KR100365291B1 - An agent for preventing development of alcoholic fatty liver comprising an active substance derived from silk fibroin - Google Patents

Abstract

The present invention relates to an alcoholic fatty liver formation preventive agent comprising silk fibroin as an active ingredient, and more particularly, to forming alcoholic fatty liver containing silk fibroin of low molecular weight which can be ingested into the human body by refining treatment and hydrolysis. It is about a preventive agent. Silk fibroin is a protein that can be obtained from cocoon and consists of 18 kinds of amino acids such as glycine, alanine, tyrosine, etc. This silk fibroin has excellent biocompatibility and has been used as cosmetic raw material, medical suture, bio material, and diabetes treatment agent from the past. Has been. However, there is no known or verified whether such silk fibroin has a protective effect against alcoholic fatty liver. The present invention finds and demonstrates that can be used as an active ingredient of alcoholic fatty liver formation preventive agent, and is an invention for a new use of silk fibroin.

Description

An agent for preventing development of alcoholic fatty liver comprising an active substance derived from silk fibroin}

The present invention relates to an alcoholic fatty liver formation preventive agent comprising silk fibroin as an active ingredient, and more particularly, an alcoholic fatty liver formation preventing agent comprising silk fibroin having a low molecular weight that can be absorbed by the human body by refining treatment and hydrolysis, etc. It is about.

Silk fibroin is a protein that can be obtained from cocoon and consists of 18 kinds of amino acids such as glycine, alanine, tyrosine, etc. The silk fibroin has high recovery rate and excellent biocompatibility. It has been used as a medical suture, bio material and diabetes treatment. However, it has not been known or verified in clinical trials whether the silk fibroin has a protective effect on alcoholic fatty liver.

The present inventors have focused on the fact that silk fibroin has an effect of promoting alcoholic metabolism, and it has been proved through preclinical experiments that the silk fibroin can be used as an active ingredient of an alcoholic fatty liver formation preventive agent. It is an invention of a new use as an agent for preventing fatty liver formation.

About 5% of normal liver is composed of fatty tissue, and triglycerides, fatty acids, phospholipids, cholesterol, and cholesterol esters are the main components of fat, but once fatty liver occurs, most of the components are replaced with triglycerides. Fatty liver is defined as when fat is observed in at least 5% of hepatocytes or at least 5 mg of fat per 100 mg of liver. Fatty liver is caused by a disorder of fat metabolism in the liver, which is known to be caused by a fat metabolism disorder in the liver cells or a defect in the process of carrying excess fat or hydrous carbon.

In general, fatty liver may simply be thought of as a buildup of fat in the liver, but considering that 50% of patients with alcoholic fatty liver and 30% of patients with nonalcoholic fatty liver already have cirrhosis, fatty liver is very It should be considered one of the serious liver diseases.

On the other hand, alcohol is not stored in the body and is completely lost by oxidation in the liver. Specifically, alcohol in the liver is largely alcohol dehydrogenase (ADH) pathway, microsomal ethanol oxidizing system It is metabolized and converted into acetaldehyde by three pathways: the MEOS pathway and the catalase pathway, which are in turn metabolized to acet salts by aldehyde dehydrogenase (ALDH). At this time, acetaldehyde is toxic and can damage liver cells, and also fatty acids are produced as a result of the metabolism of alcohol, fat is accumulated in the liver. At this time, the fatty liver accumulated in the liver is called alcoholic fatty liver.

Alcoholic fatty liver can progress to chronic liver disease. Among patients with alcoholic fatty liver, 10-35% are reported to progress to hepatitis, and 8-20% to cirrhosis, and alcoholic fatty liver patients are cirrhosis compared to patients with nonalcoholic fatty liver. It is reported that they have more and are more likely to develop hepatitis.

However, until now, no proper preventive agent has been developed for fatty liver patients.

The present inventors focused on the report that silk fibroin can promote alcohol metabolism and regulate blood cholesterol, and through preclinical experiments, it has been found that silk fibroin has a prophylactic effect on alcoholic fatty liver. It is to provide an alcoholic fatty liver formation prevention agent as an active ingredient.

