KR20170040793A - New tricin derivative and pharmaceutical use thereof - Google Patents

Abstract

The present invention relates to a novel tricin derivative isolated from Zizania latifolia and medical uses thereof. A novel tricin derivative, salcolin D, isolated from Zizania latifolia which is a natural substance, as a safe compound which does not show cytotoxicity, effectively inhibits excessive production of NO which causes inflammatory diseases compared to existing tricin derivatives and inhibits release of -hexosaminidase inducing allergy diseases. Accordingly, the tricin derivative can be provided as an anti-allergic drug or a composition for preventing or treating inflammatory allergy diseases.

Description

Novel tricine derivatives and pharmaceutical uses thereof < RTI ID = 0.0 >

The present invention relates to novel tricyclic derivatives isolated from Zizania latifolia and their uses.

Inflammation is generally a localized protective response of the body tissue to host infiltration by foreign substances or harmful stimuli. Causes of inflammation include infectious causes such as bacteria, viruses and parasites, physical causes such as burns or radiation, chemicals such as toxins, drugs or industrial preparations, immune responses such as allergies and autoimmune reactions, or conditions associated with oxidative stress Lt; / RTI >

Inflammation is regulated by complex processes such as nitric oxide (NO) produced by activated macrophages, cytokines, prostaglandins, and staged reactions of growth factors.

In congenital immunity, macrophages play an important role in initiating and expanding defense against pathogens. However, studies on effective inflammatory drugs that can reduce NO production because of NO produced by activated macrophages are exposed to endotoxin in other cells and cause rheumatoid arthritis, atherosclerosis, asthma and pulmonary fibrosis .

It is known that allergies cause abnormal immune function due to genetic and environmental factors, resulting in an abnormally excessive immune response to a harmless antigen, allergen. First, when the body is exposed to house dust mites, pollen, animal hair, cockroaches, fungi and food antigens, the allergen is recognized by circulating antigen presenting cells, Lt; / RTI > Differentiated Th2 cells secrete TNF-, IL-4, IL-5, IL-8 and IL-13, which are allergen-induced cytokines. They stimulate the development of acidic white blood cells in the bone marrow, Not only induces the migration of acidic white blood cells, but also acts on B cells to promote the production of immunoglobulin IgE and IgG1. The IgE produced by a series of mechanisms of action strongly binds to mast cells through the IgE receptor called FcR in tissues. Then, it is exposed again to the same allergen as the first, and binds to IgE bound to allergen-causing mast cells. The mast cells bind to allergic histamine, β-hexosaminidase, inflammatory prostaglandins, heparin and free radicals Which secrete various allergic disease symptoms, and these reactions lead to chronic inflammation.

Korean Patent Publication No. 2010-0097843 (2010.09.06)

The present invention is to provide a novel tricin derivative isolated from methanol extract of Zizania latifolia, which is effective for inflammation and allergy relief, and to provide it as a composition for treating an anti-inflammatory, anti-allergic or inflammatory allergic disease.

The present invention provides a tricine derivative represented by the following formula (1).

[Chemical Formula 1]

The present invention provides an anti-inflammatory agent containing the tricin derivative represented by the above formula (1) as an active ingredient.

The present invention provides an antiallergic agent containing the tricine derivative represented by the above formula (1) as an active ingredient.

The present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory allergic diseases containing the tricin derivative represented by the above formula (1) as an active ingredient.

The present invention provides a cosmetic composition for preventing or improving an inflammatory allergic disease containing the tricin derivative represented by the general formula (1) as an active ingredient.

The present invention also provides a health food for preventing or ameliorating an inflammatory allergic disease containing the tricin derivative represented by the general formula (1) as an active ingredient.

According to the present invention, the novel trichine derivative, sarcocline D, isolated from natural herbicide, effectively inhibits excessive production of NO, which is a cause of inflammatory diseases, than the previously known trichine derivatives, It has been confirmed that the effect of inhibiting the release of? -Hexosaminidase is excellent, so that it can be provided as a composition for preventing or treating an anti-inflammatory, anti-allergic or inflammatory allergic disease.

FIG. 1 shows the chemical structure of Compound 1-5 isolated from Gochang Chol, wherein Compound 1 is tricine, Compound 2 is chalcone A, Compound 3 is chalcone B, Compound 4 is chalcone C and Compound 5 is chalcone D .
Figure 2 is the major gHMBC correlation diagram of compound 5.
FIG. 3 shows the results of confirming cytotoxicity and NO production inhibitory effect of compounds isolated from goose chow in RAW 264.7 cells stimulated with LPS, and stimulated cells with LPS (100 ng / mL) for 24 hours and analyzed NO production , The values are expressed as the mean ± SEM of triplicate experiments, and the values in the other subscripts are significant differences (p <0.05) as determined by Duncan's multiple range test.
FIG. 4 shows DNP-specific IgE 450 ng / mL inhibition effect on β-hexosaminidase release of compounds isolated from goat supernatant in RBL-2H3 cells induced by IgE and DNP-BSA Stimulated with RBL-2H3 cells (2 × 10 ⁵ cells) overnight, treated with various doses of compound for 10 minutes, stimulated with 10 μg / mL of DNP-BSA for 10 minutes, Hexosaminidase release was determined and the values were expressed as mean ± SEM of triplicate experiments and the values in the other subscripts were significant differences (p < 0.05).

