KR101470613B1 - Composition comprising latifolin for preventing or treating inflammatory diseases - Google Patents

Abstract

The present invention relates to a composition for preventing or treating an inflammatory disease comprising latifolin as an active ingredient. Latofolin according to the present invention is excellent in inhibiting the inflammatory response through the expression and activity increase of hemoxysinase-1 (HO-1) enzyme in macrophages, and can be usefully used for the prophylaxis or treatment of inflammatory diseases .

Description

[0001] The present invention relates to a composition for preventing or treating inflammatory diseases,

The present invention relates to a composition for preventing or treating an inflammatory disease comprising latifolin as an active ingredient.

Along with the recent economic growth, the average life span has been increasing, and the proportion of the elderly population has been increasing. In addition, the abundant dietary life changes the typical Korean food culture which mainly uses food materials such as seafood and vegetables, and the ratio of lifestyle diseases such as cancer, diabetes, hypertension, obesity and vascular diseases is increasing every year. In addition, the incidence of inflammatory diseases caused by the persistence of inflammation caused by immunomodulation abnormality has also been increasing not only in Western countries but also in Korea in recent 10 years. As a result, interest in health and longevity has increased in recent years, and consumers' demand for health functional materials and products is increasing. This has led to the expansion of the market for health functional foods and pharmaceuticals, and research is actively underway to find new functional materials from natural resources. Among them, researches on anti-inflammatory substances, which are aimed to identify diseases that can be caused by inflammation and to prevent or treat them, are also important.

Inflammation is a mechanism of recovery to regenerate damaged areas caused by chemical substances, bacterial infections, and physical effects that cause changes in organisms or tissues. When inflammation is manifested by harmful stimuli, infections and trauma, inflammation is induced by local release of vasoactive substances such as inflammatory components. If the inflammatory reaction is continued, mucous membrane damage is promoted, resulting in redness, fever, swelling, Inflammatory diseases such as rheumatoid arthritis, arteriosclerosis, gastritis, and asthma caused by pain, dysfunction, and the like.

These inflammatory reactions involve a variety of mediators such as cytokines, prostagrandins E2 and PGE2, and free radicals. In particular, in macrophages, NF-κB (nclear), which is a transcription factor of inflammatory responses by stimulation such as cytokine, tumor necrosis factor-α, TNF-α and lipopolysaccharide (COX-2) and inducible nitric oxide synthase (iNOS), which are activated by nitric oxide (NO) and factor-κB, It produces stargalandin E2 and causes inflammation.

Macrophages are involved in the maintenance of homeostasis in response to various host reactions such as acquired immunity as well as innate immunity, and play important roles in biological defense in the early stages of infection by producing nitric oxide and various cytokines in the inflammatory reaction. The macrophages or monocytes such as RAW 264.7 have been shown to stimulate tumor necrosis factor-a, interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) by stimulation of lipopolysaccharide, an endotoxin present in the extracellular membrane of Gram- ). ≪ / RTI > The formation of these inflammatory mediators is a process in which arachidonic acid is converted to leukotriene, thromboxane, prostaglandin and the like through the action of cyclooxygenase (COX) and nitric oxide (NO) Is involved in the mass production of inflammatory mediators and plays a major role in the host is known to cause fatal consequences. COX is an enzyme that promotes the conversion of arachidonic acid from phospholipids in cell membranes to prostaglandins, and there is an isoform of cyclooxygenase-1, cyclooxygenase-2. Cyclooxygenase-1 is expressed in most tissues to maintain homeostasis of the body, while cyclooxygenase-2 is inhibited by various stimuli such as growth factors, cytokines and lipopolysaccharides, (macrophage) or monocyte (monocyte), etc., and the resulting prostaglandins inhibit tumor cell apoptosis and induce angiogenesis and contribute to tumorigenesis. Nitric oxide (NO) is produced by nitric oxide synthase (NOS) in the cell with L-arginine as a substrate and is produced by L-citruline, It is a major transmitter in the cardiovascular system that simultaneously exhibits neurotoxic and neuroprotective functions and is also reported to be involved in the development of shock and various neurodegenerative diseases. NOS is classified as neurogenic NOS (nNOS, NOS1), inductive NOS (iNOS, NOS2) and endothelial NOS (eNOS, NOS) depending on the cell type (Tsao et al. Regardless of the calcium concentration and the stimulation of calmodulin injected from the outside, it is active only in activated cells and is induced in various cells. Nuclear transcription factors-kappa B and NF-κB are also involved in the transcription of several inflammatory mediators such as TNF-α, IL-1β, IL-6, iNOS and COX-2 Which greatly influences their gene expression.

