Antioxidant and Hepatoprotective E Ect of Aqueous Extract of Germinated and Fermented Mung Bean On Ethanol-Mediated Liver Damage

Hindawi Publishing Corporation
BioMed Research International

Volume 2013, Article ID 693613, 9 pages
http://dx.doi.org/10.1155/2013/693613
Research Article
Antioxidant and Hepatoprotective Effect of
Aqueous Extract of Germinated and Fermented Mung Bean on
Ethanol-Mediated Liver Damage
Norlaily Mohd Ali,1 Hamidah Mohd Yusof,1 Kamariah Long,2

Swee Keong Yeap,3 Wan Yong Ho,1 Boon Kee Beh,4 Soo Peng Koh,2
Mohd Puad Abdullah,1 and Noorjahan Banu Alitheen1
1
Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences,
University Putra Malaysia, 43400 Serdang, Selangor, Malaysia
2
Department of Bioprocess Biotechnology, Malaysian Agriculture Research Development Institute,
43400 Serdang, Selangor, Malaysia
3
Institute of Bioscience, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia
4
Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences,
University Putra Malaysia, 43400 Serdang, Selangor, Malaysia
Correspondence should be addressed to Noorjahan Banu Alitheen; noorjahan@biotech.upm.edu.my
Received 30 August 2012; Revised 25 October 2012; Accepted 26 October 2012
Academic Editor: Andre Van Wijnen
Copyright © 2013 Norlaily Mohd Ali et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Mung bean is a hepatoprotective agent in dietary supplements. Fermentation and germination processes are well recognized to
enhance the nutritional values especially the concentration of active compounds such as amino acids and GABA of various foods.
In this study, antioxidant and hepatoprotective effects of freeze-dried mung bean and amino-acid- and GABA-enriched germinated
and fermented mung bean aqueous extracts were compared. Liver superoxide dismutase (SOD), malondialdehyde (MDA), ferric
reducing antioxidant power (FRAP), nitric oxide (NO) levels, and serum biochemical pro�le such as aspartate transaminase (AST),
alanine transaminase (ALT), triglycerides (TG), and cholesterol and histopathological changes were examined for the antioxidant
and hepatoprotective effects of these treatments. Germinated and fermented mung bean have recorded an increase of 27.9 and
7.3 times of GABA and 8.7 and 13.2 times of amino acid improvement, respectively, as compared to normal mung bean. Besides,
improvement of antioxidant levels, serum markers, and NO level associated with better histopathological evaluation indicated that
these extracts could promote effective recovery from hepatocyte damage. ese results suggested that freeze-dried, germinated, and
fermented mung bean aqueous extracts enriched with amino acids and GABA possessed better hepatoprotective effect as compared
to normal mung bean.
1. Introduction comparable to development of chronic hepatic disease in

