KR101770413B1 - A composition for the prevention or treatment of edema or dermatitis containing oriental medicine herbs oil extract as an active ingredient - Google Patents
A composition for the prevention or treatment of edema or dermatitis containing oriental medicine herbs oil extract as an active ingredient Download PDFInfo
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- KR101770413B1 KR101770413B1 KR1020150147791A KR20150147791A KR101770413B1 KR 101770413 B1 KR101770413 B1 KR 101770413B1 KR 1020150147791 A KR1020150147791 A KR 1020150147791A KR 20150147791 A KR20150147791 A KR 20150147791A KR 101770413 B1 KR101770413 B1 KR 101770413B1
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- South Korea
- Prior art keywords
- weight
- soluble fraction
- edema
- fat
- extract
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- A61K36/185—Magnoliopsida (dicotyledons)
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Abstract
The present invention relates to a pharmaceutical composition for the prevention or treatment of inflammation or edema containing as an active ingredient an extract of a fat-soluble medicinal herbaceous material, wherein the extract obtained by extracting at least one selected from the group consisting of white radish, As a component, it can alleviate myalgia and prevent and treat inflammation or swelling such as myositis and dermatitis.
Description
The present invention relates to a pharmaceutical composition containing as an active ingredient an oil-soluble fraction extract capable of alleviating muscle pain and capable of preventing or treating inflammation or edema such as muscle inflammation and dermatitis.
Modern people are suffering from muscular pain called myofascial pain syndrome due to muscle tension coming from repeated long time posture through use of computer and smart phone, driving of vehicle. Myofascial pain syndrome occurs in the fascia that connects the muscles and bones, especially in the neck and shoulders. Clinical pathologic findings are not accompanied by clinicopathologic findings, and anti-inflammatory analgesics and muscle relaxant injections are used as treatment methods, and physical therapies and massage therapies using ultrasound are performed. This is why they prefer alternative medicine therapy because of the side effects of using analgesics.
On the other hand, dermatitis is an inflammation that occurs in the skin, and is divided into atopic dermatitis, contact dermatitis and seborrheic dermatitis. Such inflammation is one of the damage of tissue, external stimulation or defense of biological tissues against various infectious agents. It is caused by enzymatic activation, inflammatory mediator secretion, inflammatory mediator secretion due to organic interactions of various inflammatory mediators and various immune cells in blood vessels and body fluids Infiltration and fluid exudation, circulatory disturbances, tissue degeneration and hyperplasia.
In the process of inflammatory reaction, macrophages are collected at the wound area at the initial stage and attack the invading bacteria. After that, plasma accumulates on the injured area and blood flow is increased, resulting in external symptoms such as fever, erythema, edema and pain . If the inflammatory reaction continues or occurs excessively, it progresses to a major pathological condition (irritable allergic disease, chronic inflammatory disease) of the disease, resulting in serious abnormal disorder.
Non-steroidal antiinflammatory drugs (NSAIDS), a widely used agent for the treatment of most inflammatory diseases, are produced from arachidonic acid, called cyclooxygenase (COX), by prostaglandin biosynthesis (Rajakariar R et al. 2006). In addition to the main treatment effect, it causes serious adverse effects such as gastrointestinal disorders, hepatic disorders, and renal dysfunction. ).
Therefore, the development of a novel anti-inflammatory analgesic agent that is excellent in anti-inflammatory efficacy is widely demanded because there is no side effect and it is not difficult to use for a long period of time. This is why research on material development through the verification of efficacy from natural resources is being activated recently.
Aroma oil, used as an alternative therapy, is extracted from plants and is known as a substance that alleviates the symptoms of certain areas of the body and enables the activation of psychological healing and physiological functions. Many studies have been reported that aromatic oils have been used for therapeutic purposes of antiinflammation, antibacterial, and pain relief for a long time.
Aromatherapy is a compound of Aroma (aroma) and Therapy (Therapy), and it absorbs volatile essential oil (Essential Oil) extracted from various ways of flowers, stems, leaves, roots, Or by applying it to the skin to heal disease or symptoms. Essential oil molecules inhaled into the body reaches the cerebral limb system that regulates the movement of the olfactory cells and hormones, autonomic nervous system and immune system, stimulates the nervous system, thereby improving psychological stability and improving concentration and memory. It also has affinity for specific organs and tissues in the body. When reaching a specific organ, it circulates throughout the body through blood vessels and body fluids, reducing pain, enhancing the body's immune function, and affecting the function of each internal organ, lymph node and hormone It balances the body's functions and restores the homeostatic ability, thereby enhancing the resistance and immunity of the outside, thereby increasing resistance to bacteria and viruses. In particular, aroma oil is becoming increasingly important because it reduces mental stress and relieves pain by loosening muscled muscles, and the aroma market is also evolving to emphasize functionality.
However, the plants used for aromatherapy are imported from abroad, and there is no essential oil made using domestic medicinal herbs for use in aromatherapy.
It is an object of the present invention to provide a pharmaceutical composition containing as an active ingredient an oil-soluble fraction extract capable of alleviating muscle pain and preventing or treating inflammation or edema such as muscle inflammation and dermatitis.
Another object of the present invention is to provide a cosmetic composition containing the fat-soluble fraction extract as an active ingredient.
Still another object of the present invention is to provide a health functional food containing the fat-soluble fraction extract as an active ingredient.
