WO2011010769A1 - Composition for treating or preventing diabetes comprising silk peptide as active ingredient - Google Patents

Abstract

The present invention relates to a GYG (Gly-Tyr-Gly) peptide and a composition for treating or preventing diabetes comprising the same as an active ingredient. More specifically, the present invention relates to a selection of materials determining anti-diabetic effects of silk peptide extracted using protease; and separation of materials modulating biological activities. A fraction showing especially outstanding anti-diabetic effects has been identified after separating silk peptides with molecular weight of 1,000 or less, which are identified as having high content of amino-nitrogen and high activity for a-glucosidase inhibition, from gel-filtration, and measuring the anti-diabetic effects for each fraction. An investigation of the in vitro anti-diabetic effects of the silk peptide of the present invention showed that the silk peptide resulted in a high rate of weight gain and thus a relatively low tendency of weight loss phenomenon due to diabetes. Thus, the composition for treating or preventing diabetes comprising the silk peptide of the present invention was identified as having activities of blood glucose reduction and antioxidation, and of being safe for the checked organs.

Description

Diabetes treatment or prevention composition comprising silk peptide as an active ingredient

The present invention relates to a GYG (Gly-Tyr-Gly) peptide and a composition for treating or preventing glucose using as an active ingredient a silk peptide containing the same.

Silk has been mainly used as a high quality material for clothing, but recently, chemical composition has been revealed, and development is actively progressed as a functional food material (Akai, H. New physiological functions of silk material. Shokuhin to Kaihatsu, 34, 45-47 1999; Alarcon, Aguilara., Rornan-Ramos, FJ and Parez-Gutierrez, R. Study of the anti-hyperglycemic effect of plants used as antidiabetics.J Ethnopharmacol 61, 101-110. 1998). In particular, silk is a natural protein with high purity and amino acid resources that can be produced in large quantities. It consists of sericin and fibroin, and when hydrolyzed, it becomes a silk peptide in the form of an oligopeptide in which two or more free amino acids and amino acids are combined.

The characteristics of silk peptides are a mixture of various amino acids and their combinations, which are components of human proteins, and have high physiological activity that shows medicinal efficacy, and the types of amino acids and their binding forms. Depending on the variety. Therefore, the material can be used for various purposes, but the composition and molecular weight of amino acids vary depending on the hydrolysis method and the method of filtering the residue, and the physiological activity peculiar to the silk peptide is also very different. Currently developed silk peptides are mostly hydrolyzed cocoons with acid and alkali, and most of them contain not only peptides but also amino acids (Chen, K., Umeda, Y. and Hirabayashi). , K. Enzymatic hydrolysis of silk fibroin.J Seric Sci. 65, 131-133. 1996).

Until now, many of the major benefits of silk peptides have been reported, and the field of research that is being studied is diabetes. Diabetes is divided into insulin-dependent (type 1) and insulin-independent (type 2). Most of these activity studies report the results of administration experiments on experimental animals. It is not enough. In particular, research results related to the antidiabetic effect of fibroin published in Korea and Japan show that the interpretation of the action of promoting insulin secretion has a contradictory result (Nam, JH and Oh, YS A study of pharmacological effect of silk fibroin.RDA J Agric Sci 37, 145-157. 1995; Park, KJ, Hong, SE, Do, MS and Hyun, C, K. Stimulation of insulin secretion by silk fibroin hydrolysate in streptozotocin-induced diabetic rats and db / db mice.Kor J Pharmacogn 33, 21-18.2002; Zhang, BB and Moller, DE New approaches in the treatment of type 2 diabetes.Cur Opin Chem Biol 4, 461-467. 2000).

The present invention aims to solve the above problems, and an object of the present invention is to select a substance having an antidiabetic activity or a marker substance of a silk peptide prepared using protease. It is another object of the present invention to provide an amino acid sequence having an antidiabetic activity of silk peptides.

In order to achieve the above object, the present invention provides a composition for treating or preventing diabetes, wherein the anti-diabetic active material of the silk peptide extracted using protease is a silk peptide having an molecular weight of 1,000 or less as an active ingredient. More preferably, the molecular weight of the silk peptide is 200 ~ 400 provides a composition for treating or preventing sugar as an active ingredient silk peptide.

In addition, the silk peptide according to the present invention provides a composition for treating or preventing diabetes, comprising a Gly-Tyr-Gly (GYG), Gly-Glu-Tyr (GEY) or Gly-Glu (GE) sequence do.

Diabetes treatment or prevention composition comprising the silk peptide according to the present invention as an active ingredient has a blood sugar lowering as described above and has the effect of treating or preventing diabetes. In addition, the weight loss due to diabetes is relatively small, suggesting the possibility of improving the blood fat component of the silk peptide, and does not appear to cause changes in the weight of the organ has a safe effect on the measured organ.

1 is a graph showing the amino nitrogen content of the silk peptide fraction by molecular weight by ultrafiltration according to the present invention.

