KR102161179B1 - Antioxidative composition comprising extract of red tea stem - Google Patents

Abstract

The present invention relates to an antioxidant composition containing as an active ingredient a black tea stem extract obtained by extracting from a black tea stem, a black tea by-product, wherein the black tea stem extract according to the present invention is a cancer, cardiovascular system that can be caused by oxygen free radicals. It can be usefully used for preventing or treating diseases, such as diseases, arthritis, Parkinson's disease, health functional foods or additives for preventing oxidation of foods, for preventing skin aging or for preventing skin damage caused by UV rays.

Description

Antioxidative composition comprising extract of red tea stem}

The present invention relates to an antioxidant composition containing, as an active ingredient, a black tea stem extract obtained by extracting from a black tea stem, a by-product of black tea.

By a variety of oxidation reactions, chemicals, food, human diseases and radiation superoxide (superoxide, O ₂ ^-, hydroxyl radicals (hydroxyl radical, · OH) and hydrogen peroxide (H ₂ O ₂₎ is reactive, such as strong radicals When free radicals and free radicals are generated in a living body, the lipids of the cell membrane containing a large amount of unsaturated fatty acids are oxidized and lipid peroxide is produced in the cell membrane. It is degraded, inhibiting the overall function of the cell, and causing local disorders on the tissues, such as changing the structure of the cell, etc. In addition, free radicals and free radicals modify or destroy cell components such as nucleic acids and sugars. As a result, it is a cause of various human diseases such as cancer, liver diseases such as alcoholic hepatitis, stroke due to cerebrovascular disorders, myocardial infarction, diabetic vascular disorders, hyperlipidemia, acute inflammation, rheumatic diseases, etc. The cause is caused by oxygen free radicals. Diseases that can be include cancer, high blood pressure, cardiovascular diseases such as arteriosclerosis or myocardial infarction, arthritis, rheumatoid arthritis, Parkinson's disease, stroke, atopic dermatitis, diabetes, cataracts, aging, autoimmune diseases, hepatitis, nephritis, Gout, brain disorders, etc. For example, free radicals damage cells to promote body aging, and when exposed to ultraviolet rays, melanin pigments are formed, but if metabolism is not done well, spots and freckles are produced. Collagen is also oxidized and skin elasticity is weakened In addition, free radicals are combined with fat to form lipid peroxide, which may accumulate in blood vessels, resulting in blood flow disorders, and inflammation may occur when lipid peroxide accumulates in the liver and kidneys. In addition, peroxidation lipids act on each layer of the skin, causing atopic dermatitis.

In addition, active oxygen reacts with unsaturated fatty acids present in a large amount of food, causing rancidity, leading to decisive defects in maintaining the safety and high quality of food. In many fields, such as a decrease in yield due to a problem caused by active oxygen.

Therefore, the discovery of a new substance having strong and non-toxic antioxidant activity will be very useful as a therapeutic agent for various diseases or as an anti-aging cosmetic additive and food additive.

Until now, synthetic antioxidants such as butylated hydroxy toluene (BHT) and butylated hydroxy anisol (BHA) have been used as lipid peroxidation inhibitors, but these BHT and BHA have excellent lipid peroxidation inhibitory activity, but have the potential to cause cancer or deformity. There was a disadvantage that it was very high and could not be used continuously. Accordingly, research is being actively conducted to separate and use substances with antioxidant effect from natural plants (Natural antioxidant from residual sources, Food Chem, 72, 145~171, 2001). Representative natural plant-derived antioxidants include rosemary extract and green tea. Extracts and the like. In addition, there are reports that potato peel extract, olive meal, and grape seed exhibit antioxidant power (Food Chem, 85, 215~220, 2004, Food Chem, 93, 197~204, 2005, Food Chem, 97, 472~ 479, 2006).

However, despite the social and technological necessity for antioxidant substances derived from various natural plants as such, the antioxidants derived from natural foods that are stably supplied in the market are limited to a few such as green tea extract and rosemary extract. There was an inadequate aspect to be used by the public due to its high manufacturing cost.

Therefore, there is no toxicity or side effects by creating new antioxidant active substances from natural products, and while exhibiting excellent antioxidant activity, it is necessary to develop eco-friendly and economical natural antioxidants by utilizing discarded waste resources and low manufacturing cost.

An object of the present invention is to provide an antioxidant composition containing a black tea stem extract (RTSE) as an active ingredient, a pharmaceutical composition for anti-aging, an antioxidant health functional food, an antioxidant cosmetic composition, and a food additive for preventing food deterioration.

In order to achieve the above object, the present invention provides a composition containing black tea stem extract (RTSE) as an active ingredient, thereby providing an antioxidant composition, an anti-aging pharmaceutical composition, an antioxidant health functional food, an antioxidant cosmetic composition, and a food deterioration prevention It can be used as a food additive.

