WO2013095027A1 - Anti-inflammation composition using vladinol f - Google Patents

Anti-inflammation composition using vladinol f Download PDF

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WO2013095027A1
WO2013095027A1 PCT/KR2012/011219 KR2012011219W WO2013095027A1 WO 2013095027 A1 WO2013095027 A1 WO 2013095027A1 KR 2012011219 W KR2012011219 W KR 2012011219W WO 2013095027 A1 WO2013095027 A1 WO 2013095027A1
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composition
inflammatory
vladinol
hexane
chronic
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PCT/KR2012/011219
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French (fr)
Korean (ko)
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윤원종
함영민
정용환
박수영
오대주
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(재)제주테크노파크
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/60—Moraceae (Mulberry family), e.g. breadfruit or fig
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • the present invention relates to anti-inflammatory compositions using Vladinol F.
  • Inflammation is the body's defense against external infections such as physical and chemical stimuli, bacteria, fungi, viruses, and allergens.
  • Inflammatory responses are part of the innate immune response, and as in other animals, the innate immune response in humans begins by recognizing and attacking non-self through patterns of cell surfaces where macrophages are specifically present in pathogens.
  • plasma accumulates at the site of inflammation, diluting the toxicity secreted by bacteria, increasing blood flow, and accompanied by symptoms such as erythema, pain, edema, and fever.
  • COX cyclooxygenase
  • NOS nitric oxide synthase
  • NOS nitric oxide
  • bacterial endotoxins such as eNOS (endothelial NOS), nNOS (neurotrophic NOS), and LPS (lipopolysaccharide), which are calcium or capmodulin dependent, or IL-1 ⁇ , TNF- ⁇ , IL
  • iNOS inducible NOS
  • IL-12 inflammatory cytokines
  • NO produced by eNOS or nNOS plays an important role in maintaining the homeostasis of the body by performing various physiological responses related to blood pressure control, neurotransmitter, learning, memory, etc., but NO produced by iNOS is associated with arthritis, sepsis and tissue It is known to be involved in various inflammatory diseases such as transplant rejection, autoimmune diseases and neuronal death (Moncade S. et al, Pharmacol. Rev., 1991, 43, 109; Nature Medicine, 2001, 7, 1138; Mu , MM, J. Endotoxic Res. 7, p341, 2001).
  • the COX enzyme has hydroperoxidase (HOX) activity with COX function and synthesizes PGG 2 and PGH 2 intermediates from arachidonic acid, and these compounds include PGE 2 , PGF 2 , PGD 2 , and prostacyclin. And thromboxin A 2 (thromboxane A2, TxA2).
  • HOX hydroperoxidase
  • PGF 2 a compound that is synthesized PGG 2 and PGH 2 intermediates from arachidonic acid
  • PGD 2 a hydroperoxidase
  • thromboxin A 2 thromboxane A2, TxA2
  • the function of PGH synthase is involved in pain and inflammatory responses through the synthesis of PGE 2 .
  • COX-1 is always expressed in most tissues, whereas COX-2 is rapidly induced by inflammatory cytokines and plays an important role in the inflammatory response.
  • NO, PGE 2 , Inhibitors such as inflammatory cytokines, iNOS inhibitors, and COX-2 inhibitors may be used as therapeutic agents for inflammatory diseases.
  • the present invention discloses regulainol F, which is an active substance isolated from a narrow leaf snail having NO production inhibitory activity, PGE 2 production inhibitory activity, and inflammatory cytokine production inhibitory activity.
  • the present invention isolates and identifies Rajin F, an active substance, from the narrow leaf zenith and extracts based on the anti-inflammatory activity of the narrow leaf zenith and extracts (see Korean Patent Application No. 2009-0113113). 2 and by confirming the inhibitory activity of the production of inflammatory cytokines (TNF- ⁇ , IL-1 and IL-6).
  • the active substance is a known substance (CAS Registry Number: 133318-48-6; Planta medica , 56 475-477, 1990; Biol. Pharm. Bull. 29 (6) 1271-274, 2006), the structural formula and IUPAC The name can be found in [Formula 1] below.
  • the anti-inflammatory composition of the present invention is completed based on the experimental results as described above, characterized in that it comprises Vladinol F as an active ingredient.
  • active ingredient means a component that can exhibit activity alone or in combination with a carrier which is not active in itself.
  • anti-inflammatory is meant to include the improvement, treatment, inhibition or delay of the development of inflammatory diseases as defined below.
  • the "inflammatory disease” may be defined as any condition specified by a local or systemic biological defense response against external physical and chemical stimuli or infection of an external infectious agent such as bacteria, fungi, viruses, and various allergens. .
  • This response activates various inflammatory mediators and enzymes associated with immune cells (eg iNOS, COX-2, etc.), secretion of inflammatory mediators (eg NO, TNF- ⁇ , IL-6, IL-1 ⁇ , PGE 2) . ), Accompanied by a series of complex physiological reactions such as fluid infiltration, cell migration, tissue destruction, etc., and are manifested externally by symptoms such as erythema, pain, edema, fever, deterioration or loss of certain functions of the body.
  • the inflammatory disease may be acute, chronic, ulcerative, allergic or necrotic, so as long as any disease is included in the definition of an inflammatory disease as above, whether it is acute, chronic, ulcerative, allergic or Irrespective of necrosis
  • the inflammatory diseases include asthma, allergic and non-allergic rhinitis, chronic and acute rhinitis, chronic and acute gastritis or enteritis, ulcerative gastritis, acute and chronic nephritis, acute and chronic hepatitis, chronic obstructive pulmonary disease, pulmonary fibrosis Irritable bowel syndrome, inflammatory pain, migraines, headache, back pain, fibromyalgia, fascia disease, viral infections (eg, type C infections), bacterial infections, fungal infections, burns, surgical or dental wounds, Prostaglandin E excess syndrome, atherosclerosis, gout, arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancre
  • the compound of Formula 1 of the present invention may be used in the form of a pharmaceutically acceptable salt, and the salt may be an acid addition salt formed by a pharmaceutically acceptable free acid.
  • Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes.
  • organic acids such as dioate, aromatic acids, aliphatic and aromatic sulfonic acids, organic acids such as acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid.
  • Such pharmaceutically toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide and iodide Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suverate , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, meth Oxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfon
  • Acid addition salt in the present invention is a conventional method, for example, a precipitate produced by dissolving the compound of Formula 1 in an organic solvent, such as methanol, ethanol, acetone, methylene chloride, acetonitrile and adding an organic or inorganic acid
  • an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile
  • the solvent may be prepared by filtration, drying, or by distillation under reduced pressure of the solvent and excess acid, followed by drying or crystallization under an organic solvent.
  • the compound of ⁇ Formula 1> of the present invention can be used to make a pharmaceutically acceptable metal salt using a base.
  • Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt.
  • corresponding silver salts are obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg, silver nitrate).
  • the compound of ⁇ Formula 1> of the present invention can be used in the form of not only pharmaceutically acceptable salts thereof, but also possible solvates, hydrates, stereoisomers and the like that can be prepared therefrom.
  • the composition of the present invention may include the active ingredient in any amount (effective amount) as long as it can exhibit the improvement activity of the inflammatory disease intended to be treated according to the use, formulation, formulation purpose, etc.
  • a typical effective amount is the total weight of the composition It will be determined within the range of 0.001% to 15% by weight based on the above.
  • the term "effective amount” as used herein refers to the amount of an effective ingredient capable of inducing the improvement, treatment, or suppression / delay of the development of such pathological symptoms in a mammal, preferably a human, to which it is applied. Such effective amounts can be determined experimentally within the range of ordinary skill in the art.
  • compositions of the invention are mammals and humans, in particular humans.
  • composition of the present invention can be used as a pharmaceutical composition in a specific embodiment.
  • the pharmaceutical composition of the present invention includes oral formulations (tablets, suspensions, granules, emulsions, capsules, syrups, etc.), parenteral formulations (sterilized), including pharmaceutically acceptable carriers, excipients, etc., in addition to the active ingredients thereof.
  • oral formulations tablets, suspensions, granules, emulsions, capsules, syrups, etc.
  • parenteral formulations stereos
  • pharmaceutically acceptable carriers including pharmaceutically acceptable carriers, excipients, etc., in addition to the active ingredients thereof.
  • Aqueous or oily suspensions for injection topical formulations (solutions, creams, ointments, gels, lotions, patches) and the like.
  • pharmaceutically acceptable means that the subject of application (prescription) does not have more toxicity (adequately low toxicity) to which the subject of application (prescription) is adaptable without inhibiting the activity of the active ingredient.
  • Examples of pharmaceutically acceptable carriers include lactose, glucose, sucrose, starch (such as corn starch, potato starch, etc.), cellulose, derivatives thereof (such as sodium carboxymethyl cellulose, ethylcellulose, etc.) malt, gelatin, talc, solids Lubricants (e.g. stearic acid, magnesium stearate, etc.), calcium sulfate, vegetable oils (e.g. peanut oil, cottonseed oil, sesame oil, olive oil, etc.), polyols (e.g. propylene glycol, glycerin, etc.), alginic acid, emulsifiers (e.g. TWEENS), wetting agents (e.g.
  • Sodium lauryl sulfate Sodium lauryl sulfate
  • colorants such as sodium lauryl sulfate
  • flavoring agents such as sodium lauryl sulfate
  • tableting agents such as sodium lauryl sulfate
  • stabilizers such as sodium lauryl sulfate
  • antioxidants such as sodium lauryl sulfate
  • preservatives water, saline, phosphate buffer solutions and the like.
  • Excipients may be selected and used according to the formulation of the pharmaceutical composition of the present invention, for example, when the pharmaceutical composition of the present invention is prepared with an aqueous suspending agent, suitable excipients are sodium carboxymethyl cellulose, methyl cellulose, hydropropylmethyl cellulose And suspending agents and dispersing agents such as sodium alginate and polyvinylpyrrolidone. Suitable excipients when prepared as injections include Ringer's solution, isotonic sodium chloride, and the like.
  • compositions of the present invention may be administered orally or parenterally, and may optionally be administered topically, as in the case of atopic dermatitis compositions.
  • the daily dosage of the pharmaceutical composition of the present invention is usually 0.001 ⁇ 150 mg / kg body weight range, it can be administered once or divided into several times. However, since the dosage of the pharmaceutical composition of the present invention is determined in view of various related factors such as the route of administration, the age, sex, weight of the patient, and the severity of the patient, the dosage may limit the scope of the present invention in any aspect. It should not be understood as.
  • composition of this invention can be grasped
  • the food composition of the present invention may include sweeteners, flavoring agents, bioactive ingredients, minerals, etc. in addition to the active ingredients.
  • Sweeteners may be used in amounts that give the food a suitable sweet taste, and may be natural or synthetic.
  • a natural sweetener is used.
  • sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose and maltose.
  • Flavoring agents can be used to enhance the taste or aroma, both natural and synthetic. It is the case of using a natural thing preferably.
  • the natural flavor may be obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, and the like, or may be obtained from green tea leaves, round leaves, jujube leaves, cinnamon, chrysanthemum leaves, jasmine and the like.
  • ginseng red ginseng
  • bamboo shoots aloe vera, ginkgo and the like can be used.
  • Natural flavors can be liquid concentrates or solid extracts.
  • synthetic flavoring agents may be used, and synthetic flavoring agents may include esters, alcohols, aldehydes, terpenes, and the like.
  • catechins such as catechin, epicatechin, gallocatechin, epigallocatechin, vitamins such as retinol, ascorbic acid, tocopherol, calciferol, thiamine, riboflavin, and the like can be used.
  • mineral calcium, magnesium, chromium, cobalt, copper, fluoride, germanium, iodine, iron, lithium, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, silicon, sodium, sulfur, vanadium, zinc and the like can be used.
  • the food composition of the present invention may contain a preservative, an emulsifier, an acidulant, a thickener, and the like, in addition to the sweetener.
