KR101540957B1 - Composition containing moojabanchromanol b for preventing or treating inflammatory disease - Google Patents

Abstract

The present invention relates to a composition for preventing or treating an inflammatory disease containing mojabanchromanol b as an active ingredient. The chromosome b of the present invention inhibits the inflammatory response by inhibiting the production of nitric oxide which is an inflammatory mediator and inhibiting the production of TNF-α, IL-6 and IL-1β, inflammatory cytokines. And thus can be usefully used for the prophylaxis or treatment of inflammatory diseases.

Description

[0001] The present invention relates to a composition for preventing or treating an inflammatory disease containing chomannol b as an active ingredient (Composition containing moojabanchromanol b for preventing or treating inflammatory disease)

The present invention relates to a composition for preventing or treating an inflammatory disease containing mojabanchromanol b as an active ingredient.

 With the recent increase in average life expectancy and changes in the industrial environment, the number of lifestyle-related diseases such as cancer, diabetes, hypertension, obesity, and vascular diseases is increasing every year as the food culture in Korea is changing. In addition, the onset of inflammation caused by immunomodulatory disorders has continued, and the incidence of inflammatory diseases caused thereby has also increased in recent decades. Although many inflammatory medicines have been developed and used, these medicines have a disadvantage in that they cause side effects such as skin atrophy, growth retardation and weakening of immunity and do not act on complex inflammatory mechanisms. Therefore, there is a need to develop a substance that can overcome these shortcomings and treat inflammation more fundamentally.

On the other hand, the inflammatory reaction is a mechanism to regenerate and repair the injured area when an invasion that brings about a physical change such as a physical action, a chemical substance, a bacterial infection, or the like is applied to a living body or tissue. It has been known that vasoactive substances such as serotonin, bradykinin, prostaglandin, hydroxyeicosatetraenoic acid (HETE) and leukotriene are liberated, resulting in increased vascular permeability and inflammation. In inflammation and immune responses, macrophages secrete nitrogen monoxide (NO), prostaglandin E ₂ (PGE ₂ ), and inflammatory cytokines through activation. LPS (lipopolysaccharide) involved in the activation of macrophages is one of the cell wall components of Gram - negative bacteria and is an external factor well known as endotoxin. LPS is an inflammatory cytokine such as TNF-alpha, IL-6, and IL-1β (interleukin-1 beta) in macrophages or mononuclear cells such as RAW 264.7 cells pro-inflammatory cytokines, which are known to increase inflammation.

In recent years, it has been reported that a substance isolated and purified from natural materials is less toxic than other organic compounds and is safe even if ingested for a long period of time. Therefore, attempts to develop a therapeutic agent using such a substance have been actively conducted, but the result is still small .

On the other hand, mojabanchromanol is an active substance isolated and purified from moth, and is known to have excellent antioxidative effect, bone health improvement and bone strengthening effect, prevention or treatment effect of bone disease, and calcium absorption enhancement effect (Antioxidant alga Sargassum siliquastrum Journal of Environmental Biology July 2008, 29 (4) 479-484 (2008)).

As described above, various pharmacological effects of mungbean chomannol are known, but the anti-inflammatory effect has not yet been known, and research on this has been limited.

Accordingly, the inventors of the present invention have been studying natural materials derived from natural materials having excellent inflammation-suppressing effects, and found that mothromochromin b has few side effects in vivo, inhibits the production of nitrogen monoxide, an inflammatory mediator, and inhibits the inflammatory cytokine TNF- alpha, IL-6, and IL-1 [beta] to inhibit the inflammatory response, thereby completing the present invention.

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases containing mojabanchromanol b as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or ameliorating an inflammatory disease containing mojabanchromanol b as an active ingredient.

The present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease containing mojabanchromanol b as an active ingredient.

The present invention also provides a food composition for preventing or ameliorating an inflammatory disease containing mojabanchromanol b as an active ingredient.

