KR20210135675A - Composition for anti-inflammation comprising extract of Rhamnus yoshinoi leaf - Google Patents

Abstract

The present invention relates to a composition for anti-inflammation. More specifically, the present invention relates to a composition for anti-inflammation which contains an extract of Rhamnus yoshinoi leaves. The present invention can provide a pharmaceutical composition, a cosmetic composition, and a food composition that can effectively prevent and alleviate inflammation. In order to achieve the above object, the present invention provides the composition for anti-inflammation derived from Rhamnus yoshinoi.

Description

Composition for anti-inflammation comprising extract of Rhamnus yoshinoi leaf

The present invention relates to an anti-inflammatory composition, and more particularly, to an anti-inflammatory composition comprising an extract of a leaf extract.

Inflammation has been considered an innate immune response to protect the body from various pathogens such as bacteria, viruses and fungi. Although inflammation is thought to be a host defense mechanism against various pathogens, the inflammatory response induces chronic inflammation, a major cause in the pathogenesis of various diseases such as inflammatory/autoimmune diseases, neurodegenerative diseases and cancer. Therefore, inhibition of inflammation has been regarded as an important target for the prevention of inflammation-mediated human diseases such as cancer, diabetes, neurological autoimmune diseases and arthritis.

Since forest resources have been used for medicinal purposes since ancient times, the development of functional materials using forest resources can be said to be a competitive industry. . The jjakjarae trees (Rhamnus yoshinoi) of the fly on the Korea Chungcheongnam-do and the rock mountain and river basins of the country, except in Gyeongsangbuk-do, Gyeonggi Province. The tips of the branches usually turn into thorns. The leaves are alternate phyllotaxis, oval or obovate in oval shape, dull-pointed at the tip, and 3-8cm long. The veins on the back side have hairs and then disappear, and there are dull sawtooths on the edges. Flowers are male and female, yellow-green, and hang 1-3 in each leaf axil. The fruit is a drupe, broad obovate round, with a hole at the bottom. Compared to buckthorn, the leaves are narrower and the bark is used for dyes, but it is rarely used as a food or pharmaceutical material.

On the other hand, in the prior art, it has been disclosed about the use of the extract of J. japonica for skin wrinkle improvement, but little is known about the use other than this.

Therefore, functional research and research on related functional ingredients are required to discover new functional edible plant resources using sagebrush. The paradigm for diseases has shifted from treatment to prevention, and the development of natural functional materials has resulted in a growing interest in well-being and health. this is being requested

Laid-open Patent Publication No. 10-2018-0068476 Laid-open Patent Publication No. 10-2018-0068904

In order to solve the above problems, the present invention is to provide an anti-inflammatory composition of natural origin effective for the prevention and improvement of inflammation.

In order to achieve the above object, the present invention provides an anti-inflammatory composition derived from Japonica.

The anti-inflammatory composition includes a pharmaceutical composition for preventing, improving, and treating inflammation, a cosmetic composition for preventing and improving inflammation, and a food composition for preventing and improving inflammation.

The composition for the prevention and improvement of inflammation derived from nature is derived from an extract of Japonica. Specifically, it may be extracted from the leaves, branches, fruits, etc. of the chamomile tree, but is preferably a leaf extract of the chamomile tree.

A polar solvent or a non-polar solvent may be used for the extract of the leaf extract of chamomile tree. The polar solvent may include water, a lower alcohol having 1 to 4 carbon atoms, acetone, dimethyl-formamide (DMFO), and dimethyl sulfoxide (DMSO). Acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1-pentene, 1 as a non-polar solvent -Chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform, dichloromethane, 1 ,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride and THF.

The solvent in the present invention may be preferably alcohol, more preferably 70% (v/v) ethanol.

The extraction method of the extract may include, but is not limited to, extracting with stirring for 24 to 72 hours, preferably for 48 hours, by adding the leaves of jasmine leaves to the solvent. If the extraction time is less than the above time, the active ingredient is not sufficiently extracted, and if the time is exceeded, there is a fear that impurities other than the active component are extracted together.

The extraction temperature is not limited, but may be performed at room temperature of 20°C to 25°C.

When included in the pharmaceutical composition, cosmetic composition, food composition, etc., the leaf extract of J. japonica may be included in a concentration of more than 0 to 300 μg/ml, preferably more than 0 to 100 μg/ml.

