KR20060064783A - Anti-inflammatory agent containing extract from abeliophyllum distichum - Google Patents

Abstract

 The present invention relates to an anti-inflammatory agent comprising the extract of S. aureus. In particular, the present invention relates to a safe extract of mysterious wood, which effectively inhibits the inflammatory response and has no cytotoxicity, and the extract of the mysterious wood can be usefully used for the treatment and prevention of inflammatory diseases.

Striations, anti-inflammatory

Description

Anti-inflammatory agent containing extract of S. aureus {ANTI-INFLAMMATORY AGENT CONTAINING EXTRACT FROM ABELIOPHYLLUM DISTICHUM}

Figure 1 shows the inhibitory activity of COX-2 expression of the extract of the leaves of the crypt (Example 2) and the fruit extract (Example 6).

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to an anti-inflammatory agent comprising the extract of the bark, more particularly to a safe anti-inflammatory agent that effectively inhibits the inflammatory response and is free of cytotoxicity.

[Private Technology]

Inflammation is a locally occurring immune response when tissues are damaged or destroyed by harmful substances or organisms from outside, which are useful defense mechanisms to protect the living body and remove products produced by tissue damage. In normal cases, the living body restores its normal structure and function by neutralizing or eliminating the pathogenic factors through the inflammatory response and regenerating the upper tissue, but also progresses to a disease state such as chronic inflammation. In addition to being able to observe the inflammatory response in almost all diseases, it is known that enzymes related to the inflammatory response play an important role in carcinogenesis.

The anti-inflammatory effects of most nonsteroidal antiinflammatory drugs (NSAIDs) are mediated by inhibition of cyclooxygenase (COX) enzyme activity (Vane et al., Annu. Rev. Pharmacol. Toxicol., 38, 97, 1998). . COX is the main enzyme involved in the biosynthesis of prostaglandin present in vivo (Smith et al., J. Biol. Chem., 271, 33157, 1996). Two isomeric enzymes, COX-1 and COX-2, exist. COX-1 is constantly present in tissues such as the stomach and kidneys, and is involved in maintaining normal homeostasis, whereas COX-2 is involved in mitogen or cytokines during inflammation and other immune responses. It is an enzyme that is expressed transiently and quickly within. Nonsteroidal anti-inflammatory drugs used in the treatment of chronic inflammatory diseases such as acute or rheumatoid arthritis are known to exhibit side effects such as gastrointestinal disorders by inhibiting COX-2 enzymes as well as COX-1 enzymes (Masferrer Et al., Proc. Natl. Acad. Sci. USA, 91, 3228, 1994; Seibert et al., Proc. Natl. Acad. Sci. USA, 91, 12013, 1994). Therefore, in order to increase the efficacy of the anti-inflammatory drugs and minimize the side effects, it is necessary to find a substance that selectively inhibits only the COX-2 enzyme and develop it as an anti-inflammatory drug.

In addition, COX-2 inhibitors are expected to play an important role in the prevention of cancer as well as in the treatment of inflammation. Reportedly, the induction of COX-2 is increased in cancer cells and malignant tumor tissues, resulting in much higher amounts of prostaglandin compared to normal cells, and most of the increase in the amount of prostaglandin in tumor tissues is mainly due to COX-2 expression. Increased by (Kargman et al. Cancer Res., 55, 2556, 1995; Ristimaki et al. Cancer Res., 57, 1276, 1997). Prostaglandins, such as prostaglandin E2 (PGE2), not only promote blood vessel production, promote cell proliferation, but also suppress immune capacity, so their overproduction creates a good environment for cancer cell growth. Give it. Moreover, excessive expression of COX-2 inhibits apoptosis and increases the metastatic capacity of cancer cells.

In this regard, recent studies have shown a 40-50% reduction in the risk of colon cancer in people who have long used NSAIDs (nonsteroidal anti-inflammatory drugs), and in patients with familial adenomatous polyposis (FAP). The development of colorectal polyposis into colorectal cancer was observed to be prevented by NSAIDs-based drugs (Peleg et al., Arch. Intern. Med., 154, 394, 1994). In addition, COX-2 expression and activity were always preceded by APC mice, a colon cancer experimental model, in the stage of modification and loss of APC gene, which is a stage of colon cancer (Prescott et al., Cell, 87, 783, 1996). In addition, various studies have shown that COX-2 has a significant effect on the developmental stage of colon cancer, and COX-2 protein is expressed more in human stomach and breast cancer tissues than in normal tissues, and COX inhibitors are expressed in human breast cancer cells. Has been reported to significantly reduce the incidence of cancer and animal model-induced cancer by COX-2 inhibitors (Noguchi et al., Fatty Acids, 53, 325, 1995; Thompson et al., Cancer Res., 57). , 267, 1997). Therefore, COX-2 selective inhibitors are not only the main targets for the development of new anti-inflammatory drugs, but also the potential for cancer prevention.

