KR100891493B1 - Pufferfish liver oil with biofunctional activities and its manufacturing method - Google Patents

Abstract

A pufferfish liver oil and a manufacturing method thereof is provided to offer muscle cell proliferation inhibitory effect to prevent arterial sclerosis by performing degumming, deoxidation, and bleaching etc of the degumming, deoxidation, bleaching. A method for manufacturing pufferfish liver oil comprises the following steps of: extracting and filtering pufferfish liver oil after heating the liver with NaOH and distilled water; degumming the extracted pufferfish liver oil while injecting pufferfish liver oil for 20 minutes; deacidifying supernatant; bleaching the deacidified pufferfish liver oil after adding activated charcoal and acid clay at 60 °C for 20 minutes(D); and deodorizing the bleached pufferfish liver oil with a steam heating under pressure method(E).

Description

Pufferfish liver oil with biofunctional activities and its manufacturing method

 The present invention relates to a blowfish liver oil extract having a physiological activity and a method for preparing the same, which are described in detail. Using puffer fish liver oil, which contains many of the same polyunsaturated fatty acids, the effect of improving liver function, treating hyperlipidemia, and the abnormal proliferation of smooth muscle cells are known to be a major risk factor for atherosclerosis. The present invention relates to a puffer fish liver oil extract having a physiological activity that is intended to be used as a raw material of functional foods and medicines for preventing and treating arteriosclerosis, and a method of manufacturing the same.

Despite the peculiar toxicity of puffer fish, the demand for fish is rapidly growing nationwide, which is rare for a single fish species.

 On the other hand, the soy sauce of blowfish has a high lipid content and relatively good freshness immediately after pretreatment, so it is highly available as a major fish oil resource. Cod liver oil and shark liver oil have been used as a functional food material for a long time, and if the pulp liver oil is developed commercially, it is expected to be highly commercialized.

 Due to the various living conditions and dietary changes of modern people, there have been many changes in the form of diseases, especially the metabolic diseases such as circulatory system and pulse system are increasing. In Korea, various adult diseases such as obesity and cardiovascular diseases are increasing to the level of developed countries due to the change of eating habits. In order to prevent cardiovascular diseases, various methods for avoiding excessive intake of calories and limiting the amount and type of fat ingested have been proposed, but in recent years, proper intake of natural foods is important for cardiovascular diseases. In this study, fish oil contains many polyunsaturated fatty acids, including EPA and DHA, which are effective for various diseases such as heart disease. In particular, fish liver oil is rich in vitamins A, D, and E. It is worth developing as a dietary supplement. The physiological functions of polyunsaturated fatty acids are effective in preventing cardiovascular diseases, antitumor effects, and memory learning loss, including improving blood flow, preventing and preventing cerebral infarction and myocardial infarction, and preventing and preventing arteriosclerosis. Various physiological effects are also expected, such as improvement of function, improvement of allergic constitution, prevention of progression of diabetes complications and improvement of skin shine.

   In 1993, Russel Ross of the University of Washington, in his cellular injury response, argued that abnormal proliferation of vascular smooth muscle cells is one of the major causes of these atherosclerosis. In fact, in atherosclerosis, atheroma is formed by the combination of early muscle atherosclerotic lesions with smooth muscle cells, vascular endothelial cells, fibrous cells, phagocytic cells and various inflammatory cells. Because of the heterogeneity of these cells in atherosclerosis, it is impossible to explain that any one cell plays a central role in the formation of lesions, but atherosclerotic lesions as a result of balloon injury in animal experiments. Abnormal proliferation of smooth muscle cells has been observed in most patients, and since then, most cardiovascular disease doctors and vascular biologists have assumed that this abnormal proliferation of smooth muscle cells is an important risk factor for atherosclerosis.

 Smooth muscle cells, which are known to be present in the mesenchymal layer of the aorta, maintain the cell cycle state at G0 / G1 while maintaining a quiescent state without cell proliferation under normal conditions. However, after injury to the inner wall, the smooth muscle cells migrate to the inner layer of the aorta, and then divide again, causing DNA replication through the G1 phase, S phase, and M phase through the G2 phase. Induce. In most cells, DNA replication beyond G1 checkpoint and mitosis beyond G2 checkpoint require the activity of a CDK (Cyclin-dependent kinase) protein. CDK alone cannot be activated and is called Cyclin. It must be combined with protein to have kinase activity. Thus, the Cyclin-CDK complex induces cell division by phosphorylating proteins involved in cell proliferation. Cyclin and CDK come in many varieties. The S phase-inducing proteins are mainly cyclin D / E and cdk4 / 6, and cdk4 / 6 phosphorylates Rb (Retinoblastoma) protein, resulting in transcriptional activity of transcription factor E2F. There are many factors that regulate the activity of the cyclin-cdk complex for cell cycle regulation, and the representative ones are p21 and p27 proteins that act when DNA is damaged at the G1 checkpoint. They bind to the cyclin-cdk complex that induces S-phase, inhibiting the kinase activity of cdk4 / 6/2, leaving the cells in G1 state.

In general, fish oil has high content of polyunsaturated fatty acids such as DHA and EPA, and various physiological functions are revealed. Among them, prevention and prevention of cerebral infarction and myocardial infarction, prevention of arteriosclerosis caused by food, antitumor effect, memory learning It is known to prevent deterioration. Despite these trends, little research has been conducted on the basic studies of cellular mechanisms such as cell inhibition and various cell cycle regulation in the prevention of atherosclerosis of fish oil.

