KR20240107069A - Composition for preventing and treating inflammatory disease comprising a purified extract or compounds isolated from the extract of Physalis alkekengi as an active ingredient - Google Patents

Abstract

The present invention relates to a composition containing as an active ingredient a fraction or compound purified and separated from the perilla extract, 3,4',5-trihydroxy-3',7-dimethoxy isolated from the perilla extract of the present invention. -Flavone, Physalin B, and Isopisalin B compounds exhibit the effect of significantly reducing the increased expression of inflammatory cytokines IL-1β and TNF-α induced by LPS, and are therefore compositions for preventing, improving, or treating inflammatory diseases It can be usefully used.

Description

Composition for preventing and treating inflammatory disease comprising a purified extract or compounds isolated from the extract of Physalis alkekengi as an active ingredient}

The present invention relates to a pharmaceutical composition and health functional food for the prevention or treatment of inflammatory diseases containing as an active ingredient a purified product or compound isolated from an extract of Physalis alkekengi var . francheti (Masters) Hort.

Inflammation is a response of living tissue to damage, and the inflammatory response not only plays a key role in the defense and healing of the organism by mobilizing the immune system, but is also a very important process that is involved in the pathogenesis of many diseases. There are countless causes of inflammation, but biological causes such as bacteria, fungi, and viruses are one of them. Additionally, during an inflammatory response, macrophages, which are immune cells, produce cytokines such as interleukin-1β (IL-1β) or tumor necrosis factor-α (TNF-α) or other inflammatory mediators such as nitric oxide (NO) or prostaglandin (PG). It plays an important role in the reaction process by producing . Production of these mediators by macrophages is found in many inflammatory tissues. For example, when a living body is exposed to an external immune stimulant such as lipopolysaccharide (LPS), a bacterial endotoxin, immune cells are activated through a biosignaling pathway, and at the same time, the expression of inflammatory factors by these cells increases. This process In the first step, the mRNA expression of the factor increases. In addition, increased cytokines and biological enzymes such as IL-1β, TNF-α, iNOS, COX-2, etc. can cause inflammation, pain control, apoptosis, tumorigenesis, and autophagy. It plays an important role in pharmacological or physiological biological reactions such as immune response (autoimmune response). Overproduction of inflammatory mediators is easily found in many inflammatory diseases such as rheumatoid arthritis, atherosclerosis, chronic hepatitis, pulmonary fibrosis, etc. and is a major cause of the pathology of these diseases. Therefore, the method of inhibiting the gene expression of these inflammation-inducing mediators can be a treatment principle and method that can prevent or suppress various inflammatory diseases.

The inflammatory response, which is described as an active biological defense process against damage to living tissue, is a series of biological processes that initiate a response when a living cell or tissue is damaged by any cause, minimize the damage, and repair the damaged area. It causes. During an inflammatory response, macrophages, which are immune cells, produce cytokines such as interleukin-1β (IL-1β) or tumor necrosis factor-α (TNF-α) or other cytokines such as nitric oxide (NO) or prostaglandin (PG). It plays an important role in the reaction process by producing inflammatory mediators. Production of these mediators by macrophages is found in many inflammatory tissues. For example, when a living body is exposed to an external immune stimulant such as lipopolysaccharide (LPS), a bacterial endotoxin, immune cells are activated through a biosignaling pathway, and at the same time, the expression of inflammatory factors by these cells increases. This process In the first step, the mRNA expression of the factor increases. Therefore, if we search for substances that inhibit the production of TNF-α, NO, prostaglandin, or IL-1β, they may turn out to be effective substances for inflammatory diseases such as edema or dermatitis.

Non-steroidal antiinflammatory drugs (NSAIDS), which are widely used as treatments for most inflammatory diseases, biosynthesize prostaglandin from arachidonic acid called cyclooxygenase (COX). It exhibits an anti-inflammatory effect by inhibiting the enzyme activity involved in, but in addition to the main therapeutic effect, it causes serious side effects such as gastrointestinal disorders, liver disorders, and renal disorders, so there are restrictions on long-term use (Rajakariar R et al. 2006). Therefore, there is a widespread demand for the development of new anti-inflammatory analgesics that have excellent anti-inflammatory effects while having few side effects, so long-term use is not difficult. This is also the reason why research on material development through verification of efficacy from natural resources has become active in recent years.

Physalis alkekengi var. francheti (Masters) Hort) is a perennial herb cultivated throughout Korea. It grows at the edge of forests below 600 m above sea level and is about 60 to 90 cm tall. There are long rhizomes that creep sideways underground. The leaves are alternate, light-ovate, have sharp ends, have tooth-like serrations, and each node produces two leaves. Flowers emerge from the leaf axil, are white, and grow one at a time. The pedicel is 3 to 4 cm long. The calyx is short and tube-shaped, and the end is shallowly divided into 5 pieces and has hairs on the edges. The fruit is a spherical axil that ripens to red. As a herbal medicine, whole plant is used and is also called Physalis Herba and Deungryong. Ingredients include physalin A, B, C, saponin, flavonoid, and alkaloid in the whole plant, and 0.12% carotenoid in the fruit, a red pigment and acetic acid. Contains corbic acid, resin, pectin, tannin, bitter substances, carotene, alkaloids, quercetin, caffeic acid, cinnamic acid, and ferulic acid, and the root contains 3-alpha-tyglooxytrophane, physalin, and zeacic acid. It contains xanthine, and the seeds contain oil whose main ingredient is linolein. However, experimental studies on the anti-inflammatory activity of fractions or compounds purified from acinar root have not yet been reported.