  • Such preservatives, emulsifiers and the like are preferably added and used in very small amounts as long as the use to which they are added can be achieved.
  • trace amount is meant numerically expressed in the range of 0.0005% to about 0.5% by weight based on the total weight of the food composition.
  • preservatives examples include sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), and the like.
  • Emulsifiers that can be used include acacia gum, carboxymethylcellulose, xanthan gum, pectin and the like.
  • acidulants examples include lead acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, and the like. Such acidulant may be added so that the food composition is at an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to the purpose of enhancing taste.
  • Thickeners that can be used include suspending implements, sedimenters, gel formers, swelling agents and the like.
  • herbal medicines may be added to enhance flavor and palatability and to add other functionalities (such as prevention of arthritis or osteoporosis).
  • Herbal medicines that may be added include tofu extract, fast extract, antler extract, safflower extract, earthworm extract, and succinate Extracts, tortoiseshell extracts, cornus extracts, goji berry extract, licorice extract, donkey extract, brown root extract, kangjinhyang extract, haphwanpi extract, mountain rump extract, lump extract, ginseng extract and the like can be exemplified.
  • the present invention can be understood as a cosmetic composition.
  • the cosmetic composition can be prepared in various forms, for example, emulsions, lotions, creams (oil-in-water, water-in-oil, multiphase), solutions, suspensions (anhydrous and Aqueous), anhydrous products (oil and glycol based), gels, masks, packs or powders.
  • composition of the present invention may include an acceptable carrier in cosmetic preparations in addition to the active ingredient.
  • acceptable carrier in cosmetic preparation means a compound or composition already known and used that can be included in a cosmetic preparation or a compound or composition which will be developed in the future and has no toxicity above the adaptable toxicity to the human body upon contact with the skin.
  • the carrier may be included in the composition of the present invention from about 1% to about 99.99% by weight, preferably from about 50% to about 99% by weight of the composition.
  • ratios limit the scope of the present invention in any aspect because the ratios vary depending on the formulation of the cosmetics as described above and their specific application site (face or hand) or their preferred application amount. It should not be.
  • examples of the carrier include alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, moisturizers, viscosity modifiers, emulsions, stabilizers, sunscreen agents, colorants, fragrances, and the like.
  • Alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, humectants, viscosity modifiers, emulsions, stabilizers, sunscreens, colorants, compounds / compositions that can be used as fragrances, etc., which can be used as the carrier are already known in the art. As a result, those skilled in the art can select and use appropriate materials / compositions.
  • the present invention relates to a method for separating Vladin F from a narrow leaf bud.
  • the separation method of the present invention comprises the steps of (a) preparing a powdery extract by immersing a narrow leaf zenith and leaves, stems or a mixture thereof in a mixed solvent of ethanol and water, and (b) suspending the powdery extract in distilled water.
  • the mixed solvent of ethanol and water of step (a) is preferably 70 to 80% (v / v) ethanol.
  • “% (v / v)” is a concentration expression by volume percentage and refers to a volume of solute dissolved in a volume of solution as known in the art. For example, 30% (v / v) means that 30 ml of solute is dissolved in 100 ml of the solution.
  • the fractionation of the step (b) sequentially means that the fractions of the distilled water layer continue to be used after the fractionation to fractionate with the solvents in the order listed above.
  • the present invention can provide an anti-inflammatory composition.
  • the anti-inflammatory composition of the present invention may be commercialized as a drug or food.
  • Figure 1 is a schematic diagram of the separation process of the active substance Vladinol F from the narrow leaf zenith and extract.
  • FIG. 2 is the 1 H NMR spectrum of vladinol.
  • FIG. 3 is a 13C NMR spectrum of Rajinol F.
  • FIG. 4 is the DEPT NMR spectrum of remindinol F.
  • FIG. 5 is the HSQC (2D) NMR spectrum of Rajinol F.
  • FIG. 6 is the HMBC (2D) NMR spectrum of Rajinol F.
  • FIG. 7 is the LC-MS / MS spectrum of Rajinol F.
  • FIG. 13 is a result showing the INOS and COX-2 production inhibitory activity of Rajinol F.
  • Narrow leaf zenith and leaf and stem mixed powder was immersed in 80% ethanol for 24 hours and extracted three times, and the extract obtained by filtration was concentrated under reduced pressure to remove the solvent to obtain a powdery extract.
  • the narrow leaf stem and the 80% ethanol extract thus obtained were suspended in 10-fold distilled water, and then sequentially fractionated using hexane (n-Hexane), dichloromethane (methylene chloride), ethyl acetate (EtOAc) and butanol (butanol). 11.2 g of the dichloromethane fraction obtained after the fractionation was coated on the surface of celite and subjected to column chromatography to obtain a total of four purified products. The column size was 7 cm ⁇ 20 cm and hexane, dichloromethane, ethyl acetate, and methanol were used as mobile phases, respectively. Normal phase silica gel column chromatography was performed on 6.6 g of the purified product No.
  • LC-MS / MS analysis was performed to determine the molecular weight of the identified material.
  • Vladinol F had a molecular weight of 360.15 and was analyzed to be 383.18 in sodium-bound form by LC-MS / MS analysis.
  • MS / MS was performed to confirm the structure more accurately to obtain a unique cleavage pattern of Rajinol F (Vladinol F) (Fig. 7).
  • Murine macrophage lines RAW 264.7 were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) to obtain 10% fetal bovine serum (FBS), penicillin (100 units / mL), and streptomycin (100 g / mL) was added and stored at Dulbeccos Modified Eagles Medium (DMEM; GIBCO Inc.). These cells were incubated at 37 ° C. in subconfluence with 95% air, 5% CO 2 humidified air conditions and subcultured every three days. The number of cells was measured using a hemocytometer, and the number of living cells was confirmed by trypan blue dye exclusion.
  • FBS fetal bovine serum
  • penicillin 100 units / mL
  • streptomycin 100 g / mL
  • DMEM Dulbeccos Modified Eagles Medium
  • RAW 264.7 cells were adjusted to 1.5 ⁇ 10 5 cells / mL using DMEM medium supplemented with 10% FBS, inoculated into 24 well plates, and treated with LPS (1 ⁇ g / mL) at the same time for 24 hours. .
  • the amount of NO produced was in the form of NO 2- present in the cell culture solution using Griess reagent [1% (w / v) sulfanilamide, 0.1% (w / v) naphylethylenediamine in 2.5% (v / v) phosphoric acid]. Measured. 100 L of the cell culture supernatant and 100 L of Griess reagent were mixed and reacted in 96 well plates for 10 minutes, and the absorbance was measured at 540 nm. The amount of NO produced was compared to standard sodium nitrite (NaNO2).
  • FIG. 8 The results are shown in [FIG. 8].
  • A, B and C is a positive control group 2-amio-4-methyl pyridine (20 uM), dexamethason (20 uM) and NS-398 (20 uM) (COX-2 inhibitor, Sigma-Aldrich, respectively) Corporation).
  • RAW 264.7 cells (1.5 ⁇ 10 5 cells / mL) were incubated in DMEM medium with LPS (1 ⁇ g / mL) for 24 hours, followed by incubation for 24 hours, followed by centrifugation at 3,000 rpm for 5 minutes.
  • the lactate dehydrogenase (LDH) assay was measured using a non-radioactive cytotoxicity assay kit (Promega). 50 L of the culture medium and 50 L of the reconstituted substrate mix obtained by centrifugation in a 96 well plate were allowed to react for 30 minutes at room temperature. After 50 L of stop solution was added, the absorbance was measured at 490 nm using a microplate reader (Bio-TEK Instruments Inc., Vermont, WI, USA). Average absorbance values were obtained for each sample group, and cytotoxicity was evaluated by comparing with the absorbance values of the control group (LDH control, 1: 5000).
  • FIG. 9 The results are shown in FIG. 9. Referring to FIG. 9, it can be seen that the treatment of Rajin F (treatment concentrations 25, 50 and 100 ⁇ g / ml) suppressed the production of PGE 2 in a concentration-dependent manner ( P ⁇ 0.05; **, P ⁇ 0.01 ).
  • A, B and C is a positive control group 2-amio-4-methyl pyridine (20 uM), dexamethason (20 uM) and NS-398 (20 uM) (COX-2 inhibitor, Sigma-Aldrich, respectively) Corporation).
  • RAW 264.7 cells (1.5 ⁇ 10 5 cells / mL) were inoculated into 24 well plates using DMEM medium and incubated 18 hours before in a 5% CO 2 incubator. Thereafter, the medium was removed and treated with fresh medium containing the sample and LPS (1 g / mL) at the same time, and incubated under the same conditions as the previous culture. After 24 hours, the culture medium was centrifuged (12,000 rpm, 3 minutes) to determine the pro-inflammatory cytokines production in the supernatant. All samples were stored below -20 ° C until quantification.
  • Pro-inflammatory cytokines were quantified using a mouse enzyme-linked immnunosorbent assay (ELISA) kit (R & D Systems Inc., Minneapolis, MN, USA). The r2 value of the standard curve for the standard was above 0.99.
  • ELISA enzyme-linked immnunosorbent assay
  • FIGS. 10 to 12 show that regulain F (treatment concentrations 25, 50 and 100 ⁇ g / ml) inhibits the production of TNF- ⁇ , IL-1 and IL-6 in a concentration dependent manner (P ⁇ 0.05; **, P ⁇ 0.01).
  • A, B and C are positive controls, 2-amio-4-methyl pyridine (20 uM), dexamethason (20 uM) and NS-398 (20 uM) (COX-2 inhibitor, Sigma-Aldrich Corporation), respectively.
  • phosphate buffered saline PBS
  • cell lysis buffer 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 2 mM EDTA, 1 mM EGTA, 1 mM NaVO 3 , 10 mM NaF, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 25 ⁇ g / mL aprotinin, 25 ⁇ g / mL leupeptin] were added and dissolved at 4 ° C. for 30 minutes and then at 4,15,000 rpm.
  • PBS phosphate buffered saline
  • Antibodies for examining the expression level of COX-2 were anti-mouse COX-2 (BD Biosciences Pharmingen, San Jose, CA, USA) was diluted 1: 1000 in TTBS solution and reacted at room temperature for 2 hours, and then washed three times with TTBS.
  • TTBS TTBS
  • anti-mouse IgG Amersham Pharmacia Biotech, Little Chalfont, UK
  • HRP horse radish peroxidase
  • the results are shown in FIG. 13.
  • the results in FIG. 13 show that Rajinol F (treatment concentrations 25, 50 and 100 ⁇ g / ml) showed iNOS and COX-2 expression inhibitory activity in a concentration-dependent manner.
  • the experimental results show the data of three or more independent experiments as mean ⁇ standard error.
  • the statistical significance test between the experimental groups was performed using student's t-test analysis (* P ⁇ 0.05, ** P ⁇ 0.01).

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Abstract

The present invention relates to an anti-inflammation composition using Vladinol F which is an active material isolated from Ficus erecta var. sieboldii (Miq.) King. Vladinol F, which is an active material, has an activity of inhibiting the generation of NO, an activity of inhibiting the generation of PGE2, and an activity of inhibiting the generation of inflammatory cytokines (TNF-α, IL-1β and IL-6), INOS and COX-2.

Description

[규칙 제26조에 의한 보정 19.03.2013]　블라디놀 F를 이용한 항염증성 조성물[Correction 19.03.2013] according to Rule 26
본 발명은 블라디놀 F를 이용한 항염증성 조성물에 관한 것이다.The present invention relates to anti-inflammatory compositions using Vladinol F.
염증은 외부의 물리·화학적 자극, 박테리아, 곰팡이, 바이러스, 각종 알레르기 유발 물질 등 외부 감염원의 감염에 대한 생체의 방어 반응이다. Inflammation is the body's defense against external infections such as physical and chemical stimuli, bacteria, fungi, viruses, and allergens.
