WO2010016675A2 - Pharmaceutical composition for preventing and treating inflammatory diseases containing extracts of the immature fruit of rhus succedanea as active ingredients - Google Patents

Abstract

The present invention relates to an anti-inflammatory composition containing extracts of the immature fruit of Rhus succedanea as active ingredients. The extracts of the immature fruit of Rhus succedanea of the present invention exhibit anti-inflammatory effects through inhibiting the generation of nitrogen oxide (NO), inhibiting the expression of inducible nitric oxide synthase (iNOS), and inhibiting the activity and the expression inhibition activity of interleukin-1ß(IL-1ß) which is pro-inflammatory cytokine, and has no cytotoxicity. Therefore, the present invention can be valuably used in medicines for preventing and treating inflammatory diseases, in health food, or in functional feed.

Description

Pharmaceutical composition for the prevention and treatment of inflammatory diseases comprising the bark of lacquer

The present invention relates to an anti-inflammatory composition and a method of treatment using the same.

Inflammation is the body's response to external invasive factors, such as infections, and protects the body by preventing external invasive factors from spreading. However, excessive inflammation can cause a variety of diseases. The development of many inflammation-related diseases is associated with the activation of macrophages and the excessive production of inflammation-related factors. Representative inflammation-related factors include IL-1β, TNF-α and nitrogen oxide (NO). They induce inflammation to remove external invaders, and when excessively produced, they destroy the tissues in vivo.

IL-1β is produced mainly by macrophages activated with pro-inflammatory cytokines and is known to be involved in various immune and inflammatory responses. NO is a free radical produced by various NOSs (eNOS, nNOS, iNOS, etc.) and is known to be involved in antibacterial, anticancer and vasodilating effects. In an inflammatory state, macrophages express large amounts of inducible nitric oxide synthase (iNOS), thus producing excess NO. Expression of IL-1β and iNOS is regulated at the transcription level, and NF-κB is a transcription factor that plays an important role in the expression of these inflammatory mediators. NF-κB is also known to play an important role in the expression of these inflammatory mediators as well as various immune and inflammatory processes.

Rhus succedanea is a dicotyledonous plant belonging to the Arachnoid family, which grows in low areas and is 13 m high. Leaves are alternate, about 30 cm long, 7-15 small leaves, long oval or elliptical basso pointed. Flowers are mixed with male and female. May bloom and yellow-green. Calyx, petals, and stamens are 5 each, and female flower has 5 small stamens, 1 ovary, and pistil is divided into 3 parts. Fruits are nucleus, flat, ball-shaped, yellow, without hairs, ripen in October, and harvested from the skin of fruit. It is distributed in Korea (Jeju Island, Jeonnam), Japan, China, Korea, and the Himalayas. It grows in the wild and is raised for ornamental purposes, but there is no record of medicinal use. Unlike the Rhus verniciflua, the lacquer does not rise.

It has been reported that sumac extract has anticancer, analgesic, antioxidant and anti-inflammatory effects (Kitts DD et al. , J Toxicol Environ Health A, 64: 357-371, 2001; Kim IT et al. , J Ethnopharmacol , 94: 165-173, 2004; Lim KT et al. , Food Chem Toxicol , 39: 229-237, 2001; Lin YM et al. , Planta Med , 65: 120-125, 1999). However, little is known about the pharmacological activity of lacquer bark.

Accordingly, the present inventors prepared the extract of the barley lacquer immature fruit, the extract is rapidly increased by inflammation, IL-1β and NO production, IL-1β and iNOS expression and NF-κB activity inhibition, dermatitis inhibition and death by sepsis The present invention was completed by confirming that it can be used as a pharmaceutical composition for anti-inflammatory as a safe substance having excellent anti-inflammatory effect and no cytotoxicity.

An object of the present invention is to provide an anti-inflammatory treatment composition and a method for treating inflammation using the composition.

In order to solve the above object, the present invention provides an anti-inflammatory composition containing an extract of Rhus succedanea immature.

In addition, the present invention provides an anti-inflammatory skin external preparation containing a barley lacquer immature fruit extract.

In addition, another object of the present invention to provide a functional food for preventing or improving inflammation containing a barley lacquer immature fruit extract.

