KR101332824B1 - Pharmaceutical Compositions for Preventing or Treating Arthritis Comprising Euphorbia ebracteolata Extracts - Google Patents

Abstract

The present invention is directed to a pharmaceutical composition having an anti-inflammatory effect comprising an Ingenol-3-palmitate or Ingenol-3-myristinate compound extracted from a mantra It is about. Euphorbia ebracteolata extract or fraction of the present invention exhibiting potent anti-inflammatory activity against arthritis is a herbal derived from natural products, with no toxicity and side effects, and nitric oxide and tumor necrosis factor, which are key mediators of the inflammatory response. It significantly inhibits the production of -α and Interleukin-1β, and has an excellent anti-inflammatory effect, such as the inhibitory effect of PGE ₂ , an inflammatory mediator, so that the composition containing it not only treats and prevents arthritis inflammatory diseases but also relieves symptoms. It can be usefully used to improve.

Description

Pharmaceutical Compositions for Preventing or Treating Arthritis Comprising Euphorbia ebracteolata Extracts}

The present invention provides a method for extracting and purifying the active ingredient from the folk tales, and ingenol-3-palmitate or ingenol-3-myristinate containing the extract. The present invention relates to a herbal composition of the present invention and a pharmaceutical composition having an antiarthritis effect using the same. Specifically, the present invention relates to extracting and concentrating bioactive components of Euphorbia ebracteolata to identify active substances having anti-inflammatory effects, and to treating arthritis compositions using the same and a method of preparing the same.

Aging and degeneration of the joints result in the inhibition of the synthesis of chondrocytes that form the joints such as type II collagen and proteoglycans, Inflammatory cytokines such as 1β, IL-1β) and tumor necrosis factor-α (TNF-α) are produced. Arthritis is a disease caused by the destruction of joint tissue due to increased synthesis and activity of matrix metalloproteinase (MMP), which breaks down the joint matrix through this mechanism.

In addition, arthritis is exacerbated by the production of nitric oxide (NO) by inflammatory cytokines and the production of self-amplifying cytokines by nitric oxide produced, which leads to the synthesis of more MMPs and promotes the degradation of joint matrix. As arthritis progresses, expression and activity of MMP-1, 2, 3, 8, 9, 13, etc. are increased (Wieland HA et al., Nat Rev Drug Discov 4: 331-344, 2005). At the same time, the inflammatory cytokine is a lipid metabolite, prostaglandin E ₂ (prostaglandin E ₂ , PGE ₂ ) increases the production of inflammatory reactions in the joints. Although the cause is uncertain, it is known that the disease is deeply related to aging or excessive weight, in which the degenerative change of articular cartilage is the first. Degenerative changes begin in articular cartilage leading to necrosis of cartilage cells, and the substrate is destroyed by cathepsin B, catepsin D, collagenase and the like. If the production of proteoglycan and collagen does not keep up with the degree of destruction, the ability of cartilage to adapt to external forces gradually decreases, resulting in fine fractures in the cartilaginous subchondral tissue. As the disease progresses, hardening of the subchondral cartilage, hyperplasia of the bone around the joint, and deformation of the joint occur, and the surface of the cartilage becomes rough and the inflammatory reaction appears repeatedly in the joint cavity wrapped in the joint membrane. Therefore, arthritis causes repetitive pain, joint stiffness, gradual movement disorder of the joint, and the like.

Arthritis increases with pain in the joints, due to the production of PGE ₂ already mentioned. PGE ₂ is synthesized by an enzyme called cyclooxygenase-2 (COX-2), which increases its activity by inflammatory cytokines such as NO, TNF-α, IL-1β or other stimuli. . In addition, as osteoarthritis progresses, cartilage is destroyed and inflammation-related factors such as cytokines, chemokines, and protease are secreted, causing inflammation of the synovial membrane, thereby increasing serum hyaluronic acid (HA) concentration. IL-1β and TNF-α can directly damage the living organisms distributed in the synovial membrane or stimulate the sensory nerve fibers and nociceptors distributed in the synovial membrane. These inflammatory cytokines release PGE ₂ and histamine from chondrocytes and mast cells, which in turn stimulate painful receptors.

Currently used treatment methods for arthritis primarily use appropriate nonsteroidal anti-inflammatory drugs (NSAIDs) to try to alleviate the inflammation and use steroid preparations if the inflammation is severe or the efficacy of NSAIDs is not good. If refractory, an immunosuppressant or surgical treatment may be used. NSAIDs, used to alleviate inflammation, mainly inhibit COX and inhibit the production of prostaglandin, which is involved in the inflammatory response, thereby exhibiting anti-inflammatory effects of arthritis but causing side effects such as gastrointestinal disorders, liver disorders, and renal dysfunction. Difficult to use Steroid preparations are also the fastest methods available to patients, and they are fast and dramatic drugs with anti-inflammatory effects of arthritis, but they are less toxic due to weakened resistance to bacterial infections, worsening diabetes, adrenal insufficiency and mental dysfunction. It is not only very serious, but it is difficult to stop the treatment when it is necessary to use caution, and if possible should be prohibited because it is known that the development of a therapeutic agent for arthritis with a strong effect and relatively low side effects is required. Therefore, considering the efficacy and side effects, it is considered that natural product formulations, which have abundant clinical experience and excellent evaluation in terms of safety, will be good candidates for the prevention and treatment of arthritis diseases.

