Red Cabbage Juice Decreases Oxidative Stress in Hyperuricemia Model Rats

Serum uric acid levels are recognized to be a substantial risk factor for developing oxidative stress-related illnesses. Red cabbage has the largest concentration of anthocyanins of any antioxidant, which contributes to oxidative stress. This study aimed to see how anthocyanins in red cabbage juice affected MDA levels in hyperuricemia model rats at different dosages and times of the intervention. A Randomized Complete Design was used to create a before and post-test group research design with 24 male Sprague-Dawley rats aged 10-12 weeks classified into four groups, the Control Group (KN) and the Treatment Group (P1-3). KN rats received standard feed, whereas P1-3 received a regular meal and dosages of red cabbage juice of 1.8, 3.6, and 7.2 ml/200g BW for 28 days. Uric acid levels are measured before and after induction, whereas MDA levels are measured before the installation, after induction (Day 0), on Day 14, and on Day 28. Data analysis was performed utilizing the One Way Anova test, Repeated Measured ANOVA, and a mixed design. Statistical testing revealed that differences in dosage and intervention time duration significantly lowered MDA levels in all treatment groups (p0.05). On day 28, the red cabbage juice intake of 7.2 ml/200grBB returned MDA levels to normal.

Serum uric acid levels are recognized to be a substantial risk factor for developing oxidative stress-related illnesses. Red cabbage has the largest concentration of anthocyanins of any antioxidant, which contributes to oxidative stress. This study aimed to see how anthocyanins in red cabbage juice affected MDA levels in hyperuricemia model rats at different dosages and times of the intervention. A Randomized Complete Design was used to create a before and post-test group research design with 24 male Sprague-Dawley rats aged 10-12 weeks classified into four groups, the Control Group (KN) and the Treatment Group (P1-3). KN rats received standard feed, whereas P1-3 received a regular meal and dosages of red cabbage juice of 1.8, 3.6, and 7.2 ml/200g BW for 28 days. Uric acid levels are measured before and after induction, whereas MDA levels are measured before the installation, after induction (Day 0), on Day 14, and on Day 28. Data analysis was performed utilizing the One Way Anova test, Repeated Measured ANOVA, and a mixed design. Statistical testing revealed that differences in dosage and intervention time duration significantly lowered MDA levels in all treatment groups (p0.05). On day 28, the red cabbage juice intake of 7.2 ml/200grBB returned MDA levels to normal. This open access article is under the CC-BY-SA license.