In addition, another object of the present invention to provide a functional feed composition for preventing or improving inflammation containing barley lacquer immature fruit extract.

In addition, the present invention provides a method for treating inflammation using the composition.

Immature lacquer extract of the present invention is characterized by inhibiting NOx production, inhibiting the expression of inducible nitrogen oxide synthase (iNOS), and inhibiting and inhibiting the expression of interleukin-1β (IL-1β), an proinflammatory cytokine. As it has anti-inflammatory effect and no cytotoxicity, it can be usefully used in medicine, dietary supplement or functional feed for the prevention and treatment of inflammatory diseases.

Figure 1 is a view showing the results of confirming the cytotoxicity of the extract of the present invention, macrophages.

2 is a diagram showing the inhibitory effect of IL-1β and iNOS gene expression induced by LPS treatment in macrophages of the extract of the present invention.

3 is a diagram showing the effect of inhibiting NF-κB activity induced by the treatment of LPS in macrophages of the extract of the present invention:

4 is a diagram showing the dermatitis inhibitory effect of the extract of the present invention induced by the application of phorbol ester in BALB / c mice.

5 is a diagram showing the inhibitory effect of death induced by LPS in BALB / c mice of the extract of the present invention.

In order to achieve the above object, the present invention provides a pharmaceutical composition for anti-inflammatory containing Rhus succedanea immature fruit extract as an active ingredient.

In addition, the present invention provides an anti-inflammatory skin external preparation containing the bark lacquer immature fruit extract as an active ingredient.

In another aspect, the present invention provides a functional food for preventing or improving inflammation containing a barley lacquer immature fruit extract as an active ingredient.

In addition, the present invention provides a functional feed composition for preventing or improving inflammation containing a barberry lacquer immature fruit extract as an active ingredient.

In addition, the present invention provides a method for treating inflammation comprising administering a pharmaceutically effective amount of immature lacquer ivy extract to an individual suffering from an inflammation-related disease.

Hereinafter, the term used by this invention is demonstrated.

As used herein, the term "anti-inflammatory" or "inhibition of inflammation" refers to chronic inflammation that has progressed to a disease state, an inflammatory response generally caused by a harmless substance such as pollen, an inflammatory response caused by an inflammatory substance such as lipopolysaccharide (LPS), and asthma. It also refers to suppression of inflammatory reactions harmful to the human body, such as inflammatory reactions caused by autoimmune reactions such as rheumatoid arthritis.

Hereinafter, the present invention will be described in detail.

The present invention provides an anti-inflammatory pharmaceutical composition containing Rhus succedanea immature fruit extract as an active ingredient.

The gossamer lacquer immature extract

1) step of extracting the dried barberry lacquer into powder and adding an extraction solvent;

2) filtering the extract of step 1); And

3) It is preferably prepared by a manufacturing method comprising the step of concentrating the extract of step 2) under reduced pressure, but not limited to the above method.

The immature lacquer can be used without limitation, such as harvested, cultured or commercially available, in a preferred embodiment, it is preferable to use after drying to remove the foreign matter and salt by washing with water after harvesting.

The bark lacquer immature fruit extract may be prepared by conventional extraction methods in the art, such as ultrasonic extraction, filtration and reflux extraction. Preferably it may be an extract extracted from the bark lacquer unripe and dried with water, a lower alcohol of C ₁ ~ C ₄ or a mixed solvent thereof, more preferably may be an extract extracted with a lower alcohol of C ₁ ~ C ₄ , Most preferably, the extract may be extracted with methanol or ethanol. In a preferred embodiment, the immature lacquer tree and immature dry matter are ground to an appropriate size, put in an extraction container, methanol is extracted, and filtered using a sonicator, and then filtered with a filter paper to obtain a methanol extract. At this time, the volume of the methanol solvent is preferably 5 to 15 times the volume of the ground sample. The extraction time is preferably 1 to 5 days, most preferably 3 days. Thereafter, a method such as concentration or lyophilization may be further roughened.

