KR102127282B1 - Composition for Anti-inflammatory Comprising Biorenovation Extract of Prunus yedoensis Matsum - Google Patents

Abstract

The present invention relates to an anti-inflammatory composition containing a Prunus × yedoensis bioconversion extract extracted from Prunus × yedoensis through a bioconversion method. The Prunus × yedoensis bioconversion extract inhibits the production of nitric oxide, prostaglandin, and pro-inflammatory cytokines by an inflammatory reaction, thereby being able to be usefully used as the anti-inflammatory composition.

Description

Composition for Anti-inflammatory Comprising Biorenovation Extract of Prunus yedoensis Matsum}

The present invention relates to a composition for anti-inflammatory comprising a Yoshino cherry bio-conversion extract extracted from Yoshino cherry tree through a bioconversion method.

Biorenovation (biorenovation) refers to a method of producing or manufacturing a desired product from precursors using enzymatic functions of microorganisms, and inducing structural changes of substances through biological methods such as microbial fermentation or enzymatic treatment. Collectively. It is newly known that the content of the active ingredient is increased or the absorption rate is improved and new functional components are generated due to the above changes, and material development using these techniques has attracted attention worldwide.

These bioconversion methods are widely used in the production of useful substances such as amino acids and vitamins, as well as the production of pharmaceutical and pharmaceutical raw materials including antibiotics and steroids. In developed countries, a number of products aimed at high value-added products are sold due to the release of products due to biotechnology, and these new biotechnology-based materials have been recognized by consumers as safety, and various biotechnological technologies have recently been systematically fused. Functional materials are emerging.

As a result, the inventors of the present invention tried to discover new materials and methods for manufacturing them, and completed the present invention by producing and utilizing a Yoshino cherry tree bioconversion extract through a bioconversion method.

Korean Registered Patent No. 10-1628939

An object of the present invention is to provide an anti-inflammatory composition comprising a Yoshino cherry tree bioconversion extract obtained through a bioconversion method as an active ingredient.

The anti-inflammatory composition according to an embodiment of the present invention may include a Yoshino cherry tree bioconversion extract as an active ingredient.

The Yoshino cherry tree ( Prunus yedoensis Matsum) is native to the southern regions of Korea and Hallasan, Jeju Island, and is now commonly inhabited nationwide. In order to obtain an extract from a Yoshino cherry tree through a bioconversion method, roots, stems, leaves, fruits, and flowers of the Yoshino cherry tree can be used without limitation. Preferably, it may be a Yoshino cherry tree flower. In this case, the Yoshino cherry tree may be used directly, or an extract extracted from the Yoshino cherry tree may be used. Yoshino cherry tree extract can be obtained by using an extraction method known in the art without limitation. Examples of the extraction method include cold immersion extraction, ultrasonic extraction, reflux cooling extraction, hot water extraction, pressure extraction, and solvent extraction. When using a solvent for extraction, an extraction solvent known in the art may be used without limitation. Examples of the extraction solvent are purified water, methanol, ethanol, propanol, n-butanol, iso-butanol, acetic acid, dimethyl formamide, dimethyl sulfoxide (DMSO), acetone, acetonitrile, ethyl acetate , Methyl acetate, pentane, hexane, chloroform, diethyl ether, carbon tetrachloride, tetrahydrofuran (THF), and the like. For example, Yoshino cherry tree roots, stems, leaves, fruits, flowers, etc. were dried at room temperature and put into an extraction tank, then purified water was added to extract them at a temperature of 80 to 140°C, and at 1 to 2 atm air pressure. An extract can be obtained.

The term "bioconversion" or "bioconversion method" used in the present invention is a method of producing and manufacturing a compound inducing structural changes such as phosphorylation and glycosylation from a substrate or precursor using microorganisms. Means The phosphorylation means that a phosphoric acid group (HOPO(OH)O-) is substituted with a hydrogen atom of a substrate by the action of a phosphatase (kinase) to bind to the substrate, and the glycosylation is a monosaccharide to the substrate, It means that sugars such as polysaccharides are added. The bioconversion may be by Bacillus amyloliquefaciens strain.

