KR101027604B1 - A composition comprising the dry powder, polar solvent extract and the insoluble residue of Anthocidaris crassispina as an active ingredient showing liver detoxification and antioxidant activity - Google Patents

Abstract

The present invention relates to a composition having liver detoxification and antioxidant activity. Specifically, the composition of the present invention containing a dry powder of sea urchin, a polar solvent soluble extract thereof, and a polar solvent insoluble residue as an active ingredient is a xenobiotics. It is useful as a pharmaceutical composition and health functional food with liver detoxification and antioxidant activity through the effect on liver damage caused by bromobenzene.

Sea urchin, liver toxicity, antioxidant

Description

Composition comprising the dry powder, polar solvent extract and the insoluble residue of Anthocidaris crassispina as an active ingredient showing liver detoxification and antioxidant activity}

Compositions containing sea urchin eggs, intestine or shell dry powder, polar solvent soluble extracts, and polar solvent insoluble residues as active ingredients exhibit liver detoxification and antioxidant activity, and thus prevent and treat diseases caused by liver damage. It is useful for this purpose.

[Reference 1] Ryo, YG, and DW Park. Anthocidaris crassispina (A.Agasiz). Bull. Fish. Res. Dev. Agency 39: 89-96. 1986

Document 2 Food consent. Academy North, Seoul. p 337-338. 1999

[Reference 3] Juhwan. <Let's know the sea> Sea urchins are nocturnal. Busan Ilbo. p 14. 1997

[4] Agriculture and Fisheries Trade Center. Agriculture and Fisheries Trade Information. Sea urchin production statistics. 2000

Nam, HK J Korean Oil Chemists' Soc 3: 33-37. 1986

[6] De la Cruz-Garcia, C., Lopez-Hernandez, J., Gonzalez-Castro, MJ, Rodriguez-Bernaldo De Qiros, Al., And Simal-Lozano, J. J Sci Food Agric 80: 1189- 1192. 2000

7 Yoo, SK, Hur, SB, and Ryu, HY Bull Korean Fish Soc 15: 345-358. 1982

8 Wui, IS, Lee, JB, and Yoo, SH Korean J Environ Biol 10: 92-99. 1992

9, Yu, CM, Cho, KA J Korean Environ Sci Soc 8: 160-164. 1999

10, Yoon, JK, Kim, GJ, and Shin, JG J Korean Toxicol 13 (4): 377-383. 1997

Reference 11 Kato, R. Xenobiotica , 7 (1-2): 25. 1977

12. Hayashi, O., Niki, E., Kondo, M. and Yoshikawa, T. Elsevier Sci Pub 1-9. 1984

13, Mitchell, J. R. and Hornig, M. G. Raven press. 23-25. 1984

Document 14 Zheng, J., and Hanzlik. RP Xenobiotica: the Fate of Foreign Compounds in Biological Systems 21 (4): 535-546. 1991

Heijine, WHJ, Slitt, AL, van Bladeren, PJ, Groten, JP, Klaassen, CD, Stierum, RH, and van Ommen, B. Toxicol Sci 79: 411-422. 2004

Reid, WD, Chistie, B., Krishna, G., Mitchell, JR, Moskowitz, J., and Brodie, BB. Pharmacology 6: 41-55. 1971

Reference 17 AOAC official Method of Analysis, 15th ed. Association of official Analytical Chemists. Washington. DC 931-943. 1990

[Document 18] Kim So-mi, Eun-hee Kim, Se-young Park, Choi Hye-hye. Anyone know our fish story. Filial piety. 2002

[19] Pryor, W. A. Elservier, Amsterdam: 331-361. 1977.9

20, Lee, H. J., Kim, G. H. and Lee, E. O. Korean J. Oriental Physiology and Pathology 17 (5): 1177-1181. 2003

[21] Ohkawa, H., N. Ohish, and K. Yaki. Analytical Biochemistry 95: 351-355. 1979

22. Casini, AF, Ferrali, M., Pompella, A., Maellaro, E., and Comporti, M. Am J Pathol 123: 520-531. 1986

23. Ellman, GL Arch. Biochem. Biophys. 82: 70. 1959

24. Lowry, OH, et al. Journal of Biological Chemistry 193: 265-275. 1951

Reitman, S., and Frankel. American Journal of Clinical Pathology 28: 58-63. 1957.

26. Karmen, A. Journal of Clinical Investigation 34: 131-133. 1955.

Document 27 Nash. T. Journal of Biological Chemistry 55: 412-416. 1953

28 Bidlack, WR and Lowry, GL Biochemical Pharmacology 31, 3: 311-317. 1982

Litwack, G., Ketterer, B., and Aris, IM Nature 234: 466-467. 1971

30 Habig, WH, MJ Pabst, and WB Jakoby. Glutathione S-transferase. Journal of Biological Chemistry 249 (22): 7130-7139

[31] Kim, S. Y., Jung, J. Y., Kim, J. R., and Kim, J. H. The Yeungnam Med. J. Vol. 11, No. 2. 1994

[32] Meister, A. and Richman, P.G. : Regeneration of γ-glutamylcystein synthetase by nonallosteric feedback inhibition by glutathione. J. Biol. Chem., 250, 1422 (1975) p 158. (1967)

33. Arrick, BA, Nathan, CF Cancer Res 44: 4224-4232. 1984

34 Mize, C. E. and R. G. Landon. 1962. Hepatic glutathione reductase: I. purification and general kinetic properties. J. Biol. Chem. 237, 1589-1595

35, Park, JC, Choi, JS, Song, SH, Choi, MR, Kim, KY, and Choi, JW Kor. J. Pharmacogn . 28 (4): 239-246. 1997

36. Kim, E. J., Lee, C. K., and Choi, J. W. Kor. J. Pharmacogn. 23 (2): 81-82. 1992

Hasegawa, LS and Hammock, BD Biochem Pharmacol 31 (11): 1979-1984. 1982

Sea urchins belong to the Phylum Echinodermata and the Class Echinoidea, and are mainly distributed from coastal to deep waters. Sea urchins are distributed throughout Korea's East Sea, China, and Japan. The sea urchins produced in the coastal waters of Korea are used for food, Anthocidaris crassispina , Hemicentrotus pulcherrimus , and Pseudocentrotus depressus . , Strongylocentrotus internmedius (Ryo, YG, and DW Park.Anthocidaris crassispina (A.Agasiz). Bull. Fish.Res . Dev. Agency 39: 89-96. 1986). Sea urchins contain water, protein, fat, B vitamins, vitamin C, iron, magnesium, and calcium. In particular, sea urchin contains more protein than sea cucumber. Sea urchins, which contain various nutrients, are not only good for tuberculosis, but also for expectorants, tonics, especially neuralgia, and are known to be useful foods with alcohol detoxification. Agree on Food Consent, Academy North, Seoul, p 337-338, 1999; Joo Hwan, <Let's Know the Sea> Sea urchins are nocturnal. However, in the case of sea urchin, about 20% of the gonads (egg) are eaten, and the remaining 80% is made up of sea urchin shells, so about 2,000 tons are discarded as waste resources based on the average annual production of 2,500 tons of sea urchins. Most of the degradable shells have been left as they are, causing environmental problems (Agriculture and Fisheries Trade Center, Agriculture and Fisheries Trade Information, Sea Urchin Production Statistics. 2000).

