KR20180065799A - A composition for anti-bacterial effect and anti-inflammation comprising unripe apple extracts and baicalin - Google Patents

Abstract

The present invention relates to a composition containing an unripe apple extract and baicalin. The composition containing a mixture of an unripe apple extract and baicalin as an effective component of the present invention has antimicrobial and anti-inflammatory effects by inhibiting skin flora and having an excellent effect on inhibiting the expression of iNOS and NO; and has no cytotoxicity and skin side effects, and thus can be used safely as a cosmetic composition, a pharmaceutical composition or a food composition.

Description

TECHNICAL FIELD The present invention relates to an antibacterial and anti-inflammatory composition comprising an immature apple extract and baicalin as an active ingredient. BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antibacterial and anti-

The present invention relates to a composition containing a mixture of unripe apple extract and baicalin, and more particularly to a composition containing an immature apple extract and an antibacterial and antiinflammatory composition comprising baicalin as an active ingredient. will be.

All aerobic respiration creatures, including humans, use oxygen to generate the energy needed to sustain life. In this process, active oxygen is produced and they are known to cause harmful actions in vivo, and various direct or indirect biological disorders related thereto have been reported.

NO (Nitric Oxide), which is a part of active oxygen species, is harmful to reactive oxygen species and causes inflammation and various diseases when the function of antioxidant system is deteriorated. In addition, NO is a very unstable gas with toxicity. It is one of the free radicals that causes cellular damage at high concentration. It causes damage of various normal cells to cause inflammation, and it causes tissue damage, genetic mutation and nerve injury .

Among them, inflammatory reaction is a mechanism to repair and regenerate the injured area when an invasion that brings about physical changes such as physical action, chemical substance, bacterial infection to the living body or tissue is performed, and once the stimulus is applied, Vasoactive substances such as serotonin, bradykinin, prostaglidin, and leukotriene are liberated, resulting in increased vascular permeability and inflammation.

Macrophages produce nitric oxide (NO) and cytokines during inflammatory reactions and play an important role in biological defense in the early stages of infection. Lipopolysaccharides, also known as endotoxins, are found in the extracellular membrane of Gram-negative bacteria and are known to increase inflammatory cytokines in macrophages such as RAW 264.7 or monocytes. Furthermore, the formation of such an inflammatory mediator leads to the process of converting arachidonic acid into frost gladin and the NO formation process due to the activity of phospholipase A2. In the body inflammation process, inflammatory factors such as excess NO and prostaglandin E ₂ (PEG ₂ ) are formed by iNOS and COX-2. Among these, NO has various physiological functions such as body defense function, signal transduction function, neurotoxicity, and vasodilation. In general, the formation of NO plays an important role in killing bacteria or eliminating tumors, but NO produced by iNOS in the inflammatory state not only promotes inflammatory responses such as vascular permeability and edema, but also stimulates inflammation mediator biosynthesis, .

The use of anti-inflammatory drugs has caused pain relief to date, and most of non-narcotic analgesics have also been used for the treatment of acute and chronic inflammatory diseases because of their anti-inflammatory effects. However, it is difficult to use it for long term due to side effects. As a result, the severity of side effects is too great for the current treatment methods, so it is essential and urgent to develop new and improved therapeutic agents. In recent years, efforts have been made to find the active ingredient in natural products in connection with the problem of overcoming such side effects.

On the other hand, it is known that unripe apple contains a large amount of flavan-3-ol based compound which exhibits various physiological activities as compared with mature apple, and Korean Patent No. 10-1065558 Discloses that the extract of the immature apple has a higher active ingredient content than the ripe apple and has excellent skin whitening effect and can be used as a pharmaceutical composition for improving the skin condition.

In addition, baicalin is a kind of flavonoid, which is a plant of the family Lamiaceae ( Scutellaria baicalensis Georgi ), and it is known to have pharmacological effects such as anti-infective, anti-allergic and antihypertensive effects.