Puffer fish has a specific toxicity, but because of its toxicity, puffer fish is distributed to food and non-edible parts by professional wholesalers after being separated and processed.

 At this time, the blowfish soy sauce separated and removed is so large that the soy sauce is about 18g per 100g of fish compared to other fish species, and the amount of lipid is much higher than that of other portions, which contains about 35g per 100g of soy sauce. Soy sauce has a high lipid content and relatively good freshness immediately after pretreatment, so it is highly available as a major fish oil resource, but studies on this area have been insufficient.

In general, fish oil has high content of polyunsaturated fatty acids such as DHA and EPA, and various physiological functions are revealed. Among them, prevention and prevention of cerebral infarction and myocardial infarction, prevention of arteriosclerosis caused by food, antitumor effect, memory learning It is known to prevent deterioration. Despite these trends, little research has been conducted on the basic studies of cellular mechanisms such as cell inhibition and various cell cycle regulation in the prevention of atherosclerosis of fish oil.

The blowfish liver has a high lipid content and needs to be used as a major fish oil resource. Since blowfish is a fish with a unique physiological function to defend its life, there is a high possibility of new functional ingredients. The effects of edible blowfish liver oil on the prevention of various adult diseases were reviewed.

 Therefore, the present invention is to remove the residual toxicity in the puffer fish liver oil, extract and purify the liver oil, safety review, physiological in order to develop a technology for manufacturing and industrializing the high value-added puffer fish liver oil from blowfish soy sauce that has not been used until now For characteristics and commercialization, blowfish soy sauce containing about 30 to 40% fat contains a large amount of n-3 polyunsaturated fatty acids EPA and DHA.

 As such, the physiological functions of polyunsaturated fatty acids, EPA and DHA, are effective in the prevention of cardiovascular diseases, prevention of anti-tumor effects, and reduction of memory learning ability, including improvement of blood flow, prevention and prevention of cerebral infarction and myocardial infarction, prevention and prevention of arteriosclerosis. In addition, various physiological effects such as improvement of eye function, improvement of allergic constitution, prevention of progression of diabetic complications and improvement of skin shine are expected. The aim of this study was to establish the effectiveness of this study and the technologies available as new dietary supplements.

The present invention relates to a puffer fish liver oil having a physiological activity and a method of manufacturing the same, using a puffer fish liver oil containing a large amount of polyunsaturated fatty acids such as n-3 fatty acids, EPA and DHA, in the liver of puffer fish. Of functional foods and medicines for the prevention and treatment of atherosclerosis by examining the proliferation of smooth muscle cells, which are known to be an important risk factor for atherosclerosis and the efficacy of hyperlipidemia and the treatment of hyperlipidemia. At the same time, it would be possible to pursue the effect of providing puffer fish liver oil having the physiological activity to be used as a raw material.

The present invention relates to a puffer fish liver oil extract having a physiological activity and a method for preparing the same, which are described in detail. Using puffer fish liver oil, which contains many of the same polyunsaturated fatty acids, the effect of improving liver function, treating hyperlipidemia, and the abnormal proliferation of smooth muscle cells are known to be a major risk factor for atherosclerosis. The present invention relates to a puffer fish liver oil extract having a physiological activity that is intended to be used as a raw material of functional foods and pharmaceuticals for the prevention and treatment of atherosclerosis, and a method of manufacturing the same.

 The present invention will be described in more detail with reference to the following Examples and Experimental Examples. However, the examples are not intended to limit the present invention only to illustrate the invention, and various modifications are possible.

 Example 1 Ingredients of Blowfish Soy Sauce

 1) Test Material

Blowfish professional processing factory (East Food Co., Ltd. in Busan Gijang County Material) milbok (Lagocephalus in gloveri Abe et Tabeta) was used to then remove the puffer fish liver separate the treatment process, with the rapid processing, immediately frozen at -35 ℃ sample.

Example 2 Blowfish liver oil production technology

 1) Extraction and Purification of Lipids by Solvent Extraction

In order to extract total lipid from black liver, 50 g of the sample was taken, 10 ml of distilled water was added, and a mixed solution of 100 ml of methanol and 50 ml of chloroform was added and ground in a homogenizer for 2 minutes. 50 ml of chloroform was added to the mixture and homogenized further for 30 seconds. The homogenate was weakly suction filtered with buchner funnel using Dongyang Filter (No. 5), the pigment was removed using activated carbon (Wako pure chemical), and then the filtrate was transferred to the separatory filter to pour ether and shake vigorously. After extracting only fat, it was removed with water through anhydrous Na 2 SO 4 to obtain a crude lipid.

 All solvents used for lipid extraction were removed by vacuum evaporator under nitrogen stream and the amount of lipid was calculated by gravimetric method. Meanwhile, the purified lipid was dissolved in a small amount of ether and filled with nitrogen gas, and then stored in a freezer at -20 ° C. and used as a fraction of the lipid and a sample for fatty acid analysis.

 2) Detoxification and Extraction of Liver Oil by Alkaline Hot Water Extraction Method

  (A) Detoxification and extraction and purification of liver oil

Liver oil was extracted from Lagocephalus gloveri Abe et Tabeta by hot water extraction to obtain a bulk extract of fish oil. In other words, 2 liters of tap water was added to 3 kg of black lactose liver, NaOH was added to a concentration of 1%, heated in a hot bath at 100 ° C. for 2 hours, and extracted with puffer fish liver oil in parallel with detoxification treatment. crude oil) was obtained. Blowfish tidal oil was stored in a freezer (-25 ° C) and used for the experiment. The purification of blowfish tidal oil was used as sample liver oil in the order of degumming, deoxidation, decolorization, and deodorization which are common fish oil refining methods.