Figure 2 is a diagram showing the absorbance measured at 340 nm by the TNBS method fractions of 1,000 molecular weight separated by gel filtration of the silk peptide according to the present invention.

Figure 3 is a graph showing the inhibition rate of α-glucosidase activity by the concentration of the F3 and F4 fraction of the silk peptide according to the present invention.

4 is a diagram showing chromatograms re-divided into F4-1 and F4-2 of the F4 fraction of the silk peptide according to the present invention.

Figure 5 is a graph showing the weight gain rate of 7 weeks of type 1 diabetes model animals administered silk peptides according to the present invention.

Figure 6 is a photograph of the type 1 diabetes model animal according to FIG.

Figure 7 is a graph showing the blood glucose measurement results of the silk peptide administration group according to the present invention measured for 7 weeks.

Figure 8 is a graph showing the results of the glucose tolerance test of the type 1 diabetes model administered silk pepta according to the present invention.

9 is a graph showing the results of dietary intake for 7 weeks between the silk peptide administration group and the diabetic control group according to the present invention.

10 is a graph showing the results of measuring the weight of the organ for 7 weeks between the silk peptide administration group and the diabetic control group according to the present invention.

11 is a graph showing the concentration of fat component of the plasma between the silk peptide administration group and the diabetic control group according to the present invention.

Figure 12 is a graph showing the weight gain rate of 7 weeks of type 2 diabetes model animals administered silk peptides according to the present invention.

FIG. 13 is photographic data of a type 2 diabetes model animal according to FIG. 12.

14 is a graph showing the blood glucose measurement result of the silk peptide administration group according to the present invention.

15 is a graph showing the results of the glucose tolerance test of the type 2 diabetes model administered silk peptides according to the present invention.

16 is a graph showing the results of dietary intake for 8 weeks between the silk peptide administration group and the diabetic control group according to the present invention.

Figure 17 is a graph showing the results of measuring the weight of the organ between the silk peptide administration group and the diabetic control group according to the present invention.

18 is a graph showing the concentration of fat component of the plasma between the silk peptide administration group and the diabetic control group according to the present invention.

19 is a graph showing the plasma insulin concentration between the silk peptide administration group and the diabetic control group according to the present invention in a type 1 diabetes model animal.

20 is a graph showing the plasma insulin concentration between the silk peptide administration group and the diabetic control group according to the present invention in a type 2 diabetes model animal.

Figure 21 is a graph showing the sugar load test results of the synthetic peptide (GYG) according to the present invention.

Figure 22 is a graph showing the sugar load test results of the synthetic peptide (GE) according to the present invention.

FIG. 23 is a graph showing the results of observing cytotoxicity by treating synthetic peptides (GYG and GE) according to the present invention at different concentrations in 3T3-L1 cell lines (adipocytes).

24 is a graph showing Glucose uptake activity of synthetic peptides (GYG and GEY) according to the present invention.

25 is a photograph showing the results of observing the inhibitory ability of TG accumulation in adipocytes of the synthetic peptides (GYG and GEY) according to the present invention.

26 is a photograph showing the results of observing the inhibitory ability of TG accumulation in adipocytes of the synthetic peptides (GYG and GEY) according to the present invention.

According to an aspect of the present invention, the present invention provides a composition for treating or preventing diabetes, wherein the anti-diabetic active substance of silk peptide extracted using protease is a silk peptide having a molecular weight of 1,000 or less as an active ingredient.

The antidiabetic active substance of the silk peptide extracted using the protease of the present invention is preferably a composition for treating or preventing diabetes, wherein the silk peptide having a molecular weight of 200 to 400 is an active ingredient.

Silk peptide of the present invention is characterized in that it comprises a sequence of any one of GYG, GEY, GE.

Through the following examples will be described the present invention in more detail. These examples are intended to explain the present invention in detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

Example 1

In vitro  Experiment

Silk peptide

Silk peptides prepared using cocoon protease were supplied from World Way.

Ultrafiltration (UF)

The 40% aqueous silk peptide solution was centrifuged at 8,000 rpm for 30 minutes at 4 ° C., and the supernatant was diluted 4 times. Ultrafiltration using a small pore pore filtration membrane (Satocon cassete, Germany) was used to obtain a fraction of 10,000 or more, fractions of 3,000 to 10,000, fractions of 1,000 to 3,000, and fractions of 1,000 or less.

Amino-nitrogen (AN) measurement

Amino nitrogen content was measured using the modified Takeuchi et al. (1990) method. To 0.125 ml of silk peptide fraction, 1 ml of 0.212 M phosphate buffer (pH 8.2) and 1 ml of 0.1% fresh prepared aqueous solution (TNBS) were added, respectively, and the light was blocked and stirred at 50 ° C. for 1 hour. 2 ml of 100 mM HCl solution was added to the reaction solution, and the resultant was allowed to stand for 20 minutes. Then, 4 ml of distilled water was added thereto. After 10 minutes, the absorbance was measured at 340 nm. As a standard substance, leucine was used.