In an embodiment of the present invention, the barley lacquer quince extract prepared by the above method inhibited IL-1β and NOx (NO) production induced by LPS (lipopolysaccharide) treatment in a concentration-dependent manner (see Table 1). The expression of the IL-1β gene and the iNOS gene, a NO producing enzyme, was concentration-dependently inhibited (see FIG. 2). In addition, it is known that NF-κB plays an important role in gene expression of inflammatory mediators such as IL-1β and iNOS. The extract of the present invention suppressed NF-κB activity induced by LPS treatment in a concentration-dependent manner. (See Figure 3).

In addition, in the embodiment of the present invention, the dermatitis induced by phorbol ester by the extract of the present invention was concentration-dependently reduced (see FIG. 4), and it was confirmed that the death of sepsis mice induced by LPS was reduced ( See FIG. 5).

In addition, in the examples of the present invention, the extract of the present invention was shown to have no cytotoxicity at all, especially at a concentration of 0.1-3 μg / ml, inducing an anti-inflammatory effect (see FIG. 1).

In view of this, the barley lacquer extract of the present invention is excellent in anti-inflammatory effect, it is judged to be very safe bar can be very useful in pharmaceutical compositions for anti-inflammatory.

The present invention contains 0.1 to 50% by weight of the barberry lacquer extract of the present invention with respect to the total weight of the pharmaceutical composition as an active ingredient, and may include a pharmaceutically acceptable carrier, excipient or diluent. Dosage forms of the pharmaceutical compositions of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection. The salt is not particularly limited as long as it is pharmaceutically acceptable, and for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, methanesulfonic acid , Benzene sulfonic acid, toluene sulfonic acid, naphthalene sulfonic acid and the like can be used.

The pharmaceutical composition of the present invention may be in various oral or parenteral formulations. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose (at least one compound). lactose) and gelatin. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Inflammation may occur in a human body such as chronic inflammation progressing to a disease state, an inflammatory response generally caused by a harmless substance such as pollen, an inflammatory response caused by an inflammation-causing substance such as LPS, or an autoimmune response such as asthma or rheumatoid arthritis. Including but not limited to all harmful inflammatory reactions.

The subject to which the pharmaceutical composition of the present invention can be applied is a vertebrate, preferably a mammal, more preferably an experimental animal such as a rat, mouse, rabbit, guinea pig, hamster, dog, cat, and most preferably Are protozoan animals such as chimpanzees and gorillas.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and when parenteral administration, it is preferable to select an external skin or intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injection method. Most preferably, it is used for external skin.

The dosage of the pharmaceutical composition of the present invention varies depending on the weight, age, sex, health status, diet, time of administration, administration method, excretion rate and severity of the disease, the daily dosage is immature lacquer 0.1 to 100 mg / kg, preferably 30 to 80 mg / kg, more preferably 50 to 60 mg / kg, and can be administered 1 to 6 times per day based on the amount of the extract.

The pharmaceutical 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.

In addition, the present invention provides an anti-inflammatory skin external preparation containing the bark lacquer immature fruit extract as an active ingredient.

In an embodiment of the present invention, the barley lacquer extract of the present invention is a concentration of IL-1β and NO production induced by LPS treatment, the expression of the iNOS gene, the IL-1β and NO producing enzyme and NF-κB activity Dependently (see Table 1 and Figures 2-3). In addition, the dermatitis induced by phorbol ester by the extract of the present invention was concentration-dependently reduced (see FIG. 4), and it was confirmed that death of sepsis mice induced by LPS was reduced (see FIG. 5). In addition, the extract of the present invention showed no cytotoxicity at all, especially at a concentration of 0.1-3 μg / ml, inducing anti-inflammatory effects (see FIG. 1).

In view of this, the bark lacquer of the present invention immature fruit extract is excellent anti-inflammatory effect, it is determined that it is very safe, it can be used very useful in anti-inflammatory skin external preparations.

Inflammation may occur in a human body such as chronic inflammation progressing to a disease state, an inflammatory response generally caused by a harmless substance such as pollen, an inflammatory response caused by an inflammation-causing substance such as LPS, or an autoimmune response such as asthma or rheumatoid arthritis. Including but not limited to all harmful inflammatory reactions.