1 is a table showing the composition of the normal diet,

2 is a table showing the composition of the alcohol diet,

3 is a chart showing the weight change of the experimental group,

Figure 4 is a chart showing the change in the plasma AST of the experimental group,

5 is a chart showing the ALT change in the plasma of the experimental group,

6 is a table showing the total cholesterol content contained in the plasma of the experimental group,

7 is a chart showing the triglyceride content contained in the plasma of the experimental group,

8 is a chart showing the high density lipoprotein cholesterol content contained in the plasma of the experimental group,

9 is a chart showing the lipid content accumulated in the liver of the experimental group,

10 is a chart showing the changes in the activity of ADH and ALDH in the liver of the experimental group,

11A to 11D are enlarged photographs of liver tissue of the experimental group.

Silk fibroin is a fibrous protein inherently insoluble high molecular compound that is not absorbed into the body by itself. Therefore, in order to digest and absorb it in the body, it is necessary to purify silk fibroin and decompose it into low molecules.

In order to purify and decompose silk fibroin, pigment, fat and other impurities are first removed from the silk fiber, and then refined to remove sericin to extract pure high molecular weight fibroin, and acid hydrolysis of the high molecular weight fibroin. To decompose into low molecular weight fibroin having an average molecular weight of 200-500. The low molecular weight fibroin is concentrated through a reduced pressure concentrator, and then automatically sterilized at 2-3 ° C. for 2-3 seconds and freeze-dried to form a powder.

This powdery fibroin has a low molecular weight and is easily digested and absorbed in the body, so that it may be eaten as it is or dissolved in a liquid such as water. The present invention provides an alcoholic fatty liver formation preventive agent comprising a low molecular weight silk fibroin having an average molecular weight of 200-500 as an active ingredient.

Hereinafter, the present invention will be described based on Examples.

(Example)

18 rats (hereinafter referred to as "mouse") were selected and randomly divided into 3 experimental groups of 6 rats each. The first group is defined as the "normal control" to feed the normal liquid diet, the second group is designated as the "alcohol intake group" to feed the alcoholic liquid diet, and the third group is "simultaneously fed the alcoholic liquid diet and silk fibroin simultaneously. Intake group ".

The composition of the diet fed to the normal control is shown in Figure 1, the composition of the diet fed to the alcohol intake group is shown in Figure 2. Comparing the diet fed to the normal control group (hereinafter referred to as the "normal diet") and the diet fed to the alcohol intake group (hereinafter referred to as the "alcohol diet"), the alcohol diet contains alcohol instead of 25.6 g of maltodextrin in the composition of the normal diet. Add 50g of calories to match your normal diet. Concomitant groups were fed 5% by weight of silk fibroin to the alcohol diet. Mice were placed in cages for the duration of the experiment and raised under artificial lighting conditions (temperature 102 ± 2 ° C., humidity 55 ± 10%, 12 hour cycle illumination).

Alcoholic fatty liver testing is determined by clinically enzymatic chemical testing of alanine aminotransferase ("ALT") and aspartate aminotransferase ("AST") in the blood. . In general, fatty liver increases aminotransferase (ALT AST) (usually 300 IU / L or less), which is characterized by an increase of AST 2-3 times compared to ALT.

In this example, the body weight, plasma ALT and AST, plasma and liver lipid levels, alcohol dehydrogenase (ADH), and aldehyde dehydrogenase (ALDH) activity in clinical trials were examined to test fatty liver formation and liver function in mice. Were examined and finally slaughtered for histopathological examination.

Figure 3 is the result of measuring the weight at intervals of one week from the start of the experiment. In Figure 3, the normal control group and the simultaneous intake group shows that the weight is gradually recovered after one week after the start of the experiment gradually recovered. The weight loss during the week is thought to be due to the change in feed (from solid food to liquid diet). The alcoholic group, however, continued to lose weight and died 100% before three weeks.

4 and 5 show AST and ALT as indicators of liver function, and blood was collected from the orbital vein of the mouse at intervals of two weeks from the beginning of the experiment, and then measured by separating the plasma by centrifugation at 3000 rpm for 10 minutes. . (Measurement of cholesterol, high density lipoprotein cholesterol and triglycerides performed in this example was also measured in the same manner)

In FIG. 4, in the alcohol intake group, AST in plasma showed a sharp rise up to two weeks and finally died. On the other hand, in the simultaneous intake group, AST was similar to that of the normal control group until 2 weeks after administration, and tended to increase slightly compared to the normal control group, but the difference was not significant.

