WO2024101570A1 - Antioxidant, anti-inflammatory, and anti-allergy composition containing stevia extract as active ingredient - Google Patents

Abstract

The present invention relates to an antioxidant, anti-inflammatory, and anti-allergy composition including a Stevia rebaudiana extract as an active ingredient.

Description

Antioxidant, anti-inflammatory and anti-allergy composition containing stevia extract as an active ingredient

Cross-reference to related applications

This application claims priority benefit to Korean Patent Application No. 2022-0148552, filed on November 9, 2022, and the contents of the patent application are hereby incorporated by reference in their entirety.

The present invention relates to an antioxidant, anti-inflammatory and anti-allergy composition containing stevia extract as an active ingredient.

Cosmetics are applied to the skin to clean, protect, and beautify the skin, and are defined as having a mild effect. Products used for this purpose include various forms such as lotion, emulsion, cream, essence, and foundation. The product is on the market. However, among the ingredients essential in cosmetics, preservatives, surfactants, pigments, ultraviolet absorbers and other ingredients irritate skin cells, cause allergic reactions, and ultimately cause inflammation in the skin, causing side effects. It could be the cause. In addition, fatty acids, higher alcohols, and proteins among sebum, sweat, and cosmetic ingredients discharged from the body are decomposed by skin flora existing on the skin, which may cause skin inflammation, and ultraviolet rays from the sun. It is a well-known fact that inflammation in the skin is also caused. Therefore, preventing skin inflammation is a very important issue in cosmetics. Representative substances used in cosmetics for this purpose include allantoin and bisabolol, and indomethacin is a representative example of substances used in pharmaceuticals. However, allantoin has poor solubility in water or oil (solubility in water at room temperature is less than 0.5 parts by weight, insoluble in oil), which limits its use as a product, and bisabolol has excellent anti-inflammatory effects but has side effects. It is known that indomethacin is a medicine and cannot be applied to cosmetics.

Meanwhile, in addition to the anti-inflammatory effect, antioxidant effects are also essential for cosmetics. Here, the antioxidant effect refers to fats and oils used as softeners in cosmetics, dissolved oxygen in the air or water dissolved in oils, and active radicals induced by ultraviolet rays. It refers to the inhibition of oxidation or decomposition by Since general cosmetics have a shelf life of about 2 to 3 years, if long-term oxidation stability is not secured, the product may deteriorate during the distribution process. Representative substances used as antioxidants in cosmetics include dibutylhydroxyanisole (hereinafter referred to as BHA), dibutylhydroxytoluene (hereinafter referred to as BHT), ascorbic acid (vitamin C), and tocopherol (vitamin C). E) and its derivatives are the most widely used. However, although BHA and BHT have excellent antioxidant effects, they are known to cause side effects, and vitamin C and vitamin E cannot be used directly due to their poor stability, and vitamin C derivatives and vitamins that have improved their stability E derivatives have the problem of having little antioxidant effect.

Allergy is a biochemical phenomenon that represents a specific and altered reaction to a substance. This is a type of hypersensitivity reaction caused by immune imbalance and is a reaction that occurs to a specific antigen called an allergen. It is a reaction induced when IgE-class antibodies specific for that antigen bind to mast cells or basophiles. am. When a large number of allergen-specific IgE antibodies are produced, these antibodies bind to Fc receptors on the surface of mast cells or basophils. When allergens bind to these cells, these cells are activated and produce various bioactive substances (cytokines, prostaglandins). , leukotrienes, histamine, bradykinin, tryptase, etc.) are secreted, leading to changes such as vasodilation, increased vascular permeability, smooth muscle contraction, and inflammatory response. These reactions can occur locally or systemically depending on the type and amount of mediators produced by these cells. Diseases include allergic atopy, systemic allergy, allergic rhinitis, asthma, food allergy, and skin allergy. If you look at allergic reactions in detail, histamine is formed by decarboxylating histidine by L-histidine decarboxylase, and is stored in the form of granules in mast cells or basophils. When an allergic reaction begins when an allergen binds to a mast cell or basophil, the cells degranulate and histamine is secreted. At this time, β-hexosaminidase is also secreted. This enzymatic activity can be used as a measure of mast cell degranulation and can be used in experiments. Allergic diseases were 0.9 per 100,000 people in 1979, but increased significantly to 1.4 per 100,000 in 1998 (approximately 55.6%), and the importance of alleviating and preventing diseases has come to the fore. Existing treatments can be broadly divided into steroidal anti-inflammatory drugs or non-steroidal anti-inflammatory drugs and antihistamines or anti-leukotriene drugs. The former mostly have a strong immunosuppressive effect, so they can have a temporary sedative effect in a short period of time, but can cause mild symptoms such as nausea. There are many problems with long-term use, ranging from side effects to serious side effects such as growth inhibition and osteoporosis. The latter may provide temporary relief, but its use is limited due to side effects such as drowsiness and dizziness. Therefore, the need for substances derived from natural extracts with fewer side effects in the body and high anti-allergy activity is emerging.