In the case of using the barley lacquer extract of the present invention as an external preparation for skin, it is additionally a fatty substance, an organic solvent, a dissolving agent, a thickening agent and a gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance. For surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or skin It may contain adjuvants commonly used in the field of dermatology, such as any other ingredients commonly used in external preparations. In addition, the ingredients may be introduced in amounts generally used in the field of dermatology.

In another aspect, the present invention provides a functional food for preventing or improving inflammation containing a barley lacquer immature fruit extract as an active ingredient.

In an embodiment of the present invention, the barley lacquer extract of the present invention is a concentration of IL-1β and NO production induced by LPS treatment, the expression of the iNOS gene, the IL-1β and NO producing enzyme and NF-κB activity Dependently (see Table 1 and Figures 2-3). In addition, the dermatitis induced by phorbol ester by the extract of the present invention was concentration-dependently reduced (see FIG. 4), and it was confirmed that death of sepsis mice induced by LPS was reduced (see FIG. 5). In addition, the extract of the present invention showed no cytotoxicity at all, especially at a concentration of 0.1-3 μg / ml, inducing anti-inflammatory effects (see FIG. 1).

In view of this, the bark lacquer of the present invention immature fruit extract is excellent anti-inflammatory effect, it is judged to be very safe bar, can be very useful for preventing or improving health functional foods.

Inflammation may occur in a human body such as chronic inflammation progressing to a disease state, an inflammatory response generally caused by a harmless substance such as pollen, an inflammatory response caused by an inflammation-causing substance such as LPS, or an autoimmune response such as asthma or rheumatoid arthritis. Including but not limited to all harmful inflammatory reactions.

The functional food according to the present invention may be, for example, various functional foods such as chewing gum, caramel products, candy, ice cream, confectionery, beverage products such as soft drinks, mineral water, alcoholic beverages, and health functional foods including vitamins and minerals. Can be.

It can be used as it is, or can be used in combination with other food or food ingredients, and can be suitably used according to a conventional method. The blending amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or hygiene). In general, during the preparation of food or beverages, the bark of the bark of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less with respect to the raw material. However, in the case of long-term intake for health and hygiene or health control purposes, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

The functional food of the present invention may contain various flavors, natural carbohydrates, and the like as additional ingredients. Natural carbohydrates described above are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin, cyclodextrin, sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate is preferably selected in the range of 0.01 to 0.04 parts by weight, preferably about 0.02 to 0.03 parts by weight, per 100 parts by weight of the health food of the present invention.

In addition to the above, the functional food of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols. And carbonation agents used in carbonated beverages. In addition, the functional food of the present invention may contain a flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. Although the ratio of such an additive is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health food of the present invention.

In addition, the present invention provides a functional feed composition for preventing or improving inflammation containing a barberry lacquer immature fruit extract as an active ingredient.

In an embodiment of the present invention, the barley lacquer extract of the present invention is a concentration of IL-1β and NO production induced by LPS treatment, the expression of the iNOS gene, the IL-1β and NO producing enzyme and NF-κB activity Dependently (see Table 1 and Figures 2-3). In addition, the dermatitis induced by phorbol ester by the extract of the present invention was concentration-dependently reduced (see FIG. 4), and it was confirmed that death of sepsis mice induced by LPS was reduced (see FIG. 5). In addition, the extract of the present invention showed no cytotoxicity at all, especially at a concentration of 0.1-3 μg / ml, inducing anti-inflammatory effects (see FIG. 1).

In view of this, the bark lacquer of the present invention immature fruit extract is excellent anti-inflammatory effect, it is judged to be very safe, it can be very useful in the functional feed composition for preventing or improving inflammation.

Poultry, livestock, and the like, the feed composition can be steadily ingested, thereby preventing inflammation and treating already occurring inflammation.

In addition, the present invention provides a method for treating inflammation comprising administering a pharmaceutically effective amount of immature lacquer ivy extract to an individual suffering from an inflammation-related disease.

In an embodiment of the present invention, the barley lacquer extract of the present invention is a concentration of IL-1β and NO production induced by LPS treatment, the expression of the iNOS gene, the IL-1β and NO producing enzyme and NF-κB activity Dependently (see Table 1 and Figures 2-3). In this result, the bark immature fruit extract of the present invention inhibits IL-1β and NO production, and effectively inhibits inflammation by inhibiting the expression of the IL-1β and NO producing enzyme iNOS gene and NF-κB activity. It can be seen that.