humans. Upon stimulation from various hepatotoxins, Kupf-
Liver is a pivotal in�ammatory organ that, involved in meta- fer cells which release proin�ammatory mediators such as
bolism, storage, and excretion of metabolites. ere are con- NO and Interferon-gamma (IFN-𝛾𝛾) will eventually result
siderable numbers of hepatotoxins that have been reported in accumulation of reactive nitrogen species (ROS). ROS
to cause a liver damage such as ethanol, paracetamol, and has been shown to cause lipid peroxidation and membrane
carbon tetrachloride [1–5]. e mice model of liver injuries degradation which will generate liver damage and in�am-
induced by various hepatotoxins showed similar trend mation [1, 3, 5, 6]. Natural sources of antioxidantssuch as
but with slight variations such as increased membrane per- green tea has been reported to increase the level of SOD and
meability, lipid peroxidation, and cell death which was FRAP in cytoplasm of rat’s liver which reverted the injury
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2 BioMed Research International
effect close to normal [7]. Polyphenols, �avonoids, and antho- Invitrogen (USA). e Rhizopus sp. strain of 5351 inocu-
cyanins have been suggested to exert strong antioxidant acti- lums was obtained from MARDI’s (Malaysian Agricultural
vity, which contribute to the protective effect of against liver Research and Development Institute) culture collection cen-
injury in rats [4, 7]. ter. Milk thistle extracts containing 80% of silybin was
Mung bean (Vignaradiata), which is mainly cultivated obtained from Lipa Pharmaceutical Pty. Ltd. (Australia).
in East Asia and South Asia regions contains rich source of
protein, essential amino acids, minerals, vitamins, and �bers. 2.2. Animals. Male Balb/c mice of 8–10 weeks old weigh-
It has been renowned of having multinutritional values as ing 20–25 g were maintained under standard condition of
well as medicinal properties. Earlier studies have demon- temperature (22 ± 5∘ C) and humidity in animal house with
strated that mung bean can act as an antioxidant [2, 8], liver 12 h of light/dark cycle. Animals were provided with food
protection [9] and antidiabetic agent due to its low glycemic and water ad libitum. Experiments were strictly conducted
index [10, 11]. Recently, it was reported that the ethanolic and approved by Animal Care and Use Committee, Universiti
extract of mung bean exhibits antiin�ammatory response by Putra Malaysia, (Ref: UPM/FPV/PS/3.2.1.551/AUP-R2).
decreasing the proin�ammatory cytokines in mouse macro-
phages [5, 12]. Germination and fermentation have been
well associated with the elevated amount of antioxidants and 2.3. Plant Material. Mung bean (Vignaradiata) seeds were
GABA content [13–16]. GABA (𝛾𝛾-amino butyric acid) is a purchased from the local store in Selangor. e mung bean
nonprotein amino acids that acts as a neuron inhibitors in seeds were allowed to undergo solid-state fermentation base
mammals, which can be extracted from plant. Numerous on our previous method [20] and germination process prior
studies have proclaimed that the roles of GABA as an anti- to extraction. For fermented mung bean [20], about 1000 g
hypertension, anticancer, and antiin�ammatory agents and of dehulled mung bean seeds were soaked in cold water at
its other healthful bene�ts [17–19]. ese aspects have stim- room temperature for 18 h. Soaked mung beans were washed
ulated the interest to generate GABA-enriched natural prod- thoroughly and steamed for 40 minutes. Aer that, steamed
ucts. Fermented soybean product, GABA-Tempeh, is a tradi- seeds were cooled to room temperature and subsequently
tional food that contains abundance of oligopeptides and free mixed with Rhizopus sp. strain of 5351 inoculums. From our
amino acids (mainly GABA) which contribute to lower level previous preliminary studies (data not shown), mung bean
of cholesterol in plasma [18]. is effect can be correlated seeds were screened with different Rhizopus sp. strains (5346,
with liver function as lipid metabolism in body. erefore, 5347, 5351, 5375, 5376, 5377, 5408, and 5410). e results
by undergoing germination and fermentation processes, revealed that Rhizopus sp. strain 5351 yielded the highest
freeze-dried mung bean aqueous extracts could contribute total amino acids and GABA content in fermented mung
to liver protective effect and other healthful bene�ts. �u bean aer 48 h of incubation at 30∘ C. Following this, the
et al. [9] were the �rst to report the hepatoprotective effects hepatoprotective effects of fermented mung bean inoculated
of mung bean. eir study compared the histological and with 5351 strain were evaluated. e inoculated beans were