The problem to be solved by the present invention is to provide an antioxidant, anti-inflammatory and anti-allergy composition containing stevia ( Stevia rebaudiana Bertoni) extract as an active ingredient.

Another problem to be solved by the present invention is to provide a pharmaceutical composition for antioxidant, anti-inflammatory and anti-allergy purposes, including a composition containing stevia extract as an active ingredient and a pharmaceutically acceptable carrier.

Another problem to be solved by the present invention is to provide an antioxidant, antibacterial and anti-allergy cosmetic composition containing a composition containing stevia extract as an active ingredient and a cosmetically acceptable carrier.

Another problem to be solved by the present invention is to provide an antioxidant, antibacterial and anti-allergy health functional food composition containing a composition containing stevia extract as an active ingredient and a foodologically acceptable carrier.

In one aspect, the present invention provides an antioxidant, anti-inflammatory and anti-allergy composition containing stevia ( Stevia rebaudiana Bertoni ) extract as an active ingredient.

In the present invention, the stevia extract may be a stevia ethanol extract, a stevia subcritical extract, or a mixture thereof.

In the present invention, the stevia ethanol extract may be an extract using high concentration ethanol of 50% (v/v) or more.

In the present invention, the stevia subcritical extract can be extracted with subcritical water for 5 to 120 minutes under conditions of 90 to 250°C and 1 to 5 MPa.

In the present invention, the stevia subcritical extract can be extracted with subcritical water for 5 to 120 minutes under conditions of 200 to 230°C and 2 to 4 MPa.

In the present invention, the minimum effective concentration of stevia extract may be 1 μg/ml.

In another aspect, the present invention provides a pharmaceutical composition for antioxidant, anti-inflammatory and anti-allergy purposes, comprising a composition containing the above-described stevia extract as an active ingredient and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides a quasi-drug composition for antioxidant, antibacterial and anti-allergy purposes, including a composition containing the above-described stevia extract as an active ingredient and a quasi-drug acceptable carrier.

In another aspect, the present invention provides an antioxidant, anti-inflammatory and anti-allergy cosmetic composition comprising a composition containing the above-described stevia extract as an active ingredient and a cosmetically acceptable carrier.

In another aspect, the present invention provides an antioxidant, antibacterial and anti-allergy health functional food composition containing a composition containing the above-described stevia extract as an active ingredient and a foodologically acceptable carrier.

The present invention can provide an antioxidant, anti-inflammatory and anti-allergic composition containing as an active ingredient a stevia extract that is non-cytotoxic, has excellent antioxidant effect, reduces NO production, and has an inhibitory effect on β-hexosaminidase secretion.

Figure 1 shows the results of ABTS radical scavenging ability analysis of Examples 1 to 4.

Figure 2 shows the results of analysis of DPPH radical scavenging ability of Examples 1 to 4.

Figure 3 shows the results of cytotoxicity analysis in Examples 1 to 4.

Figure 4 shows the NO Synthesis analysis results of Examples 1 to 4.

Figure 5 shows the results of cytotoxicity analysis in Examples 1 to 4.

Figure 6 shows the results of the β-hexosaminidase secretion assay in Examples 1 to 4.

Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings so that those skilled in the art can easily implement the present invention. However, the present invention may be implemented in many different embodiments and is not limited to the embodiments described herein. Throughout the specification, similar parts are given the same reference numerals.

In addition, in the present invention, when similar expressions such as "about", "approximately", or at least are used in relation to numerical values, ±10%, ±7% based on the numerical value. %, ±5%, ±3%, ±2%, or ±1% theoretical, experimental, statistical, or rule of thumb error is intended to be permitted.