The chromosome b of the present invention inhibits the inflammatory response by inhibiting the production of nitric oxide which is an inflammatory mediator and inhibiting the production of TNF-α, IL-6 and IL-1β, inflammatory cytokines. And thus can be usefully used for the prophylaxis or treatment of inflammatory diseases.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view showing a process for preparing an extract of Solanum tuberculosis of the present invention and a process for separating and purifying mungbean chromanol b.
FIG. 2 is a graph showing the degree of inhibition of NO production in RAW 264.7 cells induced by LPS-induced inflammation, when treated with the extracts of the present invention.
FIG. 3 is a graph showing the degree of inhibition of IL-6 production upon treatment of RAW 264.7 cells with LPS-induced inflammation, according to the present invention.
FIG. 4 is a graph showing inhibition of TNF-.alpha. Production in RAW 264.7 cells induced by LPS-induced inflammation, when treated with Solanum tuberculosis extract of the present invention.
FIG. 5 is a graph showing the degree of inhibition of IL-1β production in RAW 264.7 cells induced by LPS-induced inflammation, when treated with Solanum tuberculosis extract of the present invention.
FIG. 6 is a graph showing cytotoxicity of the extract of Solanum tuberculosis of the present invention in RAW 264.7 cells induced by LPS.
FIG. 7 is a graph showing the degree of inhibition of IL-6 production by RAW 264.7 cells induced by LPS in each solvent fraction when treated with the extract of Solanum tuberculosis of the present invention.
FIG. 8 is a graph showing inhibition of TNF-.alpha. Production by RAW 264.7 cells induced by LPS in each solvent fraction when treated with the extract of Solanum tuberculosis extract of the present invention.
FIG. 9 is a graph showing the degree of inhibition of IL-6 production in RAW 264.7 cells induced by LPS-induced inflammation, when treated with the n-hexane fraction of the present invention.
FIG. 10 is a graph showing inhibition of TNF-.alpha. Production in RAW 264.7 cells induced by LPS-induced inflammation, when treated with the n-hexane fraction of the present invention.
FIG. 11 is a graph showing the degree of inhibition of IL-6 production upon treatment of RAW 264.7 cells induced by LPS with fractions obtained by fractionating the extracts of Solanum tuberculosis of the present invention by TLC.
FIG. 12 is a graph showing the inhibition of TNF-.alpha. Production in the fraction obtained by fractionation of RAW 264.7 cells induced by LPS with TLC by the extract of Solanum tuberculosis extract of the present invention.
FIG. 13 is a graph showing the degree of inhibition of IL-6 production in the HPLC fraction treated with RAW 264.7 cells induced by LPS with the extract of Solanum tuberculosis of the present invention.
FIG. 14 is a graph showing inhibition of TNF-.alpha. Production upon treatment of RAW 264.7 cells with LPS-induced inflammation by HPLC fractionation of the extracts of Solanum tuberculosis of the present invention.
FIG. 15 is a graph showing the degree of suppression of NO production in RAW 264.7 cells induced by LPS in marmosom Chromanol b treatment according to the present invention.
FIG. 16 is a graph showing the degree of inhibition of IL-6 production in RAW 264.7 cells induced by LPS-induced inflammation, in the case of mumps chromoman b treatment according to the present invention.
FIG. 17 is a graph showing inhibition of TNF-.alpha. Production in RAW 264.7 cells induced by LPS with mumps chromomann b according to the present invention.
FIG. 18 is a graph showing the degree of inhibition of IL-1? Production in RAW 264.7 cells induced by LPS with mumps chromomann b according to the present invention.
FIG. 19 shows the cytotoxicity of mumps chromanol b according to the present invention in RAW 264.7 cells induced by LPS.

The present invention provides a composition for the prevention or treatment of inflammatory diseases containing, as an active ingredient, mojabanchromanol b represented by the following formula (1).

The composition comprises a pharmaceutical composition or a food composition

Hereinafter, the present invention will be described in detail.

The active ingredient mamvanti chromanol b in the composition of the present invention can be obtained by a conventional extraction method, and commercially available ones can be purchased and used, and they can be obtained by using a conventional organic synthesis method. Typical extraction methods include, but are not limited to, ultrasonic extraction, filtration, and reflux extraction.

In the present invention, the mother liquor Chromanol b is obtained through the following steps.

First, the alfalfa seedlings are washed, lyophilized, and then pulverized and stored at a temperature of -10 to 30 ° C. After extracting the dried and grounded Solanum tuberculosis with an organic solvent, it is concentrated under reduced pressure and lyophilized to obtain powdery Solanum tuberculosis extract in powder form. At this time, the organic solvent may include C ₁ -C ₄ alcohol, water, or a mixed solvent thereof. The C ₁ -C ₄ alcohol is preferably methanol or ethanol, but is not limited thereto.