The folk drama is a plant belonging to the Great Drama and the genus, and there are about 1,600 species in the world, and about 11 species and varieties are growing in Korea. The folk drama with Daeguk is thinner in its roots and rarely grows in the lowland grasslands and forests of Jeolla province in Korea. It is named because the new shoots coming from the ground in early spring are red like crab legs. It is called folk drama in the sense that there is no vesicle lobe in. The herbal medicine called folk rapeseed, which is the main ingredient presented in the present invention, is generally well known as a herbal medicine and has fewer side effects such as toxicity. Focusing on protecting and regenerating cartilage and bone tissue in the joints, the core of arthritis treatment, to demonstrate effective pharmacological and primary efficacy separation and scientifically proven therapeutic effects on the arthritis of medicinal herbs called folk tales In accordance with the present invention, the present invention has been completed. Therefore, the present inventors searched for the effect of inducing inflammation and removing the inflammation by using endotoxin, and the hexane extract of the subterranean pole showed a significant anti-inflammatory activity, thereby having an effect on the inflammation of macrophages. The components were followed and developed as new therapeutic agents for inflammatory diseases.

The present invention relates to a method for extracting and purifying an active ingredient from a single herbal medicine, a herbal composition containing the extract, and an anti-arthritis treatment method using the same. Specifically, the extract of Euphorbia ebracteolata is concentrated by hot water or alcoholic aqueous solution. The present invention relates to a method for treating arthritis having a protective effect on articular cartilage and a composition for the above treatment. In the RAW264.7 cell line stimulated with interferon γ / LPS, the folk extract of the present invention inhibited NO and PGE ₂ production, iNOS expression, an NO-producing enzyme, and IL-1β expression, an proinflammatory cytokine. It has excellent anti-inflammatory effects, such as inhibition of TNF-α expression, and verified the efficacy of non-toxic food ingredients, natural products or conventional herbal medicine by confirming that it is a safe substance having no cytotoxicity. In addition, the anti-inflammatory and anti-inflammatory effects of arthritis were investigated through animal experiments while maintaining the cartilage and bone tissue of the joint, which is the core of arthritis treatment, and the anti-inflammatory bioactive components of arthritis were excellent in Euphorbia ebracteolata. Extraction, fractions were prepared. In particular, the present invention was completed by separating an extract capable of effectively inhibiting inflammatory cell growth and high immune cell activating effect, excellent in vivo inflammatory cell growth in the maximal fraction, and demonstrating its efficacy.

An object of the present invention is to provide a pharmaceutical composition for treating and preventing arthritis by extracting an active substance that is effective in enhancing immune function and inflammatory diseases in Euphorbia ebracteolata.

In order to solve the above problems, according to a preferred embodiment of the present invention, Ingenol-3-palmitate, Ingenol-3-myristy extracted from Euphorbia ebracteolata as an active ingredient Provided is a pharmaceutical composition for preventing and treating arthritis, including nate (Ingenol-3-myristinate).

According to another suitable embodiment of the present invention, Ingenol-3-palmitate, Ingenol-3-myristinate is characterized in that the compound represented by the formula (1).

(Wherein R is CO (CH ₂ ) ₁₄ CH ₃ or CO (CH ₂ ) ₁₂ CH _3. )

According to another suitable embodiment of the present invention, Ingenol-3-palmitate, Ingenol-3-myristinate is added 5-20 L of water or alcohol solvent per kg of dry pole roots and left at 20-50 ° C. for 0.5-2 days. Filtering and evaporating under reduced pressure to obtain an alcohol extract, adding a solvent mixed with water and methanol 1: 1 to the alcohol extract, and then extracting an organic material by adding hexane to the separated water layer, and The organic material is characterized by being extracted by fractionation in three steps of chromatography.

According to another suitable embodiment of the present invention, the alcohol solvent is characterized in that ethanol or methanol.

According to another suitable embodiment of the present invention, the chromatography is characterized in that the one selected from the group consisting of normal phase vacuum flash chromatography, C18 normal phase semi-preparative high performance liquid chromatography.

Compounds extracted from the euphorbia ebracteolata inhibit the activity of nitric oxide and inflammatory mediators IL-1β and TNF-α and inflammatory mediator PGE ₂ in macrophages involved in the inflammatory response. The pharmaceutical composition comprising the compound may be developed as an anti-inflammatory agent and may also help in the prevention of degenerative diseases such as gastritis, colitis, hepatitis, arthritis and arteriosclerosis and cancer caused by inflammatory reactions.

FIG. 1 is an HPLC chromatogram of Ingenol-3-palmitate as an index component of Euphorbia ebracteolata extract.
2 is an HPLC chromatogram of an Ingenol-3-myristinate compound, which is an indicator component of Euphorbia ebracteolata extract.
3 is a measure of the toxicity of immune cells of the folk rapeutic extract of Comparative Examples and Examples.
Figure 4 is a measure of the amount of NO production inhibition of the folk electrode extract of Comparative Examples and Examples.
5 is a measure of the amount of inhibition of production of TNF-α of the folk electrode extract of Comparative Examples and Examples.
Figure 6 measures the amount of inhibition of production of IL-1β of the folk extract of Comparative Examples and Examples.
Figure 7 measures the amount of inhibition of production of PGE ₂ of the folk extract of Comparative Examples and Examples.
Figure 8 shows the preparation process of the folk electrode extract of the embodiment.

As used herein, the term “arthritis” or “inhibition of inflammation” refers to suppression of inflammatory reactions that are harmful to the human body, such as chronic inflammation progressing into a disease state, inflammatory reactions generally caused by harmless substances such as pollen, and inflammatory reactions such as asthma or degenerative arthritis. Means.

