INTRODUCTION:

Diabetes mellitus (DM) is a complication characterized by hyperglycemia associated with abnormalities containing carbohydrates, fats, and proteins associated with decreased insulin secretion or increased insulin sensitivity, or which is related and can also increase microvascular, macrovascular, and neuropathy. Criteria for diagnosis of diabetes mellitus are fasting blood sugar levels ≥ 126 mg / dL or at 2 hours after eating ≥ 200 mg / dL or HbA1c ≥ 8%^1-2.

World Health Organization (WHO)³ report in 2017 shows the number of diabetics in 1980 has increased from 108 million to 422 million in 2014. Diabetes is the leading cause of blindness, kidney failure, heart attack, stroke, amputation of the lower extremities, and death. Nearly half of all deaths are due to high blood glucose levels. WHO projects that diabetes will be the seventh leading cause of death by 2030⁴.

One cause of high glucose levels in people with DM is the oxidation event. Glucose can be oxidized before or after binding to a protein (glycated protein) to produce Reactive Oxygen Species (ROS)⁵. DM sufferers have higher levels of plasma lipid peroxide and Thiobarbituric Acid Reactive Substances (TBARS) compared to normal people. The combination of glycation and glucose oxidation results in the formation of AGEs (Advanced Glycogen End-products). Glycated protein and AGEs modified protein can cause oxidative stress by releasing O₂*, H₂O₂ and toxic carbonyl which can damage the protein. Natural antioxidant compounds are able to control blood glucose levels and prevent complications of diabetes. Antioxidant natural compounds are active compounds of polyphenols in plants which also have hypoglycemic effects^6-7.

Flavonoids are secondary metabolites produced by plants which belongs to a large group of polyphenols. This compound is found in all parts of the plant including leaves, roots, wood, bark, pollen, nectar, flowers, fruit and seeds⁸. Flavonoids has the ability to catch free radicals and inhibits lipid oxidation⁹. The antioxidant activity of the phenol and flavonoid components works by reducing free radicals depending on the number of hydroxy groups in their molecular structure. Red beans (Phaseolus vulgaris L) one type of beans have antioxidant activity¹⁰. Classification of red beans are on Figure I:

Figure 1 Chemical structure of Atenolol and Losartan

Regnum : Plantae

Divisio : Spermatophyta

Subdivisio : Angiosspermae

Class : Dicotyledonae

Ordo : Rosales (Leguminales)

Famili : Leguminosae (Papilionaceae)

Subfamili : Papilionoideae

Genus : Phaseolus

Spesies : Phaseolus vulgaris L.

Based on the results of Suhaling's research (2010)¹¹, the IC50 value of antioxidant activity of methanol extracts of red beans was 164.44ppm. The processed red beans in the form of tempe fermented for 36 hours have antioxidant activity with IC50 values of 90.84ppm. The fermentation process utilizes microbes to produce primary metabolites and secondary metabolites thereby increasing antioxidant activity in red beans^12-13. The selection of tempe as a processed red bean product is based on several research results where tempe is not only good for consumption, has a high nutritional content and this form can increase the antioxidant levels in the form of isoflavones, namely genestein, daizein, and 8 hydroxy daizein, SOD (Super Oxide Dismutase) and vitamin E¹⁴.

This study aims to look at the correlation of flavonoid content as a natural source of antioxidants from red beans that are prepared differently in the form of juice, ethanol extracts, and fermented processed products from red beans, namely tempe, to their diabetes activity in vitro. In vitro antidiabetic activity testing uses the anthrone-sulfate method, where reducing or non-reducing sugars will react with concentrated sulfuric acid to form fulfural or their derivatives that will react with antrone reagents to form a greenish-yellow complex^15-16. Determination of total flavonoid levels using the colorimetric method, where the AlCl₃reagent will react with flavonoids to form complex compounds that are stabilized by CH₃COOK¹⁷. Correlation of flavonoid content correlation with antidiabetic activity in red beans was analyzed by Pearson correlation method from SPSS software program¹⁸.