염증 반응은 선천성 면역 반응의 일부이며, 다른 동물에서처럼 인간의 선천성 면역 반응은 대식세포가 병원체에 특이적으로 존재하는 세포 표면의 패턴을 통해 비자기(non-self)로 인식하고 공격함으로써 시작된다. 염증 반응 시에는 염증 부위에 혈장이 축적되어 세균이 분비한 독성을 희석시키며, 혈류가 증가하고, 홍반, 통증, 부종, 발열 등의 증상이 수반되게 된다.Inflammatory responses are part of the innate immune response, and as in other animals, the innate immune response in humans begins by recognizing and attacking non-self through patterns of cell surfaces where macrophages are specifically present in pathogens. During the inflammatory reaction, plasma accumulates at the site of inflammation, diluting the toxicity secreted by bacteria, increasing blood flow, and accompanied by symptoms such as erythema, pain, edema, and fever.
이러한 염증 반응에는 다양한 생화학적 현상이 관여하는데, 특히 산화질소 합성효소(nitric oxide synthase, NOS)와 다양한 프로스타글란딘(prostaglandins)의 생합성과 관련되는 사이클로옥시제나제(cyclooxygenase, COX)가 염증 반응의 중요한 매개체로 알려져 있다. These inflammatory responses involve a variety of biochemical phenomena, particularly cyclooxygenase (COX), which is associated with the biosynthesis of nitric oxide synthase (NOS) and various prostaglandins. Known as
NOS는 세 가지 이성질체가 존재하는데, 칼슘이나 카모듈린 의존성인 eNOS(내피성 NOS)와 nNOS(신경성 NOS), 그리고 LPS(lipopolysaccharide)와 같은 세균의 내독소나 IL-1β, TNF-α, IL-6, IL-8, IL-12과 같은 여러 염증성 사이토카인에 의해 유도되는 iNOS(유도성 NOS)가 있으며, L-아르기닌(L-arginine)으로부터 산화질소(NO)를 생성한다. There are three isomers of NOS: bacterial endotoxins such as eNOS (endothelial NOS), nNOS (neurotrophic NOS), and LPS (lipopolysaccharide), which are calcium or capmodulin dependent, or IL-1β, TNF-α, IL There are iNOS (inducible NOS) induced by several inflammatory cytokines such as -6, IL-8 and IL-12, which produce nitric oxide (NO) from L-arginine.
eNOS나 nNOS에 의해 생성되는 NO는 혈압 조절 작용, 신경 전달 작용, 학습, 기억 등과 관련된 다양한 생리 반응을 수행함으로써 인체의 항상성 유지에 중요한 역할을 하지만, iNOS에 의해 생성되는 NO는 관절염, 패혈증, 조직이식거부반응, 자가면역질환, 신경세포의 사멸 등 다양한 염증성 질환에 관여하는 것으로 알려져 있다(Moncade S. et al, Pharmacol. Rev., 1991, 43, 109; Nature Medicine, 2001, 7, 1138; Mu, M. M., J. Endotoxic Res. 7, p341, 2001).NO produced by eNOS or nNOS plays an important role in maintaining the homeostasis of the body by performing various physiological responses related to blood pressure control, neurotransmitter, learning, memory, etc., but NO produced by iNOS is associated with arthritis, sepsis and tissue It is known to be involved in various inflammatory diseases such as transplant rejection, autoimmune diseases and neuronal death (Moncade S. et al, Pharmacol. Rev., 1991, 43, 109; Nature Medicine, 2001, 7, 1138; Mu , MM, J. Endotoxic Res. 7, p341, 2001).
COX 효소는 COX의 기능과 함께 하이드로퍼옥시다제(hydroperoxidase, HOX) 활성을 가지고 아라키돈산으로부터 중간체인 PGG2와 PGH2를 합성하며, 이들 화합물로 PGE2, PGF2, PGD2, 프로스타시클린 및 트롬복신A2(thromboxane A2, TxA2)를 만든다. COX의 기능 중 PGH 합성효소의 기능은 PGE2의 합성을 통해 통증과 염증 반응에 관여한다. The COX enzyme has hydroperoxidase (HOX) activity with COX function and synthesizes PGG 2 and PGH 2 intermediates from arachidonic acid, and these compounds include PGE 2 , PGF 2 , PGD 2 , and prostacyclin. And thromboxin A 2 (thromboxane A2, TxA2). Among the functions of COX, the function of PGH synthase is involved in pain and inflammatory responses through the synthesis of PGE 2 .
COX에는 두 가지 아형이 있고 COX-1은 대부분의 조직에 항시 발현되는데 비해, COX-2는 염증성 사이토카인에 의해 신속히 발현이 유도되어 염증 반응에서 중요한 역할을 한다. There are two subtypes of COX and COX-1 is always expressed in most tissues, whereas COX-2 is rapidly induced by inflammatory cytokines and plays an important role in the inflammatory response.
따라서 NO, PGE2, 염증성 사이토카인 등의 억제제나 iNOS 억제제 그리고 COX-2 억제제는 염증질환 치료제로서 활용될 수 있다.NO, PGE2, Inhibitors such as inflammatory cytokines, iNOS inhibitors, and COX-2 inhibitors may be used as therapeutic agents for inflammatory diseases.
본 발명은 NO 생성 억제 활성, PGE2의 생성 억제 활성 및 염증성 사이토카인의 생성 억제 활성을 가지는 좁은잎천선과로부터 분리한 활성물질인 블라디놀 F를 개시한다.The present invention discloses Vladinol F, which is an active substance isolated from a narrow leaf snail having NO production inhibitory activity, PGE 2 production inhibitory activity, and inflammatory cytokine production inhibitory activity.
본 발명의 목적은 좁은잎천선과로부터 활성물질인 블라디놀 F를 이용한 항염증성 조성물을 제공하는 데 있다. It is an object of the present invention to provide an anti-inflammatory composition using Vladin F, an active substance, from a narrow leaf stem.
본 발명의 다른 목적이나 구체적인 목적은 이하에서 제시될 것이다.Other and specific objects of the present invention will be presented below.
본 발명은 좁은잎천선과 추출물의 항염증 활성에 기초하여(대한민국 특허출원 제2009-0113113호 참조), 좁은잎천선과 추출물로부터 활성물질인 블라디놀 F를 분리·동정하고, 그것들이 NO, PGE2 및 염증성 사이토카인(TNF-α, IL-1 및 IL-6)의 생성 억제 활성을 확인함으로써 완성된 것이다.The present invention isolates and identifies Vladin F, an active substance, from the narrow leaf zenith and extracts based on the anti-inflammatory activity of the narrow leaf zenith and extracts (see Korean Patent Application No. 2009-0113113). 2 and by confirming the inhibitory activity of the production of inflammatory cytokines (TNF-α, IL-1 and IL-6).
상기 활성물질은 공지의 물질이며(CAS Registry Number:133318-48-6; Planta medica, 56 475-477, 1990; Biol. Pharm. Bull. 29(6) 1271-274, 2006), 그 구조식과 IUPAC 명칭은 아래의 [화학식 1]에서 확인할 수 있다. The active substance is a known substance (CAS Registry Number: 133318-48-6; Planta medica , 56 475-477, 1990; Biol. Pharm. Bull. 29 (6) 1271-274, 2006), the structural formula and IUPAC The name can be found in [Formula 1] below.
화학식 1
Figure PCTKR2012011219-appb-C000001
 Formula 1
Figure PCTKR2012011219-appb-C000001
본 발명의 항염증성 조성물은 전술한 바의 실험 결과에 기초하여 완성된 것으로서, 블라디놀 F를 유효성분으로 포함함을 특징으로 한다.The anti-inflammatory composition of the present invention is completed based on the experimental results as described above, characterized in that it comprises Vladinol F as an active ingredient.
본 명세서에서, 상기 "유효성분"의 의미는 단독으로 목적하는 활성을 나타내거나 또는 그 자체는 활성이 없는 담체와 함께 활성을 나타낼 수 있는 성분을 의미한다.In the present specification, the term "active ingredient" means a component that can exhibit activity alone or in combination with a carrier which is not active in itself.
또 본 명세서에서, "항염증"은 아래에서 정의되는 염증성 질환의 개선, 치료, 그러한 질환의 발병 억제 또는 지연을 포함하는 의미이다.In addition, in the present specification, "anti-inflammatory" is meant to include the improvement, treatment, inhibition or delay of the development of inflammatory diseases as defined below.
또 본 명세서에서, 상기 "염증성 질환"이란 외부의 물리·화학적 자극 또는 박테리아, 곰팡이, 바이러스, 각종 알레르기 유발 물질 등 외부 감염원의 감염에 대한 국부적 또는 전신적 생체 방어 반응으로 특정되는 어떠한 상태로서 정의될 있다. 이러한 반응은 각종 염증 매개 인자와 면역세포와 관련된 효소(예컨대 iNOS, COX-2 등) 활성화, 염증 매개 물질의 분비(예컨대, NO, TNF-α, IL-6, IL-1β, PGE2의 분비), 체액 침윤, 세포 이동, 조직 파괴 등의 일련의 복합적인 생리적 반응을 수반하며, 홍반, 통증, 부종, 발열, 신체의 특정 기능의 저하 또는 상실 등의 증상에 의해 외적으로 나타난다. 상기 염증성 질환은 급성, 만성, 궤양성, 알레르기성 또는 괴사성을 띨 수 있으므로, 어떠한 질환이 상기와 같은 염증성 질환의 정의에 포함되는 한 그것이 급성이든지, 만성이든지, 궤양성이든지, 알레르기성이든지 또는 괴사성이든지를 불문한다. 구체적으로 상기 염증성 질환에는 천식, 알레르기성 및 비-알레르기성 비염, 만성 및 급성 비염, 만성 및 급성 위염 또는 장염, 궤양성 위염, 급성 및 만성 신장염, 급성 및 만성 간염, 만성 폐쇄성 폐질환, 폐섬유증, 과민성 대장 증후군, 염증성 통증, 편두퐁, 두통, 허리 통증, 섬유 근육통, 근막 질환, 바이러스 감염(예컨대, C형 감염), 박테리아 감염, 곰팡이 감염, 화상, 외과적 또는 치과적 수술에 의한 상처, 프로스타글라딘 E 과다 증후군, 아테롬성 동맥 경화증, 통풍, 관절염, 류머티스성 관절염, 강직성 척추염, 호지킨병, 췌장염, 결막염, 홍채염, 공막염, 포도막염, 피부염(아토피성 피부염 포함), 습진, 다발성 경화증 등이 포함될 것이다. In the present specification, the "inflammatory disease" may be defined as any condition specified by a local or systemic biological defense response against external physical and chemical stimuli or infection of an external infectious agent such as bacteria, fungi, viruses, and various allergens. . This response activates various inflammatory mediators and enzymes associated with immune cells (eg iNOS, COX-2, etc.), secretion of inflammatory mediators (eg NO, TNF-α, IL-6, IL-1β, PGE 2) . ), Accompanied by a series of complex physiological reactions such as fluid infiltration, cell migration, tissue destruction, etc., and are manifested externally by symptoms such as erythema, pain, edema, fever, deterioration or loss of certain functions of the body. The inflammatory disease may be acute, chronic, ulcerative, allergic or necrotic, so as long as any disease is included in the definition of an inflammatory disease as above, whether it is acute, chronic, ulcerative, allergic or Irrespective of necrosis Specifically, the inflammatory diseases include asthma, allergic and non-allergic rhinitis, chronic and acute rhinitis, chronic and acute gastritis or enteritis, ulcerative gastritis, acute and chronic nephritis, acute and chronic hepatitis, chronic obstructive pulmonary disease, pulmonary fibrosis Irritable bowel syndrome, inflammatory pain, migraines, headache, back pain, fibromyalgia, fascia disease, viral infections (eg, type C infections), bacterial infections, fungal infections, burns, surgical or dental wounds, Prostaglandin E excess syndrome, atherosclerosis, gout, arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, irisitis, scleritis, uveitis, dermatitis (including atopic dermatitis), eczema, multiple sclerosis, etc. Will be included.
