KR101426873B1 - Pharmaceutical composition for prevention and treatment of inflammatory diseases comprising extract or fractions of Ardisia tinctoria Pit. as an active ingredient - Google Patents

Abstract

The present invention relates to a method for the treatment of Aldisia tinctoria tinctoria The present invention relates to a composition for preventing and treating inflammatory diseases, which comprises extracts or fractions thereof. More particularly, the present invention relates to a composition for inhibiting the production of nitric oxide, which is rapidly increased by inflammation induced by the aldicia tincturea extract or its fractions . The extracts of Aldicia tincturea significantly reduced the levels of PGE ₂ and IL-6 (interleukin-6) and IL-1beta cytokines in a concentration-dependent manner and inhibited the expression of iNOS and COX-2 genes and proteins And inhibit nuclear translocation of p65 protein and phosphorylation of signal transducer. Also, it was confirmed that the aldicia tincturea extract and the ethyl acetate fraction had an inflammation-inhibiting effect in the carrageenan-induced mouse foot model. Therefore, the above-mentioned aldicia tincture extract or its fractions can be effectively used as an active ingredient of compositions for preventing or treating inflammation-related diseases, compositions for external application for skin, cosmetic compositions and compositions for health food.

Description

[0001] The present invention relates to a pharmaceutical composition for the prevention and treatment of inflammatory diseases containing an extract of Aldicia tincturea or a fraction thereof as an active ingredient. as an active ingredient}

The present invention relates to a method for the treatment of Aldisia tinctoria tinctoria The present invention relates to a pharmaceutical composition for preventing and treating inflammatory diseases using an extract or a fraction thereof.

Inflammation refers to the pathological condition of abscesses formed by the infiltration of external infectious agents (bacteria, fungi, viruses, various allergens). Specifically, when external bacteria invade a specific tissue and proliferate, the leukocyte of the living body recognizes it and actively attacks the proliferated foreign germs. In this process, the dead cells of the leukocyte are accumulated in the invaded tissue At the same time, the cell debris of invading microorganisms killed by leukocytes melts into the invading tissues and abscess forms. The treatment of abscess due to inflammation can be promoted by the anti-inflammatory action. The anti-inflammatory action is to inhibit the growth of invading microorganisms by using an antibacterial agent or to activate the macrophage that digests foreign substances accumulated in the abscess, And enhancing the function of excretory macrophages.

In general, an inflammatory reaction is a defensive reaction process of a living body that attempts to repair and regenerate a damaged region when an invasion of biological changes occurs in the cell or tissue of the living body. Therefore, these series of reactions include local blood vessels, various tissue cells of body fluids, and immune-mediated cells. With the recent development of molecular biology, inflammatory diseases have been attempted to be understood at the molecular level of cytokine, and factors affecting these diseases are also being clarified one by one.

The inflammatory cytokines and mediators are regulated by nuclear elements. For example, NF-κB (nuclear factor-kappa B) is a nuclear protein of the Rel gene family. In the cytoplasm, NF-κB is associated with IκB (inhibitory kappa B) IκB kinase is activated by various stimuli such as chemokines and lipopolysaccharide (LPS) such as reactive oxygen and tumor necrosis factor-alpha (TNF-alpha) Out. NF-κB, composed of a heterodimer of p50 and p65, is known to promote the expression of genes (tumor necrosis factor or cyclooxide synthase) that are activated and then migrate to the nucleus to induce an inflammatory response (Oh GT et al., Artherosclerosis , 159 (1): 17-26, 2001; Epstein FH et al al . , The New England Journal of Medicine , 336 (15): 1066-1071,1997; Zhang WJ et al ., FASEB J , 15 (130): 2423-2431, 2001; Denka et al . , J. Biol . Chem . , 276 (30): 28451-28458, 2001; Sahnoun Z et al . , & Lt ; / RTI > Phsiology , 53 (4): 315-339,1998; Lindner V Pathobiology , 66 (6): 311-320, 1998; Landry DB meat al ., Am . J. Pathol ., 151 (4): 1085-1095,1997; ; Gerritsen ME et al . , Am . J. Pathol ., 147 (2): p278-292, 1995).

Nitric oxide (NO) is produced by the oxidation of L-arginine by Nitric oxide synthase (NOS), which acts as a mediator of inflammation, (Kou and Schroder, Annuals of Surgery 221, 220-235, 1995). Inducible nitric oxide synthase (iNOS) among NOS is known to be closely related to overproduction of NO in cells. Prostaglandin E ₂ (PGE ₂ ) and Leukotriene In addition, PGE ₂ is produced by the cyclooxygenase-2 enzyme (COX-2) as an inflammatory mediator produced from arachidonic acid It is mainly produced in macrophages and monocytes, and macrophages are rapidly induced by inflammatory agents such as riboplysaccharide.

Materials used in animal models for inflammation induction include adjuvant, collagen II, carrageenan, etc. Chondrus In addition to suppression of immune response, carrageenan extracted from cripus is known to have various biological actions such as acute inflammation and induction of chronic inflammation, DIC induction, tumor growth promotion, activation of Hageman factor and kinin, and complement inactivation (Chan WY et al ., J. Pharmacol . Exptl . Therap. , 147: 48,1965).

The most powerful anti-inflammatory drug developed by the first-class drug is steroids. However, long-term use is accompanied by side effects. Therefore, in the treatment of inflammation, steroids are surprisingly effective at first use, completely eliminating symptoms, but only for a short time. Symptoms of steroid use reappear with the use of steroids. Goes. Side effects of steroids include side effects such as round polyhedronic face, retention of fluid, adrenal suppression, increased susceptibility to infection, and other psychoses, cataracts, glaucoma, peptic ulcer, delayed wound healing, Have.

Therefore, it is necessary to study a substance which can be safely ingested safely without a separate purification process, which has an effect of suppressing inflammation, and which can be easily used for food, as a medicament containing a natural plant extract as an active ingredient. For example, researchers at the University of Hirosaki and the University of Osaka have found that NF-κB, a protein that regulates genes that cause inflammatory reactions in atopic dermatitis, binds to specific genes of immune cells to produce inflammatory substances, κB to inhibit the action of NF-κB by combining artificial DNA with NF-κB by administering artificial DNA similar to the gene to which κB binds. And Notabis, Switzerland, has developed and marketed "Elidel", a Pimecrolimus ingredient, a treatment for atopic dermatitis, which is a kind of inducer of ascorbic acid and selectively inhibits the release of inflammatory cytokines. In addition, Professor Jang Hyun-wook of Yeungnam University Yakult University said that extracts of Saururus chinensis and leather trees are effective in treating asthma and allergies, and signed a technology transfer agreement with Korea Pharma on the 19th, with a technical salary of 150 million won and 4% (January 22, 2006 article article).