The anti-inflammatory composition of the present invention excessively stimulates the human immune system due to harmful stimuli such as inflammatory factors, such as TNF-α (tumor necrosis factor-alpha), IL-1 (interleukin- 1), IL-6 (interleukin-6), prostaglandin (prostaglandin), leukotriene (luecotriene), or inflammation that occurs in inflammatory diseases caused by inflammatory substances (inflammatory cytokines) such as nitric oxide (NO) can be reduced or suppressed.

Examples of such inflammatory diseases include dermatitis such as acne, atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, iritis, sore throat, tonsillitis, pneumonia, gastric ulcer, pancreatitis, gastritis, Crohn's disease, inflammatory bowel disease, colitis, hemorrhoids, gout, ankylosing spondylitis, lupus, fibromyalgia, psoriasis, rheumatoid arthritis, osteoarthritis, osteoporosis, hepatitis, cystitis, nephritis, Sjogren's syndrome and multiple sclerosis, and the like.

The food composition or health functional food composition comprising the leaf extract of the present invention may be prepared in the form of food or beverage in addition to powders, granules, pills, tablets, and capsules, and additives commonly used in the art during manufacturing. , may further include additional raw materials and the like. In addition, the preparation of the food composition may be prepared by a conventional method used in the art.

The cosmetic composition comprising the leaf extract of Cordyceps extract of the present invention may include ingredients commonly used in cosmetic compositions in addition to the extract of Cordyceps leaf extract, such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances. phosphorus adjuvants, carriers, and the like.

In addition, the cosmetic composition of the present invention can be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant- It may be formulated as cleansing containing, oil, powder foundation, emulsion foundation, wax foundation, and spray, but is not limited thereto, and may be prepared by a conventional method used in the art.

The pharmaceutical composition comprising the leaf extract of Cordyceps plant of the present invention may contain a pharmaceutically acceptable carrier in addition to the extract of the leaf extract, and these carriers are commonly used in formulation, and include lactose, dextrose, sucrose, Sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate ate, talc, magnesium stearate, and the like. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components. In addition, the preparation of the pharmaceutical composition may be prepared by a conventional method used in the art.

The pharmaceutical composition may be administered by an administration method such as oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebrovascular injection, but is not limited thereto.

The present invention relates to an anti-inflammatory composition comprising an extract of a chamomile leaf extract, and it is possible to provide a pharmaceutical composition, a cosmetic composition, and a food composition capable of effectively preventing and improving inflammation.

1 shows the evaluation of NO production inhibition and inflammatory mediator inhibition activity of Experimental Example 1.
Figure 2 shows the evaluation results of the MAPK activity inhibitory effect of Experimental Example 2.
3 shows the evaluation results of the HO-1 activity inhibitory effect of Experimental Example 3.

Hereinafter, the present invention will be described in detail by way of Examples and Experimental Examples.

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

<Example>

400 ml of 70% ethanol was added to 20 g of chamomile leaves, and the mixture was extracted by stirring at room temperature for 48 hours. After extraction, it was filtered with filter paper (No. 2, Whatman Co., Maidstone, England), concentrated with a reduced pressure reflux cooling device (EYELA, Tokyo, Japan) in a bath below 40℃, and freeze-dried, -80 until the experiment. Stored at ℃. The prepared example was dissolved in dimethyl sulfoxide (DMSO) and used in the experiment.

<Experiment method>

1. Experimental Materials

Medium (DMEM)/F-12 1:1 + 2.50 mM L-glutamine, + 15 mM HEPES buffer medium (DMEM/F-12) used for culturing mouse macrophages was purchased from Lonza (Hyclone, USA). . Lipopolysaccharide (LPS) was purchased from Sigma Aldrich (St.Louis. MO, USA) and antibodies Ikb-a, p65, p-ERK1/2, total-ERK1/2, p-p38, total for Western blot analysis. -p38 and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA).

2. RAW264.7 Cell Culture

The mouse macrophage RAW264.7 used in the experiment of the present invention was purchased from ATCC and used, and DMEM/F containing 100 U/ml penicillin and 100 μg/ml streptomycin supplemented with 10% fetal bovine serum (FBS). _{It was grown in a 37% incubator (Thermo, Germany) containing 5% CO 2} as a -12 medium and subcultured using Trypsin-EDTA solution when the cell density was more than 80% saturated and used for experiments. For the sample treatment, the extracts of oleracea were dissolved in dimethyl sulfoxide (DMSO), and DMSO was used as a control.