α-glucosidase activity inhibition rate measurement

α-glucosidase activity inhibition rate was measured using the modified Ohta et al. (2002) method. α-glucosidase solution was put into 10 ml of 0.9% saline in 1 g of rat intestinal α-glucosidase extract (sigma, USA), and sonication was repeated three times for 1 minute, followed by centrifugation at 3,000 rpm for 30 minutes. One supernatant was prepared. To 100 μl of the silk peptide fraction, 2 ml of 10 mM phosphate buffer (pH 7.0) and 200 μl of 4-nitrophenyl α-D-glucopyranoside (NPG) were mixed and reacted at 37 ° C. for 5 minutes. 200 μl of α-glucosidase solution (0.15 unit / ml of buffer) was added to the reaction solution and reacted again at 37 ° C. for 1 hour. Then, 1.5 ml of Na ₂ CO ₃ was added to stop the reaction and the absorbance was measured at 405 nm. IC ₅₀ was the concentration of the sample to reduce the activity of α-glucosidase by 50% and the inhibition rate was calculated as follows.

OD _sample : with sample, OD _blank : without sample and enzyme, OD _control : without sample

Gel filtration (GF)

Analytical conditions of gel filtration for fractions of 10,000 or less silk peptide molecular weight are shown in Table 1.

TABLE 1

Conditions for Gel Filtration of Silk Peptides (Molecular Weight <1 kDa)

High performance liquid chromatography (HPLC) analysis

Analysis of standard materials and F3 and F4 fraction analysis for the selection of indicators were performed using HPLC (Varian 230, Varian Inc., USA) and the analysis conditions are shown in Table 2.

TABLE 2

Analysis of Standards and Conditions for Silk Peptide Fractions (F3, F4)

Example 2

In vivo  Experiment

Experimental animals

Experimental animals were tested in insulin-dependent diabetes model animals and insulin-independent diabetes model animals.

Insulin-dependent diabetes model animals, that is, type 1 diabetes model, was used as a 10-week-old male Sprague-Dawley rat (160 ± 10 g) supplied from a central laboratory animal (Central Lab Amimal Inc., Korea). The experimental animals were acclimated for 7 days before inducing diabetes and fasted for 12 hours before inducing diabetes, and then streptozotocin (Sigma-Aldsich, USA) was dissolved in 0.1 M citrate buffer (pH 4.5) at a concentration of 55 mg / kg (body weight). Intraperitoneal injection. On the next day of diabetes induction, a 5% glucose solution was used instead of water for 24 hours to prevent temporary hypoglycemia. Blood glucose was measured 72 hours after diabetes induction, and individuals over 250 mg / dl were selected and used as a type 1 diabetes model.

Insulin-independent diabetes model animals ie The type 2 diabetes model was male C57BL / K at 8 weeks of age from a central laboratory animal (Central Lab Amimal Inc., Korea)._SJ-db / db mouse (30 ± 3g) was used.

Breeding experimental animals

Experimental animals were kept in this study for 7 weeks (rat) and 8 weeks (mouse) after 1 week preliminary breeding. Experimental animals use breeding cages (42 cm x 28 cm) to maintain a laboratory temperature of 22-24, humidity of 60%, and to consider day and night cycles (12 hours light / 12 hours dark) controlled by environmental controls. Breeding in the university animal room. Water and feed were unlimitedly consumed during the entire experiment, and dietary intake was measured twice a week and body weight once a week during the experimental period.

Group Setup and Sample Dosing

Antidiabetic experiments using insulin-dependent (Streptozotocin diabetes-induced experimental model) and insulin-independent antidiabetic (db / db mouse) models consisted of four experimental groups. control (normal experimental model), DM-control (diabetes-induced model), SP-0.1 (0.1 g / kg silk peptide orally administered to diabetes-induced model), SP-0.2 (0.2 g / kg silk peptide orally administered to diabetes-induced model) ) And 8 experimental models were used for each group.

In the diabetes-induced experimental group, silk peptides were dissolved in DW, and 1 ml of rats and 0.5 ml of mice were orally administered at a constant time every day. Control and DM-control were orally administered distilled water at a certain time.

Blood sugar measurement

In order to measure blood glucose, blood was collected from the tail vein at a constant time after 12 hours of fasting, and blood glucose was measured by Superglucocard II (ARKRAY Inc., Japan) immediately after blood collection.

Payload inspection

The oral glucose tolerance test was performed for 12 hours at 7 weeks of breeding, after which the fasting blood glucose was measured and the sample was orally administered, and then 30 minutes after oral administration of glucose 2 g / kg (body weight). Blood glucose was measured by.

