KR101121040B1 - Composition for lowering blood sugar and cholesterol comprising high molecular weight silk fibroin and its preparation method - Google Patents

Abstract

The present invention relates to a high molecular weight silk fibroin having a blood sugar and cholesterol lowering property and a method for producing the same, more specifically, a high molecular weight silk fibroin having an average molecular weight (Da) of 190,000-400,000 and optionally sericin as an active ingredient. It relates to a blood sugar or cholesterol lowering composition and a method for producing the same.
According to the present invention, a high molecular weight fibroin rather than a low-divided silk fibroin peptide shows a better blood sugar and cholesterol lowering effect, and by combining sericin with such a high molecular weight fibroin, a better blood sugar and cholesterol lowering effect, It can be effectively used in the manufacture of a functional food or drug for treating or preventing diabetes, obesity, or hyperlipidemia. In addition, since the process of removing sericin or the process of lowering fibroin in the existing silk protein manufacturing process can be omitted, the overall manufacturing cost can be reduced.

Description

Composition for lowering blood sugar and cholesterol containing high molecular weight silk fibroin, and preparation method thereof {Composition for lowering blood sugar and cholesterol comprising high molecular weight silk fibroin and its preparation method}

The present invention relates to a high molecular weight silk fibroin having a blood sugar and cholesterol lowering property and a method for producing the same, more specifically, a high molecular weight silk fibroin having an average molecular weight (Da) of 190,000-400,000 and optionally sericin as an active ingredient. It relates to a blood sugar or cholesterol lowering composition and a method for producing the same.

Silk has been widely used as the best textile material based on its excellent strength and gloss, and the silk industry has been spotlighted as a major export industry in the 60s and 70s in Korea. The decline in price competitiveness compared to silk products in developing countries has led to a downturn. However, silkworms and the silk industry obtained therefrom are attracting much attention to functional foods using silk as the low molecular weight silk peptides obtained by hydrolyzing silkworm powder or silk are utilized for foods with enhanced blood sugar and various functionalities, and some are commercialized. ought.

Various studies have been made on the application of silk as a functional food and a method for manufacturing silk as a functional food, some of which have been filed and published in patents and papers. For example, Korean Patent No. 1994-0002871 discloses that a low molecular weight silk fibroin can be used as a food material. In addition, Korean Patent No. 0286388 discloses a method for preparing silk powder peptide using an enzyme. Korean Patent Registration No. 2011-0018167 discloses a method for producing silk peptides having increased physiological activity by irradiation with radiation and silk peptides produced by the method. Korean Patent Registration No. 2000-0010464 discloses a functional beverage containing silk peptide powder prepared by hydrolyzing fibroin with enzyme after removing sericin by high temperature and high pressure refining of cocoon in weak alkaline aqueous solution. Republic of Korea Patent No. 1048657 discloses a composition for preventing and improving brain diseases, including a silk amino acid and tyrosine as an active ingredient and a method for manufacturing the same. In addition, in the 2004 issue of the Korean Society of Radiology, results of silk fibroin enzyme hydrolyzate using silk fibroin with molecular weight of 480 ~ 14,000 are disclosed for improving the animal cognitive function.

However, the prior arts have the common feature that low molecular weight of silk fibroin is used to make silk digestible in the body, and sericin is removed from fibroin and sericin, two macromolecules that make up silk, and used as a food mainly in fibroin. Have been. In this case, a process for lowering the molecular weight of the silk is required, and since the sericin, which takes about 20-30% of the weight of silk, cannot be utilized, the overall manufacturing cost increases.

Thus, the present inventors have conducted research to overcome the problems of the prior art, and as a result of using a high molecular weight silk, it was confirmed that the blood sugar and cholesterol lowering effect is superior, the sericin is superior to the blood glucose and cholesterol lowering effect than fibroin. It confirmed and completed this invention.

Therefore, the main object of the present invention is to provide a blood glucose and cholesterol lowering composition containing a high molecular weight silk fibroin rather than a low-divided fibroin peptide as in the prior art.

Another object of the present invention is to provide a composition for lowering blood sugar and cholesterol containing high molecular weight silk fibroin and sericin together without removing sericin.

Still another object of the present invention is to provide a method for preparing a silk protein containing the high molecular weight silk fibroin and sericin.

In the specification of the present invention, silk cocoon means a house (silk cocoon) that silkworms spew and wrap their bodies by themselves. The silk released from the silk cocoon is composed of two proteins, fibroin and sericin, which are collectively called silk proteins. It is composed of sericin protein and about 3% minerals and carbohydrates.

According to an aspect of the present invention, the present invention provides a composition for lowering blood sugar or cholesterol, comprising a polymer silk fibroin having an average molecular weight (Da) of 190,000-400,000 as an active ingredient.

In the embodiment of the present invention it was demonstrated that the high molecular weight silk fibroin has a lower blood sugar or cholesterol lowering effect than the hydrolyzed low molecular weight silk peptides used in the prior art.

In the composition of the present invention, the polymer silk fibroin provides a blood sugar or cholesterol lowering composition, wherein the 10% (w / w) silk formic acid solution has a viscosity (cps) of 70-350. The viscosity is proportional to the molecular weight of the silk fibroin, it is difficult to measure, and has a certain value different from the average molecular weight that can cause a difference in the value according to the measuring method is advantageous in defining the characteristics of the polymer fibroin of the present invention Do.

In the composition of the present invention, it provides a composition for lowering blood glucose or cholesterol, preferably further comprising sericin as an active ingredient.

Conventionally, in the manufacturing process of silk protein, sericin, which accounts for about 25% of silk protein, was removed, and the usefulness of silk fibroin peptide was tested. Or demonstrated for the first time excellent cholesterol lowering effect.

In the composition of the present invention, preferably, the mixed weight ratio of the polymer silk fibroin and sericin is 80:20 to 50:50 to provide a composition for lowering blood sugar or cholesterol.

In the embodiment of the present invention, the higher the content of sericin removed during the manufacturing process of the conventional silk protein, rather proved to have an excellent effect on lowering blood sugar or cholesterol.

In the composition of the present invention, the blood sugar or cholesterol lowering composition is characterized in that it is used as a raw material of a functional food or medicine for treating or preventing diabetes, obesity or hyperlipidemia.