Thereafter, distilled water is added to the extracts of Solanum tuberculosis, and the resulting suspension is fractionated with hexane, chloroform, ethyl acetate and butanol in order to obtain solvent fractions. The n-hexane fraction having the highest anti-inflammatory activity was purified by silica gel column chromatography; Sephadex LH-20 column chromatography; Thin Layer Chromatography (TLC); Or high-performance liquid chromatography (HPLC), to obtain a single substance, maban chromanol b.

The chromosome b of the present invention inhibits the inflammatory response by inhibiting the production of nitric oxide which is an inflammatory mediator and inhibiting the production of TNF-α, IL-6 and IL-1β, inflammatory cytokines. And thus can be usefully used for the prophylaxis or treatment of inflammatory diseases.

The inflammatory disease may be selected from the group consisting of venous sinusitis, gastritis, rhinitis, conjunctivitis, asthma, bronchitis, dermatitis, atopic dermatitis, inflammatory bowel disease, inflammatory liver disease, inflammatory vascular disease, inflammatory rheumatoid arthritis, restenosis, psoriasis, Diseases, and the like, but are not limited thereto.

The composition of the present invention may further comprise at least one known active ingredient having the effect of preventing or treating inflammatory diseases together with mabunamic cholinol b.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. In addition, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral formulations such as syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method. Suitable formulations known in the art are preferably those as disclosed in Remington ' s Pharmaceutical Science, recently, Mack Publishing Company, Easton PA. Examples of carriers, excipients and diluents which may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When the composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The term "administering" as used herein is meant to provide any desired composition of the invention to a subject in any suitable manner.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the individual, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. For the desired effect, the mumps chromoman b of the present invention may be administered in an amount of 1 mg / kg to 10000 mg / kg per day, and may be administered once a day or divided into several doses.

The pharmaceutical composition of the present invention may be administered to a subject in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the prevention or treatment of inflammatory diseases.

In the present invention, the term "health functional food" refers to a food having a biological control function such as prevention and improvement of disease, bio-defense, immunity, recovery after disease and aging inhibition.

The composition of the present invention may be added to a health functional food for the purpose of preventing or improving an inflammatory disease. When the moth brown chromanol b of the present invention is used as a food additive, the moth brown chromanol b can be directly added or used together with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, the mother liquor Chromanol b of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of foods to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharine and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, A carbonating agent used in a carbonated beverage, and the like. In addition, the composition of the present invention may comprise flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of such additives is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, preferred examples, experimental examples, and formulation examples are provided to facilitate understanding of the present invention. However, the following examples, experimental examples and preparation examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples, experimental examples and preparation examples.

[Example 1: Isolation of hymenchromanol b from Solanaceae moth)

1. Preparation of extracts of Solanum tuberculosis

Solitary the dog Sargassum (Myagropsis myagroides) collected in Pusan Songjeong in 2011, and then washed thoroughly with fresh water, then lyophilized to finely pulverized and stored at -20 ℃. The dried and pulverized alfalfa seedlings were extracted with methanol to obtain methanol extract of Allium tuberculosis. Methanol extract was extracted from alfalfa ganoderma lucidum and concentrated under reduced pressure to remove methanol, followed by lyophilization to obtain powdery alfalfa ganoderma lucidum extract. The dried extracts of the Solanum tuberculosis extract were stored at 4 ° C and used in the experiment.

2. Isolation of mungbean chromanol b from extracts

2-1. Fraction of solvent system

The methanol extract of Alaska pollack gum obtained in the above step 1 was concentrated to remove methanol, and then fractionated in order of high polarity solvent and high solvent. That is, distilled water was added to the methanol extract of Allium tuberculosis, and the resulting suspension was fractionated with hexane, chloroform, ethyl acetate and butanol in order to obtain respective solvent fractions. In the case of a non-polar solvent, magnesium sulfate was added to remove moisture from the solvent layer. Each solvent fraction was concentrated and weighed. FIG. 1 shows a schematic diagram of the process for the preparation of the external mothballs of the present invention and the separation and purification process of the mothballs chromanol b.

As shown in Fig. 1, 13 g of the hexane fraction, 11 g of the chloroform fraction, 0.5 g of the ethyl acetate fraction and 4.4 g of the butanol fraction were obtained after the fractionation.

