Method for extracting non-polar natural substance

Abstract

Disclosed are: a method for extracting a non-polar natural substance from a natural raw material; a method for extracting a toxic substance; and a method for preventing or treating anti-inflammatory and autoimmune diseases, comprising an extracted non-polar natural substance as an active ingredient. The method for extracting a non-polar natural substance from a natural raw material, according to an embodiment of the present invention, comprises the steps of: extracting the natural raw material with alcohol at a first concentration; and adding a resin to the extract to adsorb the non-polar natural substance.

Description

Extraction method of nonpolar natural substances

[0001] The present invention relates to a method for extracting a non-polar natural substance from a natural raw material, a method for extracting a toxic substance, and a method for preventing or treating anti-inflammatory and autoimmune diseases, which comprises the extracted non-polar natural substance as an effective ingredient.

[0002] Extraction is the separation of components contained in a solid or liquid raw material by dissolving them with a solvent. The general extraction method is the solid-liquid extraction method when the raw material is solid, and the liquid-liquid extraction method when the raw material is liquid.

[0003] Liquid-liquid extraction (LLE) is an extraction method that fractionates a liquid mixture containing a solute by applying a solvent to the mixture according to the properties of a specific solvent.

[0004] In order to fractionate components in natural products according to polarity, a liquid-liquid extraction method is generally used. However, nonpolar solvents such as hexane, methane dichloride, and ethyl acetate are highly toxic during liquid-liquid extraction. In addition, the extraction process is very inefficient and complex due to repeated extraction processes and a drying process to remove the nonpolar solvent, and the process is long. The extraction efficiency is low, and the production cost is high as the process is long. In particular, it is not very suitable for separating or extracting trace amounts of active substances.

[0005] For example, the process for extracting Brevilin A from Centipeda minima involves extracting the raw material multiple times with methanol, fractionating the extract multiple times with hexane, fractionating the extract multiple times with chloroform, fractionating the extract multiple times with ethyl acetate, and fractionating the extract multiple times with butyl alcohol, and further performing column separation. From the dry weight of Centipeda minima, only about 27 mg of Brevilin A is extracted, so the extraction amount is extremely trace and the efficiency is very low. Therefore, a novel extraction or separation extraction method that has the productivity to extract trace amounts of active substances and can be upcycled to help the environment is required (Non-patent Document 1).

[0006] Patent Document 1 discloses an extract of the plant, and in this case, a natural substance was extracted using a supercritical carbon dioxide extraction method. However, this extraction method has the problem that the conditions for use are complicated, the extraction time is considerable, and the extraction efficiency is also very low.

[0007] There are 191 kinds of components in the plant, including 1) terpenoids: 112 kinds (22 kinds of mono / diterpenoids, 54 kinds of sesquiterpenoids, 36 kinds of triterpenoids), 2) flavonoids: 21 kinds, 3) sterols: 11 kinds, 4) phenols: 22 kinds, and 5) organic acids: 25 kinds.

[0008] Among the terpenoids, it contains four types of sesquiterpenoids as dual active ingredients: Brevilin A, Arnicolide D, Arnicolide C, and Microhelenin C, while other ingredients contain skin toxicity and toxic substances.

[0009] Therefore, it is necessary to develop a novel extraction method that can selectively, easily, and efficiently extract trace amounts of sesquiterpenoid compounds from natural products and make the extracts easily usable.

[0010] Most natural products contain active ingredients, and research is ongoing to utilize these non-polar substances and to extract them.

[0011] Anti-inflammation refers to the efficacy of preventing, suppressing, inhibiting, improving, or / and treating inflammation or / and inflammation-related diseases. Inflammation is manifested by symptoms or signs such as edema caused by an increase in body fluid between tissue cells, congestion due to vasodilation, fever due to pyrogens and vasodilation, and pain due to arachidonic acid metabolites. It can be classified into acute, subacute, and chronic inflammatory diseases according to time, and can be classified into infectious, allergic, autoimmune, toxic, metabolic, and traumatic inflammatory diseases according to pathophysiology.

[0012] For example, the inflammation may include respiratory inflammatory diseases such as rhinitis, sinusitis, otitis media, nasopharyngitis, laryngitis, bronchitis, asthma, chronic obstructive pulmonary disease, bronchiectasis, bronchiolitis, pneumonia, and pulmonary fibrosis; digestive inflammatory diseases such as stomatitis, esophagitis, gastritis, peptic ulcer, irritable bowel syndrome, inflammatory bowel disease, cholecystitis, cholangitis, pancreatitis, and hepatitis; skin inflammatory diseases such as acne, contact dermatitis, seborrheic dermatitis, atopic dermatitis, and psoriasis; cardiovascular inflammatory diseases such as endocarditis, myocarditis, pericarditis, vasculitis, arteriosclerosis, and sepsis; endocrine inflammatory diseases such as thyroiditis, hyperparathyroidism, and diabetes; urogenital inflammatory diseases such as glomerulonephritis, nephropathy, interstitial nephropathy, orchitis, oophoritis, endometritis, and vaginitis; Musculoskeletal inflammatory diseases such as rheumatoid arthritis, spondyloarthropathy, osteoarthritis, gout, systemic lupus erythematosus, systemic sclerosis, myopathy, Sjogren's syndrome, Behcet's disease, and antiphospholipid syndrome; neuropsychiatric inflammatory diseases such as vascular dementia, Alzheimer's disease, degenerative brain disease, depression, and schizophrenia.

[0013] Autoimmune diseases are diseases in which the body's immune cells attack its own organs or tissues, resulting from immune imbalance or loss of tolerance to self-antigens. Autoimmune diseases cause damage to cells or tissues through humoral immunity, cell-mediated immunity, or both. This inappropriate immune response to self-antigens is called autoimmunity. Autoimmune diseases involve numerous molecules, cells, and tissues targeted by the autoimmune response, and can be systemic or organ-specific, depending on the distribution of the target antigen. For example, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and multiple sclerosis (MS) are systemic autoimmune diseases. Alopecia areata is an autoimmune disease that causes patchy hair loss regardless of gender, age, or hair color, with a global prevalence of approximately 0.1-0.2%.

[0014] Tofacitinib is widely used as an immunosuppressant for autoimmune diseases.

[0015] Tofacitinib {3-((3R,4R)-4-methyl-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)-3-oxopropanenitrile} is a compound represented by the following chemical formula. Tofacitinib was first disclosed in International Patent Publication No. 2001 / 042246.

[0016]

[0017] Tofacitinib is a synthetic JAK (Janus kinase) inhibitor. Janus kinase (JAK) is a type of enzyme that catalyzes the transfer of phosphate groups. Cytokines, proteins secreted by immune cells, transmit signals within the cell when they bind to cytokine receptors. Cytokine receptors bind to Janus kinases such as TYK2, JAK1, JAK2, and JAK3.

[0018] Additionally, other JAK inhibitors, such as ruxolitinib, baricitinib, fedratinib, filgotinib, upadacitinib, and rillecitinib, are in development or in clinical trials.

[0019] When cytokines bind to cytokine receptors, a phosphorylation reaction occurs in JAK, activating STAT (signal transducer and activator of transcription), and signal transduction continues to cause hematopoiesis, inflammatory response, and immune response. In autoimmune diseases where a hyperimmune response occurs due to excessive production of cytokines, a JAK inhibitor inhibits the phosphorylation enzyme of the cytokine receptor to which the cytokine binds.

[0020] However, not only tofacitinib, but also JAKI and JAK inhibitors in the same series, such as baricitinib and ritilitinib, are synthetic drugs and their safety cannot be guaranteed due to the numerous confirmed side effects of the raw materials. In addition, they were developed as prescription drugs, so patient accessibility is low, and they are limited to pharmaceutical use and are limited to oral administration. When taken for a long time, especially when taken for hair loss treatment, there are problems such as serious infections (nasopharyngitis, gastroenteritis, upper respiratory tract infection) and intestinal perforation due to immunosuppression, and increased cholesterol levels, headache, rash, shingles, and anemia. In addition, tofacitinib has the problem of incurring high costs of about 6 million won per month when not covered by domestic health insurance based on the daily prescription amount of 10 mg / day.

[0021] For example, among natural raw materials, Centipeda minima (Centipeda minima, Tobangpul) is an annual herb in the Asteraceae family. In China, it is used in folk medicine to treat chronic rhinitis, nasal diseases, eye diseases, and headaches. Furthermore, a mixture of the leaves and stems, placed in the nostrils overnight, is effective against chronic malaria. In India, it is used for eye diseases, nasal diseases, and toothache. It is distributed in Korea, Japan, China, India, Australia, eastern Siberia, and North America. Little is known about the anti-inflammatory, autoimmune, and non-alcoholic fatty liver disease effects of Centipeda minima, and further research is needed.

[0022] Additionally, there is no research on the dosage and administration of Brevilin A among the extracts of the plant.

[0023] The invention was completed by finding that an extract extracted with low concentration alcohol has fewer side effects and is effective in treating anti-inflammatory and autoimmune diseases, and the extraction method according to the Republic of Korea Patent Publication No. 10-2598042 can be appropriately utilized in the present invention.