The composition of the present invention can treat or prevent diabetes by lowering blood sugar, and can treat or prevent obesity and hyperlipidemia by lowering cholesterol. Functional foods for this purpose may be prepared in the form of tablets, capsules, beverages and the like or in the form of other functional foods according to methods known in the functional food manufacturing field. In addition, pharmaceuticals for this purpose may be prepared by methods known in the pharmaceutical art, and may be mixed with themselves or with a pharmaceutically acceptable carrier, excipient, diluent, or the like to provide powders, granules, tablets, capsules, or injections. It can be prepared and used in formulations. They can also be administered orally or parenterally.

The dosage for administering the polymeric silk fibroin and sericin according to the present invention as an active ingredient may be appropriately selected according to the age, sex, condition and symptoms of the disease of the patient, preferably 500-1000 mg (adult basis) per day. Polymeric silk fibroin and sericin can be administered.

According to another aspect of the present invention, the present invention provides a method for preparing a silk protein comprising polymeric silk fibroin and sericin having an average molecular weight Da of 190,000-400,000 including the following steps:

a) refining silk cocoon for 1 hour at 90-100 ° C. in distilled water without a refining agent;

b) washing the refined silk cocoon with distilled water at a temperature of 60-80 ° C .;

c) dissolving the rinsed silk cocoon in LiBr aqueous solution or CaCl ₂ / water / ethanol (molar ratio: 1/8/2) mixed solvent;

d) dialysis of the silk protein solution with a molecular weight cutoff tube of 12,000-14,000 to remove salts; And,

e) lyophilizing the salt-free silk protein solution.

In the manufacturing method of the present invention, it is possible to prevent sericin from being removed by refining in the step a) without a refining agent.

Here, refining means removing sericin from silk, and a degumming agent means a medicament that has been used to remove sericin in the manufacturing process of conventional silk proteins, and refining of silk is possible in various ways. For example, the degree of removal of sericin may be adjusted by treating with boiling water at 115 ° C., or the degree of sericin removal may be controlled by treating at high temperature with various types of soap aqueous solutions. The soap here is Tallow, Oliver oil, Coconut oil, Caster oil, Arachis oil, Cotton seed oil, Chrysal Chrysalis oil, Sodium Laurate, Sodium Myristate, Sodium Stearate, Sodium Arachidate, Sodium Oleate, Sodium Ricinol Such as sodium ricinoleate. Furthermore, it is also possible to refine | purify in alkaline aqueous solution or acidic aqueous solution, and the refining method using an enzyme can also be applied. In addition, refining using amine chemicals (methyl amine, ethyl amine, etc.) is also applicable. Therefore, it is possible to further reduce the cost by simplifying the conventional silk protein manufacturing process, rather it is possible to produce silk protein with better blood sugar and cholesterol lowering effect by including sericin.

In the production method of the present invention, in step c), the LiBr aqueous solution solvent does not break the silk molecular chain and breaks only the hydrogen bonds between the silk molecular chains so as to dissolve the silk molecular chains, so that hydrolysis of the silk fibroin protein does not occur. In addition, in step c), the CaCl ₂ / water / ethanol mixed solvent has a molar ratio of 1-2 / 8-16 / 2-4, and the CaCl ₂ / water / ethanol mixed solvent at a temperature of 80-90 ° C. Dissolve for 3 to 60 minutes. As the dissolution time increases in the CaCl ₂ / water / ethanol mixed solvent, the molecular weight of silk fibroin decreases. Therefore, in order to obtain high molecular weight fibroin, the dissolution time is preferably 3-60 minutes, more preferably 3-30 minutes.

According to another aspect of the present invention, the present invention provides a method for preparing a silk protein comprising polymeric silk fibroin and sericin having an average molecular weight Da of 190,000-400,000 including the following steps:

a) refining silk cocoon in a refining solution in step a) of the method for preparing silk protein to remove sericin and performing the remaining steps to prepare a polymer silk fibroin having an average molecular weight Da of 190,000-400,000; And,

b) mixing sericin with the polymer silk fibroin.

According to the production method of the present invention, it is possible to increase the content of sericin more than the content of sericin contained in the original silk protein, it is possible to control the exact content ratio of silk fibroin and sericin.

In the manufacturing method of the present invention, the refining agent of step a) may be used in various ways. For example, the degree of removal of sericin may be controlled by treating with boiling water, or the degree of sericin removal may be controlled by treating at high temperature with various types of aqueous soap solutions. The soap here is Tallow, Oliver oil, Coconut oil, Caster oil, Arachis oil, Cotton seed oil, Chrysal Chrysalis oil, Sodium Laurate, Sodium Myristate, Sodium Stearate, Sodium Arachidate, Sodium Oleate, Sodium Ricinol Such as sodium ricinoleate. Furthermore, it is also possible to refine | purify in alkaline aqueous solution or acidic aqueous solution, and the refining method using an enzyme can also be applied. In addition, refining using amine chemicals (methyl amine, ethyl amine, etc.) is also applicable. Preferably, it is characterized in that the sodium oleate (sodium oleate) and sodium carbonate (sodium carbonate). In an embodiment of the present invention, sericin is removed by sequencing at 90-100 ° C. for 1-3 hours in a solution of soldium oleate of 0.3-0.6% owf (on the weight of fiber) and sodium carbonate solution of 0.1-0.3% owf. It was.

In the production method of the present invention, the sericin in step b) provides a method for producing silk protein, characterized in that 100% silk sericin extracted from silk cocoon. The method of extracting only silk sericin from silk cocoon is known in the prior art, in the embodiment of the present invention by lyophilizing an aqueous solution of sericin dissolved in a cocoon for 1 hour in boiling water to extract silk sericin of high molecular weight. Silk sericin powder was obtained.

In the production method of the present invention, the mixing weight ratio of the polymer silk fibroin and sericin in step b) provides a method for producing a silk protein, characterized in that 80:20 to 50:50.

In the embodiment of the present invention, the higher the content of sericin removed during the manufacturing process of the conventional silk protein, rather proved to have an excellent effect on lowering blood sugar or cholesterol.

As described above, according to the present invention, a high molecular weight fibroin rather than a low-differentiated silk fibroin peptide shows a better blood sugar and cholesterol lowering effect, and by combining sericin with such high molecular weight fibroin, a better blood sugar and cholesterol lowering Since it is effective, it can be effectively used in the manufacture of functional foods or pharmaceuticals for treating or preventing diabetes, obesity, or hyperlipidemia. In addition, since the process of removing sericin or the process of lowering fibroin in the existing silk protein manufacturing process can be omitted, the overall manufacturing cost can be reduced.

