KR20150034364A - Pharmaceutical compositions for prevention or treatment of autoimmune diseases comprising saucerneol D - Google Patents

Abstract

The present invention relates to a pharmaceutical composition capable of being used to effectively prevent and treat autoimmune diseases caused by an immune hypersensitivity reaction. According to the present invention, a saucerneol D compound derived from Saururus chinensis Baill. has excellent effects in inhibiting generation of nitrogen oxide caused by maturation of dendritic cells; inhibiting expression of surface molecules; inhibiting endocytosis; inhibiting cytokine secretion; inhibiting migration of dendritic cells; inhibiting expression of signal transduction substances activated in a maturation process; and inhibiting activation of T cells. Therefore, according to the present invention, a composition comprising a saucerneol D compound as an active ingredient ultimately reduces an immune reaction effectively by inhibiting maturation of dendritic cells. Accordingly, the composition can be practically used as an immunosuppressant capable of preventing or treating diseases such as autoimmune diseases caused by an immune hypersensitivity reaction. The composition comprises a saucerneol D compound represented by chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for preventing or treating autoimmune diseases,

The present invention relates to pharmaceutical compositions that can be used to effectively prevent and treat autoimmune diseases caused by immunological hypersensitivity reactions.

Dendritic cells (DCs) are powerful antigen presenting cells that induce and regulate acquired immune responses. It is present in immature form in most tissues such as lymphatic organs, skin, and digestive tract, and effectively captures and captures the antigen. Upon stimulation by lipopolysaccharide (LPS) or inflammation-inducing cytokines, they migrate to the T cell region of the nearby lymphoid tissue and mature. Mature dendritic cells have decreased antigen capture ability, while antigen presenting ability is increased and expression of various adhesion / co-stimulatory molecules (CD40, CD80, CD86) is increased. Mature dendritic cells not only present MHC I and MHC II molecule-bound antigen peptides to T cells but also bind to CD154 and CD28 molecules of T cells through increased adhesion / co-stimulatory molecules to induce activation of T cells . When dendritic cells mature, they secrete cytokines such as interleukin-12 and interferon-γ.

The response of immune cells can be divided into two categories: immune tolerance and immune response. Dendritic cells can mediate both responses. This dichotomous function of dendritic cells suggests that dendritic cells may be involved in autoimmune disease. Recent studies have shown that when artificially matured dendritic cells, T lymphocyte activation is promoted and autoimmune disease is induced . Several articles have suggested that autoimmune diseases such as psoriasis, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome and diabetes are associated with dendritic cells It is known.

On the other hand, Saururus chinensis is a perennial grassy area in Jeju Island and some parts of Mt. Jirisan. It grows in a humid and well - humid region with a distinctive smell. Its height is 50 ~ 100㎝. Leaves are 5 ~ 15㎝ long and 0.3 ~ 0.8㎝ wide. They are long oval and staggered. The surface of the leaf is light green and the back side is light white. When the flower is bloomed, the upper two or three leaves turn white. The rootstock is coarse and horizontal, and the leaves are heart-shaped or oval-shaped. Several spike-like white flowers bloom in May-August, and the fruit is rounded one by one in the bloom from September to October. Leaves and stem roots, including flowers, are used as medicines.

Studies on the ingredients of Saururus chinensis have been carried out on flavonoids, alkaloids, amino acids, fatty acids, quinones and essential oils. The main component of the outpost is methyl n-nonyl ketone, the stem contains hydrolyzable tannin, the leaves are quercetin, isoquercetrin, avicularin, , Hyperin, rutin, and hydrolyzable tannins.

On the other hand, sesquiterpene compounds are known to exist in the extract of Saururus cernuus which is a common plant distributed in the wetland of North America. (Rao et al., Tetrahedron Lett. 24 (45): 4947-4950, 1983) isolated compounds SC-8, SC-9, manastatin A, manastatin B and , And their structure was confirmed. Among them, manastantin A was confirmed to have a neuroprotective effect. In Korea, the isolation and identification of dimethyl terephthalate and quercetin have confirmed pharmacological action and antibacterial action (Kwak, Jae Wook, Ph.D. in Kyung Hee University, 1988). In addition, recently, a patent for anti-cancer activity of Saururus chinensis (Korean Patent No. 10-0248942) and anti-inflammatory activity (Korean Patent Laid-open No. 10-2004-0075135) has been filed.