A therapeutic agent for allergic diseases using an antibody against various kinds of cytokines and chemokines characteristic of allergic diseases, an agent for treating allergic dermatitis using a natural extract of fermented extract of aquatic plants, and an anti-inflammatory disease And development of therapeutic agents have been carried out. However, development of anti-inflammatory drugs using Aldicia tincture has not been developed so far.

Ardisia tinctoria Pit. Is a plant belonging to the family Myrsinaceae and is known to be used as a black dye in the civilian area. However, A. crenata and A. pusilla , which belong to the same species, have antioxidant activity (2008-001634, Japan Studies have shown that components such as syringic acid, isorhamnetin and quercetin isolated from A. elliptica plants have antibacterial activity (Salmonella) (Methin Phadungkit & Omboon Luanratana, 20 (7), 693-696, 2006, Natural Product Research). However, there have been no reports on research on the Aldichia tincturea or on the components thereof.

As a result of screening related to the inflammatory activity of the plant extracts of the present invention, the present inventors have found that the extracts of Aldrichia tincturea, NO, PGE ₂ , IL-6 (interleukin-6) and IL-1beta It was found that there was an effect of remarkably lowering the amount of cytokine. As a study of this inflammation inhibitory mechanism, it was confirmed that the aldicia tincturea extract inhibits the expression of iNOS and COX-2 gene and protein, inhibits nuclear transfer of p65 protein and phosphorylation of signal transducer, In addition, it was confirmed that the aldicia tincturea extract had an anti-inflammatory effect in the mouse footbath model. Therefore, the present invention has been accomplished by showing that the extract of Aldrichia tincturea can be used as an effective ingredient of a pharmaceutical composition for the prevention or treatment of various inflammatory diseases including allergies, which is a safe substance without plant-derived cytotoxicity.

It is an object of the present invention to provide a pharmaceutical composition, an external preparation for skin, a cosmetic composition and a composition for health food containing an extract of Ardisia tinctoria Pit. Or a fraction thereof as an active ingredient for the prevention and treatment of inflammatory diseases.

In order to accomplish the above object, the present invention provides a method for producing an antibody against Aldisia tinctoria tinctoria The present invention provides a pharmaceutical composition for preventing and treating an inflammatory disease containing an extract or a fraction thereof as an active ingredient.

The present invention also provides an external preparation for skin for the prevention and improvement of inflammatory diseases containing an aldialytic tincture extract or a fraction thereof as an active ingredient.

In addition, the present invention provides a cosmetic composition for prevention and improvement of inflammatory diseases containing an aldicia tincturea extract or a fraction thereof as an active ingredient.

In addition, the present invention provides a composition for health food for preventing and improving inflammatory diseases, which comprises an aldicia tincturea extract or a fraction thereof as an active ingredient.

The extract of Ardisia tinctoria Pit. Or the fraction thereof of the present invention has excellent anti-inflammatory activity and is almost free from cytotoxicity. Therefore, the medicament for the prevention and treatment of inflammation-related diseases, allergic diseases, Food, food additive, functional beverage, or beverage additive.

Figure 1 shows the inhibitory effect of aldicia tincturea extract on the production of nitric oxide induced by lipopolysaccharide in Raw264.7 cells (# is less than P <0.001 compared to negative control and * is less than positive control P < 0.05 or less):
-; Negative control treated with DMSO only;
+; Positive controls induced with 0.5 [mu] g / ml of LPS;
5; Treated with 5 μg / ml of Aldicia tincturea extract and then induced into LPS;
10; Treated with 10 μg / ml of Aldicia tincturea extract and then induced into LPS;
20; Treated with 20 μg / ml of Aldicia tincturea extract and then induced into LPS; And
30; After treating the extract with 30 μg / ml of Aldicia tincturea, it was induced to LPS
FIG. 2 is a graph showing the inhibitory effect of the aldicia tincturea extract on the expression of LPS-induced nitric oxide synthase:
a; Nucleic acid amplification;
b; Western blasting; And
c; Immunofluorescent staining.
Figure 3 shows the inhibitory effect of the aldicia tincturea extract on the production of prostaglandins induced by LPS (# is less than P <0.001 compared to negative control and P * 0.005 less than positive control).
Figure 4 shows the inhibitory effect of the aldicia tincturea extract on the expression of prostaglandins induced by LPS:
a; Nucleic acid amplification; And
b; Western blasting.
FIG. 5 shows the inhibitory effect of the aldicia tincturea extract on LPS-induced cytokine production (# is less than P <0.001 compared to negative control, * is less than P <0.005 compared to positive control, and ** is positive control P <0.05).
a; IL-6 (interleukin-6); And
b; IL-1beta (interleukin-1beta).
Figure 6 shows the inhibitory effect of the LPS-induced p65 nuclear transfer on the aldicia tincturea extract:
LPS; LPS induced group; And
LPS + AT; AT was treated with 30 μg / ml and induced to LPS.
FIG. 7 is a graph showing the inhibitory effect of the aldicia tincturea extract on the phosphorylation of signal transduction material induced by LPS.
Figure 8 shows the inhibitory effect of aldicia tincturea extract on carrageenan-induced mouse foot species:
Control group; PBS alone group;
Carrageenan; Induction of inflammation with carrageenan after PBS administration;
AT; Induced 40 mg / kg of the aldicia tincture extract to carrageenan;
ATE; After administration of 40 mg / kg of the aldicia tinctacterium ethyl acetate fraction, the carrageenan was induced; And
Indomethacin; Indomethacin 5 mg / kg followed by induction with carrageenan.
FIG. 9 is a graphical representation of the < RTI ID = tinctoria Pit.) TLC (Thin Layer Chromatography) of extracts and fractions:
T: Aldicia tincturea extract;
H: aldicia tincturea n-hexane fraction;
C: aldicia tincturea chloroform fraction;
E: aldicia tincturea ethyl acetate fraction;
B: aldicia tincture butanol fraction; And
W: Aldicia tincture water fraction.
Figure 10 is a graphical representation of the &lt; RTI ID = 0.0 &gt; tinctoria Pit.) Extracts and fractions thereof.

Hereinafter, terms used in the present invention will be described.

The term "inflammation" as used herein refers to the pathological condition of an abscess formed by the infiltration of an external infectious agent (bacteria, fungi, viruses, various kinds of allergens).

As used herein, the term "allergy " refers to a phenomenon in which a living body in contact with an exogenous substance exhibits a different reaction to the substance from the normal.

The term "prophylactic," as used herein, refers to any action that inhibits or delays the progression of an inflammatory disease upon administration of a composition of the present invention.

As used herein, the terms " treatment "and" improvement "refer to all actions by which administration of the composition of the invention improves or alleviates the symptoms of an inflammatory disease.