[0024] In addition, during the course of further research, the present invention was completed as a result of continued research on a method for reducing impurities in an extract and extracting an extract containing high concentration and high purity indicator substances.

[0025] Patent Document 1: Republic of Korea Patent Publication No. 10-2020-0085024

[0026] Patent Document 2: Republic of Korea Patent Publication No. 10-2598042

[0027] Non-patent literature 1: Arch Pharm Res Vol 29, No 1, 64-66, 2006.

[0028] The present invention aims to provide a method for selectively extracting non-polar natural substances from natural raw materials with high purity.

[0029] In addition, it is intended to provide a method for extracting toxic substances from natural raw materials.

[0030] In addition, the present invention aims to provide a pharmaceutical composition for preventing or treating anti-inflammatory and autoimmune diseases, which contains an extracted non-polar natural substance as an active ingredient.

[0031] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0032] A method for extracting a non-polar natural substance according to one embodiment of the present invention includes a step of extracting a natural substance raw material with alcohol of a first concentration and a step of adding a resin to the extract to adsorb the non-polar natural substance.

[0033] In one embodiment of the present invention, the natural raw material may include at least one selected from the group consisting of Centipeda minima, Litsea glutinous, plants of the genus Arnica, and plants of the genus Helenium.

[0034] In one embodiment of the present invention, a step of washing the natural raw material with purified water may be further included.

[0035] In one embodiment of the present invention, the natural raw material and the purified water can be mixed in a weight ratio of 1:5 to 20 and washed once for a period of less than 6 hours.

[0036] In one embodiment of the present invention, the first concentration of alcohol may be alcohol having a concentration of less than 50%.

[0037] In one embodiment of the present invention, the first concentration of alcohol may be 5 to 25% alcohol.

[0038] In one embodiment of the present invention, the alcohol may include at least one selected from the group consisting of methanol, ethanol, propanol, butanol, benzyl alcohol, isopropyl alcohol, cetearyl alcohol, cetyl alcohol, stearyl alcohol, lauryl alcohol, myristyl alcohol, and behenyl alcohol.

[0039] In one embodiment of the present invention, the resin may include at least one selected from the group consisting of HP-20, HLB, C18, tC18, C8, tC2, Silica, Florisil®, Accell Plus CM, Accell Plus QMA, Alumina A, Alumina B, Alumina N, Amino Propyl (NH2), Cyano Propyl (CN), and Diol.

[0040] In one embodiment of the present invention, the non-polar natural substance may include at least one selected from the group consisting of Brevilin A, Arnicolide D, Arnicolide C, and Microhelenin C as an active ingredient.

[0041] In one embodiment of the present invention, the brevilin A inhibits or suppresses the JAK-STAT signaling pathway, the arnicolide D, the arnicolide C and the microhellenin C inhibit or suppress the cytokine-cytokine receptor interaction, the MAPK signaling pathway and the PI3K-AKT signaling pathway, and the extract of the root of the Chinese yam can regulate the WNT signaling pathway (WNT / β-catenin pathway).

[0042] In one embodiment of the present invention, the step of washing with alcohol having a second concentration lower than the first concentration may be included.

[0043] In one embodiment of the present invention, the second concentration of alcohol may be alcohol having a concentration of less than 50%.

[0044] In one embodiment of the present invention, the step of dissolving the non-polar natural substance by mixing alcohol of a third concentration higher than the first concentration may be included.

[0045] In one embodiment of the present invention, the third concentration of alcohol may be alcohol having a concentration of 50% or more.

[0046] In one embodiment of the present invention, the step of adding resin to the alcohol solution from which the nonpolar natural substance has been eluted to re-adsorb the nonpolar natural substance and the step of mixing alcohol into the resin to which the nonpolar natural substance has been adsorbed to re-elute the nonpolar natural substance may be repeated at least once.

[0047] In one embodiment of the present invention, the step of mixing a lipophilic solubilizer after removing the resin may be included.

[0048] In one embodiment of the present invention, the lipophilic solubilizer is MCT OIL, BRIJ 010-SS-(RB), Capryol 90, Capryol PGMC, Gantrez ES-435, Gantrez S-97 BF, Gelucire 43 / 01, Gelucire 44 / 14, Gelucire 50 / 13, Gelucire 48 / 16, Labrafac CC, Labrafac Lipophile WL1349, Labrafac PG, Medium-chain triglycerides, Labrafil M 1944 CS, Labrafil M 2125 CS, Labrafil M 2130 CS, Lauroglycol 90, Lauroglycol FCC, Monosteol, Peceol, Pharmasolve, Plurol Oleique CC497, Span 20-LQ-(SG), Span 60-PA-(SG), upper Refined It may include at least one selected from the group consisting of Oleic Acid-LQ-(JP), Super Refined Tween 80A-LQ-(MH), Surfadone LP-300, Transcutol HP, Transcutol P, TWEEN 60-SS-(SG), TWEEN 80 HP-LQ-(MH), TWEEN 80-LQ-(SG), Vitamin E TPGS, Miglyol 812, and captax 355.

[0049] In one embodiment of the present invention, the method may include a step of obtaining a supernatant after removing alcohol.

[0050] In one embodiment of the present invention, the steps of adding resin to the obtained supernatant to re-adsorb a non-polar natural substance, mixing alcohol into the resin to re-elute the non-polar natural substance, removing the resin, mixing a lipophilic solubilizer, and removing the alcohol to obtain a supernatant may be repeated one or more times.

[0051] A method for extracting a toxic substance according to another embodiment of the present invention may include a step of extracting a natural raw material with alcohol of a first concentration, and then mixing alcohol of a higher concentration than the first concentration with the residue to extract the residue extract.

[0052] Additionally, we aim to provide the optimal dosage for achieving effects on autoimmune diseases such as hair loss by using non-polar natural substances extracted from natural raw materials.

[0053] In another embodiment of the present invention, in a method for treating anti-inflammatory and autoimmune diseases, the content of brevilin A in a composition for treating anti-inflammatory and autoimmune diseases, which comprises an extract extracted from a natural raw material with alcohol as an active ingredient, is administered in a daily dose of 0.5 to 12 mg.

[0054] In one embodiment of the present invention, the composition may be administered at least once a week.

[0055] Embodiments of the present invention relate to a method for selectively extracting non-polar natural substances from natural raw materials, which can extract non-toxic substances contained in natural raw materials with high purity.

[0056] Additionally, by controlling the concentration of the extraction solvent, non-polar natural substances can be separated and then re-extracted to extract toxic components with high purity.

[0057] In addition, before extracting natural raw materials, the extraction yield can be improved through a pretreatment process that minimizes the loss of raw materials.

[0058] In addition, the nonpolar natural substances extracted in this way can be applied in various forms such as transdermal and oral, and have anti-inflammatory, autoimmune, alopecia, and anticancer effects. When applied in the form of health functional foods including these nonpolar natural substances, they have the effect of helping relieve hangovers.

[0059] In addition, the safety of the raw material can be ensured by using the natural extract of the nonpolar natural substance extracted in this way compared to the existing synthetic drug tofacitinib, which is an autoimmune disease treatment drug. In addition, it shows a superior effect in anti-inflammatory and autoimmune diseases without side effects compared to the single prescription of brevilin A, which is known as the extract of the Chinese yam.

[0060] In addition, while existing synthetic drugs are limited to oral administration, the embodiments of the present invention utilizing natural compositions have fewer or almost no side effects compared to synthetic drugs, and are easily applied not only to drugs but also to cosmetics, foods, etc.

[0061] Figures 1 and 2 are graphs showing the average hair count change pattern and improvement rate as results of a hair loss symptom relief efficacy test according to an embodiment of the present invention.

[0062] Figure 3 is a graph showing the interaction coefficient for the hair count by repeated measures analysis of variance for the hair count by week to confirm the difference between groups in the hair count measurement results.

[0063] Figure 4 is a graph showing the results of the tester's visual evaluation of the degree of hair improvement.

[0064] Figures 5 and 6 are graphs showing the average hair count change pattern and improvement rate as a result of a hair cycle change pattern test according to an embodiment of the present invention.

[0065] Figures 7 to 10 are graphs showing the difference between groups in the results of measuring the number of normal hairs (Terminal) and the number of hairs in the growth phase (Anagen) by week, and showing the improvement rate thereof.

[0066] In a method for treating anti-inflammatory and autoimmune diseases according to one embodiment of the present invention, the content of brevilin A in the composition for treating anti-inflammatory and autoimmune diseases, which comprises an extract extracted from a natural raw material with alcohol as an active ingredient, is administered in a daily dose of 0.5 to 12 mg.

[0067] Hereinafter, a method for extracting non-polar natural substances according to embodiments of the present invention, the natural substances extracted thereby, and their uses will be described.

[0068] Before proceeding, it should be noted that the technical terminology used herein is only for the purpose of referring to specific embodiments and is not intended to limit the present invention. In addition, the singular forms used herein include plural forms as well, unless the phrases clearly indicate the opposite meaning. In addition, the meaning of "comprise" or "include" as used herein specifies a specific characteristic, area, integer, step, operation, element, or component, and does not exclude the addition of other specific characteristics, areas, integers, steps, operations, elements, or components.