Studies on sea urchins are mostly related to the components and production of sea urchins, including Nam et al. (Nam, HK J Korean Oil Chemists' Soc 3: 33-37. 1986) and De la Cruz-Garcia et al. (De la Cruz-Garcia, C., Lopez-Hernandez, J., Gonzalez-Castro, MJ, Rodriguez-Bernaldo De Qiros, Al., And Simal-Lozano, J. J Sci Food Agric 80: 1189-1192.2000) For protein, amino acid and fatty acid composition, Yoo et al. (Yoo, SK, Hur, SB, and Ryu, HY Bull Korean Fish Soc 15: 345-358. 1982) reported on the spawning and growth of sea urchins. In addition to sedimentary sediment contamination in the southwestern seawater using sea urchins (Wui, IS, Lee, JB, and Yoo, SH Korean J Environ Biol 10: 92-99. 1992) and biological evaluation of seawater (Yu, CM, Cho, KA J Korean Environ) Sci Soc 8: 160-164. 1999), but there are few reports on sea urchin's liver detoxification and antioxidant activity.

As the industry develops, industrial chemicals, which are xenobiotics, are widely used, and human health problems are being raised due to their exposure to the human body. The health hazards of industrial chemicals to humans are mainly due to the toxic effects caused by these substances, and the degree of this poisoning phenomenon is known to be greatly affected by the physiological and dietary conditions of the human body (Yoon, JK, Kim, GJ, and Shin, JG J Korean Toxicol 13 (4): 377-383. 1997). The detoxification mechanism of toxic substances that protects the living body from these foreign substances is related to physiological adaptation to maintain the homeostasis of the living body and is affected by the internal and external environment and physiological conditions (Kato, R. Xenobiotica , 7). (1-2): 25. 1977).

Intermediate metabolites of biological foreign bodies may attack tissues and cause tissue damage (Hayashi, O., Niki, E., Kondo, M. and Yoshikawa, T. Elsevier Sci Pub 1-9. 1984). Metabolism of foreign substances is mainly in the liver, and the free radicals produced during this process are known to lead to peroxidation of lipids, nucleic acid attack, degeneration of enzyme proteins, and ultimately tissue damage through biochemical chain reaction. (Mitchell, JR and Hornig, MG Raven press. 23-25. 1984). Bromobenzene, a representative biological foreign substance, is widely used as an organic solvent in the industry as an industrial chemical and is exposed to the human body to the multifunctional complex oxidase mechanism (cytochrome P450) present in the microsome of liver tissue cells. Is first metabolized and converted to highly toxic bromobenzene-3,4-oxide (epoxide), which reacts with cell membranes and macromolecules to cause biotoxicity. , J., and Hanzlik.RP Xenobiotica: the Fate of Foreign Compounds in Biological Systems 21 (4): 535-546. 1991). This electrophilic substance is known to be excreted by detoxification by binding to glutathione (GSH) by the detoxifying enzyme glutathione S-transferase (GST) (see FIG. 1), and some of the epoxide hydrolase Is detoxified by Heijine, WHJ, Slitt, AL, van Bladeren, PJ, Groten, JP, Klaassen, CD, Stierum, RH, and van Ommen, B. Toxicol Sci 79: 411-422. 2004). On the other hand, bromobenzene-3,4-oxide, an intermediate metabolite of bromobenzene, is an electrophilic substance and, if not rapidly detoxified, is known as a nucleophilic substance in vivo, such as DNA, RNA, protein and lipids. It is well known to bind and cause tissue damage (Reid, WD, Chistie, B., Krishna, G., Mitchell, JR, Moskowitz, J., and Brodie, BB. Pharmacology 6: 41-55. 1971) . Since the metabolism of bromobenzene is clearly distinguished between phase I and phase II detoxification mechanisms, it is thought that it is easy to use for modeling bromobenzene metabolism in liver damage.

In order to investigate the effects of sea urchins on the detoxification and antioxidant activity of damaged liver tissues, the present inventors investigated the physiological function of sea urchins by orally administering the sea urchin fractions into eggs, intestines and shells. After inducing acute toxicity with benzene, the present invention was completed by checking the effects on lipid peroxide and various enzymes related thereto.

In order to solve the above object, the present invention provides a pharmaceutical composition for the treatment and prevention of liver disease containing dried powder of sea urchin, polar solvent soluble extract thereof or polar solvent insoluble residue as an active ingredient.

In another aspect, the present invention provides a health functional food for preventing and improving liver disease containing dried powder of sea urchin, polar solvent soluble extract thereof or polar solvent insoluble residue as an active ingredient.

Sea urchins as defined herein are characterized as comprising anthracidaris crassispina , a horse urchin ( Hemicentrotus pulcherrimus) , a pink urchin ( Pseudocentrotus depressus) or a sea urchin ( Strongylocentrotus internmedius), preferably an anthocidaris crassispina Shall be .

       Sea urchins as defined herein are characterized as comprising eggs, intestines or shells, preferably eggs.

The dry powder as defined herein is characterized in that it comprises a lyophilized powder dried by vacuum freeze drying, hot air drying or room temperature drying, preferably vacuum freeze drying.

The polar solvent soluble extract as defined herein is a water soluble extract, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, preferably a mixed solvent of water and ethanol, more preferably an extract available in 50 to 99% ethanol. Characterized in that it comprises a.

Polar solvent insoluble residue as defined herein is characterized in that it comprises a residue except the polar solvent soluble extract.

     Liver disease as defined herein includes one or more diseases selected from fatty liver, hepatitis, cirrhosis, liver cancer and the like.

The composition comprises reduced serum AST, ALT, AD and AH activity due to liver disease; It is characterized by the effect of increasing GST, γ-glutamylcysteine, GSH reductase and epoxide hydrolase activity.

Hereinafter, a method of obtaining a sample of the present invention will be described in detail.