Figure 1 shows the changes in blood glucose levels by the dietary period of mice according to the silk fibroin / sericin mixed protein diet.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example  1-3 and Comparative example  1-2: of various molecular weight silk Fibroin  Produce

Silk fibroin of various molecular weights was prepared and tested as follows to evaluate the physiological activity efficacy using the same.

Silkworm cocoon (Korea Rural Development Administration) was used as a test material, and refined by soap refining method to remove sericin, and then silk fibroin was prepared. Refining was refined for 1 hour at boiling temperature using an aqueous solution of Sodium oleate 0.3% (owf) / Sodium carbonate 0.2% (owf) in a bath ratio 1:20, washed with distilled water and dried to obtain the final silk fibroin. The solvent and dissolution time were adjusted to obtain silk fibroin of various molecular weights. The first sample (Example 1) dissolved silk fibroin in 9.3 M LiBr aqueous solution at room temperature for 24 hours, and the other three samples contained silk fibroin in CaCl _2. / H, respectively 3 min at 85 ℃ to yokbi 1:20 using a mixture of O ₂ / EtOH (molar ratio 1/8/2) (example 2), 30 minutes (example 3), 20 hours (Comparative example 1) dissolved. After dissolution, the dialysis membrane (Molecular weight cut off = 12,000 ~ 14,000) was dialyzed in flowing distilled water for 3 days, and then filtered to produce non-woven fabrics of regenerated silk aqueous solutions of various molecular weights. The freeze-dried silk fibroin powder was prepared. Got it.

In addition, to prepare a silk fibroin peptide as a separate sample, the silk fibroin obtained by refining was added to 25% aqueous hydrochloric acid solution and hydrolyzed by treatment at 110 ° C. for 14 hours. After acid treatment, 4 M aqueous sodium hydroxide solution was added to neutralize the acid solution, and then passed through a filter and desalted to obtain silk fibroin peptide powder (Comparative Example 2).

Test Example  One: silk Fibroin  Molecular Weight and Viscosity Measurement

In order to measure the molecular weight of silk fibroin prepared according to Examples 1-3 and Comparative Examples 1-2, 4M silk urea solution was prepared by adding urea to an aqueous silk solution, followed by Fast Protein Liquid Chromatography (FPLC) (AKTA Purifier). , Amersham Biosciences, USA). The column was superdex 200 10/300 GL, flow rate was measured under the operating conditions of 0.5ml / min, the molecular weight markers GE Gel gel filtration low molecular weight and high molecular weight calibration kit (Gel Filtration LMW and HMW calibration kit) ) Was used.

The viscosity of the silk solution was measured as a secondary measurement index to easily determine the molecular weight of the difficult-to-molecular weight silk. After dissolving the silk fibroin and sericin powder in 98% formic acid for 3 hours to prepare a 10% (w / w) regenerated silk formic acid solution, shear using a rheometer (MARS III, Hakke, Germany) using plate and plate geometry Shear viscosity was measured at a rate of 10 sec ⁻¹ and 25 ° C.

As a result of the test, the molecular weight of the silk fibroin prepared by the aqueous LiBr solution was the largest, and the molecular weight decreased as the time of dissolving with CaCl ₂ / water / ethanol mixed solvent increased, and the hydrolyzed silk fibroin peptide had the highest molecular weight. Was small. Among them, high molecular weight fibroin having a molecular weight of 190,000 Da or more was set as Examples 1, 2 and 3, and the rest was set as Comparative Examples 1, 2 and 2. In addition, the viscosity of the silk solution increased proportionally as the molecular weight increased.

Test Example 2: Test related to cholesterol lowering effect by molecular weight of silk fibroin

1) Animals and Diet

40 male C57BL / 6N mice, 4 weeks old, were purchased from a central laboratory animal. The mouse has a characteristic of increasing lipids and blood sugar in the body when high fat is ingested, which is useful for experiments on lipid metabolism and glucose metabolism. They were individually housed in stainless steel cages in a room maintained at 25 ± 2 ° C, 50% relative humidity and 12 / 12h light / dark cycle, fed and fed with pelletized solid feed for 2 weeks after arrival and then randomized block 8 by 7 groups (group 1; normal diet group, group 2; high fat diet control group, group 3; silk fibroin diet group, group 4, high fat + CaCl ₂ / water / ethanol mixture prepared with high fat + LiBr aqueous solution) Silk fibroin diet group, Group 5, dissolved in solvent for 3 minutes; high fat + CaCl ₂ / water / ethanol mixed solvent, group 6, silk fibroin dissolved in 30 minutes, high fat + CaCl ₂ / water / ethanol mixed solvent for 20 hours Soluble silk fibroin diet group, group 7; high fat + silk peptide diet group).

The diet and water were freely consumed without restriction, and the weight was measured once a week. The dietary composition is shown in Table 2 below. In order to induce high fat, the AIN-76 dietary composition was used as a control group (normal diet group) and the high fat diet control group (17% Lard) was added to the AIN-76 composition. Each dietary sample prepared by adding 2% of each regenerated silk sample was fed to 8 weeks.

2) Measurement of the weight of the body and organs of the rat

For the change in body weight and feed efficiency, the body weight was measured every week with the start date of the experiment as 0day, and the weight change status of the animals was compared until the end of the experiment, and the feed efficiency was calculated from the feed intake and body weight gain.

When the experimental diet reached 7 weeks, the animals were fasted for 18 hours, and then anesthetized by injecting 1% concentration of ketamine in 0.2 ml per 100 g of body weight, followed by anesthesia. Blood was collected from the inferior vena cava with a syringe treated with heparin (1000 unit / ml), centrifuged at 1000 g for 15 minutes to separate plasma, and then stored frozen until analysis. After collecting blood, tissues (liver, kidney, heart, epididymal fat) were immediately removed, rinsed several times with PBS, and weighed by removing water with gauze. The test protocol was approved by Kyungpook National University Ethics Committee for Animal Testing.

In the following experimental examples, the experimental results are expressed as mean ± standard deviation, and the standard deviation is calculated from experimental results obtained from eight rats. ^abc means that the statistical analysis showed a significant difference at the level of p <0.05. That is, a and b can be said to be significantly different values at a level of p <0.05, and a and a can be said to be values at the same level.