The bio-converted extract of Yoshino cherry can increase the content of the active ingredient present in the Yoshino cherry tree by bio-conversion by culturing the Yoshino cherry or the extract extracted from the Yoshino cherry (Yellow cherry extract) together with the strain, and contained in the Yoshino cherry tree. The functional groups of the useful compounds can be substituted, leading to the production of new compounds that did not exist. The Yoshino cherry tree bioconversion extract may be bioconverted by the Bacillus amyloliquefaciens strain, and the Yoshino cherry bioconversion extract extracted from the Yoshino cherry tree through the bioconversion by the Bacillus amyloliquefaciens strain may cause an inflammatory reaction. Nitric dxide (NO), prostaglandin E2 (PGE2), may have activity that inhibits the production of pro-inflammatory cytokines. Specifically, in the inflammatory response induced by LPS, it is possible to inhibit the expression of mRNA, protein related to the production of nitric oxide, prostaglandin, and pre-inflammatory cytokines.

The term "composition for anti-inflammatory" as used in the present invention means a composition that prevents inflammation or acts to eliminate the inflammation. Such a composition may be used as a food composition, cosmetic composition, pharmaceutical composition, and the like.

The food composition may be provided in any formulation suitable for food additives or functional food, and may be, for example, solutions, emulsions, viscous mixtures, powders, granules, tablets, capsules, and the like. At this time, the present invention may include various bases and/or additives necessary and appropriate for the formulation of the formulation within a range that does not impair the main effect of the object, fragrances, pigments, fungicides within a range that does not impair the effect , Antioxidants, preservatives, moisturizers, thickeners, inorganic salts, emulsifiers and the like may further contain additives. The blending amount of these additives may be selected within a range that does not impair the purpose and effect of the present invention depending on the formulation or purpose of use. For example, based on the total weight of the food composition, the additive may be 0.01 to 5% by weight, more specifically 0.1 to 3% by weight.

The cosmetic composition may be provided in any formulation suitable for topical application, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsions It may be a foundation, a wax foundation, a spray, or the like. At this time, the present invention may include various bases and/or additives necessary and appropriate for the formulation of the formulation within a range that does not impair the main effect desired, a nonionic surfactant within a range that does not impair the effect, Silicone polymers, extenders, fragrances, preservatives, fungicides, oxidation stabilizers, organic solvents, ionic or nonionic thickeners, softeners, antioxidants, free radical disruptors, opacifiers, stabilizers, emollients, silicones, α-hydroxy acids, anti-foaming agents, moisturizing agents, vitamins, insect repellents, flavoring agents, preservatives, surfactants, anti-inflammatory agents, substance P antagonists, fillers, polymers, propellants, basic or acidifying agents, coloring agents may further include additives, etc. . The blending amount of these additives may be selected within a range that does not impair the purpose and effect of the present invention depending on the formulation or purpose of use. For example, based on the total weight of the cosmetic composition, the additive may be 0.01 to 5% by weight, more specifically 0.1 to 3% by weight.

The pharmaceutical composition may be provided in any formulation suitable for pharmaceuticals, and may be, for example, an oral dosage form or a parenteral dosage form in a solid, semi-solid or liquid form. Formulations for oral administration may be tablets, pills, granules, capsules, powders, granules, powders, emulsions, syrups, pellets, and the like. Formulations for parenteral administration may be injections, drops, ointments, lotions, sprays, suspensions, emulsions, suppositories, and the like. At this time, the present invention may include various bases and/or additives necessary and appropriate for the formulation of the formulation within a range that does not impair the main effect of the object, and carriers, surfactants, within a range that does not impair the effect, Additives such as excipients, colorants, spices, preservatives, stabilizers, buffers, suspending agents may be further included. Such pharmaceutical compositions may be administered orally, parenterally, rectal, topical, transdermal, intravenous, intramuscular, intraperitoneal, subcutaneous, and the like. The active ingredient of the pharmaceutical composition may vary depending on the age, gender, weight, pathology and severity of the subject to be administered, the route of administration or the judgment of the prescriber. For example, the daily dose may be from 0.01 mg/kg/day to 100 mg/kg/day, more specifically from 0.1 mg/kg/day to 50 mg/kg/day.

The Yoshino cherry tree bioconversion extract according to the present invention is extracted from the Yoshino cherry tree through a bioconversion method, and can be usefully used as an anti-inflammatory composition by inhibiting the production of nitric oxide, prostaglandin, and pre-inflammatory cytokines by an inflammatory reaction.

1 is an HPLC analysis result of a Yoshino cherry bioconversion extract (BR) extracted from a Yoshino cherry flower extract (PY) through a bioconversion method according to an embodiment of the present invention.
Figure 2 is a cell viability results of PY and PYBR for RAW 264.7 cells stimulated with LPS according to an embodiment of the present invention.
3 is a result of (a) NO production, (b) COX-2 and iNOS protein expression inhibition of PY and PYBR for RAPS 264.7 cells stimulated with LPS according to an embodiment of the present invention.
Figure 4 is a result of inhibiting the production of PEG2, IL-1β, IL-6 of PYBR for RAPS 264.7 cells stimulated with LPS according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. However, these descriptions have been presented by way of example only to help understanding of the present invention, and the scope of the present invention is not limited by these exemplary descriptions.