In Figure 5, the change in ALT in the plasma also showed a tendency similar to AST. The alcohol intake group showed a sharp increase in ALT until two weeks after administration, whereas the concurrent intake group showed a slightly higher level than the normal control group until two weeks, but it was significantly lower than the alcohol intake group ( 46.02%). At 4 weeks, the concurrent intake group was 41.00 ± 9.44 IU / L, which showed higher ALT compared to the normal control group, but all survived.

Figure 6 shows the total cholesterol content in the plasma of each group at the interval of two weeks after the start of the experiment.

In FIG. 6, the alcohol intake group showed a somewhat higher level than the normal control group and the concurrent intake group, but it was not statistically significant.

Figure 7 shows the triglycerides accumulated in the plasma at the interval of two weeks after the start of the experiment. In Figure 7, it can be seen that the triglycerides increased sharply in the alcohol intake group, while there is no significant difference between the simultaneous intake group and the normal control group. In addition, when 4 weeks passed, the triglycerides were decreased in the simultaneous intake group compared to the normal control group.

Figure 8 shows the high density lipoprotein cholesterol content after 4 weeks of the experiment. High density lipoprotein cholesterol is known to play a role in lowering the total content of cholesterol in the blood. The high-density lipoprotein cholesterol content of the alcohol intake group was 7.60 ± 5.48 mg / dl at 2 weeks after the start of the experiment, which was significantly lower than the high-density lipoprotein cholesterol of the control group and the simultaneous intake group measured at 4 weeks after the start of the experiment.

9 shows the amount of lipid accumulated in the liver. 9, the results of measuring the total cholesterol and triglyceride content accumulated by taking the liver of the alcohol intake group (immediately after death) are significantly higher than the lipid content of the normal control group and the simultaneous intake group (at the end of the experiment). It can be seen that there is almost no difference between the simultaneous intake group and the normal control group.

Figure 10 shows the activity of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in the liver. In this measurement, 20 mM pH 7.4 phosphate buffered saline containing 1 mM reduced glutathione and 1% Triton X-100 was made into a 10% liver homogenate, followed by centrifugation of the supernatant. Used.

10, the results of measuring the activity of ADH and ALDH by collecting liver immediately after the death of the alcohol intake group were significantly increased compared to those measured at the end of the experiment in the normal control group and the simultaneous intake group, but the simultaneous intake group and the normal control group were large. No difference was observed. From these results, it is thought that fibroin inhibits the formation of activated radicals by alcohol.

After the above procedure, for the histopathological observation of the liver, the alcohol intake group collected liver immediately after death. Fixed in neutral formalin for 24 hours. Subsequently, the tissue sections were stained with hematoxylin eosin (HE) and observed 100 times under an optical microscope. As a result, it was found that fatty liver was formed in the alcohol-ingested group.

However, when fibroin was ingested with alcohol at the same time, normal hepatocytes exist from around the central vein, and it can be seen that hepatocellular damage was prevented by fibroin administration. In addition, most of the liver tissues showed little alcoholic fat deposition, and thus, fatty liver was hardly observed or its distribution was very small. It was confirmed that most cells exist as normal liver cells.

Pictures of liver cells of each experimental group are shown in FIG. 11. 11A is hepatocytes of a normal control group, and B is hepatocytes of an alcohol intake group. C and D are photographs of hepatocytes in the simultaneous intake group. As shown in C and D of the photograph 9, the alcoholic fat deposition hardly occurred in the group of simultaneous intake so that there was no fear or the size of the fear was very small, and normal hepatocytes as in the photograph 9A were observed.

As seen in the examples, silk fibroin decomposed to low molecular weight to be digested and absorbed has an excellent effect in preventing alcoholic fatty liver and also in preventing alcoholic fatty liver formation. Given that the treatment for fatty liver has not yet been developed, the fatty liver formation preventive agent of the present invention using silk fibroin as an active ingredient may be of great value in medical terms.

Claims (3)

Alcoholic fatty liver formation prevention agent which uses silk fibroin as an active ingredient. 2. The alcoholic fatty liver formation preventive agent according to claim 1, wherein the silk fibroin is decomposed into a low molecular weight absorbable by the human body. The alcoholic fatty liver formation preventive agent according to claim 1 or 2, wherein the molecular weight of silk fibroin is 200-500.