In one aspect, the present invention provides an antioxidant, anti-inflammatory and anti-allergy composition containing stevia ( Stevia rebaudiana ) extract as an active ingredient.

In this specification, “active ingredient” refers to an ingredient that exhibits the desired activity or can exhibit the desired activity in combination with a carrier that is inactive on its own.

The term "stevia" used in this specification is a perennial plant belonging to the Asteraceae family and is used in herbal teas, beverages, herbal medicine preparations, health supplements, etc. Additionally, stevia leaves contain a large amount of sweet-tasting ingredients such as stevioside and rebaudioside, so they can be used as a natural sweetener. In this specification, stevia can be purchased and used commercially, or collected or cultivated in nature, and it is preferred to mainly use the leaf part, but is not limited thereto.

In this specification, “stevioside” is a natural polysaccharide substance mainly contained in stevia leaves, specifically stevioside A1, A2, steviol 1,2,3, dulcoside, rebaudioside a,b, It contains various types of sugars such as c, e, and f.

As a result of extensive research, the present inventors have confirmed that extracts obtained by extracting stevia with ethanol at a specific concentration or using subcritical extraction exhibit excellent antioxidant, anti-inflammatory and anti-allergic effects. More specifically, it was confirmed that using high-concentration ethanol as an extraction solvent or extracting under subcritical conditions can increase the antioxidant, anti-inflammatory, and anti-allergic effects of stevia extract.

Specifically, the stevia extract of the present invention exhibits ABTS radical scavenging ability and DPPH radical scavenging ability, and thus can exhibit excellent antioxidant activity. Additionally, the minimum effective concentration showing the antioxidant activity of the stevia extract of the present invention may be 1 μg/ml, and preferably 100 μg/ml or more.

In addition, the stevia extract of the present invention exhibits an anti-inflammatory effect on mouse-derived RAW264.7 macrophages stimulated with LPS, and in particular, the subcritical extract among the extracts exhibits a better anti-inflammatory effect, and the Subcritical extracts obtained under subcritical extraction conditions show particularly excellent anti-inflammatory effects.

In addition, the stevia extract of the present invention exhibits antioxidant and anti-inflammatory activities as well as anti-allergic activity, and in particular, 70% (v/v) of the ethanol extract among the extracts exhibits excellent anti-allergic effects.

As used herein, the term "extract" refers to an extract obtained by extraction treatment, a dilution or filtrate of an extract, a concentrate of a filtrate of an extract, a re-filtrate of a concentrate, an extract, a dilution, a filtrate, a concentrate, or a ash. It includes the extract itself and all formulations of extracts that can be formed using the extract, such as dried product obtained by drying the filtrate, extract, diluent, filtrate, concentrate, crude or purified product of refiltrate, or mixtures thereof.

In the present specification, drying of the extract may be performed by a known method as long as useful components from stevia are not destroyed, for example, by natural drying in the shade. Additionally, the stevia can be crushed or pulverized to the extent that useful components of stevia can be sufficiently extracted during the subsequent extraction process. The drying and crushing or grinding processes can be performed in reverse order or repeatedly as needed.

In the present specification, the stevia extract may be extracted from stevia leaves, stems, or a mixture thereof with 50 to 80% ethanol, and preferably may be extracted using 70% ethanol.

In one embodiment, the stevia extract of the present invention is extracted by adding 70% (v/v) ethanol to stevia powder, then extracted for 12 to 24 hours by rotating at a temperature of 2 to 8 ° C., impurities are removed, and the supernatant is extracted. It can be manufactured by taking it.

The term "subcritical extract" used in this specification refers to the extract itself, such as an extract obtained by subcritical extraction treatment, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, a crude product or purified product of the extract, or a mixture thereof. and extracts of all formulations that can be formed using the extract. Additionally, as used herein, the term “subcritical extract” is used interchangeably with “subcritical water extract.”

In subcritical extraction, the extraction method is not particularly limited and can be extracted according to methods commonly used in the art.

In a preferred embodiment of the present specification, the stevia subcritical extract can be extracted with subcritical water for 5 to 120 minutes under conditions of 90 to 250°C and 1 to 5 MPa, and in a more preferred embodiment, stevia subcritical extract may be extracted under conditions of 90 to 250°C and 1 to 5 MPa for 5 to 120 minutes. Under certain conditions, it can be extracted with subcritical water for 5 to 120 minutes.