Sea urchins, preferably Anthocidaris crassispina , Horse urchin ( Hemicentrotus pulcherrimus) , Pink sea urchin ( Pseudocentrotus depressus), or North sea urchin ( Strongylocentrotus internmedius), and more preferably sea urchin ( Anthocidaris crassispina) After sufficiently removing the first step of separating into shells, eggs and intestines; After grinding the shell, eggs and intestine, preferably eggs separated in the above step, 12 to 48 hours at -70 ° C to -30 ° C, preferably -60 ° C to -40 ° C, preferably 20 to 30 ° C. A second step of freezing for a period of time and then drying by vacuum freeze drying, hot air drying or room temperature drying, preferably vacuum freeze drying; Grinding it to obtain a dry powder of the present invention, preferably a lyophilized powder; Water containing about 1 to 10 times, preferably 2 to 5 times (v / w) purified water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, preferably water, of the dry powder obtained in the above step. And a mixed solvent of ethanol, more preferably 50 to 99% ethanol, followed by heat extraction for 10 minutes to 1 hour, preferably 20 minutes to 40 minutes at 10 to 50 ° C., preferably 15 to 40 ° C. A fourth step of extraction using a conventional extraction method such as an extraction method and a reflux extraction method, preferably by using an ultrasonic extraction method; Filtering it under reduced pressure, and then concentrating it at about 10 to 60 ° C., preferably 20 to 50 ° C. to obtain a polar solvent soluble extract of the present invention; And a dry powder, a polar solvent-soluble extract, and a polar solvent-insoluble residue of the present invention in the sixth step of manufacturing the insoluble residue remaining in the polar solvent-soluble extract.

The composition of the present invention comprises 0.1 to 50% by weight of the dry powder, its polar solvent soluble extract or polar solvent insoluble residue based on the total weight of the composition.

However, the composition as described above is not necessarily limited thereto, and may vary according to the condition of the patient and the type and extent of the disease.

The composition comprising the dry powder of the present invention, the polar solvent soluble extract or the polar solvent insoluble residue may further comprise suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

Compositions comprising a dry powder, a polar solvent soluble extract or a polar solvent insoluble residue thereof according to the present invention, respectively, oral formulations of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like according to conventional methods Carriers, excipients and diluents which may be used in the form of external preparations, suppositories, and sterile injectable solutions may be included in the composition comprising the compound, such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, Erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, 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 preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose, or the like. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and 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 preferred dosage of the dry powder of the present invention, its polar solvent soluble extract or polar solvent insoluble residue depends on the patient's condition and body weight, degree of disease, drug form, route of administration and duration, but may be appropriately selected by those skilled in the art. have. However, for the desired effect, the dry powder, its extract and the remaining residue after extraction is preferably administered in 0.01 mg / kg to 10 g / kg, preferably 1 mg / kg to 1 g / kg per day. The administration may be carried out once a day or divided into several doses. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The present invention provides a health functional food containing a sea urchin dry powder, a polar solvent soluble extract or a polar solvent insoluble residue as an active ingredient exhibiting a preventive and improving effect of liver disease.

The health functional food including the dry powder of the present invention, the polar solvent soluble extract or the polar solvent insoluble residue may be used in a variety of drugs, foods and beverages for the prevention and improvement of liver disease. Examples of the food to which the dry powder of the present invention, the polar solvent soluble extract or the polar solvent insoluble residue can be added include, for example, various foods, beverages, gums, teas, vitamin complexes, health supplements, and the like. It can be used in the form of granules, tablets, capsules or beverages.

The dry powder of the present invention, its polar solvent soluble extract or polar solvent insoluble residue may be added to food or beverage for the purpose of preventing and improving liver disease. At this time, the amount of the extract in the food or beverage is generally added to the health food composition of the present invention to 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 10 g based on 100 ml, preferably Can be added in a ratio of 0.3 to 1 g.

      The health beverage composition of the present invention contains, as essential ingredients, the dry powder, its polar solvent soluble extract, or its polar solvent insoluble residue, and there are no particular limitations on the liquid components, and various flavors or Natural carbohydrates and the like may be included as additional components. Examples of the above-mentioned natural carbohydrates are conventional monosaccharides such as disaccharides such as glucose and fructose, such as maltose, sucrose and the like, and polysaccharides such as dextrin, cyclodextrin and the like. Sugars and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrate 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 composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. 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 composition of the present invention.

As described above, the composition containing the dried powder of the sea urchin, polar solvent soluble extract or polar solvent insoluble residue of the present invention as an active ingredient has excellent liver detoxification and antioxidant activity in animal experiments inducing liver toxicity with bromobenzene. By showing, it can be used as a composition for the treatment and prevention of liver disease.

Hereinafter, the present invention will be described in detail by the following reference examples, examples and experimental examples. However, this is only to illustrate the present invention, the content of the present invention is not limited thereto.

Reference Example 1. Experimental Materials

Rapid freezing machine is DFS520, Ilsinsa (Korea) (Storage Capacity: 605 L / day), and Vacuum Freeze Dryer is PVTFD300R, Ilshinsa (Korea) (Water Distribution: 30 kg / day). Used.

Reference Example 2 Experimental Animal

Sprague-Dawley system with body weight of 200 ± 10 g adapted to Hyochang Science (Daegu) for 1 week under constant conditions (temperature: 20 ± 1 ^o C, humidity: 55 ± 3%, contrast: 12 hours light / dark cycle) Male rats were used.

100 and 200 mg / kg of the sample obtained in Example were administered orally for 4 weeks, and on the last day of administration, bromobenzene was suspended in 1% tween80 and 460 mg / kg was administered to the experimental group once a day for 12 hours. Intraperitoneal injection was performed for 3 days at intervals. The control group was administered the same dose of saline and 1% tween 80.

Example 1 Preparation of Dry Powder

The sea urchin ( Anthocidaris crassispina ) 24,535 g was caught on the coast of East Sea. It was purchased at Geojin Port on July 15, 2008, washed thoroughly to remove foreign substances and salts, and then supported by a sieve to minimize moisture. The shells, eggs, and intestines were separated and recovered in three parts.

1-1. Preparation of Dried Powder of Sea Urchin Shell

19295 g of the separated and recovered sea urchin shells were crushed using a blender and placed in a tray, spreaded thin and flat, completely frozen in a cryogenic freezer at -50 ° C. for 24 hours, and then lyophilized using a vacuum freeze dryer. After the mixture was ground and mixed again with a blender, 6200 g of dried powder of sea urchin shell (hereinafter referred to as 'AC-S') (yield: 32.13%) was used as a sample of the following experimental example while being stored at -30 ° C. It was.

1-2. Preparation of dried powder of sea urchin intestine

1605 g of the separated and recovered sea urchin gut was obtained in the same manner as in Example 1-1, to obtain 230 g of dried powder of sea urchin gut (hereinafter referred to as 'AC-O') (yield: 14.33%). It was used as a sample of the following experimental example while storing at.