Accordingly, the inventors of the present invention have continued to develop natural materials having antibacterial and antiinflammatory properties and fewer human side effects. As a result, when mixed with immature apple extract and mixed with bicalyin, And exhibit an excellent effect on the anti-inflammatory effect, thereby completing the present invention.

The object of the present invention is to provide a substance having excellent antibacterial and antiinflammatory properties and excellent human stability without cytotoxicity derived from natural materials.

In order to solve the above technical problems, the present invention provides an antibacterial and anti-inflammatory composition comprising an immature apple extract and baicalin as an active ingredient.

Preferably, the composition comprising the immature apple extract and baicalin in the present invention may be a cosmetic composition, a pharmaceutical composition or a food composition.

The term " immature apple " of the present invention means an apple that has been fouled one to three weeks after flowering and fertilization.

The term "extract " of the present invention means a preparation which is obtained by squeezing a herbal medicine with an appropriate leaching solution and concentrating by evaporating the leaching solution, and is not limited thereto. The extract, the diluted solution, A dried product obtained by drying, a controlled preparation thereof, or a purified product thereof.

The immature apple extract of the present invention can be extracted using a conventional extraction solvent known in the art, and the extracted solution can be used in a liquid form or can be used by concentration and / or drying. The extraction solvent may be, for example, (a) water, (b) anhydrous or hydrated lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), (c) Solvent, (d) acetone, (e) ethyl acetate, (f) chloroform or (g) 1,3-butylene glycol as the extraction solvent. Preferably, extraction is carried out using methanol, ethanol or butanol. Depending on the extraction solvent, the degree of extraction and the degree of loss of the active ingredient of the extract may differ. Therefore, an appropriate extraction solvent should be selected and used. The extraction method is not particularly limited, and examples thereof include cold extraction, ultrasonic extraction, and reflux cooling extraction.

In addition, the immature apple extract can be obtained through a conventional purification process in addition to the extraction method using the extraction solvent. For example, by separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatographies (made for separation by size, charge, hydrophobicity or affinity), and the like, An immature apple extract can also be obtained through fractionation.

In one specific embodiment of the present invention, the immature apple extract is prepared by pulverizing immature apple and then extracting a solvent selected from the group consisting of water, an anhydrous or low-boiling alcohol having 1 to 3 carbon atoms, and a mixed solvent thereof, , And the amount of the extraction solvent may be 2 to 4 parts by volume relative to immature apple.

In one specific embodiment of the present invention, the immature apple extract is prepared by pulverizing immature apples and then adding 60 to 80% ethanol (EtOH) at a volume ratio of 2 to 4 times at room temperature for 20 to 30 hours, After immersed and extracted for 26 hours, it is recovered and concentrated with a filter paper to obtain an immature apple extract.

The term " bacalin " of the present invention is a kind of flavonoid, and the bacalin may be purified from a plant, produced commercially, or produced by a microorganism.

The plant for isolating and purifying the above baicalin is a plant of the family Lamiaceae, Scutellaria baicalensis Georgi ), and may be preferably golden ( Scutellaria baicalensis ).

In one specific embodiment of the present invention, the baicalin is a plant of the family Lamiaceae ( Scutellaria baicalensis ) from the root.

In one specific embodiment, the baicalin is prepared by pulverizing the gold evenly and then adding 60 to 80% ethanol at a volume ratio of 2 to 4 times at 68 to 76 hours, preferably 70 to 74 hours at room temperature, more preferably Is dissolved in water (H ₂ O), and the resulting extract is passed through a purifier and concentrated under reduced pressure using a vacuum evaporator at a temperature of 35 to 45 ° C., preferably 40 ° C., Crystallization can be induced from the powder obtained by utilizing the temperature change to obtain high purity crystalline bacalin.

The composition of the present invention preferably contains an immature apple extract and baicalin in a weight ratio of 7: 0.5 to 1.5, more preferably 7: 1. In this case, when the baicalin content is less than 7: 0.5 by weight in the immature apple extract, the antimicrobial and anti-inflammatory effects, which are the object of the present invention, can not be obtained remarkably. When the immature apple extract contains more than 7: 1.5 by weight, The increase in the effect of the increase may be insufficient.