  (B) Animal experiment

Toxicity tests were performed in accordance with the Pufferfish Poisoning Test in the Food Code. That is, 25 ml of 0.1% acetic acid buffer solution was added to 10 g of the sample, heated in a boiling water to extract sufficient toxins, filtered through Oriental Filter (No. 5), the residue was washed again with 0.1% acetic acid solution, the filtrate was combined and quantified to 50 ml. It was made. The test solution was properly diluted with distilled water, injected into the peritoneal cavity of ICR rats (males, 19-21 g), and the toxicity values were calculated from the lethal time of TTX (tetrodotoxin) and expressed in MU (mouse unit). 1MU means TTX corresponding to an amount that can be killed within 30 minutes of injection.

  (C) HPLC method

HPLC analysis was carried out on a YMC-Pack AM-314 ODS column (6 mm ID × 300 mm) with 2 ml sodium 1-heptane sulfonate / 0.05 M potassium phosphate buffer (pH 7.0) solution and methanol mixture (99: 1) at 1 ml / min. It was eluted at a rate, eluted with 3N NaOH, converted to C ₉ -base (2-amino-6-hydroxymethyl-8-hydroxy quinazoline) at 100 ° C., and analyzed by a fluorescence detector, and detected at an excitation wavelength of 380 nm and a fluorescent wavelength of 505 nm. .

The experimental results according to the embodiment are as follows.

<Experiment 1> Lipid content of puffer fish liver

Table 1 shows the results of measuring the weight of the soy sauce and the content of lipids with respect to the total weight of the fish for the ten frozen clad fish which are the sample fish. The lipids in the liver were 29.7 ~ 37.3%, which is quite high, indicating that the liver is rich in lipids. These results suggest that blowfish soy sauce has a high potential value as a useful fish oil resource. On the other hand, in the case of the cladding used as a sample, 10 males of 26.3-31.2 cm in total length and 407-464 g in weight were relatively good fish species.

The blowfish soy sauce used as a sample was used in the experiment while the frozen fresh sample was packed at 20 kg / box and transported to the laboratory and stored in a freezer maintained at -45 ° C. Table 2 shows the results of measuring the general composition and freshness of blowfish soy sauce. As a result, the contents of moisture, crude protein and crude lipid were 44.7%, 21.5% and 31.9%, respectively, and the content and pH of VBN were 19.2% and 6.2, respectively.

<Experiment 2> Fatty acid and toxicity test of puffer fish liver oil

 (1) Total Lipid Composition and Major Fatty Acids of Liver Oil by Solvent Extraction

The total lipids of puffer fish liver oil obtained by solvent extraction method were neutral lipids, phospholipids and glycolipids of 95.46%, 1.45%, and 3.09%, respectively. Fatty acid composition of total lipid was C18: 1 23.62%, C16: 0 17.38%, C22: 6 15.99%, and these three fatty acids accounted for more than 50%, and DHA (C22: 6) and EPA (C20: 5). Were 15.99% and 3.04%, respectively, accounting for a total of 19.03% (Table 3).

 In the case of triglycerides, C18: 1 13.96%, C16: 0 20.11%, C22: 6 16.62%, these three fatty acids also accounted for more than 50%, DHA (C22: 6) and EPA (C20: 5), respectively 16,62% and 2.41%, totaling 19.03%. Phospholipids and glycolipids were excluded from the analysis because of their relatively small amounts.

 (2) Blowfish poisoning treatment by alkaline hot water extraction method

The method of extracting liver oil from blowfish soy sauce is considered to be advantageous to the industry in terms of wastewater treatment than the solvent extraction method using hexane and the like. Detoxification was performed with% NaOH in parallel. After purifying the crude oil, the toxicity test results for purified liver oil were analyzed by animal experiments and HPLC.

 (3) animal experiment

Table 4 shows the results of animal experiments to investigate the toxicity of puffer fish liver oil.

 That is, mice were divided into 5 groups of 5 mice, and the puffer fish liver oil was intraperitoneally injected to 2.0 ml per day to observe its acute toxicity and symptoms for 1 week. On day 1, acute toxicity was not observed and all were nontoxic. However, in the group injected with 2.0 ml, the oil appeared severely up to the 5th day, and all 5 animals died on the 5th day. In the 1.0 ml injection group, 2 of 5 animals died on day 6 and the symptoms were similar. From day 2, the change in body weight of the mice was different according to the group. The reason for this is that excessive lipid infusion was thought to be the result of reduced daily dietary intake due to undesirable effects on mouse metabolism or the biological system. However, there were no typical acute toxic symptoms caused by blowfish poison.

(4) HPLC

   On the other hand, to analyze the toxicity in liver oil analysis by HPLC is shown in Figure 2. In other words, the purified pufferfish poison from raw puffer liver was analyzed by HPLC, and the peaks of typical tetrodotoxin (TTX) and anhydro tetrodotoxin (anh TTX) could be seen, but the presence of blowfish poison could not be confirmed in the case of blowfish liver oil. It did not show HPLC chromatogram pattern. Although black sealed liver was non-toxic in mouse assay, HPLC analysis showed a toxic peak. This means that the black assay of non-black sealed liver is less than 10 MU / g, although it is a safe amount of less than 10 MU / g. Means that the pufferfish venom is present in the black seal. However, in the case of purified puffer fish liver oil, there was no puffer poison peak at all by HPLC analysis. This is thought to be completely removed by the detoxification process by alkaline hot water extraction method of blowfish tidal oil, and also the blowfish poison is sufficiently removed through the purification process of puffer fish liver oil.

