KR101442501B1 - The extracts of the anti-atherosclerotic and vascular diseases-preventive and inhibitive activity prepared from the bone of processing wastes of Japanese eel (Anguilla japonica) by the low temperature vacuum extraction - Google Patents

Abstract

The present invention relates to an extract of eel bone-processing by-products extracted by a low-temperature vacuum. More specifically, eel-processing by-products of the eel's head, liver, internal organs, After the selection, these eel bones and processing by-products were pulverized by low-temperature vacuum, and the extract using the pulverized powder had anti-arteriosclerosis activity and anti-vascular disease prevention and inhibitory activity, thus having aging prevention and immunity enhancing activity, The present invention relates to an extract of eel bone-processing by-products extracted with a low-temperature vacuum having anti-arteriosclerosis activity and vascular disease prevention and inhibitory activity, which can be used as health supplements.

Description

TECHNICAL FIELD The present invention relates to an extract of eel bone-processing by-products extracted with a low-temperature vacuum having anti-arteriosclerosis activity and vascular disease prevention and inhibitory activity and a method for producing the same. wastes of Japanese eel (Anguilla japonica) by the low temperature vacuum extraction}

The present invention relates to an application of a patent application No. 10-2012-0062670, and more particularly to an extract of eel bone-processing by-products extracted by low-temperature vacuum, which includes eel hair, liver, , The eel processing by-products of the epidermis were well cleaned and selected, and these eel bones and processing by-products were pulverized by low-temperature vacuum, and the extract obtained from the pulverized powder had anti-arteriosclerosis activity and vascular disease prevention and inhibitory activity The present invention relates to an extract of eel bone-processing by-products extracted with a low-temperature vacuum having anti-arteriosclerosis activity and vascular disease prevention and inhibitory activity, which can be used as a health supplement food with antioxidant and immunostimulating activity.

In general, eel is high in protein content, high in calories, high in unsaturated fatty acids, so it is recognized as the best food for prevention of adult diseases such as hypertension, frailty and restorative, so it is very popular among men in summer especially for stamina.

Eels, which are considered to be health foods, have a higher content of vitamins than ordinary fish, such as freshwater eel, conger eel, eel, and mackerel. The content of vitamin E is about 10 times that of eggs, vitamin A is a common white fish And vitamin A is effective in promoting growth, restoring visual acuity, maintaining skin and mucous membrane health, and energizing agents. The content of unsaturated fatty acids is also very high. Especially, the contents such as DHA and EPA are higher than other species. The efficacy of DHA shows that it has an excellent effect on hair, prevention of dementia, suppression of cancer, and so on, and it uses herbal medicine and natural materials from ancient times.

On the other hand, there is a protein toxin, ichtyohemotoxin, in the mucus of the blood and epidermis of the eel. This toxin causes nausea or poisoning if it enters the human body. If it enters the eye, it causes conjunctivitis, In addition to being inflamed, people with weak skin are at risk of dying if large amounts come into the body. In addition, it is difficult to completely remove this toxin. Most of the eel is not cooked by sashimi, because it is heated only at 60 ℃ or more, but it is not toxic. Instead, it is cooked in the form of a sphere or a soup.

Examples of a patent application for health foods developed using such eel are disclosed in Korean Patent Publication No. 1990-1320 (published on February 27, 1990), a method of producing nutritional food on the theme of freshwater eel, Japanese Patent Application Publication No. 1993-22955 (published on Dec. 18, 1993) discloses a health food using freshwater eel, Korean Patent Laid-Open Publication No. 1996-78 (published on January 25, 1996) It is possible that nutrients may be lost due to chemical changes caused by heat when the eel and the herbal medicine are mixed and heated.

As described above, eel-rich eels are generally used for roasting, sashimi, eel rice, hot water, concentrated liquid, and the like.

