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The Influence Water Extract of Black Soybean (Glycine max (L.) Merr.) on Reducing of Blood Glucose Level and The Superoxide

Dismutase (SOD) Activity on Diabetes Mellitus Rats Induced With Multiple Low Dose of Streptozotocin (MLD-STZ)

Luh Putu Gina¹, Chanif Mahdi¹, Aulanni’am^1,2*

1Departement of Chemistry, Faculty of Science, Brawijaya University, Malang 65145 Indonesia

2Veterinery Medicine Faculty, University of Brawijaya, Malang 65145 Indonesia

*Corresponding author: [email protected]

Received 8 May 2014; Revised 5 September 2014; Accepted 11 November 2014

ABSTRACT

Black soybean (Glycine max ( L. ) Merr.) is a tropical plant in spread widely in South East Asia including Indonesia. It contains anthocyanins and isoflavones which acts commonly as natural antioxidants. This paper discloses recent investigation on the benefits of its water extract on the oxidative stress in diabetes rat model exposed with multiple low dose of streptozotocin (MLD-STZ). It will also be reported the effect on increasing of the superoxide dismutase (SOD) activity and lowering of blood glucose levels. Groups of male Rattus novergicus strain Winstar was applied as animal model.

Key word: Black Soybean, Diabetes Mellitus, Antioxidants, MLD-STZ, Pancreatic beta cells, SOD, Blood Glucose

INTRODUCTION

Diabetes mellitus (DM) in Indonesia was commonly known as diabetes. DM has become a chronical disease due to inability of the body producing insulin. DM was also caused by ineffective metabolism of insulin, which is lead to increasing of glucose levels in the bloods [1]. The untreated of human with DM can lead to complications and spread of the serious diseases to other organs or tissues. Thus, it is very important to prevent DM. Insuline is generated in pancreas. Reduction of insulin metabolism in abnormal pancreas is one of the main factors of the rising of blood sugar. This is an important early sign for DM, and it was known as hyperglycemia. Hyperglycemia can also result the increase of free radicals in the cell. Disproportion concentration between a protective antioxidants and free radicals in the body is the beginning of the oxidative damage. It is also known as oxidative stress [2]. People with DM, and do not get proper treatment will have serious complications of disease. It can injure the other organs or tissues in the body.

Substances which are able to inhibit oxidation process known as antioxidant. It derived naturally from outside the body (exogeneous antioxidant) and also known as natural antioxidant. It can be isolated from plants, animal, fungi, and microbial. Moreover, antioxidant generated from inside the body, known as endogeneous antioxidant. Endogenous antioxidant derives from the body itself. It dominantly consists of superoxide dismutase (SOD), glutathione peroxidase, and catalase. Exogenous antioxidants are obtained from outside through food we eat to help the body counter excess free radicals. Free radicals are

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reduced the SOD activity and lead to oxidative stress. The inactivation of SOD can be prevented, and SOD activity ca be increases by addition of exogenous antioxidant [3].

Anthocyanin and isoflavone belong to secondary metabolite of flavonoids. Flavonoids are mostly polyphenols and found in the human diet. Polyphenolic compounds have been known as antioxidant phytochemicals due to their unique structure. It function as free radical scavenger, reducing the formation of hydroperoxide, and reducing the production of reactive oxygen species (ROS) [4]. Black soy contains isoflavone and anthocyanin. Both can be applied as natural antioxidants for the body [5]. Anthocyanin itself was reported have antioxidant capabilities. It had a high reactivity as hydrogen or electron donor, and derived radical to stabilize and delocalize the unpaired electrons [6]. This paper reports the effect of water extract from Indonesia black soybean in oxidative stress. The diabetes animal model was used by exposing it with multiple low dose of streptozotocin (MLD-STZ). The parameter investigated is on superoxide dismutase (SOD) and blood glucose levels.

EXPERIMENT

Chemicals and instrumentation

The chemicals used in this study mostly from Merck and was used as bought from manufacturer. Water extract of black soybean, streptozotocin (STZ), citrate Buffer pH 4.5, sodium chloride 0.9%, phosphate buffer saline (PBS), paraformaldehyde (PFA) 4% formalin, 70% ethanol 80% ethanol and 95% ethanol, distilled water, tris-HCl, PBS-Tween, and PMSF.

