KR20130013743A - Compositions for preventing, improving or treating th1- or th2-mediated immune diseases containing angelica gigas extracts - Google Patents

Abstract

PURPOSE: A composition containing an Angelica gigas extract or decursin for preventing, relieving, or treating Th1 cell- or Th2 cell-mediated immune diseases is provided to induce CD4+Foxp3+Tregs formation and to reduce Th1 or Th2 immune response. CONSTITUTION: A pharmaceutical composition for preventing or treating Th1- or Th2-mediated immune diseases contains an Angelica gigas extract or decursin of chemical formula 1 as an active ingredient. A food composition for preventing or treating the immune diseases contains the extract or decursin of chemical formula 1 as an active ingredient. A cosmetic composition for preventing or treating the immune diseases contains the extract or decursin of chemical formula 1 as an active ingredient. The active ingredient increases the amount of Foxp3. THE Th1-mediated immune diseases are atopic dermatitis, contact dermatitis, allergic rhinitis, hay fever, or allergic bronchial asthma. The Th1-mediated immune diseases are transplantation rejection, autoimmune diseases, or inflammatory diseases.

Description

Compositions for Preventing, Improving or Treating Th1- or Th2-Mediated Immune Diseases Containing Angelica gigas Extracts}

The present invention relates to a composition for preventing, ameliorating or treating a Th1 or Th2-mediated immune disease containing Angelica extract.

Foxp3 is a transcriptional factor mainly present in Regulatory T cells derived from the thymus and present in cells with CD4 + CD25 + labeled antigens, the function of which is T-expressing Foxp3. Interleukin-2 has been shown to affect T cells that are potentially responsive to antigens in CD4 + CD25- T cells that have low reactivity to the antigen and do not express Foxp3 differentiated from the thymus when the antigen is recognized. interleukin-2 (IL-2) has a role as a suppressor T cell (suppressor T cell) to suppress the production and cell division. In addition, Foxp3 is not only IL-2 but also a transcription factor NFAT (Nuclear Factor of Activated T-cell) for CD25-T cells through regulator T cells expressing Foxp3 and cell-cell contact therewith. It has been shown to function to inhibit transcriptional regulation of interleukin-4 and interferon-γ (IFN-γ), which are affected by.

In this way, artificially adding Foxp3 or administering microorganisms that promote the expression of Foxp3 is known to improve autoimmune diseases, allergic diseases and inflammatory diseases by improving various immune diseases (10-2009-0028624, 10-0913405, 10-0913406).

Meanwhile, while the use of pharmaceutical compositions derived from natural products is increasing worldwide in the field of drug development, many studies have been conducted to find useful substances for human body from herbal medicines traditionally used in Chinese medicine in Korea, China and Japan. ought. This approach has a technical advantage over the chemical synthesis approach due to the fact that the efficacy and stability of various herbals have been proven through thousands of years of empirical prescriptions and clinical trials in the private sector.

However, as a method of anti-inflammatory and itching suppression using common natural products, it is known that its effectiveness is not effective in improving various T helper cell (Th2) mediated immune diseases such as atopic dermatitis, and its practicality is not high. to be.

Among the herbal medicines, Angelica is a medicinal plant mainly produced in the high mountains of Gangwon-do, Korea. Zhuhai Meteorological Relief, On-hak Han-yeol, In-depth skin care, Lacrimal Lavender Meteorology, Evil Changyang, Geumchang (金 瘡). Consonant paper. Aka eungui (一名 乾 歸). ”(Danggui is sweet and creepy. Heals coughs and stomachs. Heals the fever and creepy symptoms caused by thermophiles. Heals uterine bleeding and infertility. When you have a variety of swelling and trauma, drink a month to drink, also known as a kerchief.) It is an herbal medicine that has been applied to the clinical practice of oriental medicine with the effect of saving blood, controlling menstruation and stopping pain, and smoothing the stool by lubricating the intestines. 184-189; et al., Medicinal Herbal Medicine Applications, 1982, 399-402; Heo Jun, Dongbobogam, 1983, 114,162,208,455,630,727). Modern clinic is widely applied to arrhythmia, ischemic stroke, senile chorea, cerebral arteriosclerosis, sickle cell anemia, arrhythmia, thromboembolic vasculitis, vascular headache, hypertension, uterine dehydration and insomnia. Chinese Pharmacology, 1997, 551-554; Myo Chamsam, Forensic Pharmacological Women's Clinical Trial, 1998,412-427; Wang, Bong-Sang, Modern Chinese Pharmacology, 1997, 1290-1304).