However, in such a case, only the edible portion having the meat quality is used according to the intended purpose, and the by-products such as the head, the internal organs, the bones, and the surface skin of the eel are discarded or used for the feed.

In order to solve the above-mentioned problems, the present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a health food The eel bones and processing by-products were pulverized by low-temperature vacuum, after the eel processing by-products of the eel's head, liver, internal organs, gallbladder and epidermis were well cleaned and selected. The extracts from the pulverized powder have anti-arteriosclerosis activity and vascular disease prevention and inhibitory activity, and have antioxidant and immune enhancing activity, and thus can be used as a health supplement food. We tried to provide extracts of eel bone-processing by-products extracted with low-temperature vacuum with disease prevention and inhibitory activity All.

As described above, the extract of the eel bone-processing by-products extracted with the low-temperature vacuum of the present invention and the fraction extract thereof have anti-arteriosclerosis activity and vascular disease prevention and inhibitory activity, and thus have antioxidant and immunostimulating activity, Can be used as therapeutic agents for hepatic function-related and immune activators. In addition, there is no toxicity, so it can be widely used as a health supplement.

Fig. 1 is a graph showing the inhibitory activity of basement membrane degrading enzyme MMP-2/9, which is an indicator of vascular stenosis and proliferation of human vascular smooth muscle cells by TNF-a
FIG. 2 is a graph showing cytotoxicity of human hot-water extract (Hx) of eel by-products to human arterial smooth muscle cell line (HASMC)

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to specific embodiments in order to facilitate understanding of the present invention. However, it should be understood that the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Preparation of eel extract using eel bones and processing by-products

The eel extract material used in the present invention is well cleaned and selected using eel processing by-products of the eel's head, liver, internal organs, gallbladder, and epidermis except for the edible part of the eel, and the eel bones and processing by- And 600 g of eel bone-by-product (hereinafter referred to as eel by-product) powders were each divided into 200 g portions and used for preparing each of the extracts as follows.

1) Eel by-product physiological saline solution (Px)

200 g of the eel by-product powder was pulverized with 300 ml of a 50 mM phosphate buffered saline solution (pH 7.2) corresponding to physiological saline buffer solution, centrifuged at 15,000 x g for 20 minutes at 4 ° C, centrifuged 100 ml was used for the activity assay.

The eel by-product powder was centrifuged with physiological saline buffer solution to extract 10 ml of physiological saline solution. The yield was 13%, and a total of 25 g was obtained.

2) Eel by-product methanol extract (Mx)

To 200 g of the eel by-product powder was added 1 liter of methanol (MeOH), and the mixture was heated at 70 ° C for 3 hours for leaching. The filtrate was collected by filtration (Oriental paper No. 1). The filtrate was collected and concentrated under reduced pressure. About 50 ml of the concentrate was obtained and lyophilized to obtain about 12 g of freeze-dried powder, and the yield was about 6%.

3) Hot water extract of eel by-products (Hx)

To prepare the hot water extract, 1 L of water was added to 200 g of the eel by-product powder, which was then kept in a heating mantle at 45 DEG C for 2 hours and filtered using a membrane filter having a size of 1 mm.

The insoluble material was removed from the filtered extract of eel by-product powder and the supernatant was concentrated to a volume of about 50% using a rotary vacuum evaporator.

The concentrate (10 ml) was dried to obtain a total yield of 24% (12%).

The yields of 1) eel by - product physiological saline (Px) and 3) eel by - product hot water extract (Hx) were similar.

[Experimental method] Measurement of anti-arteriosclerosis activity and vascular disease prevention and inhibitory activity on extracts of eel by-products extracted by low-temperature vacuum

1. Experimental animal treatment

First, Sprague-Dawley male rats weighing 50 to 70 g at 3 weeks of age were used for the experiment for 2 weeks at a temperature of 25 ° C and a relative humidity of 60%.

