KR20100032713A - Extract of sparassis crispa and its use as anti-inflammatory medicine - Google Patents

Abstract

PURPOSE: A composition for treating inflammatory diseases containing Sparassis crispa extract is provided. CONSTITUTION: A Sparassis crispa extract is cultivated by inoculating spawn of Sparassis crispa to Cryptomeria japonica. The Sparassis crispa contains 50.6% of GC content at ITS site of nucleic acid ribosome DNA. The extract is isolated using water, distilled water, ethanol, or their mixture solvent. The sequence of ITS site comprises a sequence of sequence number 1. The extract isolated by water has anti-oxidative activity. The anti-oxidative activity is activity of suppressing NO generation and inflammatory cytokine such as TNF-alpha, IL-6 and IL-1 beta.

Description

Extract of Sparassis crispa and Its Use as Anti-inflammatory Medicine

The present invention relates to a blossom mushroom extract and its use as an anti-inflammatory agent.

Inflammation is the body's defense against external infections such as physical and chemical stimuli, bacteria, fungi, viruses, and allergens.

Inflammatory responses are part of the innate immune response, and as in other animals, the innate immune response in humans begins by recognizing and attacking non-self through patterns of cell surfaces where macrophages are specifically present in pathogens. During the inflammatory reaction, plasma accumulates at the site of inflammation, diluting the toxicity secreted by bacteria, increasing blood flow, and accompanied by symptoms such as erythema, pain, edema, and fever.

These inflammatory responses involve a variety of biochemical phenomena, particularly cyclooxygenase (COX), which is associated with the biosynthesis of nitric oxide synthase (NOS) and various prostaglandins. Known as

There are three isomers of NOS, which are endotoxins of bacteria such as calcium- or capmodin-dependent eNOS (endothelial NOS), nNOS (neurotrophic NOS), and LPS (lipopolysaccharide) or IL-1β, TNF-α, IL There are iNOS (inducible NOS) induced by several inflammatory cytokines such as -6, IL-8 and IL-12, which produce nitric oxide (NO) from L-arginine.

NO produced by eNOS or nNOS plays an important role in maintaining homeostasis by performing various physiological reactions related to blood pressure control, neurotransmitter, learning, memory, etc. It is known to be involved in various inflammatory diseases such as transplant rejection, autoimmune diseases and neuronal death (Moncade S. et al, Pharmacol. Rev., 1991, 43, 109; Nature Medicine, 2001, 7, 1138; Mu , MM, J. Endotoxic Res. 7, p341, 2001).

The COX enzyme has hydroperoxidase (HOX) activity with COX function and synthesizes PGG ₂ and PGH ₂ intermediates from arachidonic acid, and these compounds include PGE ₂ , PGF ₂ , PGD ₂ , and prostacyclin. And thromboxin A ₂ (thromboxane A2, TxA2). Among the functions of COX, the function of PGH synthase is involved in pain and inflammatory responses through the synthesis of PGE ₂ .

There are two subtypes of COX and COX-1 is always expressed in most tissues, whereas COX-2 is rapidly induced by inflammatory cytokines and plays an important role in the inflammatory response.

Thus, iNOX inhibitors and COX-2 inhibitors can be used as therapeutic agents for inflammatory diseases.

Sparassis crispas , on the other hand, refers to wood fungi belonging to basidiomycetes that occur like cabbage by clustering on or near the live tree roots of conifers between summer and autumn.

It has been known that the matsutake mushrooms cause severe damage to livestock such as larch, which significantly reduces the compressive strength of wood by about 75% to 40%.

However, recently, it is known that it has a large amount of β-glucan, which is effective in hypertension, diabetes, lung cancer, colon cancer, prostate cancer, etc., and interest in its artificial cultivation method is increasing.

Republic of Korea Patent No. 449947, Republic of Korea Patent No. 483333, Republic of Korea Patent No. 474979, Republic of Korea Patent No. 816504 and the like are techniques for artificial cultivation of the mushroom mushroom.

The present invention discloses the anti-inflammatory activity and the like of the extract of the flower mushroom obtained through the artificial cultivation method developed by the present inventors.