1-3. Preparation of dried powder of sea urchin eggs

3635 g of the separated and recovered sea urchin eggs were obtained in the same manner as in Example 1-1 to obtain 810 g of dried powder of sea urchin eggs (hereinafter referred to as 'AC-R') (yield: 22.28%). It was used as a sample of the following experimental example while storing at.

Example 2. Preparation of Ethanol Extract

2-1. Preparation of Ethanol Extract of Sea Urchin Shell

800 ml of 95% ethanol was added to 200 g of AC-S obtained in Example 1-1, followed by extraction using a sonication method at 30 ° C. for 30 minutes. The filtrate was filtered using a vacuum filter, and the filtrate and ethanol extract were classified into insoluble residue. The extracted residue was removed at room temperature to remove ethanol and stored at -20 ° C. or lower, and the sample of the following Experimental Example (hereinafter referred to as 'AC-S-R'), and the ethanol extract at 40 ° C. (vacuum rotary evaporator) Concentrated to obtain an ethanol extract (hereinafter referred to as 'AC-S-E') of the sea urchin shell, which was stored below -40 ℃ was used as a sample of the following experimental example.

2-2. Preparation of Ethanol Extracts from Sea Urchins

The ethanol extract insoluble residue of sea urchin gut (hereinafter referred to as 'AC-O-R') and the sea urchin ethanol extract of AC-O obtained in Example 1-2 were the same as in Example 2-1. (Hereinafter referred to as 'AC-O-E') was obtained.

2-3. Preparation of Ethanol Extract of Sea Urchin Egg

The ethanol extract insoluble residue of sea urchin gut (hereinafter referred to as 'AC-R-R') and the sea urchin ethanol extract of AC-R obtained in Example 1-3 were the same as in Example 2-1. (Hereinafter referred to as 'AC-R-E') was obtained.

Experimental Example 1. General Component Analysis

According to the AOAC method (AOAC official method of Analysis, 15th ed. Association of official Analytical Chemists. Washington. DC 931-943. 1990), the moisture is the atmospheric pressure drying method, crude protein is Nitrogen was quantified by semi-micro Kjeldahl and calculated using a nitrogen coefficient (6.25). The ash was analyzed by dry calcination at 600 ° C. and crude fat was analyzed by Soxthlet.

Body
section Moisture Crude protein Crude fat Crude ash Carbohy-drate AC-R 2.84 ± 0.26 50.13 ± 0.29 14.18 ± 0.03 10.89 ± 0.36 21.96 ± 0.23 AC-O 2.55 ± 0.33 37.33 ± 0.40 8.64 ± 0.03 28.57 ± 0.11 22.91 ± 0.22 AC-S 1.31 ± 0.23 13.25 ± 0.76 1.33 ± 0.09 51.03 ± 1.98 33.08 ± 0.77 AC-R-E 15.15 ± 0.17 48.16 ± 0.14 6.14 ± 0.02 9.11 ± 0.07 22.44 ± 0.12 AC-O-E 17.94 ± 0.32 35.07 ± 0.36 1.83 ± 0.07 26.69 ± 0.43 18.47 ± 0.29 AC-S-E 4.33 ± 0.63 7.37 ± 0.88 0.31 ± 0.02 49.69 ± 0.57 38.30 ± 0.52

As a result of the experiment, as shown in Table 1, in the case of the sea urchin dried sample obtained in Example 1, the water content is low by 1 to 3% because the sample is freeze-dried, crude protein and crude fat is the highest content of AC-R It was confirmed that this appears high. On the other hand, it was confirmed that the ash content was the highest in sea urchin shells. This is due to the relatively high levels of AC-R and AC-O in light of the fact that proteins and lipids in sea urchins are the main ingredients (Kim So-mi, Kim Eun-hee, Park Se-young, Choi Hye-hye, Korea Fish Story Hyo-il. 2002). Understand the protein and fat content.

In addition, in the residual sample of the sea urchin ethanol obtained in Example 2, the moisture content was increased in the range of 3 to 12% for each part compared to the dry powder, it can be seen that the variation between the samples is large. The increase in can be thought to be due to water absorption during ethanol extraction along with a decrease in fat content of the residue. Crude protein showed a decrease of 2 ~ 5% in each part compared to dry powder, and the largest content change compared to dry powder was fat, which was extracted by dissolving many fat components in ethanol. do. In case of crude ash, it was confirmed that 1 ~ 2% decrease by each part compared to dry powder.

Experimental Example 3 Measurement of Lipid Peroxidation Content in Experimental Animals

In general, liver damage caused by a foreign body such as bromobenzene causes various symptoms, one of which promotes the oxidation of polyunsaturated fatty acids in large amounts in cell membranes, leading to the production of lipid peroxide (Pryor, WA Elservier, Amsterdam). 331-361. 1977). Lipid peroxide is mainly present in the form of malondialhehyde (MDA) or 4-hydroxynonenal (4-hydroxynonenal (HNE)), so it is measured to evaluate the degree of peroxidation (Lee, HJ, Kim, GH and Lee, EO Korean J. Oriental Physiology and Pathology 17 (5): 1177-1181. 2003).

The lipid peroxidation content of liver tissues was determined from the experimental animals of Reference Example 2 according to the method described in the literature (Ohkawa, H., N. Ohish, and K. Yaki. Analytical Biochemistry 95: 351-355. 1979). Was performed.

Grinding applied physiological saline in liver tissue 19 times volume per g of a town 8.1% to swaeaek sodium dodecyl sulfate and 20% acetate buffer (pH 3.5) and then added to 0.8% thiobarbituric acid for the purpose of color development at 95 ^o C 1 After reacting for a period of time, the mixture was cooled at room temperature and centrifuged for 15 minutes by adding n-BuOH: Pyridine (15: 1). A red n-BuOH: pyridine layer was taken, and its absorbance was measured at a wavelength of 532 nm. The content is expressed in malondialdehyde nmole per gram of liver tissue is shown in Table 2 below.

 Group Dose (mg / kg)  Content MDA nmoles / g of tissue  Normal           19.8 ± 2.17f  BB administration group           51.3 ± 5.49abc  AC-R 100           48.1 ± 3.18bcd 200           45.3 ± 2.47d  AC-R-E 100           46.7 ± 3.49cd 200           31.9 ± 3.17e  AC-R-R 100           49.3 ± 2.83abcd 200           48.7 ± 2.15abcd  AC-O 100           46.5 ± 3.16cd 200           45.7 ± 4.19 d  AC-O-E 100           48.9 ± 3.33abcd 200           47.6 ± 3.56bcd  AC-O-R 100           52.3 ± 3.19ab 200           49.6 ± 4.18abcd  AC-S 100           53.6 ± 2.57a 200           50.7 ± 3.17abc  AC-S-E 100           49.8 ± 2.63abcd 200           51.8 ± 3.25ab  AC-S-R 100           48.7 ± 2.16abcd 200           47.6 ± 3.18bcd 1) Values are mean ± S.D. For 6 experiments. Values followed by the same letter are not significantly different (p <0.05).