The composition comprising the immature apple extract of the present invention and the baicalin exhibits an antibacterial and anti-inflammatory effect, and has little cytotoxicity as a natural substance.

In one specific embodiment of the present invention, the present invention provides a cosmetic, pharmaceutical, or food composition comprising an immature apple extract and bicalin as an active ingredient.

The cosmetic composition of the present invention may contain, in addition to the immature apple extract and baicalin as an active ingredient, conventional additives such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers commonly added to cosmetic compositions .

The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, , Oil, powder foundation, emulsion foundation, wax foundation, spray, emulsion, multiple emulsion, liquid crystal, and the like. More specifically, it can be prepared as a nutritional cream, a convergent lotion, a soft lotion, a lotion, an essence, a nutritional gel or a massage cream.

When the formulation of the present invention is a paste, cream or gel, use is made of an animal oil, vegetable oil, wax, paraffin, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide .

When the formulation of the present invention is a powder or a spray, tosse, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, chlorofluorohydrocarbons, propane / Propane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

In the case where the formulation of the present invention is a suspension, a carrier such as 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, Castellulose, aluminum metahydroxide, bentonite, agar or tracert, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.

The pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier other than an immature apple extract and bicalin. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc., in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, and is preferably applied by parenteral administration, more preferably topical application by application.

A suitable dosage of the pharmaceutical composition of the present invention may vary depending on such factors as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate, . The dosage of the pharmaceutical composition of the present invention is in the range of 0.001-100 mg / kg on an adult basis. When the composition is an external preparation, it is preferably applied in an amount of 1.0 to 3.0 ml on an adult basis once to five times a day, and continued for 1 month or more. However, the dose is not intended to limit the scope of the present invention.

The pharmaceutical composition of the present invention may be prepared in unit dosage form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art, Into the container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in an oil or aqueous medium, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

In one specific embodiment of the present invention, the food composition contains not only an immature apple extract and baicalin as an active ingredient, but also ingredients normally added in food production such as protein, carbohydrate, fat, nutrients, . ≪ / RTI >

Examples of such carbohydrates are monosaccharides, such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavorings such as tau martin and stevia extract (e.g., rebaudioside A and glycyrrhizin) and synthetic flavors (saccharin, aspartame, etc.) may be used as flavorings.

For example, when the food composition of the present invention is prepared as a drink, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, mulberry extract, jujube extract and licorice extract are added in addition to mature apple extract of the present invention and baicalin .

Meanwhile, the immature apple extract and baicalin of the present invention are naturally harmless to the human body and have little toxicity and side effects, so that they can be safely used for a long period of use. In particular, the cosmetic composition, the pharmaceutical composition or the food composition It can be safely applied.

As described above, the composition comprising the immature apple extract of the present invention and baicalin as an active ingredient has excellent antimicrobial and anti-inflammatory effects, and is free from cytotoxicity and skin side effects, and is useful as a cosmetic composition, a pharmaceutical composition or a food composition . In addition, it is useful not only for environmental preservation but also for economic purposes, by utilizing agricultural processing by-products which are abolished in large quantities.

Figure 1 shows the production process and yield of mature apple extract.
Fig. 2 shows the production process and yield of immature apple extract.
FIG. 3 is a photograph showing the antimicrobial activity of a mixture of immature apple extract and baicalin mixed at a weight ratio of 7: 1 on E. coli with growth inhibition rings.
4 is a photograph showing the antimicrobial activity of a mixture of immature apple extract and baicalin in a weight ratio of 7: 1 to Staphylococcus epidermidis as a growth inhibitory ring.
FIG. 5 is a photograph showing the antimicrobial activity of Staphylococcus aureus in a mixture of immature apple extract and baicalin at a weight ratio of 7: 1, as inhibition of growth.
FIG. 6 is a photograph showing the antimicrobial activity against Candida albicans in a mixture of immature apple extract and baicalin at a weight ratio of 7: 1, as inhibition of growth.
FIG. 7 is a photograph showing the antimicrobial activity of Prephosphonium bacterium acnes mixed with a mixture of immature apple extract and baicalin at a weight ratio of 7: 1 by inhibition of growth.
FIG. 8 is a graph showing the NO inhibitory activity of a mixture of immature apple extract and baicalin mixed at a weight ratio of 7: 1.
FIG. 9 is a graph showing the iNOS inhibitory activity of a mixture of immature apple extract and baicalin in a weight ratio of 7: 1.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Production Example 1: Preparation of immature apple extract