The present invention relates to an antioxidant composition containing, as an active ingredient, black tea stem extract (RTSE) obtained by extracting from black tea stem, which is a black tea by-product, and the black tea stem extract according to the present invention is a cancer that can be caused by oxygen free radicals. , Cardiovascular disease, arthritis, Parkinson's disease prevention or treatment, health functional foods or additives for preventing oxidation of food, skin aging prevention or cosmetics for preventing skin damage caused by UV rays, food additives for preventing food deterioration, etc. I can.

1 is a graph showing the results of measuring the DPPH radical scavenging activity of black tea stem extract and the standard quercetin.
Figure 2 is a graph showing the results of measuring the ABTS · ⁺ radical scavenging activity of the black tea stem extract and the standard quercetin.
3 is a graph showing the results of measuring the NO-free radical scavenging activity of the black tea stem extract and the standard quercetin.
Figure 4 is a graph showing the result of measuring the superoxide-free radical scavenging activity of the black tea stem extract and the standard quercetin.
Figure 5 shows the experimental results of the oxidative DNA damage protection effect of black tea stem extract.(A) is pUC19 DNA UV photolysis when treated with hydrogen peroxide (33 mM) in the presence or absence of different concentrations of methanol extracts of black tea stems. It shows the electrophoresis pattern of, lane 1 is untreated group, lane 2 is treated group (UV & H ₂ O ₂ ), lane 3-7 is black tea stem extract (50, 100, 200, 400 and 800 ㎍/ mL), lane 8 represents a group treated with quercetin (50 μg/mL) as a positive control, and (B) represents H ₂ O _{2 in} the presence and absence of different concentrations of black tea stem extract (50-800 μg/ml). It is a graph showing the result of analyzing the supercoil and open circular DNA by Densitometry after UV-photolysis of.

The present invention provides an antioxidant composition containing the stem extract (RTSE) of black tea as an active ingredient.

In one aspect of the present invention, the stem of the black tea is obtained from a by-product of black tea ( Camellia sinensis ) and generally refers to a stem, not a leaf, used as a material for black tea. These black tea stems themselves are very inconvenient to eat and are mostly discarded as by-products, but in the present invention, these black tea stem extracts appear to have an antioxidant activity and DNA protection effect at a level comparable to or better than black tea and green tea, The composition containing the black tea stem extract can be very usefully used as an antioxidant composition.

In one aspect of the present invention, the extract may be extracted with water, a C _{1 -4} lower alcohol or a mixed solvent thereof, and more specifically, may be extracted with methanol or ethanol.

As mentioned in the background art, diseases that can be caused by oxygen free radicals include cancer, hypertension, cardiovascular diseases such as arteriosclerosis or myocardial infarction, arthritis, rheumatoid arthritis, Parkinson's disease, stroke, and atopic dermatitis. , Diabetes, cataract, aging, autoimmune disease, hepatitis, nephritis, gout, and brain disorders. For example, free radicals damage cells, promote body aging, and when exposed to ultraviolet rays, melanin pigments are formed.If metabolism is not done well, spots and freckles are produced, and skin elasticity is weakened by oxidizing collagen. In addition, free radicals are combined with lipids to form lipids peroxide, which may accumulate in blood vessels, resulting in blood flow disorders. In addition, when lipid peroxide accumulates in the liver and kidney, inflammation may occur, and lipid peroxidation may act on each layer of the skin to cause atopic dermatitis. Therefore, the antioxidant composition according to the present invention can be used for the purpose of preventing, alleviating, improving or treating the disease.

The present invention provides a pharmaceutical composition for anti-aging containing black tea stem extract as an active ingredient.

In one aspect of the present invention, the stem of the black tea is obtained from a by-product of black tea ( Camellia sinensis ) and generally refers to a stem, not a leaf, used as a material for black tea.

In one aspect of the present invention, the extract may be extracted with water, a C _{1 -4} lower alcohol or a mixed solvent thereof, and more specifically, may be extracted with methanol or ethanol.

The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. The composition including a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations contain at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. in one or more compounds. It is prepared by mixing. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc may be used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as humectants, sweeteners, fragrances, and preservatives may be included. have. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition is any selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. It can have one formulation.

The composition of the present invention is administered in a pharmaceutically effective amount.

In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type and severity of the individual, age, sex, and activity of the drug. , Sensitivity to drugs, time of administration, route of administration and rate of excretion, duration of treatment, factors including co-used drugs, and other factors well known in the medical field. However, for a desirable effect, the extract of the present invention may be administered at 1 to 200 mg/kg per day, preferably at 10 to 100 mg/kg.

The composition of the present invention may be administered as an individual therapeutic agent, or may be administered in combination with other therapeutic agents exhibiting anti-aging or therapeutic effects, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and can be easily determined by a person skilled in the art.