In addition, hemoxigenase-1 is a heme degrading enzyme that produces biliverdin, carbon monoxide and iron. The decomposition of heme by hemeoxygenase-1 has various pharmacological actions. Among them, the decomposition of iron and iron oxide in heme prevents oxidative damage of DNA through cytoprotective action and antioxidative action. Thus preventing the onset of carcinogenesis and inhibiting and protecting cancer promoting processes associated with oxidative or inflammatory cells or tissue damage. Therefore, the induction of hemeoxygenase-1 is an important part of chemical cancer prevention research. Also, decomposition of heme into carbon monoxide was found to be involved in the inflammatory reaction. In recent studies, the expression of hemoxigenase-1 was significantly elevated in rheumatoid arthritis tissue, which was significantly increased when compared to that of degenerative arthritis, and the role of hemoxigenase-1 in inflammatory arthritis (Jang, SJ et al., The effects of heme oxygenase-1 on collagen induced arthritis model, The Korean J. Anat. 39, 393-399 (2006)).

On the other hand, latifolin is a vegetable polyphenol compound isolated from the river ferment ( Dalbergia odorifera T. Chen, soybean and Leguminosae). It is well known that vegetable polyphenol compounds have excellent effects in the treatment and prevention of inflammatory diseases, and studies have been made actively on them. In addition, a pharmaceutical composition for prevention or treatment of osteoporosis comprising a strong fragrant extract and a skin external preparation (Korean Patent Laid-open No. 10-2010-0011764) containing a health food (Korean Patent Laid-Open No. 10-2001-0030064) Are known. However, studies on the new pharmacological activity of latifolin have been limited, and there has been no report on the anti-inflammatory effect of latifolin. Therefore, there is a need for a therapeutic agent for an effective inflammatory disease using a plant polyphenol component.

Therefore, the inventors of the present invention have made intensive studies to develop a composition for the prophylaxis or treatment of inflammatory diseases. As a result, it has been found that latifurin, isolated from the core of a river, has excellent inflammation through expression of hemoxysinase-1 (HO-1) Inhibitory effect, and thus the present invention has been completed.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating an inflammatory disease comprising latisin as an active ingredient.

It is still another object of the present invention to provide a food composition for preventing or ameliorating an inflammatory disease which comprises latiphorin as an active ingredient.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease comprising latisin as an active ingredient.

In addition, the present invention provides a food composition for preventing or ameliorating an inflammatory disease comprising lattiolin as an active ingredient.

Latofolin according to the present invention is excellent in inhibiting the inflammatory response through the expression and activity increase of hemoxysinase-1 (HO-1) enzyme in macrophages, and can be usefully used for the prophylaxis or treatment of inflammatory diseases .

1 is a view showing a process of separating latifolin from a core material at a strong end.
FIG. 2 is a graph showing the results of HPLC analysis for confirming the purity of latifolin separated from the core material of the river.
FIG. 3 is a graph showing the results of analysis of ¹ H-NMR (A) and ¹³ C-NMR (B) spectra for confirming the structure of latiphorin.
Fig. 4 is a graph showing cell viability for confirming cytotoxicity of latifulin to macrophages. Fig.
FIG. 5 is a graph showing the results of RT-PCR analysis of iNOS mRNA expression (A) and COX-2 mRNA expression (B) inhibitory effect on inflammation induction of latiphorin.
FIG. 6 is a graph showing the results of western blot analysis of iNOS protein expression (A) and COX-2 protein expression (B) inhibitory effect on inflammation induction of latifulin.
FIG. 7 is a graph showing the inhibitory effect on the production of NO production (A), PGE ₂ production (B), inflammation-mediated cytokines TNF-α (C) and IL-1β (D) against inflammation induction of latisin.
FIG. 8 is a graph showing the effect of IκBα degradation and IκBα phosphorylation inhibition (A), NFκB-p65 nuclear transfer inhibition (B) and NFκB-p65 binding ability inhibition (C) on inflammatory induction of latiphorin.
Fig. 9 shows the effect of latifolin on the expression ofhemoxigenase-1 protein.
Fig. 10 shows the effect of transcription of latofolin on nuclear transfer of Nrf-2, a transcription factor for the expression ofhemoxychenase-1.
11 shows the inhibitory effect of lethypoline on the production of NO production (A), PGE ₂ production (B), inflammation-mediated cytokines TNF-α (C) and IL-1β (D) by the expression of hemoxichene- Fig.