In addition, experiments were conducted using balb / c mice to confirm that the present invention was actually effective in vivo (see Examples 6 and 7). As a result, it was confirmed that the dermatitis induced by phorbol ester by the extract of the present invention was concentration-dependently (see FIG. 4), and the death of sepsis mice induced by LPS was reduced (see FIG. 5). As a result, it can be seen that the barley lacquer fruit extract of the present invention effectively inhibits inflammation in vivo.

In addition, in order for the extract to be used in vivo, it should be harmless to the living body, and cytotoxicity was measured to confirm this. As a result, the extract of the present invention showed no cytotoxicity at all, especially at a concentration of 0.1-3 μg / ml, which induces an anti-inflammatory effect (see FIG. 1).

As a result, it can be seen that the barley lacquer extract of the present invention can inhibit the effective inflammatory response in vivo.

In the treatment method of the present invention, the inflammation is also possible for inflammatory reactions other than those caused by LPS shown in the examples or inflammatory reactions with phorbol esters. In particular, the extract of the present invention inhibits IL-1β expression or inducible nitrogen oxide (NO) synthase (iNOS) expression, and thus is generally harmless, such as chronic inflammation and pollen, which can occur through this mechanism. Harmful inflammatory reactions to the human body, including inflammatory reactions by inflammatory reactions, inflammatory reactions by inflammatory agents such as LPS, and autoimmune reactions such as asthma or rheumatoid arthritis.

 The subject to which the treatment method can be applied may be a vertebrate animal such as a mouse in addition to the mouse supported in the Examples. It is preferably a mammal, more preferably an experimental animal such as a rat, a mouse, a rabbit, a guinea peak, a hamster, a dog, a cat, and most preferably the same ape species such as chimpanzees and gorillas.

 The treatment method in the embodiment shows the external skin and intraperitoneal injection method in the embodiment, but because there is no cytotoxicity, can be administered orally or parenterally in addition to the above method, rectal, vein, muscle, subcutaneous when parenteral administration Intrauterine epidural or cerebrovascular injection is preferred. Most preferably, it is used for external skin as in Example.

The dosage of the pharmaceutical composition in the treatment method may vary in the range depending on the weight, age, sex, health condition, administration time, administration method, excretion rate and severity of the patient.

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

However, the following Examples, Experimental Examples, and Production Examples specifically illustrate the present invention, and the contents of the present invention are not limited to Examples, Experimental Examples, and Production Examples.

Example 1 Preparation of Immature Lacquer and Methanol Extracts

The immature fruit of Rhus succedanea obtained in Jeju Island was washed, dried completely using a dryer, and finely chopped.

The immature fruit of the finely chopped lacquer tree was immersed in 10 times of water and extracted by hot water for 3 days. The extract was cooled, and the solvent of the supernatant obtained after filtration was evaporated in vacuo to concentrate the extract. The yield was 5.1% (w / w).

<Example 2> Preparation of the bark lacquer immature and methanol extract

The immature fruit of the finely chopped lacquer tree was immersed in 10% of 100% methanol (HPLC grade) and extracted using a sonicator (Branson Ultrasonics corporation, USA) for 3 days at room temperature. The extract was concentrated by evaporation in vacuo, yield 8.3% (w / w).

<Example 3> Preparation of bark lacquer and immature ethanol extract

The extract was obtained by the method of Example 2 using 100% ethanol (HPLC grade). The yield was 7.4% (w / w).

Experimental Example 1 Cell Culture and Extract Treatment

<1-1> Culture of RAW 264.7 Cells

RAW 264.7 macrophages from BALB / c mice in Dulbecco's modified Eagle medium (Invitrogen, USA) medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U / ml) and streptomycin (100 μg / ml) Cell lines (ATCC TIB71) were mixed and seeded in 96 well plates at a concentration of 5 × 10 ⁴ cells per well and incubated at conditions of 5% CO ₂ and 37 ° C. for 6 hours.

<1-2> Extract Treatment on RAW 264.7 Cells

After transplanting the cells to a 96 well plate at a concentration of 5 × 10 ⁴ cells per well, treating LPS (lipopolysaccharide) at a concentration of 300 ng / ml, the extract dissolved in DMSO was 0.1, 0.3, 1, and 3 Treatment was at a concentration of μg / ml.