In the experimental group, 100 mg / day and 200 mg / day of hot-water extract (Hx) from the eel by-product were induced after atherosclerosis induced by normal cholesterol, cholesterol and dietary high cholesterol, And 500 mg / day, respectively.

The amount of water and feed (Hyochang Science, Korea) was supplied without restriction during the experimental period. Dietary cholesterol was mixed with olive oil at a ratio of 4: 1 and administered for 8 weeks to induce arteriosclerosis.

(Hx) by the same method as that of the dietary high cholesterol diet, and induced cholesterol to induce atherosclerosis. Then, the hot water extract (Hx) of eel by-products and water were diluted in a ratio of 3: 7 for 4 weeks Respectively. All experimental groups were fed only with water and fasted 24 hours before treatment.

The animals were treated under ether anesthesia, and then blood was collected from the abdominal aorta and sera were collected. A certain amount of abdominal aortic vascular tissue was extracted. The liver was perfused through the portal vein with 4 ° C physiological saline, The blood was removed and the liver was then extracted. The extracted liver and abdominal aortic vascular tissues were washed with physiological saline and then pressed with a filter paper to remove the remaining physiological saline solution and then weighed and stored at -80 ° C for use as a test sample for antioxidant and oxidative stress test Respectively.

The collected blood was allowed to stand at room temperature for 30 minutes and then centrifuged at 3.000 rpm for 15 minutes to obtain serum and used as a serum lipid content measurement sample.

2. Preparation of sample

A homogenate was prepared by homogenizing four times of pH 7.4 and 0.1 M Potassium Phosphate buffer solution to a certain amount of liver tissue. The homogenate was centrifuged at 600 × g for 10 minutes, The supernatant was removed by centrifugation at 10.000 xg for 20 minutes, and the supernatant was collected and stored at -80 ° C.

The homogenate of abdominal aortic aneurysm was also used for the measurement of lipid peroxidation, xanthine oxdiase, superoxide dismutase (SOD), catalase and glutathione peroxidase activity in liver tissue and abdominal aortic vascular tissue homogenate.

3. Measurement of arteriosclerosis index

The atherosclerotic index, Al, was calculated using a method of calculating atherosclerotic index from low-density lipoprotein cholesterol (HDL-cholesterol) and high-density lipoprotein cholesterol (HDL-cholesterol) measurements.

○ Al = Δ (LDL-cholesterol - HDL-cholesterol) / HDL-cholesterol

4. Measurement of lipid peroxidation content in abdominal aortic vascular tissue

The lipid peroxidation contents in the abdominal aortic vascular tissues were measured by Ohkawa (1979) method using the content of TBA-reacting substances produced by the reaction with thiobarbituric acid (hereinafter referred to as TBA), and 1 g of the abdominal aortic vascular tissue And 5 ml of 0.05 M phosphate butter at pH 7.4. 0.5 ml of the homogenate of abdominal aortic vascular grafting is added to the test tube, 1 ml of 7% sodium dodecyl sulfate (SDS) is added, and the mixture is reacted in a 37 ° C water bath for 30 minutes. 2 ml of an equal volume of 0.67% TBA and acetic acid (1: 1) was added to the reaction mixture. The reaction mixture was heated to 98 ° C for 50 minutes, quenched, and 5 ml of n-buthanol was added thereto. Is centrifuged at 3.000 x rpm for 10 minutes.

The n-butanol layer containing the TBA reactant was then measured for absorbance at 535 nm. The MDA content was expressed in nmoles per gram of tissue.