MATERIAL AND METHODS:

Chemicals and reagents:

red beans (Phaseulus vulgaris L), aquadest, ethanol 70% p.a (Medica), methanol p.a (Bratacem), spiritus, allumunium (III) chloride p.a (Merck), potassium acetate p.a (Merck), concentrated HCl (Merck), Mg metals, NaOH (Merck), quercetin (Aldrich Chemistry), mobile phase (toluene p.a: ethyl acetate p.a: ethanol p.a (3: 3: 0.5), glucose standard (Merck), anthrone powder (Merck), concentrated sulfuric acid (Merck), tempe raprima yeast powder containing Rhizopus oligosporus and Rhizopos orrizae, Mayers, Dragondorff reagents, Ferric chloride (Merck).

Instrumentation:

powder smoothing machines, sieves, juicers, analytical scales (Ohaus, EP 214), engineering scales (Acis BC 500), maceration vessels, rotatory evaporator (IKA HB 10 basic), UV-Vis spectrophotometer (Shimadzu UV mini-1240), cuvette (HELMA), silica GF 254, chamber, glassware (pyrex), porcelain cup, blender, spray bottle, UV-cabinet, spatel, stopwatch.

Taking samples:

Red beans (Phaseulus vulgaris L.) used in this study were obtained from agricultural areas in the Tawangmangu area, Karanganyar and determined at the Center for Research and Development of Medicinal Plants and Traditional Medicines (B2P2TOOT).

Red bean juice:

Dried bean samples blended with 25mL aquadest (1:10) until smooth and homogeneous then filtered.

Red bean ethanol extract:¹⁸

Dried bean samples blended to form a powder and then macerated with 70% ethanol solvent in a ratio of 1:7.5 parts for 3x24 hours. The maceration results are then filtered, the filtrate is accommodated and the residue is macerated again with 70% ethanol solvent in a ratio of 1: 2.5 for 1x24 hours. The collected filtrate is then concentrated with a rotary evaporator at a speed of 200 rpm and at a temperature of 50⁰C to form a thick extract.

Red bean tempe extract:

Tempe red beans sliced ± 1mm thin and then dried in the oven at a temperature of 50⁰C until ready to be pollinated and then blended until smooth. Red bean tempe powder as much as 100grams was extracted with 70% ethanol solvent in a ratio of 1:7.5 parts for 3x24 hours. The maceration results are then filtered, the filtrate is accommodated and the residue is macerated again with 70% ethanol solvent in a ratio of 1:2.5 for 1x24 hours. The collected filtrate was then concentrated with a rotary evaporator at 205 rpm and at a temperature of 50⁰C, evaporation was continued in a water bath until a thick extract was formed.

Qualitative analysis:

Qualitative analysis of flavonoid compounds from red bean ethanol extract, and red bean tempeh ethanol extract were tested by comparison with quercetin, including: TLC analysis, flavonoid qualitative test using dilute NaOH reagent, test the Wilstater Cyanidin method.

Quantitative analysis:

Quantitative analysis of flavonoid compounds were tested by Total flavonoid content. The total flavonoid content (mg/mL) was determined using aluminum chloride (AlCl₃) method. The assay mixture consisting of 250mg of the red bean ethanol extract, red bean juice and red bean tempe ethanol extract dissolved in 25mL distilled water. 1mL is taken, 3mL of 70% ethanol is added, 0.2mL of 10% AlCl₃, 0.2 mL of CH₃COOK 1M, and aquadest is added to 10mL. Dissolve is left in a dark place until the Operating Time is obtained (27th minute), then the absorbance is measured on a UV-Vis spectrophotometer with a maximum wavelength of quercetin (430.0nm), replicated 3 times. Quercetin was used as a standard.

Analysis of the antidiabetic activity of kidney beans and its processed products with parameters for reducing glucose levels:

Red bean ethanol extract, red bean juice and red bean tempe ethanol extract each made in series concentration of 100ppm, 200ppm, 300ppm, 400ppm, 500ppm by piping as much as 1; 2; 3; 4; 5ml of a 1000ppm extract solution is put into a 10.0ml flask, then diluted to the mark. Each series concentration of the sample is shaken until it is homogeneous. Red bean extract sample solution 100ppm, 200ppm, 300ppm, 400ppm, 500ppm pipette 1.0ml then put in a test tube, added with 1.0ml glucose solution with a concentration of 80ppm, then add 5.0ml of anthrone reagent, closed and mixed evenly. After being mixed evenly it is heated in a 100^oC water bath during the obtained operating time (12 minutes). Cool for 1 minute and read the absorbance at the maximum wavelength obtained (628.2nm).

