KR101396387B1 - Process for preparing baicalein-6-alpha-glucoside using recombinant amylosucrase, and composition for preventing or treating of inflammatory diseases containing baicalein-6-alpha-glucoside prepared by the same - Google Patents

Abstract

The present invention relates to a preparation method of baicalein-6-alpha-glucoside using recombinant amylosucrase, and a composition to prevent or treat inflammatory diseases containing baicalein-6-alpha-glucoside as an active ingredient prepared by the same. The baicalein-6-alpha-glucoside has excellent solubility which is 16 times the solubility of that of an existing baicalein-6-alpha-glucoside; does not have cytotoxicity; has excellent Nrf2 activation; and expresses a gene associated with anti-inflammatory effect. In addition, the baicalein-6-alpha-glucoside is easily absorbed into the human body and has excellent anti-inflammatory effects without side effects because the baicalein-6-alpha-glucoside does not produce reactive oxygen species. Therefore, the baicalein-6-alpha-glucoside can be used as an advantage to prevent, alleviate or treat inflammatory diseases.

Description

A method for producing baicalein-6-alpha-D-glucoside using recombinant amylose sucrose, and a method for preventing or treating inflammatory diseases comprising baicalein-6-alpha-D-glucoside as an active ingredient 6-alpha-glucoside-based recombinant amylosucrase, and a composition for preventing or treating the inflammatory disease-containing baicalein-6-alpha-glucoside prepared by the same.

The present invention relates to a process for producing baicalein-6-alpha-D-glucoside using recombinant amylose sucrase and to a process for the prevention of inflammatory diseases comprising baicalein-6-alpha-D-glucoside as an active ingredient Or therapeutic compositions.

The inflammatory reaction is a mechanism to repair and regenerate the damaged area when an invasion that brings about physical changes such as physical action, chemical substance, bacterial infection or the like is applied to the living body or tissue. Once inflammation is applied, the inflammatory reaction locally causes histamine, serotonin, Vascular active substances such as bradykinin, prostaglandin, hydroxyeicosatetraenoic acid (HETE) and leukotriene are liberated, resulting in increased vascular permeability and inflammation.

Macrophages respond to inflammation by secretion of nitric oxide (NO), prostaglandin E ₂ (PGE ₂ ) and proinflammatory cytokines, and also act as important regulatory cells in both inflammation and immune responses. It must go through the process. Lipopolysaccharide (LPS), one of the cell wall components of Gram-negative bacteria and well known as an endotoxin, is the most well-known external factor involved in the activation of macrophages. In particular, TNF-α It is known that the pro-inflammatory cytokines such as necrosis factor-alpha, interleukin-6 and interleukin-1 beta are secreted and increased in inflammation sites.

When LPS stimulates macrophages, NO is produced from macrophages by the production of nitrogen monoxide (NO) during the process of converting L-arginine into L-citrulline by an enzyme called iNOS (inducible nitric oxide synthase). In mammals, NO is synthesized by three types of NO synthase (NOS): nNOS (neuronal NOS), eNOS (endothelial NOS) and iNOS (inducible NOS). Among these, NO produced by nNOS and eNOS is produced for normal biological function, and concentration in tissues is kept low to a certain level. However, NO produced by iNOS is overexpressed and exhibits deleterious effects on living organisms such as pathological vasodilation, cytotoxicity, and tissue damage.

Prostaglandin E ₂ (PGE ₂ ), an inflammatory factor, stimulates the phospholipid, a component of the cell membrane, stimulated by LPS to release arachidonic acid, which is released by an enzyme called phospholipase A2, as an enzyme catalyzed by COXs (cyclooxygenase) And is induced to induce an inflammatory reaction. COX is classified as a COX-Ⅰ and COX-II, COX-Ⅰ is applied to the normal physiological functions such as formation of platelets in the body, gastric protection, maintenance of the kidney function and, COX-II is synthesized inflammatory mediators of PGE ₂ do. PGE ₂ is known to be deeply involved in the development of cancer, including inflammation, inflammation, immune response, and angiogenesis, such as pain and fever.

