Effect of Moringa ( Moringa oleifera ) Leaf Flour Supplementation on Total Antioxidant Content of Sprague Dawley Rat Serum Given High-Fat Diet

Background : Moringa oleifera leaf is high in quercetin which can be a source of exogenous antioxidants. Together with endogenous antioxidants, both the antioxidants will be able to counteract oxidative stress conditions. Objectives : To analyze the effect of Moringa leaves flour supplementation on Total Antioxidants Content (TAC) of Sprague Dawley (SD) rat serum given a high-fat diet (HFD). Materials and Methods : A randomized control group post-test design was used on 24 SD rats which were divided into 4 groups, namely healthy control (K 1 ), HFD (K 2 ), supplementation with Moringa leaf flour at a dose of 100 mg/100 g BW/day (K 3 ), and a dose of 200 mg/100 g BW/day (K 4 ). After 28 days of supplementation, serum TAC was analyzed using the ELISA method. Data analysis used Paired-T Test, One Way ANOVA, and Post-Hoc Bonferroni follow-up test. Results : The results showed that the TAC of groups K1, K2, K3, and K4 respectively were 4.806 ± 0.239, 1.323 ± 0.292, 4.020 ± 0.239, and 5.123 ± 0.695. There was a significant difference in serum TAC (p=0.000) between supplementation groups. Significant differences in serum TAC were also found in the supplementation group compared to the HFD control group. Conclusion : Moringa leaves flour supplementation for 28 days at a dose of 200 mg/100 g BW/day increases serum total antioxidant content higher than at a dose of 100 mg/100 g BW/day.

groups. Furthermore, the Kruskal-Wallis test was also used to determine the same attribute among experimental animal groups. The second statistical analysis is the post-test carried out on serum TAC data, which proved that the serum TAC was normally distributed. Furthermore, the One Way ANOVA and Bonferroni Post-Hoc tests were used to examine the difference in the intervention effects in the groups. The significant difference with p-value <0.05 shows the mean ± SD and median (min ± max) for data that were normally and abnormally distributed, respectively.
The analyzed data were computerized using SPSS. Meanwhile, this research was approved by the Health Research Ethics Commission (KEPK) of the Faculty of Medicine, Diponegoro University as stated in the Ethical Clearance NO.25/EC/H/FK-UNDIP/III/2021 dated March 17, 2021.

RESULTS
The body weight characteristics of the experimental animals during the acclimatization period ranged from 182 to 187 g. Besides, none of the animals dropped out during the research and the consumption of HFD for 2 weeks led to a significant increase in weight. The results of statistical tests carried out after its administration (Table 1) showed an increase in body weight before and after being given standard feed AD II K1 and HFD (K2, K3, K4), although a significant difference was observed in the groups (p = 0.000). An insignificance difference (p = 0.282) was observed in the average body weight of the experimental animals in all groups at the beginning of the research. After 2 weeks of being fed with HFD, a significant difference was observed among the groups (p = 0.000). The results of the Kruskal-Wallis test showed that there was a significant difference in weight change among the 4 groups (p = 0.002). Descriptively, the highest percentage of weight gain was exhibited by the group that was fed with HFD (±15%) compared to the one that was only given standard feed (±6%). The results of statistical tests on experimental animal body weight before and after the administration of Moringa leaf flour ( Table 2) showed that all groups including the treatment group which was given standard feed, and HFD, experienced a significant increase in body weight (p = 0.000). The average body weight after 4 weeks of treatment was significantly different (p = 0.000). The average body weight between groups was observed after administering Moringa leaf flour for 28 days. Descriptively, the least percentage of weight gain was shown by the group with the highest dose (±12.24%). Administration of Moringa leaf flour at a dose of 200 mg higher suppressed weight gain in the treatment group compared to 100 mg. The results of the one-way ANOVA statistical test (Table 3) showed that serum total antioxidant contents were significantly different in the 4 groups (p = 0.000). The Bonferroni Post-Hoc Statistical Test showed that the comparison of serum total antioxidant contents in the K3 group to K4 was significantly different (p = 0.022) after administration of Moringa leaf flour. Treatment with a dose of 200 mg/100 g BW and 100 mg/100 g BW indicated that the total serum antioxidant contents were significantly different to the K2 group fed with HFD (p = 0.000, p = 0.000). Meanwhile, it was discovered that K3 treated with a dose of 100 mg/100 g BW was significantly different to the K1 healthy control group (p=0.001). The treatment of K4 with a dose of 200 mg/100 g BW showed a higher content compared to K3 treated with a dose of 100 mg/100 g BW, although it was not significantly different from the K1 healthy control group (p = 1.000).