<1-3> Preparation of experimental animals

BALB / c female mice of 18-22 g body weight (CORETECH, Gyeonggi-do) were cultured in the animal nursery under constant conditions (temperature: 21 ± 2 ° C., contrast: 12 hour light / dark cycle). Free intake of feed and drinking water was allowed, and sufficient water and food were provided until the start of the experiment, and the animals were purified in the animal cage for 7 days.

Experimental Example 2 Cytotoxicity Test of Immature Oak Extract

After 24 hours for the cells treated with Experimental Example 1-2, MTT test was performed using a Cell Proliferation Assay Kit (Roche, Switzerland).

Specifically, the culture solution was removed, MTT reagent was added, and the reaction was performed at 37 ° C. incubator for 4 hours, and then MTT reagent was removed. After 100 μl DMSO was added, the cell viability was measured by measuring the absorbance at 540 nm using an ELISA reader.

As a result, as shown in Figure 1, even if the extract was treated with a concentration of 3 ㎍ / ㎖ in macrophages, it was confirmed that there is no cytotoxicity.

Experimental Example 3 Confirmation of Inhibition of IL-1β and NO Production by Immature Oak Extract

<3-1> Inhibition of IL-1β Production

After cell lysate was obtained 24 hours after the cells treated with Experimental Example 1-2, IL-1β production was performed using ELISA (Enzyme-linked immunosorbent assay) method using ELISA kit (USA) of R & D systems. It was measured by.

Specifically, 100 μl of goat anti-mouse IL-1 antibody was added to a 96-well plate and left for 16 hours at room temperature. Wash three times with wash buffer (0.05% Tween 20 in PBS). 200 μl of blocking buffer (1% BSA in PBS) was added thereto, and the mixture was left at room temperature for 1 hour and washed three times using a washing buffer. 100 μl of the standard material and the sample were added to the plate, and the plate was left at room temperature for 2 hours, and then washed three times with washing buffer. 100 μl of Biotinylated anti-mouse IL-1 antibody was dispensed and left at room temperature for 2 hours and washed three times with washing buffer. After dispensing Streptavidin-HRP, it was left at room temperature for 20 minutes and washed three times with washing buffer. Subsequently, 100 μl of substrate solution was added to each well and allowed to stand for 30 minutes while keeping the light at room temperature. Then, 100 μl of a stop solution (2N H ₂ SO ₄ ) was added to stop the reaction. The concentration was calculated after measuring the absorbance at 450 nm using an ELISA reader.

As a result, as shown in Table 1, it was confirmed that IL-1β production rapidly increased by treatment with LPS was concentration-dependently reduced by the extract of the present invention.

<3-2> NO production inhibition

After 24 hours for the cells treated with Experimental Example 1-2, the amount of NO contained in the culture supernatant was measured.

Specifically, 50 μl of the culture supernatant was taken and mixed with the same volume of Griess reagent and the absorbance at 550 nm was measured. The amount of nitrite in the sample was quantified using various concentrations of sodium nitrite standard curves dissolved in the same medium.

As a result, as shown in Table 1, it was confirmed that the NO production rapidly increased by treatment in LPS is concentration-dependently reduced by the extract of the present invention.

Table 1 Example 1 Example 2 Example 3 (Μg / ml) VH 0.1 0.3 One 3 VH 0.1 0.3 One 3 VH 0.1 0.3 One 3 IL-1β production (ng /) 1.17 1.50 1.34 0.53 0.22 1.05 1.58 1.29 0.54 0.25 1.19 1.34 1.12 0.45 0.15 NO production (nmole / 10 ⁶ cells) 39.6 43.9 42.2 34.7 18.0 41.7 42.8 42.8 37.4 14.2 42.2 42.2 42.2 34.7 19.6

That is, it was confirmed that the extract of the present invention inhibits the activation of macrophages.

In addition, since the effect of the extract of Examples 1 to 3 is excellent to a similar degree, in the following experimental example was performed using only the methanol extract having the best effect of the extract.

