Red Rice Bran Extract Intervention Ability to Improve Lipid Profile and Malondialdehyde Levels in Type 2 Diabetes Mellitus Model Rats

Background: Diabetes mellitus accompanied by oxidative stress can cause cardiovascular complications. Red rice bran extract contains antioxidants that have the potential to prevent oxidative stress and improve hyperlipidemia in patients


INTRODUCTION
Diabetes mellitus accompanied by oxidative stress can cause micro and macrovascular complications 1 . One of the most common complications, also being the main cause of death in DM patients, is cardiovascular complications with a prevalence of 70% 2 .
Cardiovascular abnormalities in DM patients are caused by dyslipidemia, which is characterized by changes in lipid profiles. Profile changes that occur are caused by impaired insulin resistance which causes glucose in DM patients to not be converted into energy. This condition causes the energy produced in DM patients to be obtained from the breakdown of fat through the lipolysis process. The result of the lipolysis process is free fatty acids in the blood which will be carried to the liver to be converted into cholesterol (hypercholesterolemia) and triglycerides (hypertriglyceridemia) 3 .
On the other hand, the presence of excess free fatty acids or hyperlipidemic conditions can also cause ROS overproduction which can lead to mitochondrial DNA damage and malfunction of pancreatic cells, which will have an impact on the emergence of oxidative stress in diabetics 4 . The overproduction of ROS that occurs will also stimulate the oxidation of LDL (LDLox) which cannot be recognized by LDL receptors, so in the end, it will cause cardiovascular complications in DM patients 5 .
One of the ways to control diabetes dyslipidemia can be done with non-pharmacological and pharmacological therapies 6 . Pharmacological therapies include the administration of acarbose. However, acarbose generates side effects, namely abdominal pain, diarrhea, nausea, and vomiting 7 . Acarbose is also considered to be not effective to improve lipid profiles and reduce the formation of oxidative stresses 8,9 . There need to be alternatives that can simultaneously treat hyperlipidemia and diabetes without producing side effects 10 .
Red rice bran is a natural food ingredient that has the potential to be a therapy for diabetes mellitus and its complications. Rice bran contains antioxidant compounds consisting of vitamin E (tocopherols and tocotrienols), γ-oryzanol, phenolic compounds, and anthocyanins 11 . The antioxidant content of rice bran can potentially prevent oxidative stress formation by breaking the lipid peroxidation chain and improving hyperlipidemic conditions in DM patients by inhibiting HMG-CoA reductase 12,13 . In vitro studies also found that the antioxidant content, especially anthocyanins found in red bran, exerts a superior function compared to acarbose drug, brown rice bran and purple rice bran in treating DM 14 . In vivo studies on experimental animals also revealed that the administration of white and black rice bran extract could improve blood glucose levels in people with diabetes mellitus 15,16 .
The red rice bran used in this study comes from Magelang Regency, Central Java. Red rice bran extract in this study had an antioxidant activity of 60.14%, vitamin E content of 112.04 mg/100g, flavonoid content of 456.4 mg/100g and anthocyanin content of 340.24 mg/100g 17,18 . Red rice bran from Magelang has the advantage of higher anthocyanin content when compared to red rice bran from Texas and Bali 18 . These differences are caused by the variety, location of cultivated land and different extraction methods 17 . Red rice bran also contains phenolic compounds, flavonoid compounds, and higher total proanthocyanidins than brown and purple rice bran 14 .
Rice bran in Indonesia has not been used properly and is only used as waste. Meanwhile, red rice bran extract (RRBE) contains antioxidants that have the potential to treat people with DM dyslipidemia. To our knowledge, there has been no study on the effect of RRBE administration in vivo study of diabetes mellitus rats. This study aims to analyze the effect of RRBE intervention on lipid profile and malondialdehyde (MDA) levels of type 2 DM rats.