As a result, as shown in Table 2, the lipid peroxide content in the liver of the normal group was 19.8 ± 2.17 nmoles / g tissue, 51.3 ± 5.49 nmoles in the case of the experimental group (BB) that caused hepatotoxicity by administration of bromobenzene It was confirmed that lipid peroxide increased significantly with / g tissue.

On the other hand, in the experimental group treated with bromobenzene after pretreatment of each sea urchin extract for 4 weeks, it was confirmed that the sea urchin egg treatment group showed the most significant decrease. In particular, the reduction of lipid peroxide was the most effective in 31.9 ± 3.17 nmoles / g tissue in the 200 mg / kg AC-R-E administration group, and 46.7 ± 3.49 nmoles / g in the 100 mg / kg administration group. In AC-R-R, 49.3 ± 2.83 and 48.7 ± 2.15 nmoles / g tissues showed no difference, but overall decreased lipid peroxide.

46.5 ± 3.16, 45.7 ± 4.19 nmoles / g tissue in AC-O 100 and 200 mg / kg groups, 48.9 ± 3.33 and 47.6 ± 3.56 nmoles / g tissue in AC-OE 100 and 200 mg / kg groups, respectively. Showed a decrease.

AC-S doses were effective at 200 mg / kg rather than 100 mg / kg but did not significantly reduce lipid peroxide. In addition, sea urchin shells were also shown to be more effective at high concentrations (200 mg / kg) in all samples.

Therefore, the sea urchin contains an antioxidant component, which inhibits lipid peroxidation of the cell membrane in liver tissue, thereby inhibiting liver damage caused by lipid peroxide or restoring damaged liver.

Experimental Example 4. Quantification of Glutathione

Hepatotoxicity of brobenzine is reduced to the nucleophilic compound of the tissue by electrophilic compounds such as bromobenzene 3,4-oxide, the primary metabolite. It is known that depleted glutathione (GSH) is depleted (Casini, AF, Ferrali, M., Pompella, A., Maellaro, E., and Comporti, M. Am J Pathol 123: 520-531. 1986 ).

The experiment was carried out in the experimental animal of Reference Example 2 according to the method of the literature (Ellman, GL Arch. Biochem. Biophys. 82: 70. 1959) as follows. 0.5 ml of 4% sulfosalicylic acid was added to 0.5 ml of liver tissue homogenate, centrifuged at 2500 rpm for 10 minutes, 0.3 ml of supernatant was added, 2.7 ml of disulfide reagent was added, and the absorbance was measured at 412 nm. And calculated according to the standard calibration curve is shown in Table 3 below.

Group Dose (mg / kg) Content (μmole / g structure) Normal 5.73 ± 0.85a BB administration group 460 4.06 ± 0.37b AC-R 100 4.17 ± 0.28b 200 4.36 ± 0.41b AC-R-E 100 4.25 ± 0.33b 200 4.49 ± 0.25 b AC-R-R 100 4.09 ± 0.47b 200 4.11 ± 0.51b 1) Values represent mean ± SD (n = 6).
2) Values sharing the same superscript letter are not significantly different each other (p <0.05) by Duncan's multiple range test.

As a result, as shown in Table 3, the glutathione content of the liver tissue of the normal group was 5.73 ± 0.85 ㎛ ole / g tissue, 4.06 ± 0.37 ㎛ in the experimental group (BB) that caused hepatotoxicity by administration of bromobenzene The glutathione content was reduced to ole / g tissue, which is thought to be caused by toxic intermediate metabolites of bromobenzine.

Meanwhile, in the experimental group administered bromobenzene after pretreatment of each extract of sea urchin for 4 weeks, there was no significant increase compared to BB, but the GSH was increased in all samples. Ascending AC-RE 200 mg / kg (4.49 ± 0.25)> AC-R 200 mg / kg (4.36 ± 0.41)> AC-RE 100 mg / kg (4.25 ± 0.33)> AC-RR 200 mg / kg (4.11 ± 0.51). Higher doses (200 mg / kg) were more effective in all samples. Among the sample types, ethanol extract showed the best effect.

Therefore, the improvement of GSH-induced reduction of GSH in the experimental group administered with sea urchin extract may be related to the increased GST activity due to the continuous consumption of sea urchin extract.

Experimental Example 5. Measurement of Enzyme Activity

The experimental animals were anesthetized with CO ₂ gas, blood was collected from the abdominal aorta, and serum was separated and used for transaminase activity. The liver was perfused with physiological saline to remove foreign substances, followed by crushing and centrifugation to mitochondria. ), Cytosol and microsomal fractions were used as enzyme source.

Protein content was measured using bovine serum albumin as a standard according to the method of Lowry et al. (Lowry, OH, et al. Journal of Biological Chemistry 193: 265-275. 1951). Statistical significance was black as ^{Duncan 's multiple rage test, p} < been determined that a statistically significant when 0.05.

5-1. Measurement of AST and ALT Activity

The experimental animals of Reference Example 2 were anesthetized with CO ₂ gas, blood was collected from the abdominal aorta, and serum was separated to obtain a method such as Reitman, S., and Frankel. American Journal of clinical Pathology 28: 58-63. 1957. Using AST, ALT activity was performed as follows.

Alanine transaminase (ALT) (containing 1780 mg of DL-alanine and 29.2 mg of α-ketoglutamic acid per 100 ml) using a prepared kit (Asan Pharmaceuticals), aspartate transaminase (AST) ) (2,660 mg of L-aspartic acid and 29.2 mg of α-ketoglutamic acid per 100 ml) 1.0 ml of substrate solution was preincubated at 37 ^o C for 5 min, 0.2 ml of serum was added, and ALT was 30 min at 37 ^o C. After reacting for 60 minutes, 1.0 ml of color solution (containing 2,4-dinitrophenylhydrazine, 19.8 mg / 100 ml) was added, 1.0 ml of 0.4 N NaOH solution was added thereto, mixed, left at room temperature for 10 minutes, and absorbance was measured at a wavelength of 505 nm. The activity was expressed in Karmen units per mL of serum (Karmen, A. Journal of Clinical Investigation 34: 131-133. 1955) according to the standard calibration curve and is shown in Table 4 below.