The present invention can provide a tricine derivative represented by the general formula (1).

[Chemical Formula 1]

More specifically, the tricine derivative represented by the above formula (1) is a novel tricine derivative isolated from Zizania latifolia, and tricine-4'-O- [erythro-beta-guaiacyl- (7 & -O- methyl) -glyceryl] ether (tricin-4'-O- [erythro-beta -gaiaiacyl- (7 &quot; -O- methyl) -glyceryl] ether.

The present invention can provide an anti-inflammatory agent containing the tricin derivative represented by the general formula (1) as an active ingredient, and the tricin derivative can inhibit the production of nitric oxide (NO).

According to one embodiment of the present invention, the treatment of RAW 264.7 cells stimulated with LPS, which is a tricine derivative of the above formula (1), inhibits NO production in a concentration-dependent manner as shown in FIG. 3B Especially at the concentration of 10 μg / mL, the inhibition of NO production was superior to that of other tricine derivatives.

From the above results, it was confirmed that the tricine derivative represented by the formula (1) of the present invention inhibits NO production and exhibits an anti-inflammatory effect.

The present invention can provide an antiallergic agent containing the tricine derivative represented by the above formula (1) as an active ingredient, and the tricine derivative can inhibit the release of? -Hexosaminidase.

According to another embodiment of the present invention, the tricine derivative of Salcocolin D represented by the above formula (1) inhibits the release of β-hexamidase from DNP-BSA-induced IgE-sensitized RBL-2H3 cells, Especially at the concentration of 1 μg / mL, β-hexaminidase release inhibitory effect was superior to other tricine derivatives.

From the above results, it was confirmed that the tricine derivative represented by the formula (1) of the present invention suppresses the release of? -Hexamidase and exhibits an anti-allergic effect.

In addition, it was confirmed that the present salacoline D is a safe compound having no cytotoxicity as shown in FIGS. 3A and 4A.

Accordingly, the present invention can provide a pharmaceutical composition for the prevention or treatment of inflammatory allergic diseases containing the tricin derivative represented by the general formula (1) as an active ingredient.

The inflammatory allergic disease may be selected from the group consisting of asthma, atopic dermatitis and allergic rhinitis.

More particularly, the present invention relates to tricin-4'-O- [erythro-beta-guiacyl- (7 &quot; -O- guaiacyl- (7 &quot; -O-methyl) -glyceryl] ether.

The present invention also relates to the use of tricin-4'-O- [erythro-beta-guaiacyl- (7'-O- (7 &quot; -O-methyl) -glyceryl] ether as an active ingredient and the tricine-4'-O- [erythro- beta -guaiacyl- The present invention provides a pharmaceutical composition for preventing or treating an allergic disease characterized in that the ether inhibits the release of? -Hexosaminidase to prevent or treat an allergic disease selected from the group consisting of asthma, atopic dermatitis and allergic rhinitis can do.

In one embodiment of the present invention, the pharmaceutical composition for preventing or treating an inflammatory allergic disease containing the tricin derivative represented by the general formula (1) as an active ingredient can be administered orally, parenterally, Any one of formulations selected from the group consisting of suppositories, gels, suspensions, emulsions, drops, and liquids can be used.

In another embodiment of the present invention, the pharmaceutical composition for the prevention or treatment of inflammatory allergic diseases containing the tricine derivative represented by the general formula (1) as an active ingredient may be formulated into a pharmaceutical composition containing an appropriate carrier, excipient, disintegrant, A lubricant, a lubricant, a flavoring agent, an antioxidant, a buffer, a bacteriostatic agent, a diluent, a dispersant, a surfactant, a binder and a lubricant.

Specific examples of carriers, excipients and diluents 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. Solid formulations for oral administration may be in the form of tablets, pills, powders, granules, capsules These solid preparations can be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., into the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, syrups and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin which are commonly used simple diluents. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. 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. As the suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

According to one embodiment of the present invention, the pharmaceutical composition may be administered orally, intraarterally, intraperitoneally, intramuscularly, intraarterally, intraperitoneally, intrasternally, transdermally, nasally, inhaled, topically, rectally, &Lt; / RTI &gt; can be administered to the subject in a conventional manner.

The preferred dose of the tricine derivative may vary depending on the condition and body weight of the subject, the type and degree of disease, the type of drug, the route of administration and the period of time, and may be appropriately selected by those skilled in the art. According to one embodiment of the present invention, the daily dose may be 0.01 to 200 mg / kg, specifically 0.1 to 200 mg / kg, more specifically 0.1 to 100 mg / kg, though it is not limited thereto. The administration may be performed once a day or divided into several times, and thus the scope of the present invention is not limited thereto.

In the present invention, the 'subject' may be a mammal including a human, but is not limited thereto.

The present invention can provide a cosmetic composition for preventing or improving an inflammatory allergic disease comprising the tricin derivative represented by the general formula (1) as an active ingredient.

The cosmetic composition may contain conventional auxiliary agents such as stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers in addition to the tricin derivative as an active ingredient.

The cosmetic composition may be prepared in any form conventionally produced in the art and may be in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, oil, powder foundation, emulsion foundation, Wax foundation, spray, and the like. However, the present invention is not limited thereto. More specifically, it can be manufactured in the form of a sun cream, a flexible lotion, a convergent lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a pack, a spray or a powder.