As used herein, the term "folk larvae extract" includes all of them, without being particularly limited, as long as they are obtained by extracting an active ingredient from natural tropics. For example, the result obtained by putting a maximal pole into water or an organic solvent and eluting an effective component through means, such as standing, stirring, pressurization, or heating, is mentioned. It also includes a freeze-dried product obtained by lyophilizing the liquid extract obtained as described above. It also includes a powder obtained by pulverizing such a lyophilizate. In addition, it contains all the extracted extracts regardless of the available means for extracting the active ingredient, including those extracted and then subjected to freeze-drying and the like. Other examples include extracts obtained by conventional extraction methods such as extracting by hot water or room temperature, or extracts obtained by conventional extraction methods as described in Korean medicine or textbooks. It also includes a fraction extract obtained through a Stass Otto extraction method or various column chromatography methods, which is a special extraction method for separating specific active compounds.

The folk drama can be used without limitation, such as harvested, cultured or commercially available, it is preferable to use after drying to remove the foreign matter and salt by washing with water, for example. Mudae pole extract in the present invention may be prepared by the usual extraction methods in the art, such as ultrasonic extraction method, filtration method and reflux extraction method.

According to a preferred embodiment of the present invention, the tropic extract is added with 5-20 L of water or alcohol solvent per kg of dried root of the tropics and left at 20-50 ° C. for 0.5 hour to 2 days, followed by filtration and evaporation under reduced pressure. To obtain an alcohol extract; Adding an organic solvent mixed with water and hexane 1: 1 to the alcohol extract, and then extracting an organic material from the separated hexane layer; And extracting the organic material by fractionation with three steps of chromatography.

Hereinafter, the extraction method will be described in detail.

First, the roots of Euphorbia ebracteolata are washed with water to remove heavy metals or contaminants, dried, and then ground. Next, using about 5 to 25 times the dry weight, preferably about 20 times the volume of water, lower alcohols such as methanol or ethanol, or a mixed solvent thereof having a mixing ratio of about 1: 0.1 to 1:10 Extract. Preferably extracted with methanol at an extraction temperature of 20 to 100 ° C., preferably at 20 to 50 ° C. for about 0.5 hours to 2 days, preferably 1 hour to 1 day, and the extraction frequency is 1 to 5 times, preferably Preferably two to three consecutive extractions.

Next, the extracted material is filtered under reduced pressure, and the filtrate is concentrated under reduced pressure at 20 to 100 ° C., preferably at 50 to 70 ° C., using a vacuum rotary concentrator, followed by drying the extracted residue with a vacuum freeze dryer to obtain an alcohol extract. Each extract obtained was used for the experiment while stored at -20 ℃.

Water and hexane are added to the obtained alcohol extract in a 1: 1 ratio, mixed, and separated into a hexane layer and a water layer. The separated hexane layer was dried under reduced pressure to prepare a hexane extract. At this time, the vacuum drying conditions are as described above.

Next, the hexane extract is successively separated and purified by normal phase vacuum flash chromatography and C18 normal phase semi-preparative high performance liquid chromatography to prepare a commercial extract of the present invention. In this case, it is preferable to select an effective fraction through the anti-inflammatory efficacy test for each fraction and to separate and purify the selected effective fraction by chromatography in the next step.

In addition, the present invention provides a method for preventing or treating inflammation comprising administering the folk rapeseed extract in an amount that inhibits inflammation.

At this time, the amount of inhibiting inflammation is not limited thereto, but preferably 0.1 to 500 mg / kg and more preferably 1 to 100 mg / kg. The dosage may vary depending on the weight, age, sex, health status, diet, duration of administration, method of administration, elimination rate, severity of disease, and the like of a particular patient.

The folk rapeseed extract prepared in the present invention includes a compound represented by the following Chemical Formula 1 as an active ingredient as an Ingenane diterpene-based substance.

[Formula 1]

Wherein R is CO (CH ₂ ) ₁₄ CH ₃ or CO (CH ₂ ) ₁₂ CH ₃ .

In the formula, R is CO (CH ₂ ) ₁₄ CH ₃ Ingenol-3-palmitate, and if R is CO (CH ₂ ) ₁₂ CH ₃ Ingenol-3-pre Stinate (Ingenol-3-myristinate).

Mudae electrode extract of the present invention may also include isomers of the compound.

In order to investigate the efficacy of anti-inflammatory agents containing compounds isolated from the extract of Euphorbia ebracteolata obtained above, RAW 264.7 mouse macrophages stimulated with lipopolysaccharide (LPS) and C57BL / 6 mice As a result of the addition of the extract at various concentrations and the effect on the inflammatory response factors through molecular biological experiments with biochemical analysis, the extract according to the present invention is the TNF-, the nitrogen oxide production and the inflammatory cytokine in macrophages. a, IL-1β and the inflammation-inducing factor PGE ₂ was found to inhibit the activity excellently.

When the pharmaceutical composition containing the compound obtained by the above method was orally administered to rats and induced edema in the hind paws with carrageenan, it showed a significant inhibition rate of edema compared to the control group, and the tropic extract was orally administered to the rats. In addition, even when chronic arthritis was induced in the hind paw with Mycobacterium butyricium-containing CFA (Complete Freund's Adjuvant), it was confirmed that the anti-inflammatory effect was superior to the control group.