The immature apple used in this experiment was obtained in May 2011 in Osanri area, Yeongcheon, North Gyeongsang Province, and immature apple was used as the material after 2 weeks of flowering.

After immature apple was pulverized, 70% ethanol (EtOH) was added at a volume ratio of 3 times of the immature apple crumb, and the immersion extraction was repeated three times at room temperature for 24 hours. 1 filter paper. Each extract was concentrated at 40 ° C or lower using a rotary vacuum evaporator, and then stored in a refrigerated room after lyophilization and used as a sample of the present experiment. Figure 1 shows the production process and yield of mature apple extract. Fig. 2 shows the production process and yield of immature apple extract.

Production Example 2: Preparation of Baikalin

The gold ( Scutellaria) produced in July 2010 The resulting extract was dissolved in water (H ₂ O) and passed through a purifier. A vacuum evaporator was used at a temperature of 40 ° C to utilize the extract. After concentration under reduced pressure, crystallization was induced from the powder obtained by using the temperature change to prepare high purity crystalline bacalin.

Example 1: Preparation of a mixture of immature apple extract and baicalin

The immature apple extract prepared in Preparation Example 1 and the baicalin prepared in Preparation Example 2 were mixed in a ratio of 7: 0.2 (sample 1), 7: 0.4 (sample 2), 7: 0.6 (sample 3) 4), 7: 1 (sample 5), 7: 1.2 (sample 6), 7: 1.4 .

Test Example 1: Verification of antibacterial effect

(1) Measurement of Clear Zone

The antimicrobial activity of the causative bacteria of the skin diseases such as Staphylococcus epidermis, S. aureus, E. coli and B. subtilis was examined. The antimicrobial activity was measured by a paper disc method. That is, each strain cultured on a plate culture medium was plated at a concentration of 1 pl, cultured in 10 ml of a liquid culture medium for 22 hours, activated, and then inoculated with 0.1 ml of the culture broth in 10 ml of a liquid culture medium. After culturing for 5 hours, 10 ⁷ cells, and then homogeneously plated with a sterilized cotton swab. The sterilized filter paper disc (Tokyo, 8mm, Japan) was placed on a solid plate medium, and the sample was absorbed at a concentration of 3mg / disc and incubated at 37 ° C for 22 hours to measure the diameter ) Were measured.

In this Test Example 1, in order to examine the antibacterial effect of the immature apple extract and the golden extract baikalulin mixture, Escherichia coli coli), Staphylococcus epidermidis (Staphylococcus epidermidis), Staphylococcus aureus (Staphylococcus aureus), Candida albicans (Candida albicans , and Propionibacterium acnes . The results are shown in Table 1 and Fig. 4. As shown in the following Table 1, the sample (control) in which the immature apple extract alone was used in the test concentration (sample 3 mg / disc) showed no antimicrobial activity in all five species. The antibacterial activity against E. coli, Staphylococcus epidermidis and Candida albicans was observed in the samples (samples 1 to 9) using a mixture of immature apple extract and bicalin, and the activity against Staphylococcus aureus and P. bacterium acnes was It did not appear.

Strains Control Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Escherichia coli - - 2 mm 8 mm 11 mm 13 mm 15 mm 16 mm 16 mm 16 mm Staphylococcus epidermidis - 2 mm 4 mm 11 mm 14 mm 17 mm 18 mm 19 mm 19 mm 19 mm Staphylococcus aureus - - - - - - - - - - Candida albicans - - 3 mm 9 mm 13 mm 15 mm 16 mm 18 mm 18 mm 18 mm Prefioni
Bacterium Acne - - - - - - - - - -

Test Example  2: Anti-inflammatory effect measurement

(1) Measurement of inhibitory activity of nitrogen monoxide (Nitric oxide)

The amount of NO generated from the Raw 264.7 cell line was evaluated by Feelish's method as follows.

