KR100979889B1 - A composition comprising the powdered form or extract of ascidian Halocynthia roretzi as an active ingredient showing anti-hyperlipidemia activity - Google Patents

Abstract

The present invention is locust ( Halocynthia roretzi ) relates to a composition for the prevention and treatment of hyperlipidemia comprising a dry powder or extract of the extract as an active ingredient, in detail, the dry powder or extract of the larvae of the present invention by significantly reducing the blood lipid content of the animal model of hyperlipidemia Since it exhibits an anti-hyperlipidemic effect, the composition may be usefully used as a pharmaceutical composition or health functional food for the prevention and treatment of hyperlipidemia.

Snail, Hyperlipidemia, Cholesterol, Triton WR-1339, Poloxamer-407

Description

A composition comprising the powdered form or extract of ascidian (Halocynthia roretzi) as an active ingredient showing anti-hyperlipidemia activity}

The present invention relates to a pharmaceutical composition or health functional food for preventing and treating hyperlipidemia, which contains a dry powder or an extract of the larvae as an active ingredient.

[1] Kang Jin-kyung, Concept of lifestyle-related diseases, Journal of Korean Medical Association, March. pp188-190, 2004

[Document 2] Health Insurance Review and Assessment Service, Health Insurance Review Statistics Index, 2003

[Reference 3] Ministry of Health and Welfare, National Health and Nutrition Survey, pp49-58, 2005

Schoen FJ et al., Robbins pathologic basis of disease, 5th ed., Pp 473-484, 1994

[Reference 5] Ham IH et al., Kor . J. Herbology , 20, pp 45-52, 2005

Lee HH et al., Korean J. Anesthesiol , 40, pp 515-521, 2001

7 J Choi et al., J. Kor . Pharm . Sci . , 18, pp 240-241, 1988

8 K Kim et al., J. Kor . Pharm . Sci . , 28, pp 15-23, 1998

Nash VJ et al., Pharmacotherapy , 16, pp 10-15, 1996

10. Stellato C. et al., Br . J. Anaesth . , 67, pp 751-758, 1991

11 Wout GM et al., J. Paren . Sci . & Technol . , 46, pp 192-200, 1992

[Reference 12] Yuko, Yukok.ホ ヤ の エ キ ス 成分.化學 と 生物. 27, pp 96, 1989

[Document 13] Lee, Kang-Ho et al., Journal of the Korean Fisheries Society, 26 (1), pp8-12, 1993

[Document 14] Lee, Kang-Ho et al., Journal of the Korean Fisheries Society, 26 (2), pp150-158, 1993

[Reference 15] Lee Eung-Ho et al., Korean Journal of Food Science and Technology, 15 (1), pp1-5, 1983

16. K. Watanabe et al., Bull . Japan . Soc . Fish . 51, pp 1293, 1985

[17] CK Park et al., Nippon Suisan Gakkaishi . 56, pp 1319, 1990

18. K. Fujimato et al., Bull . Japan . Soc . Fish . 48, pp1323, 1982

[19] BD Choi et al., J. Korean . Fish . Soc . 28, pp761, 1995

[Document 20] Hong, Byung-il et al., Journal of the Korean Fisheries Society. 35 (6), pp 671-675, 2002

[Document 21] Yuk Heung Sun et al., Korean Journal of Food and Nutrition. 32 (3), pp 474-478, 2003

22 Hsi-Hsien Liu et al., J. Agric . Food Chem . 50 (12), pp3602-3606, 2002

Document 23 i.水産食品成分の機能性について.月刊Food Chemical . 1989-2, pp 52-56, 1989

Document 24 AOAC. Offical Methods of Analysis. 16th ed. Association of Official Analytical Chemists, Washington DC., Pp69-74, 1995

[Document 25] 日本 厚生 省.食品 衛生 指針 -I.日本 厚生 省 出版.東京. 30, 1960

26 W. Richmond, Clin . Chem . , 22, pp1579, 1976

27 MW Mc Gowan et al., Clin . Chem . 29, pp 538, 1983

28 H. Kusama et al., Folia pharmacol . japon 92 , pp 175-180, 1988

Modern man has lived a rich and convenient life with the development of industry. As a result, high levels of medicine have led to an increase in life expectancy, but on the other hand, a new disease called lifestyle-related disease has emerged. Lifestyle disease is a disease that has been renamed since 2003, which is commonly known as hypertension, allergies, obesity, diabetes, cirrhosis, heart disease, and cancer. The concept of lifestyle diseases was introduced because these diseases can be treated by improving eating habits, lifestyle, and environmental factors (Kang, Jin Kyung, Concept of lifestyle diseases, Korean Medical Association, March, pp. 188-19). 190, 2004).

