KR102040527B1 - Composition for Anti-inflammation Using Rhamnocitrin - Google Patents

Abstract

The present invention discloses an anti-inflammatory composition using rhamnocitrin isolated and identified in Tetracera laurei extract. The rhamnocitrin shows NO production inhibitory activity in mouse macrophage lines (RAW 264.7 cells) stimulated with lipopolysaccharide (LPS).

Description

Composition for anti-inflammatory using rhamnocitrin {Composition for Anti-inflammation Using Rhamnocitrin}

The present invention is Tetrasera low lei ( Tetracera) loureiri) it relates to a composition for anti-inflammatory with the separation from the extract, identifying the ramno Citrine (Rhamnocitrin).

Inflammation is a local biological defense response that occurs in response to physical trauma, harmful chemicals, bacteria, fungi, viruses, or pathological conditions caused by irritants in metabolites in vivo. Inflammation is triggered by various inflammatory mediators produced from damaged tissues and migrating cells. During the inflammatory reaction, plasma accumulates at the site of inflammation, diluting the toxicity secreted by bacteria, increasing blood flow, and accompanied by symptoms such as erythema, pain, edema, and fever. In normal cases, the organism restores normal structure and function by neutralizing or eliminating the pathogens through the inflammatory response and regenerating the upper tissue, but in other cases, the disease progresses to a disease state such as chronic inflammation.

Recent advances in molecular biology have led to a great deal of research into the inflammatory response at the molecular level.

Inflammatory reactions involve a variety of biochemical phenomena, but macrophages, in particular, are known to play an important role in the inflammatory response by producing nitric oxide (NO) and various inflammatory cytokines by chemical stimulation (Ito T., et. al., Curr Drug Traget Inflamm Allergy, 2 (3): 257-265, 2003). Nitric oxide is synthesized from L-arginine by the action of nitric oxide synthase (NOS). There are several isoforms of NOS. Brain NOS (brain NOS) in the brain, nNOS (neuronal NOS) in the nervous system, endothelial NOS (eNOS) in the vascular endothelial system are always expressed in a certain level in the body, a small amount produced by these monoxide Nitrogen (NO) plays an important role in maintaining the homeostasis of the body by performing various physiological reactions related to blood pressure control, neurotransmitter, learning, memory, and the like. In contrast, iNOS (induced NOS), whose expression is induced by a stimulus, produces excess NO. Nitric oxide produced by iNOS reacts with superoxide to form peroxynitrite. It acts as a powerful oxidant and damages cells and is involved in various pathological processes, including inflammation and cancer (Gupta SC et al., Exp Biol Med., 236: 658-671, 2011; Riehemann et al., FEBS Lett., 442: 89-94, 1999; Stammler et al., Science, 258: 1898-1902, 1992).

On the other hand, cyclooxygenase (COX) has hydroperoxidase (HOX) activity with COX function and synthesizes PGG ₂ and PGH ₂ intermediates from arachidonic acid, and these compounds are PGE ₂ and PGF ₂ , PGD ₂ , prostacyclin and thromboxane A ₂ (thromboxane A2, TxA2) are produced. There are also two types of isoforms in COX. While COX-1 is always expressed in most tissues and synthesizes prostaglandins (PGs), which are required for cell protective action, COX-2 is rapidly induced during the inflammatory response to produce PGE ₂ and the like. Play an important role (Weisz A., Biochem. J., 316: 209-215, 1996; (Miller MJ et al., Mediators of inflammation, 4: 387-396, 1995: Appleton L. et al., Adv) Pharmacol., 35: 27-28, 1996).

Expression of iNOS and COX-2, which leads to overproduction of NO and PGs, is regulated by the nuclear transcription factor NF-κB. NF-κB is a nuclear protein of the Rel gene family. In the cytoplasm, NF-κB binds to I-κB and is inactive.However, if I-κB kinase is activated by any factor, It is activated by the release of I-κB. NF-κB, composed of p50 and p65 heterodimers, activates and induces gene expression of iNOS and COX-2 that migrate to the nucleus and induce an inflammatory response (Oh, GT et al., Atherosclerosis, 159 (1): 17-26, 2001).