In order to accomplish the above object, the present invention provides a pharmaceutical composition for preventing or treating inflammation or edema, comprising at least one selected from the group consisting of white pine, cinnabar, and angelica guinea as an active ingredient, can do.
The fat-soluble fraction extract may be one which is obtained by mixing white roots, celadon root, and Angelica gigas Nakai at a weight ratio of 1: 1-2: 1-2.
The white pine bark, the bronze ginseng root and the true angelica gingiva may be powders dried at a moisture content of 3 to 10%.
In the supercritical extraction, the solvent may be carbon dioxide, the supercritical extraction pressure may be 300-400 bar, and the supercritical extraction temperature may be 50-70 ° C.
The main organic compounds of the fat-soluble fraction extract of the mixture of white pine, licorice and Angelica gigantea are α-Elemol, Caryophyllene oxide, Lauric acid, Ethyl Linoleate ), Cetyl alcohol, Acetovanillone, Myristic acid, 1-Eicosene, calamene, pinene, β-cardinene ( β-cadinene, α-muurolene, Caryophyllene, Hexadecanoid, 5,5-dimethyl-bicyclo [6,3,0] undeca- -Dicene-3-one (5,5-dimethyl-bicyclo [6,3,0] undeca-1,7-dien-3-one), Shyobunone, 6-butyl-1,4-cycloheptadiene, p-tolyl alcohol, 2,4,6,7,8,8a-hexahydro-5 (1H) , 6,7,8,8a-hexahydro-5 (1H) -azulenone, 5-oxo-delta-4-decahydrobenzindene, umbellulone, - Elements ( -elemene), it may be a color alkylene (calarene) and horseradish norbornene (sabinene).
The fat-soluble fraction extract comprises 0.1 to 0.4% by weight of? -Elemol, 0.1 to 1.5% by weight of caryophyllene oxide, 0.2 to 0.9% by weight of lauric acid, 0.3 To 0.7% by weight of Ethyl Linoleate, 0.3 to 0.7% by weight of Cetyl alcohol, 0.1 to 0.4% by weight of Acetovanillone, 0.2 to 0.7% by weight of Myristic acid, 0.5 to 2 wt% 1-eicosene, 5 to 13 wt% calamene, 5 to 13 wt% pinene, 5 to 13 wt% Cadinene, 4 to 11% by weight of? -Murolene, 1 to 5% by weight of caryophyllene, 1 to 2% by weight of hexadecanoid, 10 to 17% by weight of 5,5-dimethyl-bicyclo [6,3,0] undeca- 1,7-diene-3-one (5,5-dimethyl- bicyclo [6,3,0] undeca- 1,7-dien-3-one, 10-15% by weight of Shyobunone, 5 To 12% by weight of 6-butyl-1,4-cycloheptadiene, from 5 to 11% by weight of p-tolyl alcohol, from 3 to 7% by weight, Hexahydro-5 (1H) -azuranone (2,4,6,7,8,8a-hexahydro-5 (1H) -azulenone), 3-7 weight 5-oxo-delta-4-decahydrobenzindene, 3 to 6% by weight of umbellulone, 0.4 to 0.5% by weight of gamma -olefin (gamma- -alemene, from 3 to 5% by weight of calarene and from 0.2 to 0.3% by weight of sabinene.
According to another aspect of the present invention, there is provided a cosmetic composition for preventing or ameliorating inflammation or swelling, comprising at least one selected from the group consisting of white radish, May be contained as an active ingredient.
According to another aspect of the present invention, there is provided a health functional food for preventing or ameliorating inflammation or swelling, comprising at least one selected from the group consisting of white pine, white pine, and angelica orientalis, As an active ingredient.
The health functional food may be selected from the group consisting of capsules, tablets, powders, granules, liquids, rings, flakes, pastes, syrups, gels, jellies, and the like.
The pharmaceutical composition for preventing or treating inflammation or edema of the present invention contains myxiety-relieving fat-soluble fraction extract as an active ingredient and can prevent and treat inflammation or swelling such as myositis and dermatitis. Therefore, the fat-soluble fraction extract may be used to prepare a cosmetic for preventing and treating muscular pain, a cosmetic for preventing and treating muscular pain, a cosmetic for preventing and treating dermatitis, an anti-edema, a therapeutic lotion and a health functional food.
FIG. 1 is a graph showing the cell viability of normal skin cell keratinocytes (HaCaT cells) treated with lipid soluble fraction extract prepared according to an embodiment of the present invention (no LPS treatment).
FIG. 2 is a graph (LPS treatment) of cell viability of normal skin cell keratinocytes (HaCaT cells) treated with lipid soluble fraction extract prepared according to an embodiment of the present invention.
3 is a graph showing NO production of a fat soluble fraction extract prepared according to one embodiment of the present invention in human normal skin cell keratinocytes (HaCaT cells).
FIG. 4 is a photograph showing the anti-inflammatory activity of Haemophilus influenzae (Haemophilus influenzae) extracts prepared by HaCaT cells according to one embodiment of the present invention.
FIG. 5 is a photograph showing the anti-inflammatory activity of lipid soluble fraction extract prepared according to one embodiment of the present invention using HaCaT cells stimulated with LPS. FIG.
FIG. 6 is a graph showing the effect of PGE 2 on HaCaT cells treated with lipid soluble fraction extract prepared according to an embodiment of the present invention FIG.