Data on lifestyle-related diseases surveyed by the Health Insurance Review & Assessment Service showed that the number of patients using medical institutions increased from 2000 to osteoporosis (89.3%), high cholesterol (80.9%), high blood pressure (53.9%), diabetes (45.5%), and heart disease (29.0%). ). In particular, the average annual increase in the number of cases was high cholesterol (21.8%) and diabetes was 3.4% (Health Insurance Review and Assessment Service, Health Insurance Review and Statistics, 2003; Ministry of Health and Welfare, National Health and Nutrition Survey, pp49-58, 2005). Therefore, in Korea, measures for hyperlipidemia and diabetes-related diseases are urgently needed.

Hyperlipidemia is a condition in which lipid components in the blood are increased more than usual. The major components of serum lipids are composed of lipid-soluble substances such as cholesterol, triglycerides, phospholipids, and free fatty acids. It is called. Increased serum cholesterol and triglycerides are the most common causes of hyperlipidemia. Accumulation of excess fat causes blood circulation disorders and microcirculation failure, resulting in atherosclerosis, ischemic heart disease, cerebral infarction, hypertension, obesity and diabetes. May result (Schoen FJ et al., Robbins pathologic basis of disease, 5th ed., Pp 473-484, 1994).

There are many causes of atherosclerosis, but elevated plasma lipids, particularly plasma cholesterol and neutral lipids, are known as one of the most important risk factors.

Cholesterol is produced mainly in the liver and is present in the blood in the form of small round particles called lipoproteins, which carry cholesterol throughout the body. There are two types of lipoproteins, low density lipoprotein (LDL) and high density lipoprotein (HDL), and because high density lipoproteins carry cholesterol from other tissues to the liver, a lot of high-density lipoprotein removes cholesterol from blood vessels. On the other hand, low-density lipoproteins are mainly produced by the liver and transport cholesterol to other tissues of the human body, so a lot of low-density lipoproteins accumulate a lot of cholesterol in blood vessels to promote arteriosclerosis, and about 70% of blood cholesterol is present as low-density lipoproteins, which are bad cholesterol.

In fact, cholesterol is an essential ingredient for normal functioning of the human body, a nutrient necessary for making cells, and a component of various hormones such as corticosteroids, male hormones, and female hormones. Becomes In addition, cholesterol is a nutrient necessary for the human body, such as being a component of a biological membrane and being shot as a starting material for hormonal synthesis. However, excessive cholesterol intake is known to accumulate in the blood vessels and cause heart disease, and there is no preventive method except low-cholesterol diet, and taking drugs such as cholesterol-lowering drugs is effective. Use is extremely limited due to reasons such as causing side effects such as liver dysfunction.

Chitosan, phytosterol, inositol, and pectin are known substances that lower blood cholesterol in the human body, but the effects or metabolic mechanisms are not clear when phytosterol is excluded. . Phytosterol, a plant sterol due to its structural similarity to cholesterol, has already been identified as a mechanism for inhibiting metabolism of cholesterol absorption in the body through competition with low-density lipoprotein-cholesterol, which is harmful to the human body. As a food additive, the US Food and Drug Administration (FDA) Has been approved.

Another major cause of elevated plasma cholesterol is the excessive intake of fat by eating a diet that consumes large amounts of eggs, butter, meat, and the like. In the case of dietary hypercholesterolemia, it usually does not lead to severe hypercholesterolemia, but it is a problem that cholesterol gradually accumulates in blood vessels from young age and causes arteriosclerosis in adults. In addition to hypertriglyceridemia, myocardial muscle There is a risk of infarction or cerebral infarction.