METHODS
This study uses a laboratory experimental model with the type of Pretest-Posttest Control Group Design. The study was conducted in October-November at the Central Laboratory of Food and Nutrition Studies, Gadjah Mada University using five treatment groups.
The materials used in this study were 96% ethanol, bran obtained from the milling process of red rice of the Inpari 24 variety which was grown organically in Magelang Regency, Central Java. The characteristics of the bran used have a smooth texture and have a taste that is dominated by bland, slightly bitter and sweet flavors 19 . Wistar rats, comfeed standard feed, acarbose, streptozotocin (STZ), nicotinamide (NA), thiobarbituric acid (TBA) reagent, Trichloroacetic Acid (TCA), glacial acetic acid, cholesterol oxidase-p-aminophenozone reagent (CHOD-PAP), and glycerol phosphate oxidase-pamino-phenazone (GPO-PAP) reagent. The equipment used in this study was 60 mesh size, Whatman no. 1 filter paper, oven, shaker, rotary evaporator, a set of rat breeding cages, gastric probe, gloves, analytical scales, animal scales, and spectrophotometer (SP-300: Optima Japan).
The experimental design is shown in

Dosage Determination
The dose of acarbose commonly used by adult patients is 100 mg/kg BW/day 22 . The dose was converted from humans to white rats and obtained a dose of 1.8 mg/200gr/day. The dose of RRBE in this study was determined based on the anthocyanin requirement of adult humans of 12.5 mg/day and the study of giving anthocyanins of 10-20 mg could affect blood glucose, superoxide dismutase, malondialdehyde, blood antioxidant status, and inhibition of pancreatic cell damage 23,24 . The daily dose in humans of 12.5 mg was used as the median dose in this study and then converted to a rat dose and adjusted to the content contained in the RRBE with the result of 330 mg extract/kgBW/day. The first dose (165 mg extract/kgBW/day) was obtained from the second dose (330 mg extract/kgBW/day) and the third dose (660 mg extract/kgBW/day) was obtained by doubling the second dose.

Bran Ethanol Extract
The red rice bran in this study came from the milling process of red rice with the type of Inpari 21 variety and the same planting location, namely Magelang Regency. This is so that the rice obtained is homogeneous.
The red rice bran obtained was then filtered using a mesh size of 60 to have the same size of bran, then the bran that had been extracted was then heated using an oven at 105ºC for five minutes. Then, the process of maceration of the ethanol solution was carried out in a ratio of 1 to 6 with constant stirring for seven days using a shaker at 150 rpm. Furthermore, the process of filtering and concentration were done using a rotary evaporator with a temperature of 30ºC 25,26 .

Experimental Animal Induction and Treatment
Thirty-five male albino Wistar rats were divided into five treatment groups, with each group consisting of seven rats that followed the provisions of the Institutional Animal Care & Use Committee 27 . The inclusion criteria in this study were rats aged eight weeks, having a body weight between 150-200 g, fasting glucose levels > 150 mg/dL, healthy condition with normal activities and behavior. The exclusion criteria were rats that had diarrhea, rats that appeared to be unhealthy, and rats that died during treatment.
The rats were adapted for seven days in a special room with a maintenance room temperature (27-29°C), a humidity of 70-90%, with a bright light cycle and a dark light cycle every 12 hours. This is a purpose to adjust the lifestyle and prevent the rats from experiencing stress. During the adaptation, rats were fed a standard Comfeed fed AD II (Carbohydrate 51%, Protein 15%, Fiber 6%, Fat 7%).
The adapted rats were induced intraperitoneally using a high dose of 230 mg/kgBW/day nicotinamide (NA) (Sigma-Aldrich 72340) first, after 15 minutes the rats were induced using a high dose of 65 mg/kgBW/day streptozotocin (STZ) (Nacalai Tesque 32238-91). After 5 days post-induction of STZ-NA, the rats fasted for eight hours before taking blood through the retro-orbital flexus. Rats with hyperglycemic conditions with fasting blood glucose levels >150 mg/dL and changes in lipid profile were used as study samples [28][29][30] .
Administration of STZ in this study causes damage to pancreatic cells which can cause insulin receptor disturbances in experimental animals. While offering NA serves to protect pancreatic cells from the toxic effects of STZ 31,32 .

Lipid Profile
The measured lipid profile was taken from the blood serum of experimental animals. The blood sample was taken through the orbital sinus after STZ NA induction and after 21 days of intervention. The obtained rat blood was placed in the serum separator tube (SST), followed by inversion of the tubes 4-5 times. The tube was incorporated into a centrifuge and rotated for 20 minutes at 3000 rpm to obtain the blood serum. Examination of triglyceride levels using the glycerol phosphate oxidase-p-amino-phenazone (GPO-PAP) method. Examination of total cholesterol, HDL, and LDL using the cholesterol oxidase-p-aminophenazone (CHOD-PAP) method. Serum concentrations of total cholesterol, HDL, and LDL were determined using a total cholesterol assay kit, LDL assay kit, HDL assay kit and triglycerides assay kit (Human).

