diabetes mellitus is a common endocrine disease that is characterized by chronic hyperglycemia and insulin deficiency or resistance which are associated with other complications such as macroangiopathy and microangiopathy .
moreover , studies have also recorded that hyperglycemia can eventually induce the production of reactive oxygen species ( ros ) and nitric oxide ( no ) in the long run , increase  cell apoptosis , decrease  cell mass , and cause insulin deficiency and resistance . to date , the treatment for diabetes including insulin , metformin , and sulfonylureas was found to cause various side effects especially the development of resistance after a certain period of time .
thus , efforts to search for alternative and novel therapies to manage diabetes are still receiving great attention .   personalized nutritional management and physical activity have been recommended to replace the american diabetes association diet to achieve better glycemic control in diabetic patients . besides
, complementary and alternative medicine in the form of plant - based food and spices that are commonly used in traditional medicine to treat diabetes have also been recommend as better oral agents . for example , momordica charantia ( bitter melon ) is a traditional antidiabetic remedy that has been identified as a potential hypoglycemic agent in streptozocin / alloxan - induced diabetic and type ii diabetic subjects .
other than herbs , whole grains and cereals that serve as the source of energy in asian food have also been suggested as potential antidiabetic food due to their low - glycemic indices [ 6 , 7 ] .
the glycemic index of a food is defined as an effect on postprandial glucose in comparison to the reference food .
low - glycemic index foods have been proven to improve glycemic control of insulin and noninsulin - dependent diabetes mellitus .
mung bean ( vigna radiata l. ) is a food that is traditionally used to reduce fever and used for detoxification . among all types of seeds , mung bean has been recommended as an alternative food for diabetic patients due to its high - fiber content and low - glycemic index .
yao et al .   had reported that ethanolic extract of mung bean was able to reduce blood glucose , total cholesterol , and tg levels while enhancing the glucose tolerance and insulin sensitivity in type ii diabetic mice .   
fermentation is a common food processing method traditionally practiced in the east and southeast asian regions to improve the food colour quality , flavour , or even the nutrient content .
alterations made by microflora during the fermentation process may help to release the active ingredients that are beneficial to human health . fermented soybean , for example , was found to have better antidiabetic effect due to attenuation of the structures and contents of isoflavonoids and smaller bioactive peptides . although the antihyperglycemic effect of mung bean has been reported , the potential of fermentation in improving the antihyperglycemic and the antioxidant effect of mung bean is still unknown .
thus , this study compared the effects of fermented and nonfermented mung bean extracts on normoglycemic , glucose - induced hyperglycemic and alloxan - induced hyperglycemic mice .
the serum antioxidant levels of extracts treated alloxan - induced hyperglycemic mice were also evaluated in this study .
alloxan , glucose , folin - ciocalteu reagent , ascorbic acid , gallic acid , and gsh assay kit were purchased from sigma - aldrich ( usa ) .
momordica charantia was purchased from ccm pharmaceutical ( malaysia ) as positive control in this study .
strain 5351 inoculums were obtained from the culture collection center of the malaysian agricultural research and development institute ( mardi ) .
seeds of mung bean ( vigna radiate ) were subjected to solid - state fermentation before extraction .
then , the fermented bean was dried and ground into powder followed by water extraction in the ratio of 1  g of fermented seeds in 20  ml of deionised water ( 25c ) for 30 minutes .
the mixture was then centrifuged and the supernantant was freeze - dried at an operating temperature of 50c to obtain a final yield of 25% ( w / w ) .
uplc analysis was performed on a acquity uplc system ( waters corp , usa ) coupled with acquity uplc accq tag ultra column ( 2.1  100  mm , 1.7  m ) and pda detector at 55c to analyse gaba and amino acid concentrations .
gaba and amino acids were separated using a gradient mobile phase consisting of a : accq tag ultra eluent a and b : accq tag ultra eluent b with the following gradient conditions : 00.54  min , 0.19.1% b ; 5.747.74  min , 9.1%21.2% b ; and finally , reconditioning the column with 0.1% b with isocratic flow for 2.1  min after washing column with 59.6% b for 0.30  min .
one l of all samples and standards were injected at a flow rate of 0.7  ml / min .
the data were then analyzed using the waters empower 2 software . from the analysis , every 100  g of the nonfermented mung bean extract contained 0.016  0.001  g of gaba and 0.256  g of total amino acids .