 Group Dose (mg / kg)  AST  ALT  IU / L  Normal   68.3 ± 1.29e  25.3 ± 2.18e  BB administration group  183.2 ± 23.5a  60.7 ± 1.83a  AC-R 100  150.6 ± 17.6bc  55.4 ± 3.25b 200  131.7 ± 19.4 cd  49.3 ± 2.77c  AC-R-E 100  127.9 ± 20.5cd  45.7 ± 1.98cd 200  110.3 ± 18.8d  41.6 ± 2.16d  AC-R-R 100  170.2 ± 21.7ab  62.3 ± 3.17a 200  165.3 ± 20.2ab  60.9 ± 3.33a  AC-O 100  190.6 ± 30.2a  61.4 ± 2.17a 200  187.7 ± 19.7a  63.9 ± 3.16a  AC-O-E 100  180.4 ± 23.6ab  60.2 ± 2.57ab 200  169.3 ± 28.5ab  59.3 ± 3.39ab  AC-O-R 100  185.5 ± 21.8a  58.8 ± 3.16ab 200  179.3 ± 19.9ab  59.7 ± 2.98ab  AC-S 100  177.7 ± 17.6ab  61.5 ± 4.17a 200  162.5 ± 18.8ab  63.5 ± 5.19a  AC-S-E 100  189.1 ± 19.5a  62.6 ± 3.18a 200  179.3 ± 21.8ab  60.3 ± 5.10a  AC-S-R 100  193.5 ± 17.2a  59.7 ± 4.77ab 200  189.6 ± 19.9a  61.8 ± 3.98a 1) Values represent mean ± SD (n = 6).
2) Values sharing the same superscript letter are not significantly different each other (p <0.05) by Duncan's multiple range test.

As a result, the activity of aspartate transaminase (AST) and alanine transaminase (ALT) in normal livers was 68.3 ± 1.29 and 25.3 ± 2.18 IU / L, respectively. Were 183.2 ± 23.5 and 60.7 ± 1.83 IU / L, respectively, which significantly increased the enzyme activity.

On the other hand, in the experimental group treated with bromobenzene after pretreatment of each sea urchin extract for 4 weeks, both AST and ALT showed significant decreases compared to BB in both treatment groups of sea urchin and sea urchin except sea urchin. It did not appear, it was confirmed that there is no significance by dose.

However, AST significantly decreased to 150.6 ± 17.6 and 131.7 ± 19.4 IU / L in the AC-R 100 and 200 mg / kg groups, and 127.9 ± 20.5 and 110.3 in the AC-RE 100 and 200 mg / kg groups, respectively. It was confirmed that the significant reduction effect was ± 18.8 IU / L. In particular, both AC-R and AC-R-E showed more significant reductions at 200 mg / kg than 100 mg / kg. In addition, AC-R-E showed a more significant effect.

In case of ALT, 55.4 ± 3.25 and 49.3 ± 2.77 IU / L of AC-R 100 and 200 mg / kg group showed significant decrease compared to 60.7 ± 1.83 IU / L of BB, respectively. , 200 mg / kg treated group showed 45.7 ± 1.98 and 41.6 ± 2.16 IU / L, respectively. In addition, as in the case of AST, administration of 200 mg / kg was more significant than 100 mg / kg.

On the other hand, as in AC-R-R, both enzymes did not have a significant reduction effect, and it was confirmed that there was no significant difference between doses.

5-2. Aminopyrine demethylase Activity

With the slight modification of the method of Nash et al. (Nash. T. Journal of Biological Chemistry 55: 412-416. 1953), 2 mM aminopyrine in 0.1MNa ⁺ / K ⁺ phosphate buffer (pH 7.5) in 2 ml of the reaction solution, HCl, 0.5 mM NADPH, 10 mM MgCl ₂ , 150 mM KCl, 1 mM semicarbizide and enzyme solution (30-400 μg of protein) were added and the reaction solution was reacted for 30 minutes at 37 ^° C., followed by 15% ZnSO ₄ and saturated Ba (OH) was added to ₂ to terminate the reaction, and separated 10 minutes centrifuged and then allowed to stand for 5 minutes and the supernatant for 30 minutes at the purposes of the addition of Nash reagent and 60 ^o C for color development in 5 ml of reaction obtained here centrifuged again The supernatant was separated, and the absorbance was measured at a wavelength of 415 nm. The activity was calculated according to a standard curve and is shown in Table 5 below.

5-2. Activity of Aniline hydroxylase

According to the method of Bidlack et al. (Bidlack, WR and Lowry, GL Biochemical Pharmacology 31, 3: 311-317. 1982), 50 mM Tris-HCl buffer (pH) containing 10 mM MgCl ₂ and 150 mM KCl in 2 ml of the reaction. 7.4) was added to the substrate 1 mM aniline HCl, 0.5 mM NADPH and enzyme solution (300-400 μg protein) was reacted for 20 minutes at 37 ^° C and 20% trichloroacetic acid for the purpose of terminating the reaction. acid), followed by centrifugation for 10 minutes. 10% Na ₂ CO ₃ and 0.2N NaOH (containing 2% phenol) were added to the supernatant for 30 minutes at 37 ^o C. The absorbance was measured at wavelength 640 nm. Read and calculate the activity in the standard curve is shown in Table 5 below.

Group Dose (mg / kg) AD AH Normal  4.27 ± 0.18d  0.71 ± 0.105d BB administration group 460  9.38 ± 0.25a  1.43 ± 0.211a AC-R 100  9.17 ± 0.22a  1.27 ± 0.137ab 200  8.66 ± 0.15b  1.13 ± 0.125bc AC-R-E 100  8.72 ± 0.17b  1.10 ± 0.148bc 200  7.43 ± 0.14c  0.98 ± 0.131c AC-R-R 100  9.29 ± 0.21a  1.39 ± 0.213a 200  9.31 ± 0.19a  1.36 ± 0.197a 1) Values represent mean ± SD (n = 6).
2) Values sharing the same superscript letter are not significantly different each other (p <0.05) by Duncan's multiple range test.
AD: Aminopyrine N-demethylase: formaldehyde nmole / mg protein / min
AH: Aniline hydroxylase: p-aminophenol nmole / mg protein / min

As a result, as shown in Table 5, the AD enzyme activity of the normal group was 4.27 ± 0.18, and in the experimental group (BB) induced hepatotoxicity by administration of bromobenzene, the enzyme activity was 9.38 ± 0.25, compared to the normal group. Increased significantly. On the other hand, when bromobenzene was administered after pretreatment of each sea urchin extract for 4 weeks, 8.66 ± 0.15, 8.72 ± 0.17, 7.43 ± at AC-R 200 mg / kg, AC-RE 100 and 200 mg / kg, respectively. 0.14 nmols / mg / protein / min showed a significant inhibitory effect, and AC-R 100 mg / kg, AC-RR 100, 200 mg / kg showed no significant but inhibitory activity.