MDA
The MDA measurement was performed from the blood serum of experimental animals. The blood sample was taken through the orbital sinus after STZ NA induction and after 21 days of intervention, and processed to obtain the serum through centrifugation. Serum MDA levels were examined by a spectrophotometric method using the kit and protocol from the Elabscience Malondialdehyde (MDA) Assay Kit (E-BC-K025).

Data Analysis
The hypothesis in this study is that there is a dose effect (165, 330, or 660 mg/kgBW/day) of the ethanolic extract of red bran. On the levels of total cholesterol, triglycerides, HDL, LDL and MDA in rats model T2DM. The data obtained were processed using SPSS version 17. Before carrying out the analysis, the data were tested for normality using the Shapiro-Wilk test with a p value > 0.05. Then the data were analyzed using a paired t-test to analyze differences in lipid profile and MDA levels before and after treatment in all groups and an Analysis of Variance (ANOVA) test was performed to analyze the differences between the five treatment groups, followed by Post Hoc Tukey HSD. All values are reported on an average basis and ± standard deviation. P value < 0.05 was considered a statistical significance level. significantly in the K+, P1, P2, and P3 groups. The greatest decrease in cholesterol was found in the P3 treatment group (-85.37 mg/dL). The results of the ANOVA test in this study showed that there were significant differences in total cholesterol levels between each treatment group before and after the intervention. Table 2 also shows that the average difference in cholesterol levels in the group receiving RRBE (P1, P2, P3), when compared to the negative group, was statistically significant. This indicates that the RRBE was able to reduce the cholesterol levels in DM model rats and has the potential to treat DM patients who have elevated cholesterol levels. Table 2 also expresses that the positive group, when compared with the P3 group, was not statistically significant, thus the administration of RRBE with a dose of 660 mg/kg BW/day could be an alternative to acarbose in lowering cholesterol levels in T2DM rats. 0.020* 0.001* D1: Before intervention (post STZ-NA induction); D21: After intervention; K-: Negative control group; K+: positive control group given acarbose 1.8 mg/200gr/day; P1: RRBE 155 mg/kgBW; P2: RRBE 330 mg/kgBW; P3: RRBE 660 mg/kgBW; Δ Cholesterol: the difference in the mean cholesterol levels after induction and after administration of the extract for 21 days. *) significant (P<0.05); P 1 ) test paired t-test; P 2 ) one way ANOVA test; a,b,c ) Superscripts with different letters, show significant differences (Post Hoc Tukey HSD).