Example 1. Yoshino cherry tree bioconversion extract and cell preparation

1-1. Preparation of Yoshino Cherry Flower Extract

The Yoshino cherry tree flowers were obtained from the Jeju Institute of Plant Resources, and after removing the deposits, they were washed with clean fresh water and dried completely at room temperature. Dried cherry flowers were added to the extraction tank, and 1 L of 70% ethanol was added. The extract was recovered at room temperature for 24 hours to recover the primary extract, and 1 L of 70% ethanol was additionally added to the extraction tank. Secondary extract was recovered by extracting at room temperature for 24 hours. The recovered primary extract and secondary extract were mixed and filtered through a filter (TOYO, Japan) having a pore size of 5.0 μm to obtain a Yoshino cherry tree flower extract. The filtered extract was prepared by vacuum concentration to 5 brix or more.

1-2. Bioconversion method

The microorganisms used for bioconversion were Bacillus amyloliquefaciens KCTC 43033 strains, which were purchased from the Korean Collection for Type Culture (KCTC).

KCTC 43033 strain was incubated for 20 hours at a temperature of 30 °C at 1 L of nutrient liquid medium (containing 3.0 g/L of beef extract and 5.0 g/L of peptone). After incubation, microbial pellets were obtained by centrifugation at 5,000 rpm for 10 minutes.

The obtained microbial pellet was washed with PG buffer (containing 50 mM phosphate pH 7.2 and 2% glycerol) and suspended in 5 mL of PG buffer. Subsequently, a substrate (Yoshino cherry tree flower extract) was added to the suspension and incubated at 30°C for 72 hours.

After the culture was completed, the culture solution was centrifuged to collect the supernatant, concentrated under reduced pressure, and dissolved in H ₂ 0 to prepare a sample (Yellow Cherry bioconversion extract).

1-3. HPLC analysis

The sample prepared in 1-2. was confirmed whether or not a bioconversion compound was generated through HPLC (high performance liquid chromatography) analysis.

Specifically, a Prominence HPLC System (Shimadzu, Japan) apparatus was used. LC-2030/2040 PDA Detector was used as the detector, and Shim-pack GIS was used as the column. As a solvent, 0.1% TFA water and acetonitrile were used, and the analysis conditions were from 10% to 100% acetonitrile until 30 minutes. The wavelength was set to 254 nm, the flow rate was set to 1 mL/min, and the injection amount was set to 10 μl.

As a result, as shown in FIG. 1, the Yoshino cherry bioconversion extract (BR) includes a number of peaks that are not present in the Yoshino cherry flower extract (PY), and it has been confirmed that the intensity of some peaks is enhanced.

1-4. Sample and cell culture

As a sample, a Yoshino cherry flower extract (PY) and a Yoshino cherry bioconversion extract (PYBR) isolated from 1-2. were used at concentrations of 50, 100, and 200 μM.

RAW 264.7 cells were prepared using DMEM (Dulbecco's Modified Eagle Medium, Gibco, Grand Island, NY, USA) medium containing 100 units/mL penicillin-streptomycin (10%) and 10% fetal bovine serum (FBS). It was incubated at 37 °C in a% CO ₂ incubator. Cells were passaged every 2 days.

Experimental Example 1. Confirmation of cell viability

RAW 264.7 cells were cultured in a 24-well-microplate at 8.0 Х 10 ⁴ cells/mL using DMEM medium containing 10% FBS and cultured for 18 hours. Thereafter, the cells were incubated for 24 hours by simultaneously treating PY or PYBR with LPS (1 μg/mL), followed by reaction with MTT reagent (Thiazolyl Blue Tetrazolium Blue) for 4 hours. After the reaction, the cell culture was removed and DMSO was added to the remaining pellet, and absorbance was measured at 570 nm.

As a result, as shown in Figure 2, PYBR does not change the cell viability according to the treatment concentration, it was confirmed that the cell viability is superior to PY.