In order to solve the problems of the prior art, an object of the present invention is to provide an anti-inflammatory agent containing a plant extract and does not cause side effects.

It is another object of the present invention to provide an anti-inflammatory agent that can inhibit the expression of COX-2.

In order to achieve the above object, the present invention provides an anti-inflammatory agent comprising the extract of M. alba as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention relates to a extract of the bark wood to significantly inhibit the inflammatory response and an anti-inflammatory agent comprising the same.

The extract may be prepared according to the conventional method for producing a plant extract. That is, the extract may be prepared by extracting and / or fractionating a part or all of the stem wood with water or an organic solvent, such as leaves, branches, roots, fruits, stems, flowers, bark, etc. It is a method of obtaining an extract after adding a solvent. The solvent may be water, a low alcohol having 1 to 5 carbon atoms or alcohol dilution water, and the low alcohol may be ethanol, methanol, butanol, propanol, and isopropanol, but is not limited thereto. Alcohol dilution water may be a dilution of water to 50 to 99% by volume of alcohol. In addition, the extract extracted with the solvent may be further subjected to the fractionation process with a solvent selected from the group consisting of hexane, methylene chloride, acetone, ethyl acetate, chloroform and mixtures thereof. Two or more solvents may be used in the fractionation, and each solvent extract may be prepared according to the polarity of the solvent. Extract preparation temperature may be 4 to 120 ℃, but is not limited thereto. The extraction time is not particularly limited, but may be 10 minutes to 30 days, and a conventional extraction device, an ultrasonic grinding extractor, or a fractionator may be used. The prepared extract can then be filtered under reduced pressure or / and lyophilized to remove the solvent.

In one example, the extract of the camellias is extracted by adding 0.1 to 5 L of methanol per 100 g of leaves or berries to prepare a methanol extract. The methanol extract is then added to hexane or methylene chloride to obtain a fraction for each solvent to prepare a hexane extract or methylene chloride. Thereafter, ethyl acetate and butanol are sequentially added to the residue to fractionate to prepare ethyl acetate extract and butanol extract, respectively. The water fraction not extracted from the solvent is prepared as a water extract. When fractionation using the solvent, it is carried out by adding a solvent 1: 0.1 to 10 volume ratio with respect to the extract.

Among the extracts of the extract of the present invention, the extracts of the extracts of the extracts of the extracts of T. alba and M. everzilla fruit inhibit about 80% of edema formation by TPA (12- O- tetra-decanoylphorbol-13-acetate), which is an inflammatory substance. , TPA significantly inhibits the expression of COX-2 (cyclooxygenase-2) with increased expression. It was also confirmed that the extract of M. julibrissin was a safe sample without toxicity to normal cells.

The present invention provides an anti-inflammatory agent containing the extract of the aspen. Preferably, the extract of the camellias is preferably the hexane extract of the camellias, the methanol extract of the camellias, or a mixture thereof. In the case of the mixture, the mixing ratio of each extract may be 0.1 to 1: 0.1 to 1 weight ratio of the extract of the biloba leaf hexane extract: the bilberry fruit methanol.

The anti-inflammatory agent of the present invention may include the active ingredient alone, and may further include a pharmaceutically acceptable carrier or excipient according to the formulation, method of use and purpose of use. When provided in a mixture, the active ingredient may be included in the anti-inflammatory agent 0.1 to 99.9% by weight, it is usually included in an amount of 0.001 to 50% by weight. Anti-inflammatory drugs can be used for the prevention and treatment of various chronic inflammatory diseases such as rheumatoid arthritis, lupus, multiple sclerosis, various acute inflammatory diseases such as sepsis, shock, rejection of organ transplantation, ophthalmic diseases, bronchitis, or inflammatory bowel disease Do.