Angelica is a plant belonging to the genus Angelica belonging to the family Umbelliferae, Angelica gigas Nakai in Korea, Angelica sinensis (Oliv.) Diels in China, and Angelica in Japan. Angelica acutiloba (Sieb. & Zuc.) Kitagawa) (Duk-kyun Anh, Korea Headquarters of Korea, 1998, 670-671; China's Pharmacy Committee, People's Republic of China Pharmacy中華人民共和國 藥典), 1995, 109; Japan Fair Trade Association, Japanese Pharmacy Defense Commentary, 1986, 660-664.

The known components of Angelica gigas are coumarin-based decursin, decursinol, butylidenephthalide, ligustilide, and butylphthalide ( butylphthalide) are known (Biochemistry Research Society, Hyundai Biochemistry, 1994, 36-33; An Duk-kyun et al., Chinese Traditional Chinese Dictionary, 1998, 159-1168, 5688-5689; National Chinese Medicine Expatriation, Chinese Herbal Medicine, 1999, 874-875,888-889,893-903), Angelica and monogalia are n-butylidenephthalide, n-butylidenephthalide, ligustilide, butylphthalide, β-sitosterol, n-valerophenone-o-carboxylic acid, bergapten, vitamin B ₁₂ (vitamineB ₁₂ ), α-pinene, myrcene, mycene, nicotinic acid It is known to contain ingredients such as nicotinic acid, folic acid, and falcaindiol.海), 1993, 1608-1612; Wang, Bong-sang, Modern Chinese Pharmacology, 1997, 1290-1304; Wang Yak, Chinese Pharmacological Medicinal Application, 1983, 424-434; Et al., Chung-Ang University Research Application, 1997, 1807-1849).

According to various pharmacological studies on the extract of Angelica gigas, the ether extract of Angelica gigas has been shown to have an excitatory effect on the extraction and uterus of rabbits and to promote respiration. In addition, it has been found that isolated decusin and decosinol paralyze the rabbit's extraction tube and inhibit respiration, inhibit the frog extraction heart and inhibit spontaneous action in mice (Korean Patent Application No. 1998-44339). number).

On the other hand, ethanol extracts of Angelica gigas are known to contain mainly terpenoid-based coumarines, among which decosin and decosinol angelate are C ₁₉ H ₂₀ as a kind of pyranocoumarin. It is represented by the molecular formula of O ₅ and has a coumarin nucleus of the same structure. Deckerin and decosinol angelate are structural isomers, each of which has a structure in which sesenecioylic acid is bonded to an coumarin nucleus by an ester bond and angelic acid, a structural isomer of senesioyl acid. (angeloic acid) has a combined structure.

Recently, studies on the Angelica gigas have been reported to isolate the decansin from the Angelica gigas and its components, to enhance the activity of protein kinase C and to inhibit the spontaneous action (Ryu et al., 1990; Chi et al., 1998; Ahn and Kim, 1996; Kim et al., 1980), although studies on antimutagenic, antioxidant and apoptosis induction of Angelica gigas have been reported (Wn et al., 1996; Yim et al., 2005; Han et al. , 1998) There is still insufficient research on various Th 1- and Th2-mediated immune diseases such as atopic dermatitis.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made extensive efforts to develop pharmaceutical compositions for the prevention or treatment of Th1-mediated immune diseases and Th2-mediated immune diseases from natural products. As a result, it was confirmed that the decansin, the main component of Angelica extract and extract, effectively inhibited T cell-related cytokines, and applied it as a pharmaceutical composition for the prevention or treatment of Th1-mediated immune diseases and Th2-mediated immune diseases. It can be identified.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating a Th1-mediated immune disease or a Th2-mediated immune disease.