본 발명의 <화학식 1>의 화합물은 약학적으로 허용 가능한 염의 형태로 사용될 수 있으며, 염으로는 약학적으로 허용 가능한 유리산(free acid)에 의해 형성된 산 부가염을 사용될 수 있다. 산 부가염은 염산, 질산, 인산, 황산, 브롬화수소산, 요드화수소산, 아질산 또는 아인산과 같은 무기산류와 지방족 모노 및 디카르복실레이트, 페닐-치환된 알카노에이트, 하이드록시 알카노에이트 및 알칸디오에이트, 방향족 산류, 지방족 및 방향족 설폰산류와 같은 무독성 유기산, 아세트산, 안식향산, 구연산, 젖산, 말레인산, 글루콘산, 메탄설폰산, 4-톨루엔설폰산, 주석산, 푸마르산과 같은 유기산으로부터 얻는다. 이러한 약학적으로 무독한 염류로는 설페이트, 피로설페이트, 바이설페이트, 설파이트, 바이설파이트, 니트레이트, 포스페이트, 모노하이드로겐 포스페이트, 디하이드로겐 포스페이트, 메타포스페이트, 피로포스페이트 클로라이드, 브로마이드, 아이오다이드, 플루오라이드, 아세테이트, 프로피오네이트, 데카노에이트, 카프릴레이트, 아크릴레이트, 포메이트, 이소부티레이트, 카프레이트, 헵타노에이트, 프로피올레이트, 옥살레이트, 말로네이트, 석시네이트, 수베레이트, 세바케이트, 푸마레이트, 말리에이트, 부틴-1,4-디오에이트, 헥산-1,6-디오에이트, 벤조에이트, 클로로벤조에이트, 메틸벤조에이트, 디니트로 벤조에이트, 하이드록시벤조에이트, 메톡시벤조에이트, 프탈레이트, 테레프탈레이트, 벤젠설포네이트, 톨루엔설포네이트, 클로로벤젠설포네이트, 크실렌설포네이트, 페닐아세테이트, 페닐프로피오네이트, 페닐부티레이트, 시트레이트, 락테이트, β-하이드록시부티레이트, 글리콜레이트, 말레이트, 타트레이트, 메탄설포네이트, 프로판설포네이트, 나프탈렌-1-설포네이트, 나프탈렌-2-설포네이트 또는 만델레이트를 포함하나, 이에 제한되는 것은 아니다. 본 발명에서의 산 부가염은 통상의 방법, 예를 들면, <화학식 1>의 화합물을 유기용매, 예를 들면 메탄올, 에탄올, 아세톤, 메틸렌클로라이드, 아세토니트릴 등에 녹이고 유기산 또는 무기산을 가하여 생성된 침전물을 여과, 건조하여 제조되거나, 용매와 과량의 산을 감압 증류한 후 건조하거나 유기용매 하에서 결정화시켜셔 제조할 수 있다.The compound of Formula 1 of the present invention may be used in the form of a pharmaceutically acceptable salt, and the salt may be an acid addition salt formed by a pharmaceutically acceptable free acid. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. From non-toxic organic acids such as dioate, aromatic acids, aliphatic and aromatic sulfonic acids, organic acids such as acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid. Such pharmaceutically toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide and iodide Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suverate , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, meth Oxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesul Nate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1- Sulfonates, naphthalene-2-sulfonates or mandelate. Acid addition salt in the present invention is a conventional method, for example, a precipitate produced by dissolving the compound of Formula 1 in an organic solvent, such as methanol, ethanol, acetone, methylene chloride, acetonitrile and adding an organic or inorganic acid The solvent may be prepared by filtration, drying, or by distillation under reduced pressure of the solvent and excess acid, followed by drying or crystallization under an organic solvent.
또한, 본 발명의 <화학식 1>의 화합물은 염기를 사용하여 약학적으로 허용 가능한 금속염을 만들 수 있다. 알칼리 금속 또는 알칼리 토금속 염은 예를 들면 화합물을 과량의 알칼리 금속 수산화물 또는 알칼리 토금속 수산화물 용액 중에 용해하고, 비용해 화합물 염을 여과하고, 여액을 증발, 건조시켜 얻는다. 이때, 금속 염으로는 나트륨, 칼륨 또는 칼슘염을 제조하는 것이 제약상 적합하다. 또한, 이에 대응하는 은염은 알칼리 금속 또는 알칼리 토금속 염을 적당한 음염(예, 질산은)과 반응시켜 얻는다. In addition, the compound of <Formula 1> of the present invention can be used to make a pharmaceutically acceptable metal salt using a base. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. In addition, corresponding silver salts are obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg, silver nitrate).
또한, 본 발명의 상기 <화학식 1>의 화합물은 이의 약학적으로 허용되는 염뿐만 아니라, 이로부터 제조될 수 있는 가능한 용매화물, 수화물, 입체이성질체 등의 형태로도 사용될 수 있다.In addition, the compound of <Formula 1> of the present invention can be used in the form of not only pharmaceutically acceptable salts thereof, but also possible solvates, hydrates, stereoisomers and the like that can be prepared therefrom.
본 발명의 조성물은 그 유효성분을 용도, 제형, 배합 목적 등에 따라 치료를 의도하는 염증성 질환의 개선 활성을 나타낼 수 있는 한 임의의 양(유효량)으로 포함할 수 있는데, 통상적인 유효량은 조성물 전체 중량을 기준으로 할 때 0.001 중량 % 내지 15 중량 % 범위 내에서 결정될 것이다. 여기서 "유효량"이란 그 적용 대상인 포유동물 바람직하게는 사람에게서, 염증성 질환의 개선, 치료, 또는 이러한 병리적 증상의 발병 억제/지연을 유도할 수 있는 유효성분의 양을 말한다. 이러한 유효량은 당업자의 통상의 능력 범위 내에서 실험적으로 결정될 수 있다.The composition of the present invention may include the active ingredient in any amount (effective amount) as long as it can exhibit the improvement activity of the inflammatory disease intended to be treated according to the use, formulation, formulation purpose, etc., a typical effective amount is the total weight of the composition It will be determined within the range of 0.001% to 15% by weight based on the above. The term "effective amount" as used herein refers to the amount of an effective ingredient capable of inducing the improvement, treatment, or suppression / delay of the development of such pathological symptoms in a mammal, preferably a human, to which it is applied. Such effective amounts can be determined experimentally within the range of ordinary skill in the art.
본 발명의 조성물이 적용(처방)될 수 있는 대상은 포유동물 및 사람이며, 특히 사람인 경우가 바람직하다.Subjects to which the compositions of the invention can be applied (prescribed) are mammals and humans, in particular humans.
본 발명의 조성물은 구체적인 양태에 있어서는 약제학적 조성물로 이용될 수 있다.The composition of the present invention can be used as a pharmaceutical composition in a specific embodiment.
본 발명의 약제학적 조성물은 그 유효성분을 포함하는 이외에 약제학적으로 허용되는 담체, 부형제 등을 포함하여, 경구용 제형(정제, 현탁액, 과립, 에멀젼, 캡슐, 시럽 등), 비경구형 제형(멸균 주사용 수성 또는 유성 현탁액), 국소형 제형(용액, 크림, 연고, 겔, 로션, 패치) 등으로 제조될 수 있다.The pharmaceutical composition of the present invention includes oral formulations (tablets, suspensions, granules, emulsions, capsules, syrups, etc.), parenteral formulations (sterilized), including pharmaceutically acceptable carriers, excipients, etc., in addition to the active ingredients thereof. Aqueous or oily suspensions for injection), topical formulations (solutions, creams, ointments, gels, lotions, patches) and the like.
상기에서 "약제학적으로 허용되는" 의미는 유효성분의 활성을 억제하지 않으면서 적용(처방) 대상이 적응가능한 이상의 독성(충분히 낮은 독성)을 지니지 않는다 의미이다.As used herein, "pharmaceutically acceptable" means that the subject of application (prescription) does not have more toxicity (adequately low toxicity) to which the subject of application (prescription) is adaptable without inhibiting the activity of the active ingredient.
약제학적으로 허용되는 담체의 예로서는 락토스, 글루코스, 슈크로스, 전분(예컨대 옥수수 전분, 감자 전분 등), 셀룰로오스, 그것의 유도체(예컨대 나트륨 카르복시메틸 셀룰로오스, 에틸셀룰로오스, 등) 맥아, 젤라틴, 탈크, 고체 윤활제(예컨대 스테아르산, 스테아르산 마그네슘 등), 황산 칼슘, 식물성 기름(예컨대 땅콩 기름, 면실유, 참기름, 올리브유 등), 폴리올(예컨대 프로필렌 글리콜, 글리세린 등), 알긴산, 유화제(예컨대 TWEENS), 습윤제(예컨대 라우릴 황산 나트륨), 착색제, 풍미제, 정제화제, 안정화제, 항산화제, 보존제, 물, 식염수, 인산염 완충 용액 등을 들 수 있다. 이러한 담체는 본 발명의 약제학적 조성물의 제형에 따라 적당한 것을 하나 이상 선택하여 사용할 수 있다.Examples of pharmaceutically acceptable carriers include lactose, glucose, sucrose, starch (such as corn starch, potato starch, etc.), cellulose, derivatives thereof (such as sodium carboxymethyl cellulose, ethylcellulose, etc.) malt, gelatin, talc, solids Lubricants (e.g. stearic acid, magnesium stearate, etc.), calcium sulfate, vegetable oils (e.g. peanut oil, cottonseed oil, sesame oil, olive oil, etc.), polyols (e.g. propylene glycol, glycerin, etc.), alginic acid, emulsifiers (e.g. TWEENS), wetting agents (e.g. Sodium lauryl sulfate), colorants, flavoring agents, tableting agents, stabilizers, antioxidants, preservatives, water, saline, phosphate buffer solutions and the like. Such a carrier may be used by selecting one or more appropriate ones according to the formulation of the pharmaceutical composition of the present invention.
부형제도 본 발명의 약제학적 조성물의 제형에 따라 적합한 것을 선택하여 사용할 수 있는데, 예컨대 본 발명의 약제학적 조성물이 수성 현탁제로 제조될 경우에 적합한 부형제로서는 나트륨 카르복시메틸 셀룰로오스, 메틸 셀룰로오스, 히드로프로필메틸셀룰로오스, 알긴산 나트륨, 폴리비닐피롤리돈 등의 현탁제나 분산제 등을 들 수 있다. 주사액으로 제조되는 경우 적합한 부형제로서는 링거액, 등장 염화나트륨 등을 들 수 있다.Excipients may be selected and used according to the formulation of the pharmaceutical composition of the present invention, for example, when the pharmaceutical composition of the present invention is prepared with an aqueous suspending agent, suitable excipients are sodium carboxymethyl cellulose, methyl cellulose, hydropropylmethyl cellulose And suspending agents and dispersing agents such as sodium alginate and polyvinylpyrrolidone. Suitable excipients when prepared as injections include Ringer's solution, isotonic sodium chloride, and the like.
본 발명의 약제학적 조성물은 경구 또는 비경구로 투여될 수 있고, 아토피성 피부염 조성물처럼 경우에 따라서는 국소적으로 투여될 수 있다.The pharmaceutical compositions of the present invention may be administered orally or parenterally, and may optionally be administered topically, as in the case of atopic dermatitis compositions.
본 발명의 약제학적 조성물은 그 1일 투여량이 통상 0.001 ~ 150 mg/kg 체중 범위이고, 1회 또는 수회로 나누어 투여할 수 있다. 그러나, 본 발명의 약제학적 조성물의 투여량은 투여 경로, 환자의 연령, 성별, 체중, 환자의 중증도 등의 여러 관련 인자에 비추어 결정되는 것이므로 상기 투여량은 어떠한 측면으로든 본 발명의 범위를 제한하는 것으로 이해되어서는 아니 된다. The daily dosage of the pharmaceutical composition of the present invention is usually 0.001 ~ 150 mg / kg body weight range, it can be administered once or divided into several times. However, since the dosage of the pharmaceutical composition of the present invention is determined in view of various related factors such as the route of administration, the age, sex, weight of the patient, and the severity of the patient, the dosage may limit the scope of the present invention in any aspect. It should not be understood as.