The pathological model of experimental hyperlipidemia is largely divided into exogenous model and tomorrow's model. Exogenous hyperlipidemic condition models include hypercholesterolemia-induced models caused by high cholesterol dietary load, that is, hyperlipidemia by administration of vitamin D, cholesterol, olive oil, corn oil, and the like. -1339 (Triton WR-1339) model, and the like (Ham IH et al., Kor . J. Herbology , 20, pp 45-52, 2005). Among them, Poloxamer 407 is an anesthetic emulsifier (Lee HH et al., Korean J. Anesthesiol , 40, pp515-521, 2001), poorly soluble drug solubilizer (Choi JY et al., J. Kor . Pharm . Sci., 18, pp240-241, 1988), skin ointments (Kim KK et al., J. Kor. Pharm. Sci., 28, pp15-23, 1998) , but is being used as such, in the human body when administered Increased lipids and especially increased triglycerides lead to hyperlipidemia. Free fatty acids are produced in adipocytes for at least 24 hours after poloxamer injection (Nash VJ et al., Pharmacotherapy , 16, pp10-15, 1996) and interfere with the action of lipoprotein lipase (LPL). Decreases the rate of triglyceride degradation (Stellato C. et al., Br. J. Anaesth . , 67, pp751-758, 1991) and increases blood cholesterol and triglycerides in the liver, which is called 3-hydroxy-3 in the liver. -Methylglutaryl coenzyme A (HMG-CoA; 3-hydroxy-3-methylglutaryl CoA), which lowers the activity (Wout GM et al., J. Paren . Sci . Technol . , 46, pp192-200 , 1992). Triton WR-1339 is a non-specific lipase inhibitor that not only induces hyperlipidemia, but also increases serum concentrations of triglycerides, ester-bound cholesterol, phospholipids, fatty acids, etc. in vivo) and in vitro experiments (in vitro) that lie Sound end cholesterol esterase activity reduction of the (lysosomal cholesterol esterase) from the reported.

Sea urchins inhabit about 2,000 species around the world, especially Halocynthia roretzi. The species is used for reproduction in Korea and Japan. In recent years, the production of squirrel is rapidly increasing due to the spread of squirrel farming technology, and many researches on wild squirrel are being conducted on wild and aquatic farms. (27, pp96, 1989 ). Studies on the food composition of the squirrel are as follows: Chemical composition of the squirrel (Lee Kang-Ho et al., 26 (1), pp8-12, 1993), Jeong-Mee Jung (Lee, Kang-Ho et al., 26 (2), pp150-) 158, 1993;..... Lee, Eung - Ho et al., Journal of the Korea Food, 15 (1), pp1-5, 1983), the nitrogen-containing component and the seasonal variations of the amino acids (K. Watanabe et al, Bull Soc Japan 51 Fish , pp 1293, 1985; CK Park et al., Nippon Suisan Gakkaishi . 56, pp1319, 1990), and studies on precursors and production mechanisms of odorous odors (K. Fujimato et al., Bull . Japan . Soc . Fish . 48, pp1323, 1982), and Choi et al. Odor components have been analyzed and reported (BD Choi et al., J. Korean . Fish . Soc . 28, pp761, 1995).

As a report on the pharmacological effects of larvae, Hong et al. Reported that ACE (angiotensin converting enzyme) inhibits sulphate polysaccharides derived from the shells of the larvae, resulting in a decrease in blood pressure. It has been reported that serum lipids are reduced (Hong Byung-il et al., Journal of the Korean Fisheries Society. 35 (6), pp671-675, 2002; Yukheung-sun et al., Korean Journal of Food and Nutrition. 32 (3), pp474-478, 2003). In addition, Liu et al. Reported that GAGs (glycosaminoglycans), which are widely distributed in the tissues of the snail, improve immunity and promote lipid metabolism (Hsi-Hsien Liu et al., J. Agric . Food Chem . 50 (12). ), pp3602-3606, 2002).

Of aquatic functionality it is very likely to act as a composite component than the component alone as in the herbal medicine (芦己勝朗.水産食品成分の機能性について. 月刊 Food Chemical . 1989-2, pp 52-56, 1989). Even if the assumption that functional properties will be expressed by such a composite component system is not correct, the separation process of functional food materials in aquatic products is mistaken for removing well-known functional materials. For example, in the case of chitin functional material, which is well known as a functional food material, in order to obtain only the final chitin component from the skin of the red crab, the minerals such as CaCO ₃ which prevents osteoporosis may be first removed through strong HCl pretreatment, and then Strong NaOH treatment removes astaxanthin, which has antioxidant capacity, and hydroxyl protein, a typical functional nutrient.

Therefore, the present inventors first select the squirrel to look for a material in the aquatic products that makes the antihyperlipidemic effect close to the drug as a complex component system, and compares these data by examining whether it expresses the functional activity by itself without separating specific functional components. The present invention was completed by confirming the antihyperlipidemic effect through animal experiments for functional verification under the same conditions.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of hyperlipidemia, which contains a dry powder or extract of the larvae as an active ingredient.

The present invention also provides a health functional food for preventing and improving hypertension, which contains a dry powder or an extract of the larvae as an active ingredient.

Halocynthia as defined herein roretzi) is dongmulmun notochord (Phylum Chordata), ascidians (Class Asidiacea ), Side Seaweed ( Order Pleurogona ) or Sea Urchin ( Family Pyuridae ) includes whole, flesh or skin of the larvae that lives in Korea and Japan.

