KR20100045575A - Composition comprising an extract of green tea seed coat showing anti-oxidative and anti-inflammatory activity - Google Patents

Abstract

PURPOSE: A composition containing green tea seed coat extract is provided to reduce NO(nitric oxide) generation and iNOS(inducible nitric oxide synthase) generation, and enhance GSH-reductase activity. CONSTITUTION: A pharmaceutical composition which preventing and treating oxidation-relating disease or inflammatory diseases contains green tea seed coat extract as an active ingredient. The extract is isolated using water, low alcohol of C1-C4, or mixture solvent thereof. Oxidation-relating diseases are asthma, chronic pain, acute pain, pain after surgery, neuropathy, nerve injury, and irritable bowel syndrome. Inflammatory disease is gastritis, colitis, arthritis, nephritis, hepatitis, arteriosclerosis, cancer or degenerative disease. A health food for preventing and treating oxidation-relating diseases or inflammatory diseases contains green tea seed coat extract as an active ingredient. The health food is used in the form of powder, granule, tablet, capsule, or beverage.

Description

Composition comprising an extract of Green tea seed coat showing anti-oxidative and anti-inflammatory activity}

The present invention relates to a composition for the prevention and treatment of oxidation-related diseases or inflammatory diseases caused by oxidative stress containing green tea seed bark extract having antioxidant activity and anti-inflammatory activity as an active ingredient.

1, Shen et al ., Eur. J. Pharmacol ., 446 , pp 187-194, 2002

Gobert AP, et al., J. Immunol. , 168 (12) , pp6002-6006, 2002

3 Dijkstra G., et al., Scand. J. Castroenterol ., 37 (5) , pp 546-554, 2002

Document 4 Sakac V., Sakac M., Med. Pregl. , 53 , pp463-474, 2000

5 Albrecht EW, et al., Am. J. Transplant . 29 (5) , pp 448-453, 2002

Liang YC, et al ., Carcinogenesis, 20 , pp1945-1952, 1999

7 S Cho., Et al., J. Korean Soc. Food Sci. Nutr. , 32 (3) , pp458-463, 2003

8, Yen GC., Et al., Food and Chemical Toxicology , 40 (10) , pp1433-1440, 2002

9 Connye K., et al., J. Nutr., 121 , pp1562-1569, 1991

10 REFERENCES Kang WS., Et al., J. FD Hyg. Safety , 16 (1) , p41-47, 2001

[11] Green LC., Et al., Anal. Biochem ., 126 , pp131-138, 1982

Document 12 Tietze F., Anal. Biochem., 27 , pp502-522, 1969

13 Bradford MM., Et al., Ann Biochem , 72 , pp248-254, 1976

14 Aebi H., et al., Academic. press ., 150 , p121, 1984

15 Marklund S., et al., Eur. J. Biochem ., 47 , p469, 1974

16. Lawrence RA., Et al., Biochem. Biophys. Res. Comm ., 71 , p952, 1976

17 Inger C., et al., Academic. press inc ., 113 , pp484-490, 1985

18 Fautz R., et al., Mutat. Res., 253 , pp 173-179, 1991

[19] L. Renstrom Koskela., Et al ., The Juornal of Urology 180 (2) , pp737-741, 2008

20. Park SY., Et al., In vitro anti-imflammatory activity of the Artemisia fukudo extracts in murine macrophage RAW 264.7 cells, Kor. J. Food Sci. Technol. , 39 (4) , pp 464-469, 2007

Inflammation refers to the pathological state of the abscess formed by the invasion of external bacteria, the inflammatory reaction occurs through the infection or tissue damage of our body. These reactions are caused by the release of inflammatory mediators such as inflammatory cytokine, prostaglandin (PG) E ₂ , nitric oxide (NO), and thromboxane (Shen et al ., Eur. J. Pharmacol ., 446 , pp187-194, 2002). Inflammatory diseases caused by inflammatory reactions include gastritis, colitis, arthritis, nephritis, hepatitis, arteriosclerosis, cancer or degenerative diseases (Gobert AP, et al., J. Immunol. , 168 (12) , pp6002-6006, 2002; Dijkstra G., et al., Scand. J. Castroenterol ., 37 (5) , pp546-554, 2002; Sakac V., Sakac M., Med. Pregl. , 53 , pp463-474, 2000; Albrecht EW, et al., Am. J. Transplant . 29 (5) , pp 448-453, 2002).