The body weights of all the experimental groups increased for 8 weeks after feeding the diet. After comparing the weight gain after 8 weeks, the 2 groups fed the high fat diet showed the highest weight gain and did not receive the high fat diet. Significant differences were found between groups 1 and 2, but there were no significant differences between groups 3, 4, 5, 6, and 7, where silk was added to the high fat diet. On the other hand, in the case of long-term weight, soy sauce was significantly increased in the high fat diet group 2 compared to the control group 1, and the addition of silk showed the effect of high fat diet. The heart was not significantly different among all experimental groups. However, height was significantly increased in group 2 of high-fat diet group compared to control group 1, and when supplementing silk with high-fat diet, the organs showed similar long-term weight.

3) plasma lipid concentration changes

(1) Measurement of Triglyceride Concentration in Blood: It was measured using Kit (Asan Pharmaceuticals) according to the enzyme method (Blucolo & David 1973) according to Bucolo method (1973). Lipoprotein lipase was applied to triglyceride to decompose it into glycerol and fatty acid, followed by reaction with L-glycerol phosphate oxidase, amino-antipyrin and peroxidase produced by the addition of glycerol kinase (GK). At this time, the resulting quinone pigment was quantified by measuring the absorbance at 550 nm and comparing with the glycerol standard curve.

(2) Blood Cholesterol Content: As a measure of plasma total cholesterol, a test solution (Asan Pharmaceutical kit) using the enzyme method of Allain et al. (1974) was used. 1.5 mL of enzyme solution was added to 10 μL of plasma and allowed to react at 37 ° C. for 5 minutes. Plasma cholesterol exists in the form of cholesteryl ester (CE) and free cholesterol, so to quantify both, CE was converted to fatty acid and free cholesterol by cholesterol esterase. Free cholesterol is measured at, 500 nm was converted to H ₂ O ₂ and Δ4-cholestenon by cholesterol oxidase and, is the H ₂ O ₂ is mixed with peroxidase and phonol, 4-amino-antiptrine of color to red It was. At this time, the quantification was performed in comparison with the cholesterol standard solution (300 mg / dL).

(3) Plasma free fatty acid quantification: Measured according to the instructions using a commercial free fatty acid assay kit (Enzychrom, Bio-Assays Systems, CA, USA).

(4) HDL cholesterol content in blood: measured using a kit (Asan Pharmaceuticals) according to the enzyme method (Finely 1978). Dextran-sulfate-Mg (II) was reacted with plasma to precipitate LDL and VLDL excluding the HDL fraction in plasma, and then the absorbance was measured at 500 nm using the same method as the total cholesterol in blood. Quantification by comparison.

(5) plasma phospholipid determination; Plasma phospholipid quantification was measured using a measurement kit (BioAssay Systems. USA). First, 1 μl of PL-M reagent 1 was added to 20 μl of plasma, and then 1 μl of PL-M reagent 2 was added. After absorbing 1 μl of Dye reagent and 90 μl of assay buffer, the absorbance of purple quinone dyes was maintained at 26 ° C. for 15 minutes. It was measured at and compared to the phospholipid standard curve.

As shown in Table 4, the triglyceride concentration of plasma was the highest in group 2 fed high fat diet, and the groups 6 and 7 added silk to high fat diet were similar to the control group. Although the concentration of, 2, 3, 4, 5 group showed a significant triglyceride reduction effect. In the case of neutral lipids, as the molecular weight of regenerated silk increased (from group 5 to group 2), the neutral lipid reduction effect was remarkable. Plasma phospholipid content was also recovered by regenerative silk supplementation, but there was no difference in molecular weight. However, the total cholesterol content was found to be more effective the higher the molecular weight, the HDL-cholesterol content was confirmed that the recovery effect similar to the control group in the group 3 and 6 group.

4) Plsma dipokines, GOT, GPT and TBARS measurements

(1) Analysis of hepatotoxicity by measuring plasma GOT and GPT activity: colorimetric analysis of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) activities, which are closely related to hepatocellular damage by plasma isolation, by Reitman-Frankel (1957) The enzyme method according to the analysis method was applied and analyzed by spectrophotometer. The addition of L-aspartate and α-ketoglutarate for serum GOT and D, L-alanine and α-ketoglutarate for GPT converts the substrate to pyruvate by enzymes in the plasma and reacts this pyruvate with 2,4-dinitrophenyl hydrazine When 0.4N NaOH was added to the solution, the color reaction occurred. The absorbance was measured at 490-530 nm, and the activity was measured by comparing with the standard curve of lithium pyruvate. 1 mL of GOT and GPT substrate solutions were taken and allowed to stand at 37 ° C. for 5 minutes, and then 0.2 mL of plasma was added, followed by 60 minutes of GOT and 30 minutes of GPT at 37 ° C. 1 mL of Dinitrophenyl hydrazine coloring solution was added and left at room temperature for 20 minutes, and mixed with 10 mL of 0.4N NaOH solution for 10 minutes, and then absorbance was measured. At this time, the standard curve was used as a calibration curve reagent in the serum transaminase measurement reagent (Asan kit, Asan Co. Seoul, Korea).

(2) Erythrocyte TBARS measurement: Erythrocyte TBA reactive substrance (TBARS) measurements were taken according to Tarladgis (1964), 50 μl of red blood cells were diluted to 500 μl, and then 3 ml of 5% trichloroacetic acid (TCA) and 1 ml of 0.06M thiobarbituric acid (TBA). After mixing and incubating for 90 minutes in an 80 ℃ water bath. After cooling at room temperature, the supernatant was collected by centrifugation at 2,000 rpm for 15 minutes, and the absorbance was measured at 535 nm. Malondialdehyde (MDA) standard solution was prepared by hydrolysis of tetramethoxypropane (TMP). That is, 1 mmol of TMP was dissolved in 100 ml of 0.01 N HCl solution, heated at 50 ° C. water batch for 60 minutes, cooled to room temperature, and 1 ml of hydrolyzed TMP solution was diluted in 100 ml of 0.01 M Na ₃ PO ₄ (pH 7.0) buffer to MDA standard. A solution was made. Absorption spectra of MDA standard solutions were obtained and the exact concentrations were calculated from the absorbance at the maximum absorption wavelength and the extinciton coefficient of MDA. The prepared standard solution was reacted under the same conditions as red blood cells to obtain a standard curve of TBA-MDA choromopore, and from this curve, the amount of TBA reactant was calculated as MDA equivalent.