The present invention relates to a composition for preventing or treating an inflammatory disease comprising lattifolin represented by the following general formula (1) as an active ingredient.

The composition comprises a pharmaceutical composition or an improved food composition.

[Chemical Formula 1]

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

The latofolin according to the present invention can be synthesized by using a commercially available reagent or an organic synthetic method, and can be used separately from the herbal medicine. In the present invention, it was separated and used in the following manner.

First, the core material of the steel core is added with a solvent of 5 to 15 times by volume, preferably 5 to 7 times the total weight of the core material of the steel core, and the mixture is refluxed for 1 to 5 hours to obtain a strong base crude extract. The extraction solvent is not limited thereto, and at least one solvent selected from water, alcohols having 1 to 4 carbon atoms, and a mixed solvent thereof may be used. The alcohol having 1 to 4 carbon atoms is 1-100% methanol or 1-100% ethanol, and 1-100% methanol is preferred in the present invention. 1-100% methanol is added to the above crude fragrance crude extract, and a strong fragrance dichloromethane fraction is obtained using a solution-solution partitioning method in the order of hexane and dichloromethane. Six fractions (Fr1 to Fr6) were obtained by using silica gel column chromatography with silica gel column chromatography using hexane: ethyl acetate as the eluent fractions of dichloromethane. Four fractions (Fr2-1 to Fr2-4) are obtained by using silica gel column chromatography using a second fraction (Fr2) of which is a mixed solvent of hexane: acetone = 3: 1. Among them, the third fraction (Fr2-3) is subjected to silica gel column chromatography using a mixed solvent of hexane: acetone = 3: 1 to obtain the active compound, latifolin.

The latifulin of the present invention is excellent in inhibiting the inflammatory response through the expression of hemoxygenase-1 (HO-1) enzyme in macrophages, and thus can be effectively used for prevention, treatment or improvement of inflammatory diseases.

Wherein said inflammatory disease is selected from the group consisting of degenerative neuropathic arthritis, rheumatoid arthritis, neural arthritis due to physical damage, inflammatory bowelitis, ankylosing spondylitis, psoriasis, atherosclerosis, arteriosclerosis, asthma, acute pain, chronic pain, neuropathic pain, , Pain such as migraine headache and joint pain, nerve damage, irritable bowel syndrome, endotoxin-induced shock, inflammatory bowel disease, multiple sclerosis and inflammatory back pain.

The composition of the present invention may contain one or more known active ingredients having an effect of preventing or treating inflammatory diseases together with latifulin.

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 that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose , Microcrystalline 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.

Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and suspension 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 an individual by any suitable method.

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 drug form, the administration route and the period, and can be appropriately selected by those skilled in the art. For a desired effect, the composition of the present invention may be administered in an amount of 1 mg / kg to 1000 mg / kg per day. The composition may be administered once a day, or divided into several doses.

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

In the present invention, a health functional food refers to a food having a biological control function such as prevention and improvement of disease, bio-defense, immunity, recovery of disease and aging, and is harmless to human body when taken over a long period of time.

The latofolin of the present invention may be added to a health functional food for the purpose of preventing or improving an inflammatory disease. When the latifurin of the present invention is used as a food additive, the lattifolin can be directly added or used together with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, the latissolin 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 in the production of food or beverage. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

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

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

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

Hereinafter, preferred embodiments, experimental examples, and production examples are provided to facilitate understanding of the present invention. However, the following examples, experimental examples and production 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. Isolation of Latipolin from Steel Fibrous Core