In another aspect, the present invention provides a pharmaceutical composition for antioxidant, anti-inflammatory and anti-allergy purposes, including a composition containing stevia extract as an active ingredient and a pharmaceutically acceptable carrier.

In this specification, the antioxidant, anti-inflammatory and anti-allergenic properties of stevia extract are described above.

As used herein, the pharmaceutical composition may further include pharmaceutically acceptable carriers, excipients, or diluents commonly used in the manufacture of pharmaceutical compositions other than the active ingredient. At this time, the content of the stevia extract included in the pharmaceutical composition is not particularly limited, but may be 0.001% by weight to 50% by weight, preferably 0.01% by weight to 10% by weight, based on the total weight of the composition.

In the present specification, the pharmaceutical composition is any selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried agents, and suppositories. It may have a single dosage form, or may have several oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

In the present specification, the pharmaceutical composition can be administered in a pharmaceutically effective amount. In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the individual, age, gender, and type of disease. , can be determined based on factors including the activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the field of medicine.

In the present specification, the pharmaceutical composition can be administered individually or in combination with other drugs, and when administered in combination, it can be administered sequentially or simultaneously. And it can be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this amount can be easily determined by a person skilled in the art.

The appropriate total daily usage amount can be determined by treatment within the range of sound medical judgment, and is generally an amount of 0.001 to 1000 mg/kg, preferably 0.05 to 200 mg/kg, and more preferably 0.1 to 100 mg/kg. It can be administered once or in several divided doses per day.

In another aspect, the present invention provides a quasi-drug composition for antioxidant, antibacterial and anti-allergy purposes, including a composition containing stevia extract as an active ingredient and a quasi-drug acceptable carrier.

In the present specification, the quasi-drug composition may further include a pharmaceutically acceptable carrier, excipient, or diluent as needed in addition to the stevia extract of the present invention, and the pharmaceutically acceptable carrier, excipient, or diluent may achieve the effects of the present invention. It is not limited as far as possible, and may include, for example, fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, sweeteners, fragrances, preservatives, etc.

Representative examples of pharmaceutically acceptable carriers, excipients or diluents herein include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, gelatin, glycerin, gum acacia, alginate, calcium phosphate, calcium. Carbonate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, propylene glycol, polyethylene glycol, vegetable. Oils, injectable esters, Wethepsol, Macrogol, Tween 61, Cacao, Lauridge, etc. are included, but are not particularly limited thereto.

In addition, when the composition of the present invention is used as a quasi-drug, it may additionally contain one or more active ingredients that exhibit the same or similar functions as the stevia extract. For example, ingredients having known antioxidant, antibacterial, and anti-allergic activities may be additionally included. When adding active ingredients, skin safety due to combined use, ease of formulation, and stability of the active ingredients can be considered. In addition, additional active ingredients may be included in an amount of 0.0001 to 10% by weight (w/w) based on the total weight of the quasi-drug composition, and the above-described content range can be appropriately adjusted by a person skilled in the art in consideration of skin safety, ease of formulation, etc. there is.

In the present invention, quasi-drug compositions include external preparations, powders, disinfectants, toothpastes, ointments, lotions, preparations for internal use (vitamin/mineral preparations, nutritional tonics), wet tissues, spray paste, bands or patches, etc. It can be done, but is not particularly limited, and the formulation method, dosage, usage method, components, etc. of the quasi-drug can be appropriately selected by a person skilled in the art from common techniques known in the art.

In another aspect, the present invention provides an antioxidant, antibacterial and anti-allergy cosmetic composition comprising a composition containing stevia extract as an active ingredient and a cosmetically acceptable carrier.

In the present specification, the cosmetic composition may include ingredients commonly accepted in the cosmetics field without limitation, in addition to the composition containing the stevia extract of the present invention as an active ingredient, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances. It may include conventional cosmetically acceptable carriers, auxiliary ingredients, etc.

In this specification, cosmetic compositions include solution, external ointment, cream, foam, nourishing lotion, softening lotion, pack, softening water, emulsion, makeup base, essence, soap, liquid cleanser, bath agent, sunscreen cream, sun oil, suspension, and emulsion. It may be prepared in one or more formulations selected from the group consisting of liquid, paste, gel, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, patch and spray, but is limited thereto. It doesn't work.