(3) Plasma TBARS: Red blood cell TBA reactive substrance (TBARS) was measured using the same method as red blood cell TBARS, ie 50 ml of plasma was taken with 0.3 ml of 5% richloroacetic acid (TCA) and 0.06 M thiobarbituric acid (TBA) 0.1. After mixing the ml and incubated for 90 minutes in an 80 ℃ water bath. After cooling to room temperature, the supernatant was collected by centrifugation at 2000 rpm for 15 minutes, and the absorbance was measured at 535 nm.

GOT and GPT levels, which are indicators of hepatotoxicity increased by high-lipid diet (group 2), were also recovered by silk supplementation (groups 3-7). It has been shown to be effective. In particular, the highest molecular weight of silk (group 3) was found to be lower than GOT and GPT than normal test group (group 1), indicating a greater effect on restoring hepatotoxicity recovery increased by high lipid diet.

The lipid peroxidation of plasma and erythrocytes may be increased due to high lipid diet, which may lead to the problem of blocking blood vessels.In the high fat diet group (group 2), the lipid peroxidation level was higher than the normal group (group 1). It was confirmed that lipid peroxidation was decreased by the diet group supplemented with silk supplementation to the high fat diet (groups 3-7), and this effect was more effective as the molecular weight of silk increased. In particular, silk (Group 3), which has the highest molecular weight, showed similar levels of lipid peroxidation in plasma and blood.

5) Effect on lipid content in feces

Determination of lipid content in feces: Folch et al. (1957) were extracted using a method to determine fecal neutral lipids and cholesterol. 0.5 g of the dried feces were ground in a mortar, and 5 mL chloroform: methanol (2: 1, v / v) solution was added thereto, followed by extraction at 4 ° C. for 24 hours. The extract was centrifuged at 900 x g for 10 minutes at 4 ° C, the supernatant was taken, dried at 50 ° C with nitrogen gas, and dissolved in 1 mL of the same extractant. Among them, each solvent is taken for the measurement of neutral lipid and total cholesterol, dried again with nitrogen gas and dissolved in 5 mL of ethanol. Neutral lipid and cholesterol contents were quantified by the same enzyme reaction as liver tissue lipid quantification.

The experimental results show the triglyceride and cholesterol content related to lipids in the feces of rats. As these values increase, lipid-related substances in the body escape through the stool, and the lipid content in the body decreases, so increasing these contents is effective for lipid metabolism. As shown in Table 7, it can be seen that as the molecular weight of the silk increases their content increases lipid metabolism efficacy.

Test Example  3: silk Fibroin  Molecular weight lowering efficacy test by molecular weight

1) glucose concentration in blood

Blood glucose measurement: In order to measure the change in blood glucose concentration, blood glucose was collected through the tail vein at intervals of 2 weeks during the experiment, and the fasting glucose level was measured using a Glucosemeter (Accu-Chek Active Blood Glucose Test Strips-Roche Diagnostics GmbH, Germany). .

As a result, the normal rats (group 1) maintained blood sugar, but the hyperglycemic mice (group 2) showed a continuous increase in blood glucose as dietary time increased to 8 weeks. The rats fed silk fibroin were found to inhibit blood glucose increase, and the effect of increasing the molecular weight of silk fibroin increased the hypoglycemic effect. In particular, the silk diet group (group 3), which has the highest molecular weight, showed a high blood sugar level similar to that of the normal group (group 1), despite the fact that the mice had high blood sugar expression.

2) Comparison of enzyme activity related to carbohydrate metabolism

(1) Glucosekinase (GK): GK activity was measured by reducing and supplementing the method of Davidson and Arion et al. (1987) to determine the extent to which NAD + is reduced to NADH. 100 μl of 1M KCl, 600 μl of 83.33 mM Hepes-NaOH (pH 7.4), 100 μl of 2.5 mM dithio erythrit-ol (DTE), 100 μl of 75 mM MgCl ₂ , 50 μl of 10 mg / ml BSA, 50 μM NAD + 10 μl, 1000 unit glucose-6-phosphate dehydrogenase 10 μl , 10 μl of 1M glucose was added, and then 10 μl of the cytoplasmic fraction was added thereto, followed by reaction for 10 minutes at 37 ° C., and then 10 μl of 50 mM ATP was added thereto, and the absorbance change was measured at 340 nm for 10 minutes. Activity units are expressed as nmol of NADH produced for 1 min per mg of cytosolic protein. (nmol NADH oxidized / min / mg protein)

(2) Glucose-6-phosphatase (G6pase); It was measured using the method of Alegre (Alegre, 1988). Add 100 μl of 18 mM EDTA (pH 6.5), 100 μl of 265 mM glucose-6-phosphate (pH 6.5), 0.2 M NADP + to 765 μl of 131.6 mM Hepes (pH 6.5), and 0.6 IU of mutarotase (aldose-1-epimerase) and glucose dehydrogenase. / mL and 6IU / mL was added to react for 4 minutes at 37 ℃. Next, 5 μl of the microsome fraction was added and reacted at 37 ° C. for 4 minutes, and then the change in absorbance at 340 nm was measured. (nmol NADPH oxidizaed / min / mg protein)

(3) Phosphoenolpyruvate carboxykinase (PEPCK): Measured using the method of Bentle & Lardy (1976). To the sample mixture, add 100 μl of 10 mM DTT (dithiothreitol), 100 μl of 500 mM NaHCO _{3, 100} μl of 10 mM MnCl ₂ , 10 μl of 25 mM NADH to 650 μl of 76.92 mM Hepes-NaOH (pH 6.5), and add L-malate dehydrogenase, 7.2 unit, IDP ( After adding 1mM of inosine-5'-diphosphate and 2mM of PEP (phosphoenolpyruvate), 10 μl of the enzyme source cytoplasmic fraction was added to measure the change in absorbance at 25 ° C. and 340 nm for 2 minutes.