Another object of the present invention to provide a food composition for the prevention or improvement of Th1-mediated immune diseases or Th2-mediated immune diseases.

Another object of the present invention to provide a cosmetic composition for the prevention or improvement of Th1-mediated immune disease or Th2-mediated immune disease.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of Th1-mediated immune disease or Th2-mediated immune disease comprising Angelica extract or Decacin of Formula 1 as an active ingredient.

Formula 1

According to another aspect of the present invention, the present invention provides a food composition for preventing or ameliorating Th1-mediated immune disease or Th2-mediated immune disease, including Angelica extract or Decasin of Formula 1 as an active ingredient.

According to another aspect of the invention, the present invention provides a cosmetic composition for the prevention or improvement of Th1-mediated immune disease or Th2-mediated immune disease comprising a Angelica extract or Decasin of Formula 1 as an active ingredient.

The present inventors have made extensive efforts to develop pharmaceutical compositions for the prevention or treatment of Th1-mediated immune diseases and Th2-mediated immune diseases from natural products. As a result, it was confirmed that decansin, the main component of Angelica extract and extract, effectively inhibited T cell-related cytokines, and applied it as a pharmaceutical composition for preventing or treating Th1-mediated immune diseases and Th2-mediated immune diseases. It can be identified.

According to a preferred embodiment of the present invention, the Angelica is used in the present invention Angelica ( Angelica gigas Nakai). The extract extracted from Angelica gigas is decursin, decursinol, butylidenephthalide, ligustilide, butylphthalide, etc., which is a cumarine-based substance. It includes.

In the present invention, the extract has a meaning commonly used as a crude extract in the art, but broadly also includes an additional fraction (fractionation) of the extract. That is, the Angelica extract is not only obtained by using an extraction solvent, but also includes a product obtained by additionally applying a purification process. For example, fractions obtained by passing an extract through an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (made for separation according to size, charge, hydrophobicity or affinity), etc. Fractions obtained through various purification methods are also included in the extract.

Various methods such as hot water extraction, room temperature extraction, heating extraction and ultrasonic wave, supercritical, etc., which are commonly used to prepare the extract, may be used. Can be used. Preferably, a polar solvent or a nonpolar solvent can be used. Suitable as polar solvents are water, alcohols (preferably methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol, 1-pentanol, 2-butoxyethanol or ethylene glycol), acetic acid, Dimethyl-formamide (DMFO) and dimethyl sulfoxide (DMSO). Suitable as nonpolar solvents are acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1- Pentene, 1-chlorobutane, 1-chloropentane, o -xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform, dichloro Methane, 1,2-dichloroethane, anneal, diethylamine, ether, carbon tetrachloride and THF.

As a method for separating the decusin used in the present invention from true Angelica gigas, extract the dried root of Angelica gigas with alcohol, fractionate the extract with chloroform and perform silica gel column chromatography to obtain a coumarine-based mixture. Preferred is a method of obtaining the above compounds present in the ethyl acetate fraction by performing silica gel column chromatography with hexane: ethyl acetate.

On the other hand, the composition containing the Angelica extract or Decasin of the present invention as an active ingredient can be used for the prevention or treatment of various Th1-mediated immune diseases, diseases or conditions.

Th1-mediated immune disorders involve cytokines, such as IL-1β, IL-2, IL-12, IL-17, IFN-γ or TNF-α, produced by the production and / or activity of Th1 cells Means disease. Th1 cell refers to a subset of helper T cell lymphocytes that are characterized in terms of gene expression, protein secretion and functional activity. For example, Th1 cells synthesize IL-2 and IFN-y, but IL-4, IL-5, IL-10 and IL-13 do not synthesize cytokine expression patterns. Th1 cells are involved in cell-mediated immune responses, organ-specific autoimmune diseases and delayed hypersensitivity reactions to a variety of intracellular pathogens.

According to a preferred embodiment of the present invention, the Th1-mediated immune disease to which the composition of the present invention is applied is transplant rejection, autoimmune disease or inflammatory disease.