2-2. Silica gel column chromatography (silica gel column chromatography)

The methanol extract of the alfalfa mushroom obtained in the above step 1 was sequentially fractionated with hexane, chloroform, ethyl acetate and butanol, and the hexane fraction having the best anti-inflammatory activity was concentrated under reduced pressure. The concentrated hexane fractions were subjected to flash silica gel column chromatography using chloroform and chloroform-methanol (100: 1 to 1: 1, stepwise, v / v) as an elution solvent, The methanol fraction showed excellent anti - inflammatory activity.

2-3. Sephadex LH-20 column chromatography (Sephadex LH-20 column chromatography)

The chloroform-methanol (50: 1, v / v) fraction having excellent anti-inflammatory activity was concentrated in the above 2-2, and chloroform-methanol (1: 1, v / v) And subjected to Sephadex LH-20 column chromatography.

2-4. Thin Layer Chromatography (TLC)

After performing Sephadex LH-20 column chromatography in 2-3 above, the obtained eluate was analyzed by silica gel TLC (hexane-ethyl acetate = 2: 1, v / v) tubes 7 to 15 (Fr. 1) and tubes 16 to 24 (Fr. 2). Fr. 2 was subjected to chromatography using an ODS Sepak cartridge using an aqueous methanol containing 60 to 90% water as an elution solvent. As a result, Fr. 3, Fr. 4, Fr. 5, Fr. 6, Fr. 7 fractions were obtained.

2-5. High Performance Liquid Chromatography (HPLC)

The fractions obtained in 2-4 above were subjected to HPLC analysis (ODS column id 4.6 x 150 mm, 50 → 100% aqueous methanol gradient, 1 ml / min) to obtain six fractions (Fr. 4-1, Fr. 4-2, Fr. 4-3, Fr. 4-4, Fr. 4-5, Fr. 4-6). Finally, Fr. 4-3 were separated by preparative HPLC (ODS column, id 10 x 150 mm, 75% aqua methanol, 3 ml / min) to purify Mm-5 (2 mg) for retention time 38 minutes.

2-6. Identification of mothball Chromanor b

Fr. 4-3 was fractionated by preparative HPLC (ODS column, id 10 x 150 mm, 75% aqua methanol, 3 ml / min) to purify Mojabanchromanol b (2 mg) with a retention time of 38 minutes . Sargassum having the same structural formulas and below with a purified compound ¹ H NMR and ¹³ C NMR spectral analysis was identified as playing chroma b.

[constitutional formula]

[Experimental Example 1: Anti-inflammatory effect of extracts of Solanum tuberculosis mats]

In order to investigate the anti-inflammatory effect of the extracts of Solanum tuberculosis of the present invention, the following procedure was carried out.

1. Preparation and culture of RAW 264.7 cell line

RAW 264.7 cells, the macrophage line of Murine, distributed in Korean Cell Line Bank (KCLB40071), were placed in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin, and incubated at 37 ° C in 5% CO ₂ < / RTI > The culture of the cultured RAW 264.7 cells was collected with a scraper, and centrifuged at 1000 rpm, 3 minutes, and 4 ° C. The cells were diluted to 2.5 × 10 ⁵ and 1 × 10 ⁶ cells / mL by adding 10% FBS-DMEM medium. The cells were cultured in a CO ₂ incubator (MCO-15 AC, Sanyo, Osaka, Japan) for 18 to 20 hours at 37 ° C. and then washed three times with DPBS to remove floating cells 10% FBS-DMEM medium was added. Then, PBS, LPS (1 g / mL) and extracts of Solanum tuberculosis extract obtained from 1 in 1 of the above Example 1 were added to the culture medium and cultured in a CO ₂ incubator (MCO-15AC, Sanyo, Osaka , Japan) for 12 to 24 hours.

2. Measurement of nitric oxide production inhibitory activity

RAW 264.7 cells prepared in 1 above were treated with LPS to induce inflammation. Then, the extracts of Solanum tuberculosis obtained in 1 in 1, which was separated from the above Example 1 2, were treated by concentration to produce NO inhibiting activity Were measured.

Specifically, a supernatant of RAW 264.7 cells was added to a microplate and an equal amount of griess reagent (1% sulfanilamide + 0.1% naphthylendiamine dihydrochloride (1: 1) was added and reacted at room temperature for 10 minutes. Absorbance was measured at 540 nm using a microplate reader (Model 550, Bio-Rad, Richmond, USA). The concentration of nitric oxide (NO) in the cell culture fluid was calculated by comparing the standard curve with the concentration of sodium nitrite (NaNO ₂ ). The results are shown in Fig.

