INTRODUCTION:

Hyperuricemia is a condition where there is an increase in serum uric acid levels, which is above 7.0mg/dl in men and above 6.0 mg / dl in women^1,2. Uric acid levels in the body are related to a low-purine diet and decreased organ function in old age, causing an increase in the prevalence of gout^3,4.

The enzyme Xanthine Oxidase (XO) plays an important role in uric acid metabolism, where XO oxidizes hypoxanthine derived from metabolites from nucleic acid degradation into xanthine and further into uric acid which produces reactive oxygen species (ROS), especially superoxide as an important messenger in inducing inflammation and signal transduction that leads to tissue damage by increasing cyclooxygenase-2 (COX-2), early growth response protein 1 messenger ribonucleic acid (EGR-1 mRNA), and phosphorylation of extracellular signal-regulated protein kinase 1 and 2 (ERK 1/2)^5,6.

Uric acid has a role in modulating COX-2 levels in the body. Uric acid can activate the proliferation pathway in vascular smooth muscle cell (VSMC) and uric acid augmentation of VSMC proliferation in renal arteries is mediated by COX-2.Research conducted by examining the effects of uric acid on COX-2 concentration time and dose dependently on VSMCs found that uric acid increased COX-2 concentration significantly. Uric acid stimulates an increase in COX-2 levels which is very important in inflammatory mediators such as prostaglandins and VSMC proliferation⁷.

Currently, the use of traditional medicine in Indonesia is progressing quite rapidly. Traditional medicines are now starting to be reused by the community as an alternative treatment. alternative treatment, even though modern drugs or synthetic drugs are still circulatingon the market^8,9. Although traditional medicines derived from plants and natural ingredients also have side effects, the level of danger and risk of long-term use is much lower than that of chemical drugs¹⁰.

Research results that have been reported related to plants of the Etlingera genus, namely E. rubroloba, are used as traditional medicine in Southeast Sulawesi to increase endurance and relieve joint pain¹¹. Ethanol extract of E. rubroloba contains secondary metabolite compounds of flavonoids, alkaloids, saponins, tannins and terpenoids and has immunostimulatory effects^12,13, E. rubroloba stem has pharmacological activity as an antioxidant and XO inhibitor¹⁴, Ethanol extract of E. rubroloba stem also has strong antioxidant activity and potential as an anti-inflammatory with TNF-α parameters¹⁵. Research conducted on different species, namely ethanol extract of E. elatior (Jack) R.M. Smith has potential as antihyperuricemia¹⁶and as a Hepaprotector¹⁷, besides research on E. rubrostriata, E. littoralis, E. fulgens, and E. maingayi leaves, has antioxidant and antibacterial properties¹⁸. Another study mentioned that the compound Sinaphyl alcohol diacetate, and Ergosterol peroxide which was successfully isolated from the fruit of E.rubroloba, which reduced IL-12 levels and TLR-2 protein expression so that it was effective as an immunomodulation of DM infected with TB¹⁹.

Close kinship in plants of the same genus causes similarities in terms of morphology, chemical content and pharmacological potential²⁰. One of the secondary metabolite compounds in E. rubroloba and E. elatior fruits is flavonoids. Flavonoid compounds are able to reduce uric acid levels by preventing the formation of free radicals through suppression of ROS²¹. Research also shows that flavonoid compounds have XO inhibitor activity in vitro and can reduce COX-2 expression in mice so that it has potential as an anti-inflammatory²².

MATERIALS AND METHODS:

Materials:

The materials used in this study were the fruit of E. rubroloba A. D Poulsen, mice, cotton, tissue, aluminum foil, filter paper, ethanol 96%, ethanol 70%, distilled water, potassium oxonate, Na CMC 0.5% (Food Grade®), allopurinol, chloroform, aqua pro injection, absorbent paper, aquadest, uric acid testing strips (Easy Touch), handscoon, Prostaglandin Synthase-2 ELISA Kit (Elabscience), Physiological Nacl 0.9% (PT. Brataco), syringe, and needle.

