INTRODUCTION:

Diabetes mellitus is one of metabolic syndrome. Diabetes mellituscan cause several complicated diseases such as heart disease, kidney disease, neuron disease, stroke, dyslipidemia, obesity and many more. It is called metabolicsyndrome. Metabolic syndrome is a condition where a person has central obesity, dyslipidemia, high blood pressure and hyperglycemic¹.

The number of people with Diabetes Mellitus (DM) is increasing every year. According to the International Diabetes Federation (IDF) developing countries such as Indonesia ranks 4th in the number of people with diabetes mellitus after India, China and the United States². Diabetes Mellitus (DM) is the number 3 killer disease in Indonesia.

The prevalence of DM in Indonesia tends to increase, from 5.7% in 2007 to 6.9% in 2013. Based on the data obtained, it is estimated that the number of people with diabetes mellitus (DM) in Indonesia will increase significantly to 21.3 million in 2030³. Based on data from the Ministry of Health in (2017) states that 2/3 diabetics (a term for patients with diabetes) in Indonesia do not know that they have diabetes. With the high morbidity of this disease, it takes a very large cost for the treatment of diabetes mellitus.

Application of natural product is one alternative treatment that is often applied by people since then. Utilization of natural ingredients is used as traditional medicine because it has many advantages including being easy to find and reducing the undesirable effects of synthetic preparations⁴. The use of synthetic drugs has many side effects. Therefore, another treatment is needed, one of which is the utilization of waste from plants that are still very rarely used, one example is black rice bran (Oryza sativa L. Indica) and red rice bran (Oryza nivara).

Rice bran is a by-product of rice processing which is usually used for animal feed. Based on the results of previous studies, rice bran has a good nutritional composition thus it can be used as a functional food to reduce cholesterol levels in the blood, as an antioxidant, prevention of cardiovascular disease and inhibit menopause time and reduce blood glucose levels⁵.

Based on research conducted by Dwinani (2014) states that the content of anthocyanin in black rice bran has the ability to increase insulin secretion in test animals of Wistar strain rats at a dose of 200mg/kg body weight⁶. Other supporting research has been carried out by Damayanthi (2010) in red rice bran containing phenolic bioactive compounds, anthocyanin, β-carotene, tocopherol and oryzanol which can reduce free radicals (ros)⁷.

Based on this reason, the researchers wanted to know the effectiveness of the combination of red rice bran extract (Oryza nivara) and black rice bran (Oryza sativa L. Indica) when compared to the hypoglycemic potential of the combination of glibenclamide and metformin in glucose-induced white rats.

MATERIALS AND METHODS:

Chemical reagents:

The materials used in this study were red and black rice bran, 96% ethanol, aquades, animal feed, Glucovance (a combination of glibenclamide-metformin), glucose and Na.CMC, poloxamer (Pluronic-407) 200mg/200gram BB rats, Propylthiouracil 100mg, simvastatin 10mg/70 kg body weight and gemfobrozil 300mg/kg body weight.

Plant Material:

Samples of red rice bran (Oryza nivara) and black rice bran (Oryza sativa L. Indica) were obtained from Wonggeduku District, Konawe Regency, Southeast Sulawesi. The sample determination was carried out in the Biology Laboratory of the Teaching and Education Faculty (FKIP) of Halu Oleo University, Kendari.

Sample processing:

Samples of red rice bran (Oryza nivara) and black rice bran (Oryza sativa L. Indica) are obtained in the form of grain, so that the milling is first done then dried in the sun.

Extraction:

Red rice bran sample (Oryza nivara) and black gran sample (Oryza sativa L. Indica) as much as 20grams that passed the mesh 60 sieve were extracted by the mass method using 96% ethanol finder fluid with a sample ratio and 1: 4. 5 days while occasionally stirring the pulp is then re-macerated until a clear solvent is obtained. Each result of the search was put together and then evaporated with a rotary evaporator.

Antidiabetic Assay:

Selection and Preparation of Test Animals:

The test animals used in this study were male Wistar strain rats that were 8 weeks old and weighed between 150-200g and placed in separate cages according to the test group. Before being treated, the test animals were first adapted in the experimental cages one week before being treated. Experimental animals were fed standard diet pellets and ad libitum drinking water^8,9.

The assay of Antidiabetic Effects:

In this test the test animals were divided into 3 groups for oral glucose tolerance test in mice, where each group consisted of 5 mice. Each group of rats was given glucose dose of 2g/kg BW. Blood sugar levels in all groups of rats were then examined at 0, 30, 60, 90, and 120 minutes⁸. All blood samples were taken by cutting the tip of the rat's tail and his blood sugar level measured by glucometer.

Group 1. Negative Control of Na-CMC

Group 2. Positive Combination Control (Glibenclamide + Metformin) 5mg/500mg

Group 3. Combination of red rice bran and black rice bran ethanol extract (460mg/kg body weight and 200mg/kg body weight orally).