iNOS is known to induce excess NO by inducing expression by LPS (lipopolysaccharide) or various cytokines in epithelial cells or leukocytes, and most of NO production during inflammatory reactions is due to iNOS. Liang YC, et al ., Carcinogenesis, 20 , pp1945-1952, 1999)

Antioxidants that can protect the human body from reactive oxygen species (ROS) or free radicals are systems that prevent the generation of free radicals, remove the generated radicals, and are involved in the recovery of damaged tissues. You can share it. In addition, the biological defense system for removing ROS is enzymatic defense system such as superoxide dismutase, catalase, glutathione peroxidase, and chain reaction of ROS or free radicals. It is largely classified as a nonenzymatic defense system that stops or terminates. Organisms that use oxygen have superoxide dismutase (SOD), an enzyme that removes superoxide, and the living body is protected from damage caused by superoxide. Catalase exhibits a function of defending a living body by removing H ₂ O ₂ generated by enzymatic reactions such as SOD in tissues. In addition, it is reported that not only removes free radicals of reactive oxygen species generated during metabolism but also irreversibly inactivates by these free radicals, and also removes free radicals generated by fatty acidization. Glutathione (GSH, glutathione), which accounts for most of the non-protein thiol in animal tissues, plays an important role in intracellular antioxidants as a free radical scavenger and a substrate of GSH-Px that metabolizes H ₂ O ₂ and lipid peroxide. (Cho SY., Et al., J. Korean Soc. Food Sci. Nutr. , 32 (3) , pp458-463, 2003).

It is also known that the glutathone redox cycle plays an important role in protecting cells from oxidative stress by scavenging free radicals and keeping the cells in a reduced state (Yen GC., Et al., Food and Chemical Toxicology). , 40 (10) , pp1433-1440, 2002)

GSH-Px, a selenium-dependent antioxidant enzyme, has been reported to affect total dietary fat intake by catalyzing lipid peroxidation and detoxification of hydrogen peroxide (Connye K., et al., J. Nutr., 121 , pp1562). -1569, 1991).

Green tea seed shell of the present invention refers to the coat (coat) surrounding the seeds of the tea tree ( Camelia sinensis ). Green tea is a dried leaf of tea tree and has been known to strengthen blood vessels with pharmacological effects such as excitement, cardiac diuresis, antipyretic and convergence (Kang WS., Et al., J. FD Hyg.Safety , 16 (1) ) , p41-47, 2001).

Therefore, the present inventors found that green tea seed bark extract inhibited NO production and iNOS production in a concentration-dependent manner in RAW 264.7 cells stimulated with LPS (Lipopolysaccharide), and showed an excellent anti-inflammatory effect by increasing the expression of antioxidant enzymes. The present invention has been completed by confirming that there are few safe materials.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of oxidation-related diseases or inflammatory diseases caused by oxidative stress containing green tea seed peel extract as an active ingredient.

In addition, the present invention provides a health functional food for the prevention and improvement of oxidation-related diseases or inflammatory diseases caused by oxidative stress containing green tea seed peel extract as an active ingredient.

The extract as defined herein includes extracts with water, including purified water, C ₁ to C ₄ lower alcohols or mixed solvents thereof, preferably water or ethanol.

The oxidation-related diseases include atherosclerosis, rheumatoid arthritis, arthritis, asthma, acute pain, chronic pain, neuropathic pain, postoperative pain, pains such as migraine and joint pain, neuropathy, neurological damage, irritable bowel syndrome, endotoxin Shock or inflammatory bowel disease and the like, and preferably include arthritis, asthma or inflammatory bowel disease.

The inflammatory disease is arteriosclerosis, gastritis, colitis, arthritis, nephritis, hepatitis, cancer or degenerative disease, and the like, and preferably includes arteriosclerosis or cancer.