When the formulation is a paste, cream or gel, an animal oil, a vegetable oil, a wax, a paraffin, a starch, a tracer, a cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component .

When the formulation is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, chlorofluorohydrocarbons, propane / Or propellants such as dimethyl ether.

When the formulation is a solution or an emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, - butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid esters of sorbitan.

When the formulation is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspension such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, a microcrystalline cellulose , Aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

The present invention also provides a health food for preventing or ameliorating an inflammatory allergy disease containing the tricin derivative represented by the general formula (1) as an active ingredient.

More particularly, the present invention relates to a tricine-4'-O- [erythro-β-guaiacyl- (7'- 7 "-O-methyl) -glyceryl] ether as the active ingredient and the tricine-4'-O- [erythro-β-guiacyl- (7" The present invention provides a health food for preventing or ameliorating allergy disease characterized by inhibiting the release of? -Hexosaminidase to prevent or ameliorate allergic diseases selected from the group consisting of asthma, atopic dermatitis and allergic rhinitis .

The health food is used together with food or food additives other than the tricine derivative, 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 its use purpose, for example, prevention, health or therapeutic treatment.

The effective dose of the compound contained in the above-mentioned health food may be used in accordance with the effective dose of the therapeutic agent, but may be less than the above range for health and hygiene purposes or for long-term intake for health control purposes, It is clear that the component can be used in an amount of more than the above range since there is no problem in terms of safety.

There is no particular limitation on the type of the health food, and examples thereof include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, Drinks, alcoholic beverages and vitamin complexes.

The present invention also relates to the use of tricin-4'-O- [erythro-beta-guaiacyl- (7'-O- (7 &quot; -O-methyl) -glyceryl] ether as an active ingredient and inhibiting the release of? -Hexosaminidase in vitro. A reagent composition for suppressing the release of beta-hexosaminidase can be provided.

In addition, the present invention relates to a method for the production of tricin-4'-O- [erythro-beta-guiacyl- (7 &quot; -O- erythro-beta-guaiacyl- (7 &quot; -O-methyl) -glyceryl] ether in the presence of a reducing agent to inhibit? -hexaminidase release.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Experimental Example  1> Materials and Methods

1. Plant sample

The dried ground portion of Zizania latifolia was purchased from Yeongcheon City, Gyeongbuk Province.

2. Appliances and reagents

LC-Forte / R, YMC, Japan) was performed using a YMC-DispoPack (ODS-25, 80 g, and 120 g, YMC, Japan) mesh, Merck, Germany) and Sephadex LH-20 (Merck, Germany).

Thin-layer chromatography (TLC) analysis was performed using a Kiesel gel 60F254 plate (Merck Co., Darmastdt, Germany) and RP-18 F254s plates (Merck Co., Darmastdt, Germany) UV lamp and 10% H ₂ SO ₄ solution.

¹ H and ¹³ C NMR and 2D NMR data were obtained with Bruker AVANCE II 400 (1 H NMR at 400 MHz, 13 C NMR at 100 MHz) spectrometer (Bruker, Germany) using pyrimidine-d5 and TMS as internal controls.

ESI / MS was obtained with 6530 Accurate-Mass Q-TOF LC / MS (Agilent Technologies, Germany). All reagents were purchased from Sigma Chemical Co. (St. Louis, Mo., USA).

3. Separation of physiologically active substances

5 kg of dry solid powder was extracted with 45 L of 80% methanol (MeOH) at room temperature and the concentrated extract was washed successively with water (3 L), EtOAc (3 L × 3) and n-BuOH (3 L × 3) The EtOAc extract (97 g, ZLE), n-BuOH extract (240 g, ZLB) and H ₂ O extract (57 g, ZLW) were obtained by fractionation.

The EtOAc extract was applied to silica gel and eluted with CHCl ₃ : MeOH (50: 1 → 30: 1 → 20: 1 → 12: 1 → 10: 1 → 7: 1 → 5: 1 → 3: 1 → 1: L) to obtain 13 fractions (ZLE-1 to ZLE-13).

The ZLE-8 fraction (4.7 g) was analyzed using a prep-LC cartridge (YMC-DispoPack AT; ODS-25: 120 g, particle size: 25 μm). The fractions were run on a mobile phase consisting of A solvent containing 0.1% formic acid at a flow rate of 10 mL / min and B solvent containing 0.1% acetonitrile and analyzed by UV detector at 254 and 356 nm.

Min for 20 minutes and 0.2% / min for 30% B solvent at 0.2% / min rate, 30% B to 50% B at 0.2% / min and 50% B to 70% B for 20 minutes , And a gradient from 70% B to 90% B at a rate of 0.30% / min.

A total of 14 fractions (ZLE-8-14 in ZLE-8-1) were obtained and fraction ZLE-8 was obtained using a prep-LC cartridge (YMC-DispoPack AT; ODS-25: 80 g, particle size: -4 (327 mg). The mobile phase was flowed at a flow rate of 10 mL / min using a solvent A containing 0.1% formic acid and a solvent B containing 0.1% acetonitrile. The detector was maintained at 254 and 356 nm, and the gradient was 10% B The rate was 0.6% / min at 35% B and 0.5% / min at 35% B and 60% B at 35% B, respectively.

As a result, Compound 3 (27 mg, RT = 14.2 min), Compound 1 (21 mg, RT = 15.7 min) and Compound 2 (32 mg, RT = 17.8 min) were obtained.