Bacterial endotoxins such as LPS (lipopolysaccharide) activate NF-κB, which is a transcription factor by binding to toll-like receptor 4 (TLR4), and induce the expression of iNOS and COX-2 to induce NO, inflammatory cytokines, PGE _2, etc. Secrete several inflammatory modulators (Chen YC, et al, Biochem. Pharmacol., 61: 1417-1427, 2001; Dobrovolskaia MA, et al., J Immunol., 170: 508-19, 2003; Ji Y, et al., Cell Physiol Biochem., 25: 631-640, 2010). NO, Inflammatory cytokines such as TNF-α, IL-6, PGE ₂ , arthritis (Jang CH et al., Rheumatology, 2006, 45 (6): 703-710), fibromyalgia (Hernandez ME et. Al., BMC Res Notes., 2010, 3 (1): 156), Sjogren's syndrome (Baturone R. et. Al., Scand J Rheumatol., 2009, 38 (5): 386-389), etc. (Jang CH et al., Rheumatology, 45 (6): 703-710, 2006; Hernandez ME et. Al., BMC Res. Notes., 3 (1): 156, 2010; Baturone R. et. al., Scand J Rheumatol., 38 (5): 386-389, 2009).

These findings can either inhibit NO production Drugs that inhibit the production of inflammatory cytokines such as TNF-α and IL-6, and drugs that inhibit the expression of iNOS or COX-2, have the potential as effective anti-inflammatory agents (Karin M. et al., Cold Spring Harb Perspect Biol., 1, pp 1-14, 2009).

Non-steroidal anti-inflammatory drugs (NSAIDS), which are widely used as anti-inflammatory agents, are known to cause serious side effects such as gastrointestinal disorders, liver disorders and nephropathy (Rainsford KD., Subcell biochem., 42: 3). -27, 2007; Guruprasad P. Aithal., Rheumatology., 7: 139-150, 2011; Praveen PN Rao et al., Pharmaceuticals., 3: 1530-1549, 2010).

Therefore, there is still a need for the development of new drugs that have anti-inflammatory activity and have fewer side effects and lasting effects.

Tetracera laurei is a plant that has traditionally been used as a medicinal herb in Southeast Asia, including Thailand. Recently, hepatoprotective activity and antioxidant activity have been reported (Phytother Res. 17 (7): 717-21, 2003).

The present invention discloses the anti-inflammatory activity of rhamnocitrin isolated and identified in the Tetracera laurei extract.

An object of the present invention to provide an anti-inflammatory composition using rhamnocitrin.

Other and specific objects of the present invention will be presented below.

The present invention, as confirmed in the Examples and Experimental Examples below, the rhamnocitrin isolated and identified in Tetracera low-Lay extract, concentration-dependently in mouse macrophage lines (RAW 264.7 cells) stimulated with LPS (lipopolysaccharide) It was completed by confirming that NO production inhibitory activity. The rhamnocitrin is the ethyl having the highest anti-inflammatory activity among the sequential fractions obtained when the tetracera laurei 70% ethanol extract is suspended in distilled water and then sequentially partitioned into hexane, dichloromethane, ethyl acetate, and butanol (n-butanol). It was isolated and identified in the acetate fraction.

In view of the foregoing, the present invention provides, in one aspect, (i) an ethyl acetate fraction of tetracera laurei extract or (ii) a rhamnocitrin of <Formula 1> (3,4 ', 5-trihydroxy-7) -methoxyflavone) can be identified as an anti-inflammatory composition comprising as an active ingredient, and in another aspect, (i) ethyl acetate fraction of tetracera laurei extract or (ii) rhamnocitrin (3) It relates to a composition for the treatment or prevention of diseases caused by nitric oxide overproduction in vivo comprising, 4 ', 5-trihydroxy-7-methoxyflavone) as an active ingredient.