The composition of the present invention comprises from 0.1 to 50% by weight of the green tea seed shell extract obtained from the green tea seed shell extract relative to the total weight of the composition.

Hereinafter, the present invention will be described in detail.

The green tea seed extract of the present invention, after washing, drying and powdering the skin portion from which the seeds of green tea seeds are removed, is about 5 to 20 times the weight of the green tea seed powder, preferably about 3 to 15 times water, C ₁ to C ₄ lower alcohol or a mixed solvent thereof, preferably water or ethanol, at about 1 to 10 days, preferably about 2 hours to 20 to 120 ° C, preferably at 75 to 100 ° C extraction temperature. Extraction method such as cold sediment extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction for 5 hours, preferably 1 to 10 times, preferably 2 to 7 times obtained by using a reflux cooling extraction method and concentrated under reduced pressure The extract of the present invention can be obtained and then purified and concentrated.

The present invention provides a pharmaceutical composition for the prevention and treatment of oxidation-related diseases or inflammatory diseases comprising green tea seed peel extract obtained by the above method.

The green tea seed peel extract obtained by the above production method exhibits anti-inflammatory effects by inhibiting NO production and iNOS production, SOD (Superoxide dismutase), Catalase (Catalase), GSH-peroxidase and GSH-reductase By increasing the activity of (GSH-reductase), it has been confirmed to have the effect of protecting the human body from oxidative stress. have.

The composition comprising the green tea seed shell extract of the present invention may further comprise a suitable carrier, excipient or diluent according to conventional methods.

Carriers, excipients and diluents that may be included in the compositions of the present invention include 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.

The composition comprising the green tea seed shell extract obtained from the extract according to the present invention, respectively, oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterilization according to a conventional method It can be formulated and used in the form of injectable solutions.

Specifically, when formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient such as starch, calcium carbonate, water It may be prepared by mixing cross, lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups.In addition to the commonly used simple diluents, water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized 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.

Green tea seed peel extract according to the present invention may vary depending on the age, sex and weight of the patient, but generally in an amount of 0.01 to 500 mg / kg, preferably 0.1 to 100 mg / kg, divided once or several times daily May be administered. In addition, the dosage of the extract obtained from the green tea seed husk extract may be increased or decreased depending on the route of administration, degree of disease, sex, weight, age and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

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

Green tea seed bark extract of the present invention has little toxicity and side effects, so can be used with confidence even for long-term use for the purpose of prevention.

The present invention provides a health functional food for the prevention and improvement of oxidation-related diseases or inflammatory diseases caused by oxidative stress containing green tea seed peel extract as an active ingredient.

The composition comprising the green tea seed shell extract of the present invention may be used in various ways, such as drugs, foods and beverages for the prevention of oxidation-related diseases or inflammatory diseases caused by oxidative stress.

Foods to which the green tea seed skin extract of the present invention may be added include various foods, for example, beverages, gums, teas, vitamin complexes, health supplements, and the like, pills, powders, granules, tablets, capsules, and the like. Or in the form of a beverage.

At this time, the amount of the extract obtained from the green tea seed shell extract in the food or beverage can be generally added to 0.01 to 15% by weight of the total food weight in the case of the health food composition of the present invention, in the case of a health beverage composition based on 100ml Can be added at a ratio of 0.02 to 10 g, preferably 0.3 to 1 g.

The health beverage composition of the present invention has no special limitations in the liquid component except for containing the green tea seed shell extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. have. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; Polysaccharides such as dextrin, cyclodextrin; Conventional sugars such as and the like and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (tauumatin, stevia green tea seed extract (e.g., Rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) are free Can be used. The proportion of such natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the 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.

The composition containing the green tea seed peel extract of the present invention as an active ingredient inhibits NO production and iNOS production, and shows an increase in glutathione content and antioxidant enzymes, thereby preventing and treating oxidation-related or inflammatory diseases caused by oxidative stress. It can be usefully used as a pharmaceutical composition and health functional food.

Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples.

However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited by the following Experimental Examples.