본 발명의 조성물은 구체적인 양태에 있어서, 기능성 음료 등의 식품 조성물로 파악할 수 있다. The composition of this invention can be grasped | ascertained by food compositions, such as a functional drink, in a specific aspect.
본 발명의 식품 조성물에는 그 유효성분 이외에 감미제, 풍미제, 생리활성 성분, 미네랄 등이 포함될 수 있다.The food composition of the present invention may include sweeteners, flavoring agents, bioactive ingredients, minerals, etc. in addition to the active ingredients.
감미제는 식품이 적당한 단맛을 나게 하는 양으로 사용될 수 있으며, 천연의 것이거나 합성된 것일 수 있다. 바람직하게는 천연 감미제를 사용하는 경우인데, 천연 감미제로서는 옥수수 시럽 고형물, 꿀, 수크로오스, 프룩토오스, 락토오스, 말토오스 등의 당 감미제를 들 수 있다. Sweeteners may be used in amounts that give the food a suitable sweet taste, and may be natural or synthetic. Preferably, a natural sweetener is used. Examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose and maltose.
풍미제는 맛이나 향을 좋게 하기 위하여 사용될 수 있는데, 천연의 것과 합성된 것 모두 사용될 수 있다. 바람직하게는 천연의 것을 사용하는 경우이다. 천연의 것을 사용할 경우에 풍미 이외에 영양 강화의 목적도 병행할 수 있다. 천연 풍미제로서는 사과, 레몬, 감귤, 포도, 딸기, 복숭아 등에서 얻어진 것이거나 녹차잎, 둥굴레, 대잎, 계피, 국화 잎, 자스민 등에서 얻어진 것일 수 있다. 또 인삼(홍삼), 죽순, 알로에 베라, 은행 등에서 얻어진 것을 사용할 수 있다. 천연 풍미제는 액상의 농축액이나 고형상의 추출물일 수 있다. 경우에 따라서 합성 풍미제가 사용될 수 있는데, 합성 풍미제는 에스테르, 알콜, 알데하이드, 테르펜 등이 이용될 수 있다. Flavoring agents can be used to enhance the taste or aroma, both natural and synthetic. It is the case of using a natural thing preferably. In addition to flavors, the use of natural ones can be combined with nutritional purposes. The natural flavor may be obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, and the like, or may be obtained from green tea leaves, round leaves, jujube leaves, cinnamon, chrysanthemum leaves, jasmine and the like. In addition, ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo and the like can be used. Natural flavors can be liquid concentrates or solid extracts. In some cases, synthetic flavoring agents may be used, and synthetic flavoring agents may include esters, alcohols, aldehydes, terpenes, and the like.
생리 활성 물질로서는 카테킨, 에피카테킨, 갈로가테킨, 에피갈로카테킨 등의 카테킨류나, 레티놀, 아스코르브산, 토코페롤, 칼시페롤, 티아민, 리보플라빈 등의 비타민류 등이 사용될 수 있다.As the physiologically active substance, catechins such as catechin, epicatechin, gallocatechin, epigallocatechin, vitamins such as retinol, ascorbic acid, tocopherol, calciferol, thiamine, riboflavin, and the like can be used.
미네랄로서는 칼슘, 마그네슘, 크롬, 코발트, 구리, 불소화물, 게르마늄, 요오드, 철, 리튬, 마그네슘, 망간, 몰리브덴, 인, 칼륨, 셀레늄, 규소, 나트륨, 황, 바나듐, 아연 등이 사용될 수 있다.As the mineral, calcium, magnesium, chromium, cobalt, copper, fluoride, germanium, iodine, iron, lithium, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, silicon, sodium, sulfur, vanadium, zinc and the like can be used.
또한 본 발명의 식품 조성물은 상기 감미제 등 이외에도 필요에 따라 보존제, 유화제, 산미료, 점증제 등을 포함할 수 있다. In addition, the food composition of the present invention may contain a preservative, an emulsifier, an acidulant, a thickener, and the like, in addition to the sweetener.
이러한 보존제, 유화제 등은 그것이 첨가되는 용도를 달성할 수 있는 한 극미량으로 첨가되어 사용되는 것이 바람직하다. 극미량이란 수치적으로 표현할 때 식품 조성물 전체 중량을 기준으로 할 때 0.0005중량％ 내지 약 0.5중량％ 범위를 의미한다.Such preservatives, emulsifiers and the like are preferably added and used in very small amounts as long as the use to which they are added can be achieved. By trace amount is meant numerically expressed in the range of 0.0005% to about 0.5% by weight based on the total weight of the food composition.
사용될 수 있는 보존제로서는 소듐 소르브산칼슘, 소르브산나트륨, 소르브산칼륨, 벤조산칼슘, 벤조산나트륨, 벤조산칼륨, EDTA(에틸렌디아민테트라아세트산) 등을 들 수 있다. Examples of preservatives that can be used include sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), and the like.
사용될 수 있는 유화제로서는 아카시아검, 카르복시메틸셀룰로스, 잔탄검, 펙틴 등을 들 수 있다.Emulsifiers that can be used include acacia gum, carboxymethylcellulose, xanthan gum, pectin and the like.
사용될 수 있는 산미료로서는 연산, 말산, 푸마르산, 아디프산, 인산, 글루콘산, 타르타르산, 아스코르브산, 아세트산, 인산 등을 들 수 있다. 이러한 산미료는 맛을 증진시키는 목적 이외에 미생물의 증식을 억제할 목적으로 식품 조성물이 적정 산도로 되도록 첨가될 수 있다.Examples of acidulants that may be used include lead acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, and the like. Such acidulant may be added so that the food composition is at an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to the purpose of enhancing taste.
사용될 수 있는 점증제로서는 현탁화 구현제, 침강제, 겔형성제, 팽화제 등을 들 수 있다. Thickeners that can be used include suspending implements, sedimenters, gel formers, swelling agents and the like.
또한 향미나 기호성을 향상시키고 다른 기능성(예컨대 관절염 또는 골다공증 예방)을 추가하기 위하여 한약재가 추가될 수 있는데, 추가될 수 있는 한약재로서는 두충 추출물, 속단 추출물, 녹용 추출물, 홍화인 추출물, 토사자 추출물, 숙지황 추출물, 별갑 추출물, 산수유 추출물, 구기자 추출물, 감초 추출물, 당귀 추출물, 갈근 추출물, 강진향 추출물, 합환피 추출물, 산두근 추출물, 괴화 추출물, 고삼 추출물 등이 예시될 수 있다.In addition, herbal medicines may be added to enhance flavor and palatability and to add other functionalities (such as prevention of arthritis or osteoporosis). Herbal medicines that may be added include tofu extract, fast extract, antler extract, safflower extract, earthworm extract, and succinate Extracts, tortoiseshell extracts, cornus extracts, goji berry extract, licorice extract, donkey extract, brown root extract, kangjinhyang extract, haphwanpi extract, mountain rump extract, lump extract, ginseng extract and the like can be exemplified.
본 발명은 또 다른 측면에 있어서, 화장품 조성물로 파악할 수 있다.In another aspect, the present invention can be understood as a cosmetic composition.
본 발명의 조성물이 화장품 조성물로서 파악될 경우, 그 화장품 조성물은 다양한 형태로 제조될 수 있는데, 예컨대, 에멀젼, 로션, 크림(수중유적형, 유중수적형, 다중상), 용액, 현탁액(무수 및 수계), 무수 생성물(오일 및 글리콜계), 젤, 마스크, 팩 또는 분말 등의 제형으로 제조될 수 있다.When the composition of the present invention is conceived as a cosmetic composition, the cosmetic composition can be prepared in various forms, for example, emulsions, lotions, creams (oil-in-water, water-in-oil, multiphase), solutions, suspensions (anhydrous and Aqueous), anhydrous products (oil and glycol based), gels, masks, packs or powders.
본 발명의 조성물은 그 유효성분 이외에 화장품 제제에 있어서 수용가능한 담체를 포함할 수 있다. The composition of the present invention may include an acceptable carrier in cosmetic preparations in addition to the active ingredient.
여기서 "화장품 제제에 있어서 수용가능한 담체"란 화장품 제제에 포함될 수 있는 이미 공지되어 사용되고 있는 화합물 또는 조성물이거나 앞으로 개발될 화합물 또는 조성물로서 피부와의 접촉시 인체가 적응 가능한 독성 이상의 독성이 없는 것을 말한다.Herein, "acceptable carrier in cosmetic preparation" means a compound or composition already known and used that can be included in a cosmetic preparation or a compound or composition which will be developed in the future and has no toxicity above the adaptable toxicity to the human body upon contact with the skin.
상기 담체는 본 발명의 조성물에 그것의 전체 중량에 대하여 약 1 중량 % 내지 약 99.99 중량 %, 바람직하게는 조성물의 중량의 약 50 중량% 내지 약 99 중량 %로 포함될 수 있다. The carrier may be included in the composition of the present invention from about 1% to about 99.99% by weight, preferably from about 50% to about 99% by weight of the composition.
그러나 상기 비율은 화장품의 전술한 바의 제형에 따라 또 그것의 구체적인 적용 부위(얼굴이나 손)나 그것의 바람직한 적용량 등에 따라 달라지는 것이기 때문에, 상기 비율은 어떠한 측면으로든 본 발명의 범위를 제한하는 것으로 이해되어서는 안 된다. However, it is understood that the ratios limit the scope of the present invention in any aspect because the ratios vary depending on the formulation of the cosmetics as described above and their specific application site (face or hand) or their preferred application amount. It should not be.
한편, 상기 담체로서는 알코올, 오일, 계면활성제, 지방산, 실리콘 오일, 습윤제, 보습제, 점성 변형제, 유제, 안정제, 자외선 차단제, 발색제, 향료 등이 예시될 수 있다. On the other hand, examples of the carrier include alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, moisturizers, viscosity modifiers, emulsions, stabilizers, sunscreen agents, colorants, fragrances, and the like.
상기 담체로서 사용될 수 있는 알코올, 오일, 계면활성제, 지방산, 실리콘 오일, 습윤제, 보습제, 점성 변형제, 유제, 안정제, 자외선 차단제, 발색제, 향료 로 사용될 수 있는 화합물/조성물 등은 이미 당업계에 공지되어 있기 때문에 당업자라면 적절한 해당 물질/조성물을 선택하여 사용할 수 있다.Alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, humectants, viscosity modifiers, emulsions, stabilizers, sunscreens, colorants, compounds / compositions that can be used as fragrances, etc., which can be used as the carrier are already known in the art. As a result, those skilled in the art can select and use appropriate materials / compositions.
본 발명은 다른 측면에 있어서, 좁은잎천선과로부터 블라디놀 F를 분리하는 방법에 관한 것이다.In another aspect, the present invention relates to a method for separating Vladin F from a narrow leaf bud.