Accordingly, the present inventors completed the present invention by confirming the inhibitory effect of the fraction and compound isolated from the perilla extract on the expression of pro-inflammatory cytokines.

Republic of Korea Patent No. 10-1172595

The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases.

Another object of the present invention is to provide a pharmaceutical composition for preventing or improving inflammatory diseases.

Another object of the present invention is to provide a cosmetic composition for preventing or improving inflammatory diseases.

Another object of the present invention is to provide a method for preventing or treating inflammatory diseases.

In order to achieve the above purpose,

The present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, comprising a fraction of Physalis alkekengi extract or a compound isolated therefrom as an active ingredient.

In addition, the present invention provides a food composition for preventing or improving inflammatory diseases, comprising a fraction of Physalis alkekengi extract or a compound isolated therefrom as an active ingredient.

Furthermore, the present invention provides a cosmetic composition for preventing or improving inflammatory diseases, comprising a fraction of Physalis alkekengi extract or a compound isolated therefrom as an active ingredient.

Furthermore, the present invention provides a method for preventing or treating inflammatory diseases, comprising administering a fraction of Physalis alkekengi extract or a compound isolated therefrom.

In the present invention, the extract may be extracted with methanol.

In the present invention, the fraction may be fractionated using water including purified water, lower alcohols having 1 to 4 carbon atoms, ethyl acetate, chloroform, hexane, or a mixed solvent thereof, preferably water, butanol, ethyl acetate, and It may have been fractionated using hexane.

In the present invention, the compound is 3,4',5-trihydroxy-3',7-dimethoxy-flavone (3,4',5-trihydroxi-3',7-dimethoxy-flavone), Physalin B It may contain one or more compounds selected from physalin B and isophisalin B, or a pharmaceutically acceptable salt thereof.

In the present invention, the inflammatory disease includes dermatitis, allergy, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, pharyngitis, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, and lupus. , fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, tenosynovitis, peritendinitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis, and acute and chronic inflammatory diseases. It may be any one selected from the group.

The fractions purified and isolated from the perilla extract of the present invention, 3,4`,5-trihydroxy-3`,7-dimethoxy-flavone, physalin B and isophisalin B compounds are used in macrophages (RAW264.7). Since it has the effect of significantly reducing the mRNA expression of inflammatory cytokines IL-1β and TNF-α, which are increased after inducing an inflammatory response by LPS, it can be usefully used as a composition for preventing, improving, or treating inflammatory diseases.

Figure 1 is a schematic diagram showing a method for producing agar extract and fractions of the present invention.
Figure 2 shows the effect on TNF-α and IL-1β gene expression after treatment with the methanol extract (EA325M) according to the present invention in the inflammatory response using LPS.
Figure 3 shows the effect on TNF-α and IL-1β gene expression after treatment with the acinar hexane fraction (EA325H) according to the present invention in the inflammatory response using LPS.
Figure 4 shows the effect on TNF-α and IL-1β gene expression after treatment with the acinar butanol fraction (EA325B) according to the present invention in the inflammatory response using LPS.
Figure 5 shows the effect on TNF-α and IL-1β gene expression after treatment with the acinar water fraction (EA325Aq) according to the present invention in the inflammatory response using LPS.
Figure 6 shows the effect on TNF-α and IL-1β gene expression after treatment with the acinar ethyl acetate fraction (EA325E) according to the present invention in the inflammatory response using LPS.
Figure 7 shows the effect on TNF-α and IL-1β gene expression after treatment with isopisaline B (EA325E-34-15-K4) according to the present invention in the inflammatory response using LPS.
Figure 8 shows the effect on TNF-α and IL-1β gene expression after treatment with Physalin B (EA325E-34-6-K4) according to the present invention in the inflammatory response using LPS.
Figure 9 shows TNF-α and IL- after treatment with 3,4`,5-trihydroxy-3`,7-dimethoxy-flavone (EA325H-34-11-K8) according to the present invention in the inflammatory response using LPS. This shows the effect on 1β gene expression.

Hereinafter, the present invention will be described in detail.

In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating inflammatory diseases, comprising a fraction of Physalis alkekengi extract or a compound isolated therefrom as an active ingredient.

As used in the present invention, "perilla extract" refers to an extract obtained by extracting the fruit, roots, leaves, stems, etc. of perilla using a suitable solvent, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, or any of these. Includes both crude and purified forms. The perilla extract may be extracted from various organs of natural, hybrid, or mutated plants, and may be, in particular, an extract obtained from the perilla fruit (fruit), but is not limited thereto.

The perilla extract of the present invention can be prepared using conventional extraction methods in the art, such as ultrasonic extraction, filtration, and reflux extraction, and perilla can be purchased and used commercially, or collected or cultivated in nature. there is.

The perilla extract according to the present invention can be separated according to a conventional method known in the art for producing extracts from natural products, that is, using a conventional solvent under conventional temperature and pressure conditions.

The perilla extract according to the present invention may be extracted using water including purified water, lower alcohol with 1 to 4 carbon atoms, or a mixed solvent thereof. For example, 70 to 99.9% by volume of methanol aqueous solution or methanol can be used as the extraction solvent, and preferably, it may be extracted with methanol. In addition, when extracting with a solvent, the dried perilla is pulverized with a grinder, then about 1 to 20 times the amount of the pulverized material, and preferably about 1 to 15 times the amount of the extraction solvent is added, followed by heating extraction, cold extraction, or reflux cooling. Extraction, or ultrasonic extraction, may be performed. This extraction may be performed at room temperature or room temperature for 12 hours to 1 week, preferably 20 hours to 72 hours, or may be performed under low or warm temperature conditions. The extract can then be filtered and/or concentrated under reduced pressure to obtain a perilla extract. The extraction process can be repeated 2 to 4 times, and additional processes such as filtration, concentration, and freeze-drying can be performed.

