KR102408812B1 - Composition for anti inflammation comprising extract of Berchemia berchemiifolia leaf - Google Patents

Abstract

The present invention relates to an anti-inflammatory composition, and more particularly, to an anti-inflammatory composition comprising an extract of fenugreek leaves. The present invention can provide a pharmaceutical composition, a cosmetic composition, and a food composition that can effectively prevent and improve inflammation.

Description

Composition for anti-inflammatory comprising extract of Berchemia berchemiifolia leaf

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

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. . Among them, Berchemia berchemiifolia is called Metdaesari, meaning bamboo that grows in mountains in Gyeongsangbuk-do and Chungcheongbuk-do. The genetic diversity is not very high, and the number of individuals in Japan is not very high, so it is a plant that needs to be evaluated as an endangered plant. there is no situation. On the other hand, in the prior art, although it has been disclosed for the prevention and treatment of psychiatric disorders, the use of the extract of mulberry tree is known, but little is known about the use except 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 with well-being trends and growing interest in health this is being requested

Laid-Open Patent Publication No. 10-2017-0061469

In order to solve the above problems, the present invention is to provide a natural anti-inflammatory composition 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 Mangae tree.

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 the extract of Mandarin tree. Specifically, it may be extracted from the leaves, branches, fruits, etc. of the Manganese tree, but is preferably a leaf extract of the Manganese tree.

A polar solvent or a non-polar solvent may be used for the extract of the Mango leaf extract. 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 is not limited thereto, but may be extracted with stirring for 24 to 72 hours, preferably 48 hours, by putting the leaves of fenugreek in the solvent. If the extraction is less than the time, the active ingredient is not sufficiently extracted, and if the time is exceeded, there is a fear that impurities other than the active ingredient 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 mango leaf extract may be included in a concentration of more than 0 to 300 μg/ml, preferably more than 0 to 100 μg/ml.

TNF-α, IL-1, IL-1β, IL-6, iNOS secreted from immune cells such as macrophages by excessively stimulating the human immune system due to harmful stimuli such as inflammatory factors, etc. , COX-2, prostaglandin, leukotriene, or nitric oxide (NO) that can reduce or inhibit inflammation that occurs in inflammatory diseases caused by pro-inflammatory substances (inflammatory cytokines) means that

Examples of inflammatory diseases as described above 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 containing the leaf extract of the Mango tree 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 industry 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 Mango tree of the present invention may include ingredients commonly used in cosmetic compositions in addition to the extract of Mango tree leaf, for example, 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 with cleansing, 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 Mango tree of the present invention may include a pharmaceutically acceptable carrier in addition to the leaf extract of Mango tree. Such 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 fenugreek leaves, and it is possible to provide a pharmaceutical composition, a cosmetic composition, and a food composition that can effectively prevent and improve 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 NF-κB activity inhibitory effect of Experimental Example 2.
3 shows the evaluation results of the MAPK 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 Manganese 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 the experiment. For the sample treatment, the extracts of Mango tree were dissolved in dimethyl sulfoxide (DMSO), and DMSO was used as a control.

3. SDS-PAGE and Western Blot Analysis

After washing twice with 1x phosphate-buffered saline (PBS) to extract proteins from the cells treated with the mulberry tree sample, a protease inhibitor (Sigma-Aldrich Co) was added to RIPA buffer (Boston Bio Products, Ashland, MA, USA). ., St. Louis, MO, USA) and a dephosphorylation enzyme inhibitor (Sigma-Aldrich Co., St. Louis, MO, USA) were lysed to obtain a 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 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 treated with tris-buffered saline (TBS-T) containing 0.05% Tween-20 for 3 minutes for 5 minutes. washed each time. After that, the secondary antibody was dissolved in 5% nonfat dry milk 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 to treat the membrane. 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 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 three repeated measurements, 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 carried out to evaluate the inhibitory effect of the leaf extract of cyanobacteria 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).

Mouse macrophages RAW264.7 were aliquoted to 5x10 ⁵ /well in a 12-well plate and cultured for 24 hours. After 24 hours, the extract was treated with 0, 12.5, 25, 50, and 100 μg/ml concentrations and cultured for 6 hours. After 6 hours, LPS was treated at a concentration of 1 μg/ml and cultured for 18 hours. After 18 hours of treatment, NO production was measured using media and Griess reagent. RAW264.7 cells were treated with Examples at the indicated concentrations for 24 hours. Cell viability was determined using the MTT assay system and expressed as cell viability (%).

For RT-PCR, RAW264.7 cells were pretreated with Examples at the indicated concentrations for 6 hours, and then co-treated with LPS (1 μg/mL) for an additional 18 hours.