As shown in FIG. 2, it was confirmed that the extracts of the extracts of the present invention inhibited NO production in a concentration-dependent manner.

Therefore, it can be seen that the extracts of Solanum tuberculosis of the present invention inhibit the production of nitrogen monoxide, which is an inflammation mediator, and thus the effect of suppressing inflammation is excellent.

3. Measurement of inflammatory cytokine production inhibitory activity

RAW 264.7 cells prepared in the above 1 were treated with LPS to induce inflammation. In order to examine inflammatory cytokine production inhibitory activity of the extracts of the present invention, Were treated by concentration Expression of the inflammatory cytokines IL-6, TNF-a and IL-l [beta] was measured.

Specifically, the supernatant of RAW 264.7 cells was added to a microplate and the amount of secretion of TNF-α, IL-6 and IL-1β cytokines was measured using an ELISA kit (Mouse ELISA set, BD Bioscience, San Diego, USA) Were measured. First, ELISA microplates were coated with anti-mouse TNF-α, IL-6 and IL-1β antibodies (mAntibody) and blocked with 10% FBS solution. Biotinylated anti-mouse TNF-α, IL-6 monoclonal antiobodies (mAb) and streptavidin-horseradish peroxidase conjugate were added to the culture medium, Was added. For IL-1β, biotinylated anti-mouse IL-1β was added and allowed to react for 1 hour, followed by addition of streptavidin-horseradish peroxidase and reaction for 30 minutes. After that, the OPD solution was added, and the absorbance was measured at 490 nm using a microplate reader (Model 550, Bio-rad, Richmond, USA). The results are shown in Figs.

As shown in FIGS. 3 to 5, it was confirmed that the extracts of Solanum tuberculosis of the present invention inhibited the production of inflammatory cytokines IL-6, TNF-α and IL-1β in a concentration-dependent manner.

Therefore, it can be seen that the extracts of the extracts of Solanum tuberculosis of the present invention significantly inhibit the production of inflammatory cytokines in a concentration-dependent manner, so that the effect of suppressing inflammation is excellent.

[Experimental Example 2: Measurement of cell stability of extracts of Solanum tuberculosis extracts]

In order to examine the cell stability of the Solanum tuberculosis extract of the present invention, RAW 264.7 cells prepared in Experimental Example 1 were treated with Solanum tuberculosis extract obtained in 1 in 1 of Example 1, The toxicity assessment was performed according to the conventionally known MTT assay.

Specifically, RAW 264.7 cells were cultured at a concentration of 1 × 10 ⁶ cells / ml, followed by addition of PBS and culture medium, and cultivation was carried out at 37 ° C. in a 5% CO ₂ incubator. MTT (thiazol blue tetrazolium bromide, 5 mg / ml) reagent was added for 2 hours to induce fomazan crystal formation. After centrifugation at 4 ° C and 2000 rpm for 10 minutes, the supernatant was removed, and the absorbance was measured at 540 nm by adding dimethyl sulfoxide (DMSO). The proliferation rate of RAW 264.7 cells was calculated using the following formula 1, and the results are shown in FIG.

As shown in FIG. 6, it was confirmed that the extracts of Solanum tuberculosis of the present invention showed no cytotoxicity at all concentrations.

Therefore, it was confirmed that the extracts of Solanum tuberculosis of the present invention have no cytotoxicity and thus can be effectively used as medicines or food preparations having little or no side effects. The antiinflammatory effect confirmed in Experimental Example 1 is cytotoxicity Is a pharmacological effect of the unrelated gibberellin.

[Experimental Example 3: Measurement of anti-inflammatory effect of extracts from Solanum tuberculosis extract]

In order to isolate and identify a substance exhibiting anti-inflammatory activity in the extracts of Solanum tuberculosis of the present invention, the following process was carried out.

1. Anti-inflammatory effects of solvent fractions

The above embodiment of a separate one from the two of the first Solitary dog Sargassum methanol extract obtained in 1 n by using the polarity difference between the solvents hexane (n -hexane), chloroform (chloroform), ethyl acetate (ethyl acetate), butanol butanol and water solvent to obtain fractions sequentially. Each of the obtained fractions was treated with RAW 264.7 cells at a concentration of 50, 100 g / ml and IL-6 and TNF-α cytokines were secreted. The results are shown in Figures 7 and 8.