In addition, the compositions of the present invention can be used for the prevention or treatment of Th2-mediated immune diseases, diseases or conditions. Th2-mediated immune disease refers to a disease involving IgE and mast cells by the production and activity of allergen-specific Th2 cells. Th2 cells refer to a subset of helper T cell lymphocytes that are specified in terms of gene expression, protein secretion and functional activity. For example, Th2 cells exhibit IL-4, IL-5, IL-10, and IL-13 cytokine expression patterns. Th2 cells are involved in the humoral immune response.

According to a preferred embodiment of the present invention, the Th2-mediated immune disease includes atopic dermatitis, contact dermatitis, allergic rhinitis, high fever and allergic bronchial asthma.

According to a preferred embodiment of the present invention, the composition of the present invention increases the amount of Foxp3. The composition of the present invention induces the formation of CD4 ⁺ Foxp3 ⁺ Tregs and increases the inhibitory activity of naturally occurring Tregs (CD4 ⁺ CD25 ⁺ ) to induce hypo-responsiveness of T cells, resulting in Th1 or Th2 By reducing the immune response, it is effective for treating Th1 cell-mediated diseases or Th2 cell-mediated diseases. In particular, the compositions of the present invention are effective for treating Th2 cell-mediated diseases.

The composition of the present invention may be a pharmaceutical composition for the prophylactic or therapeutic composition of Th1-mediated immune disease or Th2-mediated immune disease, in which case it includes a pharmaceutically acceptable carrier in addition to the above components as an active ingredient. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and agents are Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by skin application, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration and the like.

Suitable dosages of the pharmaceutical compositions of the invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to reaction, Usually a skilled practitioner can easily determine and prescribe a dosage effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.001-10,000 mg / kg.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it with a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person skilled in the art to which the present invention belongs Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

Compositions effective for the prevention or amelioration of Th1 cell-mediated diseases or Th2 cell-mediated diseases of the invention may be prepared from foods, in particular functional food compositions. The functional food composition of the present invention includes components that are ordinarily added during the manufacture of food, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, when prepared with a drink, flavoring agents or natural carbohydrates may be included as additional ingredients in addition to the extract of hawthorn as an active ingredient. For example, natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.); Disaccharides (eg maltose, sucrose, etc.); oligosaccharide; Polysaccharides (eg, dextrins, cyclodextrins, etc.); And sugar alcohols (eg, xylitol, sorbitol, erythritol, and the like). As the flavoring agent, natural flavoring agents (e.g., taumartin, stevia extract, etc.) and synthetic flavoring agents (e.g., saccharin, aspartame, etc.) can be used. Given the ease of access to food, the food of the present invention is very useful for preventing or ameliorating Th1 cell-mediated diseases or Th2 cell-mediated diseases such as inflammatory diseases.

On the other hand, a composition effective for the prevention or amelioration of a Th1 cell-mediated disease or Th2 cell-mediated disease of the present invention may be prepared as a cosmetic composition, in particular a functional cosmetic composition. Ingredients included in the cosmetic composition of the present invention include ingredients commonly used in cosmetic compositions in addition to Angelica extract or Decusin as active ingredients, and include, for example, conventional auxiliaries such as stabilizers, solubilizers, vitamins, pigments and flavorings, and Carrier.

The skin care cosmetic composition of the present invention may be prepared in any form conventionally produced in the art and may be in the form of a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, But are not limited to, water-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations and sprays. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

In the case where the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. Especially, in the case of a spray, a mixture of chlorofluorohydrocarbons, propane / Propane or dimethyl ether.

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.

The features and advantages of the present invention are summarized as follows:

(a) The composition of the present invention is a biomaterial containing Angelica extract or Decasin as an active ingredient, and exhibits very good efficacy in treating Th1 or Th2-mediated immune diseases.

(b) The composition of the present invention induces the formation of CD4 ⁺ Foxp3 ⁺ Tregs and increases the inhibitory activity of naturally occurring Tregs (CD4 ⁺ CD25 ⁺ ) to induce hypo-responsiveness of T cells Thereby treating a Th1-mediated or Th2-mediated immune disease by reducing the Th1 immune response or Th2 immune response.

(c) The composition of the present invention is excellent in safety to the human body because it does not induce apoptosis of T cells in reducing the Th1 immune response or the Th2 immune response.