Examples of the carrier or excipient include water, dextrin, calcium carbonate, lactose, propylene glycol, liquid paraffin, saline, dextrose, sucrose, sorbitol, mannitol, ziitol, erythritol, maltitol, starch, gelatin, calcium phosphate, Calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils, but are not limited thereto. Two or more carriers or excipients may be used. In addition, when the anti-inflammatory agent is formulated, conventional fillers, extenders, binders, disintegrants, surfactants, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers or preservatives may be further included.

In addition, the anti-inflammatory agent of the present invention may further include a compound or a plant extract having a known anti-inflammatory activity in addition to the extract of the wisteria, and may be included in an amount of 5 to 20 parts by weight based on 100 parts by weight of the extract.

The formulation of the anti-inflammatory agent may be in a preferred form depending on the method of use, and may be formulated by employing methods known in the art, in particular to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. Examples of specific formulations include warnings, granules, lotions, linings, limonades, powders, syrups, ointments, liquids, aerosols, EXTRACTS, elixirs, ointments, liquid extracts, emulsions, Suspensions, premises, acupuncture, eye drops, tablets, suppositories, injections, spirits, capsules, creams, pills, soft or hard gelatin capsules.

Anti-inflammatory agents according to the present invention can be used orally or parenterally, for example, can be used through the dermis, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, epidural and oral route, the dosage thereof, the method of administration , The age, sex and weight of the recipient, and the severity of the disease, it is good to determine. In one embodiment, the anti-inflammatory agent of the present invention can be administered one or more times 0.1 to 100 mg / kg (body weight) per day based on the active ingredient. However, the above dosage is only one example to illustrate and is not limited to the above range.

The anti-inflammatory agent of the present invention can be used as a medicament or cosmetic, anti-inflammatory agent may be included in 0.001 to 50% by weight in the pharmaceutical or cosmetic composition, but is not limited thereto. The cosmetic composition may be provided for the use of basic cosmetics, makeup cosmetics, body cosmetics, hair cosmetics, scalp cosmetics, shaving cosmetics or oral cosmetics. Examples of the basic cosmetics include creams, lotions, packs, massage creams, emulsions, etc. The makeup cosmetics include foundation, makeup base, lipstick, eyeshadow, eyeliner, mascara, eyebrow pencil, and the like. Examples include soaps, liquid cleansers, baths, sunscreen creams and sun oils. Hair care products include shampoos, rinses, hair treatments, hair mousses, hair liquids, pomades, hair colorants, hair bleaches, and colorants. There are rinses, hair tonics and scalp treatments as scalp cosmetics, aftershave and shaving creams as shaving cosmetics, and toothpaste and mouth washes as oral cosmetics.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and the present invention is not limited to the following examples.

Example 1-5: Preparation of Wisteria Leaf Extract

After dipping 200 g of the leaves of Mt. biloba in 1.5 L of methanol, the mixture was refluxed three times for 4 hours. The extracts were combined and concentrated under reduced pressure to obtain 35 g of methanol extract (Example 1).

To 30.5 g of the methanol extract, a total of 200 ml of n-hexane: methanol: water was added in a mixing ratio of 10: 1: 9, followed by fractionation to obtain a hexane fraction, which was concentrated under reduced pressure to obtain 5.5 g of hexane fraction (Example 2). Obtained. Thereafter, ethyl acetate and normal-butanol were added to the residue three times, and each fraction was collected and concentrated under reduced pressure to obtain 4.2 g of ethyl acetate fraction (Example 3) and 11.3 g of normal-butanol fraction (Example 4). 9.2 g of each of the remaining water fractions (Example 5) were obtained.

Example 6-9 Preparation of Wisteria Fruit Extract

55 g of the barberry fruit was immersed in 500 ml of methanol, and then extracted three times by refluxing for a total of 4 hours. The extracts were combined and concentrated under reduced pressure to give 11.5 g of methanol extract (Example 6).

200 ml of water was added to 10.5 g of the methanol extract, and then 200 ml of methylene chloride was added and extracted three times. The extract was concentrated under reduced pressure to give 4.2 g of methylene chloride fraction (Example 7). Thereafter, 200 ml of ethyl acetate was added to the residue three times, and the mixture was concentrated under reduced pressure to obtain 0.9 g of ethyl acetate fraction (Example 8) and 4.9 g of the remaining water fraction (Example 9), respectively.

Experimental Example 1: Anti-inflammatory effect test of the extract

TPA (12- O- tetradecanoylphorbol-13-acetate) is not only a cancer promoter but also a drug that induces an inflammatory response. When treated, it significantly increases the expression of COX-2.