However, no study has been made on the prophylactic or therapeutic effect of autoimmune diseases caused by immune hypersensitivity through a mechanism of inhibiting the maturation of dendritic cells of saucerneol D derived from Saururus chinensis.

Accordingly, the inventors of the present invention have been studying a functional pharmaceutical material derived from natural materials, and found that saucerneol D compound isolated from Saururus chinensis inhibits nitric oxide production, inhibits surface molecule expression, inhibits encapsulation, Confirming that it is useful in the prevention or treatment of diseases caused by immune hypersensitivity through inhibition of cytokine secretion, inhibition of dendritic cell migration, suppression of signal transduction substance activation during maturation and effective inhibition of T cell activation, .

Korean Patent Publication No. 10-2012-0006336 Korean Patent No. 10-0248942 Korean Patent No. 10-0530274

Accordingly, an object of the present invention is to provide a composition for preventing or treating an autoimmune disease, which comprises a saucerneol D compound as an active ingredient.

Another object of the present invention is to provide a health functional food for preventing or ameliorating an autoimmune disease, which comprises a saucerneol D compound as an active ingredient.

In order to achieve the above object, the present invention provides a composition for preventing or treating an autoimmune disease, comprising a saucerneol D compound or a pharmaceutically acceptable salt thereof as an active ingredient.

In one embodiment of the invention, the compound can be isolated from Saururus chinensis.

In one embodiment of the present invention, the compound may be included in the composition at a concentration of 1 [mu] M to 100 [mu] M.

In one embodiment of the present invention, the compound may have the effect of immunosuppression through a mechanism that inhibits the maturation of dendritic cells.

In one embodiment of the present invention, the mechanism of inhibiting maturation of dendritic cells is suppressed by inhibiting nitric oxide production, inhibiting surface molecule expression, inhibiting encapsulation, inhibiting secretion of cytokines, inhibiting migration of dendritic cells, Inhibiting the expression of the signal transducer that is activated, and inhibiting T cell activation.

In one embodiment of the present invention, the autoimmune disease is selected from the group consisting of MS, multiple sclerosis, psoriasis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, type I diabetes mellitus, Disease, and systemic lupus.

The present invention also provides a health functional food for preventing or ameliorating an autoimmune disease, which comprises a saucerneol D compound as an active ingredient.

In one embodiment of the present invention, the food is selected from the group consisting of beverage, meat, chocolate, foodstuff, confectionery, pizza, ramen, other noodles, rice cake, gum, candy, ice cream, alcoholic beverage, ≪ / RTI >

The saucerneol D compound derived from Saururus chinensis according to the present invention inhibits nitric oxide production due to maturation of dendritic cells; Inhibition of surface molecule expression; Inhibition of inclusion; Inhibition of cytokine secretion; Dendritic cell migration inhibition; Inhibiting the signaling substance expression that is activated during maturation; And T cell activation inhibitory effect. Therefore, the composition of the present invention comprising a saucerneol D compound as an active ingredient ultimately effectively reduces the immune response through inhibition of dendritic cell maturation, and therefore, the composition of the present invention, which is caused by immune hypersensitivity such as autoimmune diseases Can be usefully used as immunosuppressive agents capable of preventing or treating diseases.

1 is a graph showing the inhibitory effect of Sauneherol D on nitric oxide (NO) production of dendritic cells induced by treatment with lipopolysaccharide.
2 is a graph showing the inhibitory effect of saucerol D on the expression of surface molecules of dendritic cells induced by treatment with lipopolysaccharide.
3 is a graph showing the inhibitory effect of Saucerol D on the encapsulation action of dendritic cells induced by treatment with lipopolysaccharide.
4 is a graph showing the inhibitory effect of Saucerol D on the secretion of cytokines of dendritic cells induced by treatment with lipopolysaccharide.
Fig. 5 is a graph showing the inhibitory effect of Saucer Neol D on the migration of dendritic cells induced by lipopolysaccharide treatment. Fig.
Fig. 6 is a graph showing the inhibitory effect of Sauneherol D on the signal transduction system of dendritic cells induced by lipopolysaccharide treatment. Fig.
FIG. 7 is a graph showing the inhibitory effect of Saucerol D on T cell activation of dendritic cells induced by lipopolysaccharide treatment. FIG.
8 shows the process of separating the southern neol D from the ground surface of Saururus chinensis.
Figure 9 is a HPLC-DAD chromatogram of Saussureol D; HPLC analysis was carried out using a water tm system (515 pump with 2996 photodiode array detector) and a YMC J (1: 100) solution using a mixed solvent system MeCN-H2O (20: 80 to 100: sphere ODS-H80 column (4 [mu] m, 150 x 4.6 mm ID).