biochemistry changes of acetaminophen-induced liver injury then packed into perforated plastics and incubated for 48 h
and the ameliorate properties of different Taiwan’s legumes at 30∘ C. Finally, all fermented mung bean seeds were dried
such as adzuki bean, black bean, rice bean, and mung bean. and ground into powder prior to water extraction. On the
Mung bean aqueous extract was identi�ed to exhibit the best other hand, germinated mung bean seeds was prepared by
hepatoprotective effects among the legumes against liver germinating the mung beans seeds inside the container Anae-
injury agent, acetaminophen. rocult A supplied with CO2 gas for up to 72 h. Germinated
To date, no in vivo test has been conducted to assess seeds were then allowed to dry until constant moisture
the effect of freeze-driedgerminated and fermented mung content was obtained and ground into powder prior to water
bean aqueous extracts on animal model. e purposes of extraction. For control, mung bean seeds were directly
this study wereto compare the in vivo antioxidant enzymes ground into powder without prior fermentation or germina-
content and hepatoprotective effects of freeze-dried normal tion.
mung bean, nutrient-enriched germinated, and fermented Finely ground powder was then extracted using deionised
mung bean aqueous extracts on ethanol-induced liver dam- water (1 : 20 ratio) at 25∘ C for 30 minutes and placed in
age mice model. is study also aimed to establish the an incubator shaker at 300 rpm for 30 minutes under room
correlation between the effects of fermentation and germina- temperature. Mixture was then centrifuged for 5 minutes at
tion on amino acids and GABA level of mung bean and the 10,000 rpm andthe supernatant was collected. Supernatants
hepatoprotective properties of the extracts. were furthersubjected to freeze-dry at operating temperature
of −50∘ C (yield 25%, w/w). e freeze-dried powder was
stored at 4∘ C. e assays were performed according to [20–
2. Materials and Methods 22] with slight modi�cations.
2.1. Materials. Hypoxanthine, xanthine oxidase, superox- 2.3.1. GABA and Amino Acids Determination. e freeze-
ide dismutase, Folin-Ciocalteu reagent, aluminium chloride, dried powder was dissolved in distilled water and �ltered
sodium nitrate, ascorbic acid, and gallic acid were purchased through 0.2 𝜇𝜇m syringe �lter prior to UPLC analysis. e
from Sigma-Aldrich (USA). All solvents used were either of derivatization process was done by mixing 70 𝜇𝜇L of AccQ-
analytical reagent or HPLC grade. Griess reagent was from Tag Ultra borate buffer with 10 𝜇𝜇L of �ltered extracts solution,
BioMed Research International 3
followed by adding 20 𝜇𝜇L of AccQ Fluor reagent in 1.5 mL Group 9. High dose treated group, mice (p.o.) with 100 𝜇𝜇L of
eppendorf tube. All analyses were performed on a Waters 50% (v/v) of ethanol for 7 days followed by 14 days of 100 𝜇𝜇L
Acquity UPLC system, comprised of a binary solvent man- of fermented mung bean extract (1000 mg/kg).
ager, a sample manager �tted with 2 𝜇𝜇L sample loop and
UV-PDA detector set at 260 nm. e data were analyzed At the end of the experimental period, mice were sacri-
using Waters Empower 2 soware. Acquity UPLC AccQ-Tag �ced by cervical dislocation. Blood serum was obtained
Ultra Column (2.1 mm i.d. × 100 mm × 1.7 𝜇𝜇m particle size) via cardiac puncture and subjected to serum biochemistry
was used for the determination of GABA and amino acids analysis and liver was immediately collected. Weight of liver
pro�le. e mobile phase used was AccQ-Tag Ultra Eluent was recorded and expressed as a relative organ weight [24].
A for mobile phase A and AccQ-Tag Ultra Eluent B for
mobile phase B. e gradient condition was: 0–0.54 minutes,
2.5. Serum Biochemistry. Activities of blood serum marker
0–0.1% B; 0.54–5.74 minutes, 0.1–9.1% B; 5.74–7.74 min-
enzyme including alanine transaminase (ALT), aspartate
utes, 9.1–21.2% B; 7.74–8.8 minutes, 21.2–59.6% B; 8.8–11
aminotransferase (AST), triglyceride (TG), and total choles-
minutes, 59.6–0.1% B, and �nally, reconditioning the column
terol were measured using biochemical analyzer (Hitachi
with 0.1% B with isocratic �ow for 2.1 minutes aer washing
902 Automatic Analyzer) and adapted reagents from Roche
column with 59.6% B for 0.30 minutes. e �ow rate was set
(Germany).
at 0.7 mL/minutes and the injection volumes for all samples
and standards were 1.0 𝜇𝜇L. e column temperature was set
at 55∘ C according to [20, 23]. 2.6. Liver Histopathological Evaluation. Liver was removed,
�xed in 10% formalin solution, embedded in paraffin, sec-
tioned into 4 microns thickness, and stained with haema-
2.4. In Vivo Hepatoprotective Effect-Ethanol Induced Hepa- toxylin and eosin (H&E) for assessment of histopathological
