KR20130071666A - Anti-inflammation composition using vladinol f - Google Patents

Abstract

PURPOSE: An anti-inflammatory composition using vladinol F isolated from F. erecta var. sieboldii is provided to suppress generation of NO, PGE2 (prostaglandin), and inflammatory cytokines (TNF-alpha, IL-1beta, and IL-6). CONSTITUTION: An anti-inflammatory composition contains vladinol F as an active ingredient. A method for isolating vladinol F from F. erecta var. sieboldii comprises the steps of: dipping F. erecta var. sieboldii leaves, stems, or a mixture thereof in a mixture solvent of ethanol and water and preparing a power extract; suspending the extract in distilled water, and sequentially fractioning to obtain a dichloromethane fraction; coating a celite surface with the fraction and performing column chromatography using hexane, dichloromethane, ethyl acetate, and methanol to obtain 4 purified materials; performing normal phase column chromatography of a second purified material using hexane:ethyl acetate:methanol (1:2:0.2, v/v) to obtain total 20 purified materials; performing normal phase silicagel column chromatography of 16th purified material among 20 purified materials using chloroform:methanol (10:1, v/v) and preparing total 18 purified materials; and collecting 9th purified material among 18 purified materials. [Reference numerals] (AA) MC fraction of a F. erecta var. sieboldii extract (11.2g); (BB) Column chromatography; (CC) Normal phase silica gel (5cm×60cm); (DD) Normal phase silica gel (2.0cm×50cm)

Description

Anti-inflammation Composition Using vladinol F}

The present invention relates to anti-inflammatory compositions using Vladinol F.

Inflammation is the defensive response of a living body to external infectious agents such as external physical and chemical stimuli, bacteria, fungi, viruses and allergens.

The inflammatory response is part of the innate immune response, and as in other animals, the innate immune response of humans begins by recognizing and attacking macrophages as non-self through a pattern of cell surfaces that are specific to the pathogen. During the inflammation reaction, plasma accumulates on the inflamed area, diluting the toxicities secreted by the bacteria, increasing the blood flow, and accompanied by symptoms such as erythema, pain, edema, and fever.

These inflammatory reactions involve a variety of biochemical events, in particular, cyclooxygenase (COX), which is associated with the nitric oxide synthase (NOS) and the biosynthesis of various prostaglandins, .

There are three isomers of NOS: endotoxin of bacteria such as calcium or camodanol-dependent eNOS (endothelial NOS), nNOS (neurogenic NOS), and lipopolysaccharide (IL-1β, TNF-α, IL There are iNOS (inducible NOS) induced by various inflammatory cytokines such as IL-6, IL-8 and IL-12 and produce NO from L-arginine.

NO produced by eNOS or nNOS plays an important role in maintenance of homeostasis by performing various physiological responses related to blood pressure control, neurotransmission, learning, memory, etc. However, NO produced by iNOS is associated with arthritis, sepsis, (Moncade S. et al, Pharmacol. Rev., 1991, 43, 109, Nature Medicine, 2001, 7, 1138; Mu, S., et al., Immunoprecipitation, , MM, J. Endotoxic Res. 7, p341, 2001).

The COX enzyme synthesizes PGG ₂ and PGH ₂ from arachidonic acid with hydroperoxidase (HOX) activity together with the function of COX. These compounds include PGE ₂ , PGF ₂ , PGD ₂ , prostacyclin and create a duplex thromboxane _{a 2 (thromboxane A2, TxA2)} . Among the functions of COX, the function of PGH synthase is involved in the pain and inflammation reaction through synthesis of PGE ₂ .

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.

Therefore, 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 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.

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.

Other and further objects of the present invention will be described below.

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).

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.

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.

As used herein, "anti-inflammatory" is meant to include the improvement, treatment, inhibition or delay of the onset of an inflammatory disease as defined below.

In the present specification, the above-mentioned "inflammatory disease" is defined as any state specified by a local or systemic bio-defense reaction against external physical or chemical stimulation or infection of an external infectious agent such as bacteria, fungi, viruses, . This reaction is enzyme secretion (e.g., iNOS, COX-2, etc.) activation, release of inflammatory mediators (e. G., NO, TNF-α, IL -6, IL-1β, PGE 2 related to various inflammatory mediators and the immune cells ), Bodily fluid infiltration, cell migration, and tissue destruction, and manifest externally by symptoms such as erythema, pain, edema, fever, deterioration or loss of specific function of the body. The inflammatory disease may be acute, chronic, ulcerative, allergic or necrotic, so long as it is included in the definition of inflammatory diseases as described above, it may be acute, chronic, ulcerative, allergic, Whether it is necrotic or not. 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.