Extracts as defined herein include solvents selected from water, including purified water, lower alcohols having 1 to 4 carbon atoms, or mixed solvents thereof, preferably water, ethanol, or mixed solvents thereof.

Hereinafter, the present invention will be described in detail.

A first step of washing the squirrel of the present invention; A second step of separating and cutting the skin and the meat; A third step of quenching the same at -100 ° C to 0 ° C, preferably at -50 ° C to -30 ° C for about 6 hours to 24 hours, preferably about 12 hours; Through the manufacturing method comprising the fourth step of freezing under reduced pressure can be obtained the dried powder of the present invention in a dry state.

In addition, the extract of the larvae of the present invention can be prepared as follows. A first step of cutting the snails and freeze-drying them, including body fluids and intestines; This is a solvent selected from water containing purified water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, preferably water, ethanol or a mixed solvent thereof, at 0 ° C to 100 ° C, preferably at 20 ° C to 50 ° C. A second step of extracting for minutes to 1 hour, preferably for 30 minutes; This can be obtained through the production method comprising a third step of concentration under reduced pressure and freeze-dried.

The present invention provides a pharmaceutical composition for the prevention and treatment of hyperlipidemia, which contains a dry powder or extract of the larvae obtained by the above production method as an active ingredient.

In another aspect, the present invention provides a health functional food for the prevention and improvement of hyperlipidemia, which contains a dry powder or extract of the woowoori obtained by the above method as an active ingredient.

The pharmaceutical composition for the prevention and treatment of hyperlipidemia containing the dry powder or extract of the larvae of the present invention comprises 0.1 to 50% by weight of the worms relative to the total weight of the composition.

The pharmaceutical composition containing the dry powder or extract of the larvae of the present invention may further include appropriate carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

The pharmaceutical composition containing the larvae of the present invention is in the form of powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Can be formulated and used. Carriers, excipients and diluents which may be contained in the composition containing the compound include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose, etc. Or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The amount of scavengers of the present invention may vary depending on the age, sex, and weight of the patient, but may be administered once to several times daily from 0.1 to 100 mg / kg. The dosage may also be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The pharmaceutical composition may be administered to various mammals such as mice, mice, livestock, humans, and the like. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention provides a health functional food for the prevention and improvement of hyperlipidemia, comprising a dry powder or extract of the larvae as an active ingredient. Foods to which it may be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, teas, vitamin complexes, and health functional foods.

It may also be added to foods or beverages for the purpose of preventing hyperlipidemia. At this time, the amount of dry powder or extract of sea cucumber in the food or beverage may be added at 0.01 to 15% by weight of the total food weight, the health functional beverage composition is 0.02 to 5 g, preferably 0.3 to 1 g based on 100 ml Can be added.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the dry powder or extract as essential ingredients in the indicated ratios, and contains various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. can do. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And sugars such as conventional sugars such as polysaccharides such as dextrin, cyclodextrin and the like and xylitol, sorbitol, erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tautin, stevia extract) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of such natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the dry powder or extract of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the dry powder or extract of the present invention may contain a pulp for the production of natural fruit juices and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the dry powder or extract of the present invention.

The present invention relates to a composition for the prevention and treatment of hyperlipidemia, comprising a dry powder or an extract of the larvae as an active ingredient, in detail, the dry powder or extract of the sloe larvae significantly increases the blood lipid content of an animal model of hyperlipidemia. Since the anti-hyperlipidemic effect is exhibited by reducing, the composition may be usefully used as a pharmaceutical composition or health functional food for the prevention and treatment of hyperlipidemia.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples. However, the following Examples and Experimental Examples are merely illustrative of the present invention, but the content of the present invention is not limited thereto.

Example 1 Preparation of Sample

1-1. Sample Preparation

Halocynthia roretzi was farmed off the coast of Yeongjin, Gangwon-do. On December 5, 2006, a total of 294 fish were purchased at Jumunjin Fish Market. The edible part and the shell were separated and used as a sample for the following experiment. The average weight of the larvae was 64.7 ± 10.2 g. Among them, the average weight of meat without shells was 50.5 ± 7.9 g, which was 78 ± 6.3%. The average diameter of the larvae was 48.4 ± 8.3 mm, and the average height was 106.1 ± 18.4 mm, and was used as a sample of the following experiment (hereinafter referred to as “HR”).

1-2. Preparation of Soluble Ethanol Soluble Extract

250 g of the squirts obtained in Example 1-1 were cut at 100 mm intervals, and the sample containing the bodily fluid and gut flowing out during the cutting process was freeze-dried under vacuum, and 1 L of 70% ethanol was added thereto for 30 minutes at 25 ° C. Extraction was performed once, concentrated with a vacuum concentrator, and lyophilized to obtain 8.2 g of ethanol soluble extract of squill (hereinafter referred to as "HR-1").