The present invention provides a composition for preventing or treating autoimmune diseases, comprising a saucerneol D compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

≪ Formula 1 >

The 'saucerneol D' of the present invention is a compound isolated from Saururus chinensis. The inventors of the present invention have found that the compound is useful for the prevention or treatment of diseases caused by immune hypersensitivity through a mechanism of inhibiting maturation of dendritic cells It was first described that it could be used as a health functional food as well as a pharmaceutical such as a pharmacological composition as a useful substance.

In the following Experimental Example 1 of the present invention, the immature dendritic cells were treated with the saucerneol D compound of the present invention and stimulated with lipopolysaccharide to examine changes in NO production amount. As a result, The amount of NO produced was 102, 93, 64, 36, 33, 26, 23, and 22% depending on the treatment concentration of Saussureol D in the experimental group in which the guide lipopolysaccharide and saucerneol D were combined (See Fig. 1).

In Experimental Example 2 of the present invention, immature dendritic cells were treated with the saucerneol D compound of the present invention and stimulated with lipopolysaccharide to induce dendritic cells The degree of maturation was examined. As a result, the expression level of MHC Ⅱ was dependent on the treatment concentration (1, 3, 10, 30 μM) of Sauernehol D in the experimental group treated with lipopolysaccharide and saucerneol D Was reduced to 47, 40, 31, and 23 (see Fig. 2).

In Experimental Example 3 of the present invention, the immature dendritic cells were treated with the saucerneol D compound of the present invention and stimulated with lipopolysaccharide to measure the amount of antigen introduced into dendritic cells by a flow cytometer (1, 3, 10, 30 μM) of the saponin neuron D in the experimental group treated with lipopolysaccharide and saucerneol D, 20, 17, 16, and 15%, respectively. This means that the dendritic cell maturation and antigen trapping ability by lipopolysaccharide were inhibited by Sausal Neol D (see FIG. 3).

In Experimental Example 4 of the present invention, immature dendritic cells were treated with the saucerneol D compound of the present invention, stimulated with lipopolysaccharide, and the amount of cytokine produced was measured. As a result, The combination of polysaccharide and saucerneol D inhibited the secretion of IL-12, IL-1β, TNF-α and iNOS depending on the concentration of saponin D treatment 4).

In order to investigate the effect of saucerneol D on the migration of dendritic cells, the migration of dendritic cells was investigated in Experimental Example 5 of the present invention using RANTES, a ligand of CCR7, MIP-3? And CCR5 The results showed that the migration of dendritic cells was inhibited by the combination of saucerneol D and lipopolysaccharide compared to lipopolysaccharide alone treatment. (See FIG. 5). These results suggest that Sauer Neol D inhibits lymphocyte migration in dendritic cells through the interaction of chemokine and chemokine receptors.

In Experimental Example 6 of the present invention, the amount of protein of mitogen-activated protein kinases (ERK, JNK, p38) and NF-κB (p65, p50) activated during the maturation of dendritic cells was measured by Western blot. P-p38 and p-ERK dependent on the concentration of saponin D treatment (1, 3, 10, 30 μM) in the experimental group treated with lipopolysaccharide and saucerneol D And the amounts of p65 and p50 in the nucleus were decreased (see Fig. 6).

In Experimental Example 7 of the present invention, the degree of maturation of dendritic cells was measured by the effect of dendritic cells on T cell activation. As a result, dendritic cells (dendritic cells) treated with lipopolysaccharide and saucerneol D It was confirmed that the T lymphocyte proliferating ability and cytokine production amount were suppressed (see FIG. 7).

From these results, the present inventors have experimentally proved that the saucerneol D compound can inhibit the maturation of dendritic cells by lipopolysaccharide, and ultimately, the immunosuppressive effect is excellent.