The fraction ZLE-8-6 (231 mg) was also analyzed using a prep-LC cartridge (YMC-DispoPack AT; ODS-25: 80 g, particle size: 25 μm). The mobile phase was flowed at a flow rate of 10 mL / min using A solvent containing 0.1% formic acid and B solvent containing 0.1% acetonitrile, and the detector was maintained at 254 and 356 nm. The gradient was configured at a rate of 0.5% / min from 15% B to 40% B, and a rate of 0.6% / min from 40% B to 70% B.

As a result, Compound 4 (52 mg, RT = 21.1 min) and Compound 5 (21 mg, RT = 23.4 min) were obtained.

Tree Seen (Tricin; 1) Pale yellow powder (CH ₃ OH); negative ESI / MS m / z 329 [MH] ^- ; IR (CaF2 plate) 3413, 2941, 2842, 1664, 1611 cm &lt; ^{-1 &} gt ;; ^{1 H NMR (400 MHz, pyridined} 5) δ 7.42 (2H, s, H-2 ', 6'), 7.01 (1H, s, H-3), 6.87 (1H, d, J = 2.0 Hz, H- 8), 6.74 (1H, d, J = 2.0 Hz, H-6), 3.84 (6H, s, OCH3 ', 5'). ^{13 C NMR (100 MHz, pyridine} -d 5) δ 182.5 (C-4), 165.7 (C-7), 164.4 (C-2), 162.9 (C-5), 158.3 (C-9), 149.1 ( C-3 ', 5'), 141.1 (C-4 '), 121.1 (C-1'), 104.9 (C- 99.7 (C-6), 94.8 (C-8), 56.2 (C-3 ', 5'-OCH 3).

Salcolin A (2) yellow amorphous powder (CH ₃ OH); negative ESI / MS m / z 525 [MH] ^- ; IR (CaF ₂ plate) 3369, 2938, 1652, 1611, 1588, 1512, 1495, 1356, 1263, 1159, 1124, 841 cm ^-1 ; ^{1 H NMR (400 MHz, pyridine} -d 5) δ7.59 (1H, br. S, H-2 "), 7.48 (1H, dd, J = 8.0, 1.2 Hz, H-6"), 7.31 (2H (1H, d, J = 2.0 Hz, 1H, s, H-2 ', 6'), 7.27 J = 6.0 Hz, H-7), 4.93 (1H, q, J = 6.0, Hz, H-8 &quot;), 4.44 (1H, dd, J = 12.0, 4.0 Hz, H- _{6H, s, OCH 3 ',} 5'), 3.72 (3H, s, OCH 3 ''). 13C NMR (100 MHz, pyridine- d 5) δ183.9 (C-4), 167.3 (C-7), 165.0 (C-2), 163.9 (C-5), 159.8 (C-9), 155.1 ( C-3 ', C-3'), 149.6 (C-3 ''), 148.7 (C-3), 106.3 (C-10), 106.0 (C-2 ', 6'), 101.3 (C-6), 96.3 ( C-8), 90.4 (C-8 "), 74.9 (C-7"), 63.1 (C-9 "), 57.7 (C-3 ', 5'-OCH 3) , 57.0 (C-3 "-OCH 3).

Salcolin B (3) yellow amorphous powder (CH ₃ OH); negative ESI / MS m / z 525 [MH] ^- ; IR (CaF2 plate) 3364, 2933, 1649, 1607, 1590, 1495, 1457, 1358, 1121, 832 cm ^-1 ; ^{1 H NMR (400 MHz, pyridine} -d 5) δ 7.54 (1H, d, J = 1.2 Hz, H-2 "), 7.38 (1H, dd, J = 8.0, 1.2 Hz, H-6"), 7.31 (2H, s, H-2 ', 6'), 7.27 (1H, d, J = 8.0 Hz, H- J = 4.8 Hz, H-8), 6.74 (1H, d, J = 2.0 Hz, H-6), 5.71 (1H, , 3.6 Hz, H-7 "), 4.65 (1H, dd, J = 12.0, 5.2 Hz, H- _{3.82 (6H, s, OCH 3} ', 5'), 3.73 (3H, s, OCH 3 "). ^{13 C NMR (100 MHz, pyridine} -d 5) δ 183.9 (C-4), 167.4 (C-7), 165.1 (C-2), 164.4 (C-5), 159.8 (C-9), 155.3 ( C-3 '), 149.6 (C-3 "), 148.6 (C-4"), 141.7 (C-6 "), 117.3 (C-5"), 112.9 (C-2 "), 107.1 (C-3), 106.3 C-6), 96.3 (C -8), 89.4 (C-8 "), 75.0 (C-7"), 62.9 (C-9 "), 57.7 (C-3 ', 5'-OCH 3), _{57.1 (C-3 "-OCH 3} ).

Salcolin C (4) yellow amorphous powder (CH ₃ OH); negative ESI / MS m / z 539 [MH] ^- ; IR (KBr) 3436, 2957, 2926, 2531, 1649, 1589, 1495, 1358 cm &lt; ^{-1 &} gt ;; ¹ H NMR (400 MHz, pyridine-d ₅ )? 7.34 (2H, s, H-2 ', 6'), 7.33 (1H, d, J = 1.2 Hz, H- J = 8.0 Hz, H-5 "), 7.23 (1H, dd, J = 8.0, 1.6 Hz, H- J = 6.8 Hz, H-8), 6.75 (1H, d, J = 2.0 Hz, H-6), 5.08 , 4.0 Hz, H-8 "), 4.26 (1H, dd, J = 12.0, 3.6 Hz, H- _{3.83 (6H, s, OCH 3} ', 5'), 3.59 (3H, s, OCH 3 "), 3.30 (3H, s, OCH 3 7 &quot;).