Blood analysis and organ extraction

The animals fasted for 12 hours were anesthetized with ethyl ether, sacrificed, and the thoracic cavity was opened to collect blood from the aorta. The collected blood was immediately placed in a test tube treated with heparin and centrifuged at 3,000 × g for 5 minutes at 4 ° C. to obtain supernatant plasma. The collected plasma was analyzed by the automated blood analyzer (FUJI DRI-CHEM 3500, Fuju Photo Film Co., Japan) to analyze triglyceride, total cholesterol, HDL-cholesterol, and LDL-cholesterol was calculated by Friedwald et al. (1972). Plasma insulin levels were measured using a Mouse insulin ELISA kit (Shibayagi Co., Japan). Some of the plasma was stored frozen at −20 ° C. and used for the measurements. After the blood collection, the experimental animals were dissected and the liver, kidneys and spleens were extracted and weighed. The extracted liver was washed with 0.15 M NaCl, impurities were removed with a filter paper, 5 g of the liver was taken up in 4.5 ml of 50 mM potassium phosphate buffer (pH 7.0), and ground with a homogenizer. The ground suspension was centrifuged at 4 ° C. and 13,000 × g for 4 minutes, then the supernatant was taken and stored frozen at −20 ° C. before being used for the measurement.

Statistical analysis

Data processing was performed using SPSS program (ver. 10.0). The mean and standard deviation (SD) of each item were calculated. The paired t-test was used for the before and after differences in the group, and the difference between the groups was one-way ANOVA at p = 0.05 level and the post hoc test was performed using Duncan's multiple range test.

Experiment result

In vitro  Experiment

Solid content weight and yield by molecular weight

The solid content weight and yield of the silk peptide fraction by molecular weight by ultrafiltration are shown in Table 3.

The yield of fractions with molecular weight of 10,000 or more (28.25 g) was 56.69%, the fraction of fractions between 3,000 and 10,000 (13.44 g) was 26.96%, and the molecular weights were between 1,000 and 3,000 (4.40 g) and fractions less than 1,000 (3.75 g). ) Yields were 8.82% and 7.52%, respectively.

TABLE 3

Solid Weight and Yield of Silk Peptide Fraction by Molecular Weight by Ultrafiltration

Amino nitrogen content by molecular weight

Amino nitrogen content of the silk peptide fraction by molecular weight is shown in FIG.

Amino nitrogen is the intermediate product of the breakdown of proteins into amino acids and refers to the chemical form of nitrogen present in amino acids at free amino groups. Therefore aminoaminonitrogen is also used as a food quality indicator item indicating the total amount of a compound having an amino group terminal. Among the silk peptide fractions, the amino amino nitrogen content was found to be the highest at molecular weights of 10,000 or more and 1,000 or less.

Inhibition Rate of α-glucosidase Activity by Molecular Weight

The inhibition rate of α-glucosidase activity of the silk peptide fraction by molecular weight is shown in Table 4. Carbohydrates are partially hydrolyzed by saliva amylase and pancreatic amylase to form small sugars. It is then hydrolyzed by α-glucosidase on the brush border of the mucosal epithelial cells of the duodenum and small intestine to form monosaccharides and then absorbed. Inhibition of α-glucosidase activity in the duodenum and small intestine inhibits the degradation of glucose can prevent the rapid increase in blood glucose (Joubert et al., 1987). Thus, the inhibition rate of α-glucosidase activity is used as a major indicator in controlling blood glucose. IC ₅₀ , a 50% inhibition of α-glucosidase activity, had the lowest molecular weight of 1,000 or less at 213.56 mg / ml, which was 2 to 3 times lower than other molecular weight fractions. That is, fractions with a molecular weight of 1,000 or less have a much higher inhibitory activity on α-glucosidase than other molecular weight fractions, which can be interpreted to mean that the anti-diabetic active substance of silk peptide is mainly contained in low molecular weight compounds with a molecular weight of 1,000 or less. have.

TABLE 4

Inhibition Rate of α-glucosidase Activity of Silk Peptide Fractions by Molecular Weight

Gel filtration of fractions up to 1,000 molecular weight

Fractions of 1,000 or less molecular weight, which were shown to be excellent in inhibiting α-Glucosidase activity, were separated by gel filtration again, and these fractions are shown in FIG. 2 as a graph of absorbance measured at 340 nm by TNBS method. Gel filtration fractions No. 28-33 were designated as F1, No. 34-39 as F2, No. 40-44 as F3, No. 45-49 as F4, and No. 50-56 as F5, respectively.

Yield, amino-porous water content and α-glucosidase activity inhibition rate of the gel filtration fraction

Table 5 shows the yield, amino-porous water content, and α-glucosidase activity inhibition rate for the gel filtration fraction of 1,000 or less in molecular weight. F1 was the highest at 33.7%, followed by F2 (23.7%), F3 (10.7%), F4 (5.7%), and F5 (3.4%). Amino nitrogen content, on the other hand, showed the opposite trend in yield, with F5 being 269.6 μg / mg, followed by F4 56.1 μg / mg, F3 47.5 μg / mg, F2 24.6 μg / mg, and F1 0.9 μg / mg. The mg showed little amino nitrogen. Inhibition rate of α-glucosidase, the antidiabetic indicator, was the highest in F3 (61.0%) and F4 (76.5%) at the same concentration (150 mg / ml). appear.