It is an object of the present invention to provide an extract of zinnia mushroom having anti-inflammatory activity.

Another object of the present invention to provide an anti-inflammatory composition using the extract.

Other objects and other aspects of the present invention will be set forth below.

In one aspect, the present invention relates to a blossom mushroom extract having anti-inflammatory activity.

The blossom mushroom used as an extract in the present invention is artificially cultivated by the present inventors using cedar, and as shown in Experimental Example 2 below, its genetic characteristics are artificially cultivated using Hallasan native mushroom or oak It is different from one blossom mushroom (Hanabiotech Co., Ltd., Gyeonggi-do, Korea). More specifically, the GC content of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) is 51.6% in the case of the cultivated mushrooms grown using Halla native pine mushroom or oak, but used as an extraction target in the present invention. The blossom mushrooms had a GC content of 50.6%, which was different from the above-listed blossom mushrooms.

As described above, the blossom mushroom used as an extract object in the present invention is fundamentally different from the native blossom mushroom or other artificially grown blossom mushrooms, and the specific cultivation method thereof can be found in the following reference example.

The present inventors make the four extracts using the flower mushroom as an extraction target and using water, distilled water, ethanol or a mixed solvent thereof as the extracting solvent, NO (Nitric Oxide) production inhibitory activity, inflammatory cytokine TNF-α, The inhibitory activity of IL-6 and IL-1β production, iNOS (inducible NOS) production inhibitory activity, COX-2 production inhibitory activity, and prostagladin E ₂ production inhibitory activity were examined. There was a difference, but it was confirmed that the above activities.

The present invention is provided based on these experimental results.

In the present specification, "flower mushroom" is a seed mushroom inoculated and cultivated on a sterilized cedar ( Cryptomeria japonica ) tree, and is defined as a flower mushroom having a GC content of 50.6% in the ITS region of its nuclear ribosomal DNA. Preferably, the nucleotide sequence of the ITS region is defined as a blossom mushroom having a sequence of <SEQ ID NO: 1>.

In addition, in this specification, "anti-inflammatory activity" is defined as improving activity of an inflammatory disease defined below. Specifically, NO (Nitric Oxide) production inhibitory activity, inflammatory cytokine TNF-α, IL-6 and / or IL-1β production inhibitory activity, iNOS (inducible NOS) production inhibitory activity, COX-2 production inhibitory activity and And / or prostagladin E ₂ production inhibitory activity.

In addition, in this specification, "improvement" is meant to include the improvement, treatment of pathological symptoms of an inflammatory disease and the inhibition / delay of the onset of such pathological symptoms.

In addition, in the present specification, "extract" means that it is obtained using the flower mushroom defined above as the extraction target and using water, distilled water, ethanol or a mixed solvent thereof as the extraction solvent.

When the extract is obtained using an extraction solvent, any method such as hot water, warming, cooling, ultrasonic spinning, stirring, a method of mixing them, and the like may be used. The following Examples and Experimental Examples show the difference in the degree of effectiveness, but both room temperature extraction (80% ethanol extract, etc.) and boiling water extraction have anti-inflammatory activity.

In summary, the anti-inflammatory mushroom extract of the present invention (a) is an extract object, which is grown by inoculating the seed of the mushroom mushroom in sterilized cedar and culturing the ITS site of its nuclear ribosomal DNA. Blossom mushrooms having a GC content of 50.6% are used, (b) the extraction solvent is water, distilled water, ethanol or a mixed solvent thereof, and (c) the activity is an anti-inflammatory activity, that is, an inflammatory disease improving activity, specifically NO (Nitric Oxide) production inhibitory activity, the inhibitory activity of the production of inflammatory cytokines TNF-α, IL-6, and / or IL-1β, iNOS (inducible NOS) inhibitory activity, COX-2 inhibitory activity and / or prostagula It can be said that it can be defined as having a din E ₂ production inhibitory activity.

On the other hand, as can be seen in the following Examples and Experimental Examples, the present inventors use the water as the extraction solvent and the production temperature of the four extracts of the flower mushroom while increasing the temperature, the antioxidant activity of each extract, specifically, DPPH radical The scavenging activity of Xanthine Oxidase (Xanthine Oxidase) inhibitory activity and the scavenging activity of the superoxide radical were examined, but all extracts showed the above activities although there was a difference in activity.