3. SDS-PAGE and Western Blot Analysis

In order to extract proteins from the cells treated with the P. oleracea samples, the cells were washed twice with 1x phosphate-buffered saline (PBS), and then a protease inhibitor cocktail (Sigma-Aldrich) was added to RIPA buffer (Boston Bio Products, Ashland, MA, USA). Co., St. Louis, MO, USA) and a dephosphoryase inhibitor cocktail (Sigma-Aldrich Co., St. Louis, MO, USA) were lysed to obtain the protein. After protein quantification by BCA protein analysis (Pierce Biotechnology Inc., Waltham, MA, USA), the same amount of protein was loaded into 10% SDS-acrylamide and transferred to an NC membrane (GE Helthcare life science, Germany) 5% It was blocked with skim milk powder at room temperature for 1 hour. After 1 hour, the primary antibody was dissolved in 5% skim milk powder, reacted at 4°C for 14 hours, and the membrane was 3 minutes in tris-buffered saline (TBS-T) containing 0.05% Tween-20. washed each time. After that, the secondary antibody was dissolved in 5% non-fat milk powder on the membrane and treated for 1 hour, washed 3 times for 5 minutes with TBS-T, and then ECL western blotting substrate (Amersham Biosciences Co., Little Chalfont, England) was used. Proteins were identified.

4. Reverse Transcription Polymerase Chain Reaction (RT-PCR)

Total RNA extraction was performed from sample-treated mouse macrophage RAW264.7 using RNeasy Mini kit (QIAGEN GmbH., Hilden, Germany), and cDNA was obtained by using 1 μg of total RNA with Verso cDNA kit (Thermo Fisher Scientific Inc.). , Waltham, MA, USA) was prepared using. PCR was performed using a PCR master mix kit (Promega Co., Madison, WI, USA), and the primers used are shown in Table 1. To check the amount of DNA made through PCR, it was loaded and then separated by electrophoresis after staining with Safe shine green on a 1% agarose gel. This was confirmed by the gel documentation system (Biorad, Chemidoc, MP imaging system, USA) and was used as an internal control including the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene.

Sequences of oligonucleotide primers used for RT-PCR gene name order iNOS Forward: 5′-GTGCTGCCTCTGGTCTTGCAAGC-3′ Reverse: 5′-AGGGGCAGGCTGGGAATTCG-3′ COX-2 Forward: 5'-GGAGAGACTATCAAGATAGTGATC-3' Reverse: 5'-ATGGTCAGTAGACTTTTACAGCTC-3' TNF-α Forward: 5'-TACTGAACTTCGGGGTGATTGGTCC-3' Reverse: 5'-CAGCCTTGTCCCTTGAAGAGAACC-3' IL-1β Forward: 5'-GAAGCTGTGGCAGCTACCTATGTCT-3' Reverse: 5'-CTCTGCTTGTGAGGTGCTGATGTAC-3' IL-6 Forward: 5′-GAGGATACCACTCCCAACAG-3′ Reverse: 5′-TTCACAGAGGATACCACTCC-3′ GAPDH Forward: 5'-CAGGAGCGAGACCCCACTAACAT-3' Reverse: 5'-GTCAGATCCACGACGGACACATT-3'

All results were expressed as mean ± standard deviation after repeated measurements three times, and the significance between treatments was verified by Student's t-test and was determined to be statistically significant when the p-value was less than 0.05 (Microsoft Exel 2010, Microsoft, Redmond, WA, USA).