^{13 C NMR (100 MHz, pyridine} -d 5) δ 183.6 (C-4), 167.1 (C-7), 165.0 (C-2), 164.0 (C-5), 159.5 (C-9), 154.8 ( C-3 '), 149.5 (C-3''), 148.9 (C-4''), 142.7 , 122.3 (C-6 "), 117.1 (C-5"), 112.7 (C-2 "), 106.6 (C-3), 105.9 , 101.0 (C-6), 96.0 (C-8), 87.9 (C-8 "), 86.0 _{3), 57.3 (C-3} "-OCH 3), 56.6 (C-7" -OCH 3).

Salcolin D (5) yellow amorphous powder (CH ₃ OH); positive HRESI-MS m / z 541.1691 [M + H] +, calcd for m / z C 28 H 29 O 11 541.1704; IR (KBr) 3656, 2667, 2521, 2502, 1687, 1509, 1425, 1398, 1115 cm &lt; ^{-1 &} gt ;; ^{1 H NMR (400 MHz, pyridine} -d 5)) δ 7.33 (1H, d, J = 1.6 Hz, H-2 "), 7.29 (2H, s, H-2 ', 6'), 7.26 (1H, d, J = 8.4 Hz, H-5 "), 7.19 (1H, dd, J = 8.0, 1.6 Hz, H-6" = 2.0 Hz, H-8), 6.73 (1H, d, J = 2.0 Hz, H-6), 4.99 (1H, d, J = 4.0 Hz, H- 8H), 4.50 (1H, dd, J = 12.8, 2.8 Hz, H-9 "e), 4.25 (1H, dd, J = OCH ₃ ', 5'), 3.76 (3H, s, OCH ₃ '), 3.39 (3H, s, OCH _{3 -} 7'').

^{13 C NMR (100 MHz, pyridine} -d 5) δ 183.9 (C-4), 167.4 (C-7), 165.1 (C-2), 164.3 (C-5), 159.8 (C-9), 155.2 ( C-3 ', C-3', C-3 '), 149.1 (C-4 "), 141.5 (C-6 "), 117.3 (C-5"), 113.3 (C-2 "), 107.0 (C-3), 106.3 C-6), 96.3 (C -8), 88.1 (C-8 "), 84.7 (C-7"), 62.5 (C-9 "), 58.3 (C-3 ', 5'-OCH 3), _{57.6 (C-3 "-OCH 3} ), 57.0 (C-7" -OCH 3).

< Experimental Example  2> Cell culture

Mouse macrophage cell line RAW 264.7 cells and Rat Basophilic Leukemia cell line RBL-2H3 cells were purchased from Korean Cell Line Bank (KCLB, Seoul, Korea). RAW 264.7 cells were cultured in DMEM (Gibco-BRL, Rockville, MD, USA) containing 1% antibiotic (penicillin / streptomycin) and 10% inactivated fetal bovine serum (FBS, Gibco BRL, RBL-2H3 cells were cultured in MEM (Gibco BRL, NY, USA) containing 10% FBS and 1% penicillin / streptomycin.

Both cells were grown in an incubator at 37 ° C and 5% CO 2.

< Experimental Example  3> Cell viability  Confirm

In order to confirm the cytotoxicity of the compounds, MTT analysis was performed to analyze the activity of mitochondria which reduced the penetrating formazan dye to MTT to confirm cell viability. Dividing the RBL-2H3 cells in 96-well plate at 3 × 10 ⁴ cells per well in and from the next day 37 ℃ condition was treatment of the compound of 0.1 to 10 μg / mL concentration.

Forty-eight hours later, 100 μL of MTT (5 mg / mL) was added to each well and cultured at 37 ° C for 4 hours. Then, 100 占 퐇 of DMSO was added to each well to dissolve the cells. Plates were maintained at room temperature for 5 minutes and absorbance was measured at 550 nm using a multiwell spectrophotometer (Molecular Devices, Sunnyvale, Calif.).

In addition, cytotoxicity of compounds in IgE-sensitive RBL-2H3 cells was confirmed by MTT assay. The IgE-sensitive RBL-2H3 cells were plated at 3 × 10 ⁴ cells per well and the compounds were incubated overnight with 450 ng / mL DNP-specific IgE before treatment, at 37 ° C.

< Experimental Example  4> By LPS  Identification of induced NO products

RAW 264.7 cells were plated in 96-well plates at 1 × 10 ⁵ cells per well and cultured overnight, and various concentrations of test compounds were treated in the presence of LPS (final concentration, 100 ng / mL).

After LPS stimulation for 24 hours, the NO production in the cell culture medium was measured with a Griess Reagent System.

To 100 μL of the culture supernatant was added 10% of the same amount of a 2.5% phosphoric acid solution containing Griess reagent [1% sulfanilamide and 0.1% naphthylethylendiamine] for 10 minutes, and the absorbance at 540 nm And the NO concentration was calculated from the NaNO ₂ standard curve.