5. Inhibitory Effect of Xanthine Oxidase Activity in the Abdominal Aortic Aneurysm

Xanthine oxidase activity in the abdominal aortic vascular tissues was measured by Stirpe and Corte (1969) (Stripe, F. and Corie, ED The regulation of rat liver xanthine oxidase. Conversion in vitro of enzyme activity from dehydrogenase ), J. Biol. Chem., 244: 3855, 1969). Dissolve xanthine at a concentration of 2 mM in 0.1 M potassium phosphate buffer, add 1 ml to a microcentrifuge tube, add 0.1 ml of Xanthine Oxidase and 0.1 ml of abdominal aortic vascular grafting homogenate. For the control group, add the same amount of distilled water instead of the sample. The reaction is carried out at 37 ° C for 5 minutes. The reaction is terminated by adding 1 ml of 20% trichloroacetic acid (TCA). Absorbance of uric acid produced in the reaction solution is measured at a wavelength of 292 nm. The XO activity unit is expressed in nmole by the amount of uric acid produced from the substrate xanthine by reacting the protein contained in the enzyme reaction solution for 1 minute.

6. Activation effect of SOD enzyme which is an enzyme of harmful active oxygen scavenging system

Superoxide dismutase (SOD) activity in the abdominal aortic vascular tissues was measured by modifying the method of McCord and Fridovich (1969), and 672 μl of 0.2 M sodium phosphate buffer (pH 7.4), 100 μl of 1 mM xanthine , 30 μl of 1% sodium deoxychlorate (DOC), 30 μl of 1.5 mM Potassium Cyanide (KCN), and 150 μl of 0.2 mM cytochrome C were added to 5 μl of the homogenate for abdominal aortic vascular tissue grinding and Xanthine Oxidase 10 , And the absorbance change at 550 nm is measured for 1 minute and 30 seconds.

Xanthine is used as a substrate, cytocrome C plays a role to prevent O ₂ generation, KCN is a coloring solution, and DOC is an oxidizing agent that makes xanthine hypoxanthine. Hypoxanthine induces the reaction of the superoxide anion O2 ^- to the active site of SOD. The SOD activity unit refers to the amount of protein O ₂ ^- converted to H ₂ O ₂ by the reaction of the protein mg in the enzyme reaction solution for 1 minute and 30 seconds so that O ₂ accepts one electron and is incompletely oxidized. specific activity) was expressed as an enzyme unit equivalent to 1 mg protein.

7. Effect on catalase activity of harmful active oxygen scavenging enzyme

The catalase activity in abdominal aortic vascular tissues was measured by Aebi (1974). After mixing 0.01 ml of homogenate of abdominal aortic vascular tissue homogenate with 2.89 ml of 50 mM potassium phosphate buffer (pH 7.4), 0.1 ml of 300 mM H ₂ O ₂ was added to initiate the reaction, and then the concentration was decreased at a wavelength of 240 nm for 5 minutes The amount of absorbance is measured. The catalase activity unit is expressed as the amount of H ₂ O ₂ (nmol) in which the protein contained in the enzyme reaction solution disappears for 5 minutes.

8. Effect on the activity of the harmful active oxygen-scavenging enzyme Glutathione peroxidase

Glutathione peroxidase activity in abdominal aortic vascular tissues was measured by modifying the method of Lawrence (1976). 0.1 ml of abdominal aortic vascular tissue homogenate was added to 2.6 ml of 50 mM potassium phosphate buffer (pH 7.0), 0.1 ml of 30 mM glutathione, 0.1 ml of 6 mM NADPH and 1 unit of glutathione reductase were added and mixed. Leave at room temperature for 5 minutes. Add 0.1 ml of 7.5 mM H ₂ O ₂ to initiate the reaction and measure the absorbance at 340 nm for 2 minutes. The GSH-Px activity unit is expressed as the amount (nmol) of NADPH oxidized for 2 minutes in mg of protein contained in the enzyme reaction solution.

9. Proliferation assay of smooth muscle cells (in vivo, in vitro)

In vivo smooth muscle cell proliferation experiments were performed by balloon injury modeling (same as above), and 3 μm sections were harvested from a rat grown for a period of time and evaluated by immunostaining. Arterial exposures are perfused with saline and refluxed and immersed in 4% paraformaldehyde solution to make paraffin sections. The degree of proliferation of intravascular smooth muscle cells is assessed by in vivo proliferation using immunostaining that colorizes the reaction of the proliferating cell nuclear antigen (PCNA) with the antigen in the cell nucleus.