The term "administering" as used herein is meant to provide any desired composition of the invention to an individual by any suitable method.

The term "individual" as used herein means all animals such as humans, monkeys, dogs, goats, pigs or rats having a disease in which the symptoms of inflammatory diseases can be improved by administering the composition of the present invention.

The term " pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit or risk rate applicable to medical treatment, including the type of disease, severity, activity of the drug, The time of administration, the route and rate of excretion of the drug, the duration of the treatment, factors including drugs used simultaneously and other factors well known in the medical arts.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for the treatment of Aldisia tinctoria tinctoria The present invention provides a pharmaceutical composition for preventing and treating an inflammatory disease containing an extract or a fraction thereof as an active ingredient.

The inflammatory disease is selected from the group consisting of allergies, dermatitis, atopic dermatitis, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, Wherein the disease is any one selected from the group consisting of osteoarthritis, rheumatoid arthritis, shoulder inflammation, tendinitis, hay fever, perianal inflammation, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis and acute and chronic inflammatory diseases But is not limited thereto.

The aldicia tincturea extract is preferably but not limited to be produced by a method comprising the steps of:

1) adding an extracting solvent to the aldicia tincturea extract;

2) filtering the extract of step 1); And

3) Concentrating the filtered extract of step 2) under reduced pressure and drying.

In the above method, the aldicia tinctoria of step 1) may be used without limitation such as grown or commercially available.

The aldicia tincturea is available as leaves, stems or roots.

The extraction solvent is preferably water, alcohol or a mixture thereof. As the alcohol, C ₁ to C ₂ lower alcohol is preferably used, and as the lower alcohol, ethanol or methanol is preferably used. As the extraction method, it is preferable to use shaking extraction, Soxhlet extraction or reflux extraction, but it is not limited thereto. It is preferable that the extraction solvent is added by 1 to 10 times the amount of dried aldicia tinctoria and extracted. The extraction temperature is preferably 30 to 100 DEG C, but is not limited thereto. In addition, the extraction time is preferably 10 to 48 hours, more preferably 15 to 30 hours, but is not limited thereto. In addition, the extraction number is preferably 3 to 5 times, more preferably 3 times, but not limited thereto.

In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step 3), but it is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but not always limited thereto.

The fraction of the aldicia tincture extract of the present invention is preferably produced by a method of extracting the aldicia tincturea extract with an organic solvent, but is not limited thereto.

The organic solvent is preferably n-hexane, chloroform, ethyl acetate or butanol, but is not limited thereto. As the fraction, n-hexane fraction, chloroform fraction, ethyl acetate fraction and butanol fraction, and butanol fraction obtained by adding n-hexane, chloroform, ethyl acetate and butanol stepwise and then obtaining a soluble fraction in each solvent addition step, And the water layer in which the remaining water layer is concentrated can be used, but the ethyl acetate fraction is most preferable.

In one aspect of the present invention, in order to confirm the cytotoxicity of the aldicia tincturea extract or its fraction, RAW264.7 cells, which are macrophages of mice, were inoculated in a 96-well plate and adhered, The cell viability was calculated as 100% of the negative control group treated with DMSO. As a result, it was found that the cell viability of the extracts of aldicia tincturea or its ethyl acetate fraction, butanol fraction And water fractions were not toxic to a concentration of 20 [mu] g / ml (see Table 1).

In one aspect of the invention, the inflammation of the aldicia tincturea extract In order to investigate the inhibitory effect, the amount of nitric oxide (NO) induced by lipopolysaccharide in Raw264.7 cells was measured. As a result, the amount of nitric oxide increased by LPS was measured by using aldicia tincturea extract (Table 2). In particular, it was confirmed that the ethyl acetate fraction exhibited the strongest inhibitory effect on the nitric oxide formation (69.87 ± 2.74%) (see Table 2). In addition, it was confirmed that the production of nitric oxide was significantly decreased in the concentration-dependent manner in the group in which the aldicia tincturea extract was pretreated and the inflammation was induced by LPS, compared with the group treated with LPS alone (see Fig. 1).

In one aspect of the present invention, reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and immunofluorescence staining were performed to confirm the iNOS gene and protein expression inhibitory effect of the Aldricha tincturea extract. It was confirmed that iNOS gene expression was increased in cells treated with polysaccharide and that the expression level of iNOS and the expression of iNOS protein were remarkably decreased in the cells treated with the extract of Aldrichia tinctureia 2).

In addition, in one aspect of the present invention, the inhibitory effect of the aldicia tincturea extract on the production of prostaglandin E ₂ was confirmed. In the cells treated with lipopolysaccharide only, the production of prostaglandin E ₂ was abruptly increased, It was confirmed that the production of prostaglandin E ₂ was inhibited in a concentration-dependent manner in cells pretreated with tincture extract (see FIG. 3).

In addition, in one aspect of the present invention, the inhibitory effect of the aldicia tincturea extract on COX-2 expression was examined. As a result, in the cells pretreated with the aldicia tincturea extract, the amount of COX- And the expression was markedly decreased (see Fig. 4).

In addition, in one aspect of the present invention, the inhibitory effect of the aldicia tincturea extract on cytokine production was evaluated. As a result, the amount of IL-6 and IL-beta cytokine increased by the treatment of LPS was reduced by pretreatment of the aldicia tincturea extract (See Figs. 5A and 5B).

In one aspect of the present invention, the inhibitory effect of p65 on the NF-κB protein of the aldicia tincturea extract was found to decrease by 30 minutes when the LPS alone was treated, whereas the p65 The amount was confirmed to increase. However, it was confirmed that the increase of the amount of p65 in the nucleus was weak when the aldicia tincturea was induced to LPS after pretreatment (see FIG. 6).

In addition, in one aspect of the present invention, the inhibitory effect on the phosphorylation of the signal transduction protein of the aldicia tincturea extract was examined. As a result, when the macrophage was pretreated with the aldicia tincturea extract and treated with LPS, the whole form of JNK and p38 There was no change in the protein, and the expression of the phosphorylated form of the protein was increased by LPS, and there was no change in the treatment with the Aldichia tincturea extract. On the other hand, the ERK protein showed no change in the whole type of protein when treated with the extract of Aldrichia tincturea, and the expression of the phosphorylated form of the protein was also inhibited (see Fig. 7).

In addition, in one aspect of the present invention, since the aldicia tincturea extract has excellent anti-inflammatory effect on animal cells, the anti-inflammatory effect was confirmed through animal experiments. As a result, in the model of the carrageenan-induced mouse footbath, the aldicia tincturea extract And ethyl acetate fraction showed a significant inflammation inhibitory effect (see FIG. 8).