1-1. Isolation of Latipurin

The core material (1.2 kg) was added to 2 L of methanol, and the mixture was refluxed for 2 hours to obtain 173 g of methanol extract of the strongest end group. Subsequently, 1 L of 60% methanol was added to the methanol extract of the strongest stage crude, and 20 g of the fragrance dichloromethane fraction was obtained using a solution-solution partitioning method in the order of hexane and dichloromethane. Six fractions (Fr1 to Fr6) were obtained from the obtained dichloromethane fractions by using silica gel column chromatography with varying hexane: ethyl acetate solvent ratio (10: 90 - 40: 60). Four fractions (Fr2-1 to Fr2-4) were obtained by silica gel column chromatography using a mixed solvent of hexane: acetone = 3: 1 (9 g) as the second fraction (Fr2). Of these, 7 g of the third fraction (Fr2-3) was subjected to silica gel column chromatography using a mixed solvent of hexane: acetone = 3: 1 to obtain 143.4 mg of latifolin.

The process of separating latifulin from the core material of the steel of the present invention is shown in Fig.

1-2. Structure identification of latifolin

In order to identify the structure of latissin obtained in Example 1-1, the structure was identified using ¹ H-NMR and ¹³ C-NMR. Sekine et al., Journal of Agricultural and Food Chemistry, 57, pp. 5707-5712, 2009). ^{The results of 1} H-NMR and ¹³ C-NMR spectra are shown below and shown in FIG. 2 and FIG. 3.

_{^{1 H-NMR (CD 3 OD}} , 500 MHz) δ: 3.84 (3H, s, -OCH 3), 3.86 (3H, s, -OCH 3), 4.71-5.35 (1H, m, = CH 2 trans, - _{C H -CH = CH 2, J} = 17.0, 10.0, 6.0 and 1.6 Hz), 6.2-6.57 (1H, m, -C H = CH 2 ,, J = 17.0, 10.0 and 6.0 Hz), 6.52 (1H, s, 3-H), 6.78 (1H, s, 6-H), 6.85-7.19 (4H, m, B ring).

_{^{13 C-NMR (CD 3 OD}} , 125 MHz) δ: 40.1 (CH A), 55.4 (2-OCH 3), 56.1 (4-OCH 3), 98.6 (C-3), 113.9 (= CH 2), (C-3 '), 116.20 (C-6), 118.7 (C-5'), 124.0 1 '), 139.6 (C- 5), 140.4 (CH X), 146.2 (C-4), 150.7 (C-2), 154.6 (C-2').

Experimental Example 1. Cell culture and cytotoxicity measurement

For cytotoxicity measurement of the cell line of latapholin obtained in Example 1, cell viability was measured.

1-1. Cell culture

To obtain primary macrophages isolated from the peritoneal cavity of mice, 3 ml of Thioglycolate (TG) was injected into the abdominal cavity of C57BL / 6 mice, and the mice were bred for 4 days. The mouse abdominal cavity was injected with a syringe medium (RPMI 1640 + antibiotics + 10% FBS), and the abdominal cavity was massaged for 1 minute and then the medium was collected again. The collected medium was centrifuged to discard the supernatant, and 5 ml of the medium was again placed thereon. Double samples were collected to measure the number of cells plated 10㎕ a 1x10 ⁶ cell / mL in 96-well (well), _{and, 37 ℃, 5% CO 2} , After incubation for 2-3 hours in an incubator adjusted to 95% humidity, the floating cells were discarded and the medium was changed. After the new medium was added, it was stabilized in the incubator for about 12 hours, and the reagent was treated and the experiment was carried out.

1-2. Cytotoxicity measurement

To measure the cytotoxicity of latifolin, cell viability experiments were performed.

Primary macrophages (2 x 10 ⁵ cells / well) isolated from the abdominal cavity of mice were inoculated with 10% heat-inactivated FBS, penicillin G (100 IU / ml) (Gibco, 15240062) and streptomycin The cells were cultured for 48 hours at 37 ° C in a 5% CO ₂ incubator (Sanyo, MCO175) and cultured for 10, 20, 40, 80, and 60 min in a DMEM medium containing 100 μg / ml (Gibco, 15240062) The cells were cultured in a 5% CO ₂ incubator for 48 hours at a concentration of 160 μM, and the cell viability was measured using the MTT method. In addition, all the experimental values were expressed as the average cell survival rate for the control group, and were calculated using the experiment values repeated three times. The results are shown in Fig.

