Laness.stem bark.

Lipoxygenase Inhibition Activity Test Inhibition of Lipoxygenase Activity of Baicalein Positive Control
Based on the previous testing, known baicalein have IC 50 values of 0.0012 mM (0.32 mg / mL) 13 as a competitive inhibitor of linoleic acid and 22.5 μM (6.08 mcg / mL) as a lipoxygenase inhibitor in vitro.In this test, the IC 50 values obtained from baicalein of 0.25 mg / mL was obtained by a linear equation as the data in Table 12.The difference results obtained presumably because testing procedures are different from previous stud- Note: A = Absorbance reference solution with enzyme B = Absorbance reference solution without enzymes C = Absorbance sample solution with the enzyme D = Absorbance of the sample solution without enzymes Value IC 50 samples were determined by the equation of nonlinear regression with the help of analysis software GraphPad Prism version 7, the x-axis shows the concentration of the sample that has been transformed into a logarithm and the y-axis shows the % inhibition.

Figure 1 :
Figure 1: Curve relationship borate buffer pH 0.2 M with the uptake.

Figure 2 :
Figure 2: Curve relationship downtime solution stop the uptake.

Figure 4 :
Figure 4: The calibration curve of quercetin.
at 234 nm λ bark of G. porrecta Laness.use a maximum wavelength of 435 nm.In testing the use of quercetin as a positive control and quercetin calibration curve was made to determine the linear regression equation.The linear regression equation of quercetin obtained value of 0,0431x + y = 0.1394.By testing methanol extract 6012 mg / mL, absorption generated after deducting the control sample of 0.286.Based on calculations using the linear regression equation quercetin, obtained value x (extract concentration) of 3.40 mg / mL.Levels of total flavonoids calculated using equivalent levels of quercetin and obtained flavonoid (quercetin equivalents / g) of 5.66 mgQE / g.From the results obtained, the levels of flavonoids in the methanol extract of the bark of Garcinia porrecta Laness which have antioxidant activity and inhibition of lipoxygenase enzymes highest is only 0.56%.Suspected of secondary metabolites such as group xanthones,10 triterpene and phloroglucinol provide a synergistic effect with compounds flavonoids in antioxidant activity and inhibition of enzyme lipoxygenase.9