AH enzyme activity was increased to 0.71 ± 0.105 nmols / mg / protein / min in the normal group and 1.43 ± 0.211 nmols / mg / protein / min in the BB. On the other hand, after four weeks of pre-treatment with each extract of sea urchin, it was 1.13 ± 0.125, 1.10 ± 0.148, 0.98 ± 0.131 nmols / mg / protein at AC-R 200 mg / kg, AC-RE 100 and 200 mg / kg, respectively. / min showed a significant inhibitory effect, AC-R 100 mg / kg, AC-RR 100, 200 mg / kg was not significant, but showed the same pattern as the enzyme activity of AD by inhibiting the activity.

As an enzyme to metabolize detoxification type 1 drugs, the activity of aminopyrine demethylase (AD) which forms formaldehyde based on aminopyrineHCl as a substrate and aniline to metabolize detoxification type 2 drugs In the activity of (aniline) HCl-based aniline hydroxylase (AH), which produces p-aminophenol, sea urchin raw material significantly inhibits AD and AH at a high concentration of 200 mg / kg. AC-RE significantly inhibited AD and AH at all concentrations, especially at high concentrations (200 mg / kg). On the other hand, AC-R-R was found to have no significant inhibitory effect in both AD and AH.

5-3. Glutathione S-transferase

Glutathione S-transferase (GST) is a enzyme that is involved in detoxifying an electrophilic compound with glutathione. It has been reported that GST itself binds directly to an electrophilic compound without using GSH (Litwack, G., Ketterer, B., and Aris, IM Nature 234: 466-467. 1971).

According to the method of Habig et al. (Habig, WH, MJ Pabst, and WB Jakoby.Glutathione S-transferase.Journal of Biological Chemistry 249 (22): 7130-7139), 0.1 M potassium phosphate buffer (pH 6.5) was added to 3.5 ml of the reaction solution. 1 mM glutathione, 1 mM 1-chloro-2,4-dinitrobenzene and 0.1 ml of enzyme solution were added to the reaction mixture at 25 ^° C. for 2 minutes, followed by thioether ( thioether) was read at 340 nm and the enzyme activity was calculated using an absorption coefficient of 9.6 mM ^-1 cm ^-1 and is shown in Table 6 below.

Group Dose (mg / kg) Activity (1,2-dinitro-4-nitrobenzene nmole / mg protein / min) Normal         218.6 ± 17.8a BB administration group 460         170.5 ± 18.3b AC-R 100         181.6 ± 21.3b AC-R 200         190.2 ± 19.0b AC-R-E 100         185.7 ± 15.3b AC-R-E 200         195.6 ± 20.5b AC-R-R 100         170.9 ± 21.8b AC-R-R 200         173.5 ± 22.5b 1) Values represent mean ± SD (n = 6).
2) Values sharing the same superscript letter are not significantly different each other (p <0.05) by Duncan's multiple range test.

 As a result, as shown in Table 6, the enzyme activity in the liver of the normal group was 218.6 ± 17.8 nmols / mg / protein / min, the enzyme activity of the experimental group (BB) induced the liver toxicity by administration of bromobenzene At 170.5 ± 18.3 nmols / mg / protein / min, significant activity inhibition was observed compared to the normal group.

On the other hand, in the experimental group treated with bromobenzene after pretreatment of each sea urchin extract for 4 weeks, there was no significant increase in enzyme activity compared to BB, but the overall increase was observed.

200 mg / kg and 100 mg / kg of AC-RE increased to 195.6 ± 20.5 and 185.7 ± 15.3 nmols / mg / protein / min, respectively, and 200 mg / kg and 100 mg / kg of AC-R, respectively. 190.2 ± 19.0, 181.6 ± 21.3 nmols / mg / protein / min. These results suggest that there is no significant change in the activity of this enzyme even under pathophysiological conditions such as bromobenzene administration because the absolute amount of GST protein in liver tissue is very large.

5-4. γ-glutamylcysteine synthetase

GSH is a substance composed of tripeptide of γ-glutamylcysteing glycine (γ-Glutamylcysteinglycine) and is involved in cell detoxification. In the first stage of GSH synthesis, γ-glutamylcysteine synthase acts, and this enzyme binds glutamate and cysteine, which in turn acts as GSH synthetase, resulting in glycine. By binding, the tripeptide GSH is synthesized (Kim, SY, Jung, JY, Kim, JR, and Kim, JH The Yeungnam Med. J. Vol. 11, No. 2. 1994).

Reaction in accordance with Meister and Richman's method (Meister, A. and Richman, PG: Regeneration of γ-glutamylcystein synthetase by nonallosteric feedback inhibition by glutathione. J. Biol. Chem., 250, 1422 (1975) p158. (1967)) 3.5 ml solution of 10 eseo 37 ^o C was added a 0.1M tris HCl buffer (pH8.0), 8.9mM L-glutamic acid, 0.94mM EDTA, 3.2mM MgCl 2, 1.35mM ATP and the enzyme solution (100-300 μg protein) After the reaction, the enzyme activity at 600 nm was measured using a spectrophotometer, and the results are shown in Table 7.

Group Dose (mg / kg) Activity (Pi nmole / mg protein / min) Normal            19.4 ± 1.48a BB administration group 460            14.3 ± 1.19b AC-R 100            14.8 ± 1.09b 200            14.9 ± 1.46 b AC-R-E 100            15.0 ± 1.18 b 200            15.5 ± 1.13b AC-R-R 100            14.6 ± 1.92 b 200            14.2 ± 1.41b 1) Values represent mean ± SD (n = 6).
2) Values sharing the same superscript letter are not significantly different each other (p <0.05) by Duncan's multiple range test.

As a result, as shown in Table 7, the γ-Glutamylcystein synthetase enzyme activity of the normal group was 19.4 ± 1.48 Pi nmols / mg / protein / min, and the enzyme activity of the experimental group (BB) induced hepatotoxicity by administration of bromobenzene 14.3 ± 1.19 Pi nmols / mg / protein / min decreased compared to normal group. On the other hand, in the experimental group treated with bromobenzene after pretreatment of each sea urchin extract for 4 weeks, there was no significant increase in enzyme activity in all samples, especially high concentration of 200mg / kg EtOH extract samples and raw materials. In the samples, 15.5 ± 1.13 and 14.9 ± 1.46 Pi nmols / mg / protein / min, respectively.

5-5. Glutathione Reductase Activity

GSH is oxidized by GSH peroxidase to form an oxidized type GSH disulfide (GSSG), and is reduced back to GSH by GSH reductase. This GSH-associated enzyme, GSH reductase, is sensitive to contaminants and drugs in the external environment and is used as an indication of the toxic effects of their ingestion and inhalation (Arrick, BA, Nathan, CF Cancer Res 44: 4224-4232. 1984.).