As mentioned above, inflammation can cause various serious diseases such as cancer, vascular disorders and diabetes (Schetter AJ, Heegaard NH, Harris CC. Inflammation and cancer: interweaving microRNA, free radical, cytokine and p53 pathways. Carcinogenesis 2010; 31: 37-49; Khatami M. 'Yin and Yang' in inflammation: duality in innate immune cell function and tumorigenesis. Expert Opin Biol Ther 2008; 8: 1461-72.

Recent studies indicate that the functional significance of tissue-associated macrophages in inflammatory responses is increasingly emphasized. These cells have been reported to invade chronically inflamed tissues, aggressively regulate various inflammatory molecules and promote inflammatory tissue damage (Ohno S, Inagawa H, Dhar DK, Fujii T, Ueda S, Tachibana M, et (TAM) in advanced gastric carcinoma: the impact on FasL-mediated counterattack. Anticancer Res 2005; 25: 463-70.).

Anti-inflammatory drugs are largely classified as steroids and non-steroids. Steroids bind to nuclei to synthesize antiinflammatory proteins, while nonsteroidal agents act on the activity of cyclic oxidases in the early stages of prostaglandin synthesis in arachidonic acid, inhibiting prostaglandin formation. The cyclized oxidase that produces prostaglandins is produced mainly by the pro-inflammatory transcription factor NF-κB.

In general, non-steroidal anti-inflammatory drugs have anti-inflammatory effects by inhibiting NF-κB. Recently, however, NF-κB has been shown to be a major factor involved in the anti-inflammatory effect of Nrf2 (Nuclear factor-erythroid 2-related factor 2) It acts antagonistic to NF-κB by inducing the expression of antioxidant proteins such as NADH quinone oxiodoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) . Nrf2 has been found to be involved in various inflammatory and chronic chronic diseases such as autoimmune diseases, acute pneumonia, intestinal diseases and skin inflammation. Therefore, recent studies on the method of inhibiting inflammation by activating Nrf2 have become more important. However, non-steroidal preparations can cause cirrhosis or renal failure when taken over a long period of time, and have side effects that may cause lowering of kidney function and elevation of blood pressure. The biggest side effect of nonsteroidal preparations is to inhibit the synthesis of prostaglandins protecting gastric barrier and promote gastric acid secretion.

In order to solve the side effects of such anti-inflammatory drugs, researches are actively carried out to develop physiologically active substances having anti-inflammatory functions in natural products. However, the glycosides derived from natural products have a problem in that the absorption rate in the body is low and it is difficult to realize a therapeutic effect using various absorption routes (oral, injection, spray, etc.).

Baikal lane belongs to flavon, a kind of flavonoid, abundant in the roots of gold. Baikal lane is a form of the asparaginized form of baicalin, in which a hydroxyl group is bonded to 5,6,7 carbon of the A ring of the flavored structure. Baikal lane is commonly used as an antioxidant to remove reactive oxygen species (Hamada et al., 1993), and is a representative substance that inhibits 12-lipoxygenase causing oxidative damage (Leung et al., 2007). In addition, baicalein inhibits mitochondrial lipid peroxidation and is known to prevent apoptosis due to oxidative damage (Chem et al., 2006).

Therefore, the inventors of the present invention have conducted studies to develop a new drug having no adverse effect on the living body and having an adverse effect and anti-inflammatory effect, and it has been found that baicalein-6-alpha-D- glucoside has excellent water- The present invention has been accomplished by confirming that the gene involved in anti-inflammation is expressed and the active oxygen species are not produced, so that absorption in the body is easy and the side effect is not exerted, and the inflammation-suppressing effect is excellent.

It is an object of the present invention to provide a method for producing baicalein-6-alpha-D-glucoside using recombinant amylose sucrose.

Another object of the present invention is to provide a composition for preventing or treating an inflammatory disease comprising baicalein-6-alpha-D-glucoside as an active ingredient.

The present invention provides a method for producing baicalein-6-alpha-D-glucoside using recombinant amylose sucrose.

The present invention provides a composition for preventing or treating an inflammatory disease comprising baicalein-6-alpha-D-glucoside as an active ingredient.