Assay:

In this test the test animals were divided into groups of 3 groups to test dyslipidemia in mice, where each group consisted of 5 mice. Each group of rats was given poloxamer (200mg/200g BW) as an inductor and continued with PTU 100mg as a co-inductor intraperitonially. Blood lipid levels in all groups of rats were then examined at 0, 30, 60, 90, and 120 minutes, on the first day, day 4 (after induction), and day 19 (after the therapeutic process). All blood samples were taken by cutting the tip of the rat's tail and his blood lipid level was measured by a cholesterol test kit.

Group 1. Negative Control (Na-CMC 0.5% w/v)

Group 2. Positive Control (Simvastatin 10mg/kg BW and Gemfibrozil 300mg/kg kg)

Group 3. Combination of red rice bran and black rice bran ethanol extract (460mg/200g body BW and 200mg/200g BW orally)

Data analysis

The results of the study are expressed as mean ± SEM. Data significance was analyzed by One-way Analysis of Variance (ANOVA) (SPSS 20.0 program) with post hoc LSD’s test. Data is considered significant if the p value is ≤ 0,05.

RESULT:

Measurement Results of Blood Sugar Levels in Blood of Rats

Table 1: Results of analysis of measurements of blood sugar levels in mice

Observational result	Treatments	IK 95%
Observational result	Treatments	Mean difference	P
KGDIG	Negative vs positive	-12.600	0.247
	Negative vs extract	1.200	0.910
	Positive vs extract	13.800	0.207
Minute 0	Negative vs positive	9.800	0.247
	Negative vs extract	17.800^*	0.047
	positive vs extract	8.000	0.340
Minute 30	Negative vs positive	23.000^*	0.004
	Negative vs extract	27.200^*	0.001
	Positive vs extract	4.200	0.533
Minute 60	Negative vs positive	42.200^*	0.000
	Negative vs extract	36.800^*	0.001
	Positive vs extract	-5.400	0.509
Minute 90	Negative vs positif	54.200^*	0.000
	Negative vs extract	42.000^*	0.002
	Positive vs extract	-12.200	0.259
Minute 120	Negative vs positif	63.200^*	0.000
	Negativevs extract	44.800^*	0.000
	Positive vs extract	-14.200	0.102

Note * = p < 0,05 = significantly different

Note:

FBGL : Fasting Blood Glucose Level (mg/dL)

GIBG : Glucose Induced of Blood Glucose Level (mg/dL)

Minute 0 : Minute 0 of Blood Glucose Level (mg/dL)

Minute 30 : Minute 30 of Blood Glucose Level (mg/dL)

Minute 60 : Minute 60 of Blood Glucose Level (mg/dL)

Minute 90 : Minute 90 of Blood Glucose Level (mg/dL)

Minute 120 : Minute 120 of Blood Glucose Level (mg/dL)

Table 2: The results of measurements of fasting blood levels and after induction

Treatments	IK 95%
Treatments	Mean difference;	P Value
KDPN vs SIPN	-58.000^*	0.000
KDPP vs SIPP	-50.000^*	0.000
KDPE vs SIPE	-79.800^*	0.000

Note: IK : Confidential interval

KDP : Fasting Blood Glukose Level

SIP : After Poloxamer Induction

KDPN : Fasting blood glucose of negatif

KDPP : Fasting blood glucose of positif

KDPE : Fasting blood glucoseof extract

SIPN : After poloxamer induction of negative

SIPP : After poloxamer induction of positive

SIPE : After poloxamer induction of extract

DISCUSSION:

The first step is to test the normality using the Kolmogorov-Smirnov method of blood sugar level data. If the test results indicate that the data distribution is normal, each test result is shown by the value of p (sig)> 0.05. Then proceed with the one way ANOVA test giving a p value (sig) <0.05 meaning that there is an effect of reducing blood sugar levels to the combination of red rice bran and black rice bran, then Post Hoc Test analysis is done if the one way ANOVA test results reveal significant differences, then the Post Hoc test is continued to find out the meaningful differences in each group.

Data on blood sugar levels after treatment are processed using the Kolmogorov-Smirnov Test method to determine data distribution. The results of data processing showed that the data were normally distributed so that the data processing was continued using one-way analysis of variance (One Way ANOVA) to determine differences between treatment groups. Analysis using one-way variance showed the treatment minutes 0, 30, 60, 90, and 120 with a significance value of p <0.05 which showed a significant difference in blood sugar levels in rats (Rattus norvegicus). After obtaining a significant difference in blood sugar levels in rats (Rattus norvegicus), the statistical test was continued with the Post Hoc test to find out which groups there were significant differences in blood sugar levels.

Based on the research that has been conducted, the results of statistical analysis that has been obtained at minute 0 treatment results reveal that there is no significant difference between the negative and positive control groups and between the extract and positive control treatments with significant values (p> 0.05). The results of ANOVA one-way test for blood sugar levels after induction and after treatment revealed that (P> 0.05), then statistically not significantly different means that there was no significant change between blood sugar after induction and after given treatment. Furthermore, the results of statistical analysis on the minutes 30, 60, 90, and 120 also revealed a significant difference in the negative control group with positive control and between negative control and extract with significant values respectively (p <0.05). Blood sugar levels in the treatment group decreased after being given extract treatment.