1-3. Manufacture of Dry Powder

250 g of the turmeric obtained in Example 1-1 was thoroughly washed with tap water to remove foreign substances and salt, and then supported by a sieve to minimize water, and the shell and meat were separated to be fed separately. . During the cutting process, the body fluids and intestines flow out, and the squid was included in the meat and spread flat and thin on the tray. At this time, about half the thickness of the sample in the tray, left in a fast freezing machine (DFS520) for 12 hours at -50 ℃ with a temperature sensor plugged in completely frozen, and then freeze-drying the pressure-reducing freeze dryer (Iljin, Korea Ilshinsa, PVIFD30A) ) The principle of the freeze drier is that when the tray is slowly heated, the sample is in a nearly vacuum state so that the phase changes from solid to gas. At this time, the water vapor of the sample is gradually reduced by collecting the water vapor by cooling it again. The conditions for the vacuum freeze dryer used in the experiment were vacuum up to 5 mili torr and temperature up to 30 ° C. In order to prevent the sample from bubbling (boiling and overflowing out of the tray) by rapid temperature rise, the temperature for heating the sample is gradually raised from 0 ° C. After about 42 hours, drying stops when the temperature of the sample and the tray reaches 30 ° C. To obtain a dry powder of the larvae (hereinafter referred to as "HR-2").

As a result of the experiment, as shown in Table 1, the HR obtained in Example 1-1 occupies 78.0% of the edible part, and 22.0% of the shell part of the non-edible part, whereas Example 1-3 The dry sample obtained in U.S. showed 42.8% of meat (hereinafter referred to as "HR-M") and 57.2% of shell part (hereinafter referred to as "HR-T"), resulting in a higher proportion of non-planted parts. I could confirm it.

Weight of HR (%) Weight of HR-2 (%) Yield (%) HR-M 14,836 g (78.0%) 550 g (42.8%) 5.0 HR-T 4,185 g (22.0%) 734 g (57.2%) 13.1 all 19,021 g (100.0%) 1284 g (100.0%) 6.8

Reference Example 1 Preparation of Experimental Animals and Experimental Diet

Experimental animals were fed from Hyochang Science and raised for 2 weeks under animal's constant conditions (temperature: 20 ± 2 ℃, humidity: 40 ~ 60%, contrast: 12 hours light / dark cycle). g of ICR (Institute of Cancer Research, USA) mice were used and fasted with water only for 24 hours before the experiment. At this time, the experimental animals were treated within a certain time (10:00 AM ~ 12:00 AM) in consideration of the daily variation of enzyme activity.

The experimental animals were divided into three groups of nine animals per group, and were divided into a control group fed a normal feed, a negative control group induced with hyperlipidemia, and an HR-2 administration test group. HR-M and HR-T were suspended in 10% tween80 and 100, 200 mg / kg of the animals were ingested once a day for 2 weeks and 4 weeks using an oral zone.

Experimental Example 1. General Component Analysis

In order to analyze the general components of HR obtained in Example 1, experiments were performed using the AOAC method as follows (AOAC.Offical Methods of Analysis.16th ed.Association of Official Analytical Chemists, Washington DC., Pp69- 74, 1995).

Specifically, moisture was measured by atmospheric heating drying method, crude fat was measured by Soxthlet method, and ash was measured by dry calcination method. Crude protein was measured by semimicro Kjedahl method and nitrogen coefficient (6.25) was used. Calculated.

As a result, as shown in Table 2 and Table 3, HR had a very high content of moisture in both the edible and non-edible parts, and there were a lot of crude ash and polysaccharides in meat, and a lot of crude ash in non-edible portion. The above-mentioned HR-M contained almost equal amounts of polysaccharides, minerals, fats and proteins, and HR-T contained only a high content of 74% of minerals, followed by polysaccharides, proteins, and fats. In addition, HR-2 was composed mostly of minerals and proteins, contained polysaccharides, and had a very low crude fat content. HR showed a high moisture content of 84.5 to 93.4%.

ingredient HR-M HR-T moisture 90.7 ± 0.7 84.5 ± 0.8 Crude protein 1.8 ± 0.1 1.4 ± 0.03 Crude fat 2.4 ± 0.02 0.8 ± 0.02 View minutes 2.5 ± 0.4 11.5 ± 0.3 Polysaccharide 2.6 ± 0.01 2.0 ± 0.01

ingredient HR-M HR-T Crude protein 18.9 ± 1.1 8.7 ± 0.2 Crude fat 25.8 ± 2.2 4.8 ± 0.1 View minutes 27.3 ± 4.6 73.6 ± 1.6 Polysaccharide 28.1 ± 0.1 12.9 ± 0.1

Experimental Example 2. Determination of volatile basic nitrogen (VBN) content

In order to analyze the volatile basic nitrogen content of HR obtained in Example 1-1, a microdiffusion method using a Conway unit was carried out as follows. (日本 厚生 省. 食品 衛生 指針 -I. 日本 厚生 省出版. 東京. 30, 1960).