In vitro, smooth muscle cell proliferation is obtained by purchasing human smooth muscle cells and using cultured cells (these cells are currently in the laboratory). After culturing for 24 hours, cells were treated with cell proliferation factors (PDGF, FGF, thrombin, TGF-β, etc.) at 1 × 10 ⁵ cells / ml of smooth muscle cells and cultured for 24 hours. MTT and tryphan blue exclusion The degree of proliferation is checked, and the degree of inhibition of the cell cycle of the cell cycle is evaluated using a flow cytometer.

10. MMP-2, MMP-9 zymography analysis

The inhibitory activity of metalloproteinase (MMP) (in vivo and in vitro) was determined by zymography. In the case of tissues, 5 times the amount of homogenate buffer (250 mM sucrose, 10 mM Hepes-Tris , pH 7.5) and developed on electrophoresis (120V, 20mA) on a 10% SDS-polyacrylamide gel containing 0.1% gelatin.

After development, wash with 2.5% Triton X-10 three times for 15 minutes and incubate gel in incubation buffer (50 mM Tris-HCl, 0.2 M NaCl, 10 mM CaCl ₂ , each sample) at 37 ° C for 20 hours. The gel was stained with 0.1% coomassie blue (AcOH: MeOH: H ₂ O, 1: 3: 6) and destained. The band of 74 KD and 92 KD was identified and evaluated.

<Experimental Results>

1. Inhibitory activity of eel by-product extract on human vascular smooth muscle cell-derived MMP-9

In the above Example 1, the extracts of hot-water extract of eel by-products (Hx) and methanol extract of eel by-products (Mx) were used for the inhibition of MMP-9 produced by vascular smooth muscle cells.

The results of the above experiments show that TNF-a of FIG. 1 inhibits the growth of smooth muscle cells derived from human blood vessels and the production of basement membrane degrading enzyme MMP-2/9, which is an index of vascular stenosis. / ml and methanol extract (Mx) 200 ~ 500 ug / ml.

2. Proliferation inhibitory activity (toxicity) on human arterial smooth muscle cell line (HASMC) by MTT assay according to concentration of extracts of eel byproducts (Px, Mx, Hx)

The human arterial smooth muscle cell line (HASMC) (2.4 × 10 ³ ) was dispensed in a 96-well microplate at a rate of 150 μl per well, and MTT assay was performed.

The cell proliferation inhibitory activities of eel byproduct saline (Px), eel by-product methanol extract (Mx) and eel by-product hot water extract (Hx) at concentrations of 0, 2, 5, 10, 50 and 100 ug / ml were examined .

As a result, there was no toxicity at the concentration up to 100 ug / ml, and it was recognized that there was no toxicity for use in future experiments under this concentration. In other words, there is no or low cytotoxicity and it can be used as a health functional food supplement.

FIG. 2 shows the results of cytotoxicity against human arterial smooth muscle cell line (HASMC) of hot-water extract (Hx) of eel by-products. Human arterial smooth muscle cell line (HASMC) (2.4 × 10 ³ ) (Hx) of the eel by-products were not observed in the human arterial smooth muscle cell line (HASMC), and the remaining physiological saline extract (Px) The results of the methanol extract (Mx) were also the same (not shown).

Therefore, Fig. 2 shows the hot-water extract (Hx) of eel by-products.

3. Effect of serum cholesterol on atherosclerotic index of hot-water extract (Hx)

The atherosclerotic index (AI) of cholesterol in the serum of experimental animals

The atherogenic index of cholesterol in serum was calculated as 0.7 in normal group, 1.87 in high cholesterol diet group, 1.87 in high cholesterol diet group, AI = △ [(LDL-cholesterol-HDL-cholesterol) / HDL- 0.8 in the hot water extract of eel (Hx) group and 1.23 in the cholesterol + hot water extract group (Table 1).