Therefore, the aldicia tincturea extract of the present invention or its fractions inhibits the production of nitric oxide which is rapidly increased by inflammation induction, and inhibits PGE ₂ and IL-6 (interleukin-6) and IL-1beta cytokines Inhibit the expression of iNOS and COX-2 genes and proteins, inhibit the nuclear translocation of p65 protein and phosphorylation of signal transduction materials, and also inhibit significant inflammation in carrageenan-induced mouse paw It can be used as an effective ingredient of a pharmaceutical composition for the prevention and treatment of inflammation-related diseases.

The composition containing the aldicia tincturea extract of the present invention or a fraction thereof may further contain one or more active ingredients which exhibit the same or similar functions in addition to the above components.

The composition of the present invention may further comprise a pharmaceutically acceptable additive, wherein pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose Starch glycolate, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, calcium stearate, , White sugar, dextrose, sorbitol and talc. The pharmaceutically acceptable additives according to the present invention are preferably included in the composition in an amount of 0.1 to 90 parts by weight, but are not limited thereto.

That is, the composition of the present invention can be administered in various formulations of oral and parenteral administration at the time of actual clinical administration. In the case of formulation, a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, . &Lt; / RTI &gt; Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, Sucrose, Lactose, Gelatin, or the like. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions and syrups, and various excipients such as wetting agents, sweetening agents, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used . Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

The composition of the present invention may be administered orally or parenterally in accordance with the intended method, and may be administered orally, parenterally or intraperitoneally, rectally, subcutaneously, intravenously, intramuscularly, . The dosage varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity.

The dosage of the composition of the present invention varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease, and the daily dose is the daily dose of Aldicia tincturea extract Kg, preferably 0.001 to 10 mg / kg, and may be administered 1 to 6 times a day.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention and treatment of inflammatory diseases.

The present invention also provides a method for treating an inflammatory disease comprising administering to a subject suffering from an inflammatory disease a composition comprising a pharmaceutically effective amount of an aldicia tincturea extract or a fraction thereof as an active ingredient.

The present invention also provides a method of preventing an inflammatory disease comprising administering to a subject a composition comprising a pharmaceutically effective amount of an aldicia tincturea extract or a fraction thereof as an active ingredient.

The pharmaceutically effective amount is 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, but is not limited thereto. The dose may vary depending on the weight, age, sex, health condition, diet, administration period, method of administration, rate of elimination, severity of disease, and the like of a particular patient.

The subject is a vertebrate animal, preferably a mammal, more preferably an experimental animal such as a mouse, a rabbit, a guinea pig, a hamster, a dog or a cat, and most preferably an ape-like animal such as a chimpanzee or a gorilla.

The above-mentioned administration method may be oral or parenteral administration. In the case of parenteral administration, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravenous injection, intramuscular injection, intramural injection, intracerebroventricular injection, Can be administered by injection.

The inflammatory disease is selected from the group consisting of allergies, dermatitis, atopic dermatitis, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, Wherein the disease is any one selected from the group consisting of osteoarthritis, rheumatoid arthritis, shoulder inflammation, tendinitis, hay fever, perianal inflammation, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis and acute and chronic inflammatory diseases But is not limited thereto.

The aldicia tincturea extract of the present invention or its fractions inhibits the production of nitric oxide which is rapidly increased by inflammation induction, and the amount of PGE ₂ and IL-6 (interleukin-6) and IL-1beta cytokine Significantly inhibited the expression of iNOS and COX-2 genes and proteins, inhibited the nuclear translocation of p65 protein and phosphorylation of signal transduction material, and showed a significant inflammation-inhibiting effect in carrageenan-induced mouse peduncle models , And a pharmaceutical composition for the prevention and treatment of inflammation-related diseases.

The present invention also provides an external preparation for skin for the prevention and treatment of inflammatory diseases, which comprises an aldicia tincturea extract or a fraction thereof as an active ingredient.

Inflammatory diseases include allergies, edema, dermatitis, atopy, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, ulcerative colitis, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia Selected from the group consisting of psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, hay fever, perianal inflammation, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis and various acute and chronic inflammatory diseases But the present invention is not limited thereto.

The aldicia tincturea extract of the present invention or its fractions inhibits the production of nitric oxide which is rapidly increased by inflammation induction, and the amount of PGE ₂ and IL-6 (interleukin-6) and IL-1beta cytokine Significantly inhibited the expression of iNOS and COX-2 genes and proteins, inhibited the nuclear translocation of p65 protein and phosphorylation of signal transduction material, and showed a significant inflammation-inhibiting effect in carrageenan-induced mouse peduncle models , And an external preparation for skin for the prevention and treatment of various inflammatory diseases.

When the aldicia tincture extract of the present invention or a fraction thereof is used as an external preparation for skin, it may further contain a fatty substance, an organic solvent, a solubilizer, a thickening agent and a gelling agent, a softener, an antioxidant, a suspending agent, a stabilizer, ), Perfumes, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipids Or any other ingredient conventionally used in external preparations for skin. The present invention also relates to a cosmetic composition, In addition, the components can be introduced in amounts commonly used in the dermatology field.

The dose of the aldicia tincturea extract to the external preparation for skin is 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, but is not limited thereto. The dose may vary depending on the weight, age, sex, health condition, administration period, elimination rate, severity of disease, etc. of the particular patient.

In addition, the present invention provides a cosmetic composition for prevention and improvement of inflammatory diseases containing an aldicia tincturea extract or a fraction thereof as an active ingredient.

The aldicia tincturea extract of the present invention or its fractions inhibits the production of nitric oxide which is rapidly increased by inflammation induction, and the amount of PGE ₂ and IL-6 (interleukin-6) and IL-1beta cytokine Significantly inhibited the expression of iNOS and COX-2 genes and proteins, inhibited the nuclear translocation of p65 protein and phosphorylation of signal transduction material, and showed a significant inflammation-inhibiting effect in carrageenan-induced mouse peduncle models , An effective ingredient of a cosmetic composition for prevention and improvement of inflammatory diseases.

When the aldicia tincture extract of the present invention or a fraction thereof is used as a cosmetic composition, for example, a solution, a gel, a solid or a paste anhydrous product, an emulsion obtained by dispersing an oil phase in an aqueous phase, a suspension, a microemulsion, a microcapsule, In the form of granules or ionic forms (liposomes), non-ionic follicular dispersions, creams, skins, lotions, powders, ointments, sprays or conical sticks. It can also be prepared in the form of a foam or an aerosol composition further containing a compressed propellant.

The cosmetic composition may further comprise at least one selected from the group consisting of fatty substances, organic solvents, solubilizers, thickening agents and gelling agents, softening agents, antioxidants, suspending agents, stabilizers, foaming agents, fragrances and fragrances in addition to the aldicia tincturea extract of the present invention or fractions thereof Surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, barrier agents, wetting agents, essential oils, dyes, pigments, lipophilic or lipophilic active agents, lipid vesicles or cosmetics Such as any other ingredients conventionally used in cosmetics.

