J. Pure App. Chem. Res.

, 2014,3 (3), 131-137 X

11 November 2014

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’am1,2*

1
Departement of Chemistry, Faculty of Science, Brawijaya University, Malang 65145 Indonesia
2
Veterinery Medicine Faculty, University of Brawijaya, Malang 65145 Indonesia
*
Corresponding author: aulani@ub.ac.id

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

M Area M+G Area M+G+A Area
Molecule M+1
(m/z) % (m/z) % (m/z) %
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
Blood Glucose Levels Blood Glucose Levels
Group After Induction of MLD- After Therapy (mg/dL)
STZ (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
Dose Therapy 750 mg/kgBW 290.0 ± 58.0 101.0 ± 8.00
Dose Therapy 1000 mg/kgBW 234.0 ± 30.0 107.0 ± 7.00

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.
SOD Activity Increase
Group
(unit/mL) 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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