This study used male rats (Rattus norvegicus) Wistar strain, body weight 200 gram and kept in a box measuring 20x30x40 cm cages.

The tools applied is digital glucometer (EasyTouch GCU) to check the blood glucose levels of experimental animals, SOD kit (Cal. K335-100, BioVision), reaction tube, vortex, scissors, tweezers, pasteur pipette, glass objects, spatula, miter chrom knife, analytical balance, scalpel, surgical instruments, clamp (block holder), incubator, hot plate, water bath, gloves, light microscopes, centrifuges, and mortar.

Preparation water extract from black soybean

A dried black soybean (Glycine max (L.) Merr.) was bought from local traditional market in Malang Indonesia was weighed (1000 mg). It was added 100 mL of distilled water, and heated at 80 °C for 20-60 min. The liquid part was separated to obtain total extract volume in 10 mL.

Phytochemicals test

Phytochemical test was conducted to determine the presence of anthocyanin compounds in water extracts of black soybeans. Water extract of black soybean (3 mL) was added 3 mL of solution hydrochlori acid 2 M. This mixture was to 100 °C for 5 min, and the mixture color turned to red. Then, it was added dropwise solution of sodium hydroxide 2 M until green solution was formed. This color indicates the presence of anthocyanin [7].

LC-MS Analysis

The compounds compose in water extract of black soybean (Glycine max (L.) Merr.) were analyzed and identified qualitatively toward their flavonoid contents using liquid chromatography mass spectrometry (LC-MS).

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Preparation of experiment animals

Experiment using animal model was conducted following the ethical protocol using animal model for research from Ethical Clearance Commition from University of Brawijaya.

Treatment The animal model was prepared in big groups of two month old of Rattus novergicus species rat, weigh 200 gram was adapted for 1 week. The healthy rats (negative control) were given food and a distilled water every day. Diabetic rats (positive control) were given food and water contains MLD-STZ with dosage level 20 mg/kg body weiigh (BW) five times each day for 5 days. All the animal was monitorred their blood glucose before and after induction of MLD-STZ using glucometer. Groups of rats were considered as positive control for diabetes if blood glucose levels were over 200 mg/dL.

Therapy rats were group of diabetic rats resulted from the induced MLD-STZ 20 mg/kg BW dose five times for five consecutive days. Then they were treated with water extract of black soybeans for 14 days using different dosage level such as 500 mg/kg BW, 750 mg/kg BW and 1000 mg/kg BW. The blood glucose level measurements were performed after induction of MLD-STZ and after treatment for 14 days using glucometer. It was to determine glucose levels before and after the treatment of black soybean water extract. At the end, all model rats were sacrificed by neck dislocation way, pancreas dissected and pancreatic organs were taken for SOD analysis.

Measurement of blood glucose

Blood of the model rat was taken before the MLD-STZ injection to determine the initial blood glucose levels in the pre-exposed to DM. Blood glucose levels were checked using a digital glucometer. Stick glucometer and digital glucometer were prepared in accordance with the instructions for use. Blood drip from the tail to stick glucometer and awaited the results shown on screen digital glucometer. The processes of checking glucose levels were done every survey of glucose was conducted before and after MLD-STZ injection (rats blood glucose levels exceeding 200 mg/dL expressed DMT 1), and after treatment with water extract of black soybean.

Therapy extract water black soybean (Glycine max ( L. ) Merr .)

Diabetic rats were treated with aqueous extract of black soybean orally (sonde). Rats were divided into 3 treatment groups: group 1 at a dose of 500 mg/kg BW, group 2 at a dose of 750 mg/kg BW and group 3 with a dose of 1000 mg/kg BW. Each group was treated for 2 weeks and then dissected.

Table 1. Mixure of solutions were added in sample, blank 1, blank 2 and blank 3.

Sample Blank 1 Blank 2 Blank 3

Sample 20 µL - 20 µL -

Aquadest - 20 µL - 20 µL

WST solution 200 µL 200 µL 200 µL 200 µL

Enzyme solution 20 µL 20 µL - -

Buffer - - 20 µL 20 µL

Determination of superoxide dismutase activity (SOD)

Determination of SOD activities on sample was performed using a kit Cal. K335-100 from BioVision. Each sample has 3 blank solution (as it in Table 1). Pancreatic extract (20 mL) was added into the sample wells and blank wells 2. Meanwhile for blank wells 1 and 3

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was added with 20 mL of distilled water. At the same time, 200 mL working solution was added further each with 20 mL buffer solution to blank wells 2 and 3. The 20 mL of enzyme solution was added to each of the wells and were homogenized further. It was performed by wrapping the microplate using allumunium foil and shaking them. These mixtures then were incubated at 37 °C for 20 min, and the absorbance was measured at a wavelength of 450 nm suing UV-Vis spectrophotometer.

