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Antidiabetic Effect of Soybean and Chungkukjang

Jung-In Kim, Min-Jung Kang, and Tae-Wan Kwon
School of food and Life Sciences, Food Science Institute, Inje University

Diabetes mellitus is the fourth leading cause of death amon Koreans. To control blood glucose and lipid levels are the major goals for treatment of diabetes mellitus. We investigated hypoglycemic and hypolipidemic effects of soybean and Chongkukjang in rats. Methanol extract of soybean and Chongkukjang inhibited yeast a-glucosidase activity by 43.6% and 62.7% in vitro. Respectively. Thus it appeared that soybean and Chongkukjang could suppress postprandial hyperglycemia. Consumption of autoclaved soy flour and Chongkukjang improved glucose tolerance and significantly decreased plasma triglyceride level of normal rats. The effect of chronic feeding of Chongkukjang on carbohydrate and lipid metabolism were studied in neonatal streptozotocin-induced diabetic rats, animal model of type 2diabetes. Fasting plasma glucose and insulin levels of Chongkukjang group were significantly lower than those of the control group. Feeding of Chongkukjang significantly decreased plasma triglyceride and cholesterol levels and improved fat change of liver compared with the control group. Thus it could be concluded that soybean and Chongkukjang could be helpful in controlling hyperglycemia and dyslipidemia in diabetic patients

Keywords : soybean, Chongkukjang, diabetes mellitus, glucose, triglyceride, cholesterol


The number of diabetics has been sharply growing over the recent 10 years in Korea, making diabetes mellitus rank the fourth leading cause of death among Koreans (1,2). Patients with diabetes show abnormal metabolism of carbohydrates, proteins, and lipids due to hormone imbalance, leading to hyperglycemia and dyslipidemia and finally to causing complications including cardiovascular disorders and neuropathy (3,4). In diabetic patients, the level of triglycerides and cholesterols increases, and HDL-cholesterol decreases, causing abnormal lipid metabolism and producing a risk factor of cardiovascular complications, one of the major causes of death among diabetics (3,5). As a common therapy for diabetes, a combination of medication, dietary therapy, and exercise therapy is administered, with an aim of maintaining ideal level of plasma glucose continuously and preventing and delaying diabetic complications (7,9). However, as there is no established solution for complete treatment of diabetes for now, studies have continued on medicines and foods to control hyperglycemia and dyslipidemia.
Soybeans are known for its low glycemic index (GI) having an effect on diabetes (10). Since soybeans contain physiological active substances such as dietary fibers, vitamin E, isoflavonne, phenol, saponin, trypsin inhibitor, and phytic acids, other than proteins, they are expected to be very effective to prevent chronic degenerative disorders including

atherogenesis, heart diseases, and cancers(11). In addition, chungkukjang traditional Korean fermented soy bean food not only contains physiological active substances found in soy beans of which it is made, but also is likely to have the potential to generate new physiological active substances including polyglutamate during the fermentation stage, possibly having the effect of controlling diabetes, improving lipid metabolism, lowering blood pressure, and killing bacteria (12,13). However, studies on the effect of ingestion of soy beans and chungkukjang on controlling plasma glucose and improving diabetes are still lacking. Jenkins et al.(14) reported that soybeans reduced digestion of in vitro carbohydrates, and after ingestion of soybeans, plasma glucose level increased moderately. It has been reported that traditional Chinese fermented soybean food Touchi-extract reduced activities of a-glucosidase in vitro and that effects of reducing plasma glucose and blood lipids were found among those who had the extract for a long time (15, 16). a-glucosidase is an enzyme involved in the digestion of carbohydrates, and a-glucosidase inhibitor is on the market as an oral hypoglycemic agent, because it suppresses postprandial hyperglycemia. If soybeans and chungkukjang inhibit a-glucosidase activities, they are expected to have an effect on plasma glucose control. Therefore, this study aimed to identify the effect of plasma glucose control, by measuring a-glucosidase inhibiting activity of soybeans and chungkukjang and using an animal model.
Materials and Method
  Materials for experiment
Soybeans were used in this study. They were washed to make chungkukjang and soaked in water for 18 hours before being autoclaved for 15 minutes at 121℃ and then cooled down to 60℃. Bacillus subtillus cultured at a liquid medium was inoculated to 20% of weight of the soy beans and fermented in an incubator at 37℃ for 72 hours. The autoclaved soybeans and chungkukjang were freeze-dried and ground, gone through three times of mixing, extracting and filtering with 10% methanol equal to ten times heavier, and then enriched at a reduced pressure and extracted, using rotary vacuum evaporator(EYELA, Japan).