In the present invention, the term "subject" refers to all animals including humans that have already developed or may develop a disease that can be prevented or treated through anti-aging activity, and by administering a composition containing the extract of the present invention to an individual , The disease can be effectively prevented and treated.

The administration route of the composition may be administered through any general route as long as it can reach the target tissue. The composition of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermal, oral, intranasal, intrapulmonary, or rectal, as desired, but is not limited thereto. In addition, the composition may be administered by any device capable of moving the active substance to the target cell.

The present invention also provides an antioxidant food containing the stem extract of black tea.

The food composition of the present invention may contain a health functional food.

In one aspect of the present invention, the stem of the black tea is obtained from a by-product of black tea ( Camellia sinensis ) and generally refers to a stem, not a leaf, used as a material for black tea.

In one aspect of the present invention, the extract may be extracted with water, a C _{1 -4} lower alcohol or a mixed solvent thereof, and more specifically, may be extracted with methanol or ethanol.

The health functional food of the present invention includes a black tea stem extract, but may contain an appropriate food supplementary additive.

In the present invention, the term "food supplementary additive" refers to a component that can be added auxiliary to food, and is added to the manufacture of health functional foods of each formulation, and can be appropriately selected and used by those skilled in the art. Examples of food additives include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavors and natural flavoring agents, coloring agents and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners. , pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, etc. are included, but the types of food additives of the present invention are not limited by the above examples.

In the present invention, the term "health functional food" refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and pills using raw materials or ingredients having useful functions for the human body. Here, the term “functionality” means obtaining a useful effect for health purposes such as controlling nutrients or physiological effects on the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and at the time of the production, it can be prepared by adding raw materials and ingredients commonly added in the art. In addition, unlike general drugs, it has the advantage of not having side effects that may occur during long-term use of drugs by using food as a raw material, and has excellent portability, and the health functional food of the present invention can be consumed as a supplement to enhance the antioxidant effect. Do.

The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, when preparing food, the black tea stem extract according to the present invention is added in an amount of 1 to 10% by weight, preferably 5 to 10% by weight of the raw material composition. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, the amount may be used even below the above range.

There is no particular limitation on the type of the food. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages and vitamin complexes, and it includes all health foods in the usual sense.

The health food composition of the present invention may contain various flavoring agents or natural carbohydrates as an additional component, like a conventional food. The natural carbohydrates described above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as taumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 of the composition of the present invention.

The present invention provides an antioxidant cosmetic composition containing a stem extract of black tea.

The present invention also provides an antioxidant health functional food containing the stem extract of black tea.

In one aspect of the present invention, the stem of the black tea is obtained from a by-product of black tea ( Camellia sinensis ) and generally refers to a stem, not a leaf, used as a material for black tea.

In one aspect of the present invention, the extract may be extracted with water, a C _{1 -4} lower alcohol or a mixed solvent thereof, and more specifically, may be extracted with methanol or ethanol.

In one aspect of the present invention, the cosmetic composition is characterized in that it is for preventing damage to skin DNA by ultraviolet rays.

In one aspect of the present invention, the cosmetic composition is a sunscreen, skin, lotion, cream, essence, emulsion, gel, lipstick, cleansing foam, cleansing cream, cleansing water, spray, shampoo, conditioner, treatment, body cleanser, soap , A pack, a massage agent, a face powder, a compact, a foundation, a two-way cake, and a makeup base, but may be formulated with any one selected from the group consisting of, but is not limited thereto.

The present invention provides a food additive composition for preventing food deterioration containing a black tea stem extract.

The present invention also provides an antioxidant health functional food containing the stem extract of black tea.

In one aspect of the present invention, the stem of the black tea is obtained from a by-product of black tea ( Camellia sinensis ) and generally refers to a stem, not a leaf, used as a material for black tea.

In one aspect of the present invention, the extract may be extracted with water, a C _{1 -4} lower alcohol or a mixed solvent thereof, and more specifically, may be extracted with methanol or ethanol.

Hereinafter, the present invention will be described in more detail through examples according to the present invention and comparative examples not according to the present invention, but the scope of the present invention is not limited by the examples presented below.

<Experiment preparation> Purchase of experimental materials

DPPH (1,1-diphenyl-2-picrylhydrazyl), ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid), ferric chloride, Griess reagent, nitro Nitro blue tetrazolium (NBT), xanthine, xanthine oxidase, 2,4,6-tris (2-pyridyl)-s-triazine (2,4,6-Tris( 2-pyridyl)-s-triazine, TPTZ), quercetin, sodium nitroprusside (SNP) and hydrogen peroxide were purchased from Sigma-Aldrich (St. Louis, MO, USA). .

The pUC19 plasmid DNA was obtained from New England Biolabs, UK. Methanol used in the preparation of the extract used the highest analytical grade.