in contrast , the concentration of gaba in the fermented mung bean extract increased by 7.6-fold to 0.122  0.009  g/100  g of dried powder while the amount of amino acids increased by 13 fold to 3.326  g/100  g dried powder .  
the experiments were evaluated on normoglycemic , glucose - induced hyperglycemic , and alloxan - induced diabetic mice .
the mice were subjected to 18  h of fasting before each test was carried out .  
balb / c mice ( 8 weeks old , 1822  g ) were purchased from the animal house in the institute of bioscience , university putra malaysia .
standard laboratory pellet diet and water were made available ad libitum throughout the experimental period at 22c of dark - light cycle .
this study was approved by the animal care and use committee of university putra malaysia .
mice were randomly assigned into their respective groups for normoglycemic , glucose - induced hyperglycemic , and alloxan - induced hyperglycemic studies as listed below .
blood glucose was collected from all experimental mice for analysis after 18  h of fasting .  
group i : normal control mice without any treatment ; group ii : m. charantia extract ( 200  mg / kg ) ; group iii : nonfermented mung bean extract ( 1000  mg / kg ) ; group iv and v : fermented mung bean extract ( 200  mg / kg and 1000  mg / kg , resp . ) . after 18 hours of fasting , blood glucose was determined ( 0  min ) before oral administration of distilled water or the respective extracts ( 0.3  ml / mice ) was given to each mouse .
monitoring of blood glucose level was continued at 30 , 60 , 120 , and 240  min after the administration of treatment or distilled water .  
mice were randomly assigned into five different groups ( n = 8) as listed above .
after 18  h of fasting , blood glucose level was determined at 0  min .
then , oral feeding of distilled water or the respective extract was given simultaneously with 1  g / kg ( 0.3  ml ) of glucose solution to each group .
monitoring of blood glucose level was continued at 30 , 60 , 120 , and 240  min after the oral administration .  
group 1 was the normal control mice that received distilled water as placebo while groups 26 were diabetic mice .
diabetes was induced using intraperitoneal injection of alloxan ( 100  mg / kg , 0.1  ml ) .
the hyperglycemic mice were maintained on 5% glucose solution for the next 24  h to prevent hypoglycemia and monitored for 3 days to ensure constant blood glucose levels before they were subjected to 18 hours of fasting .
after the fasting period , blood glucose was determined at 0  min , followed by oral feeding with distilled water ( group 1 and 2 ) or the respective treatment .
group 3 received m. charantia extract ( 200  mg / kg ) ; group 4 received nonfermented mung bean extract ( 1000  mg / kg ) ; group 5 and 6 received fermented mung bean extract ( 200  mg / kg and 1000  mg / kg , resp . ) .
in addition , all the mice were also fed with glucose solution once ( 1  g / kg , 0.3  ml , p.o . ) .
monitoring of blood glucose level was continued at 30 , 60 , 120 , and 240  min after administration .
treatments with distilled water and the extracts were continued for a total of 10 days . on the last day of treatment ,
all mice were fasted for 18  h before being anesthetized with ether and sacrificed by cervical dislocation .
blood was collected to obtain serum for determination of glucose , total cholesterol ( biovision , usa ) , triglyceride ( tg ) ( biovision , usa ) , low - density lipoprotein ( ldl ) ( biovision , usa ) , high - density lipoprotein ( hdl ) ( biovision , usa ) , and insulin ( mercodia , sweden ) levels .
the antioxidant level of serum was evaluated by detecting the level of malondiadehyde ( mda ) as described previously   while serum nitric oxide level was determined using the griess method ( invitrogen , usa ) .   the results for blood glucose level , serum biochemical profiles , and antioxidant level were presented as mean  s.d .
one way analysis of variance ( anova ) followed by duncan test was used in this study with p values < 0.05 being considered as significant .
overall , fermented mung bean , nonfermented mung bean , and m. charantia extracts did not produce any hypoglycemic effect but caused slight hyperglycemic effect within 2 hours of oral feeding in normal mice ( figure 1(a ) ) .
however , for the glucose - induced hyperglycemic mice , the animals from all groups were found to develop high blood glucose levels at the first 30 minutes after oral administration ( figure 1(b ) ) .
however , treatment with 1000  mg / kg body weight of fermented and nonfermented mung bean extracts could significantly reduce the elevated blood glucose level in comparison to the normal control group .
although significant effect was also shown by the m. charantia extract treatment but the effect was comparatively weaker than in the previous groups .
on the other hand , low concentrations of the fermented mung bean extract did not show any significant difference in antihyperglycemic effect when compared to the normal control .  