Example 3 Purification Technique of Puffer Fish Liver Oil

 Generally, insoluble oils, water, phospholipids, etc. are mixed in the colloidal form of crude oils obtained from the tissues of aquatic animals, and when these are not properly removed, saponifications are not separated in the deoxidation process, thereby deteriorating the purification efficiency and the quality of refined oils. Let's do it. AV was 18.8 KOH mg / g when the amount of phosphoric acid added was 0.3 ml with respect to 100 ml of cod liver oil. The yield of degumming oil was 87.5% when 0.3 ml of phosphoric acid was added (Table 5).

 This is known to be because free fatty acids, monoglycerides and diglycerides, etc., present in puffer fish liver oil, are removed together. Degum puffer fish liver oil contains a large amount of free fatty acids, and it is necessary to form free fatty acids by saponification which is difficult to maintain, to separate and remove them.

 When 14.5 ml of 20% sodium hydroxide solution was added to 100 ml of the crude oil, and the excess amount was calculated from the acid value of 2 to 3 g, the changes in acid value, pH, and yield of blowfish liver oil are shown in Table 6.

 After the degumming treatment, the decolorization treatment was carried out as follows.

 That is, the puffer fish liver oil heated to 60 ° C. was added so that the concentration of phosphoric acid was 0.3%, followed by stirring, standing, and centrifugation (2,000 × g, 20 minutes) for 30 minutes while injecting nitrogen, followed by washing with warm water, followed by dehydration. Deoxidized oil was obtained. The acid value and pH of the puffer fish liver oil deoxidized with sodium hydroxide (60 ° C., 20 minutes) were 2.5 KOH mg / g and 6.5, respectively. This is because most free fatty acids are removed by alkali treatment, and in general, the amount of sodium hydroxide for deoxidation is actually higher than the theoretical value based on the free fatty acid content due to coloring components, metal salts and trace phospholipids in degumming oil. A little excess should be used.

 Natural dyes, saponifications and metal compounds remain in the deoxidized oils, which are dark in color, and cause deterioration in the quality of oils and fats during refining and storage. And the decolorization process was performed as follows. That is, the amount of activated clay was added to 2-3% of dehydrated puffer fish liver oil, and 0.2 to 0.3% of activated carbon was added thereto, followed by heating at 100 ° C. for 20 minutes and filtration under reduced pressure to obtain depigmented oil. The bleaching effect of is shown in Table 7.

 The pH value of the bleached puffer liver oil was not significantly different according to the amount of activated clay, and the acid value was decreased. The color tone was pale yellow as the general vegetable cooking oil, and the yield was up to 75% for the deoxidized oil. On the other hand, the extraction of crude oil was compared with the acid value and pH using a solvent extraction method and hot water extraction method is shown in Table 8.

 In general, fish oil has a characteristic unpleasant odor and taste even after degumming, deoxidation and decolorization identification, and is mainly known to be due to unsaturated hydrocarbons, lower fatty acids and carbonyl compounds. In order to improve the unpleasant smell and taste of the bleached puffer fish liver oil, the deodorization process by the vacuum heating method was performed. That is, the deodorization process is shown in FIG. 3 was carried out under the same apparatus (A: Steam generator, B: Oil heater, C: Condenser, D: Draft, E: Vacuum pump.). That is, under reduced pressure steam distillation apparatus using a pump (E) to 4 torr or less under reduced pressure and heated to a temperature around 180 ℃ as a mentle was carried out deodorization process for 1 hour. After deodorization, nitrogen gas was injected from the upper part of the portion B of FIG. 3 to minimize oxidation of the sample holding after deodorization and was stored. Even after deodorization, the smell peculiar to fish oil was not completely removed, and when it was commercialized in the final stage, it was necessary to encapsulate it to be convenient for ingestion.

 Next, as a result of examining an optimization method for preparing purified puffer fish liver oil in the above process, one of the most efficient methods is as follows.

 That is, crude oil takes 3.0 kg of blowfish soy sauce, adds 2 liters of distilled water, adds NaOH to a concentration of 1% based on the weight of the blowfish soy sauce, and is heated in a water bath at around 60 ° C. After pressing for 2 hours and compressing with follicles, about 1,200 ml of crude oil was obtained. At this time, the crude oil (crude oil) showed a value of pH 4.5, AV 15.37.

 Then we go into the purification stage,

First, the degumming process was added by adding 0.3% phosphoric acid (H ₃ PO ₄ ) to the weight of the puffer fish liver oil extracted and mixed for 20 minutes while injecting nitrogen and degumming. At this time, the crude oil (crude oil) showed a value of AV 21.94.

 In the deoxidation process, the weight of puffer fish liver oil was measured and warmed to 40 ° C., followed by mixing for 20 minutes by adding NaOH to 20% concentration with respect to the weight of puffer fish liver oil, and mixing with saturated brine to 5% concentration. After stirring for 30 minutes in a 40 ° C water bath, the supernatant was separated by Winsim separation (5,000 rpm x 15 minutes). In the above, the weight of puffer fish liver oil is measured in advance in order to obtain the amount of NaOH to be 20% of the weight of liver oil and the amount of saturated saline to be 5% of the weight of liver oil.