The Effect of RRBE on Total Cholesterol Levels
The antioxidant content of rice bran is being studied intensively because it can show several beneficial biological activities both in vitro and in vivo which can potentially be used as supportive therapy in overcoming diabetes mellitus conditions while reducing the emergence of complications 33,34 . RRBE contains antioxidant compounds consisting of vitamin E (tocopherols and tocotrienols), γ-oryzanol, phenolic compounds, and anthocyanins 11 .
Based on the results of statistical analysis, the administration of RRBE in groups P1, P2, and P3 could reduce total cholesterol levels in T2DM rats and group P3 with a dose of 660 mg/dL being the best dose of RRBE in reducing total cholesterol levels compared to other groups. This is in line with in vivo and in vitro studies which found that the level of reduction in cholesterol levels was in line with the amount of bran extract given 20,35 . The decrease in cholesterol levels was higher in the P3 group probably due to the higher antioxidant content when compared to other doses.
The content of antioxidants, especially vitamin E (tocotrienol), γ-oryzanol and phenolic compounds in rice bran can function in regulating the synthesis of endogenous cholesterol by inhibiting the activity of HMG-CoA reductase 13,36,37 . HMG-CoA reductase is an enzyme that occurs during the process of converting HMG-CoA into mevalonic acid which is an important step in cholesterol biosynthesis 38 . Inhibition of HMG-CoA reductase will result in isoprene not being formed and squalene formation not occurring so that cholesterol synthesis is inhibited. This inhibited cholesterol will result in decreased cholesterol synthesis 37 . The process of inhibiting HMG-CoA reductase can reduce cholesterol levels in animal and human studies 38 .
Red rice bran also contains phenolic compounds consisting of protocatechuic acid, chlorogenic acid, vanillic acid, hydroxybenzoic acid, coumaric acid, and ferulic acid 13 . Phenol compounds found in rice bran, especially chlorogenic acid, can reduce cholesterol levels by increasing glucose uptake in skeletal muscle cells through the activation of AMPactivated protein kinase (AMPK) which can reduce fatty acid synthesis in liver cells 13,39 . This decrease will increase the uptake of cholesterol in the blood to be resynthesized which will cause a decrease in cholesterol levels in the blood 37,40 .
The results of other studies also showed that oryzanol contained in rice bran was able to prevent an increase in cholesterol levels. Oryzanol in the body plays a role in the mechanism of cholesterol absorption in the intestine by forming complex compounds with insoluble cholesterol. This causes the solubility of cholesterol in the body to be reduced in bile acid solutions. Excess cholesterol contained in the body will eventually be excreted with feces. This causes cholesterol levels in the body to be reduced 41 . In addition, the content of oryzanol and tocotrienol in rice bran can lower cholesterol levels in the blood by inducing the release of the cholesterol 7-alpha-hydroxylase (CYP7A1) enzyme which plays a role in maintaining the stability of cholesterol levels in the body by converting it into bile acids 42 .
The results of the analysis also showed a decrease in total cholesterol levels in the high P3 group when compared to the administration of acarbose in this study. This is probably due to the flavonoid content, especially anthocyanins and proanthocyanidins in the P3 group can inhibit the activity of α-glucosidase and αamylase better than high-dose acarbose drugs, according to previous in vitro studies 14 41 . Increased insulin secretion will prevent increased lipolysis and impaired lipogenesis in the liver which causes cholesterol levels in the body not to increase 43 . Table 3 shows that the triglyceride levels before and after intervention experienced a significant decrease in the K+, P1, P2, and P3 groups. The greatest decrease in triglycerides was found in the P3 treatment group (-52.39 mg/dL). The results of the ANOVA test in this study showed that there were significant differences in triglyceride levels between each treatment groups after the intervention. Table 3 also shows that the average difference in triglyceride levels in the group receiving RRBE (P1, P2, P3) when compared to the negative group, was statistically significant. This shows that RRBE can reduce triglyceride levels in DM model rats and has the potential to treat DM, which has increased triglyceride levels. Table 3 also denotes that the group given RRBE (P1, P2, P3), when compared with the positive group, has a statistically significant result. This result indicates that the administration of RRBE has been able to replace acarbose in reducing triglyceride levels in T2DM rats.  Several studies recently suggested that the decrease and increase in triglyceride levels were positively correlated with cholesterol levels. 41 This is in line with the results of statistical analysis in this study, namely the administration of RRBE can reduce cholesterol and triglyceride levels in all groups receiving RRBE and P3 groups with a dose of 660 mg/dL being the best dose of RRBE in this study.

The Effect of RRBE on Triglyceride Levels
Triglycerides are a form of fat that is absorbed by the intestine after hydrolysis which then enters the plasma in two forms, namely as chylomicrons (derived from intestinal absorption after ingestion of fat) and as very low density lipoprotein (VLDL) which is formed by the liver 44 .
The content of phenolic compounds (ferulic acid) and flavonoids (anthocyanins) found in rice bran can improve insulin resistance in people with diabetes mellitus 45,46 . improvement of insulin resistance can suppress the production of free fatty acids in the body. blood circulation by inhibiting fatty acid synthase (FAS), which is an enzyme that is very important in fat metabolism. Inhibited FAS can directly reduce the formation of fatty acids, thereby reducing the formation of triglycerides and VLDL which will prevent the occurrence of hypertriglyceridemia, postprandial hyperlipidemia and lipoprotein lipase resistance in people with diabetes mellitus 46,47 .
The antioxidant content in rice bran which has the effect of inhibiting HMG-CoA reductase can also reduce triglyceride levels in plasma. The decrease in plasma triglyceride levels occurs due to an increase in the speed of LDL catabolism, thereby reducing LDL storage in plasma which affects the decrease in triglyceride levels 48 . Table 4 show that HDL levels before and after intervention experienced a significant increase in the acarbose (K+) group and the RRBE group (P1, P2, P3) groups. The greatest increase in HDL was in the P3 treatment group (50.89 mg/dL). The results of the ANOVA test in this study showed that there were significant differences in HDL levels between each treatment group before and after the intervention Table 4 also shows that the average difference in HDL levels in the group receiving RRBE (P1, P2, P3), when compared to the negative group, was statistically significant. The results mean that RRBE can increase HDL levels in DM model rats, thus having the potential to treat DM with increased HDL levels. Table 4 also described that the P3 group when compared with the positive group was statistically insignificant. Hence, the administration of RRBE at a dose of 660 mg/kg BW/day could be an alternative to acarbose in increasing HDL levels in T2DM rats.  0.876 0.001* D1: Before intervention (post STZ-NA induction); D21: After intervention; K-: Negative control group; K+: positive control group was given acarbose 1.8 mg/200gr/day; P1: RRBE 155 mg/kgBW; P2: RRBE 330 mg/kgBW; P3: RRBE 660 mg/kgBW; Δ HDL: the difference in the mean HDL after induction and after administration of the extract for 21 days. *) significant (P<0.05); P 1 ) test paired t-test; P 2 ) one way ANOVA test; a,b,c ) Superscripts with different letters, show significant differences (Post Hoc Tukey HSD).