As used herein, the term “fraction” refers to a result obtained by a fractionation method that separates a specific component or group from a mixture containing various components. In the present invention, it refers to the result obtained by a fractionation method for separating specific components or specific groups from the perilla extract prepared as described above.

In order to obtain a perilla fraction according to the present invention, a conventional fractionation solvent known in the art, for example, water containing purified water, lower alcohol with 1 to 4 carbon atoms, ethyl acetate, chloroform, hexane, or a mixed solvent thereof is used. It may be a fraction obtained through sequential systematic fractionation, but is not limited thereto. Preferably, it may be fractionated using water, butanol, ethyl acetate, and hexane.

For example, the perilla fraction of the present invention is obtained by adding water to the extract obtained above and then performing a conventional fractionation process using n-hexane, methylene chloride, ethyl acetate, and butanol to produce n-hexane, chloroform, ethyl acetate, etc. non-polar solvent-soluble extract fraction soluble in non-polar solvent; And a polar solvent-soluble extraction fraction soluble in polar solvents such as butanol and water can be obtained.

In one embodiment of the present invention, after inducing an inflammatory response by LPS in macrophages (RAW264.7), the effect of treatment with a fraction obtained from agar extract on changes in mRNA expression of inflammatory cytokines IL-1β and TNF-α was examined. As a result of the analysis, it was confirmed that the hexane fraction, ethyl acetate fraction, butanol fraction, and water fraction of the methanol extract of Perilla perilla were superior in the inhibitory effect on IL-1β and TNF-α expression compared to the methanol extract of Perilla perilla. In particular, the water fraction was It was confirmed that the effect of reducing the expression of both inflammatory cytokines was excellent (see Examples 1 to 3).

Additionally, the perilla fraction of the present invention may also include one obtained by additionally applying a purification process. For example, fractions obtained by passing the perilla extract according to the present invention through an ultrafiltration membrane with a certain molecular weight cut-off value, separation by various chromatographs (designed for separation according to size, charge, hydrophobicity or affinity), etc. Fractions obtained through various additional purification methods are also included in the acinar fraction of the present invention.

For example, the compounds of the present invention are purified by purification methods such as column chromatography or re-curing using a silica gel column, Sephadex column, ion exchange resin, etc., by purifying the non-polar solvent-soluble fraction, preferably using a developing solvent (chloroform and water). 3,4`,5-trihydroxy-3`,7-dimethoxy-flavone of the present invention is obtained through a process of repeating the purification process using a silica gel column and recrystallization using a mixed solvent (expanding while increasing the polarity). , Physalin B and Isopisalin B compounds can be separated.

The compounds of the present invention include 3,4`,5-trihydroxy-3`,7-dimethoxy-flavone (3,4`,5-trihydroxi-3`,7-dimethoxy-flavone), Physalin B ( It may contain one or more compounds selected from physalin B) and isophisalin B, or a pharmaceutically acceptable salt thereof.

The 3,4',5-trihydroxy-3',7-dimethoxy-flavone of the present invention may be represented by the following formula (1).

The physalin B of the present invention may be represented by the following formula (2).

The isopisaline B of the present invention may be represented by the following formula (3).

In one embodiment of the present invention, after inducing an inflammatory response by LPS in macrophages (RAW264.7), the effect of treatment with acinar extract and compounds derived from it on changes in mRNA expression of inflammatory cytokines IL-1β and TNF-α As a result of the analysis, while the expression change was weak in the methanol extract of Perilla perilla, 3,4',5-trihydroxy-3',7-dimethoxy-flavone, isolated from the fraction of the methanol extract of Perilla perilla, It was confirmed that physalin B and isopisalin B compounds significantly reduce the expression of IL-1β and TNF-α even at low concentrations, and thus are excellent in preventing, improving, or treating inflammatory diseases (Examples 1 to 3 reference).

In the present invention, the inflammatory diseases include dermatitis, edema, allergy, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, pharyngitis, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, and rheumatism. Fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendonitis, tenosynovitis, peritendinitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis, and acute and chronic inflammation. It is preferable that it is one selected from the group consisting of diseases, but it is not limited thereto.

The pharmaceutical composition of the present invention is used for “prevention” and/or “treatment” of inflammatory diseases. For prophylactic use, the pharmaceutical composition of the present invention is administered to an individual suspected of having or at risk of developing a disease, disorder, or condition described herein. For therapeutic use, the pharmaceutical compositions of the present invention may be used to treat or at least partially stop the symptoms of a disease, disorder, or condition described herein in an individual, such as a patient already suffering from a disorder described herein. Administered in sufficient amount. The amount effective for such use will depend on the severity and course of the disease, disorder or condition, previous treatment, the individual's medical condition and responsiveness to the drug, and the judgment of the physician or veterinarian.

The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods. You can.

Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain at least one excipient in the fraction, such as starch, calcium carbonate, and sucrose. ) or prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. . Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.

As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerogeratin, etc. can be used.

The composition of the present invention can be administered in a pharmaceutically effective amount.

In the present invention, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the individual, age, gender, and severity of the disease. It can be determined based on factors including the type, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and can be easily determined by a person skilled in the art. The preferred dosage of the composition of the present invention varies depending on the patient's condition and weight, degree of disease, drug form, administration route and period, and may be administered once a day or in several divided doses.