 The decolorizing process is carried out by measuring the weight of deoxidized puffer fish liver oil, warming it to 60 ° C, adding acidic clay and 0.5% activated carbon to 2% by weight of puffer fish liver oil, and treating it for 20 minutes under reduced pressure at 60 ° C. Filtration gave about 800 ml of bleached oil. Here, as described above, the weight of puffer fish liver oil is measured in advance to determine the amount of activated carbon so as to add 2% of the weight of cod liver oil and 0.5% of the weight of cod liver oil.

 PH of this bleaching oil was 4.5, and AV value was 0.26. Deodorization process was selected as the optimum manufacturing process by removing the odor peculiar to fish oil by the apparatus as shown in FIG.

Example 4 Biological Activity Test of Puffer Fish Liver Oil

 In this study, we established primary cell line of smooth muscle cells in the aorta of young mouse (4 months) and investigated cell proliferation inhibition and cell cycle change by puffer fish liver oil.

(1) Improve liver function and treat hyperlipidemia

  (A) Material

The raw materials used in this experiment were black laver ( Lagocephalus ) processed every day at Pufferfish processing plant (Dongnam Food Co., Ltd.) located in Busan. gloveri Abe et collected by separation between the Tabeta) with frozen stored in a -35 ℃ were used in the experiments.

  (B) Sample liver oil

   Five times of distilled water was added to the liver of the black lacquer, NaOH was added to make it 1% concentration, and the crude oil was extracted by heating in a hot water for 1 hour. What was done was used as sample liver oil.

  (C) laboratory animals

Experimental animals are distributed from Daehan Biolink Co., Ltd. Environmental controlled rearing system that automatically controls the contrast (12 hours light / dark cycle), humidity (55 ~ 60%) and temperature (24 ± 2 ^o C) in the animal company ICR male rats weighing 20 ± 2 g and reared Sprague-Dawley male rats weighing 200 ± 10 g were used for 2 weeks. Ml / g, 0.005 ml / g and 0.01 ml / g were orally administered for 2 weeks and used for the experiment.

  (D) Swimming experiment

70% water was added to the animal experiment tank (70 × 70 × 60 cm, water temperature 24 ~ 26 ^o C), and the weight of 5% rats was hung by the tail muscles of the rats according to the method of Frenkel. Time was measured (see Table 9).

  (E) Induce hepatotoxicity and hyperlipidemia

Before inducing hepatotoxicity and hyperlipidemia, the animals were dissolved in 10% Tween 80 for 2 weeks and administered orally once a day. Carbon tetrachloride (CCl ₄ ): olive oil (1; 1 v / v) ) Was intraperitoneally administered 0.2 ml per 100 g of body weight and treated 24 hours later. Induced liver toxicity D-galactosamine was a D-galactosamineHCl (GaIN) was dissolved in physiological saline was administered intraperitoneally to the animals by 400 mg / kg, and proceeding in the same way as CCl _4. In order to induce hyperlipidemia with Poloxamer-P407, poloxamer P-407 solution was administered intraperitoneally at 300 mg / kg on the last day of sample administration, and then treated for 24 hours.

  (B) Measurement of bleeding time: Anesthetized experimental animals were cut 0.3 cm at the end of the tail, and the time until bleeding was measured by dipping 5 cm of the tail in 37.5 ° C saline solution.

Measurement of Plasma clotting time: Plaster the plastic test tube in a water bath at 37 ° C, add 100 µl of plasma, 100 µl of saline (0.15 M NaCl), and 100 µl of 25 mM CaCl ₂ , mix, and then add CaCl ₂ while shaking gently. The time from later to the coagulation of plasma was measured.

  (H) Determination of enzyme activity in serum

 Determination of Aminotransferase (AST, ALT) and Sorbitol dehydrogenase (SDH): Determination of Aminotransferase (AST, ALT) was performed by Reitman and Frankel. ) Was used.

  (I) Effect on blood lipid content

   The total cholesterol content was measured using a kit prepared by the enzyme method of Richmond et al. (AM 202-K, Asan), and the triglyceride content was measured using the kit prepared by McGowan et al. (AM 157S-K, Asan). Experiment using.

  (D) Statistical processing

   The results obtained in this experiment were expressed as mean ± standard deviation, and the statistical significance test was done by Duncan's multiple range test.

 <Experiment 1> Test result of improving liver function and treatment of hyperlipidemia of puffer fish liver oil

 (A) Effect on fatigue recovery

Black Lago ( Lagocephalus) gloveri Abe et Tabeta) present by Admiral processed to extract, in the oral administration, the pufferfish liver oil in a water bath in order to observe the effects on fatigue recovery of purified blowfish liver oil per 2 weeks capacity swimming measure the time to death from the result ( Table 9) In the normal mice, the death time was 3.59 minutes, but when puffer fish liver oil was orally administered for 2 weeks, the death time during swimming showed a tendency to increase dose-dependently. From these results, it is thought that the puffer fish liver oil component contains the component which suppresses fatigue.

 (B) Effect on liver function

Activity of ALT, AST and SDH, which are indicators of blood liver function, to observe the effects on the liver function of puffer fish liver oil extracted from puffer fish using carbon tetrachloride (CCl ₄ ) and D-galactosamine (GaIN) as inducing agents of liver injury The results of measuring the results are shown in FIGS. 4 and 5.

When puffer liver oil was orally administered for 2 weeks by dose and carbon tetrachloride and GaIN were administered, blood AST, ALT and SDH activities were significantly increased compared to the normal group. The hepatotoxic mechanism of carbon tetrachloride is metabolized to trichloromethyl free radical (CCl ₃ ), an active metabolite by the cytochrome P450 system in the vesicles of the biofilm, or CCl ₃ is combined with O ₂ molecules to form trichloromethyl peroxy radical (OOCCl ₃ ). Peroxidation of unsaturated fatty acids can disrupt membrane structure and function. On the other hand, these highly reactive radicals also bind to cytochrome P450 itself, destroying these enzymes in the endoplasmic reticulum.