In addition, the present invention provides a health food for preventing and improving inflammatory diseases containing an aldicia tincturea extract or a fraction thereof as an active ingredient.

The aldicia tincturea extract of the present invention or its fractions inhibits the production of nitric oxide which is rapidly increased by inflammation induction, and the amount of PGE ₂ and IL-6 (interleukin-6) and IL-1beta cytokine Significantly inhibited the expression of iNOS and COX-2 genes and proteins, inhibited the nuclear translocation of p65 protein and phosphorylation of signal transduction material, and showed a significant inflammation-inhibiting effect in carrageenan-induced mouse peduncle models , A composition for a health food for preventing and improving various inflammatory diseases.

Inflammatory diseases include allergies, edema, dermatitis, atopy, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, ulcerative colitis, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia Selected from the group consisting of psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, hay fever, perianal inflammation, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis and various acute and chronic inflammatory diseases But the present invention is not limited thereto.

The health food of the present invention may be used as it is, or may be used in combination with other food or food ingredients, and may be suitably used according to conventional methods.

There is no particular limitation on the kind of the health food. Examples of foods to which the Aldricha tincture extract can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, Tea, a drink, an alcoholic beverage, and a vitamin complex, all of which include health foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the health food of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, , A carbonating agent used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples.

However, the following examples and experimental examples are illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

< Example  1> Aldishia Tincture ( Ardisia tinctoria Pit .) Preparation of extract

The 100% methanol extract of Aldicia tinctoria of the present invention was purchased from the overseas plant extract bank of the International Biomaterials Hub Center, Korea Research Institute of Bioscience and Biotechnology, and the KRIB (Herbarium of the Korea Institute of Bioscience and Biotechnology, KRIB 0027026) Research Institute of Bioscience and Biotechnology).

In detail, the extracting process was as follows. After drying the aldicia tincturea (leaf, young branch, flower), 113 L of the pulverized powder sample was added with 1 L of methanol and the mixture was sonicated with an ultrasonic wave extractor (SDN-900H, SD-ULTRASONIC CO. ) For 30 times (40 KHz, 15 minutes extraction - 120 minutes stop) at room temperature. After filtration and concentration under reduced pressure, 10.33 g of an aldicia tinctoria methanol extract (hereinafter referred to as Aldicia tinctoria extract) was obtained. In the present invention, the aldicia tincturea extract was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 20 mg / ml and diluted by concentration.

< Example  2> Aldishia Tincture  menstruum Fraction  Produce

To 450 mg of the aldicia tincturea extract obtained in Example 1, distilled water was added to suspend, and the same amount of n-hexane was added to the mixture. The n-hexane soluble fraction and the water soluble fraction were separated, Filtration and concentration under reduced pressure gave 90.4 mg of n-hexane fraction. Then, the n-hexane fraction was removed and chloroform was equally added to the remaining water layer to obtain 22.0 mg of chloroform fraction. The remaining water layer was equilibrated with ethyl acetate to obtain 44.8 mg of an ethyl acetate fraction, and An equal amount of butanol was added to the remaining water layer, and 57.6 mg of a butanol fraction was obtained in the same manner. The remaining water layer was concentrated to obtain 205.2 mg of a water fraction.

< Example  3> Aldishia Tincture  Extract or its Fraction TLC  Research

The aldicia tincturea extract and fractions obtained in Example 1 and Example 2 were dissolved in methanol at a concentration of 10 mg / ml and subjected to silica gel thin layer chromatography (TLC Silica gel 60 F ₂₅₄ , Merck) Substances were identified. As a developing solvent, a mixed solvent of n-hexane: acetone at a ratio of 2: 1 was used. Silica gel reverse phase chromatography (TLC Silica gel 60 RP-18 F ₂₅₄ S, Merck) was carried out to check the polarity materials, and the developing solvent was 50% methanol (FIG. 10).

< Example  4> Aldishia Tincture  Extract or its Fraction UPLC  analysis

The aldicia tincturea extract and fractions obtained in Example 1 and Example 2 were dissolved in methanol at a concentration of 10 mg / ml and filtered through a membrane filter of 0.2 쨉 m. The HPLC was performed using an ACQUITY UPLC ^™ BEHC ₁₈ (10 mm × 2.1 mm, id, 1.7 μm, waters, USA) at 35 ° C., The mobile phase A was initially treated with water and formic acid (100: 0.1, v / v) and the mobile phase B with acetonitrile and formic acid (100: 0.1, v / v) The mobile phase B was maintained at 100% for 9 minutes, the mobile phase B was lowered to 10% for 9.5 minutes, and the mobile phase B was kept at 10% for 11 minutes. And used for analysis. The flow rate of the mobile phase was 0.4 ml / min and the sample injection volume was 3 μl.

The CAD (Charge Aerosol Detector) is a detector that measures charge by attaching a charge to materials separated from the UPLC. The relative content and the degree of separation of the material can be confirmed by chromatographic method (FIG. 11).

< Experimental Example  1> Aldishia Tincture  Extract or its In the fraction  Cytotoxicity test for

In order to confirm the effect of the aldicia tincturea extract or its fraction on the cell survival rate, Raw264.7 cells, which are macrophages of mice, were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5% fetal bovine serum , Gibco Co., Ltd.) at a concentration of 1 × 10 ⁵ cells / ml, and 100 μl of each was inoculated in a 96-well plate. After 4 hours, the aldicia tincturea extract or its fraction was treated at a concentration of 20 μg / ml, or the aldicia tincturea extract was treated at a concentration of 0, 5, 10, 20 and 30 μg / ml. After incubation for 24 hours, 10 μl of 5 mg / ml MTT solution was added to each well. After further incubation for 4 hours, supernatant was removed, 100 μl of DMSO was added, and the absorbance was measured at 570 nm , And the cell viability was calculated according to the following equation with a negative control group treated with 0.2% DMSO as 100%.

As a result, as shown in Table 1, it was confirmed that the aldicia tincturea extract, ethylacetate, butanol, and water fraction had little effect on the cell survival rate, but hexane fraction and chloroform fraction showed very strong cell survival inhibition effect Table 1).

< Experimental Example  2> Aldishia Tincture  Extract or its Fraction NO  Generation inhibitory effect

The present inventors have found that inflammation of the aldicia tincture extract In order to investigate the inhibitory effect, the amount of nitric oxide (NO) induced by lipopolysaccharide (LPS, Sigma) in Raw264.7 cells was measured.