significant changes of body weights were observed in the untreated normal mice . for all the diabetic mice , changes of body weights were not significant ( figure 2 ) .
treatments on day 15 were based on 22  g / mice while from day 6 to10 were based on 24  g / mice .
generally , diabetic mice ( groups 2 to 6 ) gained less weight than normal mice ( group 1 ) during the treatment period ( figure 2 ) . nonfermented mung bean showed a hyperglycemic effect similar to the untreated diabetic mice in group 2 .
m. charantia and fermented mung bean extracts ( 200  mg / kg body weight ) on the other hand were able to prevent drastic increases in blood sugar when compared to the untreated diabetic mice .
among all treatment groups , high concentration of fermented mung bean extract ( 1000  mg / kg body weight ) was able to reduce blood sugar level most significantly throughout the period of monitoring ( 30  min to 2 hours after feeding ) ( figure 1(c ) ) .   
the alloxan - induced diabetic mice were monitored continuously in the following 10 days with continued treatment for groups 3 to 6 .
untreated mice in group 2 maintained a high blood glucose level in comparison to mice in the other groups .
the blood sugar levels of m. charantia , nonfermented mung bean , and low concentration of fermented mung bean ( 200  mg / kg body weight ) extracts treatment groups were found to be reduced slightly at day 10 while a high concentration of fermented mung bean extract at 1000  mg / kg body weight was able to reduce blood sugar levels even at day 5 after administration ( figure 1(d ) ) .   
the serum lipid profile and the insulin level of the alloxan - induced hyperglycemic mice after 10 days of treatment were assessed .
untreated diabetic mice in group 2 showed significantly higher levels of total cholesterol and tg but lower levels of hdl and insulin .
in contrast , a high concentration of fermented mung bean extract ( 1000  mg / kg body weight ) showed lower levels of total cholesterol and tg but higher levels of insulin and hdl in comparison to the nonfermented mung bean extract ( table 1 ) .
similar to the effects on serum lipid profile and insulin levels , untreated diabetic mice in group 2 exhibited significantly higher mda and no levels . both m. charantia and
fermented mung bean extracts were able to restore the antioxidant level more effectively than the nonfermented mung bean extract .
besides , we also observed that the antihyperglycemic effect of the fermented mung bean extract was dosage dependent whereby a higher concentration of fermented mung bean ( comparing between group 5 and group 6 ) exhibited better antioxidant activity with lower no level ( table 1 ) .
previously , m. charantia   and mung bean   have been reported as potential antidiabetic agents .
low - glycemic - index mung bean was able to reduce plasma lipid level , epidilymal adipocyte volume and plasma insulin [ 68 ] .
thus , mung bean has been recommended as a food of choice for diabetic patients .
however , it is still uncertain whether fermentation could enhance the antidiabetic effect of mung bean . in this study , we have compared the antihyperglycemic effects of fermented and nonfermented mung bean extracts in normoglycemic , glucose - induced hyperglycemic , and alloxan - induced hyperglycemic mice .
normal fasting blood glucose for mice is around 90  mg / dl ( ~5  mmol / l ) .
animals having fasting blood glucose levels more than 200  mg / dl ( ~11 
changes of fasting blood glucose of normaglycaemic and glucose - induced hyperglycaemic mice which were 20% lower than those of the untreated normal control were considered as hypoglycaemia .
all the extract - treated mice in the normoglycemic study showed slight increases in blood sugar without indication of hypoglycemic effect .
this outcome may be contributed by the primary metabolites that are present in the water extract , or more specifically by the carbohydrates in this context .
however , in both glucose- and alloxan - induced hyperglycemic mice , m. charantia , fermented and non - fermented mung bean extracts were able to reduce oral glucose tolerance to prevent drastic glucose increase in the blood .
these results suggested the possibility of using fermented and nonfermented mung bean extracts for regulating blood sugar via their antihyperglycemic effects , which could enhance glucose adsorption in the gut .
continued administration of the extracts for 10 days had also been associated with blood sugar reduction ( figure 1(d ) ) . fermented mung bean extract at 1000  mg / kg body weight showed the best reductions of blood sugar levels of diabetic mice at day 5 .
m. charantia , nonfermented mung bean , and low concentration of fermented mung bean extracts showed similar trends of reduction at day 10 .
the findings from this study were in good agreement with a previous report on the assistance of m. charantia in the regulation of blood sugar via improved insulin sensitivity   and recovery of -cells in the pancreas after 12 days of treatment . in this study , fermented mung bean ( 200  mg / kg body weight ) and non - fermented mung bean exhibited a trend of blood sugar regulation similar to that of m. charantia extract .