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 an 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 ordinary skill of those skilled in the art.

The subject to which the composition of the present invention can be applied (prescription) is preferably a mammal and a person, particularly a human.

The 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. Aqueous or oily suspensions for injection), topical formulations (solutions, creams, ointments, gels, lotions, patches) and the like.

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.

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. The carrier may be selected from one or more of suitable pharmaceutical formulations according to the formulation of the pharmaceutical composition of the present invention.

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, hydropropylmethylcellulose And suspending agents and dispersing agents such as sodium alginate and polyvinylpyrrolidone. Suitable excipients when prepared from injection solutions include Ringer's solution, isotonic sodium chloride, and the like.

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.

The daily dose of the pharmaceutical composition of the present invention is usually 0.001 to 150 mg / kg body weight, and may be administered once or several times. However, since the dosage of the pharmaceutical composition of the present invention is determined in view of various related factors such as route of administration, age, sex, weight, and patient's severity of the patient, the dose is limited in any aspect to the scope of the present invention Should not be understood to be.

The composition of this invention can be grasped | ascertained by food compositions, such as a functional drink, in a specific aspect.

The food composition of the present invention may contain sweetening agents, flavoring agents, physiologically active ingredients, minerals and the like in addition to the active ingredients thereof.

Sweetening agents may be used in an amount that sweetens the food in a suitable manner, and may be natural or synthetic. Preferably, natural sweeteners are used. Examples of natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose and maltose.

Flavors may be used to enhance taste or flavor, both natural and synthetic. Preferably, a natural one is used. When using natural ones, the purpose of nutritional fortification can be performed in addition to the flavor. 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. Also, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, banks and the like can be used. The natural flavoring agent may be a liquid concentrate or a solid form of extract. Synthetic flavors may be used depending on the case, and synthetic flavors such as esters, alcohols, aldehydes, terpenes and the like may be used.

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.

As the mineral, calcium, magnesium, chromium, cobalt, copper, fluoride, germanium, iodine, iron, lithium, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, silicon, sodium, sulfur, vanadium and zinc can be used.

In addition, the food composition of the present invention may contain preservatives, emulsifiers, acidifiers, thickeners and the like as needed in addition to the above sweeteners.

Such preservatives, emulsifiers and the like are preferably added in a very small amount as long as they can attain an application to which they are added. The term " trace amount " means, when expressed numerically, in the range of 0.0005% by weight to about 0.5% by weight based on the total weight of the food composition.

Examples of the preservative which can be used include calcium sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate and EDTA (ethylenediaminetetraacetic acid).

Examples of the emulsifier which can be used include acacia gum, carboxymethyl cellulose, xanthan gum, pectin and the like.

Examples of the acidulant that can be used include acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, and phosphoric acid. Such an acidulant may be added so that the food composition has a proper acidity for the purpose of inhibiting the growth of microorganisms other than the purpose of enhancing the taste.

Agents that may be used include suspending agents, sedimentation agents, gel formers, bulking agents and the like.

In addition, herbal medicines may be added to enhance flavor and palatability and to add other functionalities (such as preventing arthritis or osteoporosis). 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.

In another aspect, the present invention can be understood as a cosmetic composition.

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.

The composition of the present invention may contain an acceptable carrier in cosmetic preparations in addition to its active ingredients.

As used herein, the term " acceptable carrier for a cosmetic preparation "refers to a compound or composition which is already known and used in the cosmetic preparation, or which is a compound or composition to be developed in the future, and which is not toxic to the human body.

The carrier may be included in the composition of the present invention in an amount of from about 1% by weight to about 99.99% by weight, preferably from about 50% by weight to about 99% by weight of the composition, based on the total weight thereof.

However, since the ratio depends on the above-mentioned formulation of the cosmetic product and its specific application site (face or hands) or the desired amount of application thereof, the ratio is to limit the scope of the present invention in any aspect It should not be.

Examples of the carrier include alcohols, oils, surfactants, fatty acids, silicone oils, humectants, moisturizers, viscosifiers, emulsifiers, stabilizers, sunscreens, coloring agents and perfumes.

The compounds / compositions which can be used as the carrier and which can be used as alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, moisturizers, viscosifiers, emulsions, stabilizers, sunscreens, A person skilled in the art can select and use appropriate substances / compositions.