The samples were prepared by mixing 10%, 25%, 50%, 75%, and 100% of the immature apple extract and immature apple extract in a weight ratio of 7: < / RTI > ug / ml. Nitric oxide (NO ₂ ) present in the cell culture was measured using a Griess reagent. The cultured cells were treated with lipopolysaccharide (LPS) to induce inflammation, and 100 μl of the cell culture supernatant, The reaction mixture was reacted for 10 minutes with the addition of 100 ul of the reagent, and the reaction was carried out for 10 minutes. Then, the absorbance was measured at 540 nm wavelength.

 As shown in FIG. 8, it was confirmed that the mixture (sample 5) in which the immature apple extract was mixed with the baicalin at a weight ratio of 7: 1 inhibited NO production more than using the immature apple extract alone. In FIG. 8, Con is a measurement of cultured cells to which no extract sample is added, and NC is a culture cell in which inflammation has not been induced without treating lipopolysaccharide.

(2) Measurement of iNOS protein expression by Western blotting

iNOS is usually not present in cells but once induced, it produces a large amount of NO over a long period of time. The generated NO promotes inflammatory reaction such as vascular permeability and swelling as well as promotes biosynthesis of inflammatory mediators to intensify inflammation It is known.

The samples were prepared so as to have concentrations of 5 ug / ml, 10 ug / ml and 25 ug / ml, respectively, using 70% ethanol, in which the immature apple extract was mixed with baicalin at a weight ratio of 7: 1. Expression of iNOS was measured using Towbin's Western blot as follows. Raw 264.7 cells were treated with lipopolysaccharide (LPS) and each concentration, and cells were lysed using a lysis buffer solution and centrifuged at 15,000 rpm for 15 minutes to obtain protein . Protein concentrations were quantified by the Bradford method. The obtained protein was electrophoresed using SDS-polyacrylamide gel, and then transblotted with a nitrocellulose membrane. The membrane was treated with an anti-iNOS polyclonal antibody as a primary antibody, and the secondary antibody anti-rabbit IgG (horseradish peroxidase) conjugated with horseradish peroxide (anti-rabbit IgG), and the expression of iNOS was measured using Enhanced Chemiluminescence Detection.

The measurement results are shown in Fig. In Fig. 9, Nor is a measurement of cultured cells not treated with lipopolysaccharide and not causing inflammation, and Con shows a case in which a sample mixed with baicalin was added to the inflammatory-induced cultured cells in the prepared immature apple extract And cultured cells were counted. As can be seen from FIG. 9, it was confirmed that a sample in which bicalin was mixed with immature apple extract inhibited inflammation by inhibiting iNOS.

Claims (7)

An anti-bacterial or anti-inflammatory cosmetic composition comprising an unripe apple extract and baicalin as an active ingredient. The method according to claim 1,
Wherein the antibacterial effect is an antibacterial effect against at least one microorganism selected from the group consisting of Escherichia coli , Staphylococcus epidermidis , and Candida albicans . Composition. 3. The method of claim 2,
Wherein the composition comprises an immature apple extract and BaCalin in a weight ratio of 7: 0.5 to 1.5. A pharmaceutical composition for antibacterial or antiinflammatory activity, comprising an immature apple extract and baicalin as an active ingredient. 5. The method of claim 4,
Wherein the antimicrobial effect is an antimicrobial effect against at least one microorganism selected from the group consisting of Escherichia coli , Staphylococcus epidermidis , and Candida albicans . Gt; 6. The method of claim 5,
Wherein said composition comprises an immature apple extract and baicalin in a weight ratio of 7: 0.5 to 1.5. An immune or anti-inflammatory food composition comprising an immature apple extract and baicalin as an active ingredient.

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