therefore , fermented and nonfermented mung bean extracts could also possibly regulate blood sugar level by enhancing the sensitivity of insulin and by the regeneration of -cells in the pancreas .  
clinical studies had reported that low - glycemic - index diet contributed small effects on the control of postprandial hyperglycemia in diabetic patients .
thus , the improved antidiabetic effect due to fermented mung bean may be contributed by the enhancement of phytochemicals during the fermentation process .
advances in the effectiveness for controlling glucose metabolism through fermentation were also reported for soy bean [ 2 , 16 ] .
solid state fermentation was predicted to improve the antidiabetic effect of mung bean via enrichment of antioxidants and phytochemicals . in this study , we have found that enhanced blood sugar regulation by a high concentration of fermented mung bean may be contributed by the enriched gaba content ( 7.6 fold ) in the extract as compared to the nonfermented mung bean .
the regenerative effect of gaba on islet -cell via activation of pi3-k / akt - dependent growth and survival pathways had been reported by soltani et al .   and tian et al .  
free amino acids ( 13 fold increase in fermented mung bean ) had also been reported as an effective oral supplement for diabetic patients . among the tested free amino acids ,
lysine which recorded a marked increase in the fermented mung bean ( 0.001  g/100  g dry weight of nonfermented mung bean and 0.134  g/100  g dry weight of fermented mung bean ) ( result not shown ) had also been reported as being an enhancer to the insulin - receptor tyrosine kinase activity in type ii diabetic patients .
these free amino acids or more specifically lysine may contribute synergistically with gaba to regulate the blood glucose of the fermented mung bean - extract treated diabetic mice .  
elevated serum lipid profile including cholesterol , tg , ldl with low hdl level is one of the pathogenesis of diabetes that also representing the risk factor for coronary heart disease . in this study ,
alloxan - treated diabetic mice was observed with high serum cholesterol , tg , and ldl when compared with normal mice .
treatment with m. charantia , fermented and nonfermented mung bean extracts were able to reduce the risk factors for coronary heart disease by restoring the healthy lipid profile in the alloxan - induced diabetic mice .
other than a higher lipid profile , the serum of diabetic patient was also indicated with higher level of mda and oxidative stress [ 22 , 23 ] .
fermentation was able to improve the in vitro antioxidant and phenolic contents of mung bean .
this effect may contributed greatly to the reduction of the mda level of the fermented mung bean extract ( 1000  mg / kg body weight ) treated diabetic mice . besides
, our results also showed that fermented mung bean extract was able to reduce the nitric oxide ( no ) level in the serum of the alloxan - induced diabetic mice in a dosage dependent manner .
where ethanolic extract of mung bean showed anti - inflammatory effect and reduced no synthesis in a macrophage cell line .
thus , the significant reduction of no by a high concentration of fermented mung bean extract may indirectly help to reduce the damage of -cell .   in this study , fermented mung bean extract further improved the antihyperglycemic effect of nonfermented mung bean extract in both glucose and alloxan - induced hyperglycemic mice .
this effect may be due to the improvement of the gaba and free amino acid contents through the fermentation process .
investigating on the details mechanism of fermented mung bean 's antihyperglycemic effect are still on - going .
 mung bean was reported as a potential antidiabetic agent while fermented food has been proposed as one of the major contributors that can reduce the risk of diabetes in asian populations . in this study , we have compared the normoglycemic effect , glucose - induced hyperglycemic effect , and alloxan - induced hyperglycemic effect of fermented and nonfermented mung bean extracts . 
 our results showed that fermented mung bean extracts did not induce hypoglycemic effect on normal mice but significantly reduced the blood sugar levels of glucose- and alloxan - induced hyperglycemic mice . 
 the serum levels of cholesterol , triglyceride ( tg ) , and low - density lipoprotein ( ldl ) were also lowered while insulin secretion and antioxidant level as measured by malonaldehyde ( mda ) assays were significantly improved in the plasma of the fermented mung bean - treated group in alloxan - induced hyperglycemic mouse . 
 these results indicated that fermentation using mardi rhizopus sp . 
 strain 5351 inoculums could enhance the antihyperglycemic and the antioxidant effects of mung bean in alloxan - treated mice . 
 the improvement in the antihyperglycemic effect may also be contributed by the increased content of gaba and the free amino acid that are present in the fermented mung bean extracts . 