totoxicity in Mice. Total of 72 Balb/c mice were randomly alterations. Histopathological changes of stained livers were
distributed into eight groups (𝑛𝑛 𝑛 𝑛). Hepatoprotective effects observed under bright-�eld microscope. Assessment of liver
of freeze-dried mung bean and fermented and germinated was graded based on vascular and necrotic changes according
mung bean aqueous extracts were assessed in ethanol- to [25]. Vascular changes include vessel congestion, leakage
induced liver damage animal model. Mice were pretreated of erythrocytes into surroundings, and hematoma formation.
orally with ethanol and plant aqueous extracts individually Necrotic changes show the appearance of necrosis, �brosis,
for up to 21 days. e experiment was designed as follows. and cell regeneration. No change (no distinguishable change,
Group 1. Normal group, mice (p.o.) with 100 𝜇𝜇L of normal 0%); mild change (30%); moderate change (31–60%); severe
saline for 14 days. change (61–90%); very severe change (91–100%).
Group 2. Ethanol untreated group, mice (p.o.) with 100 𝜇𝜇L of
50% (v/v) of ethanol for 7 days followed by 14 days of 100 𝜇𝜇L 2.7. In Vitro Antioxidants of Liver Homogenate Evaluation.
of 1 X PBS. Mice liver were meshed in ice-cold PBS and homogenized
before centrifuged at 2000 rpm for 5 minutes at 4∘ C. Super-
Group 3. Positive control group, mice (p.o.) with 100 𝜇𝜇L of natant was collected and subjected to different assays includ-
50% (v/v) of ethanol for 7 days followed by 14 days of 100 𝜇𝜇L ing superoxide dismutase (SOD) [26], malondialdehyde
of silybin (50 mg/kg). (MDA) [27], ferric reducing antioxidant power (FRAP) [28]
and nitric oxide (NO) assay [29].
Group 4. Low dose treated group, mice (p.o.) with 100 𝜇𝜇L of
50% (v/v) of ethanol for 7 days followed by 14 days of 100 𝜇𝜇L 2.7.1. Determination of Superoxide Dismutase (SOD). Brie�y,
of mung bean extract (200 mg/kg). SOD was determined following the method of evaluating
the inhibition of the reduction of nitro blue tetrazolium
Group 5. High dose treated group, mice (p.o.) with 100 𝜇𝜇L of (NBT) of liver homogenates. Brie�y, sample was added with
50% (v/v) of ethanol for 7 days followed by 14 days of 100 𝜇𝜇L 0.1 mol/L EDTA, 0.15 mg/mL sodium cyanide, 1.5 mmol/L
of mung bean extract (1000 mg/kg). NBT, 0.12 mmol/L ribo�avin, and 0.067 mol/L phosphate
buffer to a �nal volume of 300 𝜇𝜇L. e reduction was meas-
Group 6. Low dose treated group, mice (p.o.) with 100 𝜇𝜇L of ured at 560 nm and percentage of SOD inhibition as com-
50% (v/v) of ethanol for 7 days followed by 14 days of 100 𝜇𝜇L pared to the blank was determined. One unit of SOD was
of germinated mung bean extract (200 mg/kg). calculated by the amount of protein needed to achieve the
50% inhibition and hence expressed as unit SOD/mg protein.
Group 7. High dose treated group, mice (p.o.) with 100 𝜇𝜇L of
50% (v/v) of ethanol for 7 days followed by 14 days of 100 𝜇𝜇L 2.7.2. Determination of Malondialdehyde (MDA). Liver per-
of germinated mung bean extract (1000 mg/kg). oxidation was detected by measuring thiobarbituric acid-
reactive substance (TBARS). In brief, aliquot of 100 𝜇𝜇L liver
Group 8. Low dose treated group, mice (p.o.) with 100 𝜇𝜇L of homogenate was diluted with 400 𝜇𝜇L of PBS (8.1 g NaCl,
50% (v/v) of ethanol for 7 days followed by 14 days of 100 𝜇𝜇L 2.302 g Na2 HPO4 , and 0.194 g NaH2 PO4 /L) and mixed with
of fermented mung bean extract (200 mg/kg). 12.5 𝜇𝜇L butyhydroxytoulene (BHT, 8.8 mg/mL) and 250 𝜇𝜇L
trichloroacetic acid (TCA, 30%). e mixture was vortexed 3.2. In Vivo Hepatoprotective Effect
and kept on ice for 2 h. Next, mixture was centrifuged
at 2000 g for 15 min. Supernatant obtained was boiled for 3.2.1. Effect of Aqueous Extracts on Liver Function Biomarkers.
15 min along with 37.5 𝜇𝜇L 0.1 M EDTA and 125 𝜇𝜇L thio- ALT and AST are two biochemical markers normally used
barbituric acid (TBA, 1%). Aer mixture has been cooled for early stage assessment of liver injury. Table 1 shows that
down to room temperature, the absorbance of pink-colored ethanol had signi�cantly raised serum ALT and AST level
product was taken at 532 and 600 nm wavelength using in mice liver as compared to normal group indicating the
ELISA Reader (Bio-tek Instrument, USA). e difference incident of liver injury. e serum ALT level was successfully
between absorbance was measured and compared to that brought down in all posttreatment groups with high doses of
of the standard malonaldehyde tetramethyl acetal solutions mung bean, germinated and fermented mung bean extracts
of different concentrations. MDA activity was expressed as (1000 mg/kg). In contrast, the serum ALT level in all low
nmol MDA/g protein. doses of mung bean extracts (200 mg/kg) treated groupswere