Reference Example 1. Cell Culture and Reagents

RAW 264.7 cells, a murine macrophage cell line, were purchased from the American Type Culture Collection (ATCC) and were supplemented with 10% FBS (Fetal bovine serum) and L-glutamine at 37 ° C and 5% CO _2. Cultured in DMEM (Dulbecco's modified eagle media). Subcultures were performed once every 3 to 4 days, and LPS (E. coli serotype 0111: B4) was purchased from Sigma.

Reference Example 2. Statistics Processing

The analytical results of the experiments were expressed as mean ± standard error (SE), and the significance was the Duncan's multiple test using the one-way ANOVA of the SPSS / PC + package. Analyzed.

Example 1 Preparation of Green Tea Seed Ethanol Extract

The green tea seed husk was provided in the form of green tea seed from Hwagaeje (Korea), washed the outer skin from which the seeds were removed, and then dried by hot air drying at about 40 ℃ and powdered. 1 liter of alcohol (ethanol) in 100g of green tea seed dry After 4 hours of reflux extraction in an 80 ° C. water bath, the filtrate was collected with Filtration # 1 (Watman, USA), the filtrates were collected and concentrated under reduced pressure with a vacuum rotary concentrator (Rotavapor R-200, BUCHI). 10.5g was obtained, and stored at -20 ° C, and diluted in DMSO so that the final concentration was 0, 10, 25, 50, 100, 200 ㎍ / ㎖ when the cells were used in the experimental example.

Experimental Example 1. Inhibition Effect of NO Production

In order to investigate the effect of green tea seed husk alcohol extract on NO production in RAW 264.7 cells induced oxidative stress with LPS, experiments were conducted using the following method (Green LC., Et al. ., Anal. Biochem., 126 , pp131-138, 1982).

The degree of NO production was determined by measuring the amount of NO produced in the medium which is an indicator of NO production. Cells were injected and adhered to 24-well plates (8 × 10 ⁵ cells / well), and green tea seed bark extracts were treated by concentration and incubated for 2 hours, followed by incubation for 20 hours by adding LPS (1 μg / ml). . 50 μl of 1% sulfanylamide and 50 μl of 0.1% naphthylenediamine dihydrochloride were added to 100 μl of the media supernatant, followed by reaction at room temperature for 10 minutes, followed by absorbance at 550 nm. Measured. At this time, the standard curve was used to prepare NaNO ₂ for each concentration.

As a result, as shown in Figure 1, the LPS treatment group was significantly increased NO production than the normal group, green tea seed bark extract in the oxidative stress-induced state was confirmed that the concentration-dependent inhibition of NO production. At this time, the IC ₅₀ value was 113 ug / ml (see FIG. 1).

Experimental Example 2. Glutathione (GSH) content increase effect

In order to investigate the effect of green tea seed bark extract on GSH content in RAW 264.7 cells induced oxidative stress with LPS, experiments were conducted using the following procedure (Tietze F., Anal. Biochem). , 27 , pp 502-522, 1969).

GSH was measured using an enzymatic circulation procedure by Tietze et al. GSH is continuously oxidized by 5,5-dithiobis (2-nitrobenzoic acid) and reduced by glutathione reductase NADPH. Cells were injected and adhered to a 100 mm dish (5 × 10 ⁶ cells / dish), and the green tea seed bark extract was incubated for 2 hours, and then cultured for 20 hours by adding LPS (1 μg / ml). After removing the medium, the cells were washed twice with PBS (pH 7.4) and 1 ml of PBS was added to collect the cells. The number of cells per sample was equally counted, sonicated three times for 5 seconds under ice-cooling, and centrifuged at 4 ° C and 4,500 rpm for 10 minutes. 400 μl of the supernatant was mixed with 100 μl of 5% SSA (5-Sulfosalicylic acid), and then centrifuged at 4 ° C. and 4,500 rpm for 10 minutes. 50 μl of supernatant was mixed with 100 μl of reaction mixture (100 mM sodium phosphate buffer, 1 mM ethylenediaminetetraacetic acid (EDTA) pH 7.5, 1 mM dithiolnitrobenzene (DTNB), 1 mM NADPH, 1.6 units / ml GSH-reductase). The reaction was observed for changes in absorbance at 405 nm for 2 minutes. At this time, the standard curve was prepared by using the GSH concentration, and the amount of GSH of the sample was determined by substituting the absorbance change rate for 1 minute into the standard curve.