Specifically, mouse macrophages Raw264.7 purchased from ATCC were suspended in DMEM (Dulbecco's Modified Eagle Medium, Gibco) supplemented with 5% fetal bovine serum at a concentration of 2.5 × 10 ⁵ cells / ml 200 [mu] l of the solution was inoculated into a 96-well plate. After adhering for 4 hours, the aldicia tincturea extract of the present invention or its fractions were treated with a concentration of 20 μg / ml, respectively, or the aldicia tinctoria extract at a concentration of 0, 5, 10, 20, and 30 μg / After incubation for a period of time, 0.5 μg / ml of LPS was treated and cultured for 24 hours. 100 μl of the supernatant was collected and placed in a new 96-well plate, and the same amount of a grease reagent (Sigma) was added thereto. The mixture was allowed to react at room temperature for 10 minutes and then transferred to a microplate reader (Bio-Rad) The absorbance was measured at a wavelength of 540 nm. Calibration curves were prepared using sodium nitrite, and the amount of nitric oxide produced in the culture was determined based on this. The inhibition rate of each sample was calculated using the amount of nitric oxide produced in the LPS-treated group as 100% .

As a result, as shown in Table 2, it was confirmed that the amount of nitric oxide produced by LPS was strongly inhibited by the aldicia tincturea extract. In particular, the ethyl acetate fraction showed the strongest inhibitory effect on the nitric oxide formation (69.87 + 2.74%). On the other hand, the inhibitory effect of n-hexane (NO inhibition rate: 68.87 ± 2.45%, cell viability: 4.61 ± 0.26%) and chloroform (NO inhibitory rate: 72.67 ± 1.81%, cell viability: 5.59 ± 0.67% And the cell viability was decreased (Table 1 and 2).

Further, as shown in Fig. 1, it was confirmed that the production of nitric oxide was significantly reduced in the concentration-dependent group in the group in which the aldicia tincturea extract was pretreated and the inflammation was induced by LPS as compared with the group treated with LPS alone ).

< Experimental Example  3> iNOS  For expression Aldishia Tincture  Inhibitory effect of extract

<3-1> Nucleic acid amplification method ( RT - PCR )

Reverse transcription-polymerase chain reaction (RT-PCR) was performed to confirm the iNOS gene inhibitory effect of the extract of Aldicia tinctureia.

Specifically, by using the method of Experimental Example 1, a 100 mm petri dish was coated with 1 占⁶ (Invitrogen, Calif., USA). After the cells were cultured for 24 hours, the cells were removed from the culture vessel, and the cells were treated with the ribonuclease extract solution (Invitrogen, CA, USA) To homogenize the cells. After 5 minutes, the cells were collected and transferred to a centrifuge tube, and 200 μl of chloroform was added thereto. The mixture was thoroughly mixed for 15 seconds, left for 3 minutes, and then centrifuged at 14000 rpm for 15 minutes. The supernatant containing ribonucleic acid was transferred to a new tube and mixed with 500 μl of isopropyl alcohol. After 10 minutes, the supernatant was discarded. The supernatant was discarded. The supernatant was discarded, and the precipitated ribonucleic acid was dried at room temperature for 20 minutes. The supernatant was discarded and centrifuged at 10,000 rpm for 5 minutes. The dried ribonucleic acid was suspended in distilled water treated with DEPC (Diethylpyrocarbonate, Sigma). After quantification of ribonucleic acid, complementary DNA was synthesized using Omniscript RT kit (Pharmingen, USA). The synthesized cDNA was used as a template and an iNOS primer (forward primer 5'-GGA GCG ACT TGT GGA TTG TC-3 ': SEQ ID NO: 1, reverse primer 5'-GTG AGG GCT TGG CTG AGT GAG-3'; SEQ ID NO: 2) were mixed, and the degree of gene expression was confirmed using PCR Premix (Fermentas, USA).

As a result, as shown in FIG. 2A, the expression of iNOS gene was increased in the cells treated with lipopolysaccharide compared to the negative control. In the cells treated with the extract of Aldishia tincturea, the expression of nucleic acid of iNOS (Fig. 2A).

<3-2> Western blasting  ( Western blotting )

Western blotting was performed to confirm the effect of inhibiting the expression of iNOS protein of the extract of Aldicia tinctureia.

Specifically, after removing the medium from the cells treated with the sample in the same manner as in <Experimental Example 3-1>, the cells were detached from the culture container to prepare a protein containing protease inhibitor cocktail (Roche, USA) And homogenized using an elution solution (CelLytic TM-MT Tissue Lysis Reagent, Sigma, USA). The extract was centrifuged at 14000 rpm for 20 minutes and the supernatant and insoluble aggregates were separated. The protein concentration of the separated supernatant was measured using a Bio-Rad protein assay kit (Bio-Rad, USA). The supernatant was mixed with 1: 4 with 5 x SDS (0.156M Tris-HCl, pH 6.8, 2.5% SDS, 37.5% glycerol, 37.5 mM DTT) and boiled at 100 ° C for 10 minutes. 20 ㎍ protein was loaded on a SDS 4-12% SDS-PAGE gel, and electrophoresed at 125 V for 2 hours. The protein was separated by electrophoresis for one hour at 100 mA / And transferred to a PVDF membrane. An anti-iNOS antibody (1: 100, Santa Cruz Biotechnology, USA) was bound to the membrane as a primary antibody, followed by sensitization and analysis.

As a result, when the lipopolysaccharide and the aldicia tinctoria extract of the present invention were pretreated with macrophages, protein expression of iNOS was decreased in a concentration-dependent manner as shown in FIG. 2B (FIG. 2B).

<3-3> Immunofluorescent staining  ( Immunofluoresence )

About 2 x 10 < ⁵ > cells / ml Cells were adhered to Permanox chambered plastic slides (Nunc, USA) and inflammation was induced in macrophages as in Experiment 2, supernatant was removed, and the cells were washed with phosphate buffered saline (PBS) And washed. The cells were fixed with ethanol at 4 ° C for 30 minutes, washed and then blocked with 3% bovine serum albumin at room temperature for 30 minutes. After blocking, the primary antibody [anti-iNOS antibody (1: 100)] was reacted at 4 ° C for one day. After washing 3 times with phosphate buffer, the red (Texas red; Santa Cruz Biotechnology, USA) were incubated with a secondary antibody (Santa Cruz Biotechnology, USA) were reacted at room temperature for 2 hours. (ProLong Gold Antifade reagent, Invitrogen, USA) with phosphate buffer and photographed with a confocal microscope (LSM510m Carl Zeiss, Germany).

As a result, as shown in FIG. 2C, in the cells treated with lipopolysaccharide only, protein expression of iNOS was rapidly increased, whereas in the cells pretreated with the Aldichia tinctoria extract, protein expression was significantly decreased (Fig. 2C).