TABLE 5

Yield, amino-porous water content and α-glucosidase activity inhibition rate for gel filtration fractions with molecular weight of 1,000 or less

Inhibition of α-glucosidase Activity of F3 and F4 Fractions

The inhibition rate of α-glucosidase activity of each concentration of the F3 and F4 fractions is shown in FIG. 3. Inhibition rate of α-Glucosidase activity increased linearly with increasing concentration up to 60 mg / ml in both F3 and F4 fractions. The IC ₅₀ of the F3 and F4 fractions was 90.80 mg / ml and 37.25 mg / ml, respectively, indicating that F4 had higher α-glucosidase activity inhibition than F3.

Inhibition of α-glucosidase activity of F4-1 and F4-2 fractions and amino acid sequence of F4-2 fractions

The F4 fraction was again subjected to F4-1 and F4-2 fractions as shown in FIG. 4, and the inhibition of α-glucosidase activity of the fractions was measured (Table 6). The inhibitory activity of α-glucosidase at 1 mg / ml showed 18% and 34% inhibitory activity of F4-1 and F4-2, respectively. The amino acid sequences of the F4-1 and F4-2 fractions showing inhibitory activity of α-glucosidase were identified using the protein sequencing system (Table 6). The F4-1 fraction was GEY and F4-2 was identified as GYG.

TABLE 6

Inhibition of Amino Acid Sequence and α-glucosidase Activity of F4-1 and F4-2 Fractions

In vivo  Experiment

Insulin-Dependent Diabetes Model Animal Studies

Weight gain

Figure 5 shows the weight gain rate of 7 weeks of type 1 diabetes model animals.

Saline was administered to streptozotocin-diabetic rats for the control and diabetic controls, and 0.1 g / kg SP (SP-1) was used to give 0.1 g / kg / kg of silk peptide to the streptozotocin-diabetic rats. The day dose, 0.2 g / kg SP (SP-2) was applied 0.2 g / kg / day silk peptides to streptozotocin-diabetic rats, and 8 standard deviation values.

As a result, as shown in FIG. 5, the weight gain rate of the diabetic rats was less than 1 g / day compared to 2.05 g / day of the normal control group (diabetic control group: 0.29 g / day, SP-1 group: 0.32 g / day, SP). -2 group: 0.51 g / day) was significantly less (p <0.05).

Silk peptide group showed higher weight gain than diabetic control group, so weight loss due to diabetes was relatively low, but it was not statistically significant. Figure 6 shows the weight difference between the diabetic control group and silk peptide administration group.

Blood sugar change

Results of blood glucose change measured at weekly intervals for 7 weeks are shown in FIG. 7.

Saline was administered to streptozotocin-diabetic rats for the control and diabetic controls, and 0.1 g / kg SP (SP-1) was used to give 0.1 g / kg / kg of silk peptide to the streptozotocin-diabetic rats. The day dose, SP 0.2 g / kg (SP-2) is 0.2 g / kg / day silk peptide to streptozotocin-diabetic rats is the standard deviation value of eight rats.

The results showed little difference in blood glucose between the diabetic control group and the silk peptide group until 5 weeks (35 days), but the blood glucose level of the silk peptide group was significantly lower than the diabetic control group after 5 weeks (35 days). . In particular, the blood sugar level of the diabetic control group at 6 weeks (42 days) was 261.60 mg / dl, and the SP-1 and SP-2 groups were 203.25 mg / dl and 205.75 mg / dl, respectively. In the diabetic control group, 343.00 mg / dl, SP-1 and SP-2 groups showed significant differences of 233.50 mg / dl and 178.38 mg / dl, respectively (p <0.05). Could.

Payload inspection

Results of the glucose load test of the type 1 diabetes model are shown in FIG. 8.

Saline was administered to streptozotocin-diabetic rats for the control and diabetic controls, and 0.1 g / kg SP (SP-1) was used to give 0.1 g / kg / kg of silk peptide to the streptozotocin-diabetic rats. The day dose, SP 0.2 g / kg (SP-2) is 0.2 g / kg / day silk peptide to streptozotocin-diabetic rats is the standard deviation value of eight rats.

As a result, the diabetic control group increased 178.50 mg / dl compared to the initial blood glucose 120 minutes after Glucose administration, and the silk peptide group increased 168.00 mg / dl in the SP-1 group and 164.00 mg / dl in the SP-2 group. The blood sugar level was lower than that of the diabetic control group, but it was not statistically significant.

Dietary intake

Dietary intake for 7 weeks is shown in FIG. 9.

Saline was administered to streptozotocin-diabetic rats for the control and diabetic controls, and 0.1 g / kg SP (SP-1) was used to give 0.1 g / kg / kg of silk peptide to the streptozotocin-diabetic rats. The day dose, SP 0.2 g / kg (SP-2) is 0.2 g / kg / day silk peptide to streptozotocin-diabetic rats is the standard deviation value of eight rats.