Example 1. Preparation of black tea stem extract

Black tea stem ( Camellia sinensis ) was purchased from a special tea dealer (Hayang Traditional Market, Hayang-eup, Gyeongsan-si, Gyeongbuk, Korea, 2013). 200 g of black tea stems were pulverized and extracted three times for 24 hours on a shaker at room temperature using 95% ethanol. After filtration, the solvent was concentrated at 50° C. under reduced pressure using a rotary vacuum evaporator (Labota 4000, Heidolph, Germany). After the lyophilized black tea stem extract was obtained, it was dissolved in each solvent, and it was used for evaluation of antioxidant activity through various analyzes and measuring the protective effect against oxidative DNA damage.

Experimental example One. DPPH Radical Scavenging Measure

DPPH-free radical scavenging activity of black tea stem extract was measured using a somewhat modified method of Liyana-Pathirana, and Shahid (2005). 100 µl of black tea stem extract at different concentrations (5, 10, 25, 50, 100 and 200 µg/mL) were placed in different test tubes. A 0.1 mM DPPH methanol solution was prepared, 900 µl of this solution was added to each tube and shaken vigorously. Each tube was allowed to stand for 20 minutes in a dark room at 37°C. The black tea stem extract was not added as a control, and other conditions were prepared in the same manner. The absorbance of each sample was measured at 517 nm. The radical scavenging activity was expressed as the free radical inhibition rate by black tea stem extract, and was calculated using the following equation:

Radical scavenging activity (%) =

[(Absorbance of control group-absorbance of sample)/absorbance of control group] × 100

Quercetin was used as the standard antioxidant. Experiments were performed three times at all concentrations, and a graph was created as an average value. The experimental results are as shown in FIG. 1.

DPPH radical is a stable radical with a maximum absorbance at 517 nm, which is easily reduced by antioxidant molecules. The radical scavenging ability of the black tea stem extract is as shown in FIG. 1, and is expressed as a reduction rate of initial DPPH radical absorption due to the black tea stem extract of different concentrations. In this experiment, 100 μg/mL of black tea stem extract showed 68.84% DPPH radical scavenging activity, and the scavenging rate was increased depending on the concentration. As a result, in the case of black tea stem extract, it was found that the faster the absorbance decreases in terms of hydrogen atom or electron donating ability, the better the antioxidant activity is. Quercetin used as a positive control showed 81.4% DPPH radical inhibitory activity at 100 μg/mL, and the scavenging rate increased depending on the concentration. Therefore, due to the presence of bioactive molecules, black tea stem extracts are free radicals, especially in lipid peroxides or hydroperoxide radicals, which are the main propagators of lipid chain autoxidation. ), and can inhibit the proliferation stage of lipid peroxidation (Bamforth et al. 1993).

Experimental example 2. ABTS Radical Measurement of scavenging activity

ABTS radical scavenging activity by concentration of black tea stem extract was measured according to a method partially modified by Re et al. (1999).

ABTS· ⁺ was dissolved in water at a concentration of 7 mM. ABTS ⁺ radical cations were prepared by reacting ABTS stock solution with 2.45 mM potassium persulfate (final concentration), and this mixture was left in a dark room at room temperature for 12-16 hours before use. . Prior to the analysis, the solution was diluted with ethanol (about 1:89 v/v), equilibrated at 30°C, and placed in a 1 cm cuvette to show an absorbance of 0.700±0.02 at 734 nm. 100 µl of black tea stem extract at concentrations of 1.25, 2.5, 5, 10 and 20 µg/mL were added to 900 µl ABTS· ⁺ free radical solution, and incubated for 30 minutes. The OD value was measured exactly 30 minutes after initial mixing. For each analysis, an appropriate solvent blank was used.

The radical scavenging activity was expressed as the free radical inhibition rate by black tea stem extract, and was calculated using the following equation:

Radical scavenging activity (%) =

[(Absorbance of control group-absorbance of sample)/absorbance of control group] × 100

Quercetin was used as the standard antioxidant. Experiments were performed three times at all concentrations, and a graph was created as an average value. The experimental results are as shown in FIG. 2. As can be seen in Figure 2, the black tea stem extract exhibited 78.83% free radical inhibitory effect at 25 ㎍ / mL. The black tea stem extract directly removed free radicals at a concentration of 25 μg/mL, and the excellent scavenging activity of ABTS radical cations was concentration dependent. Quercetin exhibited an inhibition rate of 12.08 ± 0.65 IC ₅₀ and 72.53% (see Table 2).

The above results suggest that the black tea stem extract plays an important role in scavenging free radicals through a chain breaking reaction, and that it can be used as a very excellent functional food.