< Experimental Example  4> About prostaglandin production Aldishia Tincture  Inhibitory effect of extract

Aldi cyano tincture as experiments to determine the prostaglandin E ₂ (Prostaglandin E2) production-inhibiting effect of thoria extract, the <Experiment 2> and prostaglandin assay kit taking the culture supernatant of Raw264.7 cells treated in the same manner (PGE ₂ assay kit; using the R & D systems, Minneapolis, USA) to measure the production-inhibiting effect of prostaglandin E ₂ (prostaglandin E2).

As a result, as shown in FIG. 3, the production of prostaglandin E ₂ in the lipopolysaccharide-treated cells was abruptly increased, whereas in the cells pretreated with the aldicia tinctoria extract, the production of prostaglandin E ₂ was inhibited in a concentration-dependent manner (Fig. 3).

< Experimental Example  5> COX -2 for expression Aldishia Tincture  Inhibitory effect of extract

&Lt; 5-1 > RT - PCR )

The cDNA synthesized in Experimental Example 3-1 was used as a template, and a COX-2 primer (forward primer 5'-GAA GTC TTT GGT CTG GTG CCT G-3 '; SEQ ID NO: 3, reverse primer 5' (GTC ATG GTT GC-3 '; SEQ ID NO: 4) were mixed and PCR expression was confirmed using PCR Premix (Fermentas, USA).

As a result, as shown in FIG. 4A, COX-2 gene expression was increased in cells treated with lipopolysaccharide compared with the negative control, whereas COX-2 gene expression was increased in cells pretreated with Aldricha tincture extract, 2 was significantly reduced (Fig. 4A).

<5-2> Western blasting  ( Western blotting )

Anti-COX-2 antibody (1: 100, Santa Cruz Biotechnology, USA) was bound to the membrane-coated membrane in the same manner as in <Experimental Example 3-2>, followed by sensitization.

As a result, when the lipopolysaccharide and the aldicia tincturea extract of the present invention were pretreated with macrophages, protein expression of COX-2 decreased in a concentration-dependent manner (FIG. 4B).

< Experimental Example  6> Aldishia Tincture  Inhibitory effect of extract on cytokine production

In order to confirm the cytokine production inhibitory effect of the extract of Aldicia tinctureia, the present inventors measured the amount of IL-6 (interleukin-6) and IL-1beta induced by LPS with an enzyme immunoassay kit (mouse IL-6 ELISA set ; BD OptEIA ^TM , USA or mouse IL-1beta Enzyme Immunometric Assay Kit; Assay designs, USA).

Specifically, Raw 264.7 cells were cultured in the same manner as in Experimental Example 2, and 100 μl of the supernatant was recovered and dispensed into a 96-well plate coated with mouse immunoglobulin, followed by reaction with stirring for 2 hours. Then, the cells were washed four times with a washing solution, and then 100 μl of anti-IL-6 antibody or anti-IL-1beta antibody as the primary antibody was dispensed and reacted for 2 hours. After the reaction, the cells were washed and the secondary antibody was dispensed and reacted for 30 minutes. After washing again, the substrate was incubated for 30 minutes, and the degree of color development was measured at 450 nm with a microplate meter.

As a result, as shown in FIG. 5, it was confirmed that the amounts of IL-6 and IL-1beta cytokines increased by the treatment of LPS were decreased in a concentration-dependent manner by pretreatment of the extract of Aldricha tincturea And 5b).

< Experimental Example  7> Aldishia Tincture  Extract NF Inhibitory effect of -KB protein on migration

In order to confirm the inhibition effect of p65 of the NF-κB protein of the extract of Aldicia tincturea, Raw264.7 cells were treated with 30 μg / ml of aldicia tincturea and 30 minutes later, LPS was added at a concentration of 0.5 μg / ml (NE-PER Nuclear and Cytoplasmic Extraction Reagents, Thermo Fisher Scientific Inc., USA), respectively.

The protein was attached to the PVDF membrane in the same manner as in Experimental Example <3-2>, and the amount of protein expressed was confirmed by attaching the p65 antibody thereto.

As a result, as shown in FIG. 6, it was confirmed that the amount of p65 in the nucleus was increased while the amount of p65 in the cytoplasm was decreased to 30 minutes when LPS alone was treated. However, it was confirmed that the increase of the amount of p65 in the nucleus was weak when the aldicia tincturea was induced to LPS after pretreatment (Fig. 6).

< Experimental Example  8> Aldishia Tincture  Inhibitory effect of extracts on signal transduction protein phosphorylation

Cells treated with the method of Experimental Example 7 were detached from the culture container and homogenized using a protein eluting solution containing a protease inhibitor and a phosphatase inhibitor (Roche, Germany). The extract was centrifuged at 14000 rpm for 20 minutes and the supernatant and insoluble aggregates were separated. Proteins of the separated supernatant were quantified, and 20 μg of protein per sample was electrophoresed on SDS 10% SDS-PAGE gel and transferred to PVDF membrane. Protein-free sections of the membranes were blocked with skimmed milk powder and then used as primary antibodies for all forms of anti-ERK, anti-p38 MAPK, anti-JNK (1: 1000, Santa Cruz Biotechnology, USA) Or an antibody of the phosphorylated form of anti-p38 MAPK and anti-JNK (1: 1000, Enzo, Rarmingdale, NY) or anti-ERK (1: 1000, Cell Signaling Technology, USA) Secondary antibodies were sequenced.

As a result, as shown in FIG. 7, when the macrophage was pretreated with the LPS and treated with the LPS, JNK and p38 showed no change in the whole protein, and protein expression of phosphorylated form was increased by LPS , And there was no change in the treatment with Aldicia tincturea extract. On the other hand, the ERK protein showed no change in the whole type of protein upon treatment with the Aldicia tincturea extract, and the expression of the phosphorylated form of the protein was also inhibited (FIG. 7).

< Experimental Example  9> Carrageenan ( carrageenan ) Induced mice On foot  About Aldishia Tincture  Inhibitory effect of extract

Since the extract of Aldishia tinctureia had excellent anti-inflammatory effect on animal cells, the anti-inflammatory effect was confirmed through animal experiments.

Specifically, 4 to 5 BALB / c female mice (Koatech, Korea) weighing 20-25 g were randomly selected and divided into groups. The aldicia tincture extract or ethyl acetate fraction was dissolved in phosphate buffer (PBS) by ultrasonication at a concentration of 40 mg / kg. As a negative control, PBS was administered. Indomethacin (Sigma, Indomethacin, USA) was administered at a concentration of 5 mg / kg. After 30 minutes, 25 μl of carrageenan (1% v / v) solution dissolved in PBS was injected into the left foot of the mouse to induce inflammation. The thickness of the feet was measured with a caliper (Digital caliper, Niigata Seiki, Japan) before injection of carrageenan, and the degree of inflammation was calculated by measuring again 4 hours after the carrageenan injection.