DISCUSSION
These results indicate that the least total serum antioxidant contents were discovered in the group given HFD. The storage of excessive fat increases body weight thereby increasing the production of proinflammatory cytokines, such as Tumor Necrosis Factor-α (TNF-α) and Interleukin-6 (IL-6) 23 . Furthermore, increased inflammation causes an increase in ROS production and depletion in endogenous antioxidants that enchanged oxidative stress [24][25][26] . Endogenous antioxidants depletion was caused by its increased consumption in suppression of ROS progression 27 , thus endogenous antioxidants are required in sufficient quantities. Intake deficiency of exogenous antioxidants may cause endogenous antioxidants to decrease continuously and the body remains in a state of oxidative stress 28 . Decreased endogenous antioxidants required exogenous antioxidants obtained from food 29 . Endogenous and exogenous antioxidants synergistically maintain or rebalance antioxidants and ROS due to the presence of ROS reducing compounds in exogenous antioxidants such as flavonoids, vitamins, and minerals through mechanism induced enzymes factor transcription, scavenging process by capturing ROS to donate one electron and hydrogen, metal chelating that helps ROS to become relatively stable and unreactive to induce further oxidative stress, and also act as a cofactor of antioxidants enzymes 30,31 .
Giving Moringa leaf flour to the treatment group significantly increased the total antioxidant content of serum, therefore, Moringa leaf flour can act as a source of antioxidants that restores or normalizes serum total antioxidant content efficiently. Mabrouki (2020) analyzed the effect of administering Moringa leaf extract on endogenous antioxidants in experimental animals. Endogenous antioxidants were significantly recovered by administration with Moringa leaf extract by mechanism to reduce and maintain ROS in a balanced concentration 32 . In this research, the increased contents are also due to reduced antioxidants used in the suppression of ROS progression. The previous research also showed an increase in the constituents after the extract intervention due to reduced antioxidants use in reducing ROS and the provision of hydrogen to make it more stable 33 .
Based on the content of Moringa Leaf flour, flavonoids as a source of exogenous antioxidants are present in high quantities 34 . Furthermore, Rodríguez-Pérez (2015), and Makita (2016), reported that methanol extract of Moringa leaf contained 26, and 14 flavonoids, respectively 35,36 . Compared to vegetables, previous research discovered that the flavonoid content in the dried Moringa leaf was 3 to 12 times more high than other types of vegetables consumed by families, namely 12 times more than cauliflower, 9 times of peas, 5 times of cabbage, 4 times of spinach, and 3 times of broccoli 37 . Another research showed that the experimental animals are given HFD, and dietary intervention containing flavonoids for 4 weeks increased endogenous antioxidants through the mechanism of interacts synergistically with exogenous antioxidant system, then captured free radicals, prevent further oxidative damage, thereby maintaining a balanced ROS system 38,39 . Additionally, quercetin, a flavonol bioactive compound, is a class of flavonoids that was found high in Moringa leaf 40 . In previous studies, it was discovered to have reached approximately 50% of the total flavonoids in Moringa leaf extract 41 . Subsequently, quercetin had an antioxidant function that affected the increase of endogenous antioxidant 42 . Quercetin increased endogenous antioxidant Glutathione Peroxidase (GPx), Catalase (CAT), and Superoxide Dismutase (SOD) by directly or undirectly induced the Nrf2mediated transcription activity by increase Nrf2 expression of the antioxidants. These antioxidants are regulated by the transcriptional factor Nuclear Factor E2-Related Factor (Nrf2) which responds by binding to the Antioxidant Response Element (ARE) as a promoter of genes that code the antioxidant. Quercetin also regulates levels of endogenous antioxidant Glutathione (GSH). Superoxide Dismutase captures O2of ROS and transforms it into H2O2. Catalase and Glutathione Peroxidase further catalyze the decomposition of H2O2 to unreactive H2O. This Reaction requires GSH as a hydrogen donor On the other hand, Quercetin has the ability to act directly as free radical scavengers or hydrogen donors, hydroxyl radical scavenging, and metalchelating ability 43,44 .
Moringa leaf flour also has vitamins such as vitamin E, vitamin C, pro-vitamin A, and complete minerals such as Cu, Mn, Fe, and Zn 45 . These vitamin acts as a free radical scavenger and reduces free radical, donating electrons and hydrogen to free radical to prevents their oxidation and to generate a much less reactive species than most other free radicals 44,45 whereas minerals play an important role as cofactors of endogenous antioxidant that may increase the efficiency of endogenous antioxidant function. Endogenous antioxidants are metal ion cofactor-requiring enzymes that catalyze the dismutation of highly reactive superoxide radicals (O2 -) into unreactive and relatively stable molecular oxygen (O2) and hydrogen peroxide (H2O2) 48 . However, through this mechanism, all bioactive compounds and micronutrients in Moringa leaf flour become extremely effective so that they may improve the efficiency of endogenous antioxidant function. After 28 days of treatment, the mean increase in serum total antioxidant contents was higher at dose II than dose I, and the increase at dose II was equivalent to the healthy group.
The average weight gain in the group given HFD (±15%) was significantly different compared to the others. This is in line with previous research which proved HFD caused an increase of 9 to 23% 49 . There are two possible causes of weight gain due to the influence of HFD. First, fat as the main composition of HFD that contains high energy compared to others macronutrients 50,51 . Second, types of fat such as saturated fatty acids and cholesterol which are high in pork oil and duck egg yolk increase the HFD energy density and easy of absorption by the body, therefore, it is stored in excess which ultimately increases bodyweight [52][53][54] . Furthermore, administration of Moringa leaf flour at a dose of 100 mg and 200 mg was able to maintain and suppress the increase in body weight. This study is in accordance with previous studies which showed that administration of 200 mg and 400 mg of Moringa leaf extract were proven to be able to lose and maintain weight because of their high fiber content 55 . Moringa oleifera leaf can provide 41.20 g carbohydrate, 29.40 g protein, 5.20 g fat, and 12.50 g fiber by 100 g dry leaves whereas 100 g dried Moringa leaf powder contains approximately 38.20 g carbohydrate, 27.10 g protein, 2.30 g fat, and 19.20 g total fiber 56 . Due to high fiber content, Moringa can be used to suppress weight gain by mechanism increasing water-binding and swelling capacities. This slows gastric emptying, which in turn increases satiety, longer meal intervals and ultimately decreases food intake 57 .
However, there are some limitations associated with this research, such as difficulty in analyzing macronutrient and micronutrient content, especially specific polyphenols in Moringa oleifera leaf flour.

CONCLUSION
In conclusion, the administration of Moringa leaf flour at a dose of 100 mg/100 g and 200 mg/100 g body weight/day increased the total antioxidant content of the serum in Sprague Dawley rats. Furthermore, the dosage of 200 mg/100 g body weight/day had a better effect.