Sample Preparation:

The sample used in this study was Etlingera rubroloba A.D. Poulsen fruit (6.3kg) taken from Laiya District, South Konawe Regency, Southeast Sulawesi Province, then dried to obtain dry powder (3.3kg). Furthermore, it was macerated until a concentrated extract of 0.529kg was obtained. This research has received approval from the health research ethics committee, Institute for Research and Community Service, Halu Oleo University with number: 182/UN29.20.1.2/PG/2023. Samples were determined at the Biology Research Center of LIPI Cibinong Bogor Indonesia, to ensure that the samples used were fruits of Etlingera rubroloba A.D. Poulsen.

Antihyperuricemia Activity Test:

The antihyperuricemia activity test was performed using the lateral chromatography method that works with electrochemical detection techniques, where the test stick has a part that can draw whole blood from the location of the blood drop into the reaction zone. Uric oxidase in the reaction zone then oxidizes uric acid in the blood and the intensity of the electron current is measured by the device and read as the concentration of uric acid in the blood sample in mg/dl²³. The data obtained were then processed using statistical analysis.

Cyclooxygenase-2 inhibition assay:

This ELISA kit uses the Sandwich-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Rat PTGS2/COX-2. Standards or samples are added to the micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Rat PTGS2/COX-2 and Avidin-Horseradish Peroxidase (HRP) conjugate are added successively to each micro plate well and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Rat PTGS2/COX-2, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450nm±2nm. The OD value is proportional to the concentration of Rat PTGS2/COX-2. You can calculate the concentration of Rat PTGS2/COX-2 in the samples by comparing the OD of the samples to the standard curve^24,25.

Data Analysis:

The analysis of antihyperuricaemia activity and the correlation between uric acid levels and COX-2 levels were carried out with the Statistical Program for Social Science (SPSS) version 25. Furthermore, the homogeneity test of data for each group was used as a requirement for the ANOVA one-way analysis of variance test to see the average difference of two or more treatment group data. The test results were significant when the value (p<0.05) was obtained with a confidence level of 95%.

RESULT AND DISCUSSION:

Antihyperuricemia Activity:

In this study, antihyperuricemia activity was tested by looking at uric acid levels and COX-2 in ethanol extract of E. rubroloba A.D Poulsen fruit and seeing the correlation between uric acid levels and COX-2 levels. This study used 3 dose variations, namely 100, 200 and 300mg/kgBB. The selection of dose variations was based on previous research using E.elatior, where the results showed that doses of 100, 200, and 300mg/g bw proved to have the potential to reduce uric acid or as antihyperuricemia²⁶. Researchers took the same dose variation, because they saw aspects of taxonomic closeness between E. rubroloba and E. elatior which have the same genus and family so that it is expected to have antihyperuricemia activity in test animals.The negative control used in this study is Na CMC 0.5% which has inert properties and produces a stable suspension. In addition, Na CMC also does not contain active substances so that it cannot provide pharmacological effects on test animals²⁷. While the positive control used is allopurinol with a dose of 300 mg. The selection of allopurinol is because allopurinol is a specific inhibitor of the XO enzyme which has proven effective in reducing uric acid levels, namely doses ≤ 300mg have better effectiveness than febuxostat 40mg.²⁸.

Uric Acid Levels in Test Animals:

Measurement of uric acid levels in mice was carried out using the lateral chromatography method with a uric acid meter (Easy Touch®). This method is based on biosensor technology that is specific for uric acid measurement, the uric acid strips used have parts that can attract blood samples from the drip site into the reaction zone. Uric oxidase in the reaction zone then oxidizes the uric acid in the blood and the intensity of the electron current is measured by the device and read as the concentration of uric acid in the blood sample²⁹.

The choice of this method is because the lateral chromatography method has advantages such as requiring only a small amount of sample, not requiring special reagents, practical and easy to use and can be done by anyone without requiring special skills by showing results in a short time. Based on measurements of uric acid levels that have been carried out at the 1st, 2nd and 3rd hours, it shows a decrease in blood uric acid levels in mice given positive control treatment and extract doses. The average decrease in uric acid levels in the test group can be seen in Figure 1.