FIG. 7 is a graph showing the IL-6 concentration of HaCaT cells treated with lipid soluble fraction extract prepared according to an embodiment of the present invention.
FIG. 8 is a graph showing the thickness of ear edema of a mouse treated with the oil-soluble fraction extract prepared according to an embodiment of the present invention (diluent: acetone).
FIG. 9 is a graph showing the thickness of ear edema of a mouse treated with the oil-soluble fraction extract prepared according to an embodiment of the present invention (diluent: jojoba oil).
The present invention relates to a pharmaceutical composition for the prevention and treatment of inflammation or edema containing an oil-soluble fraction extract capable of alleviating myalgia and capable of preventing or treating inflammation or edema such as myositis and dermatitis as an active ingredient.
The dermatitis is selected from the group consisting of hypersensitivity, allergic dermatitis, contact dermatitis, atopic dermatitis, skin allergy and urticaria, but is not limited thereto.
Hereinafter, the present invention will be described in detail.
The pharmaceutical composition for preventing or treating inflammation or edema according to the present invention may contain, as an active ingredient, a lipid soluble fraction extract obtained by supercritical extraction from at least one selected from the group consisting of white radish, astragalus and Angelica gigas.
The white pine bark refers to the root bark of the white pine ( Dictamnus dasycarpus Turcz.), Which is a skin rash caused by wet heat, skin eruption, eczema, rub, itching, allergic dermatitis, It is used for jaundice caused by hepatitis, humid paralysis, and is also used for seawater, throat drying, and turning.
The Cnidium officinale Makino ( Cnidium officinale Makino) is a perennial herb that is used to treat headache, anemia, and women's diseases by removing leaves and stems and drying in the sun.
In addition, Angelica gigas NAKAI ( Angelica gigas NAKAI) is used as a medicinal herb, and can be used as a medicinal product for the treatment of physical weakness, headache, dizziness, arthralgia, abdominal pain, constipation, Bruises and the like.
It is preferable to use the above-mentioned white pine bark, Angelica japonica, and Angelica angustifolia as a mixture in order to increase the extraction efficiency and to have better efficacy. More preferably, -2, and more preferably in a weight ratio of 1: 1: 1. When the weight ratio of the cinnabar and the true angel is less than the lower limit based on the white blood, the extraction efficiency is low and the efficacy against inflammation or swelling can be lowered even when the supernatant is extracted by the supercritical extraction method. The efficacy can be significantly reduced.
The white pine, celia and Angelica gigas are washed with water and then dried in a drier at 40 to 60 ° C for 10 to 60 minutes so that the moisture content is 3 to 10%, preferably 3 to 8%. Generally, the moisture content of the medicinal herb is 11 to 15%. When the white pine bark, astragalus, and Angelica angustifolia having a moisture content of 11 to 15% are used, the aroma is poor and the efficacy against inflammation or edema is lowered. %, Preferably 3 to 8%.
The supercritical extraction is carried out in a supercritical state at a pressure of 300 to 400 bar, preferably 350 to 400 bar and a temperature of 50 to 70 ° C, preferably 60 to 70 ° C, Extracting the fat-soluble fraction from the selected one or more species to obtain a fat-soluble fraction extract. In this case, the solvent is not particularly limited as long as a fat-soluble fraction extract can be obtained from at least one selected from the group consisting of white radish, celadon and true angelica. However, in order to obtain high extraction efficiency, excellent inflammation or edema, carbon dioxide (Critical temperature of carbon dioxide: 31 DEG C, critical pressure: 74 bar).
The supercritical extraction method has superior flavor, high extraction efficiency and more excellent effect on inflammation or edema compared to extraction by other extraction methods such as steam distillation extraction method. Especially, when steam distillation extraction method is used, the inherent fragrance of white pine bark, celestria, and chrysanthemum is disappeared and an unpleasant smell which is denatured by heat is generated.
When the pressure is lower than the lower limit of the supercritical extraction, the fat-soluble fraction may be hardly extracted from the medicinal herb. If the supernatant is above the upper limit, the effect of inflammation or swelling may be lost. In addition, when the temperature is lower than the lower limit of the supercritical extraction, the extraction efficiency may be lowered. If the temperature is higher than the upper limit, the effect on inflammation or edema may be lowered.
The 'extraction efficiency' refers to the amount of the fat soluble fraction extract obtained from at least one selected from the group consisting of white pine, cinnabar and chrysanthemum.
