KR100455653B1 - Components for lowering blood cholesterol level extracted from shrimp fermentation product - Google Patents

Abstract

The present invention centrifuged for 10 minutes at 10,000 rpm fermentation broth, the precipitate was removed and the supernatant was recovered and then filtered for 18 hours at 4 ℃ by ultrafiltration membrane for molecular weight 40,000 Dalton, the filtrate was recovered, lyophilized Then, the dried powder was re-dissolved in Tris-HCl buffer and filtered with an ultrafiltration membrane having a molecular weight of 400 Dalton at 4 ° C. for 18 hours, the filtrate was removed, the remaining liquid was lyophilized, and the dried powder was extracted three times with distilled water under reduced pressure. To prepare a water-soluble fraction having a cholesterol lowering function by drying, to recover the water-insoluble fraction, extracted three times with ethanol, and dried under reduced pressure in vacuo to prepare an ethanol fraction having a cholesterol lowering function extracted from shrimp fermentation broth. . Since the shrimp fermented broth extract of the present invention reduces blood cholesterol, it can be usefully used as a preventive and therapeutic agent for various cardiovascular diseases or as a component of a health food.

Description

Components for lowering blood cholesterol level extracted from shrimp fermentation product}

The present invention relates to a method for producing water or ethanol fractions exhibiting blood cholesterol lowering function extracted from shrimp fermentation broth and blood cholesterol lowering fractions produced by the method.

The present invention provides an effective substance exhibiting a blood cholesterol-lowering function extracted from a shrimp fermentation broth, which means a fermented food containing shrimp, including shrimp salted fish. More specifically, the shrimp fermentation broth was centrifuged at 8,000 to 12,000 rpm for 10 to 15 minutes, the precipitate was removed, the supernatant was collected, and then filtered through an ultrafiltration membrane at 4 to 7 ° C. for 18 to 24 hours, and then the filtrate was recovered. After freeze-drying, the dried powder was dissolved in Tris-HCl buffer and filtered through an ultrafiltration membrane at 4-7 ° C. for 18-24 hours, the filtrate was removed, the residue was lyophilized, and the dried powder was distilled with 2-4 Extraction was performed once and dried under vacuum and extracted with distilled water. Then, the insoluble fractions were recovered, extracted 2 to 4 times using ethanol, and each fraction was dried under vacuum and dried under reduced pressure from shrimp fermentation broth. It is to provide an effective substance having a.

Coronary cardiovascular disease currently accounts for more than 30% of all deaths, and in developed countries nearly 40%. On the other hand, in Korea, due to economic development, the cause of westernization of diet, lack of exercise, and overwork are accumulating, and the mortality rate from heart disease due to rapid weight gain and blood cholesterol increase is increasing, especially cerebrovascular disease such as stroke. Mortality rate is 17%.

High blood cholesterol levels are known to cause atherosclerosis. Ross. R., Nature 362: 801-809 (1993). In particular, the amount of low-density lipoprotein (LDL) which is involved in the transport of cholesterol from the blood in proportion to the cause atherosclerosis, particularly oxidized LDL is known to be a strong atherosclerosis-induced effects [Note: SG Young and S. Parthasarathy, West J. Med 160: 153-164 (1994).

Reducing the amount of cholesterol in the blood is a way to reduce the intake of foods that contain too much fat, and continue to exercise. However, for people who already have high levels of cholesterol in the blood, eating foods containing special active ingredients may be one way to lower the level of cholesterol in the blood. Recently, there is a great deal of research to separate substances from natural products that can provide continuous treatment and preventive effects at the same time by deviating from the method of treating them with hypercholesterolemia patients.

Atherosclerosis is known to be one of the leading causes of high lipids in the blood [Goldstein, J., Schrott, H., Hazzard, E., Bierman, E. and Motuski, A. J. Clin. Invest. 52: 1544-1568 (1973)] or stroke [see: Tell, G., Crouse, JR and Furderg CD Stroke 19: 423-430 (1989)]. Drugs used for the treatment or prevention of lovastatin, HMG-CoA reductase inhibitors [Alfred, G., Theodore, W., Allan, S. and Palmer, T. The pharmacological basis of therapeutics 8th ed: 881-886 (1991)] Fenofibrate based on fibric acid (Alfred, G., Theodore, W., Allan, S. and Palmer, T. The pharmacological basis of therapeutics 8th ed: 881-886 (1991)) It is used to treat patients. Since HCP-CoA reductase inhibitors have been discovered from Penicillium citrinum , a compound called comppactin, inhibitors such as lovastatin have been developed by chemical modifications, and examples of the development of cholesterol inhibitors from natural products have been developed.