본 발명의 분리 방법은 (a) 좁은잎천선과 잎, 줄기 또는 이들의 혼합물을 에탄올과 물의 혼합용매에 침지시켜 분말상의 추출물을 제조하는 단계, (b) 그 분말상의 추출물을 증류수에 현탁시킨 후 헥산(n-Hexane), 디클로로메탄(methylene chloride), 에틸아세테이트(EtOAc) 및 부탄올(butanol)을 이용하여 순차적으로 분획한 후 디클로로메탄 분획물을 수득하는 단계, (c) 그 디클로로메탄 분획물을 셀라이트 표면에 코팅시킨 후 헥산, 디클로로메탄, 에틸아세테이트 및 메탄올 각각을 이동상으로 하여 컬럼크로마토그래피를 실시하여 총 4개의 정제물을 얻는 단계, (d) 그 총 4개의 정제물 중 2번 정제물에 대해서 헥산:에틸아세테이트:메탄올(1:2:0.2, v/v)을 이동상으로 하고 순상실리카겔 컬럼크로마토그래피를 실시하여 총 20개의 정제물을 얻는 단계, (e) 그 20개의 정제물 중 16번 정제물에 대해서 클로로포름:메탄올(10:1, v/v)을 이동상으로 하여 순상실리카겔 컬럼크로마토그래피를 실시하여 총 18개의 정제물을 얻는 단계, (f) 그 18개의 정제물 중 9번 정제물을 수득하는 단계를 포함하여 구성된다.The separation method of the present invention comprises the steps of (a) preparing a powdery extract by immersing a narrow leaf zenith and leaves, stems or a mixture thereof in a mixed solvent of ethanol and water, and (b) suspending the powdery extract in distilled water. Sequentially distilling with hexane (n-Hexane), dichloromethane (methylene chloride), ethyl acetate (EtOAc) and butanol (butanol) to obtain a dichloromethane fraction, (c) the dichloromethane fraction to celite Coating on the surface, and performing column chromatography with hexane, dichloromethane, ethyl acetate, and methanol as mobile phases to obtain a total of four purified products, and (d) the second purified product among the four purified products. Performing pure phase silica gel column chromatography using hexane: ethyl acetate: methanol (1: 2: 0.2, v / v) as a mobile phase to obtain a total of 20 purified products, (e) the 20 purified products Purification of Purification No. 16 to chloroform: methanol (10: 1, v / v) using mobile phase, followed by pure silica gel column chromatography to obtain a total of 18 purifications, (f) 9 of the 18 purifications And obtaining a purified product.
본 발명의 방법에서, 상기 (a) 단계의 에탄올과 물의 혼합용매는 70 내지 80%(v/v)의 에탄올이 바람직하다. "%(v/v)"는 부피 백분율에 의한 농도 표현으로서, 당업계에 알려진 바와 같이 일정 부피의 용액 중에 녹아 있는 일정 부피의 용질을 의미한다. 예컨대 30%(v/v)는 용액 100㎖에 용질 30㎖가 용해되어 있다는 것이 된다.In the method of the present invention, the mixed solvent of ethanol and water of step (a) is preferably 70 to 80% (v / v) ethanol. “% (v / v)” is a concentration expression by volume percentage and refers to a volume of solute dissolved in a volume of solution as known in the art. For example, 30% (v / v) means that 30 ml of solute is dissolved in 100 ml of the solution.
또 본 발명의 방법에 있어서, 상기 (b) 단계의 순차적으로 분획한다는 것은 증류수층의 분획물을 분획 후에도 계속 사용하여 상기 열거된 순서의 용매로 분획한다는 의미이다.In addition, in the method of the present invention, the fractionation of the step (b) sequentially means that the fractions of the distilled water layer continue to be used after the fractionation to fractionate with the solvents in the order listed above.
전술한 바와 같이, 본 발명에 따르면 항염증성 조성물을 제공할 수 있다.As described above, the present invention can provide an anti-inflammatory composition.
본 발명의 항염증성 조성물은 약품이나 식품으로 제품화될 수 있다.The anti-inflammatory composition of the present invention may be commercialized as a drug or food.
[도 1]은 좁은잎천선과 추출물로부터 활성물질인 블라디놀 F의 분리 과정의 모식도이다.Figure 1 is a schematic diagram of the separation process of the active substance Vladinol F from the narrow leaf zenith and extract.
[도 2]는 블라디놀의 1H NMR 스펙트럼이다.FIG. 2 is the 1 H NMR spectrum of vladinol. FIG.
[도 3]은 블라디놀 F의 13C NMR 스펙트럼이다.FIG. 3 is a 13C NMR spectrum of Vladinol F. FIG.
[도 4]는 블라디놀 F의 DEPT NMR 스펙트럼이다.FIG. 4 is the DEPT NMR spectrum of Vladinol F. FIG.
[도 5]는 블라디놀 F의 HSQC(2D) NMR스펙트럼이다.FIG. 5 is the HSQC (2D) NMR spectrum of Vladinol F. FIG.
[도 6]은 블라디놀 F의 HMBC(2D) NMR스펙트럼이다.FIG. 6 is the HMBC (2D) NMR spectrum of Vladinol F. FIG.
[도 7]은 블라디놀 F의 LC-MS/MS 스펙트럼이다.FIG. 7 is the LC-MS / MS spectrum of Vladinol F. FIG.
[도 8]은 블라디놀 F의 NO 생성 억제 활성과 특별한 세포독성이 없음을 보여주는 결과이다.8 is a result showing that NO and inhibitory activity of Vladinol F production and no particular cytotoxicity.
[도 9]는 블라디놀 F의 PGE2의 생성 억제 활성을 보여주는 결과이다.9 is a result showing the inhibitory activity of PGE 2 production of Vladinol F.
[도 10] 내지 [도 12]는 블라디놀 F의 사이토카인(TNF-α, IL-1 및 IL-6) 생성 억제 활성을 보여주는 결과이다.10 to 12 are results showing the cytokine (TNF-α, IL-1 and IL-6) production inhibitory activity of Vladinol F.
[도 13]은 블라디놀 F의 INOS와 COX-2 생성 억제 활성을 보여주는 결과이다.FIG. 13 is a result showing the INOS and COX-2 production inhibitory activity of Vladinol F. FIG.
이하 본 발명을 실시예 및 실험예를 참조하여 설명한다. 그러나 본 발명의 범위가 이러한 실시예 및 실험예에 의하여 한정되는 것은 아니다.Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited by these examples and experimental examples.
<실시예> 좁은잎천선과 추출물로부터 항염증 활성을 갖는 활성물질의 분리, 정제 및 동정EXAMPLES Isolation, Purification and Identification of Active Substances Having Anti-inflammatory Activity from Narrow Leaf Lines and Extracts
<실시예 1> 좁은잎천선과 80% 에탄올 추출물로부터 항염증 활성물질의 분리 및 정제Example 1 Isolation and Purification of Anti-inflammatory Active Substances from Narrow Leaf Lines and 80% Ethanol Extracts
좁은잎천선과 잎과 줄기 혼합 분말을 80% 에탄올에 24시간 동안 침지시켜 3회 반복 추출한 후 여과하여 얻어진 추출액을 감압 농축하여 용매를 제거하여 분말상의 추출물을 얻었다.Narrow leaf zenith and leaf and stem mixed powder was immersed in 80% ethanol for 24 hours and extracted three times, and the extract obtained by filtration was concentrated under reduced pressure to remove the solvent to obtain a powdery extract.
이렇게 얻어진 좁은잎천선과 80% 에탄올 추출물을 10 배량의 증류수에 현탁시킨 후 헥산(n-Hexane), 디클로로메탄(methylene chloride), 에틸아세테이트(EtOAc) 및 부탄올(butanol)을 이용하여 순차적으로 분획하고, 분획 후 얻어진 디클로메탄 분획물 11.2g을 셀라이트 표면에 코팅시킨 후 컬럼크로마토그래피를 실시하여 총 4개의 정제물을 얻었다. 컬럼의 크기는 7cm × 20cm 이며 헥산, 디클로로메탄, 에틸아세테이트, 메탄올을 각각을 이동상으로 사용하였다. 그 중 2번(FEM-M)의 정제물 6.6g에 대해 순상실리카겔 컬럼크로마토그래피를 실시하였다. 컬럼의 크기는 5cm × 60cm 이며 헥산:에틸아세테이트:메탄올(1:2:0.2, v/v)을 이동상으로 사용하였다. 분당 1.5ml의 속도로 이동상을 흘려주었으며 총 20개의 정제물을 얻었다. 상기 총 20개의 정제물 중 16번(FEM-MC-16)에 대해서 다시 순상실리카겔 컬럼크로마토그래피를 실시하였다. 컬럼의 크기는 2cm × 50cm 이며 클로로포름:메탄올(10:1, v/v)을 이동상으로 사용하였다. 분당 1.5ml의 속도로 이동상을 흘려주었으며 총 18개의 정제물을 얻었다. 상기 총 18개의 정제물 중 9번(FEM-MC-16-9) 정제물을 TLC(실리카겔 60 F 254 , 클로로포름;메탄올=10:1, v/v)에 전개했을 때 Rf는 0.3 이며 단일 스팟으로 나타났다.The narrow leaf stem and the 80% ethanol extract thus obtained were suspended in 10-fold distilled water, and then sequentially fractionated using hexane (n-Hexane), dichloromethane (methylene chloride), ethyl acetate (EtOAc) and butanol (butanol). 11.2 g of the dichloromethane fraction obtained after the fractionation was coated on the surface of celite and subjected to column chromatography to obtain a total of four purified products. The column size was 7 cm × 20 cm and hexane, dichloromethane, ethyl acetate, and methanol were used as mobile phases, respectively. Normal phase silica gel column chromatography was performed on 6.6 g of the purified product No. 2 (FEM-M). The column size was 5 cm × 60 cm and hexane: ethyl acetate: methanol (1: 2: 0.2, v / v) was used as the mobile phase. The mobile phase was flowed at a rate of 1.5 ml per minute and a total of 20 tablets were obtained. Normal silica gel column chromatography was performed again on No. 16 (FEM-MC-16). The column was 2 cm × 50 cm in size and chloroform: methanol (10: 1, v / v) was used as the mobile phase. The mobile phase was flowed at a rate of 1.5 ml per minute and a total of 18 tablets were obtained. Rf of 0.3 and a single spot when developing No. 9 (FEM-MC-16-9) of the total 18 purifications in TLC (silica gel 60 F 254, chloroform; methanol = 10: 1, v / v) Appeared.
[도 1]에 활성물질의 분리 과정을 나타내었다.1 shows the separation process of the active material.
<실시예 2> 물질의 동정 Example 2 Identification of Materials
분리된 활성물질의 구조를 분석하기 위해 9번 정제물 10mg을 클로로포름-d에 녹여 NMR spectrum을 측정하였다. 1H NMR 스펙트럼(도 2)에서 2개의 메틸기를 확인했으며 모두 산소와 연결된 메틸기(δ 3.88ppm, 3.86ppm ,s)임을 확인했다. 또한 6.6-6.9ppm 부근의 시그널들로 미루어 2개 이상의 벤젠고리가 있음을 예상하였다. 13C, DEPT NMR 스펙트럼(도 3 및 도 4)에서 총 20개 이상의 탄소가 존재함을 예측하였고 146.8-127.9ppm 부근의 시그널들로 미루어 2개의 벤젠고리 탄소 중 7개의 탄소가 산소(δ 146.8, 146.7, 145.8, 144.4ppm)와 탄소(δ 135.6, 133.3, 127.9ppm)로 치환되어 있음을 확인하였다. 또한 64.1, 62.2ppm이 CH2임에도 저자장으로 이동한 것으로 보아 산소와 결합하고 있음을 예상하였으며 두 개의 메톡시 탄소도 (δ 56.21, 56.22ppm) 분석되었다. 그리고 2D NMR분석 기법인 HSQC(도 5) 및 HMBC(도 6)을 통하여 9번 정제물은 탄소 20개로 구성된 리그난 형태임을 예상하였고 이상의 결과들을 문헌(Planta medica, 56 475-477, 1990)과 비교한 결과 Vladinol F 로 동정하였다. In order to analyze the structure of the isolated active material, 10 mg of No. 9 purified water was dissolved in chloroform-d and NMR spectrum was measured. In the 1 H NMR spectrum (FIG. 2), two methyl groups were identified and all were identified as methyl groups (δ 3.88 ppm, 3.86 ppm, s) connected with oxygen. It was also expected that there were more than two benzene rings due to signals around 6.6-6.9ppm. The 13C, DEPT NMR spectra (FIGS. 3 and 4) predicted the presence of more than 20 carbons in total, and from the signals around 146.8-127.9 ppm, 7 of the 2 benzene ring carbons were oxygen (δ 146.8, 146.7). , 145.8, 144.4 ppm) and carbon (δ 135.6, 133.3, 127.9 ppm) was confirmed that the substitution. In addition, 64.1 and 62.2ppm were expected to bind with oxygen, even though they were CH 2 , and two methoxy carbons (δ 56.21 and 56.22ppm) were analyzed. In addition, the 2D NMR analysis method, HSQC (FIG. 5) and HMBC (FIG. 6), predicted that No. 9 was a lignan form consisting of 20 carbons, and the results were compared with those of Planta medica , 56 475-477, 1990 As a result, it was identified as Vladinol F.