The baicalein-6-alpha-D-glucoside of the present invention shows a water-solubility that is 16 times better than that of the conventional baicalein, has no cytotoxicity, has excellent Nrf2 activity, expresses a gene involved in anti- By not producing a species, it is easy to absorb in the body, has no side effects, and has excellent anti-inflammatory effect.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the results of TLC analysis of baicalein-6-alpha-D-glucoside of the present invention. FIG.
FIG. 2 is a diagram showing a structural analysis result of Baicalene-6-alpha-D-glucoside of the present invention. FIG.
FIG. 3 is a graph showing the results of experiments of cytotoxicity of Baicalene-6-alpha-D-glucoside of the present invention.
4 is a graph showing the degree of generation of nitrogen monoxide in baicalein-6-alpha-D-glucoside of the present invention.
5 is a graph showing the results of the activity analysis of Nrf2-dependent NQO1 of the bicalnein derivative of the present invention.
6 is a graph showing the results of analysis of Nrf2 activity of baicalein-6-alpha-D-glucoside of the present invention.
FIG. 7 is a graph showing the results of observing the occurrence of reactive oxygen species in Baikalin-6-alpha-D-glucoside of the present invention.

The present invention

1) mixing recombinant amylose sucrose, baicalein and sucrose, adding the mixture to a Tris-HCl buffer solution and reacting;

2) removing sugar from the reaction mixture; And

3) Purification of the saccharide-free reaction mixture to obtain baicalein-6-alpha-D-glucoside represented by the following formula 1: Baikallan-6-alpha-D-glucoside using recombinant amylose sucrose A method for producing glucoside is provided.

[Chemical Formula 1]

The production method of baicalein-6-alpha-D-glucoside using the recombinant amylose sucrose of the present invention will be described step by step.

The above step 1) is a step of preparing a reaction mixture comprising recombinant amylose sucrose, baicalein, sucrose and a Tris-HCL buffer solution.

The recombinant amylose sucrose may be,

i) culturing E. coli BL21 containing recombinant pGEX-DGAS in an ampicillin-containing medium;

ii) measuring the absorbance of the culture medium to inoculate the culture medium with isopropyl-D-thiogalactopyranoside at an absorbance of 0.6 to induce the expression of the dgas gene;

iii) culturing the culture solution, centrifuging the cultured E. coli BL21 to collect only germs, suspending the culture in a phosphate buffer solution;

iv) ultrasound treatment of the present delivery fluid to induce cell disruption, followed by centrifugation;

v) isolating and purifying the supernatant of the culture broth centrifuged in step iv) to obtain purified recombinant amylose sucrose; And

vi) adding the thrombin to the purified recombinant amylose sucrose to remove the GST protein to prepare the final recombinant amylose sucrose.

In step 2), the reaction mixture prepared in step 1) is reacted with sugar, and the sugar chain reaction is preferably performed using sucrose as a substrate, and the sugar is removed using a C18-T cartridge .

In the step 3), the recombinant amylose sucrose is purified by purifying the sugar-removed recombinant amylose sucrose, and the recombinant amylose sucrose is preferably purified using recycle preparative HPLC Do.

It was confirmed that the baicalein-6-alpha-D-glucoside prepared by the above-mentioned preparation method was 16 times more water-soluble than the conventional baicalein.

The present invention provides a composition for preventing or treating an inflammatory disease comprising baicalein-6-alpha-D-glucoside represented by the following formula (1) as an active ingredient.

The composition comprises a pharmaceutical composition or a food composition.

Hereinafter, the composition of the present invention will be described in more detail.

The active ingredient baicalein-6-alpha-D-glucoside in the composition of the present invention can be obtained by the method described above, or can be obtained by a conventional organic synthesis method.

The baicalein-6-alpha-D-glucoside of the present invention shows a water-solubility that is 16 times better than that of the conventional baicalein, has no cytotoxicity, has excellent Nrf2 activity, expresses a gene involved in anti- By not producing a species, it is easily absorbed in the body, has no side effects and is excellent in an inflammation inhibiting effect, and thus can be usefully used for prevention, improvement or treatment of inflammatory diseases.

The inflammatory disease includes, but is not limited to, one or more inflammatory diseases selected from the group consisting of rheumatoid arthritis, restenosis, psoriasis, multiple sclerosis, surgical adhesions, tuberculosis and chronic inflammatory lung disease.