As a result, as shown in Figure 2, the green tea seed peel alcohol extract was found to increase significantly GSH content compared to the control group in the group treated with a concentration of 50㎍ / ㎖ or more, green tea seed peel alcohol extract Increasing this GSH level was confirmed to reduce the oxidative stress in the cell (see Figure 2).

Experimental Example 3. Antioxidant Enzyme Activity Increase Effect

In order to investigate the effect of green tea seed bark extract on antioxidant enzyme activity in RAW 264.7 cells induced oxidative stress with LPS, the following experiment was performed by the method described in the existing literature (1) Bradford MM., Et al. , Ann Biochem , 72 , pp248-254, 1976; 2) Aebi H., et al., Academic. press ., 150 , p121, 1984; 3) Marklund S., et al., Eur. J. Biochem ., 47 , p469, 1974; 4) Lawrence RA., Et al., Biochem. Biophys. Res. Comm ., 71 , p952, 1976; 5) Inger C., et al., Academic. press inc ., 113 , pp 484-490, 1985).

3-1. Sample Preparation

Cells were injected and adhered to a 100 mm dish (5 × 10 ⁶ cells / dish), and the green tea seed bark extract was incubated for 2 hours by concentration, followed by incubation for 20 hours by adding LPS (1 μg / ml). After removing the medium, the cells were washed twice with PBS (pH 7.4) and 1 ml of 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA was added to collect the cells. This was sonicated three times for 5 seconds under ice-cooling, centrifuged at 4 ° C. and 10,000 rpm for 20 minutes, and the cell supernatant was used for measuring the antioxidant enzyme activity.

3-2. Protein concentration

Protein concentrations were measured using the Bradford assay (Bradford MM., Et al., Ann Biochem, 72 , pp248-254, 1976).

Cell extract supernatant was diluted 10-fold and Bradford staining reagent was used 5-fold diluted. That is, 200 µl staining reagent was mixed in a 10 µl sample, and the mixture was left at room temperature for 15 minutes and absorbance was measured at 595 nm. At this time, the standard curve was used by diluting bovine serum albumin (Bovine Serum albumin) by concentration.

3-3. Catalase activity

Catalase activity was measured by the method of Abei et al. (Aebi H., et al., Academic. Press ., 150 , p121, 1984).

To 50 μl of cell extract supernatant, 600 μl of 50 mM phosphate buffer (pH 7.0) was added. 300 μl of 30 mM hydrogen peroxide (H ₂ O ₂ ) solution was added thereto, and the absorbance change was measured at 240 nm for 10 minutes at 10 second intervals. Control experiment in the substrate, H ₂ O ₂ was added in place of the 50mM phosphate buffer (pH 7.0) of 300㎕, other conditions H ₂ O was prepared by a standard curve concentration was measured for change in absorbance in the same manner as described above, _{It was} created using ₂ . The activity of the enzyme was 1 unit of the amount of enzyme that degrades 1 μM of H ₂ O ₂ for 1 minute.

3-4. Superoxide dismutase activity (SOD activity)

SOD activity was determined by automatic oxidation of pyrogallol by Marklund et al. (Marklund S., et al., Eur. J. Biochem ., 47 , p469, 1974).

To the 96 well plate was added 10 [mu] l of cell extract, 1.1 mM diamine tritetene pentacetic acid (DTPA) and 55.6 mM trizma base (pH 8.2). 50 μl of 6 mM pyrogallol [in 10 mM HCl] was added thereto, and the change in absorbance was measured at 10 seconds intervals at 405 nm for 10 minutes. One unit of SOD activity was expressed as the amount of enzyme that inhibits pyrogallol oxidation by 50%.

3-5. GSH-peroxidase activity (GSH-px activity)

GSH-px activity was measured by Lawrence et al. (Lawrence RA., Et al., Biochem. Biophys. Res. Comm ., 71 , p952, 1976).