The fat-soluble fraction extract extracted from the medicinal herbs mixed with the white pine bark, Angelica gigas, and Angelica gigas Nakai contains various volatile organic compounds. The organic compounds include 0.1 to 0.4% by weight of? -Elemol, 0.1 By weight of caryophyllene oxide, 0.2 to 0.9% by weight of lauric acid, 0.3 to 0.7% by weight of ethyl linoleate, 0.3 to 0.7% by weight of cetyl alcohol ( Cetyl alcohol, 0.1 to 0.4% by weight of Acetovanillone, 0.2 to 0.7% by weight of Myristic acid, 0.5 to 2% by weight of 1-eicosene, The composition of the present invention can be prepared by mixing 5 to 13 wt% of calamene, 5 to 13 wt% of pinene, 5 to 13 wt% of β-cadinene, 4 to 11 wt% of α-muurolene ), 1 to 5% by weight of caryophyllene, 1 to 2% by weight of hexadecanoid, 10 to 17% by weight, 5,5-dimethyl-bicyclo [6,3,0] undeca-1,7-diene-3-one (5,5-dimethyl- bicyclo [6,3,0] undeca- 3-one, 10-15 wt% Shyobunone, 5-12 wt% 6-butyl-1,4-cycloheptadiene, 5-11 wt% % P-tolyl alcohol, 3 to 7 wt% 2,4,6,7,8,8a-hexahydro-5 (1H) -azuranone (2,4,6,7, 5-oxa-delta-4-decahydrobenzindene, 3 to 6 wt% of 5-oxo-delta-4-decahydrobenzindene, Umbellulone, 0.4 to 0.5% by weight of γ-elemene, 3 to 5% by weight of calarene and 0.2 to 0.3% by weight of sabinene.
The pharmaceutical composition for the prevention and treatment of inflammation or edema can be formulated into various oral or parenteral administration forms.
Examples of formulations for oral administration include tablets, pills, hard, soft capsules, liquids, suspensions, emulsions, syrups, granules and the like. These formulations may contain, in addition to the active ingredient, a diluent such as lactose, dextrose, (For example, silica, talc, stearic acid and magnesium or calcium salts thereof and / or polyethylene glycol), in addition to the active ingredient (s). The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, optionally mixed with starch, agar, alginic acid Or a disintegrating or boiling mixture such as its sodium salt and / or an absorbent, a colorant, a flavoring agent and a sweetening agent. The formulations may be prepared by conventional mixing, granulating or coating methods.
Representative examples of formulations for parenteral administration include injectable preparations, and water, Ringer's solution, isotonic saline or suspensions may be mentioned as a solvent for the injectable preparation. The sterile, fixed oils of the injectable preparations may be used as a solvent or suspending medium, and any non-irritating fixed oils, including mono-, di-glycerides, may be used for this purpose. The injectable preparation may be a fatty acid such as oleic acid.
The dosage of the pharmaceutical composition for preventing or treating inflammation or edema containing the herbal oil-soluble fraction extract of the present invention as an active ingredient depends on the age, sex, weight and severity of the disease of the patient, The dosage is not more than 300 mg per kg body weight, preferably 100 to 200 mg per day, but not limited thereto.
The health functional food for preventing or ameliorating inflammation or edema containing the fat-soluble fraction extract of the present invention as an active ingredient contains 0.01 to 80% by weight of the fat-soluble fraction extract based on the total weight of the health functional food.
The health functional food may be in the form of a capsule, tablet, powder, granule, liquid, ring, flaky, paste, syrup, gel, jelly and jute for the purpose of supplementing nutrients that may be lacking in daily eating or supplementing functional raw materials useful in the human body And is easily manufactured and operated once.
In addition, the health functional food may contain at least one selected from the group consisting of glucose, citric acid, liquid oligosaccharide, corn syrup, soybean lecithin, butter, vegetable hardening oil, skim milk, sugar, margarine, salt, starch, And can be prepared by using commonly used components such as sodium bicarbonate and sugar ester.
The cosmetic composition containing the oil-soluble fraction extract of the present invention as an active ingredient for preventing or ameliorating inflammation or swelling can be prepared in the form of a general emulsified formulation and a solubilized formulation. Cosmetics of emulsified form include nutrition lotion, cream, essence, etc., and cosmetics of solubilized form have softening longevity.
Suitable cosmetic formulations include, for example, solutions, gels, solid or kneaded anhydrous products, emulsions obtained by dispersing the oil phase in water, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes) In the form of a dispersing agent, in the form of a cream, a skin, a lotion, a powder, an ointment, a spray or a conical stick. It can also be prepared in the form of a foam or an aerosol composition further containing a compressed propellant.
In addition, the cosmetic composition may further contain, in addition to the extract of the present invention or its fractions, a lipid, an organic solvent, a solubilizing agent, a thickening agent and a gelling agent, a softening agent, an antioxidant, a suspending agent, a stabilizer, a foaming agent, , Water, ionic or nonionic emulsifiers, fillers, sequestering agents and chelating agents, preservatives, vitamins, barrier agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or cosmetics And may contain adjuvants conventionally used in the cosmetics field, such as any other ingredients used.
It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the present invention. Such variations and modifications are intended to be within the scope of the appended claims.
EXAMPLES Example 1:
300 g of white radish was put into a supercritical extractor (ILSHIN Auto, Korea) using a recirculation method of supercritical carbon dioxide, and carbon dioxide was injected to extract a lipid soluble fraction under supercritical conditions of 60 ° C and 400 bar.
Example 2. Mixed fat-soluble fraction extract
The same procedure as in Example 1 was carried out except that a mixed fat-soluble fraction extract was extracted using a medicinal herb having 100 g of white radish, 100 g of Angelica gigasini, and 100 g of astragalus in place of 300 g of white radish.
<Test Example>
Test Example 1. Flavor analysis
The fragrance experts who had been training in the sense of smell were commissioned to five persons to perform the fragrance evaluation.