Effect of RRBE on HDL Levels
Based on the results of statistical analysis, giving RRBE to groups P1, P2, and P3 could increase HDL levels in T2DM rats. This increase in HDL is in line with in vivo study which states that bran supplementation can increase HDL levels in experimental animals 40 .
The P3 group with a dose of 660 mg/dL is the best dose of RRBE in increasing total HDL levels compared to other groups. The higher the increase in HDL levels in the body, the greater its capacity to transport cholesterol and prevent blockages in the blood vessels 49 .
Flavonoids in rice bran also specifically anthocyanins and proanthocyanidins function to increase HDL by activating peroxisome proliferatoractivated receptor alpha (PPARα) in the liver 52 . PPARα functions as a regulator of lipid metabolism which plays an important role in inducing the absorption of glucose and fatty acids by liver cells 53 . Activation of PPARα in the liver can increase the amount of apolipoprotein A-1 which helps the formation of HDL and increases HDL levels in the blood 54 . Table 5 represents the LDL levels before and after the intervention, which decrease significantly in the K+, P1, P2, and P3 groups. The greatest reduction in LDL was found in the P3 treatment group (-44.24 mg/dL). The results of the ANOVA test in this study showed that there were significant differences in LDL levels between each treatment group before and after the intervention. Table 5 indicates that the average difference in LDL levels in the group receiving RRBE (P1, P2, P3), when compared to the negative group, was statistically significant. This result implies that RRBE can depress the LDL levels in DM model rats and has the potential for DM therapy with increased LDL levels. Table 5 also demonstrates that the positive group when compared with the P3 group was not statistically significant. Therefore, the administration of RRBE at a dose of 660 mg/kg BW/day could be an alternative to acarbose in reducing LDL levels in T2DM rats. 0.221 0.001* D1: Before intervention (post STZ-NA induction); D21: After intervention; K-: Negative control group; K+: positive control group was given acarbose 1.8 mg/200gr/day; P1: RRBE 155 mg/kgBW; P2: RRBE 330 mg/kgBW; P3: RRBE 660 mg/kgBW; Δ LDL: the difference in the mean LDL after induction and after administration of the extract for 21 days. *) significant (P<0.05); P 1 ) test paired t-test; P 2 ) one way ANOVA test; a,b,c ) Superscripts with different letters, show significant differences (Post Hoc Tukey HSD).