Therefore, the composition of the present invention containing saucerneol D as an active ingredient can effectively prevent or treat the autoimmune disease caused by the immune hypersensitivity reaction.

Saucerneol D according to the present invention can be used in the form of a salt, preferably a pharmaceutically acceptable salt. The salt is preferably an acid addition salt formed by a pharmaceutically acceptable free acid, and the free acid may be an organic acid or an inorganic acid. The organic acids include, but are not limited to, citric, acetic, lactic, tartaric, maleic, fumaric, formic, propionic, oxalic, trifluroacetic, benzoic, gluconic, methosulfonic, glycolic, succinic, Glutamic acid and aspartic acid. The inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid.

The saucerneol D according to the present invention may be a commercially available product, or may be prepared from a natural product or produced by a chemical synthetic method known in the art.

When the saucerneol D of the present invention is isolated from natural products, there is no limitation on the separation method, and any known method can be used.

The composition of the present invention is a pharmaceutical composition containing Saucerneol D as an active ingredient, and can be prepared using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to such active ingredients, A disintegrant, a sweetening agent, a binder, a coating agent, a swelling agent, a lubricant, a lubricant or a flavoring agent.

The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration.

The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile solutions suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

In one embodiment of the present invention, saucerneol D of the present invention may be contained in the composition at a concentration of 1 to 300 μM, preferably at a concentration of 1 to 100 μM in the composition. The saucerneol D compound of the present invention may be contained in an amount of 0.1 to 95% by weight based on the total weight of the composition.

Autoimmune diseases in which the pharmaceutical composition of the present invention may exhibit therapeutic effects include MS, multiple sclerosis, psoriasis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, type I diabetes mellitus, inflammatory bowel disease, and systemic lupus, but are not limited thereto.

The present invention also provides the use of a composition comprising saucerneol D as an active ingredient for the manufacture of a medicament for the prevention or treatment of autoimmune diseases. The composition of the present invention containing saucerneol D as an active ingredient can be used for the preparation of a medicament for the prevention or treatment of autoimmune diseases.

The present invention also provides a method of preventing or treating an autoimmune disease comprising administering to a mammal a saucerneol D.

The term "mammal " as used herein refers to a mammal that is the subject of treatment, observation or experimentation, preferably a human.

The term "therapeutically effective amount " as used herein refers to the amount of active ingredient or pharmaceutical composition that elicits a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, The amount that induces the relief of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and the number of administrations of the active ingredient of the present invention will vary depending on the desired effect. The optimal dosage to be administered can therefore be readily determined by those skilled in the art and will depend upon the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, , Sex and diet, time of administration, route of administration and rate of administration of the composition, duration of treatment, concurrent administration of the drug, and the like.

In the therapeutic method of the present invention, the composition comprising the saucerneol D of the present invention as an active ingredient can be administered orally, rectally, intravenously, intramuscularly, intraperitoneally, intramuscularly, intrasternally, transdermally, Or via the intradermal route in a conventional manner.

The present invention also provides a health functional food for preventing or ameliorating an autoimmune disease comprising saucerneol D as an active ingredient.

The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and circles for the purpose of preventing and improving autoimmune diseases.

In the present invention, the term " health functional food " refers to foods manufactured and processed using raw materials or ingredients having useful functions in accordance with Law No. 6727 on Health Functional Foods. Or to obtain a beneficial effect in health use such as physiological action.

The health functional foods of the present invention may contain conventional food additives and, unless otherwise specified, whether or not they are suitable as food additives are classified according to the General Rules for Food Additives approved by the Food and Drug Administration, Standards and standards.

Examples of the items listed in the above-mentioned "food additives" include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as persimmon extract, licorice extract, crystalline cellulose, high color pigment and guar gum; L-glutamic acid sodium preparations, noodle-added alkalis, preservative preparations, tar coloring preparations and the like.

For example, a health functional food in tablet form can be obtained by granulating a mixture of saucerneol D, an active ingredient of the present invention, with an excipient, a binder, a disintegrant and other additives in a usual manner, Or the like, or compression-molding the mixture. In addition, the health functional food of the tablet form may contain a mating agent or the like if necessary.