2 g of HR was added to the analysis beaker of volatile basic nitrogen, 16 ml of distilled water was added, and the mixture was stirred well with a glass rod. For protein precipitation, 2 mL of 20% TCA solution was added, ground well, and left to stand for 10 minutes to obtain a supernatant, which was then used as a sample solution. 1 mL of boric acid absorbent was added to the inner chamber of the Conway unit and 1 mL of the sample solution into the outer chamber. Then, the lid was closed for about 2/3. Then, 1 mL of saturated potassium carbonate solution was added to the outer chamber. The outer chamber liquid, that is, saturated potassium carbonate and the sample solution, were mixed while the unit was slowly rotated by inserting a clip so that the inner and outer chamber liquids did not mix with each other. Thereafter, the mixture was left at 37 ° C. for 80 minutes and titrated with 0.01 N HCl solution. Titrate until the hue of the solution changes from green to colorless to scarlet. In the black test, a 20% TCA solution was added to the outer chamber in the manner of the above conditions. 1 mL of 0.01 N HCl solution used corresponds to 0.14 mg of volatile basic nitrogen. The content of volatile basic nitrogen was calculated according to the following equation.

Volatile basic nitrogen (mg%) = 0.14 × (A-B) × 20 × f × 100 / S

   S: Sample Collection

   A: Proper amount of sample (mL)

   B: Blank Proper amount of mL )

  f: 0.01 N HCl  Solution factor

VBN (Volatile basic nitrogen), that is, volatile basic nitrogen, is used to measure the freshness of fish and shellfish. In particular, it is used as a standard for the initial decay of aquatic products. 30 mg% of frozen fish, 50 mg% of sharks and skates, 20 mg% of pollack and cod, 20 mg% of shellfish and shellfish, and 30 mg% of other seafood.

As a result, the VBN value of HR obtained in Example 1-1 was 7.0 to 8.7 mg% level as shown in Table 4, all of which was found to be very fresh as it does not significantly fall below the initial decay criteria.

HR-M HR-T VBN (mg%) 8.7 ± 0.7 7.0 ± 0.2

Experimental Example  3. Poloxamer -407 ( Poloxamer Hyperlipidemia induced animal experiments

Poloxamer-407 (poloxamer-407) (300 mg / kg) was dissolved in physiological saline on an ice bath at the last dose of HR-2 obtained in Example 1-2 according to the method of Wout et al. It was lethal 24 hours after intraperitoneal administration (Wout et al., J. Paren . Sci. & Technol . 46, pp192-200, 1992).

3-1. Measurement of blood lipid content

CO ₂ was added to the experimental animals of HR-2 obtained in Example 1-2 Light anesthesia was performed with gas, blood was collected from the abdominal aorta, and the collected blood was left for 30 minutes, centrifuged at 3000 rpm for 10 minutes to separate serum and lipid content was measured.

3-2. Total cholesterol ( Total chlesterol ) Determination of content

Richmond Experiments were carried out using kits prepared by the enzymatic method (AM 202-K, Asan). That is, an enzyme reagent (containing 10.5 g / l of cholesterol esterase, 10.7 U / l of cholesterol oxidase, and 1.81 g / l of sodium hydroxide) was dissolved on ice-cooled solution (containing 13.6 g / l of potassium phosphate monobasic and 1.88 g / l of phenol). After dissolving in the solution, 3.0 ml of the enzyme solution prepared in 20 μl of the sample was added thereto, followed by incubation at 37 ° C. for 5 minutes, and the absorbance was measured at a wavelength of 500 nm using the reagent blank as a control. According to the standard calibration curve, the blood content was expressed in mg / dl (W. Richmond, Clin . Chem. , 22, pp1579, 1976).