[0069] In the present invention, terms such as first, second, etc. are used to describe various components, and the terms are used only for the purpose of distinguishing one component from another.

[0070] Furthermore, the terminology used herein is merely for the purpose of describing exemplary embodiments and is not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this specification, it should be understood that the terms "comprise," "include," or "have" indicate the presence of a feature, number, step, component, or combination thereof, but do not preclude the possibility of the presence or addition of one or more other features, numbers, steps, components, or combinations thereof.

[0071] The present invention is susceptible to various modifications and takes various forms. Specific embodiments are illustrated and described in detail below. However, this is not intended to limit the present invention to specific disclosed forms, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention.

[0072]

[0073] Hereinafter, the present invention will be described in more detail.

[0074] The present invention relates to a method for extracting a non-polar natural substance according to one embodiment of the present invention.

[0075] The above natural raw material may include at least one selected from the group consisting of Centipeda minima, Litsea glutinous, plants of the genus Arnica, and plants of the genus Helenium.

[0076] The above 'Centipeda minima' is an angiosperm belonging to the Campanulate family of dicotyledons and grows along roadsides, in fields, or near rice paddy ridges in Korea, Japan, China, India, Australia, eastern Siberia, and North America. Centipeda minima, also known as abul vinegar, is usually harvested in the summer when it flowers, washed in water, and dried in the sun for use. It has a spicy flavor and a warm nature, and is known to be effective for colds, tonsillitis, whooping cough, amoebic erysipelas, malaria, sinusitis, corneal clouding, scabies, and bruises.

[0077] The natural raw material used in the examples of the present invention was the Korean apricot, and domestically produced abul vinegar (Korean apricot, Yeongcheon) was purchased from the Goesan Medicinal Herb Organic Agricultural Products Cooperative (https: / natural-herb.co.kr).

[0078] There are many documents that Centipeda minima contains Brevilin A, and some documents also report that Litsea glutinous also contains Brevilin A. In addition, there are documents that plants of the genus Arnica (Arnica longifolia, Arctium lappa, Arnica chamissonis subsp. Foliosa, Arnica chamissonis Less.) and the genus Helenium (Helenium autumnale L, Helenium alternifolium, Helenium pinnatifidum, Helenium vernale, Helenium brevifolium, and Helenium flexuosum) also contain some Brevilin A.

[0079] In the present invention, the term "extracted material" or "extract" includes the extract itself and all formulations that can be formed using the extract, such as an extract obtained by extraction treatment, a diluted or concentrated extract, a dried product obtained by drying the extract, a conditioned or purified product of the extract, or a mixture thereof.

[0080] Additionally, drying of the extract can be performed using known methods, as long as the useful components of the extracted natural material are not destroyed. For example, drying can be performed naturally in a shaded area. Furthermore, crushing or pulverizing can be performed to a degree that allows sufficient extraction of the plant's useful components during the subsequent extraction process. The drying and crushing or pulverizing processes can be performed in reverse order or repeated as needed.

[0081] In one embodiment of the present invention, the non-polar natural substance may include at least one selected from the group consisting of Brevilin A, Arnicolide D, Arnicolide C, and Microhelenin C.

[0082] Brevilin A is represented by the following chemical formula:

[0083]

[0084] Brevilin A has a CAS number 16503-32-5, a molecular formula of C20H26O5, a molecular weight of 346.42, and is derived from Arnaca sp., Centipeda sp., and Helenium sp.

[0085] There are many documents that Centipeda minima contains Brevilin A, and some documents also report that Litsea glutinous also contains Brevilin A. In addition, there are documents that plants of the genus Arnica (Arnica longifolia, Arctium lappa, Arnica chamissonis subsp. Foliosa, Arnica chamissonis Less.) and the genus Helenium (Helenium autumnale L, Helenium alternifolium, Helenium pinnatifidum, Helenium vernale, Helenium brevifolium, and Helenium flexuosum) also contain some Brevilin A.

[0086] Arnicolide D is represented by the following chemical formula:

[0087]

[0088] Arnicolide D has a CAS number 34532-68-8, a molecular formula of C19H24O5, a molecular weight of 332.40, and is derived from Arnaca sp. and Centipeda sp.

[0089] Arnicolide C is represented by the following chemical formula:

[0090]

[0091] Arnicolide C has a CAS number 34532-68-7, a molecular formula of C16H26O5, a molecular weight of 334.41, and is derived from Arnaca sp. and Centipeda sp.

[0092] Microhellenin C is represented by the following chemical formula:

[0093]

[0094] Microhellenin C has a CAS number 63569-07-3, a molecular formula of C20H26O5, a molecular weight of 346.4, and is derived from Centipeda sp. and Helenium sp.

[0095] For example, the extract of the above-mentioned rhizome comprises at least one of brevilin A, arnicolide D, arnicolide C and microhellenin C, for example, brevilin A, arnicolide D, arnicolide C, microhellenin C alone, or a combination of brevilin A and arnicolide D, a combination of brevilin A and arnicolide C, a combination of brevilin A and microhellenin C, a combination of arnicolide D and arnicolide C, a combination of arnicolide D and microhellenin C, a combination of arnicolide C and microhellenin C, a combination of brevilin A, arnicolide D and arnicolide C, a combination of brevilin A, arnicolide C and microhellenin C, a combination of brevilin A, arnicolide D and arnicolide C, a combination of brevilin A, arnicolide C and microhellenin C, a combination of brevilin A, arnicolide D and microhellenin C, a combination of brevilin A, arnicolide C and microhellenin C, a combination of arnicolide D, arnicolide C and microhellenin C, brevilin A, arnicolide D, arnicolide It can be included in combination with C and microhellenin C.

[0096] In the present invention, the brevilin A inhibits or suppresses the JAK-STAT signaling pathway, the arnicolide D, the arnicolide C and the microhellenin C inhibit or suppress the cytokine-cytokine receptor interaction, the MAPK signaling pathway and the PI3K-AKT signaling pathway, and the extract of the herbaceous plant can regulate the WNT signaling pathway (WNT / β-catenin pathway).

[0097] Looking at the mechanism of action of each of the above-mentioned brevilin A, the above-mentioned arnicolide D, the above-mentioned arnicolide C, and the above-mentioned microhellenin C, the above-mentioned brevilin A inhibits or suppresses the JAK-STAT signaling pathway. Specifically, the natural substance brevilin A is a natural substance and is widely known to inhibit JAK (janus kinase activity) and STAT3 signaling in cancer cells (PLoS One 8 (5) (2013) e63697). In addition, it is effective in treating alopecia areata, a type of autoimmune disease, by suppressing a strong inflammatory response and various inflammation-related targets through suppression of the JAK-STAT (signal transducers and activators of transcription) pathway.

[0098] The above arnicolide D, the above arnicolide C and the above microhellenin C can inhibit or suppress cytokine-cytokine receptor interaction, MAPK signaling pathway (MAPK (mitogen-activated protein kinase) signaling pathway) and PI3K-AKT signaling pathway (PI3K (phosphatidylinositol-3-kinase)-AKT signaling pathway).

[0099] In addition, the extract of the above-mentioned midge including the above-mentioned brevilin A, the above-mentioned arnicolide D, the above-mentioned arnicolide C or the above-mentioned microhellenin C has the WNT signaling pathway (WNT / -catenin pathway) can regulate the WNT signaling pathway (WNT / β-catenin pathway) is essential for the early development or morphogenesis of animals. Specifically, Wnt is a secretory protein with a molecular weight of about 40,000, and 19 types of Wnt form subfamilies, and each binds to the single-transmembrane LRP5 / 6 (low-density lipoprotein receptor-related protein 5 / 6), which is a co-receptor of the seven-transmembrane Frizzled receptor of the cell membrane. The intracellular signaling pathway activated by the binding of Wnt and its receptor includes There are three types: the catenin pathway, the planar cell polarity pathway, and the Ca2+ pathway. In the catenin pathway By regulating the stability of catenin, it regulates the expression of various target genes. This pathway controls cell proliferation and differentiation, and abnormalities in this area can lead to cancer. When Wnt does not stimulate cells, casein kinase I and GSK3 are activated. cytoplasmic within the Axin and APC protein complex -Phosphorylate catenin, -TrCP E3 ubiquitin ligase is phosphorylated -catenin is recognized and maintained by uniquitination / proteasomal activation. When Wnt is stimulated, Axin leaves the complex and GSK3 It is decomposed through the e pathway. Through this process, it is decomposed within the cytoplasm. -catenin level is inhibited during the day - As phosphorylation of catenin is inhibited, -catenin levels increase, -catenin translocates into the nucleus and binds to T cell factor or Lymphoid Enhancer-Binding factor. This binding regulates gene expression. Therefore, Wnt / -Abnormalities in the regulation of the -catenin pathway can lead to cancer.

[0100] Accordingly, the extract extracted from the plant of the present invention includes Brevilin A, Arnicolide D, Arnicolide C or Microhelenin C, and when the extract of the plant of the present invention is used, the WNT signaling pathway (WNT / -catenin pathway) can be regulated to prevent and treat anti-inflammatory and autoimmune diseases.