In addition, Choi et al. Extracted flavonoids with antihyperlipidemic effect from causality [JS Choi. J. Nat. Prod. 54 (1): 218-224 (1991)], Noda extracted the gamma-butyrobetaine and porphyran from seaweed seaweed and confirmed the cholesterol lowering effect [Noda H J] appl. Phycol. 5: 255-258 (1993).

Until now, there have been reported active substances having cholesterol lowering effects in various natural products, but no active substances extracted from shrimp fermentation broth have been reported, and the present inventors have invented a method of extracting active substances having cholesterol lowering effect from shrimp fermentation broth. The present invention was completed by experimentally demonstrating the cholesterol lowering effect of the active substance in the blood of animals.

The technical problem to be achieved by the present invention is to provide an effective substance having a cholesterol lowering function extracted from a shrimp fermentation broth, in which the effective substance showing a cholesterol lowering function has not been isolated.

1 is an extraction separation process diagram showing a process of extracting an active substance composition showing a blood cholesterol lowering function of the present invention from a shrimp fermentation broth.

Accordingly, it is an object of the present invention to provide an active substance exhibiting cholesterol lowering function extracted from shrimp fermentation broth.

That is, after centrifuging the shrimp fermentation broth at 8,000 to 12,000 rpm for 10 to 15 minutes, the precipitate was removed, the supernatant was collected, and then filtered through a primary ultrafiltration membrane at 4 to 7 ° C. for 18 to 24 hours, and then the filtrate was recovered. After lyophilization, the dried powder was dissolved in Tris-HCl buffer and filtered through a secondary ultrafiltration membrane at 4-7 ° C. for 18-24 hours, the filtrate was removed, the residue was lyophilized, and the dried powder was distilled with 2∼ Four times extraction was carried out in vacuo under reduced pressure to prepare an aqueous fraction having a molecular weight of 400 to 40,000 Dalton having a cholesterol lowering function extracted from shrimp fermentation broth.

In addition, the water-soluble fraction was removed from the residue, the water-insoluble fraction was recovered and extracted 2-4 times with ethanol, and the fractions were dried under vacuum and dried under reduced pressure, and the ethanol having a molecular weight of 400-40,000 Dalton, which had a cholesterol lowering function extracted from the fermentation broth. Prepare fractions.

The Tris-HCl buffer is a Tris-HCl buffer of pH 7.0-8.0 with a pH of 15 to 25 mM, and the ultrafiltration membrane used in the first ultrafiltration is an ultrafiltration membrane capable of passing a molecular weight of 30,000-40,000 dalton or less, and a secondary ultrafiltration membrane. The ultrafiltration membrane used for filtration is an ultrafiltration membrane that can pass only a molecular weight of 400 ~ 500 dalton or less, the ethanol is 95 ~ 99.9% ethanol, characterized in that extracted with two solvents, water and ethanol.

In another aspect, the present invention is to provide an effective substance exhibiting a cholesterol lowering function of 500-40,000 daltons molecular weight extracted from the above two solvents in shrimp fermentation broth.

Shrimp fermentation extract of the present invention can be added to food for the purpose of lowering blood cholesterol. Shrimp fermentation broth extract can be added to the development of dietary supplements, for example, various foods, meat, beverages, chocolate, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, alcoholic beverages , Vitamin complexes and dietary supplements.

Hereinafter, the present invention will be described in detail.

It is an object of the present invention to provide an effective substance exhibiting cholesterol lowering function extracted from shrimp fermentation broth. Extraction of active substance from shrimp fermentation broth is as follows. That is, after centrifuging the shrimp fermentation broth at 8,000 to 12,000 rpm for 10 to 15 minutes, the precipitate was removed, the supernatant was collected, and then filtered through a primary ultrafiltration membrane at 4 to 7 ° C. for 18 to 24 hours, and then the filtrate was recovered. After lyophilization, the dried powder was dissolved in Tris-HCl buffer and filtered through a secondary ultrafiltration membrane at 4-7 ° C. for 18-24 hours, the filtrate was removed, the residue was lyophilized, and the dried powder was distilled with 2∼ Four times extraction was carried out in vacuo under reduced pressure, extracted with distilled water, and the insoluble fractions were recovered, and extracted two to four times using ethanol, and each fraction was dried under reduced pressure in vacuo to consist of water extraction fraction and ethanol extraction fraction from shrimp fermentation broth. It is to provide an effective substance having a cholesterol lowering function.