The hard capsule of the capsule-type health functional food can be prepared by filling a mixture of a hard capsule of the present invention with an additive such as an excipient such as saucerneol D, an effective ingredient of the present invention, And a mixture of saucerneol D with excipients such as excipients and the like may be filled in a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative and the like, if necessary.

The ring-shaped health functional food can be prepared by molding a mixture of saucerneol D, an active ingredient of the present invention, excipient, binder, disintegrant and the like by a known method, It may be coated with white sugar or other skin care agent, or the surface may be coated with a substance such as starch, talc.

The granular health functional food can be prepared by granulating a mixture of saucerneol D, an active ingredient of the present invention, with excipients, binders, disintegrants, and the like by a conventionally known method, Flavoring agents, matting agents, and the like.

The health functional food may be a beverage, a meat, a chocolate, a food, a confectionery, a pizza, a ramen, a noodle, a gum, a candy, an ice cream, an alcoholic beverage, a vitamin complex and a health supplement food.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

< Example  1>

The Southwest Neol  D ( saucerneol  D), experimental animals and Dendritic cell  Ready

<1-1> Originated from Saururus chinensis Southwest Neol  D preparation

Five kilograms of the shrub was washed three times with 15 L of methanol (MeOH) at room temperature and concentrated under reduced pressure to obtain 1.0 kg of methanol extract. Was suspended in distilled water, the methanol extract fraction as n-hexane, and the aqueous layer was fractionated again in CH ₂ Cl ₂ CH ₂ Cl ₂ to give an extract (100 g). The CH ₂ Cl ₂ fraction was fractionated into 21 fractions (SCC-1-SCC-21) by silica gel column chromatography (n-hexane: CH ₂ Cl ₂ gradient). In SCC-14, 1.0 g of saucerneol D was isolated. Compound 1 exhibited a retention time of 28.3 minutes by HPLC under a solvent condition of MeCN-H ₂ O with a YMC J'sphere ODS-H80 column.

<1-2> Preparation of experimental animals

C57BL / 6 female mice (Sumatoco) weighing 18-22 g were used as experimental animals. In the animal feeding room, free intake of feed and water was maintained under constant conditions (temperature: 21 ± 2 ° C, The animals were fed with water and food until the start of the experiment and the animals were kept in the animal breeding room for 7 days before the start of the experiment.

<1-3> Dendritic  Ready

Bone marrow cells were isolated from C57BL / 6 mice and cultured at a concentration of 2 × 10 ⁵ cells / ml. GM-CSF was added at a concentration of 2 ng / ml for 8 days, and the dendritic cells were known to be immature dendritic cells.

< Experimental Example  1>

Southwest Neol  D ( saucerneol  D) Dendritic NO  Effect on production

In this experiment, in order to investigate the effect of sauserol D on the production of NO (nitric oxide) in dendritic cells, immature dendritic cells were treated with the saponin D compound of the present invention and stimulated with lipopolysaccharide to produce NO .

In detail, the immature dendritic cells prepared in Example <1-3> were divided into 96-well plates at a concentration of 5 × 10 ⁵ cells / ml and stabilized for 2 hours. Sousneol D was added to the dendritic cells at a concentration of 1 to 100 μM, and 2 hours later, lipopolysaccharide was added at a concentration of 1 μg / ml and cultured for 24 hours. After incubation, 100 μl of griess A (g = 1: 1) was added to the supernatant (100 μl) and incubated at room temperature for 10 minutes. Absorbance was measured at 540 nm to quantitate NO production.

As a result, nitric oxide (NO) production was 24% in the control group and 100% in the lipopolysaccharide alone treatment group as shown in FIG. 1, and the combination of lipopolysaccharide and Sauer Neol D In the treated group, NO production was decreased to 102, 93, 64, 36, 33, 26, 23, and 22% depending on the concentration of Saussureol D treatment.

< Experimental Example  2>

Southwest Neol  D ( saucerneol  D) Dendritic  Effect on surface molecules

In this experiment, to investigate the effect of Saussure Neol D on the surface molecules of dendritic cells, immature dendritic cells were treated with the saponin D compound of the present invention and stimulated with lipopolysaccharide to obtain surface molecules of dendritic cells The degree of maturation of dendritic cells was examined.