4. Suppression of lipid peroxidation in the abdominal aortic vascular tissues of eel hot water extract (Hx)

High - fat diets or high cholesterol diets resulted in oxidative damage of body tissues and vascular tissues, accelerated oxidative stress in hypercholesterolemic conditions, and various changes in antioxidant activity. In the hypercholesterolemic state, the amount of antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase is decreased and the lipid peroxide content is increased while these values are improved by the supplementation of antioxidants. Were investigated. The MDA content of lipid peroxidation, a degradation product of lipid peroxidation in abdominal aortic vascular tissues, was 21.5 ± 3.5 nmol / g in the normal group and 37.3 ± 6.4 nmol / g in the high cholesterol diet group, (Hx) administration group showed 30.5 ± 2.4 nmol / g (Table 2).

The levels of MDA in abdominal aortic vascular tissues showed a significant decrease by 15% in the hot - water extract (Hx) of eel by - product compared to the group fed with high cholesterol diet.

The lipid peroxidic MDA produced as a reaction product was extracted by using lipid peroxidation reaction and the absorbance was measured at a specific wavelength of 535 nm (n = 5). There was no significant difference in the MDA treatment group, but the activity was decreased.

5. Inhibition of Xanthine Oxidase (Xo) activity, a harmful active oxygen generating enzyme in abdominal aortic vascular tissue

Xanthine oxidoreductase (XO) is a nonspecific enzyme in the body's free radical generation system. It mainly produces hypoxanthine, which is a metabolite of purine body, and xanthine, which oxidizes xanthine to produce superoxide radicals superoxide radical generation) enzyme.

Free radicals are known to produce free radicals not only in the metabolic process of xanthine oxidase but also in the metabolic process of the mixed function oxidase system of the endoplasmic reticulum which increases the phospholipase A2 activity and the foreign substances introduced into the body (Sohal, RS and Allen , RG, Oxidative stress as a cousal factor indifferentiation and aging, A unifying hypothesis, Exper, Gerontol., 25, 499, 1980). The activity of XO in the abdominal aortic vascular tissue was 21% lower than in the high cholesterol dietary intake group (3.5 ± 0.3 nmol / mg protein) compared to the control group (2.8 ± 0.2 nmol / mg protein) The uric acid produced in the XO active reaction solution was measured at an absorbance of 292 nm to exhibit XO activity.

6. Effect of hot water extract on superoxide dismutase activity in abdominal aortic vascular tissues

Superoxide dismutase (SOD) inhibits accumulation of two superoxide radicals (superoxide, O ₂ ^- ). SOD activity is measured by xanthine as substrate and cytochrome C as O ₂ inhibitor KCN is used as a coloring solution, and DOC is used as an oxidizing agent to make xanthine into hypoxanthine. Hypoxanthine induces the reaction of the superoxide anion O ₂ ^- to the active site of SOD.

As a result, the SOD activity in the abdominal aortic vascular tissues increased by 21.2% in the high cholesterol diet group (8.2 ± 1.1 unit / mg protein) and the eel by-product hot water extract (Hx) group (10.7 ± 1.5 unit / mg protein) (Table 4). The SOD activity, which converts superoxide to H ₂ O ₂ , was measured by time-scanning at an absorbance of 550 nm for 1 minute and 30 seconds.

7. Effect of hot water extraction concentrate on catalase activation in abdominal aortic vascular tissue

The activity of Catalase to remove H ₂ O ₂ was 24% higher than that of high cholesterol diet group (14.6 ± 2.6 nmol / mg protein / min) in hot water extract (Hx) group (18.9 ± 2.4 nmol / (Table 5).