Measuring the Yeast-glucosidase inhibiting activity
a-glucosidase inhibiting activity of individual extracts were measured in the method developed by Watanabe et al. Methanol extracts and acarbose of soybeans and chungkukjang were melted down in dimethylsulfoxide(DMSO) to the 5mg/mL concentration to use for this study. 50L of a-glucosidase(Sigma. USA: 0.7U/mL) and 10L of sample extract were added to the well of 96-well plate and then OD405 was measured in a microplate reader (Model 550. Biorad. USA). In 5 minutes, 50L of substrate solution (5Mm para-nitrophenyl-a-D-glucopyranoside in 0.1M phosphate buffer, pH7.0) was added to observe how the solution would response for 5 minutes at a room temperature. Then, OD405 was measured, and from changes to absorbance, enzyme inhibiting activity was calculated.

Test animal and diet
In order to examine how soybeans and chungkukjang affect glucose tolerance and lipid metabolism in normal rats, weaning male Sprague-Dawley (n=21) white rats were purchased from Bio Genomics (Daejun, Korea). Basal diet on the basis of AIN-76 diet was fed to the animals for 1 week, and then the animals were grouped into three of the same average weight using randomized block design. As a basal diet the AIN-76 diet was administered for the control group, a diet containing autoclaved freeze-dried soybeans for the soybean group, and a diet containing freeze-dried chungkukjang for the chungkukjang group, for 8 weeks. Composition of basal, autoclaved soy flour and chungkukjang diet is as shown in Table 1. Protein, fat, and total dietary fiber contents were identical among these three diets, making simple and complex glucose similar to each other in ratio. In order to examine how chungkukjang has an effect on carbohydrate and lipid metabolism, in the type II diabetes animal model, streptozotocin(STZ, Sigma Co. USA, 100mg/kg) in 0.1M citrate buffer(pH4.5) was injected to the abdominal cavity of male Sprague-Dawley(SD) white rats on the day 5 of birth. If plasma glucose measured from blood in tail vein of a 10-week old fasting white rat with a self glucose monitoring device (Glucotrend, Germany) was 150mg/dL or over, then it was regarded as having diabetes in this study. The animals (n=14) were grouped into two of the same average weight using randomized block design. The AIN-93G basal diet was fed for the control group, with a diet containing freeze-dried chungkukjang for the chungkukjang group, for 8 weeks. Table 2 shows composition of basal and chungkukjang diet. The animals were fed in a screen-bottomed cage, and ad libitum was supplied as a diet. Temperature and humidity at the cage were maintained at 20 to 25℃ and 50 to 60%, respectively. In regard to brightness, lights were on and off at 12-hour intervals.

Oral glucose tolerance test
In order to examine how ingestion of soybeans and chungkukjang affect glucose tolerance in normal rats, oral glucose tolerance test was administered for each group in week 7 of the diets. Plasma glucose was measured from blood in vein in tail of rats fasting for 12 hours over night with a self glucose monitoring device (Accutrend, Germany). After gastric intubation of glucose solution(1g/kg) to the control group as well as the soybean and chungkukjang group, plasma glucose was measured for periods of 30m, 60m, 90m, and 120m, respectively.

In 8 weeks of diet, the animals were fasted for 12 hours and then sacrificed for this study. Blood was taken from the heart before going through centrifugation at 3,000rpm and 4℃ for 15 minutes to get plasma. Liver tissue of diabetic rats were severed and fixed in 10% formaldehyde solution. Then HE(hematoxyline and eosin solution) dyeing and PAS(para-aminosalcylic acid solution) dyeing were performed to observe the tissue.