As shown in FIGS. 7 and 8, it was confirmed that the fraction of n -hexane in the fractions significantly inhibited the secretion amount of IL-6 and TNF-? Cytokine as compared with other fractions.

2. Anti-inflammatory activity of silica gel column chromatography fractions

Showed the best anti-inflammatory activity in the first n - hexane (n -hexane) mixed solvent of chloroform and methanol and the fractions (each solvent is 100: 1: 1, 50: 1, 20: 1, 5: 1, 1 Lt; / RTI > solvent mixture) were subjected to silica gel column chromatography, respectively, to obtain five fractions. Each of the obtained fractions was treated with RAW 264.7 cells induced by LPS at a concentration of 50 and 100 g / mL, respectively, and secreted amounts of IL-6 and TNF-α cytokines were measured. The results are shown in FIGS. 9 and 10. FIG.

As shown in Figures 9 and 10, the silica gel column chromatographic fraction performed with a 50: 1 mixture of chloroform and methanol significantly inhibited the secretion of IL-6 and TNF- [alpha] cytokines compared to the other fractions Respectively.

3. Antiinflammatory activity of thin-film chromatographic fractions of Solanum tuberosum

Thin film chromatography was performed on fractions obtained by performing silica gel column chromatography using a 50: 1 mixed solvent of chloroform and methanol showing the best anti-inflammatory activity in 2 above. As a result, six fractions were obtained. Each of the obtained fractions was treated with RAW 264.7 cells induced by LPS at a concentration of 50 and 100 g / mL, respectively, and secreted amounts of IL-6 and TNF-α cytokines were measured. The results are shown in FIGS. 11 and 12. FIG.

As shown in FIGS. 11 and 12, it was confirmed that the TLC No. 4 fraction significantly inhibited the secretion amount of IL-6 and TNF-α cytokines as compared with other fractions.

4. Anti-inflammatory activity of high performance liquid chromatography (HPLC)

HPLC was performed on TLC No. 4 fraction showing the best antiinflammatory activity in the above 3, and as a result, 6 peaks were obtained. Each peak was fractionated and treated with LPS-induced RAW 264.7 cells at a concentration of 50, 100 g / mL, and the secretion of IL-6 and TNF-α cytokines was measured. The results are shown in Figs. 13 and 14.

As shown in FIGS. 13 and 14, it was confirmed that the 4-3 fraction of the peak fractions significantly inhibited the secretion amount of IL-6 and TNF-α cytokines as compared with other fractions.

[Experimental Example 4: Measurement of anti-inflammatory effect of mulberry Chromanol b]

1. Measurement of nitrogen monoxide production inhibitory activity

RAW 264.7 cells prepared in Experimental Example 1 were treated with LPS to induce inflammation. Then, a single substance, Mojabanchromanol b, isolated from 1/2 of Example 1, 0.1, 1, 10, 50, 100 ㎍ / ml) to measure the inhibitory activity of nitric oxide (NO) production as an inflammatory mediator. The NO production inhibitory activity was measured in the same manner as described in Experimental Example 1, 2, and the results are shown in FIG.

As shown in Fig. 15, it was confirmed that the mothobeochroman b of the present invention suppressed NO production in a concentration-dependent manner.

Therefore, it can be seen that the mothball Chromanol b of the present invention suppresses the production of nitrogen monoxide, which is an inflammation mediator, and thus has an excellent effect of suppressing inflammation.

2. Measurement of inflammatory cytokine production inhibitory activity

In order to investigate the inhibitory activity of inflammatory cytokine production of mabornochroman b of the present invention, RAW 264.7 cells prepared in Experimental Example 1 were treated with LPS to induce an inflammatory reaction, 1, the secretion of IL-6, TNF-α and IL-1β cytokines produced was measured. Measurement of cytokine was carried out in the same manner as described in Experimental Example 1, 3, and the results are shown in Figs.

As shown in Figs. 16 to 18, it was confirmed that the mothball chromoman b of the present invention significantly reduced the amount of produced IL-6, TNF-a and IL-1? Cytokines. In particular, it was confirmed that the secretion of IL-6 was completely inhibited at the concentrations of 50 and 100 占 퐂 / ml.