RESULT AND DISCUSSION

Phytochemicals and LC-MS analysis of water extract black soybean

Analysis of flavonid contents in water extract gave a positive indication for the presence of anthocyanin. Further analysis was undertaken using mass spectrometry detection (MS). Prior detection, the componens cotains in the water extract of black soybean was separated in column of liquid chromatography (LC). LC gave chromatogram data which correlated to quantity of each anthocyanin component contain in water extract. Meanwhile the MS data provided meolecular weight of each separated substances and also clarify the molecule of anthocyanin. The analysis result was summarised in Table 2.

Table 2. Component identified contains on black soybean water extract

Molecule M

(m/z)

Area

% M+1 M+G

(m/z)

Area

%

M+G+A (m/z)

Area

% Genistein 270.24 14.0 271.24 432.24 1.00 474.24 8.60

Daidzein 254.23 8.20 255.23 416.23 7.30 458.23 4.00

Glycitein 284.26 1.60 289.26 446.26 1.40 488.26 11.1 Delphinidin-3-glucoside 338.80 29.6 339.80 500.8 6.80 542.80 4.60 Cyanidin-3-glucoside 322.80 2.50 323.80 484.8 7.80 526.80 2.30 Petunidin-3-glucoside 317.41 4.00 318.41 479.41 11.3 521.41 13.0

Note: M : molecular weight of aglycone; M+G : molecular weight plus glycoside; M+G+A : molecular weight plus acylated glycoside

The analysis results using LCMS toward water extract of black soybean (Glycine max (L.) Merr.) indicated isoflavone compounds (genestein 8.60%, 4.00% daidzein, and glycitein 11.1%) and anthocyanin compounds (delphinidin-3-glucoside 4.60%, cyanidin-3-glucoside and 2.30% petunidin-3-glucoside 13.0%). All these detected compounds was derivation of flavonoids (isoflavones and anthocyanins), and most research result was reported that they had antioxidant activity. The ability of flavonoids as antioxidants due to its structures acts as radical scavenger. The mechanisme involved ability if the flavonoid structure delocalising electron along to the molecule. In addition, the presence of a hydroxy group in position 3',4' (ortho-hydroxy) in B-ring coupled with double bond conjugation with the 2,3,4-oxo group (1,4-piron group) in the C-ring and the hydroxyl group in position 5-OH on A-ring was improved the scaveging activity toward the aromatic ring [8].

Reducing glucose level activity of water extract black soybean on DM rats

Groups of Rattus novergicus Winstar strain used as animal model for diabetes mellitus was treated using streptozotocin (STZ). This research applied multiple low dose prescription of STZ (MLD-STZ). Treatment was undergone for 5 consecutive days. Three variaton prescription doses of water extract black soybean was is applied in order to determine the optimum results activity on DM rats. The affect of this treatment was

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determined by comparing sugar level before and after theraphy. Detail of the result was presented in Table 3.

Table 3. The average of blood glucose levels on groups of healthy rats, diabetic rats and rats after got treatment with water extract of black soybean

Group

Blood Glucose Levels After Induction of MLD-

STZ (mg/dL)

Blood Glucose Levels After Therapy (mg/dL)

Healthy rats (negatif) 98.00 ± 7.00 98.00 ± 7.00

Diabetic rats (positif) 293.0 ± 93.0 293.0 ± 93.0

Dose Therapy 500 mg/kgBW 325.0 ± 45.0 305.0 ± 77.0

The results showed water extract of black soybean injected on DM rats significantly decrease glucose concentration of DM rats. Normal or healthy rat has glucose level 98.00 ± 7.00 mg/dL. This concentration was not changed even though during the course. But, for sick rat where it got treatment with STZ had higher glucose concentration (293.0 ± 93.0 mg/dL).