Liver and adipose tissue play an important role in maintaining energy metabolic balance, and in the fasting state, glycogen breakdown and glucose production are increased by the fasting state, and when food intake is increased, glycogen is accumulated in the liver. Do it. In this case, insulin uses lipids as an energy source, and controls lipids to be stored as glycogen while inhibiting sugar synthesis. In other words, it enhances gene expression encoding glycolysis and fatty acid synthesis, and inhibits gene expression of enzymes related to your life. In this example, we measured the activity of glucose-6-phosphatase, an enzyme that plays an important role in the end of glucose angiogenesis in liver tissues in which the action of insulin was inhibited by a high-lipid diet and had a negative effect on glucose metabolism. This enzyme decreases its activity when ingesting carbohydrates and maintains a stable blood sugar level on an empty stomach. Changes in the activity of these enzymes occur simultaneously with changes in glucagon and insulin in the blood. After carbohydrate intake, activity decreases with subsequent insulin secretion. Therefore, if there are abnormalities in sugar metabolism, these enzymes are likely to be activated.

Glucose-6-Phosphatase was slightly increased by high-lipid diet, but decreased in the experimental group supplemented with silk. As the molecular weight of silk increased, the activity of this enzyme decreased, which stabilized sugar metabolism. Appeared.

Example 4 Preparation of Silk Proteins Containing Silk Fibroin and Sericin

Silk cocoons were obtained from the Korea Rural Development Administration. The silkworm cocoons obtained consisted of 74% silk fibroin and 26% silk sericin, so different manufacturing conditions were used to prepare three types of silk proteins with different fibroin / sericin compositions. Pure silk fibroin was treated with sodium oleate (0.6%, on the weight of fiber [owf]) and sodium carbonate (0.4%, owf) for 2 hours at boiling temperature to remove sericin. Prepared by scouring with solution. For each extraction 40 g of cocoon was used in a solvent to solid ratio (bath ratio) of 25: 1. The refined silkworm cocoons were thoroughly rinsed with distilled water (60-80 ° C.) and dissolved in a calcium chloride / water / ethanol solution (molar ratio: 1/8/2) for 30 minutes. Silk fibroin solution was dialyzed into cellulose tubes (molecular weight cutoff = 12,000-14,000) against circulating pure water at room temperature for 5 days. The solution was then lyophilized to obtain a 100% regenerated silk fibroin sponge (F100).

In the case of the fibroin / sericin mixture, the raw silkworm cocoon was refined with distilled water without a refiner for 1 hour at boiling temperature. Refined silk consisted of 81% fibroin and 19% sericin (F81). The F81 silk protein was prepared through the same dissolution process as above. For the preparation of silk protein containing 50% fibroin and 50% sericin (F50), 100% silk sericin was extracted by boiling silkworm cocoon with distilled water for 1 hour. The pure silk sericin aqueous solution was then mixed with the F81 aqueous solution to prepare an F50 mixture. The solution was lyophilized to obtain a sponge sample. Sericin and fibroin content of each silk protein sample was determined by weight change after the refining process.

Test Example  4: Triglyceride-lowering efficacy with increasing sericin content

1) Animals and Diet

Four 40-week-old male C57BL / 6N mice weighing 17 grams were obtained from Orient Inc. It was purchased from (Seoul, Korea). They were individually housed in stainless steel cages in rooms maintained at 25 ° C., 50% relative humidity and 12/12 h light / dark cycles and fed on a pelleted chow diet for two weeks upon arrival. The mice were then randomly divided into five diet groups (n = 8). The first and second groups were fed with normal control (NC) and high fat diet (HF, 17%, w / w), respectively. The other three groups were fed on a high fat diet supplemented with F100 (HF-F100), F81 (HF-F81) or F50 (HF-F50) silk proteins. The composition of the diet (Table 16) was based on the AIN-76 semisynthetic diet. Mice were fed for 6 weeks and allowed free access to feed and water for the duration of the experiment. Feed consumption and weight gain were measured daily and weekly, respectively. The food efficiency ratio (FER) was calculated based on the average daily weight gain of mice divided by feed intake. Feces were collected during the last week to measure the cholesterol and triglyceride excretion levels of the feces.

At the end of the experiment, mice were anesthetized with ketamine-HCl after 12 hours of fasting. Blood samples were drawn from the inferior vena cava and placed in a heparin-coated tube, centrifuged at 1000 g for 15 minutes at 4 ° C. to obtain plasma. Body organs (liver, kidney and heart) and adipose tissue (didymis, perineum and visceral) were removed, rinsed with physiological saline, weighed and stored at −70 ° C. until analysis. The test protocol was approved by Kyungpook National University Ethics Committee for Animal Testing.

2) determination of lipid profile in plasma and feces

The concentrations of plasma total cholesterol, triglyceride, and high-density lipoprotein (HDL) cholesterol were determined using a commercial kit (Asan Pharmaceutical, Seoul, Korea). Excretion lipids were extracted and purified for total cholesterol and triglyceride analysis using the method described in Folch et al. (J. Biol. Chem. 1957, 26, 497509). Cholesterol and triglyceride levels in excreta were measured using the same enzyme kit used for plasma analysis.

3) Determination of Free Fatty Acid Concentration in Plasma

Plasma free fatty acid was measured according to the instructions using a commercial free fatty acid assay kit (Enzychrom, Bio-Assays Systems, CA, USA).

4) Measurement of Glutamate Oxaloacetate (GOT) and Glutamate Pyruvate Transaminase (GPT) Levels

GOT and GPT concentrations were determined using a commercial kit (Sigma Chemical Co., MO, USA) based on the method of Reitman and Frankel (Methods of Enzymatic Analysis (1975; pp 760-763)).

5) Measurement of Lipid Regulating Enzymes

Livers were homogenized according to the method described in Hulcher et al. (J. Lipid Res. 1973, 14, 625631). Briefly, the tissue was homogenized in 3-5 mL of 0.1 M triethanolamine, 0.02 M EDTA, and 0.002 M dithiothreitol (DTT) per gram of tissue and centrifuged at 1000 g for 15 minutes at 4 ° C. The pellet was then removed and the supernatant was centrifuged again at 10000 g at 4 ° C. for 15 minutes. After centrifugation, the pellet was resuspended in the same buffer used for homogenization and analyzed for carnitine palmitoyl transferase (CPT) and β-oxidation activity. The supernatant was further centrifuged at 105000 g for 1 hour at 4 ° C. The resulting supernatant was measured for glucose-6-phosphate dehydrogenase (G6PD) activity, and the pellet was resuspended in the same buffer containing triethanolamine, EDTA, and DTT and subjected to fatty acid synthase (FAS) and malic enzyme (ME) activity. Was analyzed. Meanwhile, protein content was determined using Bradford protein assay (Anal. Biochem. 1976, 72, 248254).