The characteristics of the fragrance at 5 cm from the nose after wetting the 100% stock solutions prepared in Examples 1 and 2 on the papers (0.5 cm x 5 cm) were described using fragrance evaluation terms. The flavor types used are aldehyde, animalic, balsamic, citrus, coniferous, earthy, floral, fruity, Green, herbal, medicinal, minty, marine, mossy, musky, oily, oriental, powdery, powdery, smoky, spicy, sweet, woody, and refreshing.
As a result of analyzing the fragrance, the fragrance of the oil-soluble fraction extract prepared in Example 1 was woody, oily, and earthy. In addition, the fragrance of the oil-soluble fraction extract prepared in Example 2 was woody, oily, animalic and balsamic type, and compared with the oil-soluble fraction extract of Example 1, It was confirmed that the oil - soluble fraction extract had a fragrance which did not show any difference even if it was used as a basic fragrance of a flour product.
Test Example 2. Analysis of volatile organic compounds
The volatile chemical components contained in the liposoluble fraction extract prepared according to Examples 1 and 2 were adsorbed on a solid phase microextraction (SPME) apparatus equipped with PDMS (polydimethyl siloxane) fiber, and then analyzed by gas chromatography-mass spectrometry chromatography-mass spectrometry, GC-MS). For the accuracy of analysis, mass spectrometry data of fragrance library was used by a Japanese perfume analysis agency.
The volatile chemical components contained in the fat-soluble fraction extract prepared according to Example 1 are shown in Table 1 below, and the volatile chemical components contained in the fat-soluble fraction extract prepared according to Example 2 are shown in Table 2 below .
As shown in Table 1 above, the total amount of the 25 volatile organic compounds contained in the fat soluble fraction extract prepared according to Example 1 was 7.02% by weight in total, and the major organic compounds were α-Elemol ), Caryophyllene oxide, Lauric acid, Ethyl Linoleate, Cetyl alcohol, Acetovanillone, Myristic acid and 1- It is 1-Eicosene.
As shown in Table 2 above, the total amount of the 24 volatile organic compounds contained in the fat soluble fraction extract prepared according to Example 2 was 133% by weight in total, and the main organic compound was 5,5-dimethyl-bicyclo [6,3,0] undeca-1,7-dien-3-one (5,5-dimethyl-bicyclo [6,3,0] undeca- calamene, pinene,? -cadinene,? -murolene, Caryophyllene, Shyobunone, 6-butyl-1, 4-cyclo P-tolyl alcohol, 2,4,6,7,8,8a-hexahydro-5 (1H) -azulene (2, 4,6,7,8,8a-hexahydro-5 (1H) -azulenone, 5-oxo-delta-4-decahydrobenzindene, umbellulone, Calarene,? -Elemol, and 1-eicosene.
Test Example 3. Anti-inflammatory test _ In vitro
- Cell culture
The cells to be used are HaCaT cells which are normal human skin cell keratinocytes. For cell culture, use DMEM (Dulbeccos Modified Eagle Medium) medium containing 10% FBS and 1% penicillin / streptomycin. Cells were cultured in a CO 2 incubator (37 ° C, 5% CO 2 ). The number of cells was 1 × 10 5 for a 96-well plate and 1.5 × 10 6 for a 6-well plate.
- Statistical processing of data
All experimental results were expressed as mean standard deviation (mean ± SD) and analyzed using students t- test. When the p value was less than 5%, it was judged as "significant difference".
-sample
The lipid soluble fraction extract prepared according to Example 2 was dissolved in 5 mg / ul medium and used at a concentration of 25, 50, 100 ug / ml.
- inflammatory reaction
Inflammation reactions are caused by defibrillation of biological tissues against external stimuli such as infection or internal stimuli such as metabolites in vivo and are involved in cytokines such as IL-6 and TNF-a, which are various inflammatory regulators. In particular, activation of the inflammatory response by stimulation such as LPS results in the expression of iNOS and COX-2, resulting in the unnecessary production of NO and PGE2 to produce inflammation.
3-1. MTT Assay method for cell viability and toxicity
Cell viability was assayed using the principle of MTT reduced to purple by mitochondrial dehydrogenase of living cells. HaCaT cells were inoculated at 100 μl / well in a 96-well plate at a concentration of 2 × 10 4 cells / well and stabilized in a CO 2 incubator for 24 hours. Cells were treated with LPS (25, 50, 100 ug / ml) at various concentrations without stimulation or stimulation with LPS, and then cultured for another 24 hours. 10 μl of MTT solution was added to the cells and incubated in a CO 2 incubator And reacted for 1 hour and 30 minutes. The cell viability of the control group was measured by measuring the absorbance change at 540 nm using a microplate reader.
As shown in Fig. 1, the lipid soluble fraction extract prepared according to Example 2 showed no cytotoxicity.
Also, as shown in FIG. 2, the liposoluble fraction extract prepared according to Example 2 showed an effect of inhibiting cytotoxicity induced by LPS, and the cell survival rate was increased in a concentration-dependent manner. That is, it was confirmed that the fat soluble fraction extract prepared according to Example 2 had a cytoprotective effect.
3-2. Measurement of nitric oxide (NO)
NO is known to play an important role in specific or nonspecific immune responses and accumulates in the form of NO2 and NO3 in cell culture fluids. The concentration of NO is measured by using the Griess reagent system for nitrite concentration in the culture medium. HaCaT cells were plated at a density of 2 × 10 4 cells / well on a 96-well plate. After 24 hours, liposoluble fraction extracts were pretreated by concentration (25, 50, 100 ug / ml) And cultured for 24 hours. Add the same amount of griess reagent to the culture medium, incubate for 10 minutes at room temperature, and measure the absorbance at 540 nm. The concentration of NO in the culture medium was determined using a standard curve of concentration of sodium nitrite. The HaCaT cells were used after being stabilized in a 37 ° C, 5% CO2 incubator.