However, for desirable effects, the composition of the present invention is preferably administered at 0.0001 to 1,000 mg/kg per day, preferably at 0.001 to 200 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. Therefore, the above dosage does not limit the scope of the present invention in any way.

The composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans through various routes. All modes of administration are contemplated, for example, oral, rectal or by intravenous, intramuscular, subcutaneous, intrathecal or intracerebroventricular injection.

In another aspect, the present invention provides a food composition for preventing or improving inflammatory diseases, comprising a fraction of Physalis alkekengi extract or a compound isolated therefrom as an active ingredient.

The terms used here are the same as described above.

In the present invention, the term "improvement" refers to the treatment of suspected inflammatory diseases and the treatment of the subject of the invention using a composition containing a fraction of the perilla extract of the present invention or a compound isolated therefrom (or a foodologically acceptable salt thereof) as an active ingredient. It refers to any action that improves symptoms or is beneficial.

The foodologically acceptable salt of the present invention is useful as an acid addition salt formed by a foodologically acceptable free acid or a metal salt formed by a base. As an example, inorganic acids and organic acids can be used as free acids. As inorganic acids, hydrochloric acid, sulfuric acid, bromic acid, sulfurous acid, or phosphoric acid can be used, and as organic acids, citric acid, acetic acid, maleic acid, fumaric acid, gluconic acid, methanesulfonic acid, etc. can be used. Additionally, as the metal salt, an alkali metal salt, alkaline earth metal salt, sodium, potassium or calcium salt can be used. However, it is not necessarily limited to this.

The food composition of the present invention includes the form of pills, powders, granules, precipitates, tablets, capsules, or liquids. Foods to which the composition of the present invention can be added include, for example, various foods, such as These include beverages, gum, tea, vitamin complexes, and health supplements.

Other than containing the effective ingredients of the present invention as essential ingredients that can be included in the food composition of the present invention, there are no particular restrictions on other ingredients, and like ordinary foods, various herbal extracts, food supplements, or natural carbohydrates can be used as additional ingredients. It may contain. The mixing amount of essential ingredients in the food composition can be appropriately determined depending on the purpose of use (prevention, improvement, health or therapeutic treatment).

In addition, the food auxiliary additives include food auxiliary additives common in the art, such as flavoring agents, flavoring agents, colorants, fillers, stabilizers, etc.

Examples of such natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. In addition to the above-described flavoring agents, natural flavoring agents (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.

In addition to the above, the food composition of the present invention contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its It may contain salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, it may contain pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients can be used independently or in combination.

In the present invention, the health supplements include health functional foods and health foods. The above-mentioned functional food is the same term as food for special health use (FoSHU), and is a medicine processed to efficiently exhibit bioregulatory functions in addition to nutritional supply, with high medical effects. It means food. Here, “function” means adjusting nutrients to the structure and function of the human body or obtaining useful effects for health purposes, such as physiological effects. The food of the present invention can be manufactured by methods commonly used in the industry, and can be manufactured by adding raw materials and components commonly added in the industry. Additionally, the food formulation can be manufactured without limitation as long as it is a formulation recognized as a food. The food composition of the present invention can be manufactured in various forms, and unlike general drugs, it is made from food as a raw material, so it has the advantage of not having side effects that may occur when taking the drug for a long time, and is highly portable.

In another aspect, the present invention relates to a cosmetic composition for preventing or improving inflammatory diseases, comprising a fraction of Physalis alkekengi extract or a compound isolated therefrom as an active ingredient.

The terms used here are the same as described above.

The cosmetic composition of the present invention can be prepared in the form of a general emulsified formulation and solubilized formulation. Cosmetics in emulsified formulations include nutritious lotions, creams, and essences, and cosmetics in solubilized formulations include flexible lotions. Suitable cosmetic formulations include, for example, solutions, gels, solid or pasty anhydrous products, emulsions obtained by dispersing the oil phase in the water phase, suspensions, microemulsions, microcapsules, microgranules or vesicles of ionic (liposome) or non-ionic form. It may be provided in the form of a dispersant, cream, skin, lotion, powder, ointment, spray or concealer stick. Additionally, it can be prepared in the form of foam or in the form of an aerosol composition further containing compressed propellant.

In addition to the active ingredients of the present invention, the cosmetic composition further contains fatty substances, organic solvents, solubilizers, thickeners and gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, Commonly used in water, ionic or non-ionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives, lipid vesicles or in cosmetics. It may contain auxiliaries commonly used in the cosmetic field, such as any other ingredients.

The fraction prepared from the perilla extract of the present invention or the compound isolated therefrom contains inflammatory cytokines, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Since it is a safe substance that inhibits the production or secretion of and is not cytotoxic, it can be usefully used in cosmetic compositions for preventing or improving inflammatory diseases.

In another aspect, the present invention relates to a method for preventing or treating inflammatory diseases, comprising administering a pharmaceutical composition containing a fraction of Physalis alkekengi extract or a compound isolated therefrom as an active ingredient. .

The terms used here are the same as described above.

In the present invention, the term "subject" refers to any animal that has developed or is likely to develop an inflammatory disease, and by administering the pharmaceutical composition of the present invention to an individual suspected of having an inflammatory disease, the individual can be efficiently treated.

In the present invention, the term "administration" means introducing the pharmaceutical composition of the present invention into a subject suspected of having an inflammatory disease by any appropriate method, and the route of administration may be oral or parenteral, as long as it can reach the target tissue. It can be administered through

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount, and the pharmaceutically effective amount is as described above.