Reaction mixture [50 μl 250 mM potassium phosphate buffer (pH 7.0), 25 μl 10 mM EDTA, 25 μl 10 mM NaN ₃ , 25 μl 10 mM GSH, 25 μl 2 mM NADPH, 50 μl DW into 20 μl of cell supernatant Add 0.83 Unit of glutathione reductase. Immediately after addition of 25 μl of 2.5 mM H ₂ O ₂ , absorbance changes were measured at 340 nm at 10 second intervals for 2 minutes. Standards were prepared by mixing NADPH with a mixture [50 μl potassium phosphate buffer (pH 7.0), 25 μl 10 mM EDTA, 25 μl 10 mM NaN ₃ , 125 μl DW, 1 unit of GSH-px activity 1 nM per minute NADPH is expressed as the amount of enzyme that is oxidized.

3-6. GSH-reductase activity

Glutathione reductase activity was measured by Inger et al. (Inger C., et al., Academic. Press inc ., 113 , pp484-490, 1985).

100 μl of cell extraction supernatant was added to a 96 well plate and the reaction mixture [50 μl of 0.2M phosphate buffer containing 2 mM EDTA, 5 μl of 2mM NADPH, 5 μl of 20mM GSSG, DW 40 μl] was immediately added for 10 minutes at 340 nm. The change in absorbance was measured at second intervals. One unit of glutathione reductase was defined as the amount of enzyme that catalyzes the reduction of 1 nM NADPH for 1 minute.

As a result, as shown in Table 1, the activity of catalase was not significantly different in the group to which green tea seed shell alcohol extract was added, compared to the normal group, and the concentrations of 10, 25, 50, and 100 µg / ml SOD activity was significantly increased when green tea seed bark alcohol extract was treated, and GSH-px activity was significantly increased when 10, 50, 100 ㎍ / mL extract was added. The effect was reduced when the 200 ㎍ / ㎖ concentration was found that the treatment of green tea seed bark extract was found to increase the SOD, GSH-px activity. In addition, the activity of GSH-reductase was gradually increased from the concentration of 25 ㎍ / ㎖ and the extract of green tea seed shell extract in RAW 264.7 macrophages was confirmed to be significantly higher than the normal group when treated with 200 ㎍ / ㎖ extract The antioxidant enzymes of SOD, GSH-px and GSH-reductase were activated.

Normal Green Tea Seed Peel Alcohol Extract (ug / ml) + LPS ⁵ 1ug / ml) 0 10 25 50 100 200 Catalase (umol / mg protein / min) 0.05 ± 0.02 ^NS1 0.04 ± 0.01 0.05 ± 0.02 0.05 ± 0.02 0.07 ± 0.01 0.08 ± 0.001 0.07 ± 0.01 SOD ² (unit / mg protein) 14.65 ± 0.15 ^d 12.00 ± 0.12 ^e 15.30 ± 0.16 ^c 15.38 ± 0.27 ^c 16.41 ± 0.00 ^b 17.24 ± 0.18 ^a 11.73 ± 0.21 ^e GSH-px ³ (unit / mg protein) 0.89 ± 0.09 ^bc 0.73 ± 0.02 ^c 1.01 ± 0.06 ^ab 0.93 ± 0.05 ^bc 1.20 ± 0.15 ^a 1.04 ± 0.08 ^ab 0.80 ± 0.06 ^bc GSH-reductase ⁴ (unit / mg protein) 71.03 ± 2.2 ^ab 57.12 ± 1.8 ^bc 51.06 ± 1.3 ^c 68.65 ± 0.9 ^ab 65.33 ± 4.2 ^ab 70.56 ± 7.0 ^ab 77.50 ± 6.0 ^{a 1} ; The data of this experiment were expressed as mean ± standard error (SE) of three experiments, and the same superscript had no significance (p <0.05). ² ; Superoxide dismutase ³ ; Glutathione peroxidase ⁴ ; Glutathione reductase ⁵ ; Lipopolysaccharide

Experimental Example 4. Measurement of Cytotoxicity of Green Tea Seed Peel Extract