3-3. Triglyceride ( Triglyceride ) Determination of content

Experiments were carried out using kits prepared according to the method of McGowan et al. (AM 157S-K, Asan). That is, an enzyme reagent (containing lipoprotein lipase 10800 U, glycerol kinase 5.4 U, peroxidase 135000 U, and L-α-glycero phosphooxidase 160 U) was dissolved on ice-cooled solution [N, N-bis (2-hydroxyethyl) -2- Aqueous solution dissolved in 0.427 g / dl of aminomethane sulfonic acid] was added to 20 μl of the sample, and 3.0 ml of the prepared enzyme solution was added thereto, followed by incubation at 37 ° C. for 10 minutes, and the absorbance was measured at a wavelength of 550 nm using the reagent blank as a control. The blood content was expressed in mg / dl according to the standard calibration curve (MW Mc Gowan et al., Clin . Chem . 29, pp538, 1983).

3-4. Statistical processing

As shown in Table 5, the results obtained in this experiment were expressed as mean ± standard deviation, and a significant difference test was performed by applying Duncan's multiple range test, which shows the difference between each experimental group by PC-STAT program. It was. With this PC-STAT program, there can be a minimum of 2 to 50 significant difference test treatments and up to 150 experimental values. In other words, by inputting up to 150 experimental values of several samples in a single share, two groups and more than 50 groups are compared to each other by performing two-section comparison statistical processing to determine mutual significance.

As shown in Table 5, there was no significant difference in lipid concentration reduction between the negative control group and the HR-administered test group that induced hyperlipidemia induced by poloxamer-407. However, when HR-T was administered for 4 weeks at 200 mg / kg, the total cholesterol content was decreased by 23.5% compared to the negative control group.

Test group Dose
(mg / kg) Triglyceride cholesterol (mg / dl) 2 W 4 W 2 W 4 W Control 96.3 ± 13.2 ^b 97.3 ± 12.8 ^b 86.3 ± 19.2 ^b 88.7 ± 15.6 ^c Negative control
(Poloxamer-407) 1327.5 ± 300.4 ^a 1319.3 ± 280.1 ^a 849.2 ± 93.4 ^a 839.7 ± 67.3 ^a HR-M 100 1356.1 ± 283.5 ^a 1296.8 ± 300.1 ^a 835.3 ± 89.7 ^a 813.5 ± 59.2 ^a 200 1368.7 ± 273.9 ^a 1302.4 ± 219.5 ^a 826.3 ± 81.6 ^a 819.2 ± 49.3 ^a HR-T 100 1342.5 ± 213.8 ^a 1176.2 ± 198.2 ^a 813.6 ± 80.9 ^a 793.2 ± 40.2 ^a 200 1239.3 ± 190.6 ^a 982.7 ± 107.3 ^a 823.5 ± 85.7 ^a 642.6 ± 41.3 ^b Values marked with the same letter are not significantly different from each other (p <0.05, Duncan's multiple range test)

Experimental Example  4. Triton WR -1339 ( Triton WR -1339) induced hyperlipidemic animal experiment

Fasting 16 hours before the administration of Triton WR-1339 (Triton WR-1339) according to the method of Kusama et al., And then induce hyperlipidemia by injecting 200 mg / kg of Triton WR-1339 into the tail vein, 18 hours after CO ₂ Blood was anesthetized with gas (H. Kusama et al., Folia pharmacol . japon 92 , pp 175-180, 1988).

4-1. Measurement of blood lipid content

The anesthetized experimental animals obtained in Example 1-2 were lightly anesthetized with CO ₂ , blood was collected from the abdominal aorta, left for 30 minutes, and centrifuged at 3000 rpm for 10 minutes to separate serum. And lipid content was measured.

4-2. Total cholesterol ( Total chlesterol ) Determination of content

Richmond Experiments were carried out using kits prepared by the enzymatic method (AM 202-K, Asan). That is, an enzyme reagent (containing 10.5 g / l of cholesterol esterase, 10.7 U / l of cholesterol oxidase, and 1.81 g / l of sodium hydroxide) was dissolved on ice-cooled solution (containing 13.6 g / l of potassium phosphate monobasic and 1.88 g / l of phenol). After dissolving in the solution, 3.0 ml of the enzyme solution prepared in 20 µl of the sample was added thereto, followed by incubation at 37 ° C. for 5 minutes, and the absorbance was measured at a wavelength of 500 nm using the reagent blank as a control. According to the standard calibration curve, the blood content was expressed in mg / dl (W. Richmond, Clin . Chem. , 22, pp1579, 1976).