As shown in FIG. 4, the survival rate of the primary macrophages 10, 20, 40, 80 and 160 μM, which had been separated from the abdominal cavity of the mouse, was measured for 48 hours and treated with 160 μM of latiphorin The cytotoxicity was observed and the maximum concentration was 80 μM. When treated with less than 80 μM of latifolin, the cell viability remained high irrespective of the latency.

Experimental Example 2: Effect of latioprin on mRNA and protein expression of iNOS and COX-2

In order to confirm the anti-inflammatory effects of latifolin, mRNA and protein expression of iNOS and COX-2, which are inflammatory mediators in primary macrophages isolated from the peritoneal cavity of mice, were observed.

2-1. RT-PCR

RT-PCR was performed to confirm iNOS and COX-2 mRNA expression promoting effects according to the content of latifolin and the treatment time.

The primary macrophages isolated from the abdominal cavity of mice at a concentration of 3 × 10 ⁶ cells / ml on a 60 mm dish were cultured for 12 hours, treated with 1 μg / ml of lipopolysaccharide (LPS) And cultured for 24 hours to induce an inflammatory reaction. After the above cultivation, RIPA (89900, Thermo) buffer was added and the supernatant was separated by centrifugation at 4 DEG C and 14,000Xg for 25 minutes. The isolated supernatant was extracted from the sample using QIAAmp Viral RNA Mini Kit (Qiagen). RT-PCR was performed using the primers shown in Table 1 using Agpath id one step step RT-PCR (Ambion). The RT-PCR was carried out at 95 ° C for 15 sec and at 60 ° C for 60 sec. The PCR reaction was repeated 45 times. The PCR reaction was carried out at 42 ° C for 10 min and 95 ° C for 10 min. The reaction results were confirmed after staining with EtBr (Ethidium Bromide) by oz gel electrophoresis. The results are shown in Fig.

gene Primer (base sequence) iNOS F: 5'-AGC CCA ACA ATA CAA ATG ACC CTA-3 ' R: 5'-TTC CTG TTG TTT CTA TTT CCT TTGT-3 ' cox-2 F: 5'-CAC TCA GTT TGT TGA GTC ATTC-3 ' R: 5'-GAT TAG TAC TGT AGG GTT AAT G-3

As shown in FIG. 5, mRNA expression of iNOS and COX-2, which are known as inflammatory mediators, is remarkably suppressed as the concentration of latifurin increases.

2-2. Western blot

Western blot was performed to confirm the effect of stimulating iNOS and COX-2 protein expression according to the content of latifolin and the treatment time.

After incubation for 12 hours with primary macrophages separated from the abdominal cavity of mice at a concentration of 3 × 10 ⁶ cells / ml in a 60 mm dish, latifurin was treated at a concentration of 1 μg / ml with LPS and cultured for 24 hours to induce inflammation Lt; / RTI > After the above cultivation, RIPA (89900, Thermo) buffer was added and the supernatant was separated by centrifugation at 4 ° C and 14,000 × g for 25 minutes. Protein quantification in the supernatant was performed using a BSA protein kit (Pierce Biotechnology).

The supernatant was electrophoresed using 7.5% SDS-polyacrylamide gel for 2 hours, and then transferred from the polyacrylamide gel using a nitrocellulose membrane (NC membrane). The transferred nitrocellulose membrane was stopped for 1 hour in fresh blocking buffer (0.1% Tween 20 in Tris-buffered saline) containing 5% skim milk. After the reaction was stopped, the membrane was washed with PBST (PBS, 0.1% Tween 20) three times, once every 10 minutes, followed by iNOS (SC-650, Santacruz biotechnology, USA), COX-2 (SC-1745, Santacruz biotechnology, USA) antibody (Ab) was diluted 1: 1000 and the reaction was carried out for 1 hour. After the reaction, the cells were washed three times with the PBST once every 10 minutes, and the secondary antibody (anti-rabbit IgG, SC-2004, Santacruz biotechnology, USA) was diluted 1: 1000 and reacted for 1 hour. After the reaction, the cells were washed three times with PBST once every 10 minutes, and then mixed with ECL (Amersham Pharmacia Biotech) solution at a ratio of 1: 1, and the mixture was lighted by pouring onto a nitrocellulose membrane. Actin content was measured in the same manner using an antibody against actin (SC-1616, Santacruz biotechnology, USA). The results are shown in Fig.