The folk drama of the present invention is a drug that has been used as a herbal medicine for a long time, the extracts of the present invention extracted therefrom also have no problems such as toxicity and side effects.

Based on the above results, the compound derived from folk rapeutic extract of the present invention is excellent in anti-inflammatory effect and is determined to be very safe, and can be very useful for anti-inflammatory drugs, health functional foods, functional feeds, etc. .

The pharmaceutical composition for the anti-inflammatory and inflammatory diseases containing the bioactive component of Euphorbia ebracteolata of the present invention, preferably comprises 0.1 to 50% by weight of the compound relative to the total weight of the composition. Do.

In addition, the composition comprising the bioactive component of the euphorbia ebracteolata of the present invention may further include suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

Pharmaceutical dosage forms of compounds containing bioactive ingredients that exhibit excellent efficacy in arthritis may be used in the form of their acceptable salts, or may be used alone or in combination with other pharmaceutically active compounds, as well as in suitable collections. .

The pharmaceutical compositions comprising the compounds according to the present invention may be prepared in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Can be formulated and used. Examples of carriers, excipients and diluents that can be included in the composition including the compound include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. High blood preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, or the like in the extract or compound. It is prepared by mixing sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The present invention provides a dietary supplement comprising the compound and a food acceptable food supplement additive which exhibits a prophylactic effect of inflammatory diseases or arthritis. Foods to which an extract or fraction of Euphorbia ebracteolata may be added include, for example, various foods, beverages, gums, teas, vitamin complexes, and health functional foods.

The composition according to the present invention may contain from 0.001 to 99.9% by weight of the polar composition as the active ingredient in the total weight of the composition. This is because the effect can be expected to be 0.001% by weight or more, it is difficult to exceed 99.9% by weight due to the presence of impurities.

In addition, the compound extracted from the folk larvae exhibits anti-inflammatory activity of arthritis in the molecular biological findings of arthritis, and has excellent anti-inflammatory and analgesic activity in inflammation and pain-inducing animals, and as a pharmaceutical composition and health functional food useful for the prevention and treatment of arthritis. Can be used.

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

At this time, the amount of the polar compound in the food may be added in 0.001 to 99.9% by weight of the total food weight, in the beverage may be added in a ratio of 0.001 to 0.1 g, more preferably 0.05 to 0.1 g based on 100 mL. have.

The beverage containing the folk compounds of the present invention is not particularly limited to other ingredients except those containing the folk compounds as essential ingredients at the indicated ratios, and various flavors or natural carbohydrates as additional ingredients are used as in general beverages. It may contain.

In addition to the above, the health functional food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (such as cheese and chocolate), pectic acid and salts thereof, alginic acid And salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the functional food compositions of the present invention may contain fruit flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The ratio of such additives is not so important, but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples, but the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples. .

Comparative Example 1: Preparation of Euphorbia ebracteolata Extract

To 240 g (dry weight) of dried roots of Euphorbia ebracteolata purchased from Jeonghan Corp., add 1 L of methanol and leave for 48 hours. After repeated filtering, the solvent was evaporated to dryness under reduced pressure for 3 hours. 16.81 g was obtained.

Comparative Example 2: Preparation of Euphorbia ebracteolata Fraction

Water and hexane (200 mL each) were added to and mixed with the extract illustrated in Comparative Example 1, and then the hexane layer and the water layer were separated. The separated hexane layer was dried under a reduced pressure to obtain 5.27 g of an organic substance as a hexane extract.

Example 1 Preparation of Euphorbia ebracteolata Bioactive Fractionated Compound 1

1 g of methanol was added to 240 g (dry weight) of dried roots of Euphorbia ebracteolata, and the mixture was left to stand for 48 hours, followed by filtration three times, and the solvent was evaporated to dryness under reduced pressure to obtain 16.81 g of methanol extract. . Water and hexane (each 200 mL) were added to the extract, mixed, the hexane layer and the water layer were separated, and the hexane layer was dried under reduced pressure to obtain 5.27 g of an organic material. The obtained organic material was fractionated by normal phase vacuum flash chromatography. The column is a glass filter column 100ⅹ95 (inner diameter ⅹ length, mm), the fixed phase is semi-preparative silica for TLC, 50 to 100% (v / v) hexane, ethyl acetate mixed solution as eluent 100% ethyl acetate was used as the washing solution. For each fraction, the amount of NO induced by lipopolysaccharide, an inflammation-inducing factor in RAW 264.7 cells, was measured, and the amounts of representative inflammatory cytokines, TNF-α, IL-1β and PGE ₂ , were measured. The effective fraction compounds 0.4 g and 0.19 g were selected by comparison between the positive control group treated with no saccharide and the negative control group treated with lipopolysaccharide. C18 normal-phase semi-preparative HPLC column (YMC Pack-SIL column, particle diameter 5um, 10 X 250 (inner diameter X length, mm), for elution: 70% (v / v) hexane / 30% ethyl acetate, elution rate: 3 mL / min, refractive index detector) was purified by separation, to obtain a viscous liquid component eluted at the retention time 37 minutes and 39 minutes. The obtained material was extracted with 41.23 mg of Ingenol-3-palmitate eluting with 37 minutes of retention time using Ingenol diterpene ester. HPLC chromatograms of the extracted material are shown in FIGS. 1 and 2.