Experimental example 3. NO free Radical Measurement of scavenging activity

NO (nitric oxide) radical is one of the important molecules involved in various physiological processes such as nerve signaling, vasodilation, and blood pressure regulation (Afanasev 2007). When NO radicals are generated during oxidative stress, it can be a major cause of various tumors and infectious diseases such as arthritis, ulcerative colitis, juvenile diabetes, and multiple sclerosis. During aerobic metabolism, NO (nitric oxide) molecules react with oxygen molecules to produce intermediates such as NO ₂ , N ₂ O ₄ and N ₃ O ₄ . Because these products are highly genotoxic, they induce deamination of purines and pyrimidines and denaturation of enzymes such as DNA ligase and DNA alkyltransferase (Lundberg et al. 2008). At physiological pH, SNP (sodium nitroprusside) in aqueous solution spontaneously produces NO (nitric oxide) (Marcocci et al. 1994), and reacts with oxygen to generate nitrate ions, which can be measured using a grease reagent. I can. NO scavengers compete with oxygen to minimize NO production.

SNPs (10 mM) in 0.1 mM phosphate buffer (PBS) were mixed with black tea stem extracts at different concentrations (25, 50, 100, 200, and 400 μg/mL), and then incubated at 25° C. for 150 minutes. After incubation, grease reagent A (2% sulfanylamide in 4% H ₃ PO ₄ ) and grease reagent B (0.2% naphthylethylenediamine dihydrochloride) were added and incubated for 10 minutes.

The OD value of the chromophore, that is, chromaphore, generated during the diazotization of nitrite of sulfanylamide and sequential coupling with naphthylethylenediamine was measured at 540 nm.

The radical scavenging activity was expressed as the inhibition rate of free radicals by the black tea stem extract and was calculated using the following equation:

Radical scavenging activity (%) =

[(Absorbance of control group-absorbance of sample)/absorbance of control group] × 100

Quercetin was used as the standard antioxidant. Experiments were performed three times at all concentrations, and a graph was created as an average value. The experimental results are as shown in FIG. 3.

In this experiment, the black tea stem extract competed with oxygen in reacting with NO radicals to reduce the level of nitrite, thus exhibiting an effect of protecting against oxidative damage. As shown in Fig. 3, the black tea stem extract exhibited a NO radical inhibition rate of 47.92% at 400 ㎍/mL, and the effect showed a concentration-dependent aspect. As shown in Table 2, the black tea stem extract exhibited an IC ₅₀ value of 404.38 ± 1.6. The standard quercetin showed an inhibition rate of 90.98% at 400 ㎍/mL.

FRAP Activity of Black Tea Stem Extract and Standard Quercetin Sample FRAP mmol fe(ll)/mg extract Black Tea Stem Extract 2606.85±18.7 Quercetin 8326±115.2

* FRAP-Ferric reducing antioxidant power (Concentration of substance having ferric-TPTZ reducing ability as equivalent to 1 μmol Fe (II).

Experimental example 4. Super Oxide free Radical Measurement of scavenging activity

In living organisms, highly toxic species, such as superoxide radicals, are continuously produced during metabolic and physiological processes. Super oxide free radicals can generate more toxic reactive free radicals such as hydroxyl radicals and singlet oxygen. Biological molecules such as DNA, lipids and proteins are definitive targets for these oxygen species and cause metabolic and cellular disorders (Martinez-Cayuela, 1995).

The superoxide anion radical protective activity of black tea stem extract was measured using the Kuthan et al., (1986) method, which partially modified.

The reaction mixture was in 0.1 mM potassium phosphate (pH 7.8) buffer solution, 0.8 mM xanthine 0.25 mL, 0.5 mM nitro-blue tetrazolium (NBT) 0.15 mL, and different concentrations (25, 50, 100, 200 and 400 μg/mL) It contains 0.09 mL of black tea stem extract. After incubation at 25°C for 15 minutes, 0.5 U/mL of xanthine oxidase was added to start the reaction, and the reaction mixture was allowed to stand at 25°C for 30 minutes, and then 0.5 mL of 1 N HCl was added to react the sample. Stopped. The absorbance was measured at 560 nm.

The radical scavenging activity was expressed as the inhibition rate of free radicals by the black tea stem extract and was calculated using the following equation:

Radical scavenging activity (%) =

[(Absorbance of control group-absorbance of sample)/absorbance of control group] × 100

Quercetin was used as the standard antioxidant. Experiments were performed three times at all concentrations, and a graph was created as an average value. The experimental results are as shown in FIG. 4.

4, the black tea stem extract exhibited an activity of scavenging superoxide radicals in a concentration-dependent manner, and exhibited 91.57% superoxide radical scavenging ability at 400 μg/mL. The black tea stem extract showed an IC ₅₀ of 93.67 ± 2.7 μg/mL, and the standard quercetin was 4.32 ± 0.8 μg/mL (see Table 2). From the experimental results, it can be seen that the black tea stem extract contains a bioactive substance capable of scavenging superoxide-free radicals, thereby terminating the radical chain reaction.