8. Effect of Glutathione peroxidase activity in the abdominal aortic vascular tissues in the hot-water extract (Hx) group of eel by-products

Glutathione peroxidase (GSH-Px) is found in all mammalian tissues. Glutathione is used to remove H ₂ O ₂ and organic hydroperoxides. The oxidized glutathione (GSSG) produced by glutathione reductase is replaced by glutathione To protect the cell membrane through a redox reaction that is dependent on the cell membrane.

Reduced glutathione, which acts as a therapeutic, and GSH-Px, a third antioxidant, are antioxidants that protect cells in the cytoplasm and mitochondria. Selenium, a major constituent of GSH-Px, is a kind of minerals that is essential for the body to ingest the necessary amount by diet.

GSH-Px activity in abdominal aortic vascular tissues was 3.2 ± 0.3 nmoles / mg protein / min in the high cholesterol diet group and 4.5 ± 0.3 nmoles / mg protein / min in the hot-water extract (Hx) GSH-Px activity was significantly increased by 35% in the hot-water extract (Hx) treated by eel by-product.

The rate at which the absorbance of NADPH decreased with the formation of oxidized glutathione (GSSG) was measured at 340 nm for 2 min. In other words, when H ₂ O ₂ was added in the presence of GSH-Px, GSSG was produced, and the activity of GSH-Px was calculated by observing the rate of GSSG reduction to glutathione by glutathione reductase and NADPH present in the reaction solution .

The above experiments are summarized as follows.

The blood vessels are composed of three layers. Endothelial cells, smooth muscle cells and adventitial cells are highly protective tissues that provide the reaction and elasticity of tissues. The inflammatory reaction of vascular endothelial cells is initiated when the inflammatory reaction by damage of the blood vessel wall due to various causes such as bacterial infection in blood vessels and adhesion due to oxidation of cholesterol proceeds.

Atherosclerosis, atherosclerosis, is an inflammatory process in which oxidized low density lipoprotein (LDL) particles leave the lumen of the artery and attach to the vascular endothelial cell membrane, resulting in an additional response such as platelets and a macrophage macrophage inflammatory response It is a disease that occurs. In conclusion, macrophages are bubble cells, which ingest or accumulate fat on the inner surface of the arteries, resulting in excessive cell proliferation of the smooth muscle, resulting in stenosis of the arterial wall.

Risk factors for arteriosclerosis are free radicals (oxygen radicals), which are the main causes of intracellular oxidative stress. Oxygen - containing organisms have superoxide dismutase (SOD), a primary antioxidant to remove superoxide (O ₂ ^- , superoxide radical), which is highly reactive.

Cell Defense to Eliminate H ₂ O ₂ The second antioxidant catalase is a powerful antioxidant enzyme that breaks down H ₂ O ₂ into water and oxygen to prevent the production of oxygen radicals. Catalase removes very slowly when the concentration of H ₂ O ₂ to be treated is low, and very quickly when the concentration is high. It has been reported that not only the free radicals of the active oxygen species generated during the metabolic process are removed, but they are irreversibly inactivated as compared with these active oxygen, and the free air produced by the lipid oxidation is removed. Third antioxidant Glutathione peroxidase (GSH-Px) is found in the cytoplasm inside the mitochondria of all mammals using glutathione as a substrate to remove the H ₂ O ₂ and organic hydroperoxides and this time to remove H ₂ O ₂ with Catalase The resulting oxidized glutathione (GSSG) protects the cell membrane through a glutathione reductase-dependent oxidation-reduction reaction that rely on glutathione (reduced glutathione). Reduced glutathione and GSH-Px have various actions such as not only removing H ₂ O ₂ but also repairing damaged cells in their original state and detoxifying them. Xanthine oxidoreductase (XO) is a nonspecific enzyme involved in the metabolism of purine, pyrimidine, pteridine, aldehyde and heterocyclic compounds. In vivo, Xanthine oxidoreductase (XO) Is an enzyme that catalyzes the reaction of oxidizing xanthine to produce uric acid, which in turn produces superoxide radicals.