Edema was induced by topical application of 5 nmol of TPA dissolved in acetone in the right ear of the mouse, and only solvent was applied topically in the left ear. 30 min prior to topical application of TPA, suspensions of Example 2 and 6 were suspended in acetone / DMSO (1: 1) mixed solvent at 1.0 mg or 5.0 mg each and topically applied to the right ear and only solvent to the left ear. Applied. After 4 hours after the TPA was applied, the mice were sacrificed and the right and left ears were cut out with a 6 mm diameter punch and weighed. The degree of edema generated is obtained by subtracting the weight of the left ear from the weight of the right ear, and substituting this into Equation 1 below to determine the edema inhibition rate.

(Calculation 1)

Edema inhibition rate (%) = 100-(degree of edema in the bark extract group / edema of TPA treated group) x 100

The experimental results were expressed as the mean value + SE, and statistical analysis was performed using the Student t-test. P value of less than 0.05 was evaluated as significant.

The results are shown in Table 1 below.

process density Edema degree p -value % Inhibition Negative Control                                                  Positive control group                                                  Example 2 Treatment Group                                                                                                    Example 6 Treatment Group                                                TPA 5 nmol / 50 μl 1.0 mg / 50 μl 5.0 mg / 50 μl 1.0 mg / 50 μl 5.0 mg / 50 μl 0 8.67 + 1.29 4.10 + 0.31 3.07 + 0.32 3.50 + 0.71 1.43 + 0.27 0.0267 0.0138 0.0251 0.0055 53 65 60 84

From the results of Table 1, it was confirmed that the extracts of the bark of Examples 2 and 6 of the present invention can significantly inhibit the induction of inflammation and can be used as an anti-inflammatory agent.

Experimental Example 2: Toxicity Test on Normal Cells

The toxic extracts of the hexane extract of Example 2 and the extract of the extract of the fruit of M. alba of Example 6 were confirmed using gutwa cells (normal small intestinal mucosa cells).

Intestinal crypt-derived cells, IEC-6, were used in the American Type Culture Collection, Rockville, MD, USA. DMEM / F12 (Dulbecco's modified Eagle's medium / Nutrient Mixture Ham's F12), FBS (fetal bovine serum), trypsin-EDTA, penicillin-streptomycin and selenium used in cell culture were purchased from Gibco / BRL (Gaithersburg, MD, USA). . In addition, 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT), bovine serum albumin (BSA), and transferrin are obtained from Sigma Chemical Co., St. Louis. , MO, USA).

IEC-6 cells were cultured in a 37 ° C. wet CO₂ incubator (5% CO₂ / 95% air) using DMEM / F12 medium, 10% FBS, 100 units / ml penicillin, 100 μg / in DMEM / F12 medium. Ml of streptomycin and 10 μg / ml insulin were added and used. Cells were cultured to 80% confluent, washed with monolayers of cells with PBS (pH 7.4), and subcultured with 0.25% trypsin and 2.65 mM EDTA. Medium was changed every two days.

Colorectal cancer cell proliferation inhibitory effect of the extract of S. aureus of Examples 1 to 9 was measured. IEC-6 cells were diluted in medium containing 10% FBS and aliquoted into 24 well plates at a density of 50,000 cells / well. After 24 hours, the cells were replaced with medium (DMEM / F12, transferrin 5 μg / ml, selenium 5 ng / ml, BSA 0.1 mg / ml, and FBS) for 24 hours, followed by 0, 12.5, 25 and The cultures were exchanged with the same medium contained at 50 μg / ml. The medium containing the extract of Myrrhizium was changed every two days. At this time, the extract of the crypts was used in DMSO, and the number of cells over time after the treatment of the crypts was measured by MTT analysis. The MTT assay is a test that utilizes the ability of intracellular mitochondria to reduce the yellow water-soluble MTT tetrazolium to a blue-violet water-insoluble MTT formazan crystal by dehydrogenase action. The used DMSO was added as 0.1% in the medium used as a solvent of the extract of the crypt tree, but does not significantly affect the cell culture. For MTT analysis, after removing the cell culture medium, 1 mL of DMEM / F12 medium containing 1 mg / mL of MTT was treated per 1 mL, and incubated in a 37 ° C. wet CO 2 incubator for 3 hours. Thereafter, the medium was removed, and then 0.5 ml isopropanol was added to dissolve the formazan crystal, and the cell viability was confirmed by measuring absorbance at 570 nm at a micro plate reader (BIO-RAD). The results are shown in Table 2 below.