As shown in FIG. 6, protein expression of iNOS and COX-2, which are known as inflammatory mediators, is remarkably suppressed as the concentration of latiforin increases.

Experimental Example 3: Effect of latisurine on production of NO and PGE2 and inflammation mediated cytokine

To determine the effect of latifolin on the production of inflammatory mediators NO and PGE ₂ and the production of TNF-α and IL-1β, inflammatory mediators of cytokines, in primary macrophages isolated from the abdominal cavity of mice, Western blotting was performed And analyzed quantitatively.

Lactophorin was pretreated with 10, 20, 40, and 80 μM in primary macrophages isolated from mouse peritoneal cavity and cultured for 3 hours. LPS induced inflammation and cultured in 5% CO ₂ incubator for 18 hours . The supernatants of the cultured cells were collected and subjected to Western blotting in the same manner as in Experimental Example 2-2, and the results were quantitatively analyzed. Showed inflammation inducer effect on NO and PGE ₂ production by Lahti morpholine in Fig. 7A and 7B, Figure 7C and Figure 7D inflammation mediated cytokine effects on the generation of TNF-α and IL-1β Respectively.

As shown in FIG. 7, the production of NO, PGE ₂ , TNF-α and IL-1β, which were increased when LPS was administered to the primary macrophages isolated from the abdominal cavity of mice, , PGE ₂ , TNF-α and IL-1β were inhibited in a concentration-dependent manner.

Thus, latiphorin inhibited the production of NO and COX-2 mediated by iNOS and iNOS as well as inflammation-inducing factors, as well as COX-2 mediated PGE ₂ , and the inflammatory mediators cytokines TNF-a and IL- , It was confirmed that the inhibitory effect of inflammatory reaction induced in macrophages was excellent.

EXPERIMENTAL EXAMPLE 4 Effect of LATIFORIN on Transcription Factor Activity

In order to examine the effects of latifolin on transcription factor activity, IκBα degradation, IκBα phosphorylation, NFκB-p65 nuclear transfer and NFκB-p65 binding ability were inhibited in primary macrophages isolated from peritoneal cavity of mice. Western blotting was performed in the same manner as in Experimental Example 2-2.

Lactophorin was pretreated with 10, 20, 40, and 80 μM of primary macrophages from mouse peritoneal cavity and cultured for 12 hours. LPS induced inflammation and cultured in 5% CO ₂ incubator for 1 hour . After incubation, the transcription factor activity in the cytoplasm was confirmed by degradation of IκBα (SC-371, Santacruz biotechnology, USA) and inhibition of IκBα phosphorylation (p-IκBα, SC-8404, Santacruz biotechnology, USA) Factor activity was confirmed by NFκB-p65 (SC-8008, Santacruz biotechnology, USA) nuclear transfer and inhibition of NFκB-p65 binding capacity. The results in the cytoplasm are shown in Fig. 7 (A), and the transfer results in the nucleus are shown in Fig. 8 (B) and Fig. 8 (C).

As shown in FIG. 8, IκBα degradation, IκBα phosphorylation, NFκB-p65 nuclear transfer, and NFκB-p65 binding ability, which were increased when LPS was treated with primary macrophages isolated from mouse peritoneal cavity, When treated at a concentration of 80 μM, the activity was decreased in a concentration-dependent manner. This is the result of inhibiting the transcription of p65, a transcription factor, by inhibiting the phosphorylation of IκB-α, which has the function of blocking NFκB transcription.

EXPERIMENTAL EXAMPLE 5. Effect of lathypholin on the expression of hemocytogenase-1 (HO-1) and nuclear transfer of Nrf-2

Determination of the effect of latifolin on nuclear transfer of Nrf-2, a transcription factor involved in expression of HO-1 and HO-1 protein in primary macrophages isolated from the peritoneal cavity of mice , Western blotting was carried out in the same manner as in Experimental Example 2-2.

As a positive control for primary macrophages isolated from mouse peritoneal cavity, 20 μM of cobalt protophorphyrin (CoPP), which is a strong inducer for the expression of hemoxichenease-1, an enzyme exhibiting antioxidant and anti-inflammatory effects, was induced , And 80 μM of latifolin were treated for 3, 6, 12 and 18 hours. The results are shown in Fig.