D-galactosamine, an amino sugar, inhibits the synthesis of RNA by decreasing the concentration of UTP, UDP, and UMP through metabolic disorders of galactose, inducing lipid accumulation, and altering the composition of carbohydrates and intracellular Ca ⁺⁺ concentration. It is known to cause damage to liver tissue. In case of acute poisoning of galactosamine, liver necrosis and chronic poisoning result in cirrhosis and cellular tumor. The results of this experiment suggest that pretreatment of puffer fish liver oil reduces hepatotoxicity by carbon tetrachloride and galactosamine.

 (C) Effects of Poloxamer P-407-induced hyperlipidemia and blood flow improvement

   Poloxamer P-407 (Pluroniz F-127, MW = 12.600) is a surfactant that enhances HMG-Co A reductase activity in the liver and is an enzyme that is present on the surface of epithelial cells and is involved in the hydrolysis of circulating blood triglycerides. Inhibition of lipase strongly leads to hypercholesterolemia or hyperlipidemia in rats, which has the advantage of measuring chemical enhancement effects and examining the mechanism of hyperlipidemia compared to the existing methods as a new experimental model. In this study, the results of the observation of the effects of puffer liver oil on the lipid composition and blood flow improvement in rats induced with hyperlipidemia with poloxamer P-407 were shown in Table 10, Table 11, and Table 12.

 The amount of triglyceride in serum was increased about 12-fold by the administration of poloxamer P-407 compared to the normal group, and the dose-dependently decreased in the group of puffer fish liver oil dose-dependently, and the total cholesterol content was also administered by poloxamer P-407. And significantly decreased by pretreatment of puffer fish liver oil. On the other hand, the bleeding time, which was significantly reduced compared to normal animals by treatment with poloxamer P-407, did not reach the normal level, but the bleeding time was prolonged. In the P-407-administered group, plasma hemostatic time was significantly shortened compared to the normal group.

 After examining the effect of puffer fish liver oil on the lipid-induced lipids and blood flow improvement in rats induced with hyperlipidemia with Poloxamer P-407 for 2 weeks, the plasma coagulation time was significantly reduced by the administration of poloxamer P-407. Increased significantly with the administration of. Hyperlipidemia is an index of arteriosclerosis, which is caused by the synthesis of triglycerides and secretion of chylomicron in the small intestine, the synthesis of triglycerides in the liver, the synthesis and secretion of VLDL and LDL-Cholesterol, the synthesis of HDL-Cholesterol and the decrease in lipase activity. In the present experiment, the increase of triglyceride and total cholesterol blood levels in hyperlipidemia induced by poloxamer P-407 was significantly reduced by pretreatment of puffer fish liver oil. Peroxidation of lipids by free radicals containing electrophilic substances produced by exogenous factors not only enhances permeability of cell membranes but also results in overall cytotoxicity resulting in aging and pathologies of various diseases. It is known to be detoxified by the action of a detoxification mechanism. Recently, studies on the mechanism of cholesterol-induced endotherial dysfunction caused by excessive intake of cholesterol have been actively reported. In particular, it is recognized that oxidative modification of LDL plays an important role. When LDL stays in the blood at high concentrations, it increases the aggregation ability of platelets and stimulates the formation of foam cells that damage blood vessel walls. Therefore, there is a high possibility of inducing atherosclerosis in the vascular system where the oxidation of LDL is actively progressing. Lipid-laden foam cells seen in the early stages of atherosclerosis are formed by the modification of monocyte or macrophage derived from excess LDL. LDL, which enters the tissues through the LDL receptors of each tissue, is broken down by lysosome enzymes and used to meet the cholesterol demands of the tissues, which does not form foam cells. The LDL receptor is thereby subject to feedback inhibition. As a result, intracellular LDL receptor synthesis is reduced, and tissue cells can no longer accept LDL. As a result, excess LDL remaining in the blood is chemically modified to contribute to foam cell formation. As LDL undergoes oxidative transformation, many molecules with biological activity are produced, such as lipoperoxides, isoprostanes and aldehydes. Oxidation of LDL increases lipid peroxide and oxygen free radicals, and these molecules are toxic to endothelial cells. Oxidized LDL adheres easily to blood vessel walls and damages blood vessel cells, deforming vascular tissues and at the same time, dividing the cells. It is known to promote and facilitate surrounding oxidized LDL, platelets and macrophage to attach to the vessel wall more easily. Hemostasis, on the other hand, is a series of biochemical reactions that prevent bleeding from damaged blood vessels. Local hemostasis occurs rapidly, with complex activation and inhibitory systems combined to minimize overbleeding and minimize unwanted thrombosis. These reactions are immediate and local and proceed with the fluidity of the circulating blood. Initiation of plasma coagulation begins with the activation of factor VII by tissue factor (tissue factor, tissue thromboplastin) and tissue factors (Tissue factor, tissue thromboplastin). It consists of an extrinsic pathway. Tissue factor (TF) is a lipoprotein that is mainly present in vascular endothelial cells.It is present in a small amount in the extracellular membrane and plasma under normal conditions. Blood clotting is initiated by a staged coagulation reaction of either phosphorus or endogenous. This coagulation-stimulating action is essential for the hemostatic response when blood vessels are damaged, but the blood coagulation during diseases caused by myocardial infarction, cancer or other coagulation It is known to promote. The results of this study suggest that puffer fish liver oil is effective in improving blood lipid components and blood flow in lipoxamer P-407-induced hyperlipidemic rats.