[0101] First, wash the natural raw material with purified water.

[0102] When purchasing natural raw materials, most are either raw or dried. These raw materials are typically unwashed and may contain a large amount of impurities.

[0103] If the extraction step is performed directly without removing these impurities, too many impurities will be carried over to the extract, and a separate process such as filtering will have to be added.

[0104] Accordingly, the above natural raw material goes through a washing step, and the washing step can be done by washing with water or by air washing using an air gun.

[0105] However, the air washing method causes loss of the natural raw material, and in particular, in the case of dried natural raw materials, there is a problem that a large amount of dust such as powder is generated as the raw material is damaged, polluting the working environment. In addition, there is a problem that the extract is contaminated with foreign substances during alcohol extraction after the air washing. In addition, even when washing by immersing in running water for a certain period of time, there is a problem that a large amount of raw material is lost.

[0106] Therefore, it is preferable to wash the above natural raw material by immersing it in purified water for a certain period of time.

[0107] For example, the natural raw material and the purified water can be mixed in a weight ratio of 1:5 to 20 and washed at least once within 6 hours.

[0108] If the weight ratio of the natural raw material and the purified water is less than 1:5 and outside the above range, the natural raw material is not sufficiently washed, and if it exceeds 1:20, there is a problem that a large amount of the raw material is lost.

[0109] In addition, if the number of washes is more than two, there is a problem that a large amount of raw material is lost due to washing, and if the washing time exceeds 6 hours, there is a problem that the raw material is lost and non-polar natural substances are also lost, reducing the yield.

[0110] Preferably, the natural raw material and the purified water are mixed in a weight ratio of 1:5 to 15, more preferably in a weight ratio of 1:6 to 1:13, or in a weight ratio of 1:8 to 1:12, or in a weight ratio of 1:10, and washed once for less than 1 hour, or less than 30 minutes, or less than 10 minutes.

[0111] A method for extracting a non-polar natural substance according to one embodiment of the present invention includes a step of extracting a natural substance raw material with alcohol of a first concentration and a step of adding a resin to the extract to adsorb the non-polar natural substance.

[0112] In the present invention, the alcohol may be selected from the group consisting of methanol, ethanol, propanol, butanol, benzyl alcohol, isopropyl alcohol, cetearyl alcohol, cetyl alcohol, stearyl alcohol, lauryl alcohol, myristyl alcohol, and behenyl alcohol, and ethanol may be suitably used.

[0113] Specifically, it is a method of manufacturing a primary extract by extracting natural substances from natural raw materials, and alcohol is used, and ethanol can be suitably used.

[0114] For example, natural raw materials can be cold-extracted by soaking them in cold alcohol.

[0115] For example, natural raw materials can be used in their raw form or in powder form.

[0116] In one embodiment of the present invention, the first concentration of alcohol may be alcohol having a concentration of less than 50%.

[0117] In the present invention, the extract of the natural raw material containing a non-polar natural substance can be extracted with alcohol having a concentration of less than 50%.

[0118] When the above natural raw material is extracted with alcohol having a concentration exceeding 50%, other substances such as toxic components may be extracted in addition to the intended effective ingredient, and due to these components, as a toxic substance, unexpected reactions may occur in the body when ingested orally, problems such as inflammation may occur, and when administered transdermally, problems such as skin abnormalities such as cracking or keratinization may occur.

[0119] In one embodiment of the present invention, the first concentration of alcohol may be 5 to 25% alcohol.

[0120] In the present invention, the extract of the natural raw material containing a non-polar natural substance can be extracted with 5 to 25% alcohol.

[0121] For example, if the extract of the plant is extracted with alcohol at a concentration of less than 5%, the problem may arise that the effective ingredients, including Brevilin A, Arnicolide D, Arnicolide C, and Microhelenin C, are not extracted in the desired amount. In addition, if the extraction is performed with alcohol at a concentration exceeding 25%, other substances, such as toxic ingredients, may be extracted in addition to the desired effective ingredients, and these ingredients may cause skin abnormalities, such as cracking or keratinization.

[0122] Non-polar natural substances can be adsorbed by adding resin to the extract.

[0123] For example, the resin may be used as a normal / reverse phase column material, and may not adsorb polar substances, but may adsorb / attach non-polar substances and hydrophobic substances. Preferably, the resin may be a reverse phase column material, and may be a polymer-based material such as polystyrene.

[0124] More preferably, the resin may include at least one selected from the group consisting of HP-20, HLB, C18, tC18, C8, tC2, Silica, Florisil®, Accell Plus CM, Accell Plus QMA, Alumina A, Alumina B, Alumina N, Amino Propyl (NH2), Cyano Propyl (CN), Diol. For example, in particular, the resin may include at least one selected from the group consisting of HP-20, HLB, C18, tC18, C8, tC2.

[0125] The resin is added to the extract obtained by extracting the natural material with alcohol of the first concentration and stirred, so that the non-polar natural material dissolved in the extract can be adsorbed to the resin.

[0126] In contrast, non-polar natural substances can be adsorbed to the resin in the column using a column extraction method rather than adding resin to the extract and stirring it.

[0127] The column may be, for example, a reversed phase column, whereby non-polar natural substances may be adsorbed onto the stationary phase, that is, a resin.

[0128] In the case of the above column extraction method, the extract can be passed through in a cyclic manner, or alternatively, the extract can be repeatedly passed through a cartridge column.

[0129] Afterwards, the resin on which the nonpolar natural substance has been adsorbed is washed with alcohol to remove impurities.

[0130] In one embodiment of the present invention, the step of washing with alcohol having a second concentration lower than the first concentration may be included.

[0131] In one embodiment of the present invention, the second concentration of alcohol may be alcohol having a concentration of less than 50%.

[0132] This means that when washing the resin on which the nonpolar natural substance is adsorbed with alcohol having a higher concentration than the second concentration of alcohol, the nonpolar natural substance adsorbed on the resin may be eluted and lost.

[0133] The second concentration of alcohol has a concentration of less than 50% and may be alcohol of a lower concentration than the first concentration.

[0134] For example, the second concentration of alcohol may be alcohol having a concentration of 30% or less, a concentration of 25% or less, or a concentration of 20% or less.

[0135] By mixing alcohol with the resin on which the washed nonpolar natural substance has been adsorbed, the nonpolar natural substance can be eluted.

[0136] In one embodiment of the present invention, the step of dissolving the non-polar natural substance by mixing alcohol of a third concentration higher than the first concentration may be included.

[0137] In one embodiment of the present invention, the third concentration of alcohol may be alcohol having a concentration of 50% or more.

[0138] For example, the third concentration of alcohol may be alcohol having a concentration of 30% or more, a concentration exceeding 30%, a concentration of 35% or more, a concentration of 40% or more, or a concentration of 45% or more.

[0139] The resin, which has been adsorbed with non-polar natural substances from which impurities have been sufficiently removed through the above washing process, is mixed with alcohol of the third concentration and stirred, so that the non-polar natural substances can be eluted into the alcohol of the third concentration.

[0140] The non-polar natural substance can be completely separated from the resin without loss of the non-polar natural substance by using alcohol of the third concentration higher than the first concentration.

[0141] In one embodiment of the present invention, the steps of adding resin to the alcohol solution from which the nonpolar natural substance has been eluted to re-adsorb the nonpolar natural substance and the steps of mixing alcohol with the resin to which the nonpolar natural substance has been adsorbed to re-elute the nonpolar natural substance may be repeated at least once. By repeating these steps, the purity of the target substance can be increased and extracted.

[0142] The non-polar natural substance is separated from the resin and dissolved in alcohol of a third concentration, thereby obtaining alcohol in which the non-polar natural substance is dissolved.

[0143] In one embodiment of the present invention, the step of mixing a lipophilic solubilizer after removing the resin may be included.

[0144] Embodiments of the present invention relate to a technology for extracting and concentrating / separating selective components (nonpolar / polar, poorly soluble / water-soluble, HLB) or substances among natural substances by inducing physical emulsification to rapidly dissolve and solubilize them, and to a method for extracting nonpolar natural substances using dissolution-emulsion extraction (DEE). By utilizing this, a substance having one of the properties can be selectively extracted or, conversely, removed from a mixture in which nonpolar / polar substances, poorly soluble / water-soluble substances, or substances having high HLB / low HLB are mixed.

[0145] The above non-polar natural substance is suitably referred to as a dissolution-emulsion extraction method (DEE), and according to the dissolution-emulsion extraction method, non-polar natural substances in natural raw materials are extracted, a phase-separation composition is mixed to dissolve and emulsify, and then the non-polar natural substance is extracted through phase separation.

[0146] By mixing alcohol with a resin on which a nonpolar natural substance has been adsorbed to elute the nonpolar natural substance, the nonpolar natural substance can be extracted by encapsulating the nonpolar natural substance with the lipophilic solubilizer to increase the purity of the nonpolar natural substance.

[0147] In contrast, after dissolving the nonpolar natural substance, the step of mixing it with the resin again to adsorb the nonpolar substance to the resin again and dissolving it can be repeated, thereby increasing the purity of the target substance and allowing it to be extracted.