NO was measured using the Griess reagent system (Promoga), total RNA was isolated, and RT-PCR was performed for iNOS, COX-2, TNF-α, IL-1β and IL-6. Values given are mean ± SD (n=3). The experimental results are shown in FIG. 1 .

As a result of the experiment, as a result of measuring the NO production inhibitory effect of the leaf extract of Manganese tree, when RAW264.7 cells were treated with LPS, the production of NO was inhibited in a concentration-dependent manner (FIG. 1A). MTT analysis was performed to determine the effect on the viability of RAW264.7 cells on the leaves of the magnolia tree. As a result, it was found that cytotoxicity was not observed when the extracts of Manganese leaf extract were treated by concentration (FIG. 1B). As a result of confirming the gene expression of iNOS, COX-2, TNF-α, IL-1β, IL-6, and GAPDH through RT-PCR analysis, the expression was suppressed in a concentration-dependent manner. This result is judged to show anti-inflammatory activity by inhibiting the expression of chronic inflammatory mediator genes of the mulberry extract.

<Experimental Example 2> Evaluation of NF-κB activity inhibitory effect

In order to evaluate the effect of the present invention on the activation of NF-κB signaling, the inhibition of IκB-α degradation, phosphorylation, and nuclear metastasis of p65 was investigated by inducing LPS in RAW264.7 cells. The specific experimental method is as follows. did

RAW264.7 cells were pre-treated with Example for 6 hours and then co-treated with LPS (1 μg/ml) for 40 minutes (FIG. 2A). RAW264.7 cells were pretreated with Example for 6 hours and then co-treated with LPS (1 μg/ml) for 40 minutes. After treatment, cytosol and nuclei were prepared. For Western blot analysis, cell lysates were subjected to SDS-PAGE and Western blot was performed using antibodies against IκB-α and p65. Actin was used as an internal control (FIG. 2B). The experimental results are shown in FIG. 2 .

As a result of the experiment, IκB-α degradation was induced by LPS in the cells not treated with the leaf extract, but the degradation of IκB-α was significantly inhibited in the cells treated with the leaf extract, and phosphorylation of IκB-α was inhibited. It was confirmed that the concentration-dependent inhibition by the tree leaf extract. In addition, LPS-induced nuclear metastasis of p65 was also decreased in a concentration-dependent manner. The present results show that the leaf extract of Mandarinus tree exhibits anti-inflammatory activity through inhibition of NF-κB signaling.

<Experimental Example 3> MAPK activity inhibitory effect evaluation

In order to evaluate the effect of the leaf extract of the present invention on MAPK activity, phosphorylation of ERK1/2 and p38, which are MAPK constituent factors, was investigated. RAW264.7 cells were pre-treated 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 ERK1/2 phosphorylation was rapidly inhibited in a concentration-dependent manner by the ERK1/2 phosphorylation by LPS in RAW264.7 cells, but it was confirmed that there was no inhibitory activity on the phosphorylation of p38. did The present results show that the extract of Mandarinus tree exhibits anti-inflammatory activity by inhibiting the activation of ERK1/2 in MAPK.

<110> National Institute of Forest Science <120> Composition for anti-inflammation comprising extract of Berchemia berchemiifolia leaf <130> 19-11534 <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer for iNOS <400> 1 gtgctgcctc tggtcttgca agc 23 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for iNOS <400> 2 aggggcaggc tgggaattcg 20 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer for COX-2 <400> 3 ggagagacta tcaagatagt gatc 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for COX-2 <400> 4 atggtcagta gacttttaca gctc 24 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer for TNF-alpha <400> 5 tactgaactt cggggtgatt ggtcc 25 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for TNF-alpha <400> 6 cagccttgtc ccttgaagag aacc 24 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer for IL-1beta <400> 7 gaagctgtgg cagctaccta tgtct 25 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for IL-1beta <400> 8 ctctgcttgt gaggtgctga tgtac 25 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for IL-6 <400> 9 gaggatacca ctcccaacag 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for IL-6 <400> 10 ttcacagagg ataccactcc 20 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer for GAPDH <400> 11 caggagcgag accccactaa cat 23 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for GAPDH <400> 12 gtcagatcca cgacggacac att 23

Claims (7)

In the anti-inflammatory composition comprising the leaf extract,
The Manganese leaf extract was extracted with 70% (v / v) ethanol,
The anti-inflammatory is dermatitis, atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, iritis, pharyngitis, 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 anti-inflammatory composition for reducing or inhibiting inflammation of an inflammatory disease that can be selected from multiple sclerosis. delete [Claim 2] The composition for anti-inflammatory according to claim 1, wherein the leaf extract of Manganese tree is extracted for 48 hours. delete [Claim 2] The composition for anti-inflammatory according to claim 1, wherein the extract of Mango leaf extract is in a concentration of 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. delete

Images