<Formula 1>

In the present specification, the term "tetracera laurei extract" refers to stems, leaves, berries, flowers, roots, etc. of tetracera laurei extract to be extracted, water, lower alcohols having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.) Chloride, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol or mixtures thereof An extract obtained by leaching using a solvent, carbon dioxide, pentane, etc. extract obtained by using a supercritical extraction solvent or fractions obtained by fractionating the extract, the extraction method taking into account the polarity, degree of extraction, the degree of preservation of the active material Any method, such as chilling, refluxing, heating, ultrasonic spinning, supercritical extraction, can be applied. In the case of the fractionated extract, the fraction obtained by suspending the extract in a specific solvent and mixing and standing with a solvent having a different polarity, the crude extract is adsorbed on a column filled with silica gel and the like, and then a hydrophobic solvent, a hydrophilic solvent, or a mixed solvent thereof is It is meant to include fractions obtained by mobile phase. In addition, the meaning of the extract includes a concentrated liquid extract or solid extract in which the extraction solvent is removed in a manner such as freeze drying, vacuum drying, hot air drying, spray drying, and the like. Preferably it means that obtained by extraction (immersion or distillation) with water, ethanol or a mixed solvent thereof, especially 60% to 90% aqueous solution of ethanol as the extraction solvent. More preferably, it means a fraction of any one fractional solvent layer obtained by suspending the 60% to 90% ethanol aqueous solution extract in water (distilled water) and sequentially fractionating with hexane, dichloromethane, ethyl acetate and butanol. Most preferably, the sequential fraction refers to a fraction of the ethyl acetate layer confirmed to exhibit high anti-inflammatory activity in the following experimental example. By "sequential fractionation" is meant here that the remaining water layer after fractionation is continued to fractionate with the solvents in the order listed above.

As used herein, the term "active ingredient" alone refers to a component that can exhibit the desired activity or exhibit itself with a carrier which is inactive.

In addition, in the present specification, "anti-inflammatory" is meant to include improvement (reduction of symptoms), treatment, inhibition of the onset or delay of the inflammatory disease as defined below.

In addition, in the present specification, the "inflammatory disease" refers to an inflammatory response specified as a local or systemic biological defense response against external physical and chemical stimuli or infection or autoimmunity of an external infectious agent such as bacteria, fungi, viruses, and various allergens. It can be defined as a pathological symptom that causes. These inflammatory responses include activation of various inflammatory mediators and enzymes associated with immune cells (eg iNOS, COX-2, etc.), secretion of inflammatory mediators (eg, NO, TNF-α, IL-6, etc.), fluid infiltration, It is accompanied by a series of complex physiological reactions such as cell migration and tissue destruction, and is manifested externally by symptoms such as erythema, pain, edema, fever, deterioration or loss of certain functions of the body. The inflammatory disease may be acute, chronic, ulcerative, allergic or necrotic, so as long as any disease is included in the definition of an inflammatory disease as above, whether it is acute, chronic, ulcerative, allergic or Irrespective of necrosis Specifically, the inflammatory diseases include asthma, allergic and non-allergic rhinitis, chronic and acute rhinitis, chronic and acute gastritis or enteritis, ulcerative gastritis, acute and chronic nephritis, acute and chronic hepatitis, chronic obstructive pulmonary disease, pulmonary fibrosis Irritable bowel syndrome, inflammatory pain, migraine, headache, back pain, fibromyalgia, fascia disease, viral infection (eg, type C infection), bacterial infection, fungal infection, burn, wound by surgical or dental surgery, pro Prolonged prostaglandin E, atherosclerosis, gout, arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, irisitis, scleritis, uveitis, dermatitis (including atopic dermatitis), eczema, multiple sclerosis Will be included.

In the present specification, "disease caused by in vivo nitric oxide overproduction" means the above-mentioned inflammatory diseases, asthma, Alzheimer's disease, Parkinson's disease, multiple sclerosis, sclerosis, tagase rejection, etc. to be. In this regard, more specific reference may be made to documents cited in Korean Patent Laid-Open Publication No. 1999-022174 and Korean Patent Laid-Open Publication No. 1999-022174, and both Korean Patent Publication No. 1999-022174 and the documents cited therein It is considered as part of this specification.

The anti-inflammatory composition of the present invention may include the active ingredient in any amount (effective amount) as long as it can exhibit the improvement activity of an inflammatory disease intended to be treated according to the use, formulation, formulation purpose, etc. Effective amounts will be determined within the range of 0.001% to 15% by weight, based on the total weight of the composition. The term "effective amount" as used herein means that when the composition of the present invention is administered to a mammal, preferably a human, such as a medical expert or the like, during the administration period, improvement, treatment, or inhibition of the development of such pathological symptoms It refers to the amount of active ingredients that can exhibit the intended medical and pharmacological effects, such as delays. Such effective amounts can be determined experimentally within the range of ordinary skill in the art.