continued to rise, indicating that the functions of liver have
been compromised. In all extract-treated groups of both
2.7.3. Determination of Ferric Reducing Antioxidant Power concentrations, the serum markers of AST were reduced
(FRAP). e FRAP was determined from reduction of Fe3+ to lower than the ethanol-attenuated group. Treatment with
to Fe2+ according to standard method with some modi�- fermented mung bean at high dose (1000 mg/kg) displayed
cation. Reagent was prepared by mixing 300 mM acetate the highest suppression percentage of serumALT (63.73%)
buffer (3.1 g C2 H3 NaO2 ⋅3H2 O and 16 mL C2 H4 O2 ), 10 mM and AST (69.84%) followed by germinated mung bean high
TPTZ (2, 4, 6-tripyridyl-s-triazine) solution, and 20 mM dose (1000 mg/kg), 45.25% (ALT) and 47.75% (AST), when
FeCl3 ⋅6H2 O solution in 40 mM HCl. e fresh working compared to ethanol control group.
solution was prepared by mixing 25 mL acetate buffer, 2.5 mL e above results showed that fermented mung beans
TPTZ solution, and 2.5 mL FeCl3 ⋅6H2 O solution and then at high dose (1000 mg/kg) were able to retain the serum
warmed at 37∘ C before using. Aliquot of 150 𝜇𝜇L of bioactive ALT and AST closest to the normal level and has better
extract (5 mg/mL) from mung bean, germinated, and fer- performance than the standard drug, silybin.
mented beans was allowed to react with 2850 𝜇𝜇L of FRAP
solution and shaken vigorously before being incubated in the 3.2.2. Effect of Aqueous Extracts on Serum TG and Cholesterol.
dark for 30 min. e reading of the colored product (ferrous Another hallmark to con�rm the acute alcohol-induced liver
tripyridyltriazine complex) was taken at 593 nm. e FRAP injury was indicated by elevated serum TG and cholesterol
activity was calculated from the standard FeSO4 calibration level. As shown in Table 1, treatment with extracts subsided
curve and FRAP value was expressed as 𝜇𝜇M Fe2+ /mg protein. the boosted level of TG and cholesterol with signi�cant
reduction in high dose fermented mung bean (1000 mg/kg)
with 38.4% and 23.42%, respectively.
2.7.4. Determination of Nitric Oxide. Brie�y, NO production
in liver was determined using a calorimetric Griess reaction 3.3. Effect of Aqueous Extracts on the Level of SOD, MDA,
(Invitrogen, USA). Liver homogenates (100 𝜇𝜇L) was loaded FRAP, and NO in Liver Homogenate. e effects of oral
onto microtitre plate, followed by 100 𝜇𝜇L Griess reagent (1% administration of mung bean, germinated, and fermented
sulphanilamide and 0.1% N-1-naphthylethylenediamine mung bean aqueous extracts on liver antioxidant were shown
dihydrochloride in 2.5% polyphosphoric acid). Later, the in Table 2. Aer being intoxicated with ethanol, a decline in
absorbance was taken at 540 nm wavelength using ELISA the level of superoxide dismutase (SOD) and ferric reducing
Reader (Bio-tek Instrument, USA). antioxidant power (FRAP) was observed in liver injury
groups (ethanol-induced) when compared to normal group.
2.8. Statistical Analysis. All quantitative measurements were Yet, SOD level increased back to normal in all extracts-
conveyed as mean ± SD Analyses were performed using one- treated mice with low (200 mg/kg/day) and high doses
way analysis of variance (ANOVA) and the group means (1000 mg/kg/day) of mung bean, germinated, and fermented
were compared by Duncan test. 𝑃𝑃 𝑃 𝑃𝑃𝑃𝑃 was considered as mung beans. On the other hand, MDA and NO levels were
statistically signi�cant. markedly increased in ethanol-attenuated liver, hallmarks of
lipid peroxidation, and in�ammatory response. Signi�cant
decrease in MDA and NO production were noticed in all
3. Results aqueous extract-treated groups. Fermented mung bean was
able to reduce MDA level by 3.6 times from 7.17 ± 0.17 to
3.1. GABA and Amino Acids Content. We have previously 2.00 ± 0.23 (nmol/g of protein) and NO level by 1.6 times
reported that fermented mung bean contained 7.6 times and from 14.72 ± 0.75 to 9.03 ± 0.06 (𝜇𝜇mol/mg of protein).
13.2 times higher GABA and amino acids contents as com- Meanwhile, it also elevated the SOD enzyme level and FRAP
pared to normal dried mung bean powder [20]. Similarly, activity by 2.3 and 2.2 times, respectively, which essentially
germinated mung bean also showed an increase in GABA contribute to hepatoprotective effects against free radicals.
and amino acids concentration by 27.9 times and 8.7 times ehighest dose of fermented mung bean (1000 mg/kg/day)
to 0.502 ± 0.035 g/100 g and 2.092 ± 0.117 g/100 g of dried was found to be the most comparable to normal and standard
powder, respectively. drug silybingroups.
T 1: Effect of mung bean extracts on serum ALT, AST, TG, and cholesterol in alcohol-induced acute liver toxicity in mice.
Treatment ALT (U/L) AST (U/L) TG (mmol/L) Cholesterol (mmol/L)