[0148] In addition, in contrast to this, after mixing the lipophilic solubilizer with the eluted nonpolar natural substance, alcohol is mixed with the resin on which the nonpolar natural substance is adsorbed to elute the nonpolar natural substance, and then the nonpolar natural substance is encapsulated with the lipophilic solubilizer to increase the purity of the nonpolar natural substance and can be extracted. In one embodiment of the present invention, the lipophilic solubilizer is MCT OIL, BRIJ 010-SS-(RB), Capryol 90, Capryol PGMC, Gantrez ES-435, Gantrez S-97 BF, Gelucire 43 / 01, Gelucire 44 / 14, Gelucire 50 / 13, Gelucire 48 / 16, Labrafac CC, Labrafac Lipophile WL1349, Labrafac PG, Medium-chain triglycerides, Labrafil M 1944 CS, Labrafil M 2125 CS, Labrafil M 2130 CS, Lauroglycol 90, Lauroglycol FCC, Monosteol, Peceol, Pharmasolve, Plurol Oleique CC497, Span 20-LQ-(SG), Span 60-PA-(SG), upper Refined Oleic Acid-LQ-(JP), Super Refined Tween 80A-LQ-(MH), Surfadone LP-300, Transcutol HP, Transcutol P, TWEEN 60-SS-(SG), TWEEN 80 HP-LQ-(MH), TWEEN 80-LQ-(SG), Vitamin E TPGS, Miglyol 812, captax 355, Labrasol, Crodex A-PA-(RB), Geleol, Gelot 64, Suppocire AS2X, Suppocire BML, Synperonic PE / F 127, Synperonic It may include at least one selected from the group consisting of PE / L 44 and Tefose 63.

[0149] When the alcohol in the alcohol solvent in which the above nonpolar natural substance is dissolved is removed by concentrating under reduced pressure, a layer separation occurs into an oil layer and an aqueous layer in which the nonpolar natural substance is concentrated.

[0150] In one embodiment of the present invention, the method may include a step of obtaining a supernatant after removing alcohol.

[0151] Thereafter, the supernatant can be easily separated, and an oil layer containing the nonpolar natural substance and the lipophilic solubilizer can be easily obtained.

[0152] For example, the non-polar natural substance extracted from the plant may contain as an active ingredient at least one selected from the group consisting of Brevilin A, Arnicolide D, Arnicolide C, and Microhelenin C.

[0153] Alternatively, the steps of adding a resin to the obtained supernatant to re-adsorb a non-polar natural substance, mixing alcohol into the resin to re-elute the non-polar natural substance, removing the resin, mixing a lipophilic solubilizer, and removing the alcohol to obtain a supernatant may be repeated at least once to further improve the purity of the target substance.

[0154] In another embodiment of the present invention, a method for extracting a toxic substance may include a step of extracting a natural raw material with alcohol of a first concentration, and then mixing an organic solvent with the residue from which the extract is removed to extract the residual extract.

[0155] The organic solvent may be a non-polar solvent such as alcohol, methanol, acetone, hexane, methane dichloride, ethyl acetate, etc.

[0156] The alcohol may be an alcohol having a higher concentration than the first concentration, and the alcohol may be selected from the group consisting of methanol, ethanol, propanol, butanol, benzyl alcohol, isopropyl alcohol, cetearyl alcohol, cetyl alcohol, stearyl alcohol, lauryl alcohol, myristyl alcohol, and behenyl alcohol, and methanol may be suitably used.

[0157] In one embodiment of the present invention, the alcohol used to extract the toxic substance may be an alcohol having a concentration exceeding 30%, and preferably, the alcohol may be extracted with a concentration exceeding 50%.

[0158] When the desired non-polar natural substance is extracted from the above natural raw material with alcohol having a concentration of 30% or less, and the remaining natural raw material (waste) is extracted with alcohol having a concentration of more than 30%, other substances such as toxic components may be extracted after the effective ingredient is extracted. The above toxic substances can be utilized as ingredients for pesticides, insecticides, etc.

[0159] The above toxic substances may be phenols, terpenoids or flavonoids.

[0160] For example, phenolic compounds may be benzoic acid, sinapic acid, syringic acid or tetracosanoic acid, and may have side effects such as causing dermatitis or burns in humans due to the protein-decomposing effect of phenolic derivatives, and have toxicity with antibacterial and insecticidal activities.

[0161] In addition, terpenoid compounds can be monoterpene (C10), sesquiterpene (C15), diterpene (C20), sesterterpene (C25), triterpene (C30), and tetraterpene (C40), and terpenoid compounds have toxicity with antibacterial and insecticidal activities.

[0162] Additionally, the flavonoid compounds may be Nicotiflorin, Luteolin, Apigenin, Hispidulin or Quercetin-3-methyl ether, and the flavonoid compounds have antibacterial and insecticidal toxicity, which can cause respiratory irritation and acute toxicity to insects.

[0163] In a method for preventing or treating anti-inflammatory and autoimmune diseases according to another embodiment of the present invention, the content of brevilin A in the composition for preventing or treating anti-inflammatory and autoimmune diseases, which comprises an extract extracted from a natural raw material with alcohol as an active ingredient, is administered in a daily dose of 0.5 to 12 mg.

[0164] In one embodiment of the present invention, the composition may be administered at least once a week. Furthermore, the composition may be administered orally or transdermally, and may be administered at least once a week for at least one month.

[0165] In the present invention, it was confirmed that the composition containing 0.5 to 12 mg of brevilin A has an anti-inflammatory and autoimmune disease treatment effect when administered percutaneously or orally once a day.

[0166] Preferably, the composition containing 0.5 mg of brevilin A is administered once a day, at least 5 times a week, for at least 6 months, or 1 mg is administered once a day, at least 5 times a week, for at least 3 months, or 6 mg is administered once a day, at least 4 times a week, for at least 1 month, or 12 mg is administered once a day, at least 2 times a week, for at least 3 months.

[0167] In addition, the term "prevention" of the present invention means all acts of inhibiting or delaying the occurrence, spread, and recurrence of the disease by administering the composition for preventing or treating anti-inflammatory and autoimmune diseases of the present invention, and "treatment" means all acts of improving or beneficially changing the symptoms of the disease by administering the composition for preventing or treating anti-inflammatory and autoimmune diseases of the present invention.

[0168] In the present invention, the autoimmune disease may be characterized by including at least one selected from the group consisting of hair loss, alopecia areata, rheumatoid arthritis, psoriasis, atopy, eczema, seborrheic dermatitis, scleroderma, cutaneous immune hypersensitivity, multiple sclerosis, lupus, Behcet's disease, ankylosing spondylitis, Sjogren's syndrome, Crohn's disease, and inflammatory bowel disease.

[0169] In the method for preventing or treating anti-inflammatory and autoimmune diseases according to the present invention, the pharmaceutical composition for preventing or treating anti-inflammatory and autoimmune diseases can be manufactured in the form of products such as hair tonic, hair conditioner, hair lotion, hair nutrition lotion, hair shampoo, hair rinse, hair treatment, hair cream, hair nutrition cream, hair moisture cream, hair massage cream, hair wax, hair aerosol, hair pack, hair nutrition pack, hair soap, hair cleansing foam, hair oil, hair drying agent, hair preservation treatment agent, hair dye, hair waving agent, hair bleaching agent, hair gel, hair glaze, hair dressing agent, hair lacquer, hair moisturizer, hair mousse, hair spray, etc., and as external skin agent, it can be manufactured in the form of cream, gel, patch, spray, ointment, paste, lotion, liniment, paste, cataplasma, etc., and can be appropriately utilized in the form of liquid, solid, or gas.

[0170] In addition, it can be provided as a pharmaceutical composition for preventing or treating anti-inflammatory and autoimmune diseases for preventing or treating diseases selected from the group consisting of hair loss, alopecia areata, rheumatoid arthritis, psoriasis, atopy, eczema, seborrheic dermatitis, scleroderma, cutaneous immune hypersensitivity, multiple sclerosis, lupus, Behcet's disease, ankylosing spondylitis, Sjogren's syndrome, Crohn's disease, and inflammatory bowel disease, which can be administered orally, parenterally, or transdermally, and can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods.

[0171]

[0172] Below, for more specific explanation, examples will be given to provide a detailed explanation.

[0173]

[0174] Example

[0175] Manufacturing example

[0176] The natural raw material is Brachycera japonica. Domestic Brachycera japonica (Abul vinegar, Yeongcheon) was purchased from the Goesan Medicinal Herb Organic Agricultural Products Cooperative (https: / natural-herb.co.kr) and washed under the conditions in Table 1 below. The washed Brachycera japonica was precipitated in 10 times its weight of 25% ethanol for 48 hours and then alcohol extracted to obtain a first extract. 10 g of HP-20 resin was mixed with 3,000 g of the first extract and stirred for 4 hours to encapsulate the indicator component in the resin. Afterwards, it was washed with 20% ethanol and eluted with 90% ethanol to obtain a second extract. MCT oil was mixed as a lipophilic solubilizer in the second extract at a ratio of 20 times that of the indicator substance (Brevilin-A), and then concentrated under reduced pressure to reduce the alcohol composition and induce salting out of the indicator component. Next, after leaving the mixture in a separating funnel for 60 minutes, the phase-separated supernatant was separated. The content of brevilin A, an indicator substance during extraction, was measured and shown in Table 2.