Therefore, it can be seen that the mothball Chromanol b of the present invention is excellent in the effect of inhibiting the inflammation, since it has excellent effect of inhibiting the production of inflammatory cytokines IL-6, TNF-α and IL-1β.

[Experimental Example 5: Measurement of cell stability of mulberry Chromanol b]

In order to examine the cell stability of the mothball Chromanor b of the present invention, RAW 264.7 cells prepared in Experimental Example 1 were coated with 0.1%, 1%, 10%, 50%, 100 μg / Ml, respectively, and then cytotoxicity was evaluated using MTT assay. The MTT assay was performed in the same manner as described in Experimental Example 2, and the results are shown in FIG.

As shown in Fig. 19, the cytotoxicity of mothball chromoman b of the present invention was not measured at all concentrations.

Therefore, the mothball chromanol b of the present invention has no cytotoxicity, and thus can be usefully used as a pharmaceutical or food preparation for anti-inflammation having little or no side effects. In addition, it can be seen that the anti-inflammatory effect of the moth, Chromanol b, identified in Experimental Example 4, is a pharmacological effect of mabornochroman b regardless of cytotoxicity.

Finally, it was confirmed that mabornochroman b, isolated from Solanum tuberculosis extract, is a compound capable of inhibiting inflammation without showing toxicity to RAW 264.7 cells.

Hereinafter, a pharmaceutical composition for preventing or treating an inflammatory disease and a food composition for preventing or ameliorating an inflammatory disease according to the present invention will be described. However, the present invention is not limited thereto. I would like to explain more specifically.

[Preparation Example 1: Preparation of pharmaceutical preparation]

1. Manufacturing of powder

Momochromin b 20 mg of formula 1

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

2. Preparation of tablets

Momochromin b represented by formula (1) b 10 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

3. Preparation of capsules

Momochromin b represented by formula (1) b 10 mg

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

4. Preparation of injections

Momochromin b represented by formula (1) b 10 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ 2H ₂ O 26 mg

(2 ml) per 1 ampoule in accordance with the usual injection preparation method.

5. Manufacture of liquids

Momochromin b 20 mg of formula 1

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

[Formulation example 2. Preparation of food preparation]

1. Manufacture of health food

Momochromin b represented by formula (1) b 100 mg

Vitamin mixture quantity

70 g of vitamin A acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

0.2 g of vitamin B12

Vitamin C 10 mg

Biotin 10 g

Nicotinic acid amide 1.7 mg

Folate 50 g

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

2. Manufacture of health drinks

Momochromin b represented by formula (1) b 100 mg

Vitamin C 15 g

Vitamin E (powder) 100 g

19.75 g of ferrous lactate

3.5 g of zinc oxide

Nicotinic acid amide 3.5 g

Vitamin A 0.2 g

Vitamin B1 0.25 g

Vitamin B2 0.3g

Water quantification

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The solution thus prepared was filtered and sterilized in a sterilized 2 liter container, It is used in the production of the health beverage composition of the invention.

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

Claims (3)

A pharmaceutical composition for the prophylaxis or treatment of inflammatory diseases, comprising as an active ingredient mojabanchromanol b, which is isolated from an extract of Myagropsis myagroides and represented by the following formula (1), wherein said Myagropsis myagroides extract is extracted with a hexane fraction and the hexane fraction extract is extracted with a mixed solution of chloroform: methanol = 100: 1 to 1: 1 in volume ratio.
[Chemical Formula 1]

The method of claim 1, wherein the inflammatory disease is selected from the group consisting of venous sinusitis, gastritis, rhinitis, conjunctivitis, asthma, bronchitis, dermatitis, atopic dermatitis, inflammatory bowel disease, inflammatory liver disease, inflammatory vascular disease, inflammatory rheumatoid arthritis, , Multiple sclerosis, and inflammatory lung disease. ≪ Desc / Clms Page number 24 >
A food composition for preventing or improving an inflammatory disease, comprising as an active ingredient mojabanchromanol b represented by the following formula (1) isolated from an extract of Myagropsis myagroides , wherein the Myagropsis myagroides ) Was extracted with a hexane fraction, and the hexane fraction extract was extracted with a mixed solution of chloroform: methanol = 100: 1 to 1: 1 in volume ratio.
[Chemical Formula 1]

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