As shown in FIG. 9, the expression of hemoxichene-1, an enzyme, was induced by treating cobalt protoporphyrin with macrophages. As compared with the increase in the concentration of latisflorin, hemoxicigenase- (9A), respectively. In addition, lethopoline 80 μM was treated with time to confirm the expression level of hemoxichenase-1. As a result, it was confirmed that hemoxicigenase-1 was expressed in the greatest amount at 18 hours and 24 hours (9B).

Also, in order to confirm the transfer of Nrf-2 into the nucleus, 80 袖 M of latifolin was treated for 15, 30, 60, 90, and 120 minutes by the same method as described above. The results are shown in Fig.

As shown in FIG. 10, the transcription of Nrf-2 into the nucleus was confirmed by treating 80 μM of latifolin with time. As a result, it was found that nuclei of Nrf-2, one of the transcription factors that act on the expression of hemocyanin- It was confirmed that the degree of transfer to the inside increased with time.

Experimental Example 6. Inhibition of Inflammatory Response through Expression of Hemoxigenase-1 Protein

Inhibition of NO production, inhibition of PGE ₂ production, and suppression of inflammatory mediators of TNF-α and IL-1β production were observed in primary macrophages isolated from the peritoneal cavity of mice.

The primary macrophages isolated from the abdominal cavity of mice were treated with 50 μM of the hemoxicinase-1 inhibitor SnPP (Tin protoporphyrin, Porphyrin Products, Logan, Ut.) For 3 hours and then treated with 10, 20, 40 and 80 μM After 12 hours of treatment, the cells were incubated with LPS for 24 hours in a 5% CO ₂ incubator. Inhibitory effects of inhibition of NO production (11A) and PGE ₂ production inhibition (11B) and inflammatory mediators of cytokine TNF-α (11C) and IL-1β (11D) through hemokoxygenase- Respectively.

As shown in Fig. 11, latisin had a remarkable effect of suppressing the increase of NO, PGE ₂ , TNF-? And IL-1? However, pretreatment of SnPP, an inhibitor of hemoxic progenase-1, resulted in increased NO, PGE ₂ , TNF-α and IL-1β production. This is the result of confirming that the inhibitory effect of latifolin on the production of NO (11A), PGE ₂ (11B), TNF-α (11C) and IL-1β (11D) is due to hemoxigenase-1 expression.

Therefore, it was confirmed that latisin had an excellent inhibitory effect on the inflammatory reaction induced in macrophages by increasing the expression and activity of hemoxicinase-1.

Hereinafter, a pharmaceutical composition containing the composition of the present invention and an example of a preparation for a health functional food will be described, but the present invention is not intended to be limited but is specifically described .

Formulation Example 1. Preparation of pharmaceutical composition

1-1. Manufacture of Powder

20 mg < RTI ID = 0.0 >

Lactose 100 mg

Talc 10 mg

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

1-2. Manufacture of tablets

Latiporin 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.

1-3. Preparation of capsules

Latiporin 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.

1-4. Injection preparation

Latiporin 10 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ H ₂ O 26 mg

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

1-5. Manufacture of liquid agent

Latifolin 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 Composition

2-1. Manufacture of health food

Latiporin 100 mg

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

0.2 [mu] g vitamin B12

Vitamin C 10 mg

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ of folic acid

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.

2-2. Manufacture of health drinks

Latiporin 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 for about 1 hour. The resulting solution was filtered to obtain a sterilized container, which was sealed and sterilized, It is used to make beverage composition.

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 (5)

A pharmaceutical composition for preventing or treating an inflammatory disease comprising latifolin represented by the following formula (1) as an active ingredient.
[Chemical Formula 1]

The pharmaceutical composition according to claim 1, wherein the latofolin is separated from the strong esophagus. The pharmaceutical composition for preventing or treating an inflammatory disease according to claim 1, wherein the latofolin increases the expression of hemoxigenase-1 and inhibits the inflammatory reaction. delete 1. A food composition for preventing or ameliorating an inflammatory disease comprising latifolin represented by the following formula (1) as an active ingredient.
[Chemical Formula 1]

Images