The composition of the present invention may contain at least one known active ingredient having the therapeutic effect of inflammatory diseases together with baicalein-6-alpha-D-glucoside.

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 preparations 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 are also 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 patient, 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 a preferred effect, the daily dose of baicalein-6 alpha-D-glucoside of the present invention may be administered in an amount of 1 mg / kg to 10 mg / kg per day, It may be administered several times.

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 and 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 baicalein-6-alpha-D-glucoside of the present invention is used as a food additive, the baicalein-6-alpha-D-glucoside can be directly added or used together with other food or food ingredients, Can be appropriately used. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, baicalein-6-alpha-D-glucoside 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 the foods to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen 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 embodiments, experimental examples, and production 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 production examples.

[Example 1: preparation of baicalein-6-alpha-D-glucoside]

In order to prepare the baicalein-6-alpha-D-glucoside of the present invention, the following procedure was carried out.

1. Preparation of Recombinant Amylosucrase

Amylose sucrase was isolated from Deinococcus geothermalis , and recombinant amylose sucrease was prepared using the amylosucrase gene obtained from Dienocorca geoscholarium . More specifically, E. coli BL21 harboring recombinant pGEX-DGAS was plated on 1 L of LB medium containing ampicillin at a concentration of 0.1 mg / ml and incubated at 37 ° C. When the absorbance reached 0.6, 1 mM isopropyl-D-thiogalactopyranoside (IPTG) was inoculated into the culture medium to induce the expression of the dgas gene. After 3 hours of incubation, the cultured E. coli BL21 was centrifuged to collect only germs, and then the cells were washed with phosphate-buffered saline (PBS, 5 ml / g (wet weight) Composition: 140 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , and 1.8 mM KH ₂ PO ₄ (pH 7.3)] and suspension culture. The suspension culture was sonicated to disrupt the cells, The resulting supernatant was purified with a Glutathione Sepharose ^™ High performance affinity column to remove the GST protein in the purified recombinant amylose sucrose, followed by treatment with thrombin Recombinant amylose sucrase was obtained.

2. Preparation of baicalein-6-alpha-D-glucoside

To prepare baicalein-6-alpha-D-glucoside of the present invention, the baicalein derivative was subjected to sugar transfer and purification of the baicalein derivative using the recombinant amylose sucrose prepared in 1 above. To 10 ml of the total reaction solution, 20 mM of Baikalin, 40 mM of sucrose and 1 mg of the recombinant amylose sucrose prepared in the above 1 were added and mixed, and the mixture was added to 100 mM Tris-HCl buffer (pH 8.0) And reacted at 30 DEG C for 12 hours. The reaction mixture was stripped of the sugar using a C18-T cartridge and purified using Recycling preparative HPLC. C18-T cartridge (100 mg / ml; Phenomenex Korea). Recycle aliquot HPLC was performed using W-251 (JAI, Tokyo, Japan) and RI-50 detector (JAI) combined with JAIGEL W-252 column. For elution of the derivative product, HPLC grade methanol was flowed at a flow rate of 3.0 ml / min to obtain purified baicalein-6-alpha-D-glucoside.

[Experimental Example 1: TLC analysis of baicalein-6-alpha-D-glucoside]

Whatman K6F silica gel plates (Whatman, Maidstone, UK) were used to perform TLC (thin layer chromatography) analysis on baicalein-6 alpha-D-glucoside prepared in Example 1 above , Butanol, ethanol and water were mixed at a ratio of 5: 3: 2 (v / v / v) as a developing solvent. 1 L of sample was spotted on the TLC plate and dried. The solution was developed in a developing solution for a predetermined time, and dried again in the air. The dried plate was measured at a wavelength of 254 nm using the phenomenon of light emission of the ring structure of polyphenol.

The dried plate was also rapidly dipped in a methanol solution containing 0.3% (w / v) N - (1-naphthyl) ethylenediamine and 5% (v / v) sulfuric acid (H ₂ SO ₄ ) After it was dried out completely. The mixture was baked at 110 ° C for 10 minutes. Then, the C18-T cartridge was used for the analysis of the baicalein alpha derivative, which was obtained by removing the sugar from the reaction solution (transfer product) and recycling preparative HPLC. The column of the chromatography used W251 and W252 columns, and methanol was used as the solvent. The results are shown in Fig.