(d) The composition of the present invention can provide a safe medicine, food or cosmetics because it uses a natural Angelica extract or a compound derived from a natural substance harmless to humans.

1 is a procedural diagram showing the process of fractionating Deckerin from Angelica.
FIG. 2 is a chromatogram showing the results of performing High Performance Liquid Chromatography (HPLC) to separate Decusin from Angelica extract.
Figure 3 is a chromatogram showing the results of analyzing the chemical formula of the decansin after performing the separated nuclear magnetic resonance spectra.
Figure 4 is a graph showing the results of performing the WST-1 test to confirm the cytotoxicity of Deckerin.
Figure 5 is a graph showing the difference in the expression level of histamine (Histamin) known as a pathological factor of allergy after treatment of Angelica extract.
Figure 6 is a graph showing the difference in the degree of inhibition of immunoglobulin E secretion after treatment with Angelica extract and decusin.
FIG. 7 is a graph showing the difference in the expression levels of known pathogenic cytokine in COX-2, Foxp3 and atopy after treatment with Angelica extract and Decusin.
8 is a graph showing the difference in the amount of cytokine expression in T cells after treatment with Angelica extract per concentration.
9 is a graph showing the difference in the expression level of cytokines in T cells after treatment with deckerin by concentration.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, and it is to be understood by those skilled in the art that the present invention is not limited thereto It will be obvious.

Example

Preparation of Angelica Angelica Extract Decker  detach

Angelica Angelica Extract Manufacturer

Angelica gigas Nakai) to extract the solvent fractions. Specifically, 100 g of dried dried Angelica Angelica was extracted with an ultrasonic extractor (Eyela, Japan) in a water bath three times for 1 hour with 85% methanol, followed by a rotary evapoarator (Eyela, Japan). Concentration under reduced pressure afforded a methanol extract.

Solvent from Angelica gigas By fraction  Extract separation

Chloroform and water were added to the methanol extract of Angelica gigas, which was prepared in the same amount, and the mixture was left to shake. The resulting mixture was concentrated under reduced pressure to obtain a chloroform fraction. Subsequently, ethanol acetate was added to the remaining water layer except for the chloroform layer, followed by shaking to obtain an ethanol acetate fraction. Subsequently, an ethanol acetate fraction was obtained, n-butanol was added to the remaining water layer, and the mixture was left to shake and concentrated to obtain an n-butanol fraction. Finally, the remaining water layer was concentrated under reduced pressure to obtain a water fraction (Fig. 1).

Separation of Active Components of Angelica gigas from Solvent Fraction

Yu, et al. (Sugar et al., 2000, 29 (2), 300-7; Yu Kyung-soo, et al., 1990, 21 (1) ), 64-68), the active ingredients of Angelica gigas were separated. Specifically, silica gel column chromatography (Nova pac silica) was performed using toluene: diethylether (1: 1 → 9: 1) using the chloroform fraction obtained in the above. A mixture of coumarin series was obtained. The coumarin-based mixture was subjected to silica gel column chromatography using hexane: ethyl acetate (Hexane: Ethyl acetate) (15: 1) as a solvent.

Contained in Methanol Extract of Angelica gigas Decker  analysis

High Performance Liquid Chromatography (HPLC) was performed to quantify the decusin and decosinol angelate contained in the Angelica gigas extract. Specifically, 10 mg of the decansin isolated above was dissolved in methanol to prepare a standard solution to be quantified by HPLC so that the final volume was 10 ml. The methanol solution was again purified by 500 μl of methanol to give a final volume of 2.5 mL. To prepare a sample solution containing decusin, 20 g of Angelica gigas was washed with water and dried in a shade to make a powder. Then, 150 g of ether was added to 3 g of the powder, and a reflux extractor (Duksan, Korea, extracted and cooled and filtered. After drying the filtrate under reduced pressure, the residue was dissolved in exactly 100 ml of methanol and filtered. Again 10 ml of the methanol filtrate was purged with methanol to be exactly 50 ml with methanol and filtered (0.45 μm). HPLC autoinjector system for 10 μl of the Decasin standard solution or sample prepared above; 2690 separation module, pumps; 2690 pump, detector; 996 photodiode array detector, column; TSK-gel ODS-80-TS, 250 × 4.6 mm, 4 μm, mobile phase; Acetonitrile: 0.03 M Sodium acetate = 1: 1: detect; 320 nm, flow rate; 1.2 ml / min, column temperature; Room temperature) and quantified by investigating the peak appearing according to retention time.