Analysis of blood composition
Plasma triglyceride and cholesterol levels in normal rats were measured by enzyme method. For diabetic rats, the blood glycosylated hemoglobin level was measured by chromatography, blood glucose, plasma triglyceride, cholesterol, and HDL-cholesterol levels by enzyme method, and the insulin level by radioimmunoassay, respectively.

Statistical processing
All the results were marked using mean±s.e.m. A t-test was used to test any significant difference between two groups, with ANOVA to test any significant difference among three groups. Tukey’s test was employed as a follow-up test(a=0.05).
Result and discussion
  Effect of inhibiting a-glucosidase activities of soybeans and chungkukjang in vitro
The results of an investigation of the effect of carbohydrate digestion enzyme inhibitory activities of the methanol extract of soybeans and chungkukjang in vitro revealed that the methanol extract of autoclaved soybeans and chungkukjang inhibited activities of yeast a-glucosidase at a concentration of 5mg/mL by 43.6% and 62.7%, respectively (See Table 3). Inhibitory activity of acarbose as the standard form accounted for 22.8%. The a-glucosidase inhibitor suppresses disaccharidase in microvilli of the small intestine reversibly and thereby delays absorption of carbohydrates in bowels and in turn reduces postprandial hyperglycemia. Therefore, it is expected that the soybean and chungkukjang extract with high a-glucosidase inhibitory activities in vitro will be able to prevent acute postprandial hyperglycemia in vivo. WHO has recommended the use of natural materials with fewer side effects for the treatment of diabetes mellitus, promoting the Traditional Health Care System (THCS), a program using medicinal herbs (18). There is growing interest in therapy using natural substances. Soybeans and chungkukjang, known for its excellent a-glucosidaase inhibitory activities, are expected to make a contribution to the control of blood glucose.

The effect of chronic feeding of soy beans and chungkukjang on glucose tolerance and improvement of the lipid level in normal rats
  Body weight gain and food intake
Table 4 shows body weight gain and food intake of normal rats fed basal, autoclaved soy flour and chungkukjang diet for 8 weeks. There was no significant difference in daily average body weight gain and food intake among diet groups.

Oral glucose tolerance test
The findings from the oral glucose tolerance test are described in Fig. 1. The fasting glucose level of the control group was 106.7±4.1mg/dL, and long-term ingestion of soy flour and chungkukjang had no significant influence on the fasting glucose level. The fasting glucose level of the control group after adding glucose (1g/kg) reached the highest level in 30 minutes of the addition, resulting in increases in glucose to 58.7±4.3 and 40.5±6.1mg/dL in periods of 30mins and 60mins, respectively. The soy flour or chungkukjang diet group showed a significantly lower(p<0.05) increase in the glucose level in 30 and 60 minutes of the addition of glucose, compared to the control group, which indicates that long-term intake of soy flour and chungkukjang improves glucose tolerance. Soybeans are low in glucose index (GI), and long-term intake of low GI foods has been reported to lower the glucose level of both normal persons(19) and diabetics(20,21). Madar(22) has reported that in an oral glucose tolerance test on diabetic rats fed soy bean dietary fibers for 6 weeks, the plasma glucose level decreased significantly in periods of 60 and 120 mins, compared to the control group. It is considered that intake of soy flour or chungkukjang will improve glucose tolerance and be effective in controlling postprandial glucose in normal rats.

Plasma cholesterol and triglyceride levels
Fig. 2 shows plasma cholesterol and triglyceride levels. The plasma cholesterol levels were 96.4±4.5mg/dL for the control group, 91.8±4.6mg/dL for the soy flour group, and 85.7±6.7mg/dL for the chungkukjang group, with no significant difference among the three groups. The triglyceride levels of the soy flour(67.9±5.1mg/dL) and chungkukjang(62.9±3.7mg/dL) groups were significantly lower(p<0.05) than the control group(94.1±4.7mg/dL). Soybean protein has been reported to lower the level of blood cholesterol as a cause for various adult diseases and prevent atherogenesis, myocardial infarction, stroke, and hypertension and improve diabetes (23). Costa et al.(24) have reported that intake of soybean protein has a good effect on the level of blood glucose, and Choi(25) has reported that ingestion of curd residue significantly lowered plasma HDL-cholesterol in rat as well as the cholesterol level in the liver. This study indicated that long-term intake of soy flour and chungkukjang improved high hyperglycemia, likely to make a contribution to the prevention of cardiovascular disorders.