As shown in Fig. 1, it was confirmed that baicalein-6-alpha-D-glucoside was an inherited product.

[Experimental Example 2: Structural Analysis of Baicalene-6-alpha-D-Glucoside]

To analyze the structure of the baicalein-6-alpha-D-glucoside prepared in Example 1, about 4.6 mg of the sample was dissolved in CD ₃ OD, and then analyzed by Varian Inova AS 400 MHz nuclear magnetic resonance spectroscopy ¹ H and ¹³ C spectra were measured with a spectrometer (Varian, Palo Alto, Calif., USA) and 2D NMR gHMBC analysis was performed. The results are shown in Fig.

As shown in FIG. 2, 1H-NMR analysis of the baicalein-6-alpha-D-glucoside of the present invention revealed that anomeric proton was observed at about 5.2 ppm, it was judged that the glucose was? -linked to the baicalein. gHMBC analysis of the relationship between carbon and hydrogen in the transit product was carried out in the literature (Liu et al., 1993 for Baikal lane-6-glucoside and Liu et al., 2009 for baicalein-7-glucoside) As a result of comparing with measured NMR results, it was confirmed that glucose binds to hydroxyl group 6 of A ring of Baikal lane.

Thus, it is considered that the baicalein-6-alpha-D-glucoside of the present invention has a structure having alpha-binding to baicalein and glucose linked to the 6-hydroxyl group of the bicalanine A ring.

[Experimental Example 3: Water solubility analysis of Baicalene-6-alpha-D-glucoside]

In order to carry out the aqueous analysis of the baicalein-6-alpha-D-glucoside of the present invention, excess baicalein and baicalein-6-alpha-D-glucoside were placed in each Eppendorf tube, Distilled water was added. The supernatant was then centrifuged at 13,000 rpm for 10 minutes, and then the absorbance of the supernatant was measured using a UV spectrophotometer (Gene Quan pro UV / Vis spectrophotometer, UK) 285 nm. The water solubility of the baicalein-6-alpha-D-glucoside of the present invention was analyzed, and the results of the analysis are shown in Table 1 below.

Constituent material Water solubility (μM) Baikal Lane 0.02 Baicalein-6-a-O-glucoside 0.33

As shown in Table 1 above, the water solubility of the baicalein-6-alpha-D-glucoside of the present invention was confirmed to be about 16 times better than that of baicalein.

Therefore, the baicalein-6-alpha-D-glucoside of the present invention has a high water solubility, and thus it is thought that it exhibits a better pharmacological effect by increasing the absorption rate in the body.

[Experimental example 4: Measurement of cytotoxicity of baicalein-6-alpha-D-glucoside]

In order to determine the cytotoxicity of baicalein-6 alpha-D-glucoside of the present invention, MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, and a yellow tetrazole.

More specifically, RAW 264.7 cells (5 x 10 ⁴ cells / well), a macrophage of mice, were placed in a 24-well culture plate and incubated at 37 ° C for 18 hours, mu] M concentration of baicalein or baicalein-6 alpha-D-glucoside. In addition, lipopolysaccharide (LPS) was added at 100 ng / ml 1 hour after the drug treatment in order to confirm the drug toxicity of the cells upon inflammation induction. After 12 hours of drug treatment, 200 [mu] l of cultured MTT solution (0.5 mg / ml) was dispensed into each well. After incubation at 37 ° C and atmospheric carbon dioxide 5% for 4 hours, the supernatant was removed, and the formazan crystals produced in the surviving cells were dissolved in 300 μl of DMSO. Samples of each well were measured for absorbance at 595 nm using a microplate reader, and the results are shown in FIG.

As shown in FIG. 3, it was confirmed that the baicalein-6-alpha-D-glucoside treatment group of the present invention was free from cytotoxicity. In addition, the treatment with LPS-treated baicalein-6-alpha-D-glucoside showed no significant cytopathic effect on cell proliferation.