Dietary intake for 7 weeks was increased in diabetic rats (diabetic control: 31.86 g / day, SP-1 group: 33.61 g / day, SP-2 group: 32.49 g, as shown in FIG. 9). / day) was significantly higher (p <0.05), and there was no difference in the intake between the diabetic control group and the silk peptide group, and the effect of silk peptide on the increased intake due to diabetes was not observed.

Long term weight

The weights of the kidneys, liver and spleen relative to 100 g body weight are shown in FIG. 10.

Saline was administered to streptozotocin-diabetic rats for the control and diabetic controls, and 0.1 g / kg SP (SP-1) was used to give 0.1 g / kg / kg of silk peptide to the streptozotocin-diabetic rats. The day dose, SP 0.2 g / kg (SP-2) is 0.2 g / kg / day silk peptide to streptozotocin-diabetic rats is the standard deviation value of eight rats.

The kidney and liver weights were significantly higher in diabetic rats than normal control group (p <0.05), but there was no difference between diabetic control group and silk peptide group. The spleen also indicated that silk peptides did not affect organ weights, as all mice did not show differences between groups at less than 0.20 g per 100 g body weight, suggesting that silk peptides are safe for the measured organs.

Blood lipid concentration

The concentration of fat component in plasma is shown in FIG. 11.

Saline was administered to streptozotocin-diabetic rats for the control and diabetic controls, and 0.1 g / kg SP (SP-1) was used to give 0.1 g / kg / kg of silk peptide to the streptozotocin-diabetic rats. The day dose, SP 0.2 g / kg (SP-2) is 0.2 g / kg / day silk peptide to streptozotocin-diabetic rats is the standard deviation value of eight rats.

Compared to the diabetic control group, the silk peptide group showed lower total cholesterol, triglyceride and LDL-cholesterol content and higher HDL-cholesterol content, indicating the possibility of improving the blood fat component of silk peptide, but it was not statistically significant. .

Insulin Independent Diabetes Model Animal Experiment

Weight gain rate

The weight gain rate of the type 2 diabetes model is shown in FIG. 12.

The normal control group and the diabetic control group (DM control) administered saline to db / db mice, and the SP 0.1 g / kg (SP-1) used 0.1 g / kg / of silk peptides to db / db mice. The day of administration, SP 0.2 g / kg (SP-2) is 0.2 g / kg / day silk peptide to db / db mice, the standard deviation of the eight rats.

Compared to the normal control group (0.10 g / day), the weight gain rate of diabetic rats (diabetic control group: 0.45 g / gay, SP-1 group: 0.46 g / gay, SP-2 group: 0.46 g / gay) was significantly higher. (P <0.05). However, there was no change in weight gain by silk peptide administration in diabetic rats. Figure 13 shows the weight difference between the rats of the normal control group, diabetes control group and silk peptide administration group.

Blood sugar change

Blood glucose before and after 8 weeks of treatment is shown in FIG. 14.

The normal control group and the diabetic control group (DM control) administered saline to db / db mice, and the SP 0.1 g / kg (SP-1) used 0.1 g / kg / of silk peptides to db / db mice. The day of administration, SP 0.2 g / kg (SP-2) is 0.2 g / kg / day silk peptide to db / db mice, the standard deviation of the eight rats.

The initial blood glucose level was not statistically different between the diabetic control group (154.75 mg / dl) and silk peptide group (SP-1 group: 134.20 mg / dl, SP-2 group: 136.40 mg / dl), but after 8 weeks of treatment, Silk peptide group (SP-1 group: 154.40 mg / dl), SP-2 group: 134.00 mg / dl) showed significantly lower levels than 217.00 mg / dl (p <0.05). mg / dl), which was statistically equivalent to blood glucose (p <0.05). Therefore, silk peptides are thought to have a hypoglycemic effect in type 2 diabetes.

Payload inspection

Results for the oral glucose tolerance test are shown in FIG. 15.

The normal control group and the diabetic control group (DM control) administered saline to db / db mice, and the SP 0.1 g / kg (SP-1) used 0.1 g / kg / of silk peptides to db / db mice. The day of administration, SP 0.2 g / kg (SP-2) is 0.2 g / kg / day silk peptide to db / db mice, the standard deviation of the eight rats.

In the diabetic control group, the glucose level increased continuously for 120 minutes after the glucose administration, but the silk peptide group showed the highest blood glucose level after 30 minutes after the glucose administration as in the normal control group. 120 minutes after the administration of glucose, the diabetic control group had the highest blood sugar elevation of 267.33 mg / dl compared to the initial blood glucose, whereas the silk peptide group was 84.33 mg / dl in the SP-1 group and 85.25 mg / in the SP-2 group. dl showed significantly lower blood glucose level than the diabetic control group, and after 180 minutes, the diabetic control group still showed high blood glucose of 161.00 mg / dl. In the silk peptide group, the SP-1 group was 26.00 mg / dl and the SP-2 group. The 22.50 mg / dl resulted in a significantly lower blood glucose level compared to the diabetic control group (p <0.05), which was statistically equivalent to that of the normal control group (p <0.05). Therefore, silk peptides have been shown to influence the improvement of glucose tolerance function of type 2 diabetes by glucose load test.