G6PD activity was determined based on the reduction of 6 mM NADP + by G6PD in the presence of glucose-6-phosphate. The enzyme activity was determined by monitoring the increase in absorbance of the reaction mixture at 340 nm at 37 ° C. and 340 nm.

ME activity was determined using the Ochoa (Methods Enzymol .; 1955; Vol. 1, pp 735-739.) Method. The reaction mixture contained 0.4 M triethanolamine (pH 7.4), 30 mM malic acid, 0.12 M MgCl 2, and 3.4 mM NADP. Absorbance was measured at 27 ° C. at 340 nm.

FAS activity was measured using spectrometry of Gibson and Hubbard (Biochem. Biophys. Res. Commun. 1960, 3, 531535). The measurement mixture contained 125 mM potassium phosphate buffer (pH 7.0), 10 mM EDTA, 10 mM β-mercaptoethanol, 33 μM acetyl-CoA, 100 μM malonyl-CoA, and 100 μM NADPH. Malonyl CoA was added to the mixture and the change in absorbance was recorded at 30 ° C. to 340 nm. The activities of G6PD, ME, and FAS were expressed as μmol or nmol converted NADPH / min / mg protein.

6) Determination of Plasma Adipokine Concentration

Leptin levels in plasma were measured using an enzyme immunoassay (EIA) kit (Spi-Bio, Montigny le Bretonneux, France), and the concentrations of resistin, adinopectin and TNF-R were measured using enzyme-linked immunosorbent assay (ELISA) kits. Shibayagi Co., Gunma, Japan).

7) statistical analysis

All data are expressed as mean ± SE. The data were assessed by one-way ANOVA using the Statistical Package for Social Sciences software program (SPSS Inc., Chicago, IL, USA), and the differences between means using Duncan's multiple range test. It was measured by. Statistical significance was considered at p <0.05.

8) Blood glucose measurement

Blood glucose levels were measured using the Accu-Chek Active Blood Glucose Test Strips (Roche Diagnostics GmbH, Germany) after 12 hours of fasting.

Test Result: Cholesterol lowering effect with increasing sericin content

1) weight gain

There was no significant difference in the initial body weight of the mice before the experimental diet, and the daily feed intake was similar in all animal groups (Table 11). However, at the end of the experimental period, significant body weight gain was observed in the HF and HF-F100 mouse groups compared to the NC group. In contrast, HF-F81 and HF-F50 mice showed lower body weight than HF animals. Therefore, mean weight gain and feed efficiency ratio (FER) were highest in the HF and HF-F100 groups. Dietary supplementation of F50 silk protein significantly reduced final body weight and mean weight gain compared to normal levels in high fat diet mice. The addition of F81 to the diet also resulted in a significant decrease in body weight. These indicate that diets supplemented with silk proteins, especially F81 and F50, can inhibit weight gain in high-fat-induced mice.

2) organ weight

Liver and heart weights did not differ significantly between animal groups (Table 12). Kidney weight was significantly higher in HF mice compared to HF-F50 group. The weights of epididymis, peri-renal and visceral adipose tissue were the lowest in control mice. Significant increases in body fat were observed in HF mice. However, in the F81 and F50 diets, the amount of adipose tissue was significantly reduced. In the F100 diet, the amount of visceral fat was decreased, but the fat tissue of the epididymis and kidney were not decreased in the high fat diet. Thus, the reduced body weight observed in the HF-F81 and HF-F50 groups seems to be due to a decrease in the amount of fat in the mice. Interestingly, as the sericin amount of silk protein increased and the fibroin amount decreased, both body weight and white adipose tissue weight decreased, indicating that sericin is more effective than fibroin in reducing high fat-induced body fat in mice. .

3) plasma and fecal lipids

High fat diet resulted in a significant increase in plasma triglyceride and total cholesterol levels in mice. Previous studies have shown that fat diets result in significant increases in plasma total cholesterol and triglyceride content in several experimental animals (Gallou-Kabani et al., Obesity 2007, 15, 19962005; Kim et al., Nutr. Res. ( NY) 2002, 22, 715722). However, diet of silk protein resulted in a significant decrease in plasma total cholesterol in high fat mice. Dietary supply of silk proteins F81 and F50 also resulted in lower plasma triglyceride concentrations. Moreover, they showed the highest concentrations of HDL-cholesterol and HDL-cholesterol / total cholesterol ratios (HTR). The higher the HDL-cholesterol level, the lower the risk of heart disease. HDL particles have antiatherogenic properties because they facilitate the translocation of cholesterol from peripheral tissues to the liver for metabolism (Nofer et al., Atherosclerosis 2002, 161, 116). Thus, atherosclerosis (AI), which measures the risk of coronary heart disease, was lowest in the HF-F81 and HF-F50 groups, and highest in the control and HF groups, indicating that F81 and F50 were associated with an increased risk of atherosclerosis. Indicates that it can be reduced.

Of the silk protein-supplied mouse groups, only HFF50 animals showed significantly lower free fatty acid and GOT levels than the HF group. The amount of GPT was not significantly different between groups. Plasma enzymes GOT and GPT are specific markers of liver injury (Ozaki et al., Arch. Biochem. Biophys. 1995, 318, 439445). The reduced activity of these enzymes indicates a decrease in oxidative stress of the liver under high fat diet conditions.

Although pure fibroin silk protein F100 significantly reduces total cholesterol levels and improves HTR and AI values, plasma total cholesterol, triglycerides, free fatty acid, GOT, GPT as dietary levels decrease fibroin levels and increase sericin levels. It is clear that HDL-cholesterol and HTR levels increase while levels and AI decrease. This suggests that sericin is more effective than fibroin in improving lipid metabolism in mice at high fat diet. In the test examples of the present invention, the HF-F50 mouse group showed a significant increase in fecal triglyceride and total cholesterol compared to the NC and HF groups. In addition, HF-F100 mice showed higher fecal cholesterol concentrations than HF animals. The increase in excretion triglyceride and total cholesterol content may be due to changes in cholesterol and triglyceride uptake in the intestine. This increased excretion of lipid excretion accounts for the effect of silk protein on reducing plasma triglyceride and total cholesterol levels in high fat mice.