동정된 물질의 분자량을 측정하기 위해 LC-MS/MS 분석을 실시했다. Vladinol F의 분자량은 360.15이며 LC-MS/MS 분석 결과 소듐이 결합된 형태인 383.18로 분석되었다. 좀 더 정확한 구조 확인을 위해 MS/MS를 수행한 결과 블라디놀 F(Vladinol F) 고유의 쪼개짐 패턴을 얻을 수 있었다(도 7).LC-MS / MS analysis was performed to determine the molecular weight of the identified material. Vladinol F had a molecular weight of 360.15 and was analyzed to be 383.18 in sodium-bound form by LC-MS / MS analysis. MS / MS was performed to confirm the structure more accurately to obtain a unique cleavage pattern of Vladinol F (Vladinol F) (Fig. 7).
<실험예> 블라디놀 F의 항염증 활성 실험Experimental Example Anti-inflammatory Activity of Vladinol F
<실험예 1> 세포 배양 Experimental Example 1 Cell Culture
American Type Culture Collection (ATCC, Rockville, MD, USA)로부터 생쥐(murine) 대식세포주인 RAW 264.7을 구입하여, 10% fetal bovine serum (FBS), penicillin (100 units/mL), 및 streptomycin (100 g/mL)을 첨가하여 Dulbeccos Modified Eagles Medium (DMEM; GIBCO Inc.)에서 보관하였다. 이들 세포들은 37℃ 에서 95% air, 5% CO2가습 공기 조건 하 포화 상태(subconfluence)에서 배양하였으며, 3일마다 계대배양하였다. 혈구계를 이용하여 세포의 수를 측정하였으며, trypan blue dye exclusion을 통하여 생세포의 수를 확인하였다.Murine macrophage lines RAW 264.7 were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) to obtain 10% fetal bovine serum (FBS), penicillin (100 units / mL), and streptomycin (100 g / mL) was added and stored at Dulbeccos Modified Eagles Medium (DMEM; GIBCO Inc.). These cells were incubated at 37 ° C. in subconfluence with 95% air, 5% CO 2 humidified air conditions and subcultured every three days. The number of cells was measured using a hemocytometer, and the number of living cells was confirmed by trypan blue dye exclusion.
<실험예 2> 블라디놀 F의 질소화합물(Nitric oxide) 생성 억제 효능 평가 <Experimental Example 2> of nitrogen VLADIMIR play F (Nitric oxide) Inhibitory Efficacy Assessment
RAW 264.7 세포를 10% FBS가 첨가된 DMEM 배지를 이용하여 1.5×105cells/mL로 조절한 후 24 well plate에 접종하고, 시료와 LPS (1 ㎍/mL)를 동시에 처리하여 24시간 배양하였다. 생성된 NO의 양은 Griess 시약 [1% (w/v) sulfanilamide, 0.1% (w/v) naphylethylenediamine in 2.5% (v/v) phosphoric acid]을 이용하여 세포배양액 중에 존재하는 NO2-의 형태로 측정하였다. 세포배양 상등액 100 L와 Griess 시약 100 L를 혼합하여 96 well plates에서 10분 동안 반응시킨 후 540 nm에서 흡광도를 측정하였다. 생성된 NO의 양은 sodium nitrite (NaNO2)를 standard로 비교하였다.RAW 264.7 cells were adjusted to 1.5 × 10 5 cells / mL using DMEM medium supplemented with 10% FBS, inoculated into 24 well plates, and treated with LPS (1 μg / mL) at the same time for 24 hours. . The amount of NO produced was in the form of NO 2- present in the cell culture solution using Griess reagent [1% (w / v) sulfanilamide, 0.1% (w / v) naphylethylenediamine in 2.5% (v / v) phosphoric acid]. Measured. 100 L of the cell culture supernatant and 100 L of Griess reagent were mixed and reacted in 96 well plates for 10 minutes, and the absorbance was measured at 540 nm. The amount of NO produced was compared to standard sodium nitrite (NaNO2).
결과를 [도 8]에 나타내었다. [도 8]을 참조하여 보면 블라디놀 F(처리 농도 25, 50 및 100 ㎍/㎖)가 농도 의존적으로 LPS 처리에 의해 유발된 NO 생성을 억제함을 알 수 있다(P<0.05;**,P<0.01). [도 8]에서 A, B 및 C는 양성대조군으로 각각 2-amio-4-methyl pyridine(20 uM), dexamethason(20 uM) 및 NS-398(20 uM)(COX-2 inhibitor, Sigma-Aldrich Corporation)이다.The results are shown in [FIG. 8]. Referring to FIG. 8, it can be seen that Vladin F ( treatment concentrations 25, 50 and 100 μg / ml) inhibits NO production induced by LPS treatment in a concentration-dependent manner ( P <0.05; **, P <0.01 ). In Figure 8, A, B and C is a positive control group 2-amio-4-methyl pyridine (20 uM), dexamethason (20 uM) and NS-398 (20 uM) (COX-2 inhibitor, Sigma-Aldrich, respectively) Corporation).
<실험예 3> 블라디놀 F의 세포독성 평가 - LDH assay Experimental Example 3 Cytotoxicity Evaluation of Vladinol F-LDH Assay
RAW 264.7 세포 (1.5×105cells/mL)를 DMEM 배지에 시료와 LPS (1 ㎍/mL)를 동시 처리하여 24시간 배양 한 후 배양 배지를 얻어 3,000 rpm에서 5분간 원심분리 하였다. LDH (lactate dehydrogenase) assay는 non-radioactive cytotoxicity assay kit (Promega)를 이용하여 측정했으며, 96 well plate에 원심 분리하여 얻은 배양 배지 50 L와 reconstituted substrate mix를 50 L를 넣고, 실온에서 30분 반응시킨 후 50 L의 stop solution을 넣은 후 microplate reader (Bio-TEK Instruments Inc., Vermont, WI, USA)를 사용하여 490 nm에서 흡광도를 측정하였다. 각 시료군에 대한 평균 흡광도 값을 구하였으며, 대조군 (LDH control, 1:5000)의 흡광도 값과 비교하여 세포독성을 평가하였다.RAW 264.7 cells (1.5 × 10 5 cells / mL) were incubated in DMEM medium with LPS (1 μg / mL) for 24 hours, followed by incubation for 24 hours, followed by centrifugation at 3,000 rpm for 5 minutes. The lactate dehydrogenase (LDH) assay was measured using a non-radioactive cytotoxicity assay kit (Promega). 50 L of the culture medium and 50 L of the reconstituted substrate mix obtained by centrifugation in a 96 well plate were allowed to react for 30 minutes at room temperature. After 50 L of stop solution was added, the absorbance was measured at 490 nm using a microplate reader (Bio-TEK Instruments Inc., Vermont, WI, USA). Average absorbance values were obtained for each sample group, and cytotoxicity was evaluated by comparing with the absorbance values of the control group (LDH control, 1: 5000).
결과를 [도 8]에 함께 나타내었다. 블라디놀 F(처리 농도 25, 50 및 100 ㎍/㎖)를 처리할 경우 약간의 세포 독성을 보였으나, 그 이상으로 NO 생성 억제 활성을 보임을 알 수 있다. The results are shown together in FIG. 8. Treatment with Vladinol F (treatment concentrations of 25, 50 and 100 μg / ml) showed some cytotoxicity, but more than that showed NO production inhibitory activity.
<실험예 4> 블라디놀 F의 PGE 2 (Prostaglandin E 2 )생성 억제 효능 평가 Experimental Example 4 Evaluation of PGE 2 (Prostaglandin E 2 ) Production Inhibitory Effect of Vladinol F
RAW 264.7 세포를 DMEM 배지를 이용하여 1.5×105cells/mL로 조절한 후 24 well plate 에 접종하고, 5% CO2항온기에서 18시간 전 배양 하였다. 이후 배지를 제거하고 시료와 LPS (1 g/mL)를 동시 함유한 새로운 배지를 처리하여 전배양과 동일 조건에서 배양하였다. 24시간 후 PGE2를 측정하기 위해 배양 배지를 원심분리 (12,000 rpm, 3 min)하여 상층액을 얻었다. PGE2의 측정은 PGE2ELISAkit(R&DSystemsInc.,Minneapolis,MN,USA)를 이용하여 정량하였으며 standard 에 대한 표준곡선의 r2값은 0.99 이상이었다.Using the RAW 264.7 cells DMEM medium and incubated 1.5 × 10 5 cells / mL and then adjusted to inoculate 24 well plate, 18 hours ago in 5% CO 2 thermostat. Thereafter, the medium was removed, and a new medium containing the sample and LPS (1 g / mL) was treated and incubated under the same conditions as the preculture. After 24 hours, the culture medium was centrifuged (12,000 rpm, 3 min) to measure PGE 2 to obtain a supernatant. PGE 2 was measured using PGE 2 ELISAkit (R & D Systems Inc., Minneapolis, MN, USA) and the r2 value of the standard curve for the standard was greater than 0.99.
결과를 [도 9]에 나타내었다. [도 9]를 참조하여 보면 블라디놀 F(처리 농도 25, 50 및 100 ㎍/㎖)를 처리할 경우 농도 의존적으로 PGE2의 생성이 억제되었음을 알 수 있다(P<0.05;**,P<0.01). [도 8]에서 A, B 및 C는 양성대조군으로 각각 2-amio-4-methyl pyridine(20 uM), dexamethason(20 uM) 및 NS-398(20 uM)(COX-2 inhibitor, Sigma-Aldrich Corporation)이다.The results are shown in FIG. 9. Referring to FIG. 9, it can be seen that the treatment of Vladin F ( treatment concentrations 25, 50 and 100 μg / ml) suppressed the production of PGE 2 in a concentration-dependent manner ( P <0.05; **, P < 0.01 ). In Figure 8, A, B and C is a positive control group 2-amio-4-methyl pyridine (20 uM), dexamethason (20 uM) and NS-398 (20 uM) (COX-2 inhibitor, Sigma-Aldrich, respectively) Corporation).
<실험예 5> 블라디놀 F의 사이토카인(TNF-α, IL-1 및 IL-6) 생성 억제 효능 평가 Experimental Example 5 Evaluation of Inhibitory Effect of Vladin F on Cytokine (TNF-α, IL-1 and IL-6) Production
RAW 264.7 세포 (1.5×105cells/mL)를 DMEM 배지를 이용하여 24 well plate 에 접종하고, 5% CO2 항온기에서 18 시간 전 배양하였다. 이후 배지를 제거하고 시료와 LPS (1 g/mL)를 동시 함유한 새로운 배지를 처리하여 전 배양과 동일 조건에서 배양하였다. 24 시간 후 배양 배지를 원심분리 (12,000 rpm, 3 분)하여 얻어진 상층액의 pro-inflammatory cytokines 생성 함량을 측정하였다. 모든 시료는 정량 전까지 -20℃ 이하에 보관하였다. pro-inflammatory cytokines 정량은 mouse enzyme-linked immnunosorbent assay (ELISA) kit (R&D Systems Inc., Minneapolis, MN, USA)를 이용하여 정량하였으며 standard 에 대한 표준곡선의 r2값은 0.99 이상이었다.RAW 264.7 cells (1.5 × 10 5 cells / mL) were inoculated into 24 well plates using DMEM medium and incubated 18 hours before in a 5% CO 2 incubator. Thereafter, the medium was removed and treated with fresh medium containing the sample and LPS (1 g / mL) at the same time, and incubated under the same conditions as the previous culture. After 24 hours, the culture medium was centrifuged (12,000 rpm, 3 minutes) to determine the pro-inflammatory cytokines production in the supernatant. All samples were stored below -20 ° C until quantification. Pro-inflammatory cytokines were quantified using a mouse enzyme-linked immnunosorbent assay (ELISA) kit (R & D Systems Inc., Minneapolis, MN, USA). The r2 value of the standard curve for the standard was above 0.99.