In the body, inflammatory factors such as nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) are formed by induction of NO synthase (iNOS) and cyclooxygenase (COX) -2. Among these, NO has various physiological functions such as body defense function, signal transduction function, neurotoxicity, and vasodilation. Nitric oxide synthase (NOS), which forms NO, is divided into three kinds of isoenzymes such as type Ⅰ, Ⅱ and Ⅲ according to physicochemical properties. Type Ⅰ (neuronal NOS, nNOS) and type Ⅲ (endothelial, eNOS) are classified into constitutive NOS because they are continuously present in the cells. In some cells, specific stimulants such as LPS, cytokine and bacterial toxin And inductive NOS (iNOS) type III, which is expressed only when exposed. These NOSs formed NO by converting l-argine to l-citrulline. These NOS expressions by iNOS are absolutely high, and they have a pathologically important effect. In general, the formation of NO plays an important role in killing bacteria or eliminating tumors, but the formation of excessive NO by pathologic causes causes inflammation, causing tissue damage, gene mutation and nerve damage.
As shown in FIG. 3, experiments were conducted by dividing the group treated with LPS and the group treated with LPS and the oil-soluble fraction extract of Example 2 at the same time. The stimulation of HaCat cells with LPS increased the production of NO, an inflammatory mediator, and showed that the significantly increased NO by LPS treatment tended to be inhibited by the lipid soluble fraction extract of Example 2.
3-3. COX-2, INOS Protein expression measurement (Western Blotting Assay) and RT- PCR Gene expression measurement
① protein quantification
HaCaT cells were plated on a 6-well plate at a density of 1 × 10 5 for 24 h. The samples were pre-treated for 1 h in a concentration-dependent manner and treated with LPS at a concentration of 10 g / ml. After 24 hours of incubation, cells were washed with 1 ml of Dulbecco's phosphate buffered saline (PBS), and 1 ml of PBS was added. The cells were collected using a scraper, centrifuged at 13,200 rpm for 5 minutes, . Add 50 μl of lysis solution (protease
② Electrophoresis
All samples should be prepared at a concentration of 30 μg / lane, electrophoresed on 10-15% SDS polyacrylamide gel, and transferred to polyvinylidenefluoride (PVDF) membrane by Semi Dry Transfer method. After transfer, wash with TNT buffer (Trizmabase, Nacl, Tween 20) three times for 5 minutes each time. Transfer the transferred PVDF membrane to primary antibody (1: 1000, COX2, iNOS, other antibody) diluted with 5% nonfat dry milk solution and react overnight at 4 ℃. After washing three times for 5 minutes on the following day, the cells were incubated with secondary antibody (1: 2000, Antirabbit IgG) for 1 hour, developed with ECL advance western blotting detection reagents and developed using
③ RT- PCR Gene expression measurement
RT-PCR was performed to investigate gene expression of iNOS expressed in HaCaT cells treated with the samples. First, HaCaT cells are seeded in a 60 mm dish at 4 × 10 6 cells / well and cultured for 24 hours. After the sample is added, it is treated with LPS and cultured for 24 hours. Cells are harvested, centrifuged at 4 ° C for 5 minutes at 2,000 rpm, and total RNA is isolated using Easy blue reagent. CDNA was synthesized using a reverse transcription kit for the separated RNA. To perform PCR on the synthesized cDNA, 1 μl of primers and 10 × buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCL, 0.1% TritonX-100), 250 μM dNTP and 1 U Taq polymerase were mixed with the
For the amplification of iNOS, sense primer: 5'-CCCTTCCGAAGTTTCTGGCAGCAGC-3 'and anti-senseprimer: 5'-GGCT GTCAGAGCCTCGTGGCTTTGG-3' were used. For amplification of COX-2, sense primer: 5-CAAAAGCTGGGAAGCCTTCT- sense primer: 5-CCATCCTTGAAAAGGCGCAG-3, sense primer: 5'-CACTCACGGCAAATTCAACGGCAC-3 'and anti-sense primer: 5'-GACTCCACGACATACTCAGCAC-3' were used for amplification of GAPDH.
As shown in Fig. 4 and Fig. 5, it was confirmed that the liposoluble fraction extract of Example 2 suppressed the expression of the protein similarly to the inhibition of NO production. LPS-induced cells cause inflammation, which increases the expression of iNOS and COX-2 proteins, known as inflammation-inducing proteins. When treated with the liposoluble fraction extract of Example 2, the iNOS protein was inhibited in a concentration-dependent manner.
2 on the expression of COX-2, which is an inducer of PGE 2 production. As a result, it was confirmed that the oil-soluble fraction extract of Example 2 showed an inhibitory effect on the expression of COX-2.
In addition, in order to examine the effect of the liposoluble fraction extract of Example 2 on the expression of iNOS and COX-2 mRNA, RT-PCR showed that the expression amount of the liposoluble fraction extract of Example 2 was similar to that of the positive control Respectively.