Normal untreated 14.09 ± 1.53 98.16 ± 1.99 1.48 ± 0.23 3.14 ± 0.39
50% EtOH (placebo) 48.11 ± 1.78 367.30 ± 1.10 2.37 ± 0.14 3.80 ± 0.20
50% EtOH + silybin (50 mg/kg) 26.72 ± 1.20∗ 171.70 ± 3.79∗ 2.77 ± 0.16 4.20 ± 0.36
50% EtOH + mung bean (200 mg/kg) 63.44 ± 2.73∗ 294.50 ± 6.28∗ 2.06 ± 0.22∗ 3.28 ± 0.31∗
50% EtOH + germinated
57.57 ± 3.60∗ 308.61 ± 1.33∗ 2.21 ± 0.06∗ 3.40 ± 0.01∗
mung bean (200 mg/kg)
50% EtOH + germinated
26.34 ± 3.50∗ 191.93 ± 1.51∗ 1.84 ± 0.35∗ 3.13 ± 0.06∗
50% EtOH + fermented
56.26 ± 4.71∗ 232.48 ± 1.52∗ 2.26 ± 0.12∗ 3.18 ± 0.21∗
50% EtOH + fermented
17.45 ± 1.88∗ 110.77 ± 6.96∗ 1.46 ± 0.76∗ 2.91 ± 0.19∗
Values are mean ± SEM of 8 animals each in a group and signi�cantly different from the 50% EtOH (Placebo) (∗ 𝑃𝑃 𝑃 𝑃𝑃𝑃𝑃) by ANOVA and followed by Duncan’s
multiple range test.
T 2: Effect of mung bean extracts on SOD, MDA, FRAP, and NO levels in liver homogenate of alcohol-induced acute liver toxicity in
mice.
SOD MDA (nmol/g of FRAP NO
Treatment
(U/mg of protein) protein) (U/mg of protein) (𝜇𝜇mol/mg of protein)
Normal untreated 16.58 ± 0.58∗ 3.02 ± 0.16∗ 9.40 ± 1.04∗ 9.97 ± 0.25∗
50% EtOH (placebo) 9.17 ± 0.79 7.17 ± 0.17 5.33 ± 0.04 14.72 ± 0.75
50% EtOH + silybin (50 mg/kg) 17.06 ± 0.01∗ 4.92 ± 0.20∗ 14.97 ± 0.08∗ 9.39 ± 2.70∗
50% EtOH + mung bean (1000 mg/kg) 17.07 ± 3.77∗ 3.78 ± 0.33∗ 5.63 ± 0.01 10.29 ± 0.11∗
50% EtOH + germinated mung bean
16.64 ± 0.73∗ 2.54 ± 0.20∗ 9.83 ± 0.02∗ 9.54 ± 0.04∗
(200 mg/kg)
50% EtOH + germinated mung bean
17.11 ± 1.26∗ 2.31 ± 0.26∗ 5.74 ± 0.02 8.84 ± 0.42∗
(1000 mg/kg)
50% EtOH + fermented mung bean
18.00 ± 0.34∗ 3.22 ± 0.32∗ 5.52 ± 0.02 10.78 ± 0.03∗
(200 mg/kg)
50% EtOH + fermented mung bean
21.35 ± 0.44∗ 2.00 ± 0.23∗ 11.92 ± 0.03∗ 9.03 ± 0.06∗
(1000 mg/kg)
Values are mean ± SEM of 8 animals each in a group and signi�cantly different from the 50% EtOH (placebo) (∗ 𝑃𝑃 𝑃 𝑃𝑃𝑃𝑃) by ANOVA and followed by Duncan’s
multiple range test.
3.4. Histopathological Evaluation. Histopathology assess- necrosis features. Moderate necrosis changes were noticed
ment of liver was performed for all groups. Figure 1(a) shows in all low doses of mung bean extracts-treated groups
that there was no pathological abnormality observed in (Figures 1(d), 1(f), and 1(h)). e high dose of mung bean,
the liver of normal mice and thus showing the absence of germinated, and fermented mung bean aqueous extracts-
vascular or necrosis changes. Figure 1(b) shows that ethanol treated groups illustrated mild necrosis and in�ammatory
induced severe necrosis changes and substantial changes in changes, with less severity than changes observed aer
liver section such as ballooning, microvesicular steatosis, ethanol administration (Figures 1(e), 1(g), and 1(i)). Reduced
increase in sinusoidal space (SS) dilation and central vein, degree of sinusoidal and central vein dilations, ballooning,
and lymphocytes cells in�ltration in sinusoids in ethanol- and hepatocytes necrosis were noticed particularly in germi-
untreated group as compared to normal group. e striking nated and fermented mung beanat high dose (1000 mg/kg)
feature observed in ethanol-induced liver was in various (Figure 1(g)).
stages of cytoplasmic condensation, microvesicular steatosis,
and hepatocytes necrosis indicating early phases of liver inju-
ry. On the other hand, livers of mice in all aqueous extracts-
4. Discussion
treated groups showed noticeable recovery from ethanol- Ethanol has been reported as an eminent contributor to
induced liver damage when compared to ethanol untreated liver and kidney injury in humans and animals who have
group with less microvesicular steatosis and hepatocytes been exposed to excess ethanol for a certain period of
CV
CV
CV
(a) (b) (c)
CV
CV
CV
(d) (e) (f)
CV CV
CV
(g) (h) (i)
F 1: e photomicrographs (40 × 10) of liver section taken from mice. Normal group (a) received saline as a normal control group, shows
a normal structure of central vein surrounded by hepatic cells, (b) received saline aer being induced with 50% ethanol as a ethanol control
group, shows a steatosis and hepatocyte necrosis; (c) received Silybin (50 mg/kg body wt.) aer being induced with 50% ethanol; (d) received
mung bean (200 mg/kg body wt.) aer being induced with 50% ethanol; (e) received mung bean (1000 mg/kg body wt.) aer being induced
with 50% ethanol; (f) received germinated mung bean (200 mg/kg body wt.) aer being induced with 50% ethanol; (g) received germinated
mung bean (1000 mg/kg body wt.) aer being induced with 50% ethanol; (h) received fermented mung bean (200 mg/kg body wt.) aer