[0177]

[0178] Loss of indicator substances according to raw material washing method

[0179] Content of the midge grass (g) Content of purified water (g) Washing time (min) Number of washes Manufacturing example 1100500301 Manufacturing example 21001,000101 Manufacturing example 31001,000301 Manufacturing example 41001,000301 Manufacturing example 51001,0003601 Manufacturing example 61001,000302 Manufacturing example 71001,000303 Manufacturing example 81002,000301 Manufacturing example 9100Running water301 Manufacturing example 10100---Manufacturing example 11100Air wash301

[0180] BA content in raw material (mg) BA content after washing (mg) BA loss (%) Extract status Manufacturing example 1312.12254.90 18.33 Good Manufacturing example 2312.12271.01 13.17 Good Manufacturing example 3312.12257.71 17.43 Good Manufacturing example 4312.12307.64 1.44 Good Manufacturing example 5312.12257.71 17.43 Good Manufacturing example 6312.12227.70 27.05 Good Manufacturing example 7312.12207.60 33.49 Good Manufacturing example 8312.12250.85 19.63 Good Manufacturing example 9312.12251.7819.33 Good manufacturing example 10312.12310.700.45 Foreign matter and contamination manufacturing example 11312.12280.7010.07 Foreign matter and contamination

[0181] Manufacturing Example 10 was a case where the raw material was extracted as is without a pretreatment process, and although there was almost no loss of the indicator substance, it was confirmed that the extract including the indicator substance contained foreign substances and was contaminated. In the case of Manufacturing Example 11, where the pretreatment to remove foreign substances was performed with an air wash instead of purified water, not only was the loss of the indicator substance more than 10%, but it was confirmed that the extract contained foreign substances and was contaminated. That is, in the case of Manufacturing Examples 1 to 8, which were washed by soaking in purified water for a certain period of time, and Manufacturing Example 9, which was washed with running water, it was confirmed that no foreign substances or contamination occurred in the extract. However, in the case of Manufacturing Examples 6 and 7, which were washed more than twice even when soaking in purified water and washing, it was confirmed that the loss of the indicator substance was too great, at more than 25%. Therefore, washing once regardless of the content of purified water and washing time could minimize the loss of the indicator substance. However, preferably, washing with purified water 5 to 10 times the amount of raw material for less than 6 hours can reduce the loss to less than 20%.

[0182]

[0183] Extraction amount of indicator substance according to extraction solvent concentration

[0184] Experimental example

[0185] According to the above Manufacturing Example 1, 3,000 g of the washed raw material of the middle head grass was precipitated in ethanol with a concentration of 10 times the weight in Table 3 below for 48 hours, and then alcohol extracted to obtain a first extract. In Examples 1 to 9 and 16 to 21, resin was mixed with 3,000 g of the first extract under the conditions of Table 4 below and stirred to encapsulate the index component in the resin. In contrast, in Examples 10 to 15, 1,000 g (Examples 10 to 12) and 50 g (Examples 13 and 14) of the first extract were extracted using a column extraction method and encapsulated in the resin in the column.

[0186] Afterwards, the resin containing the indicator substance was washed with 20% ethanol and eluted with 90% ethanol to obtain a second extract. MCT oil was mixed as a lipophilic solubilizer in the second extract at a ratio of 20 times that of the indicator substance (Brevilin-A), and then concentrated under reduced pressure to reduce the alcohol composition and induce salting out of the indicator component. Next, after leaving it in a separatory funnel for 60 minutes, the phase-separated supernatant was separated. The contents of the indicator substances, Brevilin A (BA) and Arnicolide D (AD), were measured during extraction and are shown in Table 5.

[0187] Ethanol concentration (%) Extracted BA content (mg / g) Extracted AD content (mg / g) Original content of the plant (g) Total BA in the primary extract (mg) Total AD in the primary extract (mg) Experimental example 1250.1260.07930,0003,7802,370 Experimental example 2300.1230.07730,0003,6902,310 Experimental example 3500.1890.07920,0003,7801,580 Experimental example 4750.1580.0817,0002,6861,360 Experimental example 5900.2520.08515,0003,7801,275 Experimental example 6950.2570.08415,0003,8551,260

[0188] As shown in Table 3, it was confirmed that the extraction amounts of brevilin A and arnicolide D gradually increased when extracted with high concentrations of ethanol. However, as described below, even if the extraction amount of indicator substances increases with increasing alcohol concentration, it is expected that substances other than indicator substances are also extracted in large quantities.

[0189] The amount of indicator substances included in the extract according to the resin adsorption conditions

[0190] 1st extract resin type and contentStirring time (hr)Experimental example 1Experimental example 1HP-20, 10g4Experimental example 2HP-20, 10g4Experimental example 3Experimental example 3HP-20, 10g4Experimental example 4HP-20, 10g4Experimental example 5Experimental example 5HP-20, 10g4Experimental example 6HP-20, 10g4Experimental example 7Experimental example 1HP-20, 10g1Experimental example 8Experimental example 1HP-20, 10g6Experimental example 9Experimental example 1HP-20, 10g12Experimental example 10Experimental example 1SPE(HP-20), 6g1(circulation)Experimental example 11Experimental example 1SPE(HP-20), 6g3(circulation)Experimental example 12Experimental example 1SPE(C18), 6g1 (circulation)Experimental example 1Experimental example 1 Within HLB_SPE_cartridge1, repeated 5 timesExperimental example 14Experimental example 1 Within C18_SPE_cartridge1, repeated 5 timesExperimental example 1 SPE (HP-20), within 6g1, repeated 5 timesExperimental example 1 MCT OIL 1L + HP-20, 10g4Experimental example 17Experimental example 1 MCT OIL 2L + HP-20, 10g4Experimental example 18Experimental example 1 MCT OIL 3L + HP-20, 10g4Experimental example 19Experimental example 3 HP-20, 15g4Experimental example 20Experimental example 3 HP-20, 20g4Experimental example 21Experimental example 3 HP-20, 50g4

[0191] Examples 1 to 6 were performed by mixing 10 g of HP-20 as a resin in the first extract according to Experimental Examples 1 to 6 and stirring for 4 hours. Examples 7 to 9 were performed in the same manner as Example 1 but with the stirring time changed. Examples 10 to 12 were performed by column extraction using a solid phase column extraction method using the first extract according to Experimental Example 1 and with different types of resin or circulation times. Examples 13 to 15 were performed by repeated extraction 5 times using a cartridge column. Examples 16 to 18 were performed by mixing MCT oil and resin and stirring, and Examples 19 to 21 were performed in the same manner as Example 3 using the extract according to Experimental Example 3 but with different amounts of resin.

[0192] BA content of residual extract after adsorption (mg)AD content of residual extract after adsorption (mg)Incorporated BA content (mg)BA inclusion amount (%)Incorporated AD content (mg)AD inclusion amount (%)Example 1540570324085.7180075.9Example 2690900300081.3141061.0Example 31560940222058.764040.5Example 426521003341.335726.3Example 53300108048012.719515.3Example 63405118545011.7756.0Example 79301080285075.4129054.4Example 8570540321084.9183077.2 Example 9570510321084.9186078.5 Example 10690870309081.7150063.3 Example 11660900312082.5147062.0 Example 12420360336088.9201084.8 Example 13600780318084.1159067.1 Example 14720570306081.0180075.9 Example 15270330351092.9204086.1 Example 168701170291077.0120050.6 Example 1710801140270071.4123051.9 Example 1812301230255067.5114048.1 Example 191440900234061.968043.0 Example 20960820282074.676048.1 Example 21780540300079.4104065.8

[0193] Referring to Table 5, the content of the indicator substance in the residual extract after stirring or column extraction with the resin was measured, and accordingly, the content and inclusion amount of the indicator substance adsorbed on the resin could be inferred. In the case of Examples 3 to 6, when the resin was added to the extract extracted with alcohol having a concentration exceeding 30% to include the indicator substance, it was found that the content of BA and AD in the extract was high when extracted with high-concentration alcohol, but inclusion did not occur. This is expected to be because, when extracting with high-concentration alcohol, in addition to the indicator substance targeted in the present invention, many toxic substances or other substances are extracted, and these tend to be included first in the resin, and it was found that the inclusion amounts of BA and AD were reduced.

[0194] In contrast, when the content and inclusion amount of indicator substances BA and AD adsorbed to the resin were measured by stirring the resin in the first extract extracted with less than 30% alcohol or by extracting using a column method, it was found that the inclusion amount was significantly increased compared to the inclusion amount according to Examples 3 to 6 extracted with more than 30% alcohol.