In detail, the immature dendritic cells prepared in Example <1-3> were treated with 1 to 30 μM of Sausal Neol D for 2 hours, added with 1 μg / ml of lipopolysaccharide, and cultured for 24 hours . Cells were harvested at the end of incubation and stained with APC-CD11c, FITC-CD40, FITC-CD80, FITC-CD86, FITC-MHC I and II antibodies and analyzed using flow cytometry Results were expressed as Mean Fluorescence Intensity (MFI). The higher the maturation of dendritic cells, the stronger the expression of the molecule and the higher the MFI. CD11c is a surface molecule selectively expressed in dendritic cells. It is known that CD40, CD80, CD86, MHC-I, and II are expressed more as mature dendritic cells.

As a result, as shown in Fig. 2, the expression level of CD40 was 20 in the control group and increased to 65 in the lipopolysaccharide-treated group. In the experimental group treated with the lipopolysaccharide and the saponin D, CD40 expression was decreased to 54, 53, 43 and 33 depending on the D treatment concentration (1, 3, 10, 30 μM).

The expression level of CD80 was 19 in the control group and increased to 45 in the lipopolysaccharide-treated group. In the experimental group treated with the lipopolysaccharide and the saponin D, , 10, 30 μM), the expression of CD80 decreased to 34, 31, 25, and 20, respectively.

The expression level of CD86 was 21 in the control group and increased to 72 in the lipopolysaccharide-treated group. In the experimental group treated with the lipopolysaccharide and the saponin D, , 10, 30 μM), the expression of CD86 decreased to 59, 57, 50, and 39, respectively.

The expression level of MHC Ⅰ was 19 in the control group and increased to 46 in the lipopolysaccharide treatment group. In the experimental group treated with the lipopolysaccharide and the saponin D, 3, 10, and 30 μM), the expression of MHC Ⅰ decreased to 38, 36, 31, and 21, respectively.

The expression level of MHC Ⅱ was 17 in the control group and increased to 52 in the lipopolysaccharide treatment group. In the experimental group treated with the lipopolysaccharide and the saponin D, 3, 10, and 30 μM), the expression level of MHC Ⅱ decreased to 47, 40, 31, and 23, respectively.

< Experimental Example  3>

Southwest Neol  D ( saucerneol  D) Dendritic On the inclusion  Impact

Immature dendritic cells have a high antigen capture ability, but mature dendritic cells are known to have decreased ability to capture antigens and increased antigen presentation capability. Therefore, in order to investigate the effect of Saussure Neol D on the dendritic cell function of dendritic cells, immature dendritic cells were treated with the seeding compound D of the present invention and stimulated with lipopolysaccharide, The amount of the introduced antigen was measured using a flow cytometer.

In detail, the immature dendritic cells prepared in Example <1-3> were supplemented with 1 to 30 μM of Sausal Neol D and 1 μg / ml of LPS, and then cultured for 48 hours. After culturing, FITC-dextran was treated with cells at a concentration of 0.4 mg / ml for 1 hour. Cells were harvested and washed with 0.5% BSA / PBS to remove excess FITC-dextran, and the amount of antigen introduced into the dendritic cells was measured using a flow cytometer. The CD11c-PE antibody selectively binding to dendritic cells was stained similarly to selectively isolate dendritic cells, and the amount of antigen introduced was analyzed.

As a result, as shown in Fig. 3, immature dendritic cells (control group) had an antigen acquisition rate of 53% when cultured at 37 DEG C for 2 hours, and the degree of antigen acquisition of dendritic cells treated with lipopolysaccharide was 30% (1, 3, 10, 30 μM) of the saponin neuron D in the experimental group treated with 1 to 30 μM of saponin D and 1 μg / ml of lipopolysaccharide And it was confirmed that the values were decreased to 20, 17, 16 and 15%. This means that the ripening of dendritic cells by lipopolysaccharide and the ability to trap the antigen were inhibited by Saucer Neol D. When cultured at 4 ° C, the cells were completely stopped from acquiring the antigen, and 2 to 3% of all the cells showed low antigen acquisition. This means that antigen acquisition is an active immune response.