Dietary intake

Eight weeks of dietary intake is shown in FIG. 16.

The normal control group and the diabetic control group (DM control) administered saline to db / db mice, and the SP 0.1 g / kg (SP-1) used 0.1 g / kg / of silk peptides to db / db mice. The day of administration, SP 0.2 g / kg (SP-2) is 0.2 g / kg / day silk peptide to db / db mice, the standard deviation of the eight rats.

As a result, diabetic rats (diabetic control group: 5.85 g / day, SP-1 group: 5.61 g / day, SP-2 group: 5.64 g / day) compared to the normal control group (4.06 g / day) as shown in FIG. Was significantly higher (p <0.05). There was no difference in the intake between the diabetic control group and the silk peptide group, and the effect of silk peptide on the increased intake of diabetes was not observed as in the type 1 diabetes experiment. It was.

Long term weight

Height and liver weight relative to 100 g body weight are shown in FIG. 17.

The normal control group and the diabetic control group (DM control) administered saline to db / db mice, and the SP 0.1 g / kg (SP-1) used 0.1 g / kg / of silk peptides to db / db mice. The day of administration, SP 0.2 g / kg (SP-2) is 0.2 g / kg / day silk peptide to db / db mice, the standard deviation of the eight rats.

As a result, the kidney and liver weights were lower in diabetic rats than in the normal control group. In particular, the kidneys were diabetic rats (diabetic control group: 0.78 g, SP-1 group: 0.71 g, and SP-2 group) compared to the normal control group (1.36 g). 0.81 g) was significantly less (p <0.05), indicating a decrease in kidney weight due to diabetes. Both kidneys and liver did not differ between diabetic rats, so silk peptides did not affect organ weight as in type 1 diabetes model.

Blood lipid concentration

The concentration of fat component in plasma is shown in FIG. 18.

The normal control group and the diabetic control group (DM control) administered saline to db / db mice, and the SP 0.1 g / kg (SP-1) used 0.1 g / kg / of silk peptides to db / db mice. Day dose, SP 0.2 g / kg (SP-2) is the standard deviation of 8 rats fed 0.2 g / kg / day silk peptide to db / db mouse.

As a result, silk peptide administration group showed lower total cholesterol, triglyceride and LDL-cholesterol content and higher HDL-cholesterol content than diabetic control group. It was not statistically significant.

Insulin experiments in diabetic model animals

Insulin-Dependent Diabetes-Induced Model Animals

Insulin concentrations in plasma of the type 1 diabetes-induced model are shown in FIG. 19.

The normal control group and the diabetic control group (DM control) received saline in streptozotocin-diabetic rats, and SP 0.1 g / kg (SP-1) added 0.1 g / kg / kg of silk peptide to streptozotocin-diabetic rats. The daily dose and SP 0.2 g / kg (SP-2) are standard deviations of 8 rats treated with 0.2 g / kg / day silk peptides in streptozotocin-diabetic rats.

As shown in FIG. 19, insulin tended to be higher in the silk peptide administration group (SP-1 group: 0.685 ng / ml, SP-2 group: 0.651 ng / ml) compared to the diabetic control group (0.586 ng / ml), but statistically significant level. Was not. Therefore, silk peptides tend to promote insulin secretion in type 1 diabetes, suggesting the possibility of antidiabetic activity of type 1 diabetes.

Insulin-Independent Diabetes Model Animals

Plasma insulin concentrations of the type 2 diabetes model are shown in FIG. 20.

The normal control group and the diabetic control group (DM control) administered saline to db / db mice, and the SP 0.1 g / kg (SP-1) used 0.1 g / kg / of silk peptides to db / db mice. Day dose, SP 0.2 g / kg (SP-2) is the standard deviation of 8 rats fed 0.2 g / kg / day silk peptide to db / db mouse.

As shown in FIG. 20, the silk peptide administration group (SP-1 group: 1.72 ng / ml, SP-2 group: 1.12 ng / ml) was significantly lower than the diabetic control group (2.7 ng / ml) (p <0.05) This may be interpreted as enhancing insulin resistance by enhancing insulin action.

In this experiment, type 1 diabetes model promoted insulin secretion by silk peptide and type 2 diabetes model showed lower insulin levels in plasma by enhancing insulin action by silk peptide.