In addition, the cosmetic composition herein may additionally include one or more types of cosmetically acceptable carriers that are mixed with general skin cosmetics, and include common ingredients such as oil, water, surfactants, moisturizers, low-grade Alcohol, thickener, chelating agent, colorant, preservative, fragrance, etc. may be appropriately mixed, but are not limited thereto. Cosmetically acceptable carriers included in the cosmetic composition of the present invention vary depending on the formulation.

In the present specification, when the dosage form is an ointment, paste, cream or gel, the carrier ingredients include animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or Mixtures of these can be used.

In the present specification, when the formulation is powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silica, polyamide powder, or mixtures thereof may be used as carrier ingredients, and especially in the case of spray, additional May contain propellants such as chlorofluorohydrocarbons, propane/butane or dimethyl ether.

When the formulation herein is a solution or emulsion, a solvent, solubilizing agent, or emulsifying agent is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-Butylglycol oils may be used, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitan. there is.

When the formulation herein is a suspension, the carrier ingredients include water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, and miso. Crystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used.

In the present specification, when the formulation is soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolysates, isethionates, lanolin derivatives, fatty alcohols, vegetable oils, glycerol, sugars, etc. may be used as carrier ingredients. You can.

In the present specification, when the formulation is a surfactant-containing cleansing agent, the carrier ingredients include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcositate, and fatty acid amide. Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester can be used.

In the present specification, the cosmetic composition may contain 0.01 to 10% by weight of a composition containing stevia extract as an active ingredient, preferably 1 to 5% by weight of the total cosmetic weight.

In another aspect, the present invention provides an antioxidant, antibacterial and anti-allergy health functional food composition containing a composition containing the above-described stevia extract as an active ingredient and a foodologically acceptable carrier.

The term “health functional food” used in this specification refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and pills using raw materials or ingredients with functional properties useful to the human body. Here, function means controlling nutrients for the structure and function of the human body or obtaining useful effects for health purposes, such as physiological effects.

In this specification, the health functional food can be manufactured by a method commonly used in the industry, and can be manufactured by adding raw materials and ingredients commonly added in the industry, and can be manufactured with a foodologically acceptable carrier. can be used In addition, unlike general drugs, it is made from food, so it has the advantage of not having any side effects that may occur when taking the drug for a long time, and it can be highly portable.

The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing food, the stevia extract according to the present invention may be added in an amount of 1 to 50% by weight, preferably 5 to 10% by weight, but is not limited thereto. However, in the case of long-term intake for health and hygiene purposes or health control, the amount may be used below the above range.

There is no particular limitation on the type of food in this specification, and examples of foods to which the above substances can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, and ice cream. It includes dairy products, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and includes all health functional foods in the conventional sense.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

<Example>

preparation of ingredients

Stevia was purchased from Korea Stevia Co., Ltd., and 70% ethanol was purchased from Daejeong Chemical.

Assorbic acid, L-NIL, ABTS solution, and DPPH solution were purchased from Sigma-aldrich.

RAW 264.7 Cell was obtained from the Korea Cell Line Bank, and Houttuynia cordata was purchased from Gapdang Herbal Medicine Co., Ltd.

MTT reagent, DMSO, PIPES, a Siraganian buffer component, and 4-p-nitrophenyl-N-acetyl-β-D-glucosaminide and sodium citrate, the main components of the substrate buffer, were all purchased from Sigma-aldrich.

CaCl _2, MgCl _2, Dextrose Anhydrous, NaCl, and KCl were purchased from Daejeong Chemical, and BSA was purchased from BOVOGEN.

Example 1: Preparation of high-concentration ethanol extract of stevia

80 ml of 70% (v/v) ethanol was added to 8 g of stevia powder and extracted by rotating at a temperature of 4°C for one day. After extraction, impurities were removed using a centrifuge, and the supernatant was taken to prepare a stevia extraction solution.

Example 2: Preparation 1 of stevia subcritical extract

1 g of stevia powder was filled into a 50 mL subcritical extraction cell and then installed in a subcritical extraction device (Speed Extractor E-916, BUCHI, Switzerland). Subcritical extraction conditions were about 90°C and about 3MPa for 30 minutes, and 100% distilled water was used as the extraction solvent. The obtained subcritical extract was dried under reduced pressure and freeze-dried to prepare a subcritical extract in powder form.