Cell viability was determined by the method of Fautz et al. (Fautz R., et al., Mutat. Res., 253 , pp173-179, 1991) using the characteristic that live cells are stained with neutral red reagent. Inject the cells into a 24-well plate (8 × 10 ⁵ cells / well), and attach the green tea seed alcohol extract of 10, 25, 50, 100, and 200 µg / ml to the cells, respectively. Negative control (untreated) and positive control (control) were added DMSO (0 μg / ml). After 2 hours of incubation, LPS (1 μg / ml) was added to the experimental and positive control groups and not treated to the negative control group. After incubation for 20 hours, the 24-well medium was removed, 0.5 ml of medium containing 1.14 mmol / L of neutral red reagent was added thereto, and incubated for 3 hours, followed by washing twice with PBS (phosphate buffer saline, pH 7.4). . 1 ml of cell lysis buffer (50 mM / L Tris-HCl (pH 7.4), 150 mM / L NaCl, containing acetic acid (1%, v / v) and ethanol (50%, v / v) in the cells, 5mM / ℓ dithiothereitol, Triton X-100 (1%, v / v)) was incubated for 15 minutes to release neutral red from the cells, and centrifuged for 10 minutes at 3,000 rpm for cell lysis products to measure the degree of staining. The supernatant was then taken and the absorbance measured at 540 nm.

As a result, as shown in Figure 3, compared with the cell survival rate of the positive control group (0 ㎍ / ml concentration), both the negative control group (normal group), green tea seed shell alcohol extract group had high cell survival rate, so the green tea seed shell alcohol extract cells It was confirmed that there is no toxicity (see FIG. 3).

Experimental Example 5. Measurement of the influence on the production of iNOS

In order to investigate the effect of green tea seed bark extract on iNOS expression in RAW 264.7 cells induced oxidative stress with LPS, experiments were performed using the method described in the literature (L. Renstrom Koskela., et al ., The Juornal of Urology, 180 (2) , pp737-741, 2008).

5-1. Sample Preparation

A RAW 264.7 mouse macrophage cell line was prepared with DMEM (Dulbecco's modified) supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine at 37 ° C and 5% CO _2. incubated in eagle medium). Cells were attached to 100 mM dishes (5 × 10 ⁶ cells / dish). After attaching the cells, the samples were treated at each concentration (final concentration 0, 10, 25, 50, 100, 200 µg / ml) and incubated for 2 hours, and then LPS (1 µg / ml) was used to induce oxidative damage. ) Was added and incubated for 20 hours. After removing the medium, rinsed twice with PBS (phosphate buffer saline, pH 7.4), and then removed. Cells were collected by adding 1 ml of PBS, sonicated three times for 5 seconds under ice-cooling, centrifuged at 4 ° C. and 10,000 rpm for 20 minutes, and the cell supernatant was discarded, followed by PRO-PREP ^™ Protein Extraction Solution (iNtRON Biotechnology, Cat.No. 17081, Korea) 400 μl was added and mixed well. Concentrations were measured using the Bradford assay (Bradford MM., Et al., Ann Biochem, 72 , pp248-254, 1976).

5-2. Protein Electrophoresis and Western Blotting

25㎍ separated protein is mixed with loading buffer of proper ratio, heated at 70 ℃ for 10 minutes, denatured and maintained on ice for 5 minutes. 10% mini SDS-PAGE (Poly Acrylamide Gel Electrophoresis) was separated at 50V for 30 minutes, 100V at 1 hour and transferred to Nitrocellulose transfer membrane (Whatman, England) at 15V for 2 hours. After transfer, soak in 5% formaldehyde for 10 minutes and wash with distilled water for 10 minutes. After rinsing with TBST (10mM Tris-HCl, pH 8.0, 150mM NaCl, 0.05% Tween 20) solution, it was blocked with blocking solution (TBST containing 5% skim milk) for 2 hours or more. After rinsing three times for 10 minutes with TBST solution, the primary antibodies (BD Transduction, USA) of iNOS and GAPDH were diluted with blocking solution in a ratio of 1,000 and reacted in the refrigerated state for more than 12 hours. Wash 4 times. Dilute goat anti-rabbit (Cell Signaling, USA) with IgG AP, a secondary antibody, in a blocking solution at a ratio of 1: 3,000, rinse for 3 or 4 times with TBST for 5 minutes, and then BCIP / NBT Color Development Substrate (Promega, Switzerland) color reagent (16.5 μl BCIP [50 mg / ml], 33 μl NBT [5 mg / ml]) was added to 5 ml Alkaline phosphatase buffer (100 mM Tris-HCl, pH 9.0, 150 mM NaCl, 1 mM MgCl _2). After dilution, add to membrane and rinse in distilled water, dry well and analyze result. (Park SY., Et al., In vitro anti-imflammatory activity of the Artemisia fukudo extracts in murine macrophage RAW 264.7 cells, Kor. J. Food Sci. Technol. , 39 (4) , pp464-469, 2007) Quantities were plotted using the BIO-RAD Quantity One program.