(2) Smooth muscle cell  Effect on proliferation

 (A) Cell line and culture

Aortic-derived smooth muscle cells used in the experiment were used to establish primary cell lines in the aorta of 4 month old mice. For cell proliferation, add 0.2% sodium bicarbonate, 5% fetal bovine serum and DMEM medium with antibiotics to the tissue culture flask (75㎠) at 37 ℃, 5% CO ₂ Incubate until incubated in the thermostat.

 (B) Reagent

   Polyclonal antibodies cyclin D1, cyclin E, CDK2, CDK4, p21 and p27 were purchased from New England Biolabs. XTT assay kit and cell death ELISA kit were purchased from Roche.

 (C) Pufferfish liver oil (PLO)

The sample used in this experiment was a black lago ( Lagocephalus). gloveri Abe et Tabeta) the morning oil was extracted by heating a water bath while distilled water was added to a 5-fold after the addition of NaOH to be a concentration of 1% of the time between 1. Purification of cod liver oil was carried out in the order of degumming, deoxidation, decolorization and deodorization which are common fish oil purification methods.

 (D) MTT assay

   100ul of the medium containing the appropriate cell number was added to 96well, followed by washing with PBS, followed by incubation for 12 hours after the addition of PLO. Thereafter, 50ul of MTT labeling mixture was added thereto, followed by incubation for 12 hours, and the absorbance was measured by ELISA reader at absorbance between 450 and 500 nm.

(E) [ ³ H] thymidine incorporation

Cells cultured in 24 well plates were treated with PLO at an appropriate concentration for 12 hours, and then added ¹ uCi [ ³ H] thymidine. After 12 hours, the cells were washed with PBS and 5% trichloroacetic acid, solubilized with 0.5N NaOH and measured with a liquid scintillation counter.

 (F) Apoptosis assay

   After 24 hours of treatment with the appropriate concentration of PLO, cells cultured in 24well plates were lysed and centrifuged, and the supernatant containing cytoplasmic histone-associated DNA fragments was transferred to 96well plates pretreated with streptavidin, followed by biotin. React with a mixture of pretreated anti-histone antibody and a mixture of anti-DNA antibodies bound to peroxidase. Then, after adding a substrate of peroxidase, the absorbance was measured at 405nm.

 Western blot

   After cell lysates were electrophoresed by SDS-PAGE, they were transferred to nitro cellulose membranes and reacted with 5% skim milk for 1 hour to reduce nonspecific binding. After washing four times with TBS buffer (10mM Tris-Cl, pH 7.4, 0.15M NaCl), the reaction was performed for 1 hour with a secondary antibody, and the band was detected by an ECL kit.

 (H) Immunoprecipitation

   Cell lysates (500ug) were reacted overnight at 4 ° C. with a desired antibody, followed by addition of 25ul of protein A-Sepharose, followed by reaction for 1 hour. Immunoprecipitated proteins were released by reaction in Laemmli buffer at 95 ° C.

 Immune complex kinase assay

After reacting the cell lysates (500ug) overnight with the desired antibody at 4 ℃, add 25ul of protein A-Sepharose, wash the immunoprecipitated protein with lysis buffer, and then wash 1 ug glutathione S- transferase (GST) pRb C-terminal. (pRb amino acids 769 to 921) fusion protein (Santa Cruz Biotechnology) or 5 ug histone H ₁ (Life Technologies), 20 uM / L ATP, and 5 uCi [( ^-32 P] ATP (4500 Ci / mmol; ICN) After reacting for 20 minutes at 30 ° C. in a kinase buffer containing the reaction solution, the reaction solution was released by adding Laemmli buffer and reacting at 95 ° C. Then, the band was detected after drying in a gel dryer after SDS-PAGE.

<Experiment Result 2> Smooth muscle cell  Blowfish liver oil for multiplication PLO Test result of efficacy of

 (A) Effect of proliferation of smooth muscle cells

   First, experiments were conducted on how PLO affects cell growth in smooth muscle cell proliferation. Smooth muscle cells grown in medium containing 10% FBS (fetal bovine serum) were treated with EPA, followed by MTT assay, DNA synthesis and apoptosis assay. MTT assay is a measure of the absorbance of formazan produced by mitochondrial dehydrogenase in living cells to reduce MTT reflects the concentration of live and metabolically active cells, as shown in Figure 6 high concentration of PLO The absorbance was decreased by the treatment, indicating that the proliferation of smooth muscle cells was inhibited by the treatment of PLO.

 Next, the DNA synthesis ability was measured as another measure of the proliferation capacity of smooth muscle cells. After bling thymidine, one of the DNA base sequences, with radioisotopes, PLO was treated to measure radiation content with a liquid scintilation counter to quantify DNA synthesis. In comparison, after PLO treatment, it was reduced by 50% (FIG. 7). These results provide evidence that the treatment of PLO inhibits the proliferation of smooth muscle cells, and the cell biological mechanisms thereof are shown in the next experiment.

 (B) Induction of apoptosis in smooth muscle cells following PLO treatment

 To determine whether PLO treatment induces apoptosis of smooth muscle cells, we used a cell death ELISA kit to quantitatively evaluate the amount of cytoplasmic histone-asociated DNA fragments. As shown in FIG. 8, the amount of cytoplasmic DNA-histone complex was increased by more than 60% as compared to the control without PLO, and the result showed that PLO was clearly apoptotic in smooth muscle cell proliferation. It proves its effectiveness.