The effect of chungkukjang controlling plasma glucose and improving lipid metabolism in animals with type II diabetes
  Body weight and food intake of test animals
Body weight gain after 8 weeks in the chungkukjang diet group (1.3±0.3g/day) had no significant difference from the control group (1.2±0.2g/day). Food intake of the control group was 27.0±1.1g/day, with no significant difference from the chungkukjang group (28.8±1.4g/day).

Fasting plasma glucose and insulin levels
Plasma glucose and insulin levels after 8 weeks of diet were 202.1±17.6mg/dL and 13.2±2.0ng/mL, respectively in the chungkukjang group, significantly lower than the control group (253.9±11.7mg/dL, 21.9±2.8ng/mL)(See Table 6. P<0.05). It has been reported that intake of a-glucosidase inhibitor acarbose lowered fasting plasma glucose and glycosylated hemoglobin levels in diabetic animals and diabetes patients(26,27). According to some studies, Touchi-extract was found to be highly effective in inhibiting a-glucosidase, and if long-term intake of it lowered fasting plasma glucose and glycosylated hemoglobin levels in animals and patients with type II diabetes (26,27). Long-term intake of chungkukjang lowered the plasma glucose in type II diabetes model, which seems to be due to its high a-glucosidase inhibitory activity (28).

Fasting plasma triglyceride and cholesterol levels
Plasma triglyceride and cholesterol levels were 67.3±4.3mg/dL and 98.8±8.5mg/dL, respectively, in the chungkukjang group, significantly lower than the control group (80.8±4.0, 137.4±11.0mg/dL), and the HDL-cholesterol level had no significant difference from the control group (See Table 7. P<0.05). Type II diabetes lowers insulin sensitivity and fails to control postprandial glucose, causing hyperglycemia and raising the plasma lipid level (29). A study reported that increased level of plasma neutral lipid lowered the HDL-cholesterol level and raised chiromicron residue content, causing vessel diseases in most cases(30). Being sold as a a-glucosidase inhibitor, acarbose improves carbohydrate metabolism, gradually increase plasma glucose, and relieves postprandial hyperglycemia and hyperinsulinemia. It has been also reported to reduce triglyceride biosynthesis in liver and lower fasting plasma glucose level, by improving insulin resistance (31). Long-term ingestion of fermented soybean product Touch-extract significantly lowered cholesterol and triglyceride levels in diabetic animals (15,16). According to a study (32), this is related to increasing insulin sensitivity which improved hyperglycemia and hypercholesterolemia. In chronic hyperinsulinemia patients, insulin controls VLDL secretion from the liver, and lower insulin level after meal reduces triglyceride and cholesterol levels. Zavaroni et al reported that acarbose lowered plasma insulin and triglyceride levels in animals and that plasma insulin and triglyceride had a correlation with each other (33). Therefore, long-term ingestion of chungkukjang is considered to have controlled plasma glucose and thereby improved insulin sensitivity and as a result lowered plasma triglyceride and cholesterol levels. Accordingly, chungkukjang is expected to be effective in improving carbohydrate and lipid metabolism and thereby preventing diabetes complications in diabetic animals.

Observation of fat change of liver
Whereas fat change of liver was found in the control group, there was no fat change found in the chungkukjang group (Fig. 3). Muto et al have reported that long-term treatment with complete Freund’s adjuvant (CFA) on a KK-Ay mouse lowered the plasma triglyceride level and blocked accumulation of lipomicrons in the liver, which was because CFA improved fat change. In this study, long-term ingestion of chungkukjang is considered to have improved lipid metabolism in diabetic rats and relieved fat change of liver.


We would like to thank the Korean Science and Engineering Foundation (KOSEF) for this thesis, which was part of the project performed with the 2000-2003 Basic Science Programs grant provided by the KOSEF.


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