In addition to the active ingredient, the anti-inflammatory composition of the present invention may be administered for the purpose of increasing or enhancing the anti-inflammatory effect or through the addition of similar activities such as anti-allergic activity, skin protective activity (inhibition of skin damage caused by ultraviolet rays, skin moisturizing, etc.), In order to enhance the convenience of ingestion, it may further comprise any compound or natural extract that has already been proven safe in the art and known to have the corresponding activity.

Such compounds or extracts include compounds, extracts, and medicines contained in procedures such as the Pharmacopoeia of each country ("Korea Pharmacopoeia" in Korea) and the Health Functional Foods Code of Korea ("Health Functional Food Standards and Standards" in Korea). Laws governing the manufacture and sale of compounds, extracts or health functional foods licensed under the laws of each country governing the manufacture and sale of pharmaceutical products ("Pharmaceutical Acts in Korea"). And compounds or extracts which have been individually recognized for their functionality. For example, MSM (dimethylsulfonylmethane) with 'improving arthritis' functionality in Korea Health Functional Food Code, N-acetylglucosamine with 'improving arthritis' functionality and 'skin moisturizing' function, etc. Enterococcus faecalis heat treated dry powder, guava leaf extract complex, complex including guacamole leaf extract, leaflet extract, picaopreto powder, PLAG (1-palmitoyl-2-linoleoyl- 3-acetyl-rac-glycerol), L. sakei Probio 65, which has been individually recognized for its improvement in hypersensitivity skin condition, oil-containing fatty acid gamma-linolenic acid, L.plantarum CJLP133, a vegetable-derived lactic acid bacterium, and probiotics ATP. Corresponds to the extract.

Such compounds or natural extracts may be included in one or more of the anti-inflammatory composition of the present invention and the like with the active ingredient.

The anti-inflammatory composition of this invention etc. can be grasped | ascertained as a food composition in a specific aspect.

The food composition of the present invention may be prepared in any form, for example, beverages such as tea, juice, carbonated beverages, ionic beverages, processed oils such as milk, yogurt, gums, rice cakes, sweets, bread, sweets, noodles, and the like. Foodstuffs, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, bars, and the like can be prepared as functional food preparations.

In addition, the food composition of the present invention can distinguish any product as long as it conforms to the enforcement regulations at the time of manufacture and distribution in the legal and functional divisions. For example, confectionery, soybeans, teas, beverages according to each food type, or health functional foods in accordance with the Act on Health Functional Foods, or in the Food Code of the Korean Food Sanitation Act , Special purpose foods, and the like.

The food composition of the present invention may include food additives in addition to the active ingredient. Food additives can generally be understood as substances which are added to the food, mixed or infiltrated in the manufacture, processing or preservation of the food, which must be ensured because of its daily and long-term intake with the food. The Food Additives Code of Korea ("Food Sanitation Act" in Korea) governs the manufacture and distribution of foods, and food additives with guaranteed safety are limited in terms of ingredients or functions. In the Korean Food Additives Code (KFDA Notification and Standards of Food Additives), food additives are classified into chemical synthetics, natural additives, and mixed preparations in terms of ingredients. These food additives are sweeteners, flavors in terms of function. Agent, preservative, emulsifier, acidulant, thickener and the like.

Sweeteners are used to impart a suitable sweetness to foods, either natural or synthetic, which can be used in the compositions of the present invention. Preferably, a natural sweetener is used. Examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose and maltose.

Flavoring agents can be used to enhance the taste or aroma, both natural and synthetic. It is the case of using a natural thing preferably. In addition to flavors, the use of natural ones can be combined with nutritional purposes. The natural flavor may be obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, and the like, or may be obtained from green tea leaves, round leaves, jujube leaves, cinnamon, chrysanthemum leaves, jasmine and the like. In addition, ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo and the like can be used. Natural flavors can be liquid concentrates or solid extracts. In some cases, synthetic flavoring agents may be used, and synthetic flavoring agents may include esters, alcohols, aldehydes, terpenes, and the like.

Sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. may be used as a preservative, and as an emulsifier, acacia gum, carboxymethylcellulose, xanthan gum, Pectin etc. can be mentioned, As acidic acid, acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, etc. can be used. The acidulant may be added so that the food composition is at an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to the purpose of enhancing the taste.

As the thickener, suspending implementers, sedimenting agents, gel formers, swelling agents and the like can be used.

In addition to the food additives described above, the food composition of the present invention may include a bioactive substance or minerals known in the art for the purpose of supplementing and reinforcing the functionality and nutritional properties and ensuring the stability as a food additive.

Examples of such physiologically active substances include catechins, vitamin B1, vitamin C, vitamin E, vitamin B12 and the like, tocopherol, dibenzoylthiamine, and the like contained in green tea. Examples of the minerals include calcium preparations such as calcium citrate and magnesium stearate. Magnesium preparations such as iron, iron preparations such as iron citrate, chromium chloride, potassium iodine, selenium, germanium, vanadium, zinc and the like.

In the food composition of the present invention, the food additive as described above may be included in an amount that can achieve the purpose of addition according to the product type.

Regarding other food additives that may be included in the food composition of the present invention, reference may be made to each country's food or food additives.

The composition of the present invention may be regarded as a pharmaceutical composition in another specific embodiment.

The pharmaceutical compositions of the present invention may be prepared in oral or parenteral formulations according to the route of administration by conventional methods known in the art, including pharmaceutically acceptable carriers in addition to the active ingredient. The route of administration here can be any suitable route, including the topical route, the oral route, the intravenous route, the intramuscular route, and direct absorption through mucosal tissue, and may be used in combination of two or more routes. An example of a combination of two or more routes is where a drug of two or more formulations according to the route of administration is combined, for example when one drug is administered first by the intravenous route and the other by the local route.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or formulation, and specific reference may be made to the pharmacopoeia of each country, including "Korea Pharmacopoeia."

When the pharmaceutical composition of the present invention is prepared in an oral dosage form, powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, suspensions, wafers according to methods known in the art with suitable carriers It may be prepared in a formulation such as. Examples of suitable carriers include lactose, glucose, sucrose, dextrose, sorbitol, sugars such as mannitol, xylitol, starch such as corn starch, potato starch, wheat starch, cellulose, methyl cellulose, ethyl cellulose, sodium carboxymethyl cellulose, Celluloses such as hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, and Cerrol etc. are mentioned. If formulated, appropriate binders, lubricants, disintegrants, colorants, diluents and the like may be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch ferrite, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweeteners, sodium alginate, polyethylene glycol, waxes, and the like. Sodium, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, magnesium salts thereof, calcium salts, polydetylene glycol and the like, and the disintegrating agents include starch, methyl cellulose And agar, bentonite, xanthan gum, starch, alginic acid or its sodium salt and the like. Moreover, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine etc. are mentioned as a diluent.

When the pharmaceutical compositions of the present invention are prepared in parenteral formulations, they may be formulated in the form of injections, transdermal administrations, nasal inhalants and suppositories with suitable carriers according to methods known in the art. When formulated as an injection, a suitable carrier may be an aqueous isotonic solution or suspension. Specifically, an isotonic solution such as phosphate buffered saline (PBS) containing triethanol amine, sterile water for injection, or 5% dextrose may be used. . When formulated as a transdermal administration, it may be formulated in the form of an ointment, cream, lotion, gel, external solution, pasta, linen, aerosol, and the like. Nasal inhalants can be formulated in the form of aerosol sprays using suitable propellants, such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc. witepsol), tween 61, polyethylene glycols, cacao butter, laurin paper, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, sorbitan fatty acid esters and the like.

Specific formulations of pharmaceutical compositions are known in the art and can be found, for example, in Remington's Pharmaceutical Sciences (19th ed., 1995). The document is considered part of this specification.

Preferred dosages of the pharmaceutical compositions of the present invention range from 0.001 mg / kg to 10 g / kg per day, preferably 0.001 mg / kg to 1 g, depending on the condition, body weight, sex, age, severity of the patient and route of administration. It can range from / kg. Administration can be done once a day or divided into several times. Such dosage should not be construed as limiting the scope of the invention in any aspect.