The pharmaceutical composition of the present invention is not particularly limited and can be applied to any object for the purpose of preventing or treating inflammatory diseases. For example, non-human animals such as monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cows, sheep, pigs, goats, birds and fish can be used, and the pharmaceutical composition can be administered parenterally, subcutaneously, etc. It can be administered intraperitoneally, intrapulmonaryly and intranasally, and for topical treatment, if necessary, by any suitable method, including intralesional administration. The preferred dosage of the pharmaceutical composition of the present invention varies depending on the individual's condition and weight, degree of disease, drug form, administration route and period, but can be appropriately selected by a person skilled in the art. For example, it may be administered orally, intraperitoneally, rectally or by intravenous, intramuscular, subcutaneous, intrauterine intrathecal or intracerebrovascular injection, but is not limited thereto.

The appropriate total daily usage amount can be determined by the treating physician within the range of sound medical judgment, generally at a concentration of 0.0001 to 1000 mg/kg, specifically at a concentration of 0.001 to 1000 mg/kg, and more specifically at a concentration of 1 to 200 mg/kg. The concentration of mg/kg can be administered once to several times a day.

Hereinafter, the present invention will be described in more detail in the following examples and preparation examples. However, these examples are only intended to aid understanding of the present invention, and the present invention is not limited thereto.

Example 1. Preparation of purified acorns

1-1. Preparation of perilla crude extract

Dried alkekengi ( Physalis alkekengi L. var. francheti ) collected in Icheon-si, Gyeonggi-do were purchased from Chodamchae Company. 8kg of the fruits were washed, cut, placed in 10L of 100% methanol, and extraction was repeated three times at room temperature for 24 hours. This was performed, and the extracts were collected, filtered, and concentrated to obtain 793g of perilla extract (crude extract, hereinafter referred to as “EA325M”).

1-2. Separation of purified products soluble in non-polar and polar solvents

793 g of the crude extract obtained in Example 1-1 was suspended by adding 2 L of distilled water, mixed with 2 L of hexane, and then fractionated three times by separating only the components dissolved in the nucleic acid layer in a fractionation funnel and vacuum drying them. Thus, a hexane layer was obtained. 2L of ethyl acetate was added to the remaining water layer (aqueous layer), and only the components dissolved in the ethyl acetate layer were separated through a fractionation filter, and the fractions were vacuum dried three times to obtain an ethyl acetate layer. Through the same process, 2L of butanol was added to the remaining water layer and fractionation was performed three times to obtain a butanol layer. The hexane layer, ethyl acetate layer, butanol layer, and remaining water layer recovered through the above fractionation process were each freeze-dried to obtain 85 g of hexane fraction (hereinafter referred to as “EA325H”) and 17 g of ethyl acetate fraction (hereinafter referred to as “EA325E”). , 46 g of butanol fraction (hereinafter referred to as “EA325B”) and 150 g of water fraction (hereinafter referred to as “EA325Aq”) were obtained and used as samples for the following experiments. The preparation process of each fraction from the perilla extract is as shown in Figure 1.

Example 2. Isolation of compounds from perilla extract

2-1. Isolation of 3',7-dimethylquercetin (1)

The hexane soluble fraction (EA325H, 85 g) of Example 1 was subjected to silica gel column chromatography using an elution solvent (hexane:ethyl acetate mixed solvent concentration gradient 1:0 → 0:1). Silica gel 60, 230 to 400 mesh, Merck) was used to obtain 14 small fractions EA325H-34-9-F1 to F14. Among these small fractions, the 8th fraction separated from the first column, i.e., EA325H-34-9-F8 (1.10 g), was eluted with silica gel using an elution solvent (hexane:acetone mixed solvent concentration gradient 1:0→0:1). Column chromatography (silica gel column chromatography; silica gel 60, 230 to 400 mesh, Merck) was performed to produce small fractions EA325H-34-11-1 to 10 and EA325H-34-11-K8 (40) showing the following physical properties. mg) was obtained. This EA325H-34-11-K8 was identified as ³ ', ⁷ -dimethylquercetin (3,4',5- Trihydroxy-3`,7-dimethoxy-flavone; 3,4`,5-trihydroxi-3`,7-dimethoxy-flavone; hereinafter, “EA325H-34-11-K8” The structure was analyzed and its structure was confirmed by comparison with the literature, and it was confirmed that it was the first compound isolated and reported from plants of the genus Physalis (AK Umbetova, Sh. Zh. Esirkegenova, IM Chaudri, VB Omurkamzinova, and Zh. A. Abilov, FLAVONOIDS OF PLANTS FROM THE GENUS Tamarix , Chemistry of Natural Compounds , 2004, 40(3), 297-298.).

3',7-dimethylquercetin (EA325H-34-11-K8; 3',7-dimethylquercetin) (1)

Molecular formula: C ₁₇ H ₁₄ O ₇

Molecular Weight: 330

^1H NMR (200MHz, CDCl ₃ ) δ ppm: 12.13 (1H, s, OH), 7.92 (d, J = 2.0 Hz, H-2'), 7.85 (1H, dd, J = 8.8. 2.0 Hz, H -6'), 7.02 (1H, d, J = 2.0 Hz, H-5'), 6.72 (1H, d, J = 2.0 Hz, H-8), 6.33 (1H, d, J = 2.0 Hz, H -6), 3.94 (3H, s, OCH ₃ -3'), 3.93 (3H, s, OCH ₃ -7).