Example 2 Preparation of Euphorbia ebracteolata Bioactive Fraction Compound 2

1 g of methanol was added to 240 g (dry weight) of dried roots of Euphorbia ebracteolata, and the mixture was left to stand for 48 hours, followed by filtration three times, and the solvent was evaporated to dryness under reduced pressure to obtain 16.81 g of methanol extract. . Water and hexane (each 200 mL) were added to the extract, mixed, the hexane layer and the water layer were separated, and the hexane layer was dried under reduced pressure to obtain 5.27 g of an organic material. The obtained organic material was fractionated by normal phase vacuum flash chromatography. The column is a glass filter column 100ⅹ95 (inner diameter ⅹ length, mm), the fixed phase is semi-preparative silica for TLC, 50 to 100% (v / v) hexane, ethyl acetate mixed solution as eluent 100% ethyl acetate was used as the washing solution. For each fraction, the amount of NO induced by lipopolysaccharide, an inflammation-inducing factor in RAW 264.7 cells, was measured, and the amounts of representative inflammatory cytokines, TNF-α, IL-1β and PGE ₂ , were measured. The effective fraction compounds 0.4 g and 0.19 g were selected by comparison between the positive control group treated with no saccharide and the negative control group treated with lipopolysaccharide. C18 normal-phase semi-preparative HPLC column (YMC Pack-SIL column, particle diameter 5um, 10 X 250 (inner diameter X length, mm), for elution: 70% (v / v) hexane / 30% ethyl acetate, elution rate: 3 mL / min, refractive index detector) was purified by separation, to obtain a viscous liquid component eluted at the retention time 37 minutes and 39 minutes. The obtained material was extracted 37.42 mg of Ingenol-3-myristinate eluted at 39 minutes holding time. HPLC chromatograms of the extracted material are shown in FIGS. 1 and 2.

Example 3 Preparation of Bioactive Active Fraction Compounds of Euphorbia ebracteolata 3

1 g of methanol was added to 240 g (dry weight) of dried roots of Euphorbia ebracteolata, and the mixture was left to stand for 48 hours, followed by filtration three times, and the solvent was evaporated to dryness under reduced pressure to obtain 16.81 g of methanol extract. . Water and hexane (each 200 mL) were added to the extract, mixed, the hexane layer and the water layer were separated, and the hexane layer was dried under reduced pressure to obtain 5.27 g of an organic material. The obtained organic material was fractionated by normal phase vacuum flash chromatography. The column is a glass filter column 100ⅹ95 (inner diameter ⅹ length, mm), the fixed phase is semi-preparative silica for TLC, 50 to 100% (v / v) hexane, ethyl acetate mixed solution as eluent 100% ethyl acetate was used as the washing solution. For each fraction, the amount of NO induced by lipopolysaccharide, an inflammation-inducing factor in RAW 264.7 cells, was measured, and the amounts of representative inflammatory cytokines, TNF-α, IL-1β and PGE ₂ , were measured. The effective fraction compounds 0.4 g and 0.19 g were selected by comparison between the positive control group treated with no saccharide and the negative control group treated with lipopolysaccharide. C18 normal-phase semi-preparative HPLC column (YMC Pack-SIL column, particle diameter 5um, 10 x 250 (internal diameter x length, mm) for the selected effective fractions, 70% (v / v) hexane / 30% ethyl acetate, elution rate: 3 mL / min, refractive index detector) was purified by separation, to obtain a viscous liquid component eluted at the retention time 37 minutes and 39 minutes. Each of the obtained materials was an Ingenol diterpene ester series, ingenol-3-palmitate eluted at 37 minutes of retention time, and ingenol-3-myristinate eluted at 39 minutes of retention time. Ingenol-3-myristinate) was combined to form compositions of 1: 1, 1: 2, 1: 4, 2: 1, and 4: 1, respectively. HPLC chromatograms of the extracted material are shown in FIGS. 1 and 2.

[Experimental Material]

DMEM culture, Fetal bovine serum (FBS), penicillin and streptomycin were purchased from Life Technologies Inc., Grand Island, NY. 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, inner salt: MTS (a)]

[3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, inner salt; MTS (a)], dimethyl sulfoxide (DMSO), and E. Coli Lipopolysaccharide (Lipopolysaccharide, LPS) were purchased from Sigma Chemical Co., Mo., USA. Griess reagent system kit for Nitric Oxide was purchased from Promega, WI, USA and ELISA kit for measuring TNF-α and IL-1β cytokine levels Was purchased from ebioscience, CA, USA.

Experimental Example 1. Cell Culture

RAW 264.7 mouse macrophage (Macrophage) contains fetal bovine serum (FBS, 10%) and antibiotics (antibiotics-antimycotics, 100 U / mL penicillin G sodium, 100 ug / mL streptomycin sulfate and 0.25 ug / mL amphotericin B ) In DMEM medium was passaged every other day at 37 ℃, 5% CO ₂ conditions.

Experimental Example 2. Cytotoxicity Test (MTS assay)

RAW 264.7 mouse macrophage (Macrophage) was dispensed in 96-well plate at 5 × 10 ³ per well incubated for 24 hours at 37 ℃, 5% CO ₂ conditions, Comparative Example 1, comparative example 2, ingenol-3-palmitate of Example 1, ingenol-3-myritate of Example 2, and ingenol-3-palmitate and ingenol-3-myristi of Example 3 Nate was mixed at a ratio of 1: 1, 1: 2, 2: 1, 1: 4, 4: 1, and celecoxib and aceclofenac, respectively, at 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 Administration is at a concentration of μg / mL. After administration again incubated for 48 to 72 hours at 37 ℃, 5% CO ₂ conditions. After incubation, [3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, inner salt; MTS (a)] solution is added to 20 μl of each well and allowed to react while incubating at 37 ° C., 5% CO ₂ for 1 to 4 hours. After the reaction was stopped, absorbance was measured using an absorbance spectrometer at 490 nm. The positive control group without the test substance was converted to 100% survival rate, and the survival rate in the test group for the test substance was calculated and shown in FIG. 3.