As a result, it was confirmed that the separated decusin had a purity of about 97%, and the decusin contained 129.71 mg per 3,002 mg of sample, which had a 4.23% content (SD = 0.008, CV (%) = 0.09) and a retention time of 22.350 The major peak in minutes appeared (FIG. 2).

Decker  Structural analysis

In addition to the HPLC results of the decosin isolated above, ¹³ C-nuclear magnetic resonance (NMR) and ¹ H-nuclear magnetic resonance spectroscopy were analyzed to analyze the structure.

As a result, the decansin in the isolated compound observed peaks in the ¹ H-nuclear magnetic resonance spectra, presumably the doublet coupling of the ortho-positional electrons of the aromatic ring at 6.23 and 7.59. At 6.80 and 7.16, single peaks were observed and were thought to be peaks that did not couple with ambient electrons in the aromatic ring. In addition, 6 mol of electrons of the geminal singlet, presumed to be methyl groups at 1.36 and 1.39, were observed with peaks of 5.09 (t), 2.87 (dd) and 3.20 (dd) ppm, respectively. This showed a pattern very similar to the pyranokumarin decusin (Table 1).

¹³ C-nuclear magnetic resonance spectrum analysis together with the ¹ H-nuclear magnetic resonance spectrum showed that peaks believed to be due to the aromatic benzene of the coumarin nucleus were 112 to 161 ppm due to the methyl group. Peaks were observed at 20 to 27.3 ppm, respectively (Table 2, FIG. 3), presuming that they were pyranocoumarin-based compounds commonly found in plants of the genus Angelica. The Korean Journal of Food and Nutrition Science, 2000, 29 (2), 300-7; and Yoo, Kyung-Soo et al., Korean Journal of Pharmacognosy, 1990, 21 (1), 64-68).

Therefore, as a result of analyzing ¹ H-nuclear magnetic resonance spectra and ¹³ C-nuclear magnetic resonance spectra (Table 2, FIG. 3), it was confirmed that deckerin was a compound represented by Chemical Formula 1.

Electronic Decker 3 6.23, d (9.5) 4 7.59, d (9.5) 5 6.80, s 8 7.16, s 3 ' 5.09, t (4.8) 4'a 2.87, dd (17.6,4.8) 4'b 3.20, dd (17.6,4.8) gem (CH ₃ ) a 1.36, s gem (CH ₃ ) b 1.39, s 2" 5.76, m 3 " - 2 "-CH ₃ - 3 "-CH ₃ 2.15, d (1.2) 4" 1.88, d (1.2)

carbon Decker 2 161.3 3 113.2 4 143.2 5 128.7 6 115.9 7 156.4 8 104.6 9 164.1 10 112.8 2' 27.6 3 ' 76.6 4' 69.0 gem (CH ₃ ) a 23.2 gem (CH ₃ ) b 25.0 One" 165.7 2" 115.6 3 " 158.5 4" 27.5 2 "-CH ₃ - 3 "-CH ₃ 20.3

Angelica Angelica Extract and Decker  Impact on immune regulation

Cytotoxicity test

Prior to confirming the efficacy of Angelica gigas extract and Decusin in vitro, a cytotoxicity test was conducted to determine the most suitable concentration. Cell death without toxicity to cells through WST-assay (water-soluble tetrazolium salt assay), a kind of MTT assay (3- (4,5-dimethylthiazol-2yl) -2,5-diphenyl-2H-tetrazolium bromide assay) The optimal concentration that did not cause cell death was identified. To this end, splenocytes were isolated from the spleen of the mouse, red blood cells were removed, and two concentrations (0, at 25, at 5 mg / ml) of decosin were treated in 5X10 ⁵ cells per sample. Incubated for 48 hours at 37 ℃ incubator. After 48 hours, a reagent (reagent) was treated for incubation for a WST-1 test, which is a type of MTT assay, and then cultured in a 37 ° C. incubator, and the OD was measured at 440 nm when the appropriate color of the culture medium was changed. Through this, it was possible to confirm the survival of the cell and to confirm the concentration of deckerin, which does not show toxicity to the cells, and then applied the in vitro experiment using the determined concentration.