¹³ C NMR (50MHz, CDCl ₃ ) δ ppm: 176.8 (C-4), 166.8 (C-7), 162.0 (C-5), 157.8 (C-8a), 149.9 (C-4'), 148.4 ( C-3'), 147.3 (C-2), 137.1 (C-3), 123.6 (C-1'), 123.0 (C-6'), 116.2 (C-5'), 112.2 (C-2') ), 104.9 (C-4a), 98.5 (C-6), 92.9 (C-8), 56.5 (OCH ₃ -3', OCH ₃ -7).

2-2. Separation of physalin B (2) and isophisalin B (3)

The ethyl acetate fraction (EA325E, 17g) of Example 1 was subjected to silica gel column chromatography using an elution solvent (hexane-EtOAc=99:1 → 1:1). Fractions EA325H-34-9-F1 to F14 were obtained, and among these subfractions, EA325H-34-9-F8 (1.10 g) was subjected to silica gel column chromatography using an elution solvent (hexane-acetone=4:1). By conducting silica gel column chromatography, subfraction EA325H-34-11-1~10 and physalin B (2, 90 mg) and isophysalin B (3, 7 mg) showing the following physical properties were obtained, respectively. The structure was obtained and analyzed, and the structure was confirmed by comparison with the literature.

Physalin B (EA325E-34-6-K4; physalin B)(2)

Molecular formula: C ₂₉ H ₃₂ O _8C28 H ₃₀ O ₉

Molecular Weight: 509

Isophysalin B (EA325E-34-15-K4; isophysalin B)(3)

Molecular formula: C ₂₉ H ₃₂ O _8C28 H ₃₀ O ₉

Molecular Weight: 511

Reference Example 1. Preparation of experimental materials

Cell culture reagents such as cell culture medium RPMI-1640, Dulbecco's Modifide Eagle Medium (DMEM), 10% (v/v) fetal bovine serum (FBS), penicillin, and streptomycin are supplied by Welgene (LS202-02, S-001-04). , LM001-01, Korea), and the reagent Ezcytox was purchased from Daeilab (ez 3000, Korea). The rotary evaporator was purchased from (BUCHI, R-124, German), the freeze dryer was purchased from Iishin (FDU-2200, Korea), TRIzol ^TM was purchased from GIBCO BRL (MD, USA), and the ELISA reader was purchased from Anthos ( Multiread 400, Austia), spectrophotometer from GE healthcare Life sciences (Ultraspec 2100 pro, Sweden), Primeacript Rtase from TaKaRa (Shiga, Japan), and PTC-100 Programmable Thermal Controller ^TM from GE healthcare Life sciences (Ultraspec 2100 pro, Sweden). MJ Research was purchased from Waltham (MA, USA), and ImageMaster TotalLab ^™ was purchased from Amersham Biosceinces (Piscataway, NJ).

Example 3. Effect on mRNA expression of cytokines (IL-1β and TNF-α) in inflammatory response using LPS

3-1. cell culture

RAW264.7, a mouse macrophage cell line, was purchased from ATCC. RAW264.7 was grown in the laboratory using high glucose DMEM medium containing 10% (v/v) fetal bovine serum (FBS) and antibiotics of 100U/ml of penicillin and 100μg/ml of streptomycin, supplied under 37°C and 5% CO ₂ supply conditions. were cultured in an animal cell incubator equipped with .

3-2. Sample processing

Raw 264.6 macrophage cells were placed in 6 wells at 2×10 ⁶ cells/well and cultured for 24 hours. After 24 hours, cultured in serum-free medium for 4 hours, treated with LPS (1 μg/ml), and then treated with LPS (1 μg/ml), and then added to 1, 10 or 10 of the acinar extracts, fractions or compounds derived from them, respectively, obtained in Examples 1 and 2. Treated at a concentration of 100 μg/ml and incubated for 24 hours.

3-3. RNA extraction and PCR analysis

To confirm the effect of the perilla extract, fractions, and compounds isolated therefrom obtained in Examples 1 and 2 on IL-1β and TNF-α, which are proinflammatory cytokines using lipopolysaccharide (LPS), the following We experimented together.

Total RNA was extracted from RAW264.7 cells cultured by the method of Examples 3-1 and 3-2 using TRIzol ^™ (Invitrogen, Carlsbad, CA, USA), and nano drop (NanoDrop 2000c; Thermo Scientific, Wilmington) , DE) and then stored at -80°C. After denaturing 2μg of total RNA at 65°C for 10 minutes, reverse transcription was performed using Primeacript Rtase 0.2μl (TaKaRa, Shiga, Japan) in a reaction mixture with a final volume of 20μl to obtain a cDNA mixture. cDNA was amplified using the PTC-100 Programmable Thermal Controller ^TM (MJ Research, Waltham, MA, USA) in 20 μl of reaction mixture containing 10X PCR buffer, each primer, and 0.5 U of Taq polymerase (TaKaRa, Shiga, Japan). . Each primer was produced by selecting an appropriate region based on the base sequence recorded in Genbank, and each base sequence and amplification conditions are shown in Table 1 below. The amplified DNA was confirmed by electrophoresis on a 1.2% agarose gel. The band intensity on the gel was analyzed using ImageMaster TotalLab ^™ (Amersham Biosceinces, Piscataway, NJ), and the quantitative expression level of the gene was corrected using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal standard.