 (C) G1 cell cycle arrest by PLO treatment in smooth muscle cell proliferation

   In order to elucidate the mechanism of inhibiting the proliferation ability of smooth muscle cells by PLO treatment, the protein expression levels of cyclinD1, cyclinE, cdk2 and cdk4, which are the major components of the G1 cell cycle, were evaluated by western blot experiment. Independently, the protein expression of G1 cell cycle components cyclinD1, cyclinE, cdk2 and cdk4 gradually decreased (FIG. 9).

 In the case of most cells, cell cycle transition requires cyclin-cdk complex to divide and these components need phosphorylation activity. Therefore, an experiment was conducted to determine whether cdk-associated phosphorylation activity was inhibited by PLO treatment. Using anti-cdk antibodies in cell lysates, the cdk complex was immunoprecipitated, and cdk-associated phosphorylation activity was measured using RB protein and histone H1 as substrates for immunoprecipitated proteins. As shown in FIG. 10, PLO treatment concentration-dependently inhibited phosphorylation activity against cyclinD1, cyclinE, cdk2, cdk4.

 (D) Effect of p21 on G1 cell cycle arrest induced by PLO

  p21 and p27 are negative regulators known to be involved in G1 cell cycle arrest. Since PLO induced G1 cell cycle arrest in the proliferation of smooth muscle cells, we examined whether p21 and p27 were related to G1 cell cycle arrest induced by PLO. As shown in FIG. 11, Western blot results after 24 hours with PLO treatment showed a clear increase in p21 protein compared to the control group not treated with PLO. However, the protein expression of p27 did not increase under the same experimental conditions. In conclusion, PLO inhibited the proliferation of smooth muscle cells, induced apoptosis, and suggests that the mechanism of cell cycle regulator p21 binds to the complex of cyclinD1 / cdk4 and cyclinE / cdk2 to inhibit phosphorylation activity. have.

Although the present invention has been described in detail only with respect to the described embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made within the technical spirit of the present invention, and such modifications and modifications belong to the appended claims .

The refined fish oil for health foods has a very high raw material price of 14,000 won / kg for EPA / DHA and 35,000 won / kg for squalene. Not only is it expected to be used as a functional material, refined fish oil products are imported from 14,976kg (approximately 6.8 billion won) to 142,010kg (approximately 62.3 billion won) annually, and the consumption market is very large. In addition, the average unit price per unit weight (1 kg) refined fish oil-related products are high value-added products of 680,000 won, and the industrial value of health supplements reinforced with functional materials is becoming more important. Therefore, the purpose of the present invention is to provide an effect of expanding the industrial aspect and range of use.

1 is a method for extracting and purifying puffer fish liver oil

Figure 2 Analysis of tetrodotoxin (TTX) using HPLC

Figure 3 is a view showing a reduced pressure steam distillation apparatus used in the deodorization process of puffer fish liver oil

Figure 4 shows the activity of ALT, AST and SDH which is an indicator of blood liver function.

5 is the effect on the liver function of puffer fish liver oil extracted from puffer fish using carbon tetrachloride (CCl ₄ ) and D-galactosamine (GaIN) as a drug causing liver injury

Figure 6 MTT even stone absorbance of smooth muscle cells following PLO treatment

Figure 7 DNA synthesis of smooth muscle cells following PLO treatment

Figure 8 induction of apoptosis of smooth muscle cells following PLO treatment

9 is a mechanism of inhibiting the proliferative capacity of smooth muscle cells by PLO treatment

10 is phosphorylation activity against cyclinD1, cyclinE, cdk2, cdk4 by PLO treatment

11 shows the effect of p21 on G1 cell cycle arrest induced by PLO

Claims (5)

In the extraction of puffer fish liver oil, 2 liters of distilled water was added to 3.0 kg of puffer fish soy sauce, NaOH was added to a concentration of 1% based on the weight of the raw material, and the puffer fish liver oil was extracted by heating the bath at 60 ° C. for 2 hours. Extraction step (A) of the crude oil extracted by compressing and filtration with follicles (crude oil) and phosphoric acid (H ₃ PO ₄ ) to 0.3% to the weight of the puffer fish liver oil and adding nitrogen for 20 minutes Degumming step (B) of mixing and degumming, and the weight of the deguffered puffer fish liver oil was measured, and warmed to 40 ℃, mixed with 20 minutes NaOH to 20% concentration relative to the weight of puffer fish liver oil, 5% concentration After the addition of saturated brine, the mixture was stirred for 30 minutes in a 40 ° C water bath, followed by Winsim separation at a rotational speed of 5,000 minutes for 15 minutes to separate and deoxidize the supernatant to deoxidize puffer fish. After weighing the cod liver oil, go to 60 And the addition of acidic clay and 0.5% activated charcoal to 2% to the weight of puffer fish liver oil, followed by 20 minutes under reduced pressure filtration under reduced pressure to take the discolored oil (D) and the smell of the puffer fish liver oil And a deodorizing step (E) for deodorizing by reduced pressure steam heating to improve the taste of the puffer fish liver oil. Blowfish liver oil, characterized in that the puffer fish liver oil is produced by the method of producing puffer fish liver oil of claim 1. delete The puffer fish liver oil according to claim 2, wherein the puffer fish liver oil has a smooth muscle cell proliferation inhibitory effect. The puffer fish liver oil according to claim 2, wherein the puffer fish liver oil can be used as a health food material for preventing atherosclerosis by inhibiting smooth muscle cell proliferation.

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