<Experimental Example 1> Evaluation of NO (nitric oxide) production inhibition and inflammatory mediator inhibition activity

An experiment was conducted to evaluate the inhibitory effect on NO production through inhibition of iNOS, COX-2, TNF-α, IL-1β and IL-6 expression in LPS-stimulated RAW264.7 cells. NO was measured by changing the method of Namkoong et al. (Namkoong et al., 2015), and the experimental results are shown in FIG. 1 . RAW264.7 cells were treated with LPS (1 μg/ml) for 18 hours after treatment of the leaves of jasmine leaf by concentration for 6 hours. We investigated the effect of serrata leaf on the production of inflammatory mediators induced by LPS stimulation. As a result of examining whether or not increased NO production was inhibited by LPS stimulation in RAW264.7 cells, the amount of NO secreted increased by LPS stimulation was significantly decreased with increasing concentration of the leaf extract of A. japonica (FIG. 1A). MTT assay was carried out to determine the effect of the leaf of jasmine leaf on the viability (%) of RAW264.7 cells. As a result, it was found that no cytotoxicity was observed when the leaves of the jasmine leaf were treated by concentration (FIG. 1B). As a result of RT-PCR analysis on the effect of the leaf extract of C. japonica on the expression of COX-2, which is involved in NO production, the gene expression of IL-1β, IL-6, COX-2, TNF-α, iNOS, and GAPDH. As a result, expression was inhibited in a concentration-dependent manner (FIG. 1C). In addition, it was found that the expression of the gene increased by LPS stimulation was reduced in a concentration-dependent manner through the extract pretreatment (FIG. 1D).

<Experimental Example 2> MAPK activity inhibitory effect evaluation

The activity of MAPK was evaluated in order to confirm the effect of the leaf extract of the present invention on the intracellular signal transduction system related to the antioxidant and anti-inflammatory effects. The phosphorylation of ERK1/2 and p38, which are MAPK components, was investigated. Effect of Examples on MAPK signaling activation in RAW264.7 cells induced by LPS. RAW264.7 cells were pretreated with Example for 6 hours and then co-treated with LPS (1 μg/mL) for 40 minutes. For Western blot analysis, cell lysates were subjected to SDS-PAGE and Western blot was performed using antibodies against p-ERK1/2, p-p38 and total p-38, total ERK1/2 as internal control. was used. The experimental results are shown in FIG. 3 .

It was confirmed that the LPS-induced ERK1/2 phosphorylation in RAW264.7 cells was rapidly inhibited in a concentration-dependent manner by the extract of the leaf extract of C. japonica of the present invention.

<Experimental Example 3> HO-1 activity inhibitory effect evaluation

In order to determine whether the antioxidant effect of the leaf extracts of jasmine leaf extract is related to the activation of the HO-1 signal transduction pathway, the expression of HO-1 in RAW264.7 cells was evaluated.

As a result, the expression of HO-1 was gradually increased according to the treatment concentration of J. japonica leaf (FIG. 3A). And HO-1 expression was shown at 10 hours after treatment with the leaf extract of jasmine jasmine ( FIG. 3B ). (C) RAW264.7 cells were pre-treated with SB203580, a p38 inhibitor, and PD98059, an ERK1/2 inhibitor, for 2 hours, and then treated with an extract of a chamomile leaf extract for 10 hours to confirm the results. It was confirmed that the activation was inhibited when treated with the leaf extract (FIG. 3C).

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Claims (7)

An anti-inflammatory composition comprising a leaf extract of jasmine leaf. [Claim 2] The composition for anti-inflammatory according to claim 1, wherein the extract of the leaf extract of J. japonica is extracted with 70% (v/v) ethanol. [Claim 2] The composition for anti-inflammatory according to claim 1, wherein the extract of the leaf extract of Japonica is extracted for 48 hours. According to claim 1, wherein the anti-inflammatory is dermatitis, atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, iritis, sore throat, tonsillitis, pneumonia, gastric ulcer, pancreatitis, gastritis, Crohn's disease, inflammatory bowel disease, colitis, hemorrhoids, An anti-inflammatory composition for reducing or inhibiting inflammation of an inflammatory disease selected from gout, ankylosing spondylitis, lupus, fibromyalgia, psoriasis, rheumatoid arthritis, osteoarthritis, osteoporosis, hepatitis, cystitis, nephritis, Sjogren's syndrome and multiple sclerosis . [Claim 2] The composition for anti-inflammatory according to claim 1, characterized in that the concentration of the leaf extract of J. japonica is greater than 0 to 100 μg/ml. The anti-inflammatory composition of claim 1 is an anti-inflammatory composition, characterized in that any one selected from a pharmaceutical composition, a cosmetic composition, and a food composition. A method for preventing, improving or treating inflammation using the anti-inflammatory composition of any one of claims 1 to 5.

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