The composition of this invention can be grasped | ascertained as a cosmetic composition in another specific aspect. When the composition of the present invention is identified as a cosmetic composition, its use may be understood as a use for inhibiting inflammatory skin troubles, alleviating inflammatory skin irritation, and the like.

Even when the composition of the present invention is identified as a cosmetic composition, the cosmetic composition can be classified into any product according to its purpose and law, and specifically, a functional cosmetic having a use such as skin trouble improvement, atopic dermatitis improvement, and non-functionality. General cosmetics, and the like. The product form can also be in any product form, specifically solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, waxes You can find product types such as foundation and spray. In a specific product form, it may be a softening lotion, a nourishing lotion, a nourishing 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 formulation.

The cosmetic composition of the present invention may include, in addition to the active ingredient, components conventionally used in the cosmetic composition, for example, conventional adjuvants such as stabilizers, solubilizers, surfactants, vitamins, pigments and pharmaceuticals, and carriers.

When the formulation of the present invention is a paste, cream or gel, animal oils, vegetable oils, waxes, paraffins, starches, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide may be used as carrier components. Can be.

When 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, in particular in the case of a spray, additionally chlorofluorohydrocarbon, propane Propellant such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as the carrier component, specifically water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol, fatty acid ester of sorbitan and the like can be used.

When the formulation of the present invention is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, polyoxyethylene sorbitan ester, and microcrystals are used as carrier components. Castle cellulose, aluminum metahydroxy, bentonite, agar and the like can be used.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is 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 and the like can be used.

The cosmetic composition of the present invention can be prepared according to the manufacturing method of the cosmetic composition usually carried out in the art, except for including the active ingredient exhibiting anti-inflammatory activity.

In another aspect, the present invention relates to a process for the preparation (separation) of rhamnocitrin from tetracera laurei extract.

The production method of the present invention comprises the steps of (a) extracting by extracting the tetracera laurei in an extraction solvent mixed with ethanol and water to remove the extraction solvent to prepare a solid extract, (b) the solid extract Suspended in water and then sequentially fractionated with normal hexane, dichloromethane, ethyl acetate and normal butanol, and then concentrated under reduced pressure to obtain a fraction of ethyl acetate, and (c) chromatography on the fraction of ethyl acetate. It comprises a step of separating the rhamnocithin by performing.

In the preparation method of the present invention, the extraction solvent in which the ethanol and water are mixed is preferably 60% to 900% ethanol.

In the preparation method of the present invention, the step (c) is preferably 30%, 40%, 50%, 60%, 70%, 80% and 100% methanol with respect to the fraction of the ethyl acetate as a mobile phase. Performing reversed phase chromatography on a silica gel packed column and dividing it into six fractions; Chromatographing the sixth of the six fractions to separate the rhamnocitrin.

As described above, according to the present invention can provide a composition for anti-inflammatory using rhamnocitrin (Rhamnocitrin) separated and identified in Tetracera low lei extract.

The anti-inflammatory composition of the present invention may be commercialized as foods, cosmetics, drugs, etc. for the use of improvement of inflammatory diseases, the use of alleviating inflammatory skin irritation, and the like.

1 is an isolated schematic diagram of rhamnocitrin.
2 is ¹ H NMR data and ¹³ C NMR data of rhamnocitrin.
3 is a ¹ H NMR spectrum of rhamnocitrin.
4 is a ¹³ C NMR spectrum of rhamnocitrin.
5 is the COSY spectrum of rhamnocitrin.
6 is the HMBC spectrum of rhamnocitrin.
7 is the HSQC spectrum of rhamnocitrin.
8 is an MS result of rhamnocitrin.
9 is an anti-inflammatory test result of rhamnocitrin.
10 is an anti-inflammatory test result of the ethyl acetate fraction of tetracera laurei extract.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these examples and experimental examples.