4) Lipid Regulatory Enzyme Activity

The fat cell enzymes, G6PD, ME and FAS, tended to increase slightly in the fat diet HF compared to the normal diet, but the activity of enzymes decreased as silk protein was added to the fat diet. As the sericin content increased, the degree of enzyme activity was decreased.

Lipoforming enzymes are essential for fatty acid and cholesterol biosynthesis (Park et al., Mol. Cell. Biol. 2005, 25, 51465157), and reduced activity of these enzymes can reduce the availability of fatty acids required for liver triglycerides synthesis. . Thus, the results in Table 14 indicate that as silk proteins F81 and F50 decrease the activity of adipogenic enzymes, they can reduce cholesterol and triglyceride levels in high fat mice by enhancing fatty acid oxidation in adipocytes and partially inhibiting adipogenesis. It means that increasing the sericin content is helpful for lipid metabolism.

5) Plasma Adipokine Concentration

Adipose tissue produces a variety of hormones called adipokines that are involved in the regulation of glucose and lipid metabolism (Silha et al., Obesity 2006, 14, 13201329). Previous studies have shown that the expression, production and release of adipokines such as leptin, resistin and TNF-R increase with obesity (Trayhurn and Wood, Biochem. Soc. Trans. 2005, 33, 10781081). Adiponectin, on the other hand, has been shown to be reduced in obesity and has anti-atherosclerosis, anti-inflammatory and hepatoprotective functions (Wang et al., Drug Dev. Res. 2006, 67, 677686.). In the test examples of the present invention, a significant increase in leptin, resistin and TNF-R levels was observed in high fat mice compared to the control (Table 15). However, silk protein F50 diet offset the increase in adipokine concentrations. F100 and F81 also significantly increased resistin levels in mice. Both HF-F50 and HF-F81 mouse groups showed significantly larger amounts of adiponectin compared to NC and NF groups. This means that the reduction of leptin, resistin and TNF-R and the increase of plasma adiponectin by silk protein may contribute to the antihyperlipidemic effect of mice under high fat diet. In addition, the results also indicate that, among the silk proteins analyzed, F50 is most effective in regulating the production of plasma adipokines, while F100 is the least effective, which also means that sericin will have a greater anti-obesity effect than fibroin. Imply that it can.

6) Change in blood sugar level

The initial fasting glucose level of each diet group was 80 mg / dl except for HF group and started with normal blood glucose, and the concentration change measured every two weeks during the eight-week experiment period is shown in FIG. 1. As the 4th week, the blood sugar levels of the HF group, the normal diet group and the F100 group gradually increased, whereas the blood sugar levels of the F81 and F50 groups remained almost the same. At week 6, however, blood glucose levels increased in all groups, with the steepest increase in HF. This indicates that the blood glucose increase decreases as the sericin content is higher than that of the HF group.

Test examples of the invention demonstrated that dietary supply of silk protein significantly inhibited weight gain, decreased body fat amount, and improved lipid metabolism in high fat mice. The antihyperlipidemic effect was due in part to inhibition of adipokine production and regulation of adipokine production in liver and adipocytes. Silk protein has also been shown to be effective in lowering blood sugar. Moreover, of the fibrillated F100 of pure fibroin among the analyzed silk proteins, F50 with the highest sericin content and low fibroin content significantly improves lipid metabolism, so that the higher fat-induced hyperlipidemia and obesity with increasing sericin in silk protein composition It has been shown to be more effective and to be more effective in lowering blood sugar. Thus, silk protein may be useful as a raw material in the development of functional foods or therapeutics for obesity, hyperlipidemia, diabetes and related diseases.

Claims (13)

A composition for lowering blood sugar or cholesterol, comprising a polymer silk fibroin having an average molecular weight (Da) of 190,000-400,000 as an active ingredient.
The composition of claim 1, wherein the polymer silk fibroin has a viscosity (cps) of 70-350 in a 10% (w / w) silk formic acid solution.
The composition for lowering blood sugar or cholesterol according to claim 1, further comprising sericin as an active ingredient.
According to claim 3, wherein the weight ratio of the polymer silk fibroin and sericin is 80:20 to 50:50, the composition for lowering blood sugar or cholesterol.
Functional food for improving or preventing diabetes mellitus, obesity or hyperlipidemia, comprising as a raw material a composition for lowering blood sugar or cholesterol according to any one of claims 1 to 4.
Method for preparing silk protein comprising polymeric silk fibroin and sericin having an average molecular weight Da of 190,000-400,000 including the following steps:
a) refining silk cocoon for 1-3 hours at 90-100 ° C. in distilled water without a degumming agent;
b) washing the refined silk cocoon with distilled water at a temperature of 60-80 ° C .;
c) dissolving the washed silk cocoon in LiBr aqueous solution or CaCl ₂ / water / ethanol mixed solvent;
d) dialysis of the silk protein solution with a molecular weight cutoff tube of 12,000-14,000 to remove salts; And,
e) lyophilizing the salt-free silk protein solution.
The method of claim 6, wherein in step c), the CaCl ₂ / water / ethanol mixed solvent has a molar ratio of 1-2 / 8-16 / 2-4.
7. The method of claim 6, wherein the c) is dissolved in a CaCl ₂ / water / ethanol mixed solvent at 80-90 ° C. for 3 to 60 minutes.
Method for preparing silk protein comprising polymeric silk fibroin and sericin having an average molecular weight Da of 190,000-400,000 including the following steps:
a) refining silk cocoon in a refining solution in step 6) to remove sericin and performing the remaining steps to produce a polymeric silk fibroin having an average molecular weight Da of 190,000-400,000; And,
b) mixing sericin with the polymer silk fibroin.
10. The method of claim 9, wherein the degumming agent of step a) is sodium oleate and sodium carbonate.
10. The method of claim 9, wherein the sericin in step b) is a method for producing silk protein, characterized in that 100% silk sericin extracted from silk cocoon.
The method of claim 9, wherein the mixing weight ratio of the polymer silk fibroin and sericin in step b) is 80:20 to 50:50.
A pharmaceutical for treating or preventing diabetes mellitus, obesity or hyperlipidemia, comprising as a raw material a composition for lowering blood sugar or cholesterol according to any one of claims 1 to 4.

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