Effect of RRBE on LDL Levels
Low Density Lipoprotein (LDL) is a lipoprotein molecule consisting of a combination of protein and fat synthesized by the liver containing 45% cholesterol, which can affect the incidence of heart disease 54 . Reducing LDL levels in T2DM rats is in line with in vitro studies of bran in humans which stated that the antioxidant content in bran can reduce LDL levels through the mechanism of inhibiting LDL oxidation 41 .
The LDL oxidation process occurs due to damage to plasma lipoproteins caused by fat oxidation in people with diabetes mellitus. This oxidation process in LDL will increase LDL cholesterol levels in the blood and cause blood viscosity to become thicker and cause the risk of blockage of blood vessels (atherosclerosis) to be higher 35,37 .
The antioxidant content of rice bran, especially vitamin E (112.04 mg/100g) which is quite high in this study can inhibit the oxidation process through the peroxyl radical scavenging process which is included in  41 .
The antioxidant content in rice bran, especially phenolic compounds and oryzanol, can reduce LDL levels by inhibiting HMG-CoA reductase 37,50 . Inhibition of HMG-CoA reductase will result in cholesterol resistance which can reduce the formation of very low density lipoprotein (VLDL) in the liver. This inhibition of VLDL synthesis will automatically cause a decrease in the amount of LDL in the blood 37 .
The results of the analysis in this study also showed that the P3 group with a dose of 660 mg/dL was the best dose of RRBE in reducing LDL levels compared to other groups with lower RRBE doses. This is in line with several studies which state that the consumption of rice bran is in line with a decrease in LDL levels in experimental animals 42 . Table 6 denotes the MDA levels before and after the intervention, which decrease significantly in the K+, P1, P2, and P3 groups. The greatest MDA levels reduction was found in the P3 treatment group (-5.95 nmol/mL). The MDA levels alleviation differs slightly when compared to the positive control group (-5.73 nmol/mL) and the P2 group (-4.84 nmol/mL). The results of the ANOVA test in this study showed that there were significant differences in MDA levels between each treatment group before and after the intervention. Table 6 describes that the average difference in MDA levels in the negative group, in comparison to the group receiving RRBE (P1, P2, P3), was statistically significant. This conveys that RRBE can suppress the MDA levels in DM model rats and has the potential for DM therapy. Table 6 also exhibits that the P3 group when compared with the positive group was statistically insignificant. Therefore RRBE at a dose of 660 mg/kg BW/day could be an alternative to acarbose in reducing MDA levels in T2DM rats. 0.704 0.001* D1: Before intervention (post STZ-NA induction); D21: After intervention; K-: Negative control group; K+: positive control group was given acarbose 1.8 mg/200gr/day; P1: RRBE 155 mg/kgBW; P2: RRBE 330 mg/kgBW; P3: RRBE 660 mg/kgBW; Δ MDA: the difference in the mean MDA after induction and after administration of the extract for 21 days. *) significant (P<0.05); P 1 ) test paired t-test; P 2 ) one way ANOVA test; a,b,c ) Superscripts with different letters, show significant differences (Post Hoc Tukey HSD).

Conditions
of hyperglycemia and hyperlipidemia in patients with diabetes mellitus will cause overproduction of ROS, which in turn can cause damage to mitochondrial DNA and malfunction of pancreatic cells, all of which will have an impact on the emergence of oxidative stress and decreased antioxidant capacity in patients with diabetes mellitus 15,55 . Measurement of MDA levels in this study aims to determine the incidence of oxidative stress that occurs in people with diabetes mellitus. MDA is an indicator of the presence of free radical compounds in the body 56 .
Based on the results of statistical analysis, the administration of RRBE in groups P1, P2, and P3 could reduce MDA levels in T2DM rats. The decrease in MDA levels in the P3 group in this study had the best effectiveness and the decrease was not much different when compared to the P2 and P+ groups with acarbose administration. This is in line with a study that states that giving anthocyanins as much as 10-20 mg can reduce malondialdehyde levels in vivo studies on hyperglycemic rat models 23,24 . The P3 and P2 groups in this study contained anthocyanin levels of 12.5 and 25 mg.
The decrease in blood MDA levels that occurs proves that the antioxidant content of RRBE administration can inhibit excess free radical activity in rat models of diabetes mellitus. Lipid peroxide is a reaction that occurs because free radicals bind to lipids through several stages of initiation, propagation and termination with the final result in the form of MDA 57 .
Lipid peroxide that occurs in rats can be inhibited by the antioxidant content found in rice bran. Phenolic compounds found in rice bran are radical scavengers that will inhibit lipid peroxide at the initiation stage that occurs in rats after STZ-NA induction so that MDA is not produced 57 . Meanwhile, anthocyanin compounds in rice bran act as SOD enzymes which will inhibit ROS activity so that they cannot bind to lipids in forming lipid peroxide reactions so that MDA is not produced 58 .
The content of phenolic compounds, vitamin E (tocopherols and tocotrienols) and anthocyanins in rice bran can reduce MDA levels by protecting cells from free radical damage by adding one free electron to a free radical or accepting an unstable electron so that it becomes more stable [59][60][61][62] . The addition can block the oxidation reaction at the initiation or propagation stage so that MDA is not produced 15 .