IC ₅₀ values of DPPH, ABTS, NO and superoxide radical scavenging of black tea stem extract and standard quercetin Sample IC ₅₀ (㎍/mL) DPPH ABTS NO(Nitric oxide) Super oxide Black Tea Stem Extract 68.88±1.1 12.08±0.65 404.38±1.6 93.6±2.7 Quercetin 48.41±0.7 13.62±0.4 29.34±1.3 4.32±0.8

Experimental example 5. FRAP (Ferric-reducing/antioxidant power) analysis

Antioxidants can be described as reducing agents and inactivators of oxidation accelerators. Some previous studies have reported that reducing power can be used as an important indicator of the potential for antioxidant activity. It has been suggested that antioxidant activity is related to reducing power. Therefore, the potential of black tea stem extract as an antioxidant can be measured by evaluating its ability to convert the TPTZFe (III) complex into TPTZFe (II). Meanwhile, the FRAP assay is used to measure the antioxidant activity of various food samples (Pellegrini et al. 2003). Halvorsen et al. (2006) suggests that most of the secondary metabolites are redox-active compounds and can be measured by FRAP analysis.

The antioxidant activity of black tea stem extract was determined by Pulido et al. It was measured by the (2000) method. The absorbance of the reaction mixture was read at 593 nm. Values were expressed in mmol Fe (II)/g extract.

The ferric reducing ability of the black tea stem extract was 2606.85 mmol Fe (II)/g extract, indicating that it had remarkable FRAP activity (see Table 1).

Experimental example 6. In vitro DNA damage protection effect of black tea stem extract

The DNA damage protective activity of the black tea stem extract was checked using pUC19 plasmid DNA ( E. coli ER2272, New England Bio labs, UK). Plasmid DNA in the presence of different concentrations of black tea stem extract H ₂ O ₂ + UV treatment to oxidize, Russo et al. The method according to (2000) was slightly modified and checked on 1% agarose.

Experiments were carried out in a 10 μL volume containing 186 ng of pUC19 plasmid DNA in 1 X TE buffer (10 mM Tris-Cl and 1 mM EDTA) in a microfuse tube, pH 8.0, in a tube without black tea stem extract and 3 μL H ₂ O ₂ was added at a final concentration of 33 mM to the tube containing the black tea stem extract. The reaction was started by UV-C irradiation, and irradiation was continued for 35 seconds at the surface of the trans-illuminator, at room temperature, at 280 nm, at 22500 W/cm ² . After irradiation, the reaction mixture (10 μL) was loaded onto a 1% agarose gel with a gel loading die (6X), electrophoresed in 1 X TBE buffer solution, 50 V, and untreated pUC19 plasmid DNA was used as a control. The gel was stained with an aqueous solution of ethidium bromide and visualized with a UV trans illuminator.

Quercetin (50 μg/ml) was used as a positive control. The gel was scanned with a gel documentation system (Gel Doc-XR, Bio-rad, CA, USA). Bands were quantified using a Gel-Pro Analyzer (Media Cybernetics, USA).

The hydroxyl radical is H ₂ O ₂ used in this experiment. And it can be produced in cells by various reactions such as decomposition by UV, and can cause skin diseases such as skin cancer by damaging macromolecules such as lipids, proteins and DNA (Halliwell and Gutteridge 1990). In this experiment, H ₂ O ₂ of black tea stem extract And DNA damage protection against pUC19 plasmid DNA by hydroxyl radicals generated by UV photolysis was measured.

5 shows the electrophoresis pattern of pUC19 plasmid DNA in the presence and absence of black tea stem extract at different concentrations (50, 100, 200, 400 and 800 μg/mL). In gel electrophoresis, the pUC19 plasmid DNA showed two bands in lane 1, the fast-moving sharp band corresponds to the native supercoiled circular (Sc) DNA, and the slow-moving very faint band is the open circular (open circular) band. circular) corresponds to DNA (Oc).

Then in the presence of H ₂ O ₂ exposure to UV, H ₂ O ₂ And the supercoil DNA is cleaved by OH-free radicals generated by UV photolysis, and it is clearly transformed into an open circular DNA band (lane 2).