RESULT AND DISCUSSION:

In this study three types of samples made from red beans were used, ranging from juice, red bean extract and red bean tempe extract. The purpose of making these three samples is to look at the form of red bean preparations that are able to provide a maximum flavonoid content so that its use as an antidiabetic is more appropriate in its selection and simplifies its application to the community. This method is very good for the flavonoid content search process because it does not use a heating process and uses relatively simple equipment. Selection of 70% ethanol as a solvent in the extraction process because this solvent has relatively polar properties in accordance with the nature of flavonoids, besides being inert and non-toxic¹². The yield of red bean extract was 15.2%; 14.6% and 15.4% while the yield of red bean tempe extract was 9.7%; 10.1% and 9.9%.

Total Content of Flavonoid Red Bean Juice, Red Bean Extract, Red Bean Tempe Extract:

In a qualitative test through the TLC test with quercetin standards, the sample showed content that had an Rf value parallel to the standard. Flavonoid test using NaOH reagents, the results showed that the three samples contain positive flavonoids, which were yellow because the derivatives of the flavon/flavonol compound would be decomposed by the base to become an acetophenone molecule (breaking bonds in the isoprene structure)^17,19. The flavonoid content of the three samples was also supported by the Cyanidin Wilstater test which showed a reddish color due to the oxidation reduction event between Mg metals in an acid chloride atmosphere with benzopiron nuclei in the flavonoid structure thus forming flavilium salt²⁰.

Scheme I Reaction Flavonoid with NaOH

Scheme II Cyanidin Wilstater test Reaction in Flavonoid

The total content of flavonoids in the three samples used the colorimetric method in which the samples were reacted with AlCl₃ and CH₃COOK. Flavonoids will form a colored complex with AlCl₃. This color complex causes a shift in wavelength towards the visible (visible) marked with a more intensive yellow color stabilized by CH₃COOK^21.

Scheme III Quercetin Reaction with AlCl₃

The total flavonoid content on the Table I of the three samples was different. Red bean juice has a total flavonoid content of 0.21%, red bean extract has a total flavonoid content of 0.54% and the largest total flavonoid content is owned by tempe extract which is 1.39%. Tempe was made using microorganism through the fermentation process. The fermentation process that utilizes microbes can increase the production of primary metabolites and secondary metabolites including flavonoids²².

Table I. Total levels of red bean flavanoids and their processed products

Sampel	Replication	Total flavanoid level	Average content	%KV
Red bean juice	1	0,21%	0,21%	0,57%
	2	0,21%
	3	0,21%
Red bean extract	1	0,54%	0,54%	0,64%
	2	0,54%
	3	0,53%
Tempe red bean extract	1	1,39%	1,39%	0,13%
	2	1,39%
	3	1,39%

Antidiabetic Activity of Red Bean Juice, Red Bean Extract, Red Bean Tempe Extract:

In vitro antidiabetic activity testing uses the antrorone-sulfate method, where reducing or non-reducing sugars will react with concentrated sulfuric acid to form fulfural or their derivatives that will react with antrone reagents to form a greenish-yellow complex²³. Antidiabetic activity data showed by looking at the ability to reduce glucose by 50% (EC50) after giving treatment from the three samples are on Table II.

Table II. The value of antidiabetic activity of red beans and its processed products

Sample	Replication	EC50 (ppm)	Average EC50 levels (ppm)	%KV
Red bean juice	1	455,4029	458,0415	0,57%
	2	458,0683
	3	460,6532
Red bean extract	1	436,3788	435,1279	0,27%
	2	434,1105
	3	434,8945
Red bean tempe extract	1	107,4451	108,0131	0,74%
	2	107,6641
	3	108,9300

Correlation of Total Flavonoid Levels with Antidiabetic Activity of Red Bean Juice, Red Bean Extract, Red Bean Tempe Extract:

This study aims to look at the correlation of the total content of flavonoids as a source of natural antioxidants from red beans that are prepared differently in the form of juice, ethanol extracts, and fermented processed products from red beans, namely tempe, to their diabetes activity in vitro. The selection of tempe as a processed red bean product is based on several research results where tempe is not only good for consumption, has a high nutritional content and this form can increase the antioxidant levels in the form of isoflavones, namely genestein, daizein, and 8 hydroxy daizein; SOD (Super Oxide Dismutase) and vitamin E²⁴. The correlation of total flavonoid levels with antidiabetic activity of red bean products and their processed products are on Table III.