Experimental Example 4 Inhibition of IL-1β and iNOS Gene Expression of Immature Oak Extracts

After 6 hours of cells treated with Experimental Example 1-2, after obtaining total RNA, IL-1β and iNOS gene expression was measured using the reverse transcriptase-polymerase chain reaction (RT-PCR) method.

Specifically, the cells were recovered after the end of the culture and extracted with total RNA using RNeasy mini kit (QIAGEN, USA), and then 1 ㎍ of total RNA and 1 ol of oligo-d (T) (Invitrogen, USA, 0.5 ㎍ / μl) After filling up to 50 μl of distilled water, the reaction mixture was placed in AccuPower RT-premix (Bioneer, Korea) for RT-PCR reaction. 5 minutes at 70 ° C, 5 minutes at 4 ° C, 60 minutes at 42 ° C, 5 minutes at 94 ° C, and 5 minutes at 4 ° C to obtain cDNA. PCR was performed using the primer pairs shown in Table 2 using the cDNA (3-5 μl) as a template. For distribution equal to the accuracy of PCR, make a master mix containing Taq polymerase, 2.5 mM dNTPs, primer pairs and 10X buffer, divide into each reaction tube, and add the template DNA or distilled water (negative control) as a template. PCR was performed. More specifically, denature for 5 minutes at 95 ℃, turn 35 revolutions at the conditions of 95 ℃ 45 seconds, 65 ℃ 45 seconds, 72 ℃ 45 seconds, and then stretched for 5 minutes at 72 ℃, then at 4 ℃ It was carried out in the order of cooling. The PCR product generated after the completion of the reaction was electrophoresed on 1.5% agarose gel, and the size of the band was confirmed using a transilluminator.

TABLE 2 Gene name Genebank no. SEQ ID NO: Primer pairs Sequence IL-1β NM_008361 One Forward direction 5'-TGCAGAGTTCCTACATGGTCAACCC-3 ' 2 Reverse 5'-GTGCTGCCTAATGTCCCCTTGAATC-3 ' iNOS NM_010927 3 Forward direction 5'-CTGCAGCACTTGGATCAGGAACCTG-3 ' 4 Reverse 5'-GGGAGTAGCCTGTGTGCACCTGGAA-3 ' β-actin NM_007393 5 Forward direction 5'-TGGAATCCTGTGGCATCCATGAAAC-3 ' 6 Reverse 5'-TAAAACGCAGCTCAGTAACAGTCCG-3 '

As a result, as shown in Figure 2, it was confirmed that IL-1β and iNOS expression rapidly increased by treatment in LPS concentration-dependently reduced by the extract of the present invention. Expression of the control β-actin did not decrease.

Experimental Example 5 Confirmation of NF-κB Inhibition of Immature Oak Extracts

PNF-κB-SEAP (secreted alkaline phosphatase; Han MH et al. , Int Immunopharm , 7: 1651-1658, 2007) using Lipofectamine Plus reagent (Invitrogen, USA) on cells cultured by the method of Experimental Example 1-1 ) Were transduced.

The cells were transplanted into 96 well plates at a concentration of 5 × 10 ⁵ cells per well, and then treated with LPS at a concentration of 300 ng / ml, and then the extracts were prepared at concentrations of 0.1, 0.3, 1 and 3 μg / ml. Treated.

After 24 hours, the supernatant was obtained from the treated cells, and then SEAP activity was measured using the Great EscAPe Chemiluminescence Detection Kit (Clontech, USA) and VICTOR ^™ Light (Perkin Elmer, USA).

As a result, as shown in Figure 3, it was confirmed that the NF-κB activity increased by treatment in LPS concentration-dependently inhibited by the extract of the present invention.

NF-κB is known to play an important role in gene expression of inflammatory mediators such as IL-1β and iNOS. In other words, the inhibition of IL-1β and iNOS gene expression by the extract of the present invention was found to be due to the inhibition of NF-κB activity.

Experimental Example 6 Determination of Dermatitis Inhibition of Immature Oak Extract

Phorbol esters (20 μl, 15 mg / ml), known to cause dermatitis, were applied to the ears of BALB / c mice prepared by the methods of Experimental Examples 1-3. Then, the extract dissolved in AOO (acetone: olive oil = 4: 1) was treated with 0.05, 0.5 and 5 ㎍ / ear.