First, the principle of the WST-1 assay is based on the conversion of tetrazolium salt (WST-1) to formazan pigment by mitochondrial dehydrogenase in living cells. The tetrazolium salt added to the medium is converted into formazan pigment by succinate-tetrazolium-reductase, which is present in the respiratory chain of mitochondria and is active only in living cells. As the number of living cells increases, the total activity of the mitochondrial dehydrogenase in the sample increases, which increases the production of formazan pigment. Therefore, the number of cells with metabolic activity in the formazan pigment and the medium is linearly correlated. To show relationships. Therefore, the higher the concentration, the more live cells can be said.

As shown in FIG. 4, the Angelica gigas extract and Decusin were found to show no cytotoxicity at various concentrations.

Histamine secretion experiment

After treatment with HMC-1, a human mast cell cell line, Histamine (Histamin), known as a pathological factor of allergy in culture, was enzymelinked immumosorbent assay (ELISA) Kit (Oxford Biomedical). Research, Inc. Rochester) was used to quantify expression according to the protocol.

As a result, as shown in Figure 5 it was confirmed that the histamine is reduced when the true Angelica extract extract at a concentration of 0.25 mg / ㎖.

IgE  Generation inhibition experiment

Immunoglobulin E (IgE), the largest known target for Th1 and Th2-mediated immune diseases, is immunoglobulin E (IgE). It was. To confirm this, U266B1 was used as a cell line. This cell line is a B cell line derived from humans that secrete a lot of immunoglobulin E among various antibody isotypes. When B cells are differentiated and become plasma cells, they basically secrete immunoglobulin M from various immunoglobulins and switch to various isotypes by class switching reaction. do. In this isomer determination, various cytokines secreted by T cells play an important role, and many cytokines in each environment are changed into various isotypes such as immunoglobulin A, immunoglobulin G, and immunoglobulin E. . Among these, the conversion to immunoglobulin E requires stimulation with lipopolysaccharide (LPS) / anti-CD40 and interleukin-4. Therefore, B cells stimulate the cells with lipopolysaccharide and interleukin-4, which are the most suitable conditions to make especially high secretion of immunoglobulin E, and treat various concentrations of Angelica extract and Decusin together while stimulating the cells in an incubator at 37 ° C for 72 hours. It was. After harvesting the cells for 3 days (harvest), the concentration of interleukin E present in the culture medium was quantitatively confirmed by enzyme-linked immunosorbent assay to determine the degree of immunoglobulin E reduced by the Angelica gigas extract and decusin. Confirmed.

As shown in FIG. 6, as a result of confirming the degree of inhibition of immunoglobulin E secretion against the control and the Angelica gigas extract and decusin, it was confirmed that the concentration was significantly reduced.