being induced with 50% ethanol; (i) received fermented mung bean (1000 mg/kg body wt.) aer being induced with 50% ethanol. Signi�cant
hepatoprotective effects are seen in extracts-treated particularly germinated and fermented mung bean. Arrow indicates a condition of
microvesicular steatosis in liver injury, which mainly occurs in ethanol-induced group. Circle indicates hepatocytes necrosis. Centrilobular
vein (CV).
time [1, 30]. Ethanol metabolism can trigger protein, lipid, also observed [4, 33]. Furthermore, numerous studies have
and DNA degradation due to free radicalsformation. e reported the association of antioxidant in the protection
result of the present study supports the work of previous against oxidative liver injury [5, 34, 35].
published reports using natural extracts to treat ethanol- To assess the hepatoprotective properties of extracts, in
induced mice, as a model for acute liver disease [31, 32]. Fea- vivo study were performed to measure the serum markers
tures of ethanol-attenuated hepatocytes include in�amma- and chemokine presence in it. According to Koch et al.
tion, apoptosis, and necrosis including cirrhosis. In addition, [33], ethanol catabolism will result in surplus of NADH and
prolonged exposure to ethanol has been shown to increase acetyl-CoA thus causing lipogenesis of cholesterol and TG
the level of TNF-𝛼𝛼, a proin�ammatory cytokines, which in and also the leakage of cellular enzymes into plasma asso-
turn can trigger other in�ammatory chemokine, explic- ciated with serum ALT and AST. ese will eventually
itly, NO. Decrease in antioxidant defense and elevation of contribute to liver injury. us, by restoring the level of serum
serum markers such as AST, ALT, TG, and cholesterol were ALT, AST, cholesterol, and TG back to normal, high dose
of fermented mung beans has certi�ed its hepatoprotective staining were related to the results obtained. Administration
effects at least in part. Moreover, in the present study, of ethanol in mice animal model revealed that elevated level
hepatoprotective effects of mung bean was compared against of liver function biomarkers ALT, AST, TG, and cholesterol
germinated and fermented mung beans extracts. Higher levels were detected along with the decrease of antioxi-
degree of reduction in serum ALT, AST, cholesterol, and TG dant activity and severe necrosis histopathological changes.
content were observed in germinated and fermented mung However, possible hepatoprotective effect of germinated and
bean groups as compared to mung bean groups. Nonetheless, fermented mung bean extracts was observed when attenuated
mung bean extracts also contributed to slight decline of those liver was treated with extracts. Previous study has reported
serum biomarkers. Our result was in agreement with previous the hepatoprotective properties of their extract to reduce
works done on fermented food products where the induced microvesicular steatosis and hepatocytes necrosis in chronic
serum markers were signi�cantly restored back to normal liver injury, which is in agreement with our study [9]. Liver
through in vivo [36] and in vitro [19] studies. injury hallmark such as in�ammation, lymphocytes in�ltra-
e antioxidant properties of extracts were examined tion, necrosis and ballooning effects were restored back close
in mice liver tissue via MDA, SOD, FRAP, and NO assay. to normal aer administration of high dose of germinated
Increased amount of MDA in ethanol-induced liver signi�es and fermented mung bean extracts-treatment, supported
the enhance degree of lipid peroxidation, which can lead by the decrease in ALT, AST, TG, cholesterol, NO, MDA
to liver damage. On contrary, SOD and FRAP levels in and increase in FRAP and SOD activities. e correlation
ethanol-induced group were decreased. A decrease in both between liver biomarkers and histopathological changes sug-
activities in liver tissue of ethanol-induced group was largely gested that they can be used for early detection of acute
due tothe impairment of antioxidant enzymes that safeguard liver damage. Reduction of biochemical and histological
cells against reactive oxygen species [31]. On the other hand, damage was exerted by fermented and germinated mung
increase in SOD and FRAP levels and decrease in MDA bean, conforming their hepatoprotective properties.
formation in fermented and germinated extracts-treated No studies have been conducted on histopathological
groups were as expected. High total phenolic content and changes of fermented and germinated mung bean extracts