After 4 hours, the thickness of the ear was measured and the degree of edema was measured. The thickness of the ear was measured using a digital thickness gauge (Digimatic Indicator, Japan).

As a result, as shown in Figure 4, the inflammation and edema was induced, the ear thickness was significantly increased, but by applying the extract of the present invention, it was confirmed that the ear thickness was reduced in a concentration-dependent manner.

Experimental Example 7 Inhibition of Sepsis of Immature Oak Extracts

BALB / c mice prepared by the methods of Experiments 1-3 were intraperitoneally injected with 15 mg / kg LPS, 10 mg / kg of extract dissolved in phosphate buffered saline (PBS) added with 5% Tween 80, and PBS added with Tween 80. Injection.

As a result, as shown in FIG. 5, all mice died within 2 days after LPS treatment, but in the extract treatment group of the present invention, about 70% of the mice died on day 2, and about 10% of mice after 7 days. Survived.

In other words, it can be seen that the extract of the present invention inhibits sepsis caused by LPS.

The present invention relates to an anti-inflammatory composition containing Rhus succedanea immature fruit extract as an active ingredient and a method for treating inflammation using the same, the immature lacquer fruit of the present invention exhibits an anti-inflammatory effect, and cytotoxicity is Therefore, it can be usefully used in medicines, nutraceuticals or functional feed for the prevention and treatment of inflammatory diseases.

Claims (17)

Rhus succedanea anti-inflammatory pharmaceutical composition containing an immature fruit extract as an active ingredient. According to claim 1, wherein the extract is an anti-inflammatory pharmaceutical composition, characterized in that extracted with water, alcohol or a mixture thereof. According to claim 2, wherein the alcohol is an anti-inflammatory pharmaceutical composition, characterized in that methanol. The anti-inflammatory pharmaceutical composition according to claim 1, wherein the barley lactose immature fruit extract has an inhibitory effect on IL-1β expression. The anti-inflammatory pharmaceutical composition according to claim 1, wherein the extract of immature lacquer bark has an inhibitory effect on the expression of inducible nitrogen oxide (NO) synthase (iNOS). The method of claim 1, wherein the inflammation includes chronic inflammation progressing to a disease state, an inflammatory response generally caused by a harmless substance, an inflammatory response caused by an inflammation causing substance, and an inflammatory response caused by an autoimmune response. Pharmaceutical compositions. Anti-inflammatory skin external preparation containing Rhus succedanea immature fruit extract as an active ingredient. 8. The anti-inflammatory treatment according to claim 7, wherein the inflammation comprises chronic inflammation progressing into a disease state, an inflammatory response generally caused by a harmless substance, an inflammatory response caused by an inflammation causing substance, and an autoimmune response. Skin external preparations. Rhus succedanea Functional food for preventing or improving inflammation containing immature fruit extract as an active ingredient. 10. The method of claim 9, wherein the inflammation comprises chronic inflammation progressing into a disease state, generally an inflammatory response by a harmless substance, an inflammatory response by an inflammation causing substance and an inflammatory response by an autoimmune response. Functional food for improvement. Rhus succedanea ( Rhus succedanea ) A functional feed composition for preventing or improving inflammation containing an immature fruit extract as an active ingredient. 12. The method of claim 11, wherein the inflammation comprises chronic inflammation progressing into a disease state, generally an inflammatory response by a harmless substance, an inflammatory response by an inflammation causing substance and an inflammatory response by an autoimmune response. Functional feed composition for improvement. A method of treating inflammation comprising administering a pharmaceutically effective amount of immature sumac extract to an individual suffering from an inflammation related disease. The method of claim 13, wherein the inflammation comprises chronic inflammation progressing into a disease state, generally an inflammatory response by a harmless substance, an inflammatory response by an inflammation causing substance, and an inflammatory response by an autoimmune response. .  14. The method of claim 13, wherein said inflammation is inflammation caused by LPS. The method of claim 13, wherein the inflammation is dermatitis.  14. The method of claim 13, wherein the method of inhibiting IL-1β expression or inducible nitrogen oxide (NO) synthase (iNOS) expression.

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