The NO production inhibitory activity of the compound was expressed by the IC ₅₀ value (the concentration required to inhibit NO production by 50%).

< Experimental Example  5 > antigen-induced &lt; RTI ID = 0.0 & Hexosaminidase ( hexosaminidase ) Release confirmation

The inhibitory effect of the extract on the release of? -Hexosaminidase was confirmed in RBL-2H3 cells. First, dividing the RBL-2H3 cells in a 24-well plate at 2 × 10 ⁵ cells per well and it was stimulated overnight with anti-37 ℃ -DNP IgE (450 ng / mL) .

The cells were then washed with Siraganian buffer and incubated for 20 min at 37 ° C with the addition of 160 μL of incubate buffer. 20 μL of the extract was then treated for 10 min with 20 μL of antigen (DNPBSA, 10 μg / mL) Was added to stimulate the cells to form granules at 37 占 폚 for 10 minutes. Thereafter, the reaction was stopped by soaking in an ice water bath for 10 minutes.

Transfer 25 μL of the supernatant to a 96-well plate and add 25 μL of 0.1 M citrate buffer (pH 4.5) in which the substrate (1 mM p-nitrophenyl-Nacetyl-β-D-glucosaminide) And reacted for 1 hour. Then, 200 μL of stop solution (0.1 M Na2CO3 / NaHCO3, pH 10.0) was added to stop the reaction and absorbance was measured at 405 nm using a microplate reader.

The inhibitory activity of the compound for β-hexosaminidase release was expressed by the IC ₅₀ value (the concentration required to inhibit 50% inhibition of β-hexosaminidase release).

< Example  1> tricine ( tricin ) Isolation and Identification of Derivatives

The aerial parts extract with 80% methanol and the concentrated extract was partitioned successively using EtOAc, n-BuOH and H ₂ O.

The EtOAc fractions were subjected to sequential repeated SiO ₂ and ODS-A column chromatography to purify four kinds of flavonolignans including flavone and novel flavonolignan, (3), tricin-4'-O- (threo-β-guaiacylglyceryl) ether, salcolin A (2), tricin-4'-O- (erythro-β -guaiacylglyceryl ether, salcolin B (3), and tricin-4'-O- [threo-β-guaiacyl- (7 "-O-methyl) -glyceryl] ether and salcolin C (4). All of these compounds are the first to be isolated in Z.latifolia.

The compound (5), which is a yellow amorphous powder, was identified by TLC, followed by spraying with 10% H ₂ SO ₄ and heating to a dark yellow color.

It was expected that the molecular weight would be 540 as the false molecular ion peak in the negative ESI / MS results for this molecule was identified as m / z 539 [MH] ^- .

Further, ¹ H NMR spectral results showed that 1,2,4-trisubstituted benzene rings and five olefinic methine proton signals [δ 7.29 (2H, s, H-2 ', 6'), 6.99 7.33 (1H, d, J) because of 1H, s, H-3), 6.85 (1H, d, J = 2.0Hz, H-8) and 6.73 = 1.6 Hz, H-2 "), 7.26 (1H, d, J = 8.4 Hz, H-5") and 7.19 (1H, dd, J = 8.0, 1.6 Hz, H- ) A flavonoid moiety such as a methine proton signal was identified.

(1H, d, J = 12.8, 2.8 Hz, H-7) and 4.97 (1H, m, H- (6H, s, OCH ₃ ') and two methoxy proton signals [δ 3.79 (6H, s, OCH ₃ ' , 5 '), 3.76 (3H, s, OCH ₃ '), 3.39 (3H, s, OCH ₃ 7 &quot;)] was confirmed.

The relative arrangement of the asymmetric carbons of C-7 "and C-8" in compound 5 was the binding constant (J = 4.0 Hz) between the oxygenated methine signals of H-7 "(δH 4.99) and H- ) Was predicted to be an erythro type.

The J _H -7 "and 8" binding constants of proton resonance in the guaiacylglyceryl moiety were used to distinguish between erythro and threo types, with J values of 4.0-5.0 and 6.0-7.0 Hz, respectively.

In addition, ¹³ C NMR spectrum in the 4-methoxy-carbon [δ58.3 (C-3 ', 5'-OCH 3), 57.6 (C-3''- OCH 3), 57.0 (C-7''- OCH ₃ )], compound 5 was found to be a flavonoid with a phenylpropanoid moiety.

The conjugated ketone carbon δ183.9 (C-4) and the seven oxygenated olefine quaternary carbons [δ167.4 (C-7), 165.1 (C-2), 164.3 ), 159.8 (C-9), 155.2 (C-3 ', 5'), 141.5 10) and five olefinic carbon atoms [? 107.0 (C-3), 106.0 (C-2 ', 6'), 101.3 (C-6), 96.3 From these signals the presence of a flavone moiety was confirmed.

In addition, two oxygenated olefinic quaternary carbons [? 149.8 (C-3 "), 149.1 (C-4")], one olefinic quaternary carbon [? 131.7 (C-8 &quot;), 84.7 (C-8 &quot;), 7 &quot;)] and one oxygenated methylene carbon 62.5 (C-9 &quot;) signal were confirmed, and the presence of phenylpropanoid moiety was confirmed through these signals.