This is in line with several other studies which state that the compounds contained in red rice bran and black rice bran extracts have antidiabetic properties. While the results of the analysis in the positive control group compared to the extract treatment revealed no significant difference either at 0, 30, 60, 90 and 120 minutes, with a significant value (p <0.05). This is appropriate because the mechanism of action of positive control and extracts are both able to reduce blood sugar levels.

Data from the One Way ANOVA analysis using one-way variance showed that in minute 0 treatment there was no significant difference with a significant value (p> 0.05). For the treatment at minute 30, 60, 90 and 120 there was a significant difference with the significance value also showing (p <0.05). The data obtained showed that H0 was rejected, meaning that there were significant differences from the rat blood sugar test (Rattus norvegicus). Post Hoc test results measuring blood sugar levels show that there are significant differences in the positive control and extract treatment groups compared to the negative control group. This shows that the combination of red rice bran extract (Oryza nivara) and black rice bran (Oryza sativa L. Indica) has a hypoglycemic effect on blood sugar levels of white rats (Rattus norvergicus) at doses of 460mg/kg BW and 200 mg/kg BW orally Based on the description above, it can be seen that the combination of red bran extract (Oryza nivara) and black rice bran (Oryza sativa L. Indica) at doses of 460mg/kg body weight and 200mg/kg body weight orally can reduce blood sugar levels in rats (Rattus norvegicus) white diabetes-induced wistar strain. The effect of red bran extract (Oryza nivara) in reducing blood sugar levels occurs because the red bran extract contains phenolic bioactive compounds, anthocyanin, β-carotene, tocopherol and oryzanol which can reduce free radicals (ros). black contains components that are useful for the management of diabetes mellitus. Kaneda, et al (2006) report that the main content of black rice bran ethanol extract is anthocyanin which contains the active compound cyanidine-3-glucoside¹⁰. This compound can improve the state of hyperglycemia and insulin sensitivity¹¹. In addition, anthocyanin also acts as an antioxidant that act by protecting the pancreatic beta cells against increased Reactive Oxygen Species (ROS) in hyperglycemia conditions and at the same time reduce oxidative stress in diabetics. So the use of antioxidants can prevent the complications of diabetes mellitus¹².

The positive control group compared to the negative control revealed a significant difference after treatment on the 7th and 14th days at the 0th, 30th, 60th, 90th, and 120th minutes with significant values respectively (p <0.05). While positive control compared to extracts revealed no significant difference which means positive control with extracts because it has the same mechanism so that it gives the same effect in reducing total cholesterol levels whose value is not too significant.

Based on the LSD test results, the results of this study revealed a significant difference between extracts compared to negative controls after treatment at 0, 30, 60, 90 and 120 minutes on the 7^th and 14^th days with significant values (p <0.05). Total cholesterol levels have decreased after being given extract treatment. This proves that the administration of test preparations can cause a decrease in rat cholesterol levels, this occurs because in red rice bran extract there are flavonoid compounds with a mechanism of inhibiting HMG-CoA reductase which acts to synthesize cholesterol. Inhibition of HMG-CoA reductase will reduce cholesterol synthesis in the liver thereby reducing APO B synthesis and increasing LDL receptors on the surface of the liver so that it can reduce total cholesterol, triglyceride and LDL levels of wistar rats. Whereas the administration of black bran extract containing oryzanol and anthocyanin which can reduce levels of total cholesterol, triglycerides, and LDL as well as increase HDL levels in Wistar rats. This is in accordance with research conducted by Hendra et al. In this study using black rice and brown rice which contain compounds as an anti-dyslipidemia effect.

Previous studies also used red rice bran and black rice extract in reducing total cholesterol given in a single form. In that study there was a decrease that revealed the group given red rice bran extract dose of 460mg/200g BW had decreased cholesterol levels from 211.03mg/dl to 137.19mg/dl and those given black rice extract experienced decreased cholesterol levels from 98.95mg / dl to 85.75mg/dl. Based on previous research, the combination of red rice bran extract (Oryza nivara) and black rice bran (Oryza sativa) is also more effective compared to single, although the difference between the two is not too significant, namely a decrease in value from 192.2mg/dl to 100.4mg/dl.

Decrease in total cholesterol levels in this study showed that the content of red rice bran extract (Oryza nivara) and black rice bran (Oryza sativa) has antioxidant activity. Antioxidants are compounds that can inhibit or prevent the process of fat oxidation. When fat oxidation occurs, cholesterol becomes easy to pass through the artery walls and block it. Antioxidants stabilize free radicals by completing the lack of electrons possessed by free radicals and inhibit the chain reaction of free radical formation.

ACKNOWLEDGEMENT:

The authors wish to express gratitude to Kemenristek DIKTI as financial support for implementing this study.

CONFLICT OF INTERESTS:

There are no conflicts of interests.

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Received on 22.11.2019 Modified on 06.01.2020

Research J. Pharm. and Tech. 2020; 13(11):5134-5138.

DOI: 10.5958/0974-360X.2020.00898.7