Example 3: Preparation 2 of Stevia subcritical extract

The same procedure as Example 2 was performed, but subcritical extraction was performed at a temperature of 150°C.

Example 4: Preparation 3 of stevia subcritical extract

The same procedure as Example 2 was performed, but subcritical extraction was performed at a temperature of 210°C.

<Experimental example>

Experimental Example 1: Analysis of DPPH radical inhibition effect and ABTS radical inhibition effect

1.1 ABTS radical inhibition effect analysis

The ABTS radical inhibition effect of stevia 70% ethanol extract and subcritical extract prepared according to Examples 1 to 4 was measured at each concentration.

Examples 1 to 4 used concentrations of 100, 20, and 1㎍/㎖, and ascorbic acid 100㎍/㎖ was used as a positive control.

For the ABTS solution, 10 ml of 7mM ABTS and 176 ㎕ of 140mM Potassium peroxodis㎕fate were mixed, sonicated for 10 minutes, and then reacted for 24 hours. Each sample and ascorbic acid at each concentration were mixed 1:1 with 100 ㎕ of ABTS solution. Mixed and reacted for 15 minutes, and absorbance was measured at 734 nm.

Table 1 and Figure 1 show the results.

Referring to Table 1 and Figure 1, it can be seen that all the treated samples showed an antioxidant effect as a result of repeated tests three times. At the highest concentration, Example 4 had the best antioxidant effect, Example 3, Example 2, Appeared in the order of Example 1.

1.2 Analysis of DPPH radical inhibition effect

The DPPH radical inhibition effect of stevia 70% ethanol extract and subcritical extract prepared according to Examples 1 to 4 was measured at each concentration.

Examples 1 to 4 used concentrations of 400, 200, 100, and 20㎍/㎖, ascorbic acid 100㎍/㎖ was used as a positive control, and electron donating ability (EDA) was determined according to Blois' method. DPPH 200mM (100% ethanol) was prepared, 100㎕ of each concentration sample and DPPH solution were mixed 1:1, reacted for 30 minutes, and absorbance was measured at 517nm.

It is shown in Table 2 and Figure 2.

Referring to Table 2 and Figure 2, it can be seen that all of them showed antioxidant effects, and Example 4 showed the best antioxidant effect, followed by Example 1, Example 3, and Example 2.

Experimental Example 2: Anti-inflammatory effect analysis

2.1 Cell culture

To measure the anti-inflammatory activity of the 70% ethanol extract and subcritical extract of stevia prepared according to Examples 1 to 4, RAW264.7 cells, a murine macrophage cell line, were incubated with 100 U/mL penicillin, The cells were inoculated into DMEM medium containing 100 ㎍/mL streptomycin and 10% FBS, then cultured in a 5% CO ₂ thermostat at 37°C, and subcultured at 2-day intervals.

2.2 MTT assay

Cytotoxicity evaluation of Examples 1 to 4 was measured using MTT assay.

RAW 264.7 cells were distributed at 5 x 10 ⁴ cells/well in a 24 well plate and incubated for 24 hours. Example 1 was treated to have a concentration of 1000, 500, and 200㎍/ml, the positive control was treated to have a concentration of 20uM L-NIL, and Examples 2 to 4 were treated to have a concentration of 200㎍/ml.

After further culturing for 24 hours, 600 ㎕ of medium was removed, MTT reagent was added to 1/10 of the medium, and then incubated for 3 hours.

Finally, all medium was removed, 700 ㎕ of DMSO was dispensed to elute the Formazan dye, 40 ㎕ each was duplicated in a 96 well plate, and the absorbance was measured at 550 nm.

Table 3 and Figure 3 show the results.

Referring to Table 3 and Figure 3, it can be seen that none of them exhibit cytotoxicity.

2.3 NO assay

To measure the anti-inflammatory activity of Examples 1 to 4, RAW 264.7 cells were distributed at 5 x 10 ⁴ cells/well in a 24 well plate and incubated for 24 hours.

Example 1 was treated to have a concentration of 1000, 500, and 200 μg/ml, the positive control was treated to have L-NIL 20 μM, and Examples 2 to 4 were treated to have a concentration of 200 μg/ml.