[0195] Here, referring to Examples 1 and 2, 25% alcohol and 30% alcohol do not show a large difference in their inclusion amounts, and referring to Examples 1 and Examples 7 to 9, when the stirring time is 4 hours or more, the inclusion amount is formed at almost the same level, and referring to Examples 3 and Examples 19 to 21, when the resin is mixed into the first extract and stirred, the inclusion amount tends to increase as the resin content increases, but it can be seen that the inclusion amount of alcohol with a concentration exceeding 30% is still low, and referring to Examples 1 and Examples 10 to 15, there is no large difference in the inclusion amount depending on the method of mixing the resin into the extract or column extraction of the extract.

[0196] Referring to Example 1 and Examples 16 to 18, it can be seen that when MCT oil is mixed with the resin in the extract, the inclusion amount decreases compared to Example 1 where MCT oil is not mixed, and the inclusion amount tends to decrease further as the content of MCT oil increases, indicating that MCT oil interferes with the inclusion of the indicator substance.

[0197] Therefore, rather than mixing MCT oil, a lipophilic solubilizer, with the resin, it can be applied separately from the resin in a separate step, or the steps of mixing with the resin, then eluting with alcohol, dissolving and emulsifying the eluted target substance with the lipophilic solubilizer, and inducing phase separation are repeatedly applied in a circular manner to improve the purity of the target substance extraction. Here, the resins can remove unnecessary water-soluble components in the extract, and the lipophilic solubilizer can remove unnecessary fat-soluble components, thereby improving the purity of the extract.

[0198]

[0199] Elution rate of indicator substances according to washing and elution conditions

[0200] After extraction according to the conditions in Table 6 below, the content of the incorporated indicator substance was measured.

[0201] Example 221st extract Experimental example 1 Resin type and content HP-20, 12g Stirring time (hr) 4 Extracted BA content (mg / g) 0.126 Extracted AD content (mg / g) 0.079 Content of the original plant of the Chinese yam (g) 3,000 Total BA in the 1st extract (mg) 378 Total AD in the 1st extract (mg) 237 Residual BA content in the extract after adsorption (mg) 30 Residual AD content in the extract after adsorption (mg) 66 Incorporated BA content (mg) 348.0 BA inclusion amount (%) 92.1 Incorporated AD content (mg) 171.0 AD inclusion amount (%) 72.2

[0202] The resin having the indicator substance adsorbed thereon, manufactured according to Example 22 of Table 6 above, was washed according to the conditions of Table 7 below, and the content of the indicator substance eluted into the washing solution after washing was measured and described.

[0203] Washing solution Dissolved BA content (mg) BA dissolution rate (%) Dissolved AD content (mg) AD dissolution rate (%) Example 23 Water, 1L 2.0 0.6 0.9 0.5 Example 24 Water, 1L 1.0 0.3 1.0 0.6 Example 25 Water, 1L 0.5 0.1 0.2 0.1 Example 26 10% ethanol, 1L 2.0 0.6 0.9 0.5 Example 27 15% ethanol, 1L 7.0 2.0 14.0 8.2 Example 28 20% ethanol, 1L 9.0 2.6 19.0 11.1 Example 29 25% ethanol, 1L 195.5 29.0 17.0

[0204] The resin mixture washed according to the above Example 22 was adjusted to have an ethanol concentration according to Table 8 below as an eluent, and the content of the eluted indicator substance was measured accordingly. Examples 31, 31-1, and 31-2 indicate that the indicator substance was eluted three times, and the content thereof was measured and described.

[0205] Dissolved BA content (mg) BA dissolution rate (%) Dissolved AD content (mg) AD dissolution rate (%) Example 30 80% ethanol 29 18 3.6 126.0 7 3.7 Example 31 90% ethanol 30 3.0 8 7.1 135.0 7 8.9 Example 31 - 190% ethanol 27 7.8 10.2 6.0 Example 31 - 290% ethanol 30.9 1.2 0.7

[0206] Referring to Table 8, it was found that the dissolution rates of BA and AD increased as higher concentrations of alcohol were used as the dissolution solution.

[0207] Indicator material process yield according to elution conditions

[0208] Total amount of BA in the first extract (mg) Total amount of BA eluted (mg) BA eluted (%) After the first extraction, BA yield (%) Example 3037829183.677.0 Example 3137833395.788.1

[0209] Total amount of AD in the first extract (mg) Total amount of AD eluted (mg) AD eluted rate (%) After the first extraction, AD yield (%) Example 3023729173.753.2 Example 31237146.485.661.8

[0210] Referring to Tables 9 and 10, the elution rates of BA and AD, which are indicator substances according to Examples 30 and 31, can be known, and accordingly, the yield of each indicator substance can be known compared to the indicator substance contained in the first extract. Therefore, it can be seen that as the concentration of the eluate increases, the elution rate of the indicator substance increases throughout the process, thereby increasing the process yield.

[0211]

[0212] Usage examples

[0213] Effects according to dosage and frequency of administration

[0214] According to the present invention, a product was provided that administered BA in various dosages, and a survey was conducted on a total of 113 users to investigate their respective dosage regimens and their satisfaction with the same.

[0215] User Dosage Frequency BA Dosage Dosage Period 1 Week 1 time 6mg 3 months 22 months 345 63mg 1 month 789 10 weeks 2 times 12mg 3 months 1112 1314 156mg 2 months 1617 1819 20211 months 222 324 2526 weeks 3 times 12mg 3 months 276mg 2 months 2829 30311 months 323 334 3536 3738 3940 weeks 4 times or more 12mg 3 months 412 months 424 36mg 1 month 4445 4647 4849 3mg 505 152 weeks 5 times More than 6mg6 months535 months542mg3 months5556575859601mg616263646566676869700.5mg3 months717273747576777879801 month818785836mg3 months108899091939798868292882 months84949596100107991041051 month102103106101OtherNo responseNo response109No responseNo response110No responseNo response111No responseNo response112No responseNo response113No responseNo response

[0216] The administration method of a total of 113 users was investigated according to the dosage, number of times, and period of administration in Table 11 above, and a sensory evaluation of the effect was conducted through a questionnaire on the presence or absence of hair loss improvement effect as shown in Table 12 below.

[0217] If there was a hair loss care effect, a hair loss reduction effect, a hair growth effect, increased hair strength, increased hair thickness, increased hair volume, or if the product according to the composition of the present invention was repurchased, it was marked as O, and 1 point was given for each, and the scores were evaluated accordingly.

[0218]

[0219] According to the method for preventing or treating anti-inflammatory and autoimmune diseases of the present invention, the content of brevilin A in the composition for preventing or treating anti-inflammatory and autoimmune diseases, which contains as an effective ingredient an extract extracted with alcohol from a natural raw material, is administered in a daily dose of 0.5 to 12 mg.

[0220] In one embodiment of the present invention, the composition may be administered at least once a week. Furthermore, the composition may be administered orally or transdermally, and may be administered at least once a week for at least one month.

[0221] In the present invention, it was confirmed that the composition containing 0.5 to 12 mg of brevilin A has an anti-inflammatory and autoimmune disease treatment effect when administered percutaneously or orally once a day.

[0222] Preferably, the composition containing 0.5 mg of brevilin A is administered once a day, at least 5 times a week, for at least 6 months, or 1 mg is administered once a day, at least 5 times a week, for at least 3 months, or 6 mg is administered once a day, at least 4 times a week, for at least 1 month, or 12 mg is administered once a day, at least 2 times a week, for at least 3 months.

[0223] That is, the number of administrations and the administration period can be adjusted depending on the content of Brevilin A.

[0224]

[0225] Human Application Test Results 1_Hair Loss Symptom Relief Efficacy Test

[0226] The extract concentrate according to Example 1 was used as a sample. Before use, the sample was shaken well, and an appropriate amount (0.5 to 1 mL) was evenly applied to the scalp once a day, and then massaged with fingers until sufficiently absorbed. The sample containing 12.0 mg / mL of the active ingredient, brevilin A, was used as the sample.

[0227] A double-blind method was used, and the test and control samples were distributed in the same container so that neither the research subjects nor the investigator knew the contents of the samples. The sample code used for classification was sealed and not exposed, and a classification code (A / B) was randomly marked on it.

[0228] The evaluation method was based on the "Guidelines for Human Application Tests of Cosmetics that Help Alleviate Hair Loss Symptoms" (published in May 2017) of the Ministry of Food and Drug Safety, and included a survey of research subjects and a visual evaluation by researchers through photography, and the validity was verified through a phototrichogram.

[0229] In order to analyze and compare hair characteristics, the number and type of hair per unit area must be quantitatively measured for a sufficient number of hair groups to obtain statistically meaningful results.

[0230] For this purpose, phototrichogram, which takes a photo of a certain area of ​​hair cut short and compares it with another photo of the same area after a certain period of time, can quantitatively measure various indices of hair noninvasively, has a small data error, and is relatively easy to analyze. In this study, a hair and scalp magnifying glass, Folliscope® (Lead M, Korea), was used to apply this phototrichogram method. Folliscope uses a high-resolution color video microscopic camera and a computer equipped with separate Folliscope software to analyze the main characteristics of hair, such as density, thickness, growth rate, and spacing between hairs.

[0231] After cutting the hair in the area to be evaluated, a small dot tattoo with a diameter of 1 mm was made or certain coordinates were recorded, and a phototrichogram was performed each time with the dot tattoo or certain coordinates as the center.