As a result, as shown in FIG. 8, the thickness of the foot was 0.85 ± 0.12 mm increased by carrageenan in the negative control group, but 0.32 ± 0.05 mm was increased in the group treated with the aldicia tincturea extract and the aldicia tincturea ethyl acetate fraction And 0.38 ± 0.07 mm in the pretreated group, indicating that both the aldicia tincturea extract and the fraction had an inhibitory effect on carrageenan-induced mouse foot species (FIG. 8).

< Manufacturing example  1> Preparation of pharmaceutical preparations

<1-1> Sanje  Produce

2 g of the aldicia tincturea extract of the present invention or a fraction thereof

Lactose 1 g

The above components were mixed and packed in airtight bags to prepare powders.

<1-2> Preparation of tablets

100 mg of the aldicia tincturea extract of the present invention or a fraction thereof

Corn starch 100 mg

100 mg of milk

Magnesium stearate 2 mg

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

&Lt; 1-3 > Preparation of capsules

100 mg of the aldicia tincturea extract of the present invention or a fraction thereof

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

&Lt; 1-4 >

1 g of the aldicia tincturea extract of the present invention or a fraction thereof

Lactose 1.5 g

Glycerin 1 g

0.5 g of xylitol

After mixing the above components, they were prepared so as to be 4 g per one ring according to a conventional method.

<1-5> Preparation of granules

150 mg of the aldicia tincturea extract of the present invention or its fraction

Soybean extract 50 mg

200 mg of glucose

600 mg of starch

After mixing the above components, 100 mg of 30% ethanol was added and the mixture was dried at 60 캜 to form granules, which were then filled in a capsule.

< Manufacturing example  2> Manufacturing of food

<2-1> Production of flour food

0.5 to 5.0 parts by weight of the aldicia tincturea extract of the present invention or its fractions were added to wheat flour and the mixture was used to prepare breads, cakes, cookies, crackers and noodles.

<2-2> soup  And juicy ( gravies )

0.1 to 5.0 parts by weight of the aldicia tincturea extract of the present invention or its fractions were added to soups and juices to prepare health promotion meat products, noodle soups and juices.

<2-3> Ground Beef  Produce

10 parts by weight of the aldicia tincturea extract of the present invention or its fraction was added to ground beef to prepare ground beef for health promotion.

<2-4> Dairy products ( dairy products )

5 to 10 parts by weight of the aldicia tincturea extract of the present invention or its fractions were added to milk and various dairy products such as butter and ice cream were prepared using the milk.

<2-5> Solar  Produce

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh.

Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer.

The aldicia tincture extract or its fraction of the present invention was concentrated under reduced pressure in a vacuum concentrator, dried by spraying, and dried with a hot air drier, and the resulting dried product was pulverized to a size of 60 mesh with a pulverizer to obtain a dry powder.

The grains, seeds and the aldicia tincturea extract of the present invention prepared above were blended in the following proportions.

(30 parts by weight of brown rice, 15 parts by weight of yulmu, 20 parts by weight of barley)

Seeds (7 parts by weight of perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds)

The aldicia tincturea extract or fraction thereof (3 parts by weight) of the present invention,

(0.5 part by weight),

Rhubarb (0.5 parts by weight).

< Manufacturing example  3> Manufacturing of beverage

<3-1> Health drink  Produce

(5%) of the extract of the present invention or fractions of the present invention, such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5% After sterilization, they were packaged in small containers such as glass bottles and plastic bottles.

<3-2> Preparation of vegetable juice

Vegetable juice was prepared by adding 5 g of the aldicia tincturea extract of the present invention or its fraction to 1,000 ml of tomato or carrot juice.

<3-3> Production of fruit juice

Fruit juice was prepared by adding 1 g of the aldicia tincturea extract of the present invention or a fraction thereof to 1,000 ml of apple or grape juice.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Pharmaceutical composition for prevention and treatment of          inflammatory diseases comprising fractions of Ardisia          tinctoria Pit. as an active ingredient <130> 12p-06-17 <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> iNOS forward primer <400> 1 ggagcgactt gtggattgtc 20 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> iNOS reverse primer <400> 2 gtgagggctt ggctgagtga g 21 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> COX-2 forward primer <400> 3 gaagtctttg gtctggtgcc t 21 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> COX-2 reverse primer <400> 4 gtctgctggt ttggaatagt tgc 23

Claims (12)

Ardisia tinctoria Pit.) Extract or a fraction thereof as an active ingredient for the prevention and treatment of inflammatory diseases.
The method according to claim 1, wherein the aldicia tincture comprises water, C ₁ To C ₂ lower alcohol or a mixture thereof is used as a solvent.
The pharmaceutical composition according to claim 2, wherein the lower alcohol is ethanol or methanol.
[Claim 2] The pharmaceutical composition according to claim 1, wherein the fraction is a fraction obtained by adding n-hexane, chloroform, ethyl acetate or butanol to the aldicia tincturea extract.
The pharmaceutical composition according to claim 4, wherein the fraction is an ethyl acetate fraction.
The method of claim 1, wherein the inflammatory disease is selected from the group consisting of allergies, dermatitis, atopy, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, ankylosing spondylitis, rheumatic fever, from the group consisting of fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, hay fever, tendinitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis and acute and chronic inflammatory diseases Or a pharmaceutically acceptable salt or solvate thereof.
A pharmaceutical composition for the prevention and treatment of edema containing an extract of Aldicia tincturea or a fraction thereof as an active ingredient.
A composition for a health food for prevention and improvement of edema containing an extract of Aldicia tincturea or a fraction thereof as an active ingredient.
A cosmetic composition for preventing and improving an inflammatory disease containing an extract of Aldicia tinctureya or a fraction thereof as an active ingredient.
10. The method of claim 9, wherein the inflammatory disease is selected from the group consisting of allergies, dermatitis, atopy, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, ankylosing spondylitis, Characterized in that it is any one selected from the group consisting of psoriasis arthritis, osteoarthritis, rheumatoid arthritis, shoulder inflammation, tendonitis, hay fever, periuritis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis and acute and chronic inflammatory diseases Wherein the cosmetic composition is a cosmetic composition for preventing and improving an inflammatory disease.
A composition for a health food for preventing and improving an inflammatory disease containing an aldicia tincturea extract or a fraction thereof as an active ingredient.
12. The method of claim 11, wherein the inflammatory disease is selected from the group consisting of allergies, dermatitis, atopy, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, ankylosing spondylitis, Characterized in that it is any one selected from the group consisting of psoriasis arthritis, osteoarthritis, rheumatoid arthritis, shoulder inflammation, tendonitis, hay fever, periuritis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis and acute and chronic inflammatory diseases Wherein the composition is a composition for preventing and / or improving inflammatory diseases.

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