In the present invention, the cholesterol lowering effect of the water fraction or the ethanol fraction extracted from the fermentation broth by the above method was confirmed in the animal experiment using a rat.

For artificial cholesterol induction in the experimental group, Triton WR-1339 was dissolved in physiological saline at a concentration of 120 mg / ml, and then cholesterol was induced by intraperitoneal injection of mice at 600 µg / g. In order to confirm the cholesterol lowering effect, the water fraction or ethanol fraction extracted from the shrimp fermentation broth was dissolved in physiological saline after drying under reduced pressure in a physiological saline solution and administered orally at 10 mg / 100 g.

First, 30 rats were divided into 5 groups of 6 rats, and the experiment was conducted in the following manner.

Iii) The control group was orally administered with only 1ml / 100g saline without causing cholesterol.

Ii) Triton group was orally administered to the cholesterol-induced rats in the ratio of 1ml / 100g only to the rats.

로) The lovastatin group dissolves 2 tablets of mebaco (Lovastatin preparation: lovastatin 20mg / tablet) purchased in Sino-Pharmaceutical Drugs in 10ml of saline solution and orally administered to rats artificially causing cholesterol. It was.

V) Water fraction group was orally administered 1 ml per 100 g of rat body weight which artificially induced cholesterol by dissolving the water fraction extracted from shrimp fermentation broth at 10mg / ml.

Iii) The ethanol fraction group was orally administered 1 ml per 100 g of rat body weight which artificially induced cholesterol by dissolving the ethanol fraction extracted from shrimp fermentation broth at 10 mg / ml.

The average cholesterol concentration in the blood of all the rats used in the present invention was measured immediately before the experiment, and the concentration was set to the normal cholesterol concentration, and after the oral administration of each water fraction and the ethanol fraction, blood was collected at intervals of 5 hours up to 30 hours. The change in concentration was measured.

The measurement of cholesterol concentration in all experimental groups was performed by taking 100 μl of blood from the tail vein of each hour, centrifuging for 5 min at 5,000 rpm, and taking 10 μl from the plasma except for the submerged blood cells. The total cholesterol measurement solution (AM-202-K) purchased from was reacted at 37 ° C. for 5 minutes and measured at an absorbance of 500 nm using a spectrophotometer.

Through this experiment, it was confirmed that the blood cholesterol concentration decreased when the water fraction or ethanol fraction extracted from the shrimp fermentation broth was orally administered to the mice, respectively.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples do not limit the scope of the present invention.

Example 1 Extraction of an Active Substance Showing Cholesterol Lowering Function

1 shows a method for extracting an active substance showing cholesterol lowering function from shrimp fermentation broth according to the present invention. As shown in Figure 1, the shrimp fermentation broth was centrifuged at 10,000 rpm for 10 minutes, the precipitate was removed, the supernatant was collected, and then filtered by ultrafiltration membrane at 4 ° C. for 18 hours. Thereafter, the filtrate was recovered and lyophilized, and the dried powder was dissolved in 20 mM Tris-HCl buffer (pH 7.5), which was then filtered through an ultrafiltration membrane at 4 ° C. for 18 hours, the filtrate was removed, and the residue was lyophilized and dried powder. Extracted three times with distilled water and dried under vacuum to obtain a water fraction having a cholesterol-lowering function extracted with water. An active substance having a cholesterol lowering function was prepared.

Example 2 Extraction of an Active Substance Showing Cholesterol Lowering Function

The water fraction prepared in Example 1 was removed, the water-insoluble fraction was recovered, extracted three times with 95 to 99.9% ethanol, and the supernatant was dried under reduced pressure under vacuum to obtain an ethanol fraction extracted from shrimp fermentation broth with ethanol. An active substance having a cholesterol lowering function was prepared.

Example 3 Preparation of Experimental Animal for Verification of Efficacy of High Cholesterol Lowering Agent

In order to confirm the cholesterol lowering effect, an experimental rat artificially increased the concentration of cholesterol in the blood was prepared and used in the present invention. More specifically, Triton WR-1339, purchased from Sigma, was dissolved in physiological saline at a concentration of 120 mg / ml and intraperitoneally injected to a concentration of 600 µg per 1 g of the mouse. Blood was collected from the tail vein at intervals of 5 hours up to 30 hours after intraperitoneal injection, and total cholesterol concentration in blood was measured using total cholesterol measurement solution purchased from Asan Pharmaceutical.