In biological systems, O ₂ · · OH radical generated by the reaction between the H ₂ O ₂ is a major cause of oxidative damage (Guitteridge, 1984). However, when different concentrations of black tea stem extract are pre-cultured with plasmid DNA pUC19, the degree of DNA damage induced by UV photolysis of H ₂ O ₂ decreases (lane 3-7), and the concentration-dependent native supercoil circular (super coiled circular, Sc) showed the effect of remarkably protecting DNA. Quercetin (50 μg/mL) was used as a positive control (lane 8), and was shown to reduce the amount of open circular DNA. The results of quantitatively analyzing the amounts of supercoil and linear DNA bands by densitomytri are shown in FIG. 7. In the case of the untreated group, the density of the supercoil circular (Sc) DNA is 694.78±4.12, and H ₂ O ₂ And decreased to 275.53±3.78 after exposure to UV. However, the density of supercoil (Sc) DNA was increased in the presence of different concentrations of black tea stem extract (0, 100, 200, 400 and 800 µg/mL). In the case of 800 μg/mL black tea stem extract, the density of the supercoil DNA (Sc) was 631.76±2.45, and the quercetin-treated supercoil DNA was 662.98±1.59. From the above experimental results, it can be seen that the black tea stem extract will exhibit an effect of protecting against oxidative DNA damage caused by harmful effects caused by solar UV irradiation.

<Statistical Analysis>

The experimental values were analyzed by ANOVA (one-way analysis of variance), and the significance of the difference was determined by Duncan's multiple test (P<0.05) using statistica (Statsoft Inc., Tulsa, USA). Was determined by The experimental values shown are the average values of the measured values ± standard deviations tested three times.

< Manufacturing example >

Preparation Example 1. Preparation of powder

10mg black tea stem extract

100mg sucrose

10mg talc

The ingredients are powdered, mixed, and then filled into an airtight cloth to prepare a powder.

Preparation Example 2. Preparation of tablets

10mg black tea stem extract

100mg starch

100mg sucrose

2mg magnesium stearate

After mixing the ingredients according to a conventional tablet manufacturing method, tablets are prepared by tableting them.

Preparation Example 3. Preparation of capsule

10mg black tea stem extract

3mg crystalline cellulose

15mg lactose

1mg magnesium stearate

The above ingredients are mixed according to a conventional capsule preparation method, and then filled in a gelatin capsule to prepare a capsule.

Preparation Example 4. Preparation of granules

10mg black tea stem extract

50mg soybean extract

200mg glucose

500mg starch

After mixing the above ingredients, 100 mL of 30% ethanol was added and dried at 60° C. to form granules, and then filled into a cloth to prepare granules.

Preparation Example 5. Preparation of pills

20mg black tea stem extract

1,500mg lactose

1,500mg glycerin

980mg starch

After mixing the above ingredients, it is prepared to be 4g per pill according to a conventional method for preparing pills.

Preparation Example 6. Preparation of injection

10mg black tea stem extract

180mg mannitol

2,780mg sterile distilled water for injection

Na ₂ HPO ₄ 12H ₂ O 30mg

It is prepared by mixing the above ingredients so as to be (3 mL) per ampoule according to a conventional injection preparation method.

Preparation Example 7. Preparation of liquid

10mg black tea stem extract

10,000mg isomerized sugar

5,000mg mannitol

Purified water appropriate amount

It is prepared by dissolving the above ingredients in purified water according to a conventional liquid preparation method, adding an appropriate scent, filling the bottle and sterilizing it.

Preparation Example 8. Preparation of anti-aging skin

Black Tea Stem Extract 0.001 (v/v)% Paraoxybenzoic acid methyl 0.2% Sodium ethylenediamine tetraacetate 0.02% 1,3-butylene glycol 0.02% Allantoin 3% Sodium Hyaluronic Acid 5% Carbomer 0.1% Cetostearyl alcohol 0.7% Propyl paraoxybenzoate 0.1% Sorbitanolivate 1.5% Soy lecithin 0.2% Dimethicone 0.2% Three-chill octanoate 0.2% Shea Butter 0.2% Sodium polyacrylate 3% Triethanolamine 0.1% Imidazolidinyl urea 0.3% Spices Appropriate amount Pigment Appropriate amount Purified water Balance

Using the above ingredients, it is prepared according to a conventional method in the field of cosmetic production for skin production.

Preparation Example 9. Preparation of anti-aging serum

Black Tea Stem Extract 0.001 (v/v)% Jojoba 5% Black Sesame 2 % Sweet Almond 3% Emulsing Firing Wax One % Vitamin E One % glycerin 2 % Hyaluronic acid One % Marine elastin One % Purified water Balance

Using the above ingredients, it is prepared according to a conventional method in the field of cosmetics manufacturing for serum production.

Preparation Example 10. Preparation of anti-aging lotion

Black Tea Stem Extract 0.001 (v/v)% Paraoxybenzoic acid methyl 0.2% Sodium ethylenediamine tetraacetate 0.02% 1,3-butylene glycol 0.02% Allantoin 3% Sodium Hyaluronic Acid 5% Carbomer 0.1% Cetostearyl alcohol 0.7% Propyl paraoxybenzoate 0.1% Sorbitanolivate 1.5% Soy lecithin 0.2% Dimethicone 0.2% Three-chill octanoate 0.2% Shea Butter 0.2% Sodium polyacrylate 3% Triethanolamine 0.1% Imidazolidinyl urea 0.3% Spices Appropriate amount Pigment Appropriate amount Purified water Balance

It is prepared according to a conventional method in the field of cosmetics manufacturing for the manufacture of lotions using the above ingredients.