The measurement results are shown in FIG. 3. As can be seen in Figure 3, the anti-polar extracts ingenol-3-palmitate and ingenol-3-myristinate of the present invention are cytotoxic at a concentration of 50 ug / mL, which strongly induces anti-inflammatory effects. It was confirmed that no.

Referring to Figure 3, Comparative Example 1 and Comparative Example 2 Folk extracts in RAW 264.7 mouse macrophage cells, Ingenol-3-palmitate of Example 1, Ingenol-3- of Example 2 Between the myrinate and the compound in which the ingenol-3-palmitate of Example 3 was mixed with the ingenol-3-myristinate in a ratio of 1: 1, 1: 2, 2: 1, 1: 4, 4: 1 The cytotoxic effect was found to be a safe substance that does not affect cell survival as compared to the control, no celecoxib and aceclofenac used as a positive control.

Experimental Example 3. Measurement of Nitric Oxide (NO) Formation

RAW 264.7 cells were suspended in DMEM medium containing 10% FBS free of phenol red and placed in 5 × 10 ⁵ cells for each well of a 24-well plate at 37 ° C., 5 Incubated for 24 hours at% CO ₂ culture conditions. Folk extracts of Comparative Examples 1 and 2 and ingenol-3-palmitate of Example 1, ingenol-3-myristinate of Example 2 and ingenol-3- of Example 3 in RAW 264.7 cells 5 μg / mL and 10 μg of the compound containing cemitoxib and aceclofenac mixed with a mixture of palmitate and ingenol-3-myristinate at a ratio of 1: 1, 1: 2, 2: 1, 1: 4, and 4: 1 / mL, 20 μg / mL, 50 μg / mL and incubate for 1 hour. After incubation, lipopolysaccharide, an inflammation-inducing factor, was treated at a concentration of 1 μg / mL and cultured for 24 hours. After incubation for 24 hours at 37 ° C. and 5% CO ₂ , 50 μl of all cell cultures were taken from the grease reagent (0.1% naphthylethylenediamine solution and 1% sulfanilamide in 5). The absorbance was measured at 540 nm by reacting with 180 μl of a solution of H ₃ PO ₄ ). The calibration curve was prepared using sodium nitrite solution, and the absorbance average was converted into the amount of nitrite. Based on the amount of nitrite in the negative control group treated with only lipopolysaccharide, the mononitric oxide production inhibitory activity of the test substance treated group was determined using a non-linear regression analysis. _It is shown in Figure 4 comparing the effect between the test materials in a way to determine ₅₀ (concentration that inhibits 50% of mononitrogen oxide production).

As confirmed in Figure 4, RAW 264.7 mouse macrophage cells (mouse macrophage cells) in Comparative Example 1 and Comparative Example 2 folkpoge extract and Example 1 Ingenol-3- palmitate, Example 2 Ingenol- 3-Ministinate and the ingenol-3-palmitate of Example 3 mixed with ingenol-3-myrinateate in a ratio of 1: 1, 1: 2, 2: 1, 1: 4, 4: 1 As a result of comparing the amount of Nitric Oxide (NO) produced when the compound was administered, Examples 1, 2, and 3, which are pure anti-inflammatory active substances, were mostly celecoxib, which is a conventional anti-inflammatory agent, It was confirmed that it showed an excellent inhibitory effect compared to aceclofenac. Example 1 showed higher anti-inflammatory efficiencies of about 62.5%, 44.3%, and 31.6% than negative controls, celecoxib, and aceclofenac, respectively. Example 2 showed higher anti-inflammatory efficiencies of about 58%, 38%, and 23% than negative controls, celecoxib, and aceclofenac, respectively, and in Example 3, compounds having high composition ratios generally showed excellent anti-inflammatory efficiencies.

Experimental Example 4 Measurement of TNF-α and IL-1β Cytokine Levels

RAW 264.7 cells were cultured by dispensing 5 × 10 ⁵ cells per well into a 24-well plate, and the maximal pole extracts of Comparative Examples 1 and 2, and Ingenol of Example 1 3-palmitate, the ingenol-3-myristinate of Example 2 and the ingenol-3-palmitate and ingenol-3-myristinate of Example 3 were 1: 1, 1: 2, 2: Compounds, celecoxib, and aceclofenac, mixed at a ratio of 1, 1: 4, and 4: 1, were all administered at concentrations of 5 μg / mL, 10 μg / mL, 20 μg / mL, and 50 μg / mL and incubated for 1 hour. do. After incubation, lipopolysaccharide, an inflammation-inducing factor, was treated at a concentration of 1 μg / mL and cultured for 24 hours. After incubating for 24 hours at 37 ° C. and 5% CO ₂ , 50 μl of all the cell cultures were taken and the TNF-α and IL-1β contents were measured using an enzyme immunoassay kit (EIA). 50 μl of all cell cultures were added to a 96-well plate to which capture antibodies for each target cytokine were attached, and diluted at a ratio of 1 / 250X to 1X assay diluent after 24 hours reaction. TNF-α, IL-1β antibody and horseradish peroxidase-bound secondary antibody were each reacted for 2 hours at room temperature. After the reaction, the mixture was washed 5 times with 1 × phosphate buffered saline tween (1 × PBST), and then reacted for 30 minutes by adding the following enzyme substrate. Then 2N-sulfuric acid was added to stop the reaction. The absorbance developed after the reaction was stopped at 450 nm wavelength using an absorbance spectrometer. The contents of TNF-α and IL-1β were converted using a standard curve obtained from the reaction of the standard.