< Example > Tetracera Lowley  Isolation and Identification of Active Compound Rhamnocitrin from Extracts

Example 1 Isolation of the Active Compound from Tetracera Low-Leary Extract

Dried Tetrasera Lowreyri loureiri ) 1 L of 70% ethanol was added to 100 g of stem and leaves and extracted twice at room temperature. The filtrate was filtered and the filtrate was obtained using a rotary vacuum concentrator to obtain a powdery ethanol extract. The extract was suspended in distilled water, and then the solvent was fractionated with the same amount of hexane, dichloromethane, ethyl acetate, and saturated butanol (n-butanol). It was. Among these fractions, the ethyl acetate layer (# 153-E), which was found to exhibit high anti-inflammatory activity through the following experiment (FIG. 10), was evaporated under reduced pressure to obtain an ethyl acetate fraction, and as a distilled water-methanol mobile phase, Reversed-phase silica gel (RP-18) column chromatography was performed by sequential elution (30%, 40%, 50%, 60%, 80%, 100% MeOH) and divided into six subfractions (# 153E-1 to 6). ). Fraction # 153-6 was purified by silica gel column MPLC using gradient elution with dichloromethane-methanol (1: 0 1: 1, 45min) and divided into five subfractions (# 153E-6M-1 to 5). For subfraction # 153E-6M-1-2 prep. HPLC (preparative ODS-column) was performed to separate compound 1 (FIG. 1).

Example 2 Identification of Active Compounds

In order to identify the structure of Compound 1 separated and purified in Example 1, a nuclear magnetic resonance group (NMR, Bruker Co., Ascend III 700 MHz), mass spectrometry (MS) and the like were performed. The compound was dissolved in DMSO- d ₆ in NMR-only Merck Co. and placed in a 5 mm NMR tube, and hydrogen and carbon nuclear magnetic resonances were measured. Each solvent peak was used as an internal standard or based on a peak of tetramethylsilane (TMS). Measured as. The purified compound 1 was identified as rhamnocitrin, a compound of Formula 1, based on the results of NMR analysis of FIGS. 3 to 9.

< Experimental Example > Anti-inflammatory Activity Test of Rhamnocitrin-Evaluation of NO Production Inhibitory Activity

To evaluate the intracellular anti-inflammatory activity of samples such as rhamnocitrin in RAW264.7 cells, which are mouse macrophages, the cells were dispensed in 96 well plates at 5 × 10 ⁴ cells / well for 24 hours at 37 ° C and 5% CO ₂ . Incubated for After treating the sample in a 96 well plate for 1 hour by concentration, 1 μg / ml of LPS (Lipopolysaccharide) was treated and incubated for 24 hours to evaluate anti-inflammatory activity. Anti-inflammatory activity evaluation by NO assay was performed using a griess A, B reagent, 1% sulfanilamide, 0.1% N- (1-naphtyl) ethylenediamine dihydrochloride reagent in a 1: 1 ratio. After the sample was processed in a 96 well plate, the supernatant of the medium incubated for 24 hours at 37 ° C. and 5% CO ₂ was mixed with 50 μl of griess (A + B) reagent in a 1: 1 ratio and stored at room temperature for 10 minutes. Afterwards, the NO production inhibitory activity was measured by ELISA measurement at 540 nm.

Cytotoxicity was measured by MTT method. Specifically, the remaining medium in the 96 well plate was removed, and 100 μl of serum free medium containing 10% of 5 mg / ml MTT solution was added to each well at 37 ° C. and 5% CO ₂ incubator for 2 hours. After removing the medium, DMSO was added to 100µl / well and dissolved in the shaker for 15 minutes, and the absorbance was measured at 540nm using an ELlSA reader to determine cell viability.

The results of NO production inhibition and cell viability measurement results are shown in FIG. 9.

Rhamnocitrin showed NO production inhibitory activity in a concentration-dependent manner, and showed some cytotoxicity only at a concentration of 100 μM.

Claims (5)

Tetracera loureiri ( Tetracera loureiri ) Anti-inflammatory composition comprising the ethyl acetate fraction of the stem and leaf mixture extract as an active ingredient.
delete The method of claim 1,
The composition is an anti-inflammatory composition, characterized in that the pharmaceutical composition.
Tetracera loureiri ( Tetracera loureiri ) Food composition for inhibiting inflammatory response comprising ethyl acetate fraction of the stem and leaf mixed extract as an active ingredient.
Tetracera loureiri ( Tetracera loureiri ) A cosmetic composition for reducing inflammatory skin irritation comprising an ethyl acetate fraction of the stem and leaf mixture extract as an active ingredient.

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