After 1 hour of treatment, LPS was treated to a final concentration of 1㎍/㎖, and after incubation for 24 hours, 100㎕ of the supernatant was transferred to a 96 well plate, Griess reagent was dispensed 1:1 and reacted for 10 minutes, 540nm The absorbance was measured.

Table 4 and Figure 4 show the results.

Referring to Table 4 and Figure 4, it can be seen that Example 1 exhibits anti-inflammatory activity by suppressing NO production in a concentration-dependent manner, Example 2 showed almost no anti-inflammatory effect, and Example 3 showed 15% showed anti-inflammatory activity, and Example 4 showed similar activity to Example 1 when the concentration was 1,000 μg/ml. In addition, it can be confirmed that the efficacy was better than L-NIL and that the anti-inflammatory activity was superior to Example 1 at the same concentration.

Experimental Example 3: Analysis of itching inhibition effect

3.1 MTT assay

RBL-2H3 cells were distributed in ^a 24 well plate at 2 ml, and Examples 2 to 4 were treated to have a concentration of 200 ㎍/ml.

After further culturing for 24 hours, 600㎕ of medium was removed, MTT reagent was added to 1/10 of the medium, and incubation was performed for 3 hours.

Finally, all medium was removed, 700 ㎕ of DMSO was dispensed to elute the Formazan dye, 40 ㎕ each was duplicated in a 96 well plate, and the absorbance was measured at 550 nm.

Table 5 and Figure 5 show the results.

Referring to Table 5 and Figure 5, it can be seen that cell viability is not impaired under all conditions.

3.2 β-hexosaminidase secretion assay

RBL-2H ₃ cells were distributed in a ²⁴ well plate at 2 The positive control was treated to have a concentration of 1 mg/ml of Houttuynia cordata, and Examples 2 to 4 were treated to have a concentration of 200 ㎍/ml.

After 1 hour of treatment, wash twice with siraganian buffer, then treat with A23187 (1uM) and PMA (50nM) to induce degranulation for 30 minutes, transfer 20㎕ of the supernatant to a 96 well plate, dispense 80㎕ of substrate buffer, and leave for 30 minutes. The reaction was performed at 37°C.

Finally, 200 ㎕ of Stop solution was dispensed to terminate the reaction, and then the absorbance was measured at 405 nm.

Table 6 and Figure 6 show the results.

Referring to Table 6 and Figure 6, it can be seen that Example 1 showed an itching inhibition effect similar to the positive control at all concentrations.

Claims (10)

1. An antioxidant, anti-inflammatory and anti-allergy composition containing stevia ( Stevia rebaudiana ) extract as an active ingredient.
2. According to paragraph 1,

   The stevia extract is an ethanol extract of stevia or a subcritical extract of stevia, a composition for antioxidant, anti-inflammatory and anti-allergy.
3. According to paragraph 2,

   Stevia ethanol extract is an antioxidant, anti-inflammatory and anti-allergy composition that is an extract extracted using more than 50% (v/v) ethanol.
4. According to paragraph 2,

   Stevia subcritical extract is an antioxidant, anti-inflammatory and anti-allergy composition extracted with subcritical water for 5 to 120 minutes at 90 to 250°C and 1 to 5 MPa.
5. According to paragraph 2,

   Stevia subcritical extract is an antioxidant, anti-inflammatory and anti-allergy composition extracted with subcritical water for 5 to 120 minutes under conditions of 200 to 230°C and 2 to 4 MPa.
6. According to paragraph 1,

   A composition for antioxidant, anti-inflammatory and anti-allergy, wherein the minimum effective concentration of the stevia extract is 1㎍/㎖.
7. An antioxidant, anti-inflammatory and anti-allergy pharmaceutical composition comprising the composition of any one of claims 1 to 6 and a pharmaceutically acceptable carrier.
8. A quasi-drug composition for antioxidant, antibacterial and anti-allergy purposes, comprising the composition of any one of claims 1 to 6 and an acceptable carrier.
9. An antioxidant, antibacterial and anti-allergy cosmetic composition comprising the composition of any one of claims 1 to 6 and a cosmetically acceptable carrier.
10. An antioxidant, antibacterial and anti-allergy health functional food composition comprising the composition of any one of claims 1 to 6 and a foodologically acceptable carrier.

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