3-4. Cytokine Production Quantity Measurement PGE2 , IL-6)
HaCaT cells were plated in 96-well plates at a density of 1 × 10 5 cells / well, cultured for 24 hours, treated with LPS for 1 hour, and cultured for 24 hours. PGE 2 and IL-6 contained in the cultures cultured for 24 hours were measured using an ELISA kit.
6 and 7, in order to examine the effects on IL-6 and PGE 2 secretion as inflammatory cytokines, HaCaT cells were treated with LPS alone or with LPS and the liposoluble fraction extract of Example 2 As a result, LPS-stimulated cells increased pro-inflammatory cytokine secretion such as IL-1β, IL-6 and TNF-α, but the lipid soluble fraction extract of Example 2 secreted IL-6 and PGE 2 ug / ml.
Thus, the liposoluble fraction extract of Example 2 inhibited the production of NO induced by LPS and inhibited the expression of iNOS protein in HaCaT cells, and also had the effect of suppressing the secretion amount of inflammatory cytokines IL-6 and PGE 2 Respectively.
Test Example 4. Anti-inflammatory test _In vivo (Ear Edema Test)
- Evaluate the anti-inflammatory effect on the skin surface using an animal model using Croton oil (persimmon oil), which induces inflammation and edema by stimulation when applied to skin.
- Sample
The samples used in the animal model were diluted with acetone at a certain percentage (v / v) ratio. Hydrocortisone (2 mg / ear) was used as a positive control.
- Experimental method _ Ear Edema Test (Ear Edema Test)
20 μl of the test drug is uniformly applied to the back of the left ear of a normal mouse (male, 8 weeks old) without drug treatment using a pipette, and left for 1 hour. At this time, the normal group is coated with the same amount of acetone used as a solvent. Thereafter, a 5% croton oil solution is homogeneously applied to the back of the ear using a pipette by 30 μl each. At this time, the normal group is coated with the same amount of acetone used as a solvent.
At 0, 3 and 5 hours after the application of 5% Croton oil, the thickness of the constant area of the left ear of the experimental animal is measured using a vernier caliper. Seven animals were used in each experimental group. Ear swelling of each animal was expressed as an increased percentage value of the ear thickness measured at each time zone with the reference value (100%) at 0 time zone measurement value.
In order to confirm the effect of the sample solvent, the final product was treated with jojoba oil instead of acetone.
- Statistical processing of data
All experimental results were expressed as mean ± SD (mean ± SD) and analyzed using student`s t- test. When the p value was less than 5%, it was judged as "significant difference".
As shown in FIG. 8, the 5% Croton oil induced edema in the ear of the normal state to increase the ear thickness to about 230% at the third hour (edema), and to about 260% at the fifth hour Respectively. Hydrocortisone, a positive control, applied 2 mg to each ear, resulting in a decrease in ear thickness.
The liposoluble fraction extract of Example 2 showed a tendency to decrease ear thickness in general, and the ear thickness decreased significantly at 3 hours after 50% and 70% concentration treatment. This pattern continued until 5 hours.
In addition, as a result of using jojoba oil instead of acetone as a diluent (FIG. 9), 5% Croton oil induced edema in a normal ear and increased ear thickness to about 190% at 3 hours, To 230%. This is a slight decrease compared to the previous test with acetone, which seems to be due to the absorption inhibition of 5% Croton oil by applying jojoba oil. 2 mg of Hydrocortisone, a positive control, was applied to each ear, resulting in a significant reduction in ear thickness.
The fat-soluble fraction extract of Example 2 showed a tendency of decreasing the thickness of the ear as a whole. Especially, the effect of decreasing the thickness of the ear was significant from the third hour, and continued until 5 hours.
Test Example 5. Measurement of total polyphenol and total flavonoid content
The total polyphenol content was determined from the standard curve using tannic acid by applying the Folin-Denis method. The total flavonoid content was also measured by the method of Nieva et al. That is, 100 μl of the liposoluble fraction extract of Example 2 was diluted in 900 μl of 80% ethanol, and then 100 μl of the extract was mixed with 4.3 ml of 80% ethanol containing 10% aluminum nitrate and 1 μM potassium acetate for 40 minutes at room temperature The absorbance was measured at 415 nm. The content of total flavonoids was determined from the standard curves prepared using quercetin.
Phenolic compounds are widely distributed in various plants and are known to be antioxidants. Because they have a phenoloc hydroxyl (-OH) group, they bind easily to proteins and other macromolecules; Antioxidant, anti-cancer, anti-inflammatory and anti-allergic effects. In this study, phenolic compounds showed anti - inflammatory activity by regulating enzyme activity as well as enzyme activity involved in inflammatory reaction. The total polyphenol and flavonoid contents in the oil-soluble fraction extract of Example 2 were compared using tannic acid and quercetin as reference materials, and the results are shown in Table 3 below.
As shown in Table 3 above, it was confirmed that the fat soluble fraction extract prepared according to Example 2 of the present invention contained total polyphenols and total flavonoids in a high content.