Glucose tolerance test by synthetic peptide

In the anti-diabetic activity evaluation of the peptides GEY and GYG, which were identified as active substances in silk peptides through α-Glucosidase inhibition experiments, they were tested with similar Gly-Glu (GE) instead of GEY. To evaluate antidiabetic activity, streptozotocin 40 mg / kg was administered to ICR mouse (20 g, 4 weeks old, male) for 5 days intraperitoneally to induce diabetes. Glucose test was performed for 30 hours after oral administration of GYG (10 mg / kg, 50 mg / kg) and GE (100 mg / kg, 200 mg / kg) to 12-hour fasted mice. g / kg) was orally administered to measure blood glucose over time. The results of the Glucose tolerance test are shown in FIG. 21.

The control group and the diabetic control group (DM control) administered saline to streptozotocin-diabetic rats, and the GYG-1 and GYG-2 showed synthetic peptides (Gly-Tyr-Gly) to streptozotocin-diabetic rats, respectively. 10 mg / kg and 50 mg / kg, GE-1 and GE-2 were administered 100 mg / kg and 200 mg / kg of synthetic peptide GE (Gly-Glu) to streptozotocin-diabetic rats, respectively. The deviation value.

As a result, blood glucose increased up to 90 minutes after glucose administration and then gradually decreased. GYG was the diabetic control group (GYG-1 & GYG-2) in the oral administration group of 10 and 50 mg / kg. There was a clear trend in decreasing blood glucose levels. In particular, the oral administration of 50 mg / kg was higher than that of GYG 10 mg / kg, but there was no significant difference.

In the GE oral administration group, 100, 200 mg / kg oral administration was used in a relatively high amount compared to the GYG group, and as shown in FIG. 22, the blood glucose was decreased after 90 minutes as in the GYG administration group, and GE 100 mg / kg. The difference in diabetic reduction effect was small between (GE-1) and 200 mg / kg administration group (GE-2).

These results suggest that GYG and GE have antidiabetic activity, and GYG lowering blood glucose was more effective than GE.

Antidiabetic Effect by GYG Synthetic Peptides

Sample solubility

The solubility of the synthesized two tripeptides Gly-Tyr-Gly (GYG) and Gly-Glu-Tyr (GEY) was low solubility in distilled water, but high solubility in DMSO (Dimethyl Sulfoxide). Therefore, the sample was dissolved in DMSO and used in the blood glucose control ability confirmation experiment.

Cytotoxicity

The results of observing cytotoxicity by treating the samples at different concentrations (1000 μM to 62.5 μM) in 3T3-L1 cell lines (adipocytes) are shown in FIG. 23. It was observed that both peptides GYG and GEY were toxic upon treatment of 1000 μΜ. Therefore, the maximum treatment concentration of blood glucose control ability was set to 500 μΜ.

 Glucose uptake activity

The glucose uptake capacity of the synthesized two peptides Gly-Tyr-Gly (GYG) and Gly-Glu-Tyr (GEY) is shown in FIG. 24. Although it was not observed in 125 μM, it was confirmed that glucose uptake activity was observed in 250 μM and 500 μM. In particular, GYG peptide was confirmed to be similar to the activity of 10 nM insulin at 500μM. In the case of PG used in the clinic, the activity was also observed at 2 nM, and it was confirmed that the activity similar to 10 nM insulin when 20 nM treatment.

Triglyceride (TG) accumulation

The two peptides Gly-Tyr-Gly (GYG) and Gly-Glu-Tyr (GEY) synthesized were observed to inhibit the TG accumulation in adipocytes. As a result, the photographic material is shown in FIG. 25 and the graph in FIG. 26. Both peptides Gly-Tyr-Gly (GYG) and Gly-Glu-Tyr (GEY) were found to reduce the accumulation of fat globules upon 250 μM treatment.

Claims (9)

1. An antidiabetic active substance of a silk peptide extracted using a protein hydrolase, and a composition for treating or preventing diabetes, comprising a silk peptide having an average molecular weight of 1,000 or less as an active ingredient.
2. An antidiabetic active substance of silk peptides extracted using proteolytic enzymes, and a composition for treating or preventing diabetes, comprising a silk peptide having an average molecular weight of 200 to 400 as an active ingredient.
3. A diabetic treatment or prevention composition comprising an antidiabetic active substance comprising a Gly-Tyr-Gly sequence as an active ingredient.
4. A diabetic treatment or prevention composition comprising an antidiabetic active substance comprising a Gly-Glu-Tyr sequence as an active ingredient.
5. A diabetic treatment or prevention composition comprising an antidiabetic active substance comprising a Gly-Glu sequence as an active ingredient.
6. The method according to any one of claims 3 to 5,

   The antidiabetic active substance is a composition for treating or preventing diabetes, characterized in that derived from silk peptides.
7. The method according to claim 6,

   The silk peptide is a composition for treating or preventing diabetes, characterized in that the extraction using a proteinase.
8. The method according to claim 6,

   The anti-diabetic active substance of the silk peptide is a composition for treating or preventing diabetes, characterized in that the silk peptide with an average molecular weight of 1000 or less.
9. The method according to claim 6,

   The anti-diabetic active substance of the silk peptide is a composition for treating or preventing diabetes, characterized in that the silk peptide with an average molecular weight of 200 ~ 400.

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