As confirmed in FIG. 5, the folk extract of Comparative Example 1 and Comparative Example 2, Ingenol-3-palmitate of Example 1, and Ingenol of Example 2 in RAW 264.7 mouse macrophage cells -3-myristinate and the ingenol-3-palmitate of Example 3 and the ingenol-3-myrinateate in a ratio of 1: 1, 1: 2, 2: 1, 1: 4, 4: 1 In the case of administration of the mixed compounds, the expression concentrations of the inflammatory cytokines TNF-α (Tumor necrosis factor-α) were compared. As a result, the compounds of Examples 1, 2, and 3 significantly reduced tumor necrosis by concentration. It was confirmed that the release of the factor was suppressed. Example 1 showed an excellent anti-inflammatory efficiency of 36%, 18%, 17% than the negative control, celecoxib, aceclofenac, respectively, and Example 2 was 36%, 19 than the negative control, celecoxib, aceclofenac, respectively. Excellent anti-inflammatory efficiency of about 18%. Compounds of Example 3 showed an excellent anti-inflammatory effect in the compounds showed a generally high composition ratio.

As can be seen in Figure 6, Comparative Example 1 and Comparative Example 2 folk-polar extract of RAW 264.7 mouse macrophage cells, ingenol-3-palmitate of Example 1, ingenol of Example 2 -3-myristinate and the ingenol-3-palmitate of Example 3 and the ingenol-3-myrinateate in a ratio of 1: 1, 1: 2, 2: 1, 1: 4, 4: 1 In the case of administration of the mixed compound, the expression levels of the inflammatory cytokine IL-1β (Interleukin-1β) were compared, and as a result, Example 1, Example 2, and Example 3 significantly inhibited IL-1β by concentration. It was confirmed. Example 1 showed better anti-inflammatory efficiencies of 48%, 21%, and 7% than the negative control, celecoxib, and aceclofenac, respectively, and Example 2 showed 55% and 31, respectively, than the negative control, celecoxib, and aceclofenac. Excellent anti-inflammatory efficiency of about 19%. Compounds of Example 3 showed an excellent anti-inflammatory effect in the compounds showed a generally high composition ratio. It can be confirmed that the inhibition of TNF-α and IL-1β expression in FIGS. 5 and 6 shows an excellent anti-inflammatory effect than the positive control group without addition of lipopolysaccharide (LPS) or celecoxib, aceclofenac, which does not cause inflammation. there was.

Experimental Example 5. Prostaglandin E ₂ (Prostaglandin E _{2 ,} PGE ₂ ) Production Measurement

RAW 264.7 cells were suspended in DMEM medium containing 10% FBS and 5 × 10 ⁵ cells were added to each well of a 24-well plate for 24 hours at 37 ° C. and 5% CO ₂ culture conditions. Incubated for RAW 264.7 cells were washed once with phosphate buffered saline (PBS) and replaced with fresh DMEM medium, followed by folk extract of Comparative Example 1 and Comparative Example 2, ingenol-3-palmitate of Example 1, Inzenol-3-myristinate of Example 2 and ingenol-3-palmitate and ingenol-3-myristinate of Example 3 were 1: 1, 1: 2, 2: 1, 1: 4, Compounds, celecoxib, and aceclofenac mixed at a ratio of 4: 1 were all treated simultaneously at concentrations of 5 μg / mL, 10 μg / mL, 20 μg / mL, and 50 μg / mL and incubated for 1 hour. After incubation, lipopolysaccharide was treated at a concentration of 1 μg / mL and incubated for 24 hours at 37 ° C. and 5% CO ₂ culture conditions. PGE ₂ antibody plate (PGE ₂₎ by diluting supernatant antibody plate) and treated with PGE ₂ -AchE tracer and incubated for 18 hours at room temperature. PGE ₂ antibody plate after incubation (PGE ₂ The antibody plate) was washed 5 times with 0.05% Tween in PBS for 1 minute, and then incubated at room temperature for 7 hours by treatment with Ellman's reagent. After measuring absorbance at 405 nm, PGE ₂ PGE ₂ is substituted into the calibration curve prepared with the standard solution. The production amount was converted. PGE ₂ in groups treated with LPS only The production inhibitory activity was compared by using a non-linear regression analysis to determine the IC ₅₀ (concentration that inhibits PGE ₂ production by 50%) of each test substance.