Example 4 Use of Standards to Validate High Cholesterol Lowering Agents

Mebaco (Lovastatin preparation) purchased from Sino Pharmaceutical was used as a standard substance for confirming the cholesterol lowering effect on the water fraction or ethanol fraction extracted from the shrimp fermentation broth used in the present invention. Mebaco 2 tablets (lovastatin 40mg) was dissolved in 10ml of distilled water evenly and administered orally at a concentration of 600㎍ / kg.

Example 5 Measurement of Total Cholesterol Concentration in Blood

In the present invention, the total cholesterol measurement solution (AM-202-K) purchased from Asan Pharmaceutical Co., Ltd. was used to measure the total cholesterol concentration in the blood. To describe in detail how to measure total cholesterol in the blood, first, 100 μl of blood is collected from the tail vein of the rat, and then centrifuged at 5000 rpm for 10 minutes. After centrifugation, the plasma portion was recovered, and 10 µl of which was added to 3 ml of total cholesterol measurement solution, mixed well, and reacted at 37 ° C. for 5 minutes. After the reaction, the absorbance is measured at the absorbance of 500 nm using a spectrophotometer, and the total cholesterol concentration in the blood is obtained by comparing with the cholesterol standard curve. Meanwhile, the method for preparing the cholesterol standard curve is the total cholesterol measurement solution (AM-202-K) and Using the included cholesterol standard solution in a concentration range of 100 mg / dL to 1000 mg / dL, add 10 µl of the cholesterol standard solution and 3 ml of total cholesterol measurement solution, and mix well. Let. After the reaction, the absorbance at 500 nm was measured using a spectrophotometer, and the absorbance corresponding to each concentration was calculated to prepare a cholesterol standard curve.

Example 5 Effect of Shrimp Fermentation Extract on Plasma Cholesterol Concentration in Rats

The experimental rats used in the present invention used Sprague-Dawley species of 3 weeks old, purchased and bred in experimental animal feed for 1 week, and 4 weeks old (90-110 g) when the egg mass (randomized) The experimental group was divided and used as a block design. The method for confirming the cholesterol lowering effect on the water fraction or the ethanol fraction prepared by the method of Example 1 and Example 2 is described in detail as follows.

First, four-week-old rats were divided into control group, cholesterol-inducing group, lovastatin group, and water fraction group or ethanol fraction group. In the control group, only 1 ml of saline was administered orally, and the cholesterol-induced group artificially induced cholesterol using Triton WR-1339. It was. Cholesterol-induced + water fraction and cholesterol-induced + ethanol fraction were induced artificially using Triton WR-1339, respectively, and then dissolved 10mg of water and 10mg of ethanol in 1ml of saline solution. 1 ml was administered orally.

In the whole experimental group, blood cholesterol concentration was measured in the tail vein of each rat before oral administration, and then the normal cholesterol level was measured, and then each sample (physiological saline, Triton WR-1339, Triton WR-1339 + lovastatin, Triton) was measured. WR-1339 + water fraction or ethanol fraction) was orally administered and collected in the tail vein of the rat at 5 hour intervals for up to 30 hours to measure blood cholesterol levels.

When the water fraction extracted from the shrimp fermentation broth was orally administered, as shown in Table 1, the average cholesterol concentration of the control group was 165 ± 13 to 198 ± 10 mg / dl. In the case of artificially inducing cholesterol, normal blood cholesterol level was shown up to 5 hours after induction, but increased after 10 hours, and decreased after reaching the highest concentration at 25 hours. Oral administration of lovastatin, used as a cholesterol-lowering standard, suppressed the increase in blood cholesterol levels and suppressed the increase in blood cholesterol levels of about 40.9% at the highest cholesterol level in the blood 25 hours after oral administration.

The water fraction showed a significant inhibitory effect on the increase of cholesterol in the cholesterol-induced group compared to the change in the cholesterol level in the blood, and showed an inhibitory effect on the increase of 79.8% in 25 hours after oral administration. In addition, the ethanol fraction showed lower cholesterol growth inhibition effect than the water fraction, but showed a higher cholesterol suppression effect of 63.8% than lovastatin used as a standard substance at 20 hours and 25 hours after oral administration.