In the HMBC spectrum, the oxygenated methine protons δH 4.97 (1H, m, H-8 ") showed accurate cross-peaks with the oxygenated olefin quaternary δC 141.5 (C-4 '), The phenylpropanoid moiety was found to be connected via an ether bond between δC C-8 "and δC C-4 ', and the oxygenated methine proton signal δH 4.99 (1H, d, J = 4.0 Hz, H- 7 &quot;) and the methoxy carbon signal of delta C 57.0 was confirmed by the fact that the methoxy group was connected to the C-7 &quot; hydroxyl of the phenylpropanoid moiety.

From the above results, the structure of Compound 5 is shown to be tricin-4'-O- [erythro-beta-guanacyl- (7 &quot; -O- -guaiacyl- (7 &quot; -O-methyl) -glyceryl] ether, and is 7 &quot; -methoxy salcolin B.

The high resolution positive ESI-MS showed a false positive peak [M + H] ⁺ at m / z 541.1691, which was 541.1704, confirming C ₂₈ H ₂₉ O ₁₁ .

The structure of the compound 5 is a novel substance which has not been reported so far, and it is called Salcoline D.

< Example  2> From Zizania latifolia  Identification of inhibitory effect of isolated components on NO production

 In order to confirm the anti-inflammatory effect of the components isolated from Zizania latifolia, NO production and cytotoxicity were confirmed in RAW 264.7 cells stimulated with LPS.

RAW 264.7 cells were treated with LPS (100 ng / mL) in the presence of compounds 1 to 5 and cultured for 24 hours.

As a result, it was confirmed that the NO production was suppressed depending on the concentration of all the compounds as shown in FIG. 3B. In particular, the cells treated with 0.1, 1, 5 and 10 μg / 3.18, 13.86 ± 2.51, 21.47 ± 2.62 and 59.14 ± 0.78%, respectively.

In other words, tricine (1), salicolin A (2), salcolin B (3), salcolin C (4) and salcolin D (5) at concentrations of 10 μg / mL were 23.29 ± 3.21, 32.23 (IC ₅₀ = 0.24 μg / ml) in the IC ₅₀ values required to inhibit NO production by 50%, while the inhibition of NO production by the IC ₅₀ values was ± 1.93, 34.25 ± 2.76, 43.76 ± 2.82 and 59.14 ± 0.78% tree Seen _{(IC 50 = 12.51 μg / ml} ), live Colin _{A (IC 50 = 4.91 μg /} ml), live Colin _{B (IC 50 = 5.18 μg /} ml) and live Colin _{C (IC 50 = 0.69 μg /} ml) And showed excellent effect as compared with those of Comparative Example.

Further, as a result of confirming cell viability through MTT analysis, it was confirmed that NO production inhibition was not related to cell death. From the results, it was found that the test compound and 0.1% DMSO did not affect cell survival Respectively.

< Example  3> From Zizania latifolia  The β- Hexosaminidase (β-hexosaminidase) release inhibitory effect

The inhibitory effect of each extract on β-hexosaminidase release was confirmed in DNP-BSA-induced IgE-sensitized RBL-2H3 cells and the value obtained from the DNP-BSA-treated group was used as a control.

As a result, the cells treated with tricine (1), salicolin A (2), salcolin B (3), salcolin C (4) and salcolin D (5) Inhibition of β-hexosaminidase release was observed at 24.34 ± 2.89, 25.06 ± 1.36, 27.69 ± 5.61, 70.55 ± 0.60, and 89.43 ± 2.36%, respectively, and especially at concentrations of 0.1, 1 and 10 μg / Inhibited the release of? -Hexosaminidase by 42.83 ± 3.15, 89.43 ± 2.36 and 82.97 ± 6.96%, respectively, in the treated cell group. Also, β- hexyl sosami is the choline Kinase-emitting live in IC ₅₀ value represents the concentration needed to inhibit _{50% D (IC 50 = 0.45} μg / ml) is a new tree _{(IC 50 = 11.99 μg / ml} ), choline live A (IC ₅₀ = 5.18 μg / ml), salcolicin B (IC ₅₀ = 4.91 μg / ml) and salcolin C (IC ₅₀ = 0.69 μg / ml).

From the above results, it was confirmed that the effect of inhibiting? -Hexosaminidase release of sarcolin D was the most excellent.

In addition, as shown in FIG. 4A, the compounds did not show cytotoxicity on IgE-sensitized RBL-2H3 cells under the same conditions as described above. From the results, it was confirmed that the compounds did not show toxicity to cells.

Meanwhile, the tricine derivative according to the present invention can be formulated into various forms according to the purpose. Hereinafter, some formulation methods in which the tricine derivative according to the present invention is contained as an active ingredient are exemplified, and the present invention is not limited thereto.

Formulation Example 1 Prescription Example of Pharmaceutical Composition

&Lt; Formulation Example 1-1 > Preparation of injection

10 mg of SALCOLINE D, 3.0 mg of sodium metabisulfite, 0.8 mg of methylparaben, 0.1 mg of propylparaben and an appropriate amount of sterile distilled water for injection are mixed and made to a final volume of 2 mL by a conventional method, The ampoules were filled and sterilized to prepare an injection.

&Lt; Formulation Example 2-1 > Preparation of tablet

10 mg of salacoline D, 100 mg of lactose, 100 mg of starch and an appropriate amount of magnesium stearate, and tableted according to a conventional tablet preparation method.