It was almost three fold higher glucose level than that in norma rats. Promisingly, the result gave important data when lowering glucose level was observed for theraphy using water extract of black soybean with dosage of 750 and 1000 mg/kg BW. These result closes to the gluocose level for healthy rats. This fact summarise that theraphy of black soybean water extract cures the DM rats model. However, for treatment using 500 mg/kg BW dosage did not give any significant result.

Theoretically, mechanism for reducing glucose occurs due to insulin secretion. The decrease of blood glucose levels after treatment with extract of black soybean possibly can improve pancreatic tissues to increase insulin secretion. This secretion occurs in the presence of GLUT-2 (glucose transporter) in pancreatic-β-cells in charge for carrying glucose from the blood into the cells. Glucose enters pancreatic-β-cells to further process for glycolysis and phosporilation produce adenosine triphospat (ATP). The resulted ATP is required to activate the closing of kalium-channels in pancreatic-β-cells membrane. This wa followed by opening of calsium-channels. The opening of Ca-channels directs calsium ions enter the cell and increase concentration of calsium ions in cell. A high concentration of calsium in cell are required by pancreatic-β-cells to secrete insulin [9].

Improvement of superoxide dismutase (SOD) activity of DM rats with water extract of black soybeans

The activity of SOD was derived directly using enzyme isolated from pancreatic organ of groups animal model. Analysis and procedure undertaken following ethical protocol using animal as research model. In this study, the water extract of black soybean is exogenous antioxidant required to cure the work of enzyme SOD to overcome free radicals after MLD-STZ induction. The higher SOD activity indicates the more superoxide can be neutralized by exogenous antioxidants. Sumarry of SOD measurement in the rat pancreas are presented in Table 4.

In general, healthy rat as reference has normal SOD activity. It was recorded in 83.16

± 2.90 unit/mL. However, for unhealthy rats such on DM rat has SOD value 50.80 ± 3.08 unit/mL. The research discovered that theraphy using water extract of black soybean

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significantly improve the SOD activity in pancreatic cell or DM rats. Increasing of theraphy dosages improved the SOD activity. Using prescription 500 mg/kg BW of rats improve SOD activity to 63.64 ± 2.44 unit/mL. It improved to 74.06 ± 1.84 unit/mL using 1000 mg/kg BW dosage. This significant improvement was also cofirmed with statistical analysis with significant level (p< 0.01).

Table 4. Activity of superoxide dismutase (SOD) pancreatic healthy rats, diabetic rats and therapy rats treated black soybean water extract.

Group SOD Activity (unit/mL)

Increase SOD Activity (%)*

Healthy rats (negative control) 83.16 ± 2.90 - Diabetic rats (positive control) 50.80 ± 3.08 -

Therapy 500 mg/kg BW 63.64 ± 2.44 25.28

Therapy 750 mg/kg BW 68.18 ± 1.22 34.21

Therapy 1000 mg/kg BW 74.06 ± 1.84 45.79

Note: *Percent increase of SOD activity derived from a comparison of therapy with an average SOD activity in diabetic rats.

Superoxide dismutase is one of the enzymatic defense systems in body when free radicals presence. This system prevents cellular DNA damage due to reaction with free radicals. It has function to neutralize free radicals such as hydrogen peroxide (H₂O₂) and radical oxygen that will be converted into H2O by gluthathion peroxidase (GSH). Enzymatic defense systems in body will decrease if there is immbalance condition between the amount of free radicals and endogenous antioxidants. Exogenous antioxidants was injected or consumed in daily food intake probably could serve the equilibria in body. Flavonoids contained in water extract of black soybean significantly can act as exogeneous antioxidants that reduce and prevent the occurrence of a chain reaction of free radicals leading to tissue damage [10].

CONCLUSION

The water extract of black soybean (Glycinemax (L.) Merr.) in doses variations 500;

750 and 1000 mg/kg of rat BW for 14 days shows important activity by increase SOD activity. The optimum therapeutic dose of aqueous extract of black soybean to increase SOD activity was 1000 mg/kg BW. Moreover, water extract of black soybeans also has activity reduing in blood glucose levels of diabetic rats.

ACKNOWLEDGMENT

The authors would thank to Drs. I Made Sujana, M.Sc, English Department, School of Education, Mataram University, Lombok, NTB for kindly reading this article.

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