Cholesterol inhibitory effect (%) = {(B-A) / highest cholesterol concentration in the cholesterol-induced group} × 100

A: Maximum cholesterol concentration in the experimental group—initial cholesterol concentration in the experimental group

B: Maximum Cholesterol Concentration in Cholesterol-Induced Group-Initial Cholesterol Concentration in Cholesterol-Induced Group

Blood collection time (h) Total Cholesterol Concentration (mg / dl) Control Cholesterol-inducing group Lovastatin Water fountain Ethanol fraction group 0 166 ± 30 156 ± 33 150 ± 20 158 ± 17 170 ± 29 5 175 ± 10 166 ± 40 189 ± 30 168 ± 25 165 ± 11 10 170 ± 25 309 ± 37 249 ± 15 176 ± 10 201 ± 15 15 198 ± 10 474 ± 45 352 ± 37 184 ± 49 250 ± 35 20 178 ± 10 601 ± 30 471 ± 40 280 ± 35 331 ± 11 25 180 ± 34 672 ± 46 455 ± 10 262 ± 25 357 ± 34 30 165 ± 13 593 ± 35 360 ± 15 244 ± 19 349 ± 21

Example 6 Oral Toxicity Test of Shrimp Ferment Extract

In order to examine the acute toxicity of the water fraction or ethanol fraction extracted in Examples 1 and 2, 5 mice were used as a group, and each fraction was prepared at 100 mg / ml using saline solution. Orally administered in ml. Each fraction was administered orally once and observed for 30 days by the number and visual observation of dead mice from the next day after administration. In addition, the animals were killed and dissected at 30 days of administration, and the internal organs were visually examined.

As shown in Table 2, in the case of acute oral toxicity, no lethal animals were observed for 30 days at a dose of 1,000 mg / kg shrimp extract. An autopsy of the surviving animals was performed 30 days later, and there was no gross finding on the lesions. Normal weight gain was observed for 30 days after oral administration without any weight loss. The purpose of this study was to provide information on available dose in general pharmacology and efficacy test and to obtain basic data on toxicity by identifying the degree of acute toxicity in the oral route of shrimp fermented broth extract.

Oral Toxicity Test Results Dosage 1,000mg / kg Shrimp Ferment Extract Water fraction Ethanol fraction Number of rats killed for 30 days 0 0

The effect of the present invention is to provide an extract having a cholesterol-lowering effect using the two kinds of shrimp fermentation broth and at the same time to develop medicines and foods (health food) for the treatment and prevention of hypercholesterolemia.

Claims (5)

The shrimp fermentation broth was centrifuged at 8,000 to 12,000 rpm for 10 to 15 minutes, the precipitate was removed, the supernatant was collected, and then filtered through a primary ultrafiltration membrane at 4 to 7 ° C. for 18 to 24 hours. The filtrate was recovered and lyophilized. Then, the dried powder was dissolved in Tris-HCl buffer and filtered through a secondary ultrafiltration membrane at 4-7 ° C. for 18-24 hours, the filtrate was removed, the residue was lyophilized, and the dried powder was distilled with 2-4 times. Water-soluble fraction substance with molecular weight of 400 to 40,000 Dalton having cholesterol lowering function extracted from shrimp fermentation broth prepared by vacuum drying under reduced pressure Cholesterol lowering function extracted from shrimp fermentation broth prepared by removing the water-soluble fraction from the residual solution prepared according to the method of claim 1, recovering the water-insoluble fraction, extracting 2-4 times with ethanol, and then drying the fraction under reduced pressure under vacuum. Ethanol fraction with molecular weight 400-40,000 Dalton The tris-HCl buffer according to claim 1 or 2, wherein the Tris-HCl buffer is a Tris-HCl buffer having a pH of 7.0 to 8.0 at 15 to 25 mM, and the ultrafiltration membrane used in the first ultrafiltration may pass only molecular weight of 30,000 to 40,000 dalton or less. It is an ultrafiltration membrane which can be used, and the ultrafiltration membrane used for the second ultrafiltration is an ultrafiltration membrane which can pass only 400 to 500 daltons of molecular weight or less, and the ethanol is a cholesterol lowering function extracted from shrimp fermentation broth, characterized in that 95 to 99.9% ethanol. Fractional substance with Oral cholesterol lowering agent containing an effective amount of the shrimp fermentation broth extract fraction material having the cholesterol lowering efficacy of claim 1 or 2 delete