Table III. Total flavonoid levels and antidiabetic activity of kidney beans and their processed products

Sample	Average total flavonoid levels	Antidiabetic activity (EC50) in ppm
Red bean juice	0,21%	458,0415
Red bean extract	0,54%	435,1279
Tempe red bean extract	1,39%	108,0131

CONCLUSION:

Our study showed that The total content of flavonoids in red beans and their processing products is different. Red bean juice has a total flavonoid content of 0.21%, red bean extract has a total flavonoid content of 0.54% and the largest total flavonoid content is owned by tempe extract which is 1.39%. The value of antidiabetic activity in EC50 produced by these three samples is different where red bean juice is 458.0415ppm, red bean extract is 435.1279ppm, and red bean tempe extract is 108.0131 ppm. There is a significant correlation P<0,001 (p <0.05) between flavonoid content and antidiabetic activity in red beans (Phaseulus vulgaris L.) and its processed products.

ACKNOWLEDGEMENT:

The author acknowledges to Lembaga Penelitian dan Pengabdian Masyarakat (LPPM) Nasional College of Health Science through the research scheme of Penelitian Dosen Internal year 2019.

CONFLICT OF INTEREST:

The authors have declared “no conflicts of interest with respect to the research, authorship, and/or publication of this article”.

REFERENCES:

1. American Diabetes Association. Diagnosis and Classification of Diabetes Mellitus, Diabetes Care, 2010; 33(1).

2. Rupesh Gautam, S.C. Sharma. Effect of Punica granatum Linn. (Peel) on Blood Glucose Level in Normal and Alloxan- Induced Diabetic Rats. Research J. Pharm. and Tech. 5(2): Feb. 2012; Page 226-227.

3. World Health Organization (WHO), Definition, Diagnosis and classification of diabetes mellitus and its complications, Part 1: Diagnosis and classifications of diabetes mellitus, Geneva: Department of Non-communicable Disease Surveillance; 1999.

4. Shom Prakash Kushwaha, Sunil Kumar Rawat, Pavan Kumar, Abhishek, Kishu Tripathi. Coupling Antioxidant and Antidiabetic assets of 2, 4-Thiazolidinedione Derivatives. Asian J. Pharm. Ana. 1(4): Oct.-Dec. 2011; Page 71-73.

5. Preeti K. Suresh, Abhishek K. Sah, Sanjay J. Daharwal. Role of free radicals in ocular diseases: An overview. Research J. Pharm. and Tech. 7(11): Nov. 2014 Page 1330-1344.

6. Sadowska-Bartosz I and Bartosz G. Prevention of Protein Glycation by Natural Compound, Molecules, 2015; 20: 3309-3334.

7. Shom Prakash Kushwaha, Sunil Kumar Rawat, Pavan Kumar, Abhishek, Kishu Tripathi. Coupling Antioxidant and Antidiabetic assets of 2, 4-Thiazolidinedione Derivatives. Asian J. Pharm. Ana. 1(4): Oct. - Dec. 2011; Page 71-73.

8. Preeti Tiwari, Rakesh K. Patel. Estimation of Total Phenolics and Flavonoids and Antioxidant Potential of Ashwagandharishta Prepared by Traditional and Modern Methods. Asian J. Pharm. Ana. 3(4): Oct. - Dec. 2013; Page 147-152.

9. Kumar S and Pandey AK. Chemistry and Biological Activities of Flavonoids: An Overview. The Sci. World J., 2013; 16.

10. Krishna Kondragunta. V, Karuppuraj. V, Perumal. K. Antioxidant activity and Folic acid content in indigenous isolates of Ganoderma lucidum. Asian J. Pharm. Ana. 2016; 6(4): 213-215.