INTRODUCTION
Hyperuricemia is a non-communicable condition characterized by elevated serum uric acid levels in the blood (Aletaha et al., 2010) . According to the World Health Organization (WHO), the prevalence of hyperuricemia is increasing yearly, with an incidence rate of 1-4% of the general population. This rise is also seen in emerging nations like Indonesia (Kumar & Lenert, 2016). According to Basic Health Research statistics from 2018, the prevalence of hyperuricemia illness is 7.3% of the entire population of Indonesia. According to age characteristics, the most immense majority of hyperuricemia patients at the age of 75 years was 18.95%, with a greater prevalence for women (8.46%) than males (6.13%), and the majority continues to rise with age (Fitriyah et al., 2011).
Hyperuricemia is defined as serum uric acid levels that are higher than usual, with male limits of 7.0 mg/dL and female limits of 6.0 mg/dL, due to increased activity of the enzyme xanthine oxidase (XO), which results in the accumulation of monosodium urate (MSU) crystals in blood cells and the development of inflammatory reactions (Cha et al., 2019;Fitriyah et al., 2011). Long-term collection of MSU crystals can lead to forming free radicals via reactive oxygen species (ROS) (Eleftheriadis et al., 2017;Sharaf El Din et al., 2017). As a result of increased uric acid synthesis, it produces superoxide radicals (Fabbrini et al., 2014). Free radicals can destroy cell membranes, resulting in lipid peroxidation and oxidative stress in the body (Maiuolo et al., 2016). Malondialdehyde (MDA) is the end product of lipid peroxidation and is a sign of oxidative stress (Renaudin et al., 2020) To minimize oxidative stress in the body, hyperuricemia situations necessitate using exogenous antioxidants. Theories discuss the effects of exogenous antioxidants found in food items that can replace electrons in unstable cells, preventing cell membrane breakdown (Ali et al., 2020;Deng et al., 2021;Forman & Zhang, 2021). Wulandari et al. (2021) found that providing red beet flour 3.12 g/kg BW/day rich in antioxidants may lower MDA levels to normal for 28 days in hyperuricemia model male rats.
Aside from red beet flour, red cabbage is a typical antioxidant-rich food. Red cabbage is a widely known agricultural product among the general people. Lukitasari et al. (2017) discovered that red cabbage has high antioxidant activity, with an IC50 of 725.65 0.09 ppm. The studies showed that vitamin C levels in red cabbage ranged from 41 to 146 mg/100g, total phenolics ranged from 31 to 337 mg/100g, and carotenoids from 0.37 to 5.42 mg/100g (Bahorun et al., 2004;Franke et al., 2004;Wu et al., 2004). In summary, red cabbage also has a high level of anthocyanin antioxidants. Anthocyanins are antioxidant chemicals produced from flavonoids that give purplish red hues and are the most abundant antioxidant in red cabbage (Ghareaghajlou et al., 2021;Podse˛dek et al., 2006). Because anthocyanins are polar and would dissolve in polar solvents, producing them in juice form is preferable for use in our investigation (Alappat & Alappat, 2020).
To this certain extent, researchers have not observed the effect of red cabbage juice in suppressing oxidative stress by reducing Malondialdehyde levels in hyperuricemia model rats. This study aims to test the dose variation and time variation of red cabbage juice administration to reduce MDA levels in potassium oxonate-induced rats.

Animal Model and Research Design
This was an experimental laboratory study on rats with a pre and post-test design with a control group. The experimental setup is entirely randomized. Male Sprague-Dawley (SD) white rats aged 10-12 weeks were used in the study and were obtained from the Center for Food and Nutrition Studies at Gadjah Mada University in Indonesia. Rats were placed in cages of iron blocks 80x23.5x19 cm in size, divided into six tiny chambers, each occupied by one rat, and adapted for seven days. Temperature control (27-29oC), 12 hours of bright and 12 hours of dark lights were used for rat upkeep.

Sample Size and Study Protocol
The sample of 24 SD male white rats was determined based on the sample calculation by the E-value formula with degrees of freedom in the "resource equation" statistic (Ab, 2017). Rats were randomly divided into four groups: the Negative Control group (KN) and the Treatment Group (P1-3). Each rat group was supplemented with standard confessed, and adequate Treatments groups were supplemented with 1.8, 3.6 and 7.2 ml/200g BW of red cabbage juice daily in the order of treatment groups. The procedure for producing red cabbage juice was published at the 4th International Conference on Social Determinants of Health, Uhamka, Indonesia.
Animal procedures and experimental protocols in this study have been approved by the ethics committee of the Faculty of Medicine, Sebelas Maret University protocol number 83/UNS27.06.11/KEP/EC/ 2022.

Doses of Red Cabbage Juice
The dosages are calculated using a human anthocyanin consumption of 12.5 mg (Sebastian et al., 2015). It was also adjusted with a rat-to-human conversion factor to the anthocyanin content of red cabbage juice (2.84 g/100gram) (0.018). Red cabbage juice dosages in the trial were 1.8, 3.6, and 7.2 ml/200g body weight. The intermediate dosage (3.6 ml/kg BW/day) had anthocyanin content equal to the daily need based on experimental animal conversion. The low and high dosages were calculated by halving or doubling the standard dose.