In another aspect, 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. 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.

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 the solute is dissolved in 100 ml of the solution.

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.

As described 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.
FIG. 3 is a 13C NMR spectrum of Vladinol F. FIG.
FIG. 4 is the DEPT NMR spectrum of Vladinol F. FIG.
FIG. 5 is the HSQC (2D) NMR spectrum of Vladinol F. FIG.
FIG. 6 is the HMBC (2D) NMR spectrum of Vladinol F. FIG.
[Figure 7] is the LC-MS / MS spectrum of Vladinol F.
8 is a result showing that NO and inhibitory activity of Vladinol F production and no particular cytotoxicity.
9 is a result showing the inhibitory activity of PGE ₂ production of Vladinol F.
10 to 12 are results showing the cytokine (TNF-α, IL-1 and IL-6) production inhibitory activity of Vladinol F.

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.

< Example > Narrow Leaf Lines  Isolation, Purification and Identification of Active Substances with Anti-inflammatory Activity from Extracts

Example 1 Isolation and Purification of Anti-inflammatory Active Compounds from Narrow Leaf Lines and 80% Ethanol Extracts

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. Size of the column was used to 7cm × 20cm and hexane, dichloromethane, ethyl acetate, methanol, respectively (Please confirm) mobile phase. 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. 16 (FEM-MC-16) of the total 20 purified product was subjected to pure silica gel (please confirm) column chromatography again . 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.

1 shows the separation process of the active material.

Example 2 Identification of Materials

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. And 2D NMR analysis of HSQC method (FIG. 5) and through the HMBC (6) document the results of more than 9 times purified water was estimated that lignans form composed of 20 carbon (Planta medica , 56 475-477, 1990) was identified as Vladinol F.

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).

< Experimental Example > Vladinol  F anti-inflammatory activity experiment

Experimental Example 1 Cell Culture

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.

<Experimental Example 2> A nitrogen compound of Cape Town play F (Nitric oxide) Inhibitory Efficacy Assessment

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 produced NO Griess reagent [1% (w / v) sulfanilamide, 0.1% (w / v) naphylethylenediamine in 2.5% (v / v) phosphoric acid] by using the NO ² present in the cell culture ^- in the form of Respectively. 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).

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).

Experimental Example 3 Cytotoxicity Evaluation of Vladinol F- LDH assay

RAW 264.7 cells (1.5 × 10 ⁵ cells / mL) were cultured in DMEM medium for 24 hours at the same time with LPS (1 μg / mL) and cultured for 3 min at 3,000 rpm for 5 min. 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). The average absorbance values for each sample group were determined and compared with the absorbance values of the control (LDH control, 1: 5000) to evaluate cytotoxicity.

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.

For <Experiment 4> Cape Town play F PGE ₂ (Prostaglandin E ₂ ) Evaluation of production inhibitory efficacy

RAW 264.7 cells were adjusted to 1.5 × 10 ⁵ cells / mL using DMEM medium, and then inoculated in 24 well plates, and cultured 18 hours before in a 5% CO ₂ incubator. 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 supernatant was obtained by centrifuging the culture medium (12,000 rpm, 3 min) to measure PGE ₂ . PGE ₂ was measured using PGE ₂ ELISA kit (R & D Systems Inc., Minneapolis, MN, USA) and the r2 value of the standard curve for the standard was 0.99 or more.

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).

<Experiment 5> cytokines play VLADIMIR F (TNF -α, IL -1 and IL -6) Inhibitory Efficacy Assessment

RAW 264.7 cells (1.5 × 10 ⁵ cells / mL) were inoculated in 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.

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.

Statistical processing

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 method (* P <0.05 , ** P <0.01 ).

Claims (6)

Anti-inflammatory composition comprising Vladin F as an active ingredient.
The method of claim 1,
The anti-inflammatory refers to amelioration, treatment, inhibition of onset or delayed on the inflammatory disease,
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.
The method according to claim 1 or 2,
The composition is an anti-inflammatory composition, characterized in that the pharmaceutical composition.
The method according to claim 1 or 2,
The composition is an anti-inflammatory composition, characterized in that the food composition.
The method according to claim 1 or 2,
The composition is an anti-inflammatory composition, characterized in that the cosmetic composition.
(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) 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) 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) 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) 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) obtaining the ninth of the eighteen purifieds
A method for separating vladinol F from a narrow leaf herb.

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