Thus, the baicalein-6-alpha-D-glucoside of the present invention has no cytotoxicity and is considered to have no side effects in the body.

[Experimental Example 5: Analysis of anti-inflammatory effect of baicalein-6-alpha-D-glucoside]

To investigate the anti-inflammatory effect of baicalein-6-alpha-D-glucoside of the present invention, nitric oxide (NO) method was performed. The NO method measures the amount of nitrogen monoxide, which is an early defense product in mammalian cells. Using the Greiss reagent for measuring nitrogen monoxide, the degree of nitric oxide production can be confirmed. More specifically, RAW 264.7 cell line, a mouse macrophage, was cultured in a 24-well plate for 18 hours at a concentration of 5 x 10 ⁴ cells / well, and then treated with Baikal lane and Baicalain-6-alpha -D-glucoside and treated with lipopolysaccharide (LPS) at 100 ng / ml after 1 hour. 24 hours after the treatment, the medium and the Greiss solution were reacted at a ratio of 1: 1, and the absorbance at 540 nm was measured. The results are shown in Fig.

As shown in FIG. 4, it was confirmed that the treatment with baicalein-6-alpha-D-glucoside of the present invention showed an excellent nitric oxide generation inhibiting effect at 40 μM.

Therefore, the baicalein-6-alpha-D-glucoside of the present invention has a high inhibitory effect on nitrogen monoxide and thus is considered to have excellent anti-inflammatory effect.

[Experimental Example 6: Activity analysis of Nrf2-dependent NQO1 of baicalein-6-alpha-D-glucoside]

To examine the activity of Nrf2, an indicator protein for the anti-inflammatory activity of baicalein-6-alpha-D-glucoside of the present invention, the activity of Nrf2-dependent NQO1 was assayed. The above analysis was carried out through a luciferase assay using the Nrf2-dependent NQO1 luminescent enzyme receptor cell line constructed by the present inventors. More specifically, Nrf2 luminescent enzyme receptor cell lines were cultured in a 24-well culture plate at 5 x 10 ⁴ cells / well for 18 hours, and then Baikal lane and Baikal lane-6-alpha-D- To 40 [mu] M. Eight hours after the treatment, the cells were dissolved and reacted with the luminescence enzyme to measure the degree of luminescence. The results are shown in Fig.

As shown in FIG. 5, in the group treated with baicalein-6-alpha-D-glucoside of the present invention, the anti-inflammatory effect was 1.2 times better than that of the conventional baicalin at a concentration of 20 μM. In particular, baicalein-6-alpha-D-glucoside showed almost the same level of activity as sulforaphane used as a positive control. Therefore, the baicalein-6-alpha-D-glucoside of the present invention inhibits the activity of Nrf2-dependent NQO1, and thus the anti-inflammatory effect is superior.

[Experimental example 7. Analysis of Nrf2 activity of baicalein-6-alpha-D-glucoside]

In order to analyze the Nrf2 activity of baicalein-6-alpha-D-glucoside of the present invention, the following procedure was performed.

Specifically, 3 ml of RAW 264.7 cell line was added to a 60 mM culture dish at a concentration of 1 × 10 ⁶ cells / ml, and after 18 hours, baicalein or baicalein-6-alpha-D-glucoside Respectively. After 8 hours of treatment, the cells in the incubator were dissolved and the amount of Nrf2 in the nuclei was measured by Western blotting. The results are shown in Fig.

As shown in FIG. 6, in the baicalein-6-alpha-D-glucoside treatment group of the present invention, Nrf2 in the nucleus after 8 hours was most abundant at a concentration of 20 μM and Baikalin- Both D-glucoside and baicalein treatment groups were found to be similar.

Therefore, the baicalein-6-alpha-D-glucoside of the present invention inhibits the activity of Nrf2, and thus it is judged that the anti-inflammatory effect is excellent.

[Experimental example 8] Measurement of active oxygen species in Baikalin-6-alpha-D-glucoside [

In order to measure the active oxygen species of baicalein-6-alpha-D-glucoside of the present invention, a fluorescence-activated cell sorter or flow cytometry (FACS) was used. Nrf2 involved in the inflammation development is not only increased by the drug treatment but also may be increased by the presence of reactive oxygen species (Reactive Oxygen Species).