Name Primer sequence Product size (bp) Annealing temperature (℃) Cycle numbers IL-1β 5'-tgcagagttccccaactggtacatc-3' 387 60 30 5'-gtgctgcctaatgtccccttgaatc-3' TNF-α 5'-cctgtagcccacgtcgtagc-3' 374 60 30 5'-ttgacctcagcgctgagttg-3' GAPDH 5'-atcccatcaccatcttccag-3' 579 60 30 5'-cctgcttcaccaccttcttg-3'

3-4. Inhibitory efficacy of methanol extract of Perilla on the expression of inflammatory cytokines

The results showing TNF-α and IL-1β gene expression according to treatment with acinar methanol extract (EA325M) after inducing an inflammatory response in Raw 264.7 cells using LPS are shown in Figure 2.

Compared to the group given only medium, the gene expression of TNF-α (F _4,14 =191.8, P<0.001) and IL-1β (F _4,14 =159.8, P<0.001) increased significantly in the group given LPS. did. On the other hand, when treated with 10-100 μg/ml of EA325M from the methanol extract of Perilla, compared to the LPS-treated group, TNF-α and IL-1β were decreased except for the decrease in IL-1β gene expression in the 100 μg/ml treated group. There was no significant effect on gene expression.

3-5. Inhibitory efficacy of acinar hexane fraction on inflammatory cytokine expression

The results showing TNF-α and IL-1β gene expression according to treatment with acinar hexane fraction (EA325H) after inducing an inflammatory response in Raw 264.7 cells using LPS are shown in Figure 3.

Compared to the group given only medium, the gene expression of TNF-α (F _4,14 =120.2, P<0.01) and IL-1β (F _4,14 =128.4, P<0.001) increased significantly in the group given LPS. did. On the other hand, when treated with the hexane fraction EA325H at 10-100 μg/ml, it was confirmed that TNF-α showed a statistically significant decrease as the concentration of the drug increased compared to the LPS-treated group, and IL-1β Gene expression was also confirmed to be reduced.

3-6. Inhibitory efficacy of acinar butanol fraction on inflammatory cytokine expression

The results showing TNF-α and IL-1β gene expression according to treatment with acinar butanol fraction (EA325B) after inducing an inflammatory response in Raw 264.7 cells using LPS are shown in Figure 4.

Compared to the group given only medium, the gene expression of TNF-α (F _4,14 =174.8, P<0.001) and IL-1β (F _4,14 =12.6, P<0.001) increased significantly in the group given LPS. (F=25.1, P<0.01). On the other hand, when treated with the butanol fraction of EA325B at 10-100 μg/ml, it was confirmed that TNF-α gene expression showed a statistically significant decrease in the EA325B-treated group compared to the LPS-treated group.

3-7. Inhibitory efficacy of acini water fraction on inflammatory cytokine expression

The results of TNF-α and IL-1β gene expression according to treatment with acinar water fraction (EA325Aq) after inducing an inflammatory response in Raw 264.7 cells using LPS are shown in Figure 5.

Compared to the group given only medium, the gene expression of TNF-α (F _4,14 =180.2, P<0.001) and IL-1β (F _4,14 =159.8, P<0.001) increased significantly in the group given LPS. (F=25.1, P<0.01). On the other hand, when treated with 10-100 μg/ml of the water fraction EA325Aq, it was confirmed that compared to the LPS-treated group, TNF-α and IL-1β gene expression showed a statistically significant decrease in the EA325Aq-treated group. I was able to.

3-8. Inhibitory efficacy of acinar ethyl acetate fraction on inflammatory cytokine expression

The results showing TNF-α and IL-1β gene expression according to treatment with acinar ethyl acetate fraction (EA325E) after inducing an inflammatory response in Raw 264.7 cells using LPS are shown in Figure 6.

Compared to the group given only medium, the gene expression of TNF-α (F _4,14 =13.3, P<0.01) and IL-1β (F _4,14 =65.1, P<0.001) increased significantly in the group given LPS. (F=25.1, P<0.01). On the other hand, when treated with the ethyl acetate fraction of EA325E at 1, 100 μg/ml, it was confirmed that TNF-α gene expression showed a statistically significant decrease in the EA325E-treated group compared to the LPS-treated group, and IL -1β gene expression was also confirmed to be reduced.

3-9. Inhibitory efficacy of isophisalin B on inflammatory cytokine expression

The results showing TNF-α and IL-1β gene expression according to isophisalin B (EA325E-34-15-K4) treatment after inducing an inflammatory response in Raw 264.7 cells using LPS are shown in Figure 7.

Compared to the group given only medium, the gene expression of TNF-α (F _4,14 =52.7, P<0.001) and IL-1β (F _4,14 =435.6, P<0.001) increased significantly in the group given LPS. did. On the other hand, when treated with 1-100 μg/ml of isophisalin B (EA325E-34-15-K4), a compound derived from agaris, TNF-α gene expression was EA325E-34-15-K4 compared to the LPS-treated group. It was confirmed that there was a statistically significant decrease in the group treated with K4.

3-10. Inhibitory efficacy of Physalin B on inflammatory cytokine expression

The results of TNF-α and IL-1β gene expression according to physalin B (EA325E-34-6-K4) treatment after inducing an inflammatory response in Raw 264.7 cells using LPS are shown in Figure 8.

Compared to the group given only medium, the gene expression of TNF-α (F _4,14 =74.1, P<0.001) and IL-1β (F _4,14 =35.3, P<0.001) increased significantly in the group given LPS. did. On the other hand, when treated with 1-100 μg/ml of physalin B (EA325E-34-6-K4), a compound derived from agaris, TNF-α gene expression decreased compared to the LPS-treated group. It was confirmed that there was a statistically significant decrease in the treated group.

3-11. Inhibitory efficacy of 3',7-dimethylquercetin on inflammatory cytokine expression

The results showing TNF-α and IL-1β gene expression according to 3',7-dimethylquercetin (EA325H-34-11-K8) treatment after inducing an inflammatory response in Raw 264.7 cells using LPS are shown in Figure 9. .