strong antioxidant activity were claimed in fermented [37, on ethanol-attenuated liver. e biochemical and histopatho-
38] and germinated mung bean [14, 39]. is may be the logical changes of attenuated liver aer being treated with
reason for the increase of SOD and FRAP activities in ger- fermented and germinated mung bean were as expected since
minated and fermented mung bean at high dose as compared they contain more bioactive compounds compared to mung
to mung bean, which consequently reduced the MDA level. bean. Results prove that germinated and fermented mung
In addition, it has been reported that mung bean extract bean exert better effects on liver injury than normal mung
contains volatile antioxidant which was able to inhibit mal- bean. is implies that the increase in amino acids, GABA,
onaldehyde formation in blood plasma [40]. phenolic content, and other bioactives compounds during
NO is an in�ammatory mediator and highly reactive germination and fermentation processes contribute to the
oxidant produced by iNOS, which is released by kupffer hepatoprotective effects of mung bean to ameliorate liver
cells upon exposure to hepatotoxins [5, 41]. In all extracts- injury. Overall, fermented mung bean possessed the best
treated groups, NO level was reverted to normal level. High antioxidant and hepatoprotective effect. is result gives us
dose fermented mung bean extracts were the most effective an idea that amino acid may play a more important role than
extracts to revert the elevation of NO level aer induction GABA since we have observed better improvement of amino
with ethanol followed by germinated mung bean extracts. acid level in fermented than germinated mung bean but vice
us, by suppressing NO production in liver, germinated and versa for GABA content.
fermented mung beans depicted their potential properties as
hepatoprotective agent.
A plausible justi�cation for hepatoprotective and antioxi- 5. Conclusion
dant effects of germinated and fermented mung beans at high
dose may be due to the presence of �avonoids and phenolic To the best of our knowledge, no comparison studies have
acids bioactive compounds, which were highly detected been made speci�cally between mung bean and germinated
particularly in fermented and germinated products [14, 42]. or fermented mung bean aqueous extracts in terms of their
Besides, many studies have reported on the increased content liver hepatoprotective and antioxidant enzyme properties.
of GABA and amino acids in commercial legumes aer Freeze-dried fermented and germinated mung bean aqueous
undergoing germination [43–46] and fermentation [21, 47] extracts at 1000 mg/kg body weight showed potential hep-
processes. Elevation of amino acids and GABA in our atoprotective effects on ethanol-induced liverinjury based on
germinated and fermented mung bean extracts may be well serum biochemical pro�le and histology evaluation of mice
added to liver protection properties since GABA amino acids liver. is could be largely due to the amino acids content
have been known to carry liver protection through the and antioxidant properties possessed by these extracts with
mechanism of maintenance of intracellular polyamines levels regard to FRAP scavenging activity and oxidant-related
of ethanol and CCl4 -exposed hepatic injury effects [48, 49]. factor, SOD. In conclusion, fermentation and germination
In the present study, histological evaluation was under- increased the nutritional and medicinal values of mung bean.
taken to support the biochemistry pro�les. e pathological Moreover, the results are comparable with silybin, a standard
changes observed in the ethanol-treated liver through H&E drug typically prescribed to treat liver disease. erefore,
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TG: Triglycerides
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Antioxidant and Hepatoprotective E Ect of Aqueous Extract of Germinated and Fermented Mung Bean On Ethanol-Mediated Liver Damage

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Hindawi Publishing Corporation

BioMed Research International

Norlaily Mohd Ali,1 Hamidah Mohd Yusof,1 Kamariah Long,2

Correspondence should be addressed to Noorjahan Banu Alitheen; noorjahan@biotech.upm.edu.my

Received 30 August 2012; Revised 25 October 2012; Accepted 26 October 2012

Academic Editor: Andre Van Wijnen

1. Introduction comparable to development of chronic hepatic disease in

Treatment ALT (U/L) AST (U/L) TG (mmol/L) Cholesterol (mmol/L)

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

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