Test Example 6. Electron donating ability (electron donating ability) measurement
The electron donating ability (EDA) was measured by modifying the Blois method. To 2 ml of the liposoluble fraction extract of Example 2, 1 ml of 0.2
[Equation 1]
DPPH is a chemically stabilized water-soluble substance with free radicals, which is reduced by ascorbic acid, tocopherol, and polyhydroxy aromatic compounds, resulting in discoloration of deep purple color, Which is widely used to search for antioxidants. Reactive oxygen species (ROS) are largely eliminated by the body's defense system, but if not eliminated, they react with biomolecules rapidly to cause protein denaturation, lipid peroxidation and DNA damage of the biological membrane, Lipid peroxides accelerate new radical reactions and cause various diseases.
The DPPH free radical scavenging activity of the lipid soluble fraction extract prepared according to Example 2 of the present invention was found to be very excellent at 90.24 ± 1.9% at a concentration of 1,000 ug / ml.
Hereinafter, formulation examples of the composition containing the extract of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.
Preparation Example 1. Preparation of powder
In Example 2, 500 mg of the liposoluble fraction extract
The above components are mixed and filled in airtight bags to prepare powders.
Formulation Example 2. Preparation of tablets
In Example 2, 300 mg of the liposoluble fraction extract
After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.
Formulation Example 3. Preparation of capsules
In Example 2, 200 mg of the liposoluble fraction extract
Crystalline cellulose 3 mg
Lactose 14.8 mg
Magnesium stearate 0.2 mg
The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.
Formulation Example 4. Preparation of injection
In Example 2, 600 mg of the lipid soluble fraction extract
180 mg mannitol
Sterile sterilized water for injection 2974 mg
Na 2 HPO 4 · 12H 2 O 26 mg
It is prepared by the above-mentioned component content per ampoule according to the usual injection preparation method.
Formulation Example 5. Preparation of a liquid preparation
In Example 2, 4 g of the fat soluble fraction extract
10 g per isomer
5 g mannitol
Purified water quantity
Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 g with purified water, To prepare a liquid agent.
Preparation Example 6 Preparation of Granules
In Example 2, 1,000 mg of the liposoluble fraction extract
Vitamin mixture quantity
Vitamin A acetate 70 mg
Vitamin E 1.0 mg
Vitamin B1 0.13 mg
0.15 mg of vitamin B2
Vitamin B6 0.5 mg
Vitamin B12 0.2 mg
Biotin 10 mg
Nicotinic acid amide 1.7 mg
Calcium pantothenate 0.5 mg
Mineral mixture quantity
1.75 mg of ferrous sulfate
0.82 mg of zinc oxide
Magnesium carbonate 25.3 mg
Potassium monophosphate 15 mg
Secondary calcium phosphate 55 mg
Potassium citrate 90 mg
Magnesium chloride 24.8 mg
The composition ratio of the above-mentioned vitamins and minerals is comparatively comparatively mixed with the granules according to the preferred embodiment. However, the blending ratio may be arbitrarily changed, and the above components are mixed according to the ordinary granule preparation method, Can be prepared and used in the manufacture of a health functional food composition according to a conventional method.
Preparation Example 7. Preparation of functional beverage
In Example 2, 1,000 mg of the liposoluble fraction extract
Citric acid 1,000 mg
100 g of oligosaccharide
Plum concentrate 2 g
Taurine 1 g
Purified water was added to the flask to obtain a total of 900 mL
The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The solution thus prepared was filtered and sterilized in a sterilized 2 L container, It is used in the production of the functional beverage composition of the invention.
Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.
Formulation Example 8. Preparation of cosmetic product of emulsified formulation
Cosmetics of emulsified form such as nutrient lotion, cream, essence, and cosmetics of solubilized form such as softened longevity were prepared.
Emulsifier type cosmetics were prepared with the compositions shown in Table 4 below. The production method is as follows.
1) A mixture of
2) The starting material of 10 was added to the mixture of step 1).
3) The mixture of raw materials 11 to 13 was completely dissolved by heating to 65 to 70 占 폚.
4) While the above step 3) was carried out, the mixture of step 2) was gradually added and emulsified at 8,000 rpm for 2 to 3 minutes.
5) The raw material of 14 was dissolved in a small amount of water and then added to the mixture of step 4) and further emulsified for 2 minutes.
6) The raw materials of 15 to 17 were each weighed, and then put into the mixture of step 5) and further emulsified for 30 seconds.
7) The mixture of step 6) was degassed after emulsification and cooled to 25-35 占 폚 to prepare an emulsifier-type cosmetic.
Monolauric acid ester
Formulation example 9. Solubilization Manufacture of Cosmetics for Formulation
Cosmetic products of the solubilized formulations were prepared with the compositions shown in Table 5 below. The production method is as follows.
1) 2 to 6 raw materials were put into 1 raw material (purified water) and dissolved using a mixer.
2) Raw materials 8 to 11 were completely dissolved in 7 raw materials (alcohol).
3) The mixture of step 2) was slowly solubilized by adding it to the mixture of step 1).
Hydro genide ester
Claims (11)
The pharmaceutical composition for preventing or treating inflammation or edema according to any one of claims 1 to 3, wherein the white pine bark, astragalus and Angelica gigas are powder having a moisture content of 3 to 10%.
The cosmetic composition for prevention or improvement of inflammation or swelling according to any one of claims 1 to 3, wherein the white pine bark, astragalus and Angelica gigas are powder having a moisture content of 3 to 10%.
Wherein the white pine bark, astragalus and Angelica gigas are powder dried at a moisture content of 3 to 10%, wherein the health functional food for preventing or ameliorating inflammation or edema.
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