[0232] Average hair count (N / cm) 2 ) The change pattern and improvement rate were measured and shown in Table 13 and Figures 1 and 2 below.

[0233] Category Week 0 Week 8 Week 16 Week 24 Test group 43.44 ± 9.33 44.59 ± 9.06 45.29 ± 9.19 46.68 ± 9.14 Control group 42.50 ± 8.00 42.38 ± 8.16 42.19 ± 8.10 42.69 ± 8.54

[0234] The test site of the test group using the sample according to the example for a total of 24 weeks was the primary efficacy evaluation index, hair count (N / cm 2 ) increased to a statistically significant level (p<0.05) compared to before the test 8 weeks after the test, but no statistically significant change (p<0.05) was confirmed in the control area using the control sample.

[0235] In addition, as a result of the comparison and contrast of hair count between groups, the interaction analysis comparing the change pattern of hair count by week between groups confirmed that there was a statistically significant difference (p<0.05) in the change pattern of hair count by week between the test group and the control group, and in the comparison of the change in hair count, it was confirmed that there was a statistically significant difference (p<0.05) in the change in hair count between the test group and the control group after 8 weeks. Therefore, it is judged that the test sample used in this study has the efficacy to help alleviate hair loss symptoms.

[0236] Referring to Figure 3, the hair count (N / cm 2 ) In order to confirm the difference between groups in the measurement results, the hair count by week was verified through repeated measures analysis of variance. As a result of the analysis, the hair counts of the test group and the control group did not show a statistically significant difference (p<0.05) between the groups. However, in the interaction analysis comparing the change in hair count by week between the groups, it was confirmed that the change in hair count by week between the test group and the control group had a statistically significant difference (p<0.05).

[0237] The degree of hair improvement was visually evaluated by the tester and the scores are shown in Table 14 and Figure 4.

[0238] Classification Week 8 Week 16 Week 24 Test group - 0.13 ± 0.57 0.65 ± 0.54 0.81 ± 0.59 Control group - 0.13 ± 0.28 - 0.02 ± 0.56 0.06 ± 0.61

[0239] (3: Very good, 2: Better, 1: Slightly better, 0: No change, -1: Slightly worse, -2: Worse, -3: Very bad)

[0240] When 6 to 12 mg was administered at a time, the number of hairs increased from the 8th week, and a statistically significant difference was confirmed from the 16th week.

[0241]

[0242] Human application test results 2_Test of changes in the hair cycle

[0243] The extract concentrate according to Example 1 was used as a sample, and after shaking the sample well before use, an appropriate amount (0.5 to 1 mL) was evenly applied to the scalp once a day, and then massaged with fingers until sufficiently absorbed. Rather than applying to the entire scalp in one day, two areas were divided into two days and applied to half of the area alternately each day. The active ingredient brevilin A was used as a sample containing 12.0 mg / mL of the sample.

[0244] The remaining test conditions and evaluation methods are the same as those in the human application test results 1_hair loss symptom relief efficacy test.

[0245] Average hair count (N / cm) 2 ) The change pattern and improvement rate were measured and shown in Table 14 and Figures 5 and 6 below.

[0246] Category Week 0 Week 8 Week 16 Week 24 Test group 47.18±9.05 48.03±9.15 48.38±9.12 49.47±9.21 Control group 45.00±7.76 45.09±7.56 44.78±7.30 43.94±7.54

[0247] In the test area of ​​the test group using the sample according to the example for a total of 24 weeks, the total hair count increased at a statistically significant level (p<0.05) compared to before the test after 8 weeks of the test, and in the control area using the control sample, the total hair count decreased at a statistically significant level (p<0.05) compared to before the test after 16 weeks of the test. In addition, as a result of the comparison and contrast of the total hair count between groups, the interaction analysis comparing the change in the total hair count by week between groups confirmed that the change in the total hair count by week between the test group and the control group had a statistically significant level (p<0.05). Therefore, it is judged that the test sample used in this test has a positive effect in helping the hair cycle change, such as increasing the total hair count, the number of normal hairs, and the number of growth hairs.

[0248] Referring to Figures 7 to 10, in order to confirm the difference between groups in the results of measuring the number of anagen hairs, the number of anagen hairs by week was verified through repeated measures analysis of variance. As a result of the analysis, the number of anagen hairs in the test group and the control group showed a statistically significant difference (p<0.05) between the groups. In the interaction analysis comparing the pattern of change in the number of anagen hairs by week between the groups, it was confirmed that the pattern of change in the number of anagen hairs by week between the test group and the control group had a statistically significant difference (p<0.05) between the groups.

[0249] When 6 to 12 mg was administered at a time, hair transitioned to the growth phase from the 8th week, and a statistically significant difference was observed from the 16th week. In particular, it was found that the amount of hair loss was significantly reduced in the 8th week.

[0250] Although exemplary embodiments of the present invention have been described above, the present invention is not limited thereto, and those skilled in the art will understand that various changes and modifications are possible within the scope and spirit of the claims set forth below.

Claims

1. A method for preventing or treating anti-inflammatory and autoimmune diseases, comprising a composition for treating anti-inflammatory and autoimmune diseases, comprising an extract extracted from natural raw materials with alcohol as an active ingredient, and a composition containing 0.5 to 25 mg of brevilin A as a daily dosage.

2. A step of extracting the natural raw material with alcohol of the first concentration; and A method for extracting non-polar natural substances, comprising the step of adding resin to an extract solution to adsorb non-polar natural substances.

3. In paragraph 2, A method for extracting a non-polar natural substance, characterized in that the above natural material raw material comprises at least one selected from the group consisting of Centipeda minima, Litsea glutinous, plants of the genus Arnica, and plants of the genus Helenium.

4. In paragraph 2, It includes a step of washing the natural raw material with purified water before extracting the natural raw material; A method for extracting nonpolar natural substances, comprising mixing the above natural raw material and the above purified water in a weight ratio of 1:5 to 20 and washing once for less than 6 hours.

5. In paragraph 2, A method for extracting a non-polar natural substance, wherein the alcohol comprises at least one selected from the group consisting of methanol, ethanol, propanol, butanol, benzyl alcohol, isopropyl alcohol, cetearyl alcohol, cetyl alcohol, stearyl alcohol, lauryl alcohol, myristyl alcohol, and behenyl alcohol.

6. In paragraph 2, The above resin is a method for extracting a non-polar natural substance, comprising at least one selected from the group consisting of HP-20, HLB, C18, tC18, C8, tC2, Silica, Florisil®, Accell Plus CM, Accell Plus QMA, Alumina A, Alumina B, Alumina N, Amino Propyl (NH2), Cyano Propyl (CN), and Diol.

7. In paragraph 2, A method for extracting a non-polar natural substance, characterized in that the non-polar natural substance comprises at least one selected from the group consisting of Brevilin A, Arnicolide D, Arnicolide C, and Microhelenin C as an active ingredient.

8. In paragraph 2, A method for extracting a non-polar natural substance, comprising: a step of washing with alcohol having a second concentration lower than the first concentration; 9. In paragraph 2, A method for extracting a non-polar natural substance, comprising: a step of mixing alcohol having a third concentration higher than the first concentration to elute the non-polar natural substance.

10. In paragraph 9, A step of re-adsorbing the non-polar natural substance by adding resin to the alcohol solution from which the non-polar natural substance has been eluted; and A method for extracting a non-polar natural substance, comprising the steps of mixing alcohol into a resin on which the non-polar natural substance is adsorbed and re-eluting the non-polar natural substance; and repeating the steps at least once.

11. In paragraph 2, A method for extracting a non-polar natural substance, comprising the step of mixing a lipophilic solubilizer after removing the above resin.

12. In paragraph 2, The lipophilic solubilizers are BRIJ 010-SS-(RB), Capryol 90, Capryol PGMC, Gantrez ES-435, Gantrez S-97 BF, Gelucire 43 / 01, Gelucire 44 / 14, Gelucire 50 / 13, Gelucire 48 / 16, Labrafac CC, Labrafac Lipophile WL1349, Labrafac PG, Medium-chain triglycerides, Labrafil M 1944 CS, Labrafil M 2125 CS, Labrafil M 2130 CS, Lauroglycol 90, Lauroglycol FCC, Monosteol, Peceol, Pharmasolve, Plurol Oleique CC497, Span 20-LQ-(SG), Span 60-PA-(SG), upper Refined Oleic Acid-LQ-(JP), Super A method for extracting a non-polar natural substance comprising at least one selected from the group consisting of Refined Tween 80A-LQ-(MH), Surfadone LP-300, Transcutol HP, Transcutol P, TWEEN 60-SS-(SG), TWEEN 80 HP-LQ-(MH), TWEEN 80-LQ-(SG), Vitamin E TPGS, Miglyol 812, and captax 355.

13. In paragraph 2, A method for extracting a non-polar natural substance, comprising the step of obtaining a supernatant after removing alcohol.

14. A method for extracting a toxic substance, comprising the steps of extracting a plant with alcohol of a first concentration, and then mixing alcohol of a higher concentration than the first concentration with the residue to extract the residue extract.

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