결과를 [도 10] 내지 [도 12]에 나타내었다. [도 10] 내지 [도 12]의 결과는 블라디놀 F(처리 농도 25, 50 및 100 ㎍/㎖)가 농도 의존적으로 TNF-α, IL-1 및 IL-6의 생성을 억제함으로 보여준다(P<0.05;**,P<0.01). 여기서도 A, B 및 C는 양성대조군으로 각각 2-amio-4-methyl pyridine(20 uM), dexamethason(20 uM) 및 NS-398(20 uM)(COX-2 inhibitor, Sigma-Aldrich Corporation)이다.The results are shown in FIGS. 10 to 12. The results in FIGS. 10-12 show that Vladin F ( treatment concentrations 25, 50 and 100 μg / ml) inhibits the production of TNF-α, IL-1 and IL-6 in a concentration dependent manner (P <0.05; **, P <0.01). Here, A, B and C are positive controls, 2-amio-4-methyl pyridine (20 uM), dexamethason (20 uM) and NS-398 (20 uM) (COX-2 inhibitor, Sigma-Aldrich Corporation), respectively.
<실험예 6> iNOS 및 COX-2 발현 억제 효능 평가(western-blotting) <Experiment 6> iNOS and COX-2 expression inhibitory effect evaluation (western-blotting)
배양이 끝난 세포를 수집하여 2~3회 phosphate buffered saline (PBS)로 세척 한 후 세포 용해 버퍼 [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 2 mM EDTA, 1 mM EGTA, 1 mM NaVO3, 10 mM NaF, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 25 ㎍/mL aprotinin, 25 ㎍/mL leupeptin]를 첨가하여 30분간 4℃에서 용해시킨 후 4, 15,000 rpm에서 15분간 원심 분리하여 세포막 성분 등을 제거하였다. 단백질 농도는 bovine serum albumin (BSA)를 표준화하여 Bio-Rad Protein Assay Kit를 사용하여 정량하였다. 분리된 단백질 20~30 ㎍를 8~12% mini gel SDS-PAGE로 변성 분리하여, 이를 PVDF (polyvinylidene difluoride) membrane (BIO-RAD, Richmond, CA, USA)에 200 mA로 2시간 동안 transfer하였다. 그리고 membrane의 blocking은 5% skim milk가 함유된 TTBS (0.1% Tween 20 + TBS) 용액에서 상온에서 2시간 동안 실시하였다. iNOS의 발현 양을 검토하기 위한 항체로는 anti-mouse iNOS (Calbiochem, La Jolla, CA, USA)를 COX-2의 발현 양을 검토하기 위한 항체로는 anti-mouse COX-2 (BD Biosciences Pharmingen, San Jose, CA, USA)를 TTBS 용액에서 1:1000으로 희석하여 상온에서 2시간 반응시킨 후 TTBS로 3회 세정하였다. 2차 항체로는 HRP (horse radish peroxidase)가 결합된 anti-mouse IgG (Amersham Pharmacia Biotech, Little Chalfont, UK)를 1:5000으로 희석하여 상온에서 30분 간 반응시킨 후, TTBS로 3회 세정하여 ECL 기질 (Amersham Biosciences, Piscataway, NJ, USA)과 1~3분 간 반응 후 X-ray 필름에 감광하였다.After incubation, the cells were collected and washed 2-3 times with phosphate buffered saline (PBS), followed by cell lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 2 mM EDTA, 1 mM EGTA, 1 mM NaVO 3 , 10 mM NaF, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 25 μg / mL aprotinin, 25 μg / mL leupeptin] were added and dissolved at 4 ° C. for 30 minutes and then at 4,15,000 rpm. Cell membrane components and the like were removed by centrifugation for 15 minutes. Protein concentration was quantified using Bio-Rad Protein Assay Kit by standardizing bovine serum albumin (BSA). 20-30 μg of the isolated protein was denatured by 8-12% mini gel SDS-PAGE and transferred to PVDF (polyvinylidene difluoride) membrane (BIO-RAD, Richmond, CA, USA) at 200 mA for 2 hours. The blocking of the membrane was performed for 2 hours at room temperature in TTBS (0.1% Tween 20 + TBS) solution containing 5% skim milk. Antibodies for examining the expression level of iNOS were anti-mouse iNOS (Calbiochem, La Jolla, CA, USA). Antibodies for examining the expression level of COX-2 were anti-mouse COX-2 (BD Biosciences Pharmingen, San Jose, CA, USA) was diluted 1: 1000 in TTBS solution and reacted at room temperature for 2 hours, and then washed three times with TTBS. As a secondary antibody, anti-mouse IgG (Amersham Pharmacia Biotech, Little Chalfont, UK) conjugated with HRP (horse radish peroxidase) was diluted 1: 5000 and reacted at room temperature for 30 minutes, and then washed three times with TTBS. After reacting with ECL substrate (Amersham Biosciences, Piscataway, NJ, USA) for 1 to 3 minutes, the X-ray film was photosensitive.
결과를 [도 13]에 나타내었다. [도 13]의 결과는 블라디놀 F(처리 농도 25, 50 및 100 ㎍/㎖)가 대체로 농도 의존적으로 iNOS 및 COX-2 발현 억제 활성을 보임을 보여준다. The results are shown in FIG. 13. The results in FIG. 13 show that Vladinol F ( treatment concentrations 25, 50 and 100 μg / ml) showed iNOS and COX-2 expression inhibitory activity in a concentration-dependent manner.
통계처리Statistical processing
실험결과는 3번 이상의 독립적인 실험의 데이터를 mean ± standard error 값으로 나타내었다. 실험군 사이의 통계적 유의성 검정은 student's t-test 분석방법을 사용하였다(* P<0.05, ** P<0.01).The experimental results show the data of three or more independent experiments as mean ± standard error. The statistical significance test between the experimental groups was performed using student's t-test analysis (* P <0.05, ** P <0.01).

Claims (6)

  1. 블라디놀 F 또는 그것의 약학적으로 허용가능한 염을 유효성분으로 포함하는 항염증성 조성물.An anti-inflammatory composition comprising Vladin F or a pharmaceutically acceptable salt thereof as an active ingredient.
  2. 제1항에 있어서,The method of claim 1,
    상기 항염증은 염증성 질환의 개선, 치료, 발병 억제 또는 발병 지연을 의미하며, The anti-inflammatory refers to amelioration, treatment, inhibition of onset or delayed on the inflammatory disease,
    상기 염증성 질환은 천식, 알레르기성 및 비-알레르기성 비염, 만성 및 급성 비염, 만성 및 급성 위염 또는 장염, 궤양성 위염, 급성 및 만성 신장염, 급성 및 만성 간염, 만성 폐쇄성 폐질환, 폐섬유증, 과민성 대장 증후군, 염증성 통증, 편두퐁, 두통, 허리 통증, 섬유 근육통, 근막 질환, 바이러스 감염(예컨대, C형 감염), 박테리아 감염, 곰팡이 감염, 화상, 외과적 또는 치과적 수술에 의한 상처, 프로스타글라딘 E 과다 증후군, 아테롬성 동맥 경화증, 통풍, 관절염, 류머티스성 관절염, 강직성 척추염, 호지킨병, 췌장염, 결막염, 홍채염, 공막염, 포도막염, 피부염, 아토피성 피부염, 습진 및 다발성 경화증 중 하나인 것을 특징으로 하는 항염증성 조성물.The inflammatory diseases include asthma, allergic and non-allergic rhinitis, chronic and acute rhinitis, chronic and acute gastritis or enteritis, ulcerative gastritis, acute and chronic nephritis, acute and chronic hepatitis, chronic obstructive pulmonary disease, pulmonary fibrosis, irritability Bowel Syndrome, Inflammatory Pain, Migraine, Headache, Back Pain, Fibromyalgia, Fascia Disease, Viral Infection (eg, Type C Infection), Bacterial Infection, Fungal Infection, Burn, Surgical or Dental Surgery, Prostar Gladin E hyperplasia, atherosclerosis, gout, arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, irisitis, scleritis, uveitis, dermatitis, atopic dermatitis, eczema and multiple sclerosis Anti-inflammatory composition.
  3. 제1항 또는 제2항에 있어서,The method according to claim 1 or 2,
    상기 조성물은 약제학적 조성물인 것을 특징으로 하는 항염증성 조성물.The composition is an anti-inflammatory composition, characterized in that the pharmaceutical composition.
  4. 제1항 또는 제2항에 있어서,The method according to claim 1 or 2,
    상기 조성물은 식품 조성물인 것을 특징으로 하는 항염증성 조성물.The composition is an anti-inflammatory composition, characterized in that the food composition.
  5. 제1항 또는 제2항에 있어서,The method according to claim 1 or 2,
    상기 조성물은 화장품 조성물인 것을 특징으로 하는 항염증성 조성물.The composition is an anti-inflammatory composition, characterized in that the cosmetic composition.
  6. (a) 좁은잎천선과 잎, 줄기 또는 이들의 혼합물을 에탄올과 물의 혼합용매에 침지시켜 분말상의 추출물을 제조하는 단계, (a) preparing a powdery extract by immersing the narrow leaf stem and leaves, stems or a mixture thereof in a mixed solvent of ethanol and water,
    (b) 그 분말상의 추출물을 증류수에 현탁시킨 후 헥산(n-Hexane), 디클로로메탄(methylene chloride), 에틸아세테이트(EtOAc) 및 부탄올(butanol)을 이용하여 순차적으로 분획한 후 디클로로메탄 분획물을 수득하는 단계, (b) The powdery extract was suspended in distilled water, and then sequentially fractionated with hexane (n-Hexane), dichloromethane (methylene chloride), ethyl acetate (EtOAc) and butanol, to obtain a dichloromethane fraction. Steps,
    (c) 그 분획물을 셀라이트 표면에 코팅시킨 후 헥산, 디클로로메탄, 에틸아세테이트 및 메탄올 각각을 이동상으로 하여 컬럼크로마토그래피를 실시하여 총 4개의 정제물을 얻는 단계, (c) coating the fractions on the surface of celite and performing column chromatography on hexane, dichloromethane, ethyl acetate and methanol as mobile phases to obtain a total of four purified products,
    (d) 그 총 4개의 정제물 중 2번 정제물에 대해서 헥산:에틸아세테이트:메탄올(1:2:0.2, v/v)을 이동상으로 하고 순상실리카겔 컬럼크로마토그래피를 실시하여 총 20개의 정제물을 얻는 단계, (d) Purified phase 2 silica gel column chromatography was performed on hexane: ethyl acetate: methanol (1: 2: 0.2, v / v) with respect to the second purified product from the total 4 purified products. Getting it,
    (e) 그 20개의 정제물 중 16번 정제물에 대해서 클로로포름:메탄올(10:1, v/v)을 이동상으로 하여 순상실리카겔 컬럼크로마토그래피를 실시하여 총 18개의 정제물을 얻는 단계, (e) performing pure silica gel column chromatography on purified product 16 of the 20 purified products using chloroform: methanol (10: 1, v / v) as a mobile phase to obtain a total of 18 purified products,
    (f) 그 18개의 정제물 중 9번 정제물을 수득하는 단계를 포함하는(f) obtaining the ninth of the eighteen purifieds
    좁은잎천선과으로부터 블라디놀 F를 분리하는 방법.A method for separating vladinol F from a narrow leaf herb.
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