Figure 1: Uric acid levels measurement results

The results of the average blood uric acid levels in Figure 1, show that there is a decrease in uric acid levels of test animals after treatment in the positive control group and extracts with doses of 100, 200 and 300mg/g bw. The results of the post hoc test showed a comparison between the negative control and the extract dose group at the 1st, 2nd and 3rd hours there were significant differences (p<0.05) which means that the extract dose groups of 100, 200 and 300mg/g bw have the potential as antihyperuricemia by reducing uric acid levels.

Decreased levels of COX-2:

COX-2 levels were tested using the ELISA method with the prostaglandin endopherpxide synthase-2 (PTGS2/COX-2) kit. This ELISA uses the Sandwich-ELISA principle where the microELISA plate provided in this kit has been coated with antibodies specific to Mouse PTGS2/COX-2. The results of measuring COX-2 levels (Figure 2), showed that doses of 100, 200 and 300mg/g bw had a decreasing effect.As the treatment dose increases, the COX-2 level decreases closer to normal control. The results of the COX-2 post hoc test showed that the comparison between the negative control and the extract dose variation group had a significant difference (p<0.05), which means that the extract dose group has the potential to reduce COX-2 levels. The results of research that have been reported previously are compounds that are thought to reduce COX-2 expression in test animals are flavonoids. Flavonoids, especially quercetin, can suppress COX-2 expression in test animals, which indicates that plants containing flavonoids have potential as anti-inflammatory^30,31. Flavonoid compounds as COX-2 inhibitors using molecular docking show the results of flavonoids have the best free bond energy so that they have potential as COX-2 inhibitors³².

Figure 2: Measurement results of Cox-2 levels

Relationship between Uric Acid Levels and COX-2:

Correlation testing of uric acid levels to COX-2 levels was carried out to determine the relationship and between the decrease in uric acid levels and COX-2 levels in test animals. The correlation test results show that the correlation coefficient value is 1, which means that there is a positive correlation between uric acid levels and COX-2 levels. This shows that the decrease in uric acid levels is directly proportional to the decrease in COX-2 levels in mice. The results of the correlation test can be seen in Table 1.

Table 1: Pearson correlation value of uric acid levels with COX-2 levels

Correlations
		Uric Acid Levels	COX 2 Levels
Uric Acid Levels	Pearson Correlation	1	0.550**
	Sig. (2-tailed)		0.005
	N	24	24
COX 2 Levels	Pearson Correlation	0.550**	1
	Sig. (2-tailed)	0.005
	N	24	24

** Correlations is significant at the 0.01 Level (2-tailed)

Uric acid has a role in modulating COX-2 levels in the body by activating the proliferation pathway in VSMCs and uric acid augmentation of VSMC proliferation in renal arteries is mediated by COX-2^7,33.

CONCLUSION:

The results of the study can be concluded that ethanol extract of E. rubroloba fruit has antihyperuricemia activity and reduces COX-2 levels, and there is a positive correlation between uric acid levels and COX-2.

CONFLICT OF INTEREST:

None declared.

ACKNOWLEDGMENTS:

The authors acknowledge the Direktorat Penelitian dan Tim Peningkatan Reputasi UGM to Universities of World Class Kantor Jaminan Mutu UGM for 2022 Post Doctoral funding with grant number 5061/UNI/ DITLIT/Dit-Lit/PT.01.02/2022, with assignment letter No. 1119/UN1.P.II/KPT/ HUKOR/2022. The authors would like to thank Universitas Halu oleo, Politeknik Bina Husada Kendari and Universitas Muhammadiyah Kalimatan Timur for their cooperation in this research.

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Received on 01.06.2023 Modified on 14.08.2023

Research J. Pharm. and Tech 2024; 17(4):1627-1631.

DOI: 10.52711/0974-360X.2024.00257