As a result, as shown in FIG. 4, iNOS was not expressed in the normal group that did not cause inflammation when the green tea seed shell alcohol extract was administered after inducing inflammation with LPS, and iNOS was found in the positive control group (0 µg / ml). It was expressed and showed a tendency to decrease iNOS depending on concentration. As a result, it was confirmed that green tea seed ethanol extract inhibits the expression of iNOS, an inflammatory factor (see FIG. 4).

Examples of the formulation of the pharmaceutical composition comprising the extract of the present invention will be described, but the present invention is not intended to be limited to this, but only specifically described.

Formulation Example 1 Preparation of Powder

Green Tea Seed Peel Extract 300 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

Green Tea Seed Peel Extract 50 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

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

Formulation Example 3 Preparation of Capsule

Green Tea Seed Peel Extract 50 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

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

Green Tea Seed Peel Extract 50 mg

Appropriate sterile distilled water for injection

pH adjuster

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

Green Tea Seed Peel Extract 100 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method of preparing a liquid solution, each component is added to the purified water to dissolve, the lemon flavor is appropriately added, the above components are mixed, purified water is added, the whole is adjusted to 100 ml by the addition of purified water, and then concentrated in a brown bottle. The solution is prepared by sterilization.

Formulation Example 6 Preparation of Health Food

Green Tea Seed Peel Extract 1000mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral 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 Health Beverage

Green Tea Seed Peel Extract 1000mg

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

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

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

1 is a diagram showing the effect of inhibiting the production of nitric oxide (Nitric Oxide (NO)) in RAW 264.7 macrophages induced oxidative stress with LPS of green tea seed bark extract,

2 is a diagram showing the effect of inhibiting GSH production in RAW 264.7 macrophages induced oxidative stress with LPS of green tea seed bark extract,

3 is a diagram showing the cytotoxicity of green tea seed peel extract,

Figure 4 is a diagram of the effect of inhibiting iNOS production in RAW 264.7 macrophages induced oxidative stress with LPS of green tea seed bark extract.

Claims (7)

Pharmaceutical composition for the prevention and treatment of oxidation-related diseases or inflammatory diseases caused by oxidative stress containing green tea seed extract as an active ingredient. The pharmaceutical composition according to claim 1, wherein the extract is an extract available in a solvent selected from water including purified water, C ₁ to C ₄ lower alcohol mixed solvents thereof. The method of claim 1, wherein the oxidation-related disease is atherosclerosis, rheumatoid arthritis, arthritis, asthma, acute pain, chronic pain, neuropathic pain, pain after surgery, pain such as migraine and joint pain, neuropathy, nerve damage, irritability Pharmaceutical composition which is bowel syndrome, shock caused by endotoxin or inflammatory bowel disease. The pharmaceutical composition according to claim 1, wherein the inflammatory disease is gastritis, colitis, arthritis, nephritis, hepatitis, arteriosclerosis, cancer or degenerative disease. The pharmaceutical composition of claim 1, wherein the extract comprises 0.1 to 50% by weight of the total weight of the composition. Health functional food for the prevention and improvement of oxidation-related diseases or inflammatory diseases caused by oxidative stress containing green tea seed skin extract as an active ingredient. 7. The dietary supplement of claim 6 which is a powder, granule, tablet, capsule or beverage.

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