Compared to the group given only medium, the gene expression of TNF-α (F _4,14 =423.5, P<0.001) and IL-1β (F _4,14 =243.0, P<0.001) increased significantly in the group given LPS. did. On the other hand, when treated with 1-100 μg/ml of 3',7-dimethylquercetin (EA325H-34-11-K8), a compound derived from agaris, TNF-α and IL-1β gene expression increased compared to the LPS-treated group. It was confirmed that a statistically significant decrease was observed in all groups treated with EA325H-34-11-K8.

Below, a formulation example of a composition containing the extract of the present invention is described, but the present invention is not intended to be limited, but merely explained in detail.

Formulation Example 1. Preparation of powder

Agar-derived compounds 20 mg

lactose 100 mg

Talc 10mg

The above ingredients are mixed and filled into an airtight bubble to prepare a powder.

Formulation Example 2. Preparation of tablets

Agar-derived compounds 10mg

corn starch 100mg

lactose 100mg

Magnesium stearate 2mg

After mixing the above ingredients, tablets are manufactured by compressing them according to a typical tablet manufacturing method.

Formulation Example 3. Preparation of capsules

Agar-derived compounds 10mg

crystalline cellulose 3mg

lactose 14.8mg

magnesium stearate 0.2mg

Capsules are prepared by mixing the above ingredients and filling them into gelatin capsules according to a typical capsule manufacturing method.

Formulation Example 4. Preparation of injection

Agar-derived compounds 10mg

Mannitol 180mg

Sterile distilled water for injection 2974mg

Na ₂ HPO ₄ ,12H ₂ O 26 mg

Prepare with the above ingredients per ampoule (2 ml) according to the usual manufacturing method for injections.

Formulation Example 5. Preparation of liquid formulation

Agar-derived compounds 20 mg

Iseonghwadang 10 g

mannitol 5 g

Purified water Appropriate amount

According to the usual liquid preparation method, add and dissolve each ingredient in purified water, add an appropriate amount of lemon flavor, mix the above ingredients, add purified water, adjust the total to 100 ml by adding purified water, and fill it in a brown bottle. Sterilize to prepare a liquid.

Formulation Example 6. Preparation of health food

Agar-derived compounds 1000 mg

vitamin mixture Appropriate amount

Vitamin A Acetate 70 ㎍

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

Vitamin B12 0.2 μg

Vitamin C 10 mg

biotin 10 μg

Nicotinic acid amide 1.7 mg

folic acid 50 μg

Calcium Pantothenate 0.5 mg

mineral mixture Appropriate amount

Ferrous sulfate 1.75 mg

zinc oxide 0.82 mg

Magnesium Carbonate 25.3 mg

Potassium Phosphate Monobasic 15 mg

Dibasic Calcium Phosphate 55 mg

potassium citrate 90 mg

calcium carbonate 100 mg

Magnesium chloride 24.8 mg

The composition ratio of the above vitamin and mineral mixture is a mixture of components relatively suitable for health food in a preferred embodiment, but the mixing ratio may be modified arbitrarily. The above ingredients are mixed according to a typical health food manufacturing method, and then , granules can be manufactured and used to manufacture health food compositions according to conventional methods.

Formulation Example 7. Preparation of health drink

Agar-derived compounds 1000 mg

citric acid 1000 mg

oligosaccharide 100g

plum concentrate 2g

taurine 1g

Add purified water Total 900 ml

After mixing the above ingredients according to a typical health drink manufacturing method, stirring and heating at 85° C. for about 1 hour, the resulting solution was filtered, obtained in a sterilized 2-liter container, sealed, sterilized, and refrigerated, followed by the present invention. It is used in the production of health drink compositions.

The composition ratio is a preferred embodiment of mixing ingredients relatively suitable for beverages of preference, but the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as demand class, country of demand, and intended use.

Claims (8)

3,4`,5-trihydroxy-3`,7-dimethoxy-flavone (3,4`,5-trihydroxi-3`,7-dimethoxy-flavone), physalin B and isopy A pharmaceutical composition for preventing or treating inflammatory diseases, comprising at least one compound selected from isophysalin B, or a pharmaceutically acceptable salt thereof.
The composition of claim 1, wherein the compound is isolated from Physalis alkekengi extract.
The composition according to claim 1, wherein 3,4',5-trihydroxy-3',7-dimethoxy-flavone is isolated from the hexane fraction of Physalis alkekengi extract.
The composition according to claim 1, wherein the physalin B and isophisalin B are isolated from the ethyl acetate fraction of the Physalis alkekengi extract.
The composition according to any one of claims 2 to 4, wherein the extract is extracted with methanol.
According to paragraph 1,
The inflammatory diseases include dermatitis, edema, allergy, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, pharyngitis, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus, and fibromyalgia. (fibromyalgia), psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, tenosynovitis, peritendinitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis, and acute and chronic inflammatory diseases. A composition, any one of which is selected.
3,4`,5-trihydroxy-3`,7-dimethoxy-flavone (3,4`,5-trihydroxi-3`,7-dimethoxy-flavone), physalin B and isopy A food composition for preventing or improving inflammatory diseases comprising at least one compound selected from isophysalin B.
3,4`,5-trihydroxy-3`,7-dimethoxy-flavone (3,4`,5-trihydroxi-3`,7-dimethoxy-flavone), physalin B and isopy A cosmetic composition for preventing or improving inflammatory diseases comprising at least one compound selected from isophysalin B.