Treatments and Data Collection Procedures
To establish that rats were already in hyperuricemia, blood samples for uric acid levels were taken twice, before and after the induction of potassium oxonate. Blood was drawn four times to assess MDA levels: before the installation of potassium oxonate, after the inauguration of potassium oxonate, or on day 0 (Pre Test), day 14 (Post-test 1), and day 28. (Post-test 2). 1 ml of rat blood was extracted from the retro-orbital sinus. The enzymatic approach was used to determine blood uric acid levels utilizing uric acid FS reagent and the premise of enzymatic photometric test TBHBA (2,4,6 -tribromo -3 -hydroxybenzoic acid). In contrast, MDA levels were examined by measuring Thio Barbuturic Acid Reactive Substance (TBARS) levels (Bridgers et al., 2010).

Data analysis
The data were statistically evaluated using SPSS version 25 and provided as mean SD. Statistical analysis was performed to determine the effect of differences in dose variations using a one-way ANOVA test, as well as statistical analysis to assess the impact of differences in duration of administration using a repeated measured ANOVA test and to determine the effect of the interaction of dose and duration of administration using a mixed design. The statistical significance value is p-value 0.05.

RESULTS AND DISCUSSION
After 15 days of calcium oxonate induction at a dosage of 250 mg/kg BW/day, uric acid levels demonstrated the effectiveness of hyperuricemia modelling, which was accompanied by elevated MDA levels. The P2 group had the most excellent mean uric acid levels at 8.28 mg/dL and the lowest in the P3 group at 8.03 mg/dL, whereas the KN group had the highest mean MDA levels at 10.20 nmol/mL and the weakest in the P1 group at 9.77 nmol/mL. The success of modelling is determined by the random allocation of rats and the selection of the male sex since they have no hormone estrogen. The estrogen hormone can aid in the excretion of uric acid through urine, allowing it to return to normal (Suhendi, 2011). Induction is performed using potassium oxonate, as described by Dhouibi et al. (2021), by injecting potassium oxonate intraperitoneally. The side effect of potassium oxonate induction is a decrease in endogenous antioxidant capacity, which causes oxidative stress by increasing the formation of ROS via enzymatic oxidation reactions via three main pathways, namely the xanthine oxidase system (XO) pathway, the NADPH-oxidase pathway, and the endothelial nitric oxide synthase (eNOS) enzyme system pathway (Hisatome et al., 2020). One was demonstrated in this study, in which the induction of potassium oxonate at a dosage of 250 mg/KgBB/day for 15 days caused hyperuricemia in the rat model, with an average rise in uric acid levels of 6.70 mg/dL and a substantial increase in MDA levels (Table 1). This demonstrates the importance of exogenous antioxidants in reducing oxidative damage caused by hyperuricemia.
MDA levels were assessed three times during the intervention: on day 0, day 14, and day 28. The intervention was administered in red cabbage juice at 1.8 ml/200g BW for group P1, 3.6 ml/200g BW for group P2, and 7.2 ml/200g for group P3. Table 2 depicts the changes in mean MDA levels according to dosage and time. From Table 2, based on the dose of administration between groups, there were significant differences on day 14 and day 28 (p<0.05). A comparison of dose variations between all groups can be found in Table 3. The mean MDA levels of the red cabbage juice intervention group (P1-3) differed significantly from the KN group on days 14 and 28. The P1 group had nearly the same levels as the P2 group on day 14; however, there was still a significant. Compared to the control group, The reduction in MDA levels was most substantial in P3, which received red cabbage juice at a dosage of 7.2 ml/200g BW. On the 28th day of observation, the average MDA level of the P3 group exhibited the most significant decline of all groups. 