Camphor Extract Processing time (hr) 0 μg / mL 12.5 μg / mL 25 μg / mL 50 μg / mL Example 2 0 0.318 + 0.003 0.318 + 0.003 0.318 + 0.003 0.318 + 0.003 24 0.516 + 0.008 0.478 + 0.009 0.461 + 0.006 0.450 + 0.002 48 0.752 + 0.011 0.742 + 0.032 0.720 + 0.021 0.712 + 0.012 72 0.927 + 0.029 1.054 + 0.038 1.026 + 0.019 0.976 + 0.024 Example 6 0 0.318 + 0.003 0.318 + 0.003 0.318 + 0.003 0.318 + 0.003 24 0.501 + 0.006 0.490 + 0.005 0.471 + 0.003 0.486 + 0.006 48 0.740 + 0.012 0.747 + 0.005 0.732 + 0.020 0.713 + 0.026 72 0.961 + 0.009 0.948 + 0.001 1.001 + 0.022 0.938 + 0.050

As shown in Table 2, the extracts of the bark of Example 2 and 6 did not inhibit the proliferation of normal small intestine cells, it was confirmed that there is no cytotoxicity.

Experimental Example 3: Verification of the inhibitory effect on COX-2

Five-week-old female mice were purchased from Polars International (Seoul, South Korea) and adapted to light and dark environments every 12 hours. Mice at 6 to 7 weeks of age were used for the experiment.

Two days after the hairs of the mice and the like were removed, 1 and 5 mg of the extract of Example 2 and the fruit extract of Example 6 were dissolved in 200 μl of solvent (acetone: DMSO = 1: 1), respectively, and then the skin of the mice was removed. Topical application. After 30 minutes TPA (10 nmol / 200 μl) was dissolved in acetone and applied topically and mice were sacrificed 4 hours later. Only the solvent was applied topically to the control group. The skin of the mice was cut out to remove fat tissue from the ice, liquid nitrogen was added and powdered using pestle. Protein lysis solution (150 mM NaCl, 0.5% Triton X-100, 50 mM Tris-HCl, pH 7.4, 20 mM EGTA, 1 mM DTT, 1 mM Na ₃ VO ₄ , protease inhibitor cocktail tablet) It was added, left on ice for 3 hours, and then centrifuged (12000 rpm, 10 min) to obtain a supernatant. Supernatant proteins were quantified using BIO-Rad protein assay solution.

The supernatant was then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the separated proteins were transferred to PVDF membranes. PVDP membranes were soaked in 5% skim milk powder dissolved in phosphate buffer for 3 hours, and then reacted with primary antibodies against COX-1, COX-2 and β-actin diluted 1: 1000 at 4 ° C for 12 hours. The secondary antibody reacted specifically with the antibody. At the end of each step, 0.1% PBST (phosphate buffered salin Tween 20) secondary antibody was attached, reacted with ECL (Enhanced chemiluminesence) solution, and exposed to x-ray film to change COX-1, COX-2 and β-actin. It was confirmed.

Figure 1 shows the COX-2 expression inhibitory activity of the leaves extract (Example 2) and fruit extract (Example 6), the TPA-only group is significantly more expression of COX-2 than the control group only solvent treatment. Increased. However, the pretreatment with 1 and 5 mg of hexane extracts from the leaves of M. alba and M. alba extracts from the extracts of M. julibrissin prior to topical application of TPA decreased COX-2 expression by TPA. No effect was observed. All. Therefore, it can be seen that the extract of the myrtle tree has anti-inflammatory effect by inhibiting the activity of COX-2.

As described above, the extracts of the mysterious tree of the present invention are safe samples without cytotoxicity that effectively inhibit the inflammatory response, and can be usefully used for the treatment and prevention of inflammatory diseases.

Claims (3)

Anti-inflammatory agent containing the extract of S. aureus as an active ingredient. The anti-inflammatory agent according to claim 1, wherein the extract of the camellia japonica is extracted from the leaves or berries of the camellia japonica with water or an alcohol having 1 to 5 carbon atoms. The method of claim 2, wherein the extract of the camellia sinensis is fractionated with one or more solvents selected from the group consisting of ethyl acetate, hexane, acetone, methylene chloride, chloroform, and diluting water thereof. It is an anti-inflammatory agent.

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