Specifically, 3 ml of RAW 264.7 cell line was added to a 6-well culture dish at a concentration of 1 × 10 ⁵ cells / ml, followed by addition of a positive control LPS and 20 μM of Baikalin-6-alpha-D-glucoside, 40 The cells were treated with DMSO in μM baicalein and treated. The collected cells were washed with PBS, treated with H2DCF-DA at a concentration of 100 μM, and reacted at 37 ° C for 30 minutes. This was washed once more with PBS, and then the number of cells was measured using FACS. The results are shown in Fig.

As shown in FIG. 7, in the baicalein-6-alpha-D-glucoside treatment group of the present invention, 9% active oxygen species production was measured at a concentration of 20 μM and 11% of active oxygen species production was measured at a concentration of 40 μM . On the other hand, in the case of cells treated with DMSO alone, 10% of active oxygen species were measured, and in the case of positive control (LPS), 85.5% of the treated cells were analyzed for active oxygen species.

Therefore, the baicalein-6-alpha-D-glucoside of the present invention does not produce active oxygen species, and thus it is judged that the anti-inflammatory effect is excellent.

[Preparation Example 1: Preparation of pharmaceutical preparation]

1. Manufacturing of powder

Baicalene-6-alpha-D-glucoside 20 mg

Lactose 100 mg

Talc 10 mg

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

2. Preparation of tablets

Baicalene-6-alpha-D-glucoside 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

Baicalene-6-alpha-D-glucoside 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

Baicalene-6-alpha-D-glucoside 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

Baicalene-6-alpha-D-glucoside 20 mg

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

Baicalene-6-alpha-D-glucoside 100 mg

Vitamin mixture quantity

Vitamin A acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

Vitamin B12 0.2 μg

Vitamin C 10 mg

Biotin 10 μg

Nicotinic acid amide 1.7 mg

Folic acid 50 μg

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

Baicalene-6-alpha-D-glucoside 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 (6)

1) mixing recombinant amylose sucrose, baicalein and sucrose, adding the mixture to a Tris-HCl buffer solution and reacting;
2) removing sugar from the reaction mixture; And
3) Purification of the saccharide-free reaction mixture to obtain baicalein-6-alpha-D-glucoside represented by the following formula 1: Baikallan-6-alpha-D-glucoside using recombinant amylose sucrose ≪ / RTI >
[Chemical Formula 1]

2. The method according to claim 1, wherein the recombinant amylose sucrase in step 1)
i) culturing E. coli BL21 containing recombinant pGEX-DGAS in an ampicillin-containing medium;
ii) measuring the absorbance of the culture medium to inoculate the culture medium with isopropyl-D-thiogalactopyranoside at an absorbance of 0.6 to induce the expression of the dgas gene;
iii) culturing the culture solution, centrifuging the cultured E. coli BL21 to collect only germs, suspending the culture in a phosphate buffer solution;
iv) ultrasound treatment of the present delivery fluid to induce cell disruption, followed by centrifugation;
v) isolating and purifying the supernatant of the culture broth centrifuged in step iv) to obtain purified recombinant amylose sucrose; And
vi) adding thrombin to the purified recombinant amylose sucrose to remove the GST protein, thereby producing a final recombinant amylose sucrose. ≪ / RTI > glucoside.
The method of claim 1, wherein the saccharide is removed using a C18-T cartridge in step 2).
A pharmaceutical composition for preventing or treating an inflammatory disease comprising baicalein-6-alpha-D-glucoside represented by the following formula (1) as an active ingredient.
[Chemical Formula 1]

5. The method of claim 4, wherein said inflammatory disease comprises one or more inflammatory diseases selected from the group consisting of rheumatoid arthritis, restenosis, psoriasis, multiple sclerosis, surgical adhesions, tuberculosis and chronic inflammatory lung disease. A pharmaceutical composition for preventing or treating diseases.
A food composition for preventing or ameliorating an inflammatory disease comprising baicalein-6-alpha-D-glucoside represented by the following formula (1) as an active ingredient.
[Chemical Formula 1]

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