1,75 ± 0,24 0,001 2,55 ± 0,08 0,001 P2 dan P3 0,99 ± 0,39 0,001 1,13 ± 0,09 0,001 Note: P: One way anova-post hoc tukey Table 2 also informs the effect of the duration of administration on MDA levels in all groups. Although there was a significant difference (p 0.05) in the KN group that did not get any treatment, the MDA levels hardly changed (approximately 10), and the difference in decline was less than 0.5 g, while in the treatment group showed a significant decrease as shown in Table 4. The mean MDA levels seen from the duration of administration explained that the P3 group tended to experience the highest decrease among other treatment groups, namely the P1 and 2 groups, on day 14 and day 28 of intervention. The interaction of dose and duration of administration also showed significant differences (p<0.05) between groups ( Table 2). As shown in Figure 4.1, dose groups P1 and 3 had the same impact on lowering MDA levels on day 2. On day 5, the dosage of group P1 had the same impact as group P2. The value of the decline is yet unknown because it is beyond the scope of researchers' observation. Similarly to the previous conclusion, the P3 group had the most remarkable decrease in this test. Among the other dosages, the analytical test revealed that providing red cabbage juice at a dose of 7.2ml/200gr BW had the most dominant effect on lowering MDA levels. Intervention within 14 days lowered MDA levels by 3.90 nmol/dL, and this trend continued until day 28, reaching 6.97 nmol/dL. Increasing the dose in the research seeks to find the adequate amount but must still consider the toxicity implications (Chinedu et al., 2013). It is worth noting that the usage of dosages in this study did not result in abnormalities in rats, such as weakness, prolonged sleep, coma, or death, until the conclusion of the intervention. According to Cruz et al. 2016, providing red cabbage water extracts at a dosage of 500 mg/kg twice daily provides a hypolipidemic effect in hyperlipidemic rats without harm. According to FDA 2022 standards, any drug is deemed a low-level potential hazard when the oral dosage is more significant than 2000 mg/kg BW, and the fatal toxicity among the tested rats is less than 50%. As a result, this study's administration of red cabbage juice in varied amounts was safe.
Red cabbage juice is a vegetable that has high antioxidant activity (Cruz et al., 2016;Lukitasari, 2017). Anthocyanins are the most potent antioxidants, supplying purplish red pigments that are good for health. (Ghareaghajlou et al., 2021;Podse˛dek et al., 2006). According to preliminary research, 100g of red cabbage contains 283 mg of anthocyanins or 2.8%. Because of the qualities of anthocyanins in red cabbage juice, the mechanism of the beneficial effect happened in all treatment groups. The P1 and 2 therapy groups significantly decreased MDA levels by an average of 4-6nmol/dL.
Further proof is that in the P3 group, the administration of 7.2ml/200g BW was able to reduce MDA levels to normal limits on the 28th day of intervention (< 4nmol/mL). The mechanism of anthocyanin compounds acts both directly and indirectly to lower MDA levels. Presently, by collecting ROS and providing electrons to bind, as well as indirectly, by stimulating antioxidant enzymes, blocking prooxidant enzymes, and creating phase II detoxifying enzymes and antioxidant enzymes (Situmorang & Zulham, 2020). According to numerous ideas, it is explained that oxidative stress can cause lipid peroxidation reactions, proteins including enzymes and DNA. If this condition continues, cell damage and death will occur. With a decrease in oxidative stress, oxidative stress-related diseases can be inhibited due to antioxidant and anti-inflammatory activities (Hassan & Samah et al., 2013).

LIMITATION OF THE STUDY
The study's limitation is that we did not undertake a renal histopathology test to detect glomerular injury and tubular ischemia caused by increased oxidative stress due to elevated uric acid levels.

CONCLUSIONS AND SUGGESTIONS
Red cabbage juice reduces oxidative stress in hyperuricemia rats by lowering MDA levels. This is demonstrated by administering a 7.2 ml/200gr BW on day 28, which can lower MDA levels to normal. While dosages of 1.8 and 3.6 ml/200g BW can lower MDA levels, they remained under oxidative stress conditions on days 14 and 28. The effective dosage to lower MDA levels in this investigation was 7.2 ml/200g BW.