< Experimental Example  4>

Southwest Neol  D ( saucerneol  D) Dendritic  Effects on cytokine production

Immature dendritic cells secrete many cytokines as they mature after antigen stimulation. The expression of cytokines in dendritic cells plays an important role in stimulating T cells, and IL-12, in particular, is an important indicator of matured dendritic cells. Therefore, in order to examine the effect of treatment with Saussure Neol D on the degree of maturation of dendritic cells, the immature dendritic cells were treated with the seeding compound D of the present invention and stimulated with lipopolysaccharide The amount of cytokine was measured.

In detail, Saucerol D was added to the immature dendritic cells prepared in Example <1-3> at a concentration of 1 to 30 μM for 2 hours, lipopolysaccharide was added at a concentration of 1 μg / ml, and after 24 hours Lt; / RTI &gt; After cultivation, culture medium was collected and the amount of cytokine released from dendritic cells was measured by enzyme-linked immunoassay.

As a result, as shown in FIG. 4, the control group (UN) hardly secreted IL-12, IL-1β, TNF-α and iNOS, and IL-12, IL-1β and TNF -α and iNOS secretion were increased, but the secretion of IL-12, IL-1β, TNF-α and iNOS was dependent on the concentration of saponin D treatment when lipopolysaccharide and Sauer Neol D were treated together . In contrast, IFN-α and IFN-β were significantly increased in the lipopolysaccharide-treated group compared to the control group, but not in the lipopolysaccharide-treated group and the Sauce Neol D group. These results indicate that Sauernaeol D inhibits the maturation of dendritic cells by lipopolysaccharide.

< Experimental Example  5>

Southwest Neol  D ( saucerneol  D) Dendritic  Influence on movement

When dendritic cells mature, they migrate to lymphocytes to activate T cells. At this time, the interaction between chemokine released from lymphocytes and chemokine receptor expressed by dendritic cells plays an important role in migration to lymphocytes. Immature dendritic cells express the chemokine receptors CCR1 and CCR5, and upon maturation, the expression level of the chemokine receptor CCR7 increases. Therefore, in order to examine the effect of treatment with Saussure Neol D on the degree of maturation of dendritic cells, the immature dendritic cells were treated with the seeding compound D of the present invention and then stimulated with lipopolysaccharide to obtain a dendritic cell The migration of dendritic cells was analyzed using transwell for MMP-3β, a ligand for CCR7, and RANTES, a ligand for CCR5, in cell migration.

In detail, the seeded dendritic cells prepared above in Example <1-3> were supplemented with Saucer Neol D and lipopolysaccharide at a concentration of 1 μg / ml for 24 hours. In order to verify the migration ability of dendritic cells by Saussure Neol D, the dendritic cell movement was analyzed using transwell for the ligands of CCR7, MIP-3? And RANTES, a ligand of CCR5. Dendritic cells were placed in the upper chamber, and MIP-3β and RANTES were added to the lower chamber at concentrations of 30 to 300 ng / ml, respectively, and cultured in a 37 ° C. incubator for 8 hours. The number of cells transferred to the lower chamber was then measured using a flow cytometer.

As a result, as shown in Fig. 5A, the expression of CCR1 and CCR5 was high and the expression of CCR7 was low in immature dendritic cells, whereas the expression level of CCR7 was significantly increased in the lipopolysaccharide-treated group. On the other hand, the expression of CCR7 was decreased and the expression levels of CCR1 and CCR5 were increased in the group treated with Sauer Neol D and lipopolysaccharide.

In addition, as shown in FIG. 5B, it was confirmed that the control group (UN) had no migration by MIP-3? And the cell migration was increased in a concentration-dependent manner to MIP-3? When treated with lipopolysaccharide. On the other hand, it was confirmed that the group treated with Sauer Neol D and lipopolysaccharide inhibited cell migration by MIP-3? Compared to the group treated with lipopolysaccharide. The cell migration of the control (UN) by RANTES was increased and the cell migration was less than that of the control group when Sauer Neol D was treated with lipopolysaccharide.

These results suggest that Sauer Neol D inhibits lymphocyte migration in dendritic cells through the interaction of chemokine and chemokine receptors.

< Experimental Example  6>

Southwest Neol  D ( saucerneol  D) Dendritic  Influence on signal transduction system

Dendritic cells are known to activate mitogen-activated protein kinases such as ERK, JNK, p38, and NF-κB during maturation. In this experiment, to examine the effect of treatment with Saussure Neol D on the degree of maturation of dendritic cells, immature dendritic cells were treated with the Sausseleol D compound of the present invention and stimulated with lipopolysaccharide to be phosphorylated The amount of protein of ERK, JNK and p38 was measured by western blot method.