4-3. Triglyceride ( Triglyceride ) Determination of content

Experiments were carried out using kits prepared according to the method of McGowan et al. (AM 157S-K, Asan). That is, an enzyme reagent (containing lipoprotein lipase 10800 U, glycerol kinase 5.4 U, peroxidase 135000 U, and L-α-glycero phosphooxidase 160 U) was dissolved on ice-cooled solution [N, N-bis (2-hydroxyethyl) -2- Aqueous solution dissolved in 0.427 g / dl of aminomethane sulfonic acid] was added to 20 μl of the sample, and 3.0 ml of the prepared enzyme solution was added thereto, followed by incubation at 37 ° C. for 10 minutes, and the absorbance was measured at a wavelength of 550 nm using the reagent blank as a control. The blood content was expressed in mg / dl according to the standard calibration curve (MW Mc Gowan et al., Clin . Chem . 29, pp538, 1983).

4-4. Statistical processing

As shown in Table 6, the results obtained in this experiment were expressed as mean ± standard deviation, and a significant difference test was performed by applying Duncan's multiple range test, which shows the difference between each experimental group by PC-STAT program. It was. With this PC-STAT program, there are at least 2 to 50 significant difference test treatments, and up to 150 experimental values can be processed. In other words, by inputting up to 150 experimental values of several samples in a single share, two groups and more than 50 groups are compared to each other by performing two-section comparison statistical processing to determine mutual significance.

Experimental results, as shown in Table 6, compared to the control group induced hyperlipidemia with Triton WR-1339, the test group administered HR-M showed very insignificant changes in lipid content or showed little. However, the test group treated with HR-T showed a significant difference at 4 weeks, and in particular, the administration of HR-T 200 mg / kg showed 20.9% decrease in neutral lipid and 24.4% total cholesterol compared to the negative control. there was.

Test group Dose
(mg / kg) Triglyceride cholesterol (mg / dl) 2 W 4 W 2 W 4 W Control 96.2 ± 15.2 ^b 98.3 ± 18.7 ^d 81.6 ± 10.5 ^b 79.3 ± 9.78 ^d Negative control
(WR-1339) 793.2 ± 67.4 ^a 800.1 ± 55.6 ^a 184.9 ± 27.6 ^a 190.3 ± 16.2 ^a HR-M 100 784.6 ± 50.1 ^a 779.3 ± 50.4 ^ab 189.3 ± 20.2 ^a 179.3 ± 14.7 ^ab 200 768.5 ± 68.2 ^a 769.1 ± 48.3 ^ab 200.1 ± 21.4 ^a 170.5 ± 17.7 ^ab HR-T 100 781.2 ± 45.5 ^a 718.4 ± 45.6 ^b 185.3 ± 19.5 ^a 156.3 ± 15.1 ^bc 200 753.6 ± 52.8 ^a 632.8 ± 40.7 ^c 179.9 ± 28.1 ^a 143.8 ± 13.9 ^c Values marked with the same letter are not significantly different from each other (p <0.05, Duncan's multiple range test)

It describes a formulation example of a composition comprising a dry powder or an extract of the present invention, the present invention is not intended to limit it, but is intended to explain in detail only.

Formulation example  One. Powder  pharmacy

HR-M 20 mg

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation example  2. Preparation of Tablets

HR-T 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are usually prepared by tableting according to the method for preparing tablets.

Formulation example  3. Manufacture of capsule

HR-M 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation example  4. Preparation of injections

HR-T 10 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

_{Na 2 HPO 4 · 12H 2 O} 26 mg

It is usually prepared in the above ingredient content per ampoules (2 ml) according to the preparation method of the injection.

Formulation example  5. Liquid  Produce

HR-M 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified Water Proper Amount

Normally, each component is added to the purified water to dissolve according to the preparation method of the liquid solution, lemon flavor is added, the above ingredients are mixed, purified water is added, the whole is adjusted to 100 ml by the addition of purified water, and then filled into a brown bottle and sterilized. To prepare a liquid solution.

HR-T 1000 mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

10 μg biotin

Nicotinamide 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5 mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

15 mg potassium monophosphate

Dicalcium Phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is a composition that is relatively suitable for the health functional food, the composition is mixed in a preferred embodiment, but the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health functional food manufacturing method. Then, the granules may be prepared and used for preparing the nutraceutical composition according to a conventional method.

Formulation example  7. Manufacture of health drinks

HR-M 1000 mg

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

900 ml of purified water whole

After mixing the above components according to the conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored Used to prepare the healthy beverage composition of the invention.

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Claims (6)

A pharmaceutical composition for the prevention and treatment of hyperlipidemia, containing 50% to 80% ethanol soluble extract of the bark of the larvae as an active ingredient. delete delete delete Health functional food for the prevention and improvement of hyperlipidemia containing 50% to 80% ethanol soluble extract of the bark of the larvae as an active ingredient. delete