In this experiment, xanthine oxidase activity, which is involved in the production of harmful free radicals, and SOD, catalase and GSH-Px enzymes involved in the removal of harmful free radicals were measured.

XO activity in abdominal aortic vascular tissues was significantly lower than that of high cholesterol diets in the group treated with hot - water extract (Hx) of eel by - product. XO activity studies have shown that the inhibitory effect of the flavonoid components present in the plant system depends on the position of the hydroxyl groups in the molecule, and it is reported that the flavonoid compounds containing the gallate group are competitively inhibited. It has also been reported that XO inhibition by polyphenol compounds of luteolin and tea was separated from flowers and eyes of red beans.

In the present experiment, the significant decrease in the hot-water extract (Hx) of the eel by-product was attributed to the fact that the polyphenol compound constituting the hot-water extract (Hx) of the eel by- .

SOD and catalase activities in the abdominal aortic vascular tissues were significantly increased by the addition of high-cholesterol diet (Hx), compared with those of the high-cholesterol diets, which resulted in increased production of atherosclerosis caused by excessive intake of high cholesterol As a physiological adaptation phenomenon to eliminate active oxygen, SOD, which is an enzyme for removing active oxygen, is activated.

In addition, the catalase activity of the hot - water extract (Hx) - treated group was higher than that of the high cholesterol diet group in that the catalase activity was increased in order to decompose H ₂ O _{2 produced} by autoxidation of fat and oxidation of organic matter. In addition, it has been reported that flavonoid compounds affect the activity of antioxidant enzymes and reduce tissue damage caused by harmful free radicals

The activity of Glutathione peroxidase (GSH - Px), a third antioxidant in the abdominal aortic vascular tissue, was significantly increased in the hot - water extract (Hx) group of eel by - product compared to the group of high cholesterol diet. It is reported that GSH-Px activity is more affected by total dietary fat intake than dietary fatty acid composition. In addition, when the tissue is damaged, compensatory action increases glutathione synthesis ability, promotes GSH-Px activity, increases the turnover rate of glutathione, and increases the release of glutathione into the blood

The significant increase in GSH-Px activity in the hot-water extract (Hx) group of eel by-products in this experiment was due to the prevention of oxidative cell damage and accumulation of hydrogen peroxide in the abdominal aortic vascular tissue caused by atherosclerosis induced by high cholesterol intake The GSH-Px activity was relatively increased

Intracellular lipid peroxidation may also cause atherosclerosis and aging. Lipid peroxidation accumulates due to increased intracellular oxidative stress due to increased free radical formation and weakened antioxidative defense. The lipid peroxide content decreases when the protective mechanism such as increased GSH content is strengthened.

The lipid peroxide content in the abdominal aortic vascular tissues was significantly lower than that in the high cholesterol dietary intake group and the MDA content of the eel by-product hot water extract (Hx) group was significantly lower than that of the high cholesterol dietary group. extract (Hx) and this thought to inhibit the production of H ₂ O ₂ to generate lipid peroxide the results are shown results similar to studies of Sung, ^etc.]. In other words, the administration of hot-water extract (Hx) of eel by-products effectively inhibited the induction of atherosclerosis, effectively inhibiting lipid peroxidation in the vascular aortic vascular tissue, reducing the level of lipid peroxidation (MDA), and preventing and improving oxidative stress.

Claims (1)

The eel bones and processing by - products were pulverized by low - temperature vacuum after the eel processing by - products of the eel head, liver, internal organs,

One liter of water was added to 200 g of the pulverized eel by-product powder, which was kept in a heating mantle at 45 ° C for 2 hours and filtered using a membrane filter having a size of 1 mm,
The extract of the filtered eel by-product powder was removed and the supernatant was concentrated to a volume of 50% with a rotary vacuum evaporator to prepare a hot-water extract (Hx) of eel by-products. Method for producing an extract of by-products

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