Preparation Example 11. Preparation of anti-aging essence

Black Tea Stem Extract 0.001 (v/v)% Allantoin 0.05% Sodium ethylenediamine tetraacetate 0.02% Triethanolamine 0.2% Sodium Hyaluronic Acid 7% Imidazolidinyl urea 0.15% Sodium polyacrylate 0.4% Carbomer 0.2% ethanol 3% Monostearate polyoxyethylene sorbitan 0.2% Paraoxybenzoic acid methyl 0.2% Spices Appropriate amount Pigment Appropriate amount Purified water Balance

It is prepared according to a conventional method in the field of cosmetics manufacturing for the manufacture of essences using the above ingredients.

Preparation Example 12. Preparation of anti-aging cream

Black Tea Stem Extract 0.001 (v/v)% Sodium ethylenediamine tetraacetate 0.02% Allantoin 0.1% glycerin 5% Paraoxybenzoic acid methyl 0.2% Sodium Hyaluronic Acid 6% Carbomer 0.1% Cetostearyl alcohol 1.7% Polydecene 2 % Squalane 2 % Propyl paraoxybenzoate 0.1% Butylene glycol dicaprylate 3% Cetyl Octanoate 5% Microcrystalline lead 0.1% Triethylpentanediol 0.1% Shea Butter 0.2% Sorbitanolivate 0.3% Cyclomethicone 0.3% Stearyl dimethicone 0.5% Imidazolidinyl urea 0.15% Spices Appropriate amount Pigment Appropriate amount Purified water Appropriate amount

Using the above ingredients, it is prepared according to a conventional method in the field of cosmetic production for cream production.

Preparation Example 13. Preparation of anti-aging pack

Black Tea Stem Extract 0.001 (v/v)% Sodium ethylenediamine tetraacetate 0.02% Betaine 3% Glyceryl polymethacrylate 2 % Allantoin 0.1% Sodium Hyruronic Acid 2 % glycerin 3% Dipropylene glycol 5% Paraoxybenzoic acid methyl 0.2% Polyvinyl alcohol 10% Polyoxyethylene sorbitan monooleate 0.9% Sesquioleic acid 0.3% Jojoba ester 2 % Cetearyl alcohol 1.5% Petrolatium 0.5% Spices Appropriate amount Pigment Appropriate amount Purified water Appropriate amount

Using the above ingredients, it is prepared according to a conventional method in the field of manufacturing cosmetics for manufacturing a pack.

Preparation Example 14. Preparation of anti-aging massage cream

Black Tea Stem Extract 0.001 (v/v)% Purified water Appropriate amount glycerin 4.0% vaseline 3.5% Triethanol amine 0.5% Liquid paraffin 24.5% Squalane 2.5% Beeswax 2.1% Tocopheryl Acetate 0.1% Kabapol 1.0% Sorbitansquioleate 3.1% incense a very small amount antiseptic a very small amount

It is prepared according to a conventional method in the field of cosmetics manufacturing for the manufacture of a massage cream using the above ingredients.

Preparation Example 15. Preparation of UV-blocking and anti-aging makeup base

Black Tea Stem Extract 0.001 (v/v)% Purified water Appropriate amount Coated silica One % Silica 10% Titanium dioxide 8 % Zinc oxide 3% Pigment One % Plate powder Balance

Using the above ingredients, it is prepared according to a conventional method in the field of manufacturing cosmetics for preparing a makeup base.

Preparation Example 16. Preparation of UV-blocking and anti-aging powder pact

Black Tea Stem Extract 0.001 (v/v)% Purified water Appropriate amount Mica 15% Titanium dioxide 7% Silicone oil 3% Ester oil 3% Pigment Appropriate amount Spices Appropriate amount Talc Balance

Using the above ingredients, a powder pact is prepared according to a conventional method in a cosmetic manufacturing field for powder pact manufacturing.

Preparation Example 17. Manufacture of UV-blocking and anti-aging two-way cake

Black Tea Stem Extract 0.001 (v/v)% Jeong-soo Appropriate amount Mica 15% Titanium dioxide 12% Silicone oil 3% Ester oil 5% Pigment Appropriate amount Spices Appropriate amount Talc Balance

Using the above ingredients, it is prepared according to a conventional method in the field of manufacturing cosmetics for making a two-way cake.

Claims (3)

delete Antioxidant health functional food containing ethanol extract of black tea stem as an active ingredient.
Cosmetic composition for antioxidant containing ethanol extract of black tea stem as an active ingredient.

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