In detail, the immature dendritic cells prepared in Example <1-3> were treated with lipopolysaccharide and saponin D for 15 minutes, and the cells were disrupted to separate the proteins. The amount of ERK, JNK, and p38 protein phosphorylated using total protein was measured by western blot method. The amount of NF-κB (p65, p50) transferred into the nucleus using the nuclear protein was measured by Western blotting. We also tested whether there is an inhibitory effect on IkB degradation.

As a result, as shown in FIG. 6A, the amounts of phosphorylated p-JNK, p-p38 and p-ERK were increased in the lipopolysaccharide alone treatment group, while the lipopolysaccharide and the saponin D were treated In one group, p-JNK, p-p38 and p-ERK levels were decreased in a dose dependent manner (1, 3, 10, 30 μM)

6B, the amounts of p65 and p50 in the nucleus depend on the treatment concentration (1, 3, 10, 30 [mu] M) of saponin D in the group treated with lipopolysaccharide and saponin D .

< Experimental Example  7>

Southwest Neol  D ( saucerneol  D) Dendritic  Effect on T cell activation

The important immune function of dendritic cells is to activate T cells. When dendritic cells mature, they migrate to lymphocytes and present predated antigens to T cells. T cells that recognize the antigen divide and proliferate and secrete cytokines such as IL-2 and IFN-γ to induce an immune response. In this experiment, the degree of dendritic cell maturation by treatment with Saussure Neol D was measured by the effect of T cell activation on dendritic cells.

In detail, the immature dendritic cells prepared above in Example <1-3> were supplemented with 40 μM concentration of Saussureol D and 1 μg / ml of lipopolysaccharide, and cultured for 24 hours. After cultivation, the cells were harvested and mixed with allogeneic T cells for 3 days and 5 days (FIGS. 7A and 7B). Cell culture For the last 16 hours, [3H] -thymidine was treated to infiltrate radioactive material into the DNA of proliferating T cells, and cell proliferation was measured.

As a result, as shown in FIGS. 7A and 7B, dendritic cells alone treated with lipopolysaccharide only showed strong T lymphocyte proliferative activity, whereas dendritic cells treated with lipopolysaccharide and Sausal Neol D showed T lymphocyte proliferation ability .

The amount of IFN-γ and IL-2 secreted by T cells was measured. As a result, as shown in Fig. 7C and Fig. 7D, cytokine production was strongly increased in T cells co-cultured with dendritic cells alone treated with lipopolysaccharide, while lipopolysaccharide and saucerol D were treated in the same manner T lymphocytes co-cultured with dendritic cells inhibited cytokine production.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (8)

A composition for preventing or treating an autoimmune disease comprising, as an active ingredient, a saucerneol D compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
&Lt; Formula 1 >

The method according to claim 1,
Wherein the compound is isolated from Saururus chinensis. The method according to claim 1,
Wherein the compound is contained in the composition at a concentration of 1 μM to 100 μM. The method according to claim 1,
Wherein said compound has an immunosuppressive effect through a mechanism of inhibiting maturation of dendritic cells. 5. The method of claim 4,
The mechanism of inhibiting the maturation of dendritic cells is inhibited by nitric oxide production, surface molecule inhibition, encapsulation inhibition, cytokine secretion inhibition, dendritic cell migration inhibition, signal transduction inhibition and T And inhibiting cell activation. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; 6. The method according to any one of claims 1 to 5,
The autoimmune disease is selected from the group consisting of MS, multiple sclerosis, psoriasis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, diabetes mellitus, inflammatory bowel disease and systemic lupus 1 &lt; / RTI &gt; or less. A health functional food for preventing or ameliorating an autoimmune disease which comprises a saucerneol D compound as an active ingredient. 8. The method of claim 7,
Wherein the food is selected from the group consisting of beverage, meat, chocolates, foods, confectionery, pizza, ramen, other noodles, rice cakes, gums, candy, ice cream, alcoholic beverages, alcoholic beverages, A health functional food for preventing or ameliorating a disease caused by immune hyperresponsiveness.

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