Experimental Example 2. Confirmation of NO inhibitory activity

2-1. NO generation

RAW 264.7 cells were cultured in a 24-well-microplate at 8.0 Х 10 ⁴ cells/mL using DMEM medium containing 10% FBS and cultured for 18 hours. Subsequently, the cells were incubated with PY or PYBR and LPS (1 μg/mL) for 24 hours. The generated NO was measured in the form of NO 2 ⁻ present in the cell culture using a Grease reagent. 100 μL of the cell culture solution and the same amount of the grease reagent were mixed and reacted at room temperature for 10 minutes, and then absorbance was measured at 540 nm using an ELISA reader.

As a result, as shown in FIG. 3A, PYBR was confirmed to inhibit NO production according to the treatment concentration similar to PY.

2-2. Protein expression of COX-2 and iNOS

RAW 264.7 cells were cultured for 18 hours by dispensing in a 6-well microplate at 1.0 Х 10 ⁵ cells/mL using DMEM medium containing 10% FBS. Subsequently, the cells were incubated for 24 hours by simultaneously treating PYBR and LPS (1 μg/mL). Cells were lysed with a lysis buffer for 1 hour and centrifuged to separate the protein-containing supernatant. Protein concentration in the supernatant was quantified with a BSA kit (Bio-Rad, USA). The quantified protein was electrophoresed on an 8-12% polyacrylamide gel, transferred to a PVDF membrane (poly-vinylidene difluoride membrane) (Milipore, USA) at 200 mA for 2 hours, and then the membrane was blocked. (blocking). Subsequently, the primary antibody to be blocked is an anti-COX-2 antibody (Anti-COX-2 (Rabbit) Antibody, RK100-401-226, Rockland Immunochemicals, Inc., USA), an anti-iNOS antibody (Rabbit Anti iNOS, BRAHP2399, BIO-RAD, USA) for 24 hours at 4° C., secondary antibody (HRP Anti-Rabbit IgG (H&L), RK611-103-122, Rockland Immunochemicals, Inc., USA; Anti-mouse IgG (H&L), RK610-103-121, Rockland Immunochemicals, Inc., USA) was diluted 1:5,000 or 1:10,000 and reacted at room temperature for 1 hour. Protein bands were confirmed on an imaging densitometer using an ECL kit (Bio-Rad, USA).

As a result, as shown in FIG. 3B, PYBR was confirmed to inhibit protein expression of iNOS and COX-2.

Experimental Example 3. Confirmation of PGE2 and pre-inflammatory cytokine inhibitory activity

RAW 264.7 cells were cultured in a 24-well-microplate at 8.0 Х 10 ⁴ cells/mL using DMEM medium containing 10% FBS and cultured for 18 hours. Subsequently, PYBR and LPS (1 μg/mL) were simultaneously treated to the cells, followed by incubation for 24 hours, followed by centrifugation of the cell culture (12,000 rpm, 3 minutes) to measure the content of PGE2 and pre-inflammatory cytokines in the supernatant obtained. Did. All measurement samples were stored frozen until quantification (-20°C. PGE2 is a PGE2 ELISA Kit (MousePGE2, R&DSystems, MN, USA). Pre-inflammatory cytokines are Mouse IL-6 ELISA Kit (San diego, California, USA), Mouse IL-1β/IL-1F2 (R&D Systems, MN, USA).

As a result, as shown in FIG. 4, PYBR was confirmed to inhibit the production of PGE2, IL-1β, and IL-6 depending on the treatment concentration.

Overall, it was found that the Yoshino cherry bioconversion extract extracted through the bioconversion method has excellent anti-inflammatory activity.

Claims (8)

As an anti-inflammatory composition comprising a Yoshino cherry tree bioconversion extract as an active ingredient,
The bioconversion is by Bacillus amyloliquefaciens strain,
The composition has an activity of inhibiting the production of nitrogen oxide (NO), prostaglandin (PGE2), pro-inflammatory cytokine (pro-inflammatory cytokine) by the inflammatory reaction, the composition.
The method according to claim 1,
The Yoshino cherry tree is one or more compositions selected from the group consisting of roots, stems, leaves, fruits and flowers.
delete delete The method according to claim 1,
The composition is at least one selected from the group consisting of food compositions, cosmetic compositions and pharmaceutical compositions.
The method according to claim 5,
The food composition is one or more formulations selected from the group consisting of solutions, emulsions, viscous mixtures, powders, granules, tablets and capsules.
The method according to claim 5,
The cosmetic composition is at least one selected from the group consisting of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations and sprays A composition that is a formulation.
The method according to claim 5,
The pharmaceutical composition is a solid, semi-solid or liquid form of the oral administration or parenteral administration.

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