&Lt; Formulation Example 3-1 > Preparation of capsules

10 mg of saccharin D, 50 mg of lactose, 50 mg of starch, 2 mg of talc, and magnesium stearate were mixed and filled in gelatin capsules according to a conventional capsule preparation method to prepare capsules.

&Lt; Formulation Example 4-1 > Preparation of ointment preparation

After mixing 100 mg of salcolin D, 250 mg of PEG-4000, 650 mg of PEG-400, 10 mg of white petrolatum, 1.44 mg of methyl p-hydroxybenzoate, 0.18 mg of propyl pamooxybenzoate and the remaining amount of purified water, .

&Lt; Formulation Example 2 > Formulation Example of Cosmetic Composition

&Lt; Formulation Example 2-1 > Preparation of nutrition lotion

3.0 parts by weight of propylene glycol, 0.1 part by weight of carboxy polymer, purified water of a small amount of preservative and remaining amount were heated to 80 to 85 ° C while stirring and mixing, and then charged into a manufacturing part. Then, an emulsifying agent was allowed to act. 1.0 part by weight of Polysorbate 60, 0.5 parts by weight of sesquioleate, 10.0 parts by weight of liquid paraffin, 1.0 part by weight of sorbitan stearate, 0.5 part by weight of glycerin monostearate as a parent type, 1.5 parts by weight of stearic acid, 1.0 part by weight of glyceryl stearate / PEG-400 stearate, And 0.2 parts by weight of amine were heated to 80 to 85 DEG C and then emulsified. After the emulsification was completed, the mixture was thermally cooled to 50 ° C with stirring using an agitator, and then a trace amount of a flavor was added thereto. The mixture was cooled to 45 ° C and added with a trace amount of dye. Salcolin D was added thereto at 35 ° C, .

<Formulation Example 2-2> Preparation of nutritional cream

0.3 parts by weight of carboxy polymer, 5.0 parts by weight of butylene glycol, 3.0 parts by weight of glycerin

And a remaining amount of purified water were heated to 80 to 85 占 폚 while being mixed and stirred. Then, the mixture was put into a production part and then an emulsifier was allowed to react. 2.0 parts by weight of stearic acid, 2.0 parts by weight of cetyl alcohol, 2.0 parts by weight of glyceryl monostearate, 0.5 part by weight of tanninostearate, 0.5 part by weight of sorbitan sesquioleate, 1.0 part by weight of glyceryl monostearate / glyceryl stearate / polyoxyethylene stearate, 1.0 part by weight of wax, 4.0 parts by weight of liquid paraffin, And 4.0 parts by weight of caprylic / capric triglyceride were heated to 80 to 85 占 폚 and then added with 0.5 part by weight of triethanolamine to emulsify. After the emulsification was completed, the mixture was cooled to 35 DEG C with stirring using an agitator, and then chalcone D was added thereto, followed by cooling to 25 DEG C and aging.

&Lt; Formulation Example 3 > Preparation of health food

Vitamin B 12, vitamin B 12 0.1 mg, vitamin B 2 0.15 mg, vitamin B 6 0.5 mg, vitamin B 12 0.2 쨉 g, vitamin C 10 mg, and vitamin C 10 mg), vitamin K acetate (70 쨉 g, vitamin E 1.0 mg, vitamin B 1 0.13 mg, 10 mg of biotin, 1.7 mg of nicotinic amide, 50 mg of folic acid, 0.5 mg of calcium pantothenate) and an appropriate amount of mineral mixture (1.75 mg of ferrous sulfate, 0.82 mg of zinc oxide, 25.3 mg of magnesium carbonate, 15 mg of potassium phosphate, 55 mg of calcium, 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride) were mixed to prepare a granule, and a health food was prepared according to a conventional method.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (5)

(7 &quot; -O-methyl) -glyceryl] ether (tricin-4'-O- [erythro-beta-guaiacyl- -methyl) -glyceryl] ether. (7 &quot; -O-methyl) -glyceryl] ether (tricin-4'-O- [erythro-beta-guaiacyl- (7'-O-methyl) -glyceryl] ether as an active ingredient and the tricine-4'-O- [erythro- beta -glycyl- A pharmaceutical composition for preventing or treating an allergic disease, which is characterized by inhibiting the release of? -Hexosaminidase and thereby preventing or treating an allergic disease selected from the group consisting of asthma, atopic dermatitis and allergic rhinitis. (7 &quot; -O-methyl) -glyceryl] ether (tricin-4'-O- [erythro-beta-guaiacyl- (7'-O-methyl) -glyceryl] ether as an active ingredient and the tricine-4'-O- [erythro- beta -glycyl- A health food for preventing or ameliorating an allergic disease characterized by inhibiting the release of? -Hexosaminidase to prevent or ameliorate allergic diseases selected from the group consisting of asthma, atopic dermatitis and allergic rhinitis. (7 &quot; -O-methyl) -glyceryl] ether (tricin-4'-O- [erythro-beta-guaiacyl- -methyl) -glyceryl] ether as an active ingredient and inhibiting the release of? -hexosaminidase in vitro. The β-hexamidase (β- hexosaminidase) release inhibitor. Human isolated cells were treated with tricine-4'-O- [erythro-beta-guaiacyl- (7'-O-methyl) -glyceryl] ether - (7 &quot; -O-methyl) -glyceryl] ether in the presence of a reducing agent.

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