0.1 M potassium phosphate in 3.0 ml of the reaction according to the method of Mize and Langdon (Mize, CE and RG Landon. 1962. Hepatic glutathione reductase: I. purification and general kinetic properties. J. Biol. Chem. 237, 1589-1595). buffer (pH 7.5), 0.94 mM EDTA, 4.6 mM oxidized glutathione, After adding 0.16 mM NADPH and enzyme solution (400-600 μg protein) for 10 minutes at 37 ^° C., the amount of glutathione measured at 340 nm was measured and shown in Table 8 below.

Group Dose (mg / kg) Activity (glutathione nmole / mg protein) Normal           23.9 ± 4.25a BB administration group 460           16.8 ± 1.43b AC-R 100           16.2 ± 2.11b 200           17.6 ± 2.36b AC-R-E 100           16.3 ± 1.89 b 200           18.7 ± 1.99b AC-R-R 100           15.9 ± 2.06b 200           16.0 ± 2.31 b 1) Values represent mean ± SD (n = 6).
2) Values sharing the same superscript letter are not significantly different each other (p <0.05) by Duncan's multiple range test.

As a result, as shown in Table 8, the GSH reductase activity of the normal group was 23.9 ± 4.25 nmole / mg, and the experimental group (BB) induced hepatotoxicity by administration of bromobenzene (BB) is normal to 16.8 ± 1.43 nmole / mg It was confirmed that the activity inhibition phenomenon compared to the group.

Enzyme activity was increased in AC-R-E and AC-R at 18.7 ± 1.99 nmole / mg at AC-R-E 200 mg / kg and 17.6 ± 2.36 at AC-R.

5-6. Epoxide hydroxylase activity

The epoxide detoxification system is important during liver detoxification. Bromobenzene is commonly used as an epoxide toxicity model (Park, JC, Choi, JS, Song, SH, Choi, MR, Kim). , KY, and Choi, JW Kor. J. Pharmacogn . 28 (4): 239-246. 1997). Bromobenzene is converted to highly toxic bromobenzene-3,4-oxide by a mixed function oxidation system, and an enzyme that metabolizes this epoxide to metabolize non-toxic bromobenzene-3,4-oxide It is an epoxide hydrolase, which is excreted in bromobenzene glutathione by other detoxifying glutathione S-transferase (Kim, EJ, Lee, CK, and Choi, JW Kor. J. Pharmacogn. 23 (2): 81 -82. 1992).

According to Hammock et al. (Hasegawa, LS and Hammock, BD Biochem Pharmacol 31 (11): 1979-1984. 1982) trans-stilbene oxide as a substrate in 0.1 M potassium phosphate buffer (pH 7.0) (TSO), 3 mM) and an enzyme source (100-200 µg protein) were added to make the reaction solution 3.0 ml. The reaction solution was reacted at 37 ° C. for 20 minutes, and the absorbance at 229 nm was measured for the amount of TSO lost.

Group Dose
(mg / kg) Activity
(TSO nmle / mg protein / min) Normal           14.2 ± 1.16a BB administration group 460           4.16 ± 0.53e AC-R 100           5.27 ± 0.77de 200           6.37 ± 0.92c AC-R-E 100           5.86 ± 0.63cd 200           7.77 ± 0.65b AC-R-R 100           4.33 ± 0.82e 200           4.52 ± 0.71e 1) Values represent mean ± SD (n = 6).
2) Values sharing the same superscript letter are not significantly different each other (p <0.05) by Duncan's multiple range test.

As a result, as shown in Table 9, in the normal group, the epoxide hydrolase activity in the liver tissue was 14.2 ± 1.16 nmol / mg, but in the case of BB, the enzyme activity was 4.16 ± 0.53 nmol / mg, which was significantly higher than that of the normal group. An inhibition phenomenon could be observed.

On the other hand, 6.37 ± 0.92 nmol / mg, 5.86 ± 0.63 nmol / mg, 7.77 ± 0.65 nmol / mg at AC-R 200 mg / kg, AC-RE 100 and 200 mg / kg, respectively. The concentration of hydrolase was restored. In particular, it was confirmed that AC-R-E showed the greatest effect at 200 mg / kg.

Hereinafter, the formulation examples of the composition of the present invention will be described, but the present invention is not intended to limit the present invention but merely to explain in detail.

Formulation Example 1 Preparation of Powder

AC-R 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 Tablet

AC-R-E 10 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

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

Formulation Example 3 Preparation of Capsule

AC-R-R 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 Injection

AC-R 10 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO _4, 12H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation Example 5 Preparation of Liquid

AC-R-E 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding a proper amount of lemon aroma, and then mixing the above components, adding purified water and adjusting the whole to 100 ml by adding purified water and filling into a brown bottle. The solution is prepared by sterilization.

Formulation Example 6 Preparation of Healthy Food

AC-R-R 1000 mg

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg vitamin B1

0.15 mg of vitamin B2

0.5 mg vitamin B6

0.2 [mu] g vitamin B12

10 mg vitamin C

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation Example 7 Preparation of Healthy Drink

AC-R 1000 mg

Citric acid 1000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to a total of 900 ml

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.

[National R & D project supporting this invention]

[Project unique number] B0010517-2008-01 / RRC00200-0000-00 / R01-2007-000-20704-0

[Department name] Ministry of Knowledge Economy / Ministry of Knowledge Economy / Korea Science Foundation

[Name of Research Project] Regional Industry Promotion Project / Regional Cooperation Center Project / Specific Basic Research

[Research Project Name] Sokcho Wellbeing Salted Fish Product Development Project / East Coastal Marine Bioresources Research Center Performance Utilization Project / Verification of health functionalities of fish and shellfish, seaweed and identification of key coumpounds and functional materialization

[Organization] Gangneung-Wonju National University / Gangneung-Wonju National University / Gangneung-Wonju National University

[Research Period] July 1, 2008-June 30, 2011 / January 1, 2009-December 30, 2009 / September 01, 2007-August 31, 2010

1 is a diagram showing a bromobenzene metabolic pathway.

Claims (10)

Anthocidaris crassispina : A pharmaceutical composition for the treatment and prevention of fatty liver or hepatitis, which contains a soluble extract of water, ethanol, and ethanol mixed solvent of eggs, intestines or shells. delete delete delete delete delete delete Purple sea urchin ( Anthocidaris crassispina) Health functional food for the prevention and improvement of fatty liver or hepatitis containing water and ethanol mixed solvent soluble extract of egg, intestine or shell as an active ingredient. delete delete

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