As can be seen in Figure 7, the comparative extracts of Comparative Example 1 and Comparative Example 1 in the macrophage cells of RAW 264.7 folk extract, Ingenol-3-palmitate of Example 1, Ingenol of Example 2 -3-myristinate and the ingenol-3-palmitate of Example 3 and the ingenol-3-myrinateate in a ratio of 1: 1, 1: 2, 2: 1, 1: 4, 4: 1 In the case of administration of the mixed compounds, the concentration of prostaglandin (E ₂ ), which is one of the causes of inflammation, was compared. Examples 1, 2, and 3 showed excellent inhibition corresponding to celecoxib and aceclofenac. The effect was confirmed. Example 1 showed higher anti-inflammatory efficiencies of 70%, 47%, and 47% than negative controls, celecoxib, and aceclofenac, respectively, and Example 2 was 58%, 27 than negative controls, celecoxib, and aceclofenac, respectively. Excellent anti-inflammatory efficiency of about 27%. Compounds of Example 3 showed a generally higher anti-inflammatory efficiency of the compound having a higher composition ratio than the negative control, celecoxib, aceclofenac. This is PGE ₂ in FIG. It was confirmed that expression suppression showed an anti-inflammatory effect better than the positive control group, celecoxib, and aceclofenac without addition of LPS (Lipopolysaccharide) which causes inflammation.

Preparation Example 1 Preparation of Capsule

200 mg of the folk extract extract of Example 1, Example 2, and Example 3 (1: 1: 1)

Lactose 50 mg

Starch 50 mg

Talc 2 mg

Magnesium stearate proper amount

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Preparation Example 2 Preparation of Tablet

200 mg of the folk extract extract of Example 1, Example 2, and Example 3 (1: 1: 1)

Lactose 100 mg

Starch 100 mg

Talc 2 mg

Magnesium stearate proper amount

Tablets are prepared by mixing and tableting the components according to conventional tablet production methods.

Preparation Example 3 Preparation of Liquid

1000 mg of folk extract extracts (1: 1: 1) of Examples 1, 2 and 3

2000 mg of sugar

Isomerized sugar 2000 mg

Lemon incense quantity

After adding purified water to make a total of 1000 mL, the components are mixed according to a conventional liquid preparation method, and then filled into a brown bottle and sterilized to prepare a liquid preparation.

Preparation Example 4 Preparation of Health Functional Food

1000 mg of folk extract extracts (1: 1: 1) of Examples 1, 2 and 3

<Vitamin mixture>

Vitamin A Acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Nicotinamide 1.8 mg

Folic acid 50 μg

Calcium Pantothenate 0.5 mg

<Inorganic mixture>

0.82 mg of zinc oxide

Magnesium carbonate 25 mg

Potassium citrate 90 mg

Calcium carbonate

The above ingredients may be mixed and granules prepared in accordance with a conventional health food preparation method, and may be used for preparing a health food composition according to a conventional method. The composition ratio of the above-mentioned vitamin and mineral mixture may be arbitrarily modified, and the above ingredients may be mixed according to a conventional health food manufacturing method, then granules are prepared and prepared in a health food composition according to a conventional method. Can be used.

Preparation Example 5 Preparation of Health Functional Drink

1000 mg of folk extract extracts (1: 1: 1) of Examples 1, 2 and 3

Citric acid 1000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

900 mg total, including purified water

After mixing the ingredients according to a conventional healthy beverage production method, and stirred and heated at 80 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 ml container, sealed sterilization and refrigerated. Used to prepare healthy beverage compositions.

Preparation Example 6 Preparation of Capsule

200 mg of the folk extract extract of Example 1, Example 2, and Example 3 (1: 1: 1)

50 mg of conventional arthritis medications (NSAIDs)

Lactose 50 mg

Starch 50 mg

Talc 2 mg

Magnesium stearate proper amount

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules. As the NSAIDs, known nonsteroidal anti-inflammatory drugs can be used, and the content thereof can be appropriately adjusted.

Preparation Example 7 Preparation of Ointment

1.0 wt% of the extracts of the cathode electrodes of Example 1, Example 2, and Example 3 (1: 1: 1)

20.0 wt% urea

15.0 wt% white petrolatum

Hard Flow Paraffin 6.0 wt%

Cetanol 3.0 wt%

Stearyl Alcohol 3.0 wt%

Glyceryl monostearate 5.0 wt%

Fragrance

Preservative

1.0 wt% buffer

Purified water remaining amount

The ointment is prepared by mixing the above components according to a conventional ointment preparation method.

Preparation Example 8 Preparation of Injection

300 mg of the folk extract extract of Example 1, Example 2, and Example 3 (1: 1: 1)

180 mg mannitol

Sterile distilled water for injection 2,974 mg

Na ₂ HPO ₄ 12H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Preparation Example 9 Preparation of Lotion

Folk extract compounds of Examples 1, 2 and 3 (1: 1: 1) 4.00 (%)

Brown algae extract 1.00 (%)

Sodium Citrate 0.10 (%)

Citric acid 0.05 (%)

1,3-butylene glycol 3.00 (%)

The whole amount was 100 with purified water, and a lotion was prepared at the above blending ratio (%).

Claims (5)

Ingenol-3-palmitate or Ingenol-3-myristinate extracted from Euphorbia ebracteolata as an active ingredient in a concentration of 1 to 100 mg in the composition It relates to a pharmaceutical composition for preventing and treating inflammation, characterized in that it contains so / kg,
The ingenol-3-palmitate and ingenol-3-myrinate materials were added after adding 5-20 L of water or ethanol solvent per kg of dry pole roots and left at 20-50 ° C. for 0.5-2 days before filtration. And evaporation under reduced pressure to obtain an intermediate extract,
Water and ethanol were mixed 1: 1 to the intermediate extract to separate the water and the ethanol layer, and then hexane was added to the separated water layer to extract the organic material,
A pharmaceutical composition for preventing and treating inflammation, which is obtained from a method of fractionating and extracting the organic material by chromatography. delete delete delete delete

Images