11. Suhaling, S., 2010, Uji Aktivitas Antioksidan Ekstrak Metanol Kacang Merah (Phaseolus vulgaris L.) dengan Metode DPPH, Skripsi, Fakultas Ilmu Kesehatan, Universitas Islam Negeri Alaudin, Makasar.

12. Racio IL, Elena P, Torinus MI, Martinez-Villaluenga C, Duenas M, Frias J. Fermentation Enhances the Content of Bioactive Compounds in Kidney Bean Extracts, Food Chem., 2015; 343-352.

13. Mahesh S. Patil, Shrikant R. Kulkarni. Studies on Ethanol Production from the Fruit Waste Using Ultrasound Assisted Fermentation. Asian J. Research Chem. 4(3): March 2011; Page 429-433.

14. Astuti M, Meliala A, Dalais FS, Wahlqvist ML. Tempe, a Nutritious and Healthy Food from Indonesia, Asia Pacific J. Clin. Nutr., 2000; 9(4): 322-325.

15. M Sugumaran, T Vetrichelvan, S Darlin Quine. Antidiabetic potential of aqueous and alcoholic leaf extracts of Pithecellobium dulce. Asian J. Research Chem. 2(1): Jan.-March, 2009; Page 83-85.

16. Mr. Israel Jeba Prabu, Mrs. Janet Anbumani. A Study to Assess the effectiveness of Video Assisted Teaching Programme on the level of knowledge regarding Blood Donation among GNM 1st year students of AMT School, Jammu. Int. J. of Advances in Nur. Management. 2020; 8(2):127-132.

17. Al-kayyis HK and Susanti H. Perbandingan Metode Somogyi-Nelson dan Antrone-Sulfat pada Penetapan Kadar Gula Pereduksi dalam Umbi Cilembu (Ipomoea batatas L.), Jurnal Farmasi Sains dan Komunitas, 2016; 13:2.

18. Chang CC, Yang MH, Wen HM, Chern JC. Estimation of Total Flavonoid Content in Propolis by Two Complementary Colorimetric Methods, J. Food Drug Anal, 2002; 10.

19. Rini RTP. 2015, Uji aktivitas Antioksidan Kombinasi Ekstrak Etanol Kayi Secang (Caesalpinia sappon L.) dan Kacang Merah (Phaseolus vulgaris L.) dengan Menggunakan Metode DPPH (1,1-Diphenyl-2-picrylhydrazyl), Skripsi, Fakultas Matematika dan Ilmu Pengetahuan Alam, Universitas Sebelas Maret, Surakarta.

20. Zhang Q, Lin LG, Ye WC. Techniques for Extraction and Isolation of Natural Products: a Comprehensive Review, Chin. Med, 2018; 13:20

21. Eirene GF, Matheis FJDP, Tanasale, Jolantje L, Dominggus M, Regy T. Phytochemical Screening of Water Extract of Gayam (Inocarpus edulis) Bark and Its Amylase Inhibitor Activity Assay, IOP Conf Ser: Mater Sci Eng, 2019; 509 012074.

22. Marliana SD, Suryanti V, Suyono. Skrining Fitokimia dan Analisis Kromatografi Lapis Tipis Komponen Kimia Buah Labu Siam (Sechium edule Jacq. Swartz) dalam Ekstrak Etanol, Jurnal Biofarmasi, 2005; 3(1): 26-31.

23. Malgorzata W, Joanna H, Konrad PM. Effect of Solid-State Fermentation with Rhizopus oligosporus on Bioactive Compound and Antioxidant Capacity of Raw and Roasted Buckwheat Groats. Ital. J. Food Sci, 2015; 27.

24. Soares LAL, Bassani VL, Ortega GG, Petrovick PR. Total Flavonoid Determination for The Quality Control of Aqueous Extractive from Phyllanthus Niruri L, Lat. Am. J. Pharm, 2003; 22(3): 203-7.

Received on 12.05.2020 Modified on 01.06.2020

Research J. Pharm. and Tech. 2021; 14(3):1293-1297.

DOI: 10.5958/0974-360X.2021.00229.8