ex vivo Inhibition of Cytokine Expression in T Cells in Rats

T cells were isolated from MAC lymphocytes (Miltenyi Biotec, Bergisch Gladbach) from lymph nodes of atopy-induced mice in a situation close to Th1 and Th2-mediated immune diseases. To examine the effect on the expression levels of cytokines secreted from T cells, T-cells were isolated from allergen-induced mice, followed by phorbol 12-mi with 0.25 mg / ml of Angelica extract and 50 μM of decansin. Stimulation was carried out with Phorbol 12-myristate 13-acetate (PMA) and ionomycin for 4 hours. RNA was isolated from these cells (Tri reagent; Molecular Research Center Inc, Cincinnati) and cDNA was synthesized (Oligo-dT, Mgcl _{2 ,} RNase inhibitor, Improm-II reverse transcriptase, Improm-II 5X reaction buffer; Promega, Madison , dNTP; Takara, Tokyo) (step 1; 70 ° C 5min, 4 ° C 5min, step 2; 25 ° C 5min, 42 ° C 1hr, 70 ° C 15min) Pathogenic cytokine known from COX-2, Foxp3 and atopy ) MRNA expression levels of interleukin-4, interleukin-5, interleukin-13, interferon-γ, tumor necrosis factor (TNF-α) were determined by real time PCR (SYBR premix Ex Tag; Takara, Real time PCR machine; Chromo4 detector, Bio-rad, CA) (primers; Foxp3 (F; TTC CTT CCC AGA GTT CTT CC, R; CTC AAA TTC ATC TAC GGT CCA), IL-4 (F; ACA GGA GAA GGG ACG CCA T, R; GAA GCC GTA CAG ACG AGC TCA), IL-5 (F; AGC CAC TGG ATT AGC TCA AGT, R; GGG GGT CAC ACA TTT GCT TT), IL-13 (F; GCA ACA TCA CAC AGG ACC AGA, R; GTC AGG GAA TCC AGG GCT AC), INFg (F; GAG CCA GAT TAT CTC TTT CTA CC, R; GTT GTT GAC CTC AAA CTT GG), TNFα (F; CCC TCA CAC TCA GAT CAT CTT CT, R; GCT ACG ACG TGG GCT ACA G) Hypoxanthine phosphoribosyltransferase, which is a housekeeping gene; HPRT) was used as an internal control and the relative expression levels for the control group were plotted.

As shown in FIG. 7, the Angelica gigas extract and Decasin increase the expression of Foxp3, an immune modulator, and interleukin-4, interleukin-5, interleukin-13, interferon-γ, tumors, which are important for COX-2 and atopy-induced inflammatory factors It was confirmed that all of the cytokines such as necrosis factor-α significantly reduced.

Cytokine Expression Quantitative Experiment

T-cells were isolated from atopy-induced mice, followed by enzyme-linked immunosorbent adsorption kits (eBioscience, San Diego). The amount of expression was quantified according to the protocol using.

As can be seen in FIG. 8, cytokine in T cells treated with Angelica gigas extract was significantly reduced in concentration-dependent manner, and as can be seen in FIG.

references

Ahn KS, Sim WS, Kim IH. 1996. A cytotoxic agent and protein kinase C activator from the root of Angelica gigas. Planta Med 62: 7-9.

Kim HS, Park HJ, Chi HJ. 1980. A study of effects of the root components of Angelica gigas. Korean J Pharmacogn 11: 11-14.

Salikhova RA, Poroshenko GG. 1995. Antimutagenic properties of Angelica archangelica L. Vestn Ross Akad Med Nauk 1: 58-61.

Wn H, Kong L, Wu M, Xi P. 1996. Effects of different processed products of radix Angelica sinensis on clearing out oxygen free radicals and anti-lipid peroxidation. Chung Kuo ChungYoo Tsa Chin 21: 599-601.

Yim D, Singh RP, Agarwal C, Lee S, Chi H, Agarwal R. 2005. A novel anticancer agents, decursin, induces G1 arrest and apoptosis in human prostate carcinoma cells. Cancer Res 65: 1035-1044.

Claims (7)

Pharmaceutical composition for the prophylaxis or treatment of Th1-mediated immune disease or Th2-mediated immune disease comprising Angelica extract or Decacin of Formula 1 as an active ingredient:
Formula 1

Food composition for preventing or ameliorating Th1-mediated immune diseases or Th2-mediated immune diseases, including Angelica extract or Decasin of Formula 1 as an active ingredient.
Formula 1

Cosmetic composition for preventing or ameliorating Th1-mediated immune diseases or Th2-mediated immune diseases, including Angelica extract or Decasin of Formula 1 as an active ingredient.
Formula 1

The method according to claim 1, wherein the donkey is Angelica gigas Nakai).
The composition according to any one of claims 1 to 3, wherein the active ingredient increases the amount of Foxp3.
The composition of any one of claims 1 to 3, wherein the Th2-mediated immune disease is atopic dermatitis, contact dermatitis, allergic rhinitis, hyperthermia or allergic bronchial asthma.
The composition according to any one of claims 1 to 3, wherein the Th1-mediated immune disease is transplant rejection, autoimmune disease or inflammatory disease.

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