The DPPH is a water-soluble material having a chemically stable free radical, and has a maximum absorbance at around 515 nm to 520 nm, and when used in the screening of antioxidants, the radical is extinguished by giving electrons when it encounters a material with antioxidant activity. Santin oxidase is an enzyme that produces superoxide radicals in the process of producing uric acid by using xanthine as a substrate, and superoxide radacal is produced by xanthine oxidase and causes damage to cells. It is a substance that causes oxidative stress.

In view of the foregoing, the flower mushroom extract of the present invention may be defined as having an additional antioxidant activity in addition to the anti-inflammatory activity, when water is preferably used as the extraction solvent. The antioxidant activity specifically means scavenging activity of DPPH radical, xanthine oxidase inhibitory activity and / or superoxide radical scavenging activity.

In another aspect, the present invention relates to a composition for improving inflammatory disease comprising the extract as described above as an active ingredient.

In the present specification, the "inflammatory disease" may be defined as any condition specified as a local or systemic biological defense response against external physical and chemical stimuli or infection of an external infectious agent such as bacteria, fungi, viruses, and various allergens. This response activates various inflammatory mediators and enzymes associated with immune cells (eg iNOS, COX-2, etc.), secretion of inflammatory mediators (eg NO, TNF-α, IL-6, IL-1β, PGE ₂₎ . ), Accompanied by a series of complex physiological reactions such as fluid infiltration, cell migration, tissue destruction, etc., and are manifested externally by symptoms such as erythema, pain, edema, fever, deterioration or loss of certain functions of the body. The inflammatory disease may be acute, chronic, ulcerative, allergic or necrotic, so as long as any disease is included in the definition of an inflammatory disease as above, whether it is acute, chronic, ulcerative, allergic or Irrespective of necrosis Specifically, the inflammatory diseases include asthma, allergic and non-allergic rhinitis, chronic and acute rhinitis, chronic and acute gastritis or enteritis, ulcerative gastritis, acute and chronic nephritis, acute and chronic hepatitis, chronic obstructive pulmonary disease, pulmonary fibrosis Irritable bowel syndrome, inflammatory pain, migraines, headache, back pain, fibromyalgia, fascia disease, viral infections (eg, type C infections), bacterial infections, fungal infections, burns, surgical or dental wounds, Prostaglandin E hyperplasia, atherosclerosis, gout, arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, iris, scleritis, uveitis, dermatitis, eczema, multiple sclerosis, and the like.

As used herein, the term "active ingredient" alone refers to a component that can exhibit the desired activity or exhibit itself with a carrier which is inactive.

In another aspect, the present invention relates to an iNOS inhibitor composition comprising the extract as described above as an active ingredient.

As mentioned above, inhibition of iNOS can inhibit the production of NO leading to improvement of inflammatory disease.

The following experimental example of the present invention shows that the mushroom extract of the present invention inhibits the production of NO and inhibits expression at the iNOS gene and protein levels, and inhibits the production of cytokines known to induce the expression of iNOS. Is showing.

As used herein, "iNOS inhibition" is meant to include inhibition of expression of iNOS gene and inhibition of iNOS production, and meaning including inhibition and / or reduction of NO production whatever the intermediate mechanism.

In another aspect, the present invention relates to a COX-2 inhibitor composition comprising the extract as described above as an active ingredient.

As mentioned above, inhibition of COX may inhibit the production of PGE ₂ resulting in improvement of inflammatory disease.

The following example of the present invention shows that the matsutake mushroom extract of the present invention inhibits the production of PGE ₂ and inhibits expression at the COX-2 gene and protein levels, and is known to induce the expression of COX-2. It has been shown to inhibit the production of cain.

As used herein, the term "COX-2 inhibition" is meant to include inhibition of expression of COX-2 gene and inhibition of COX-2 production, and meaning including inhibition and / or reduction of the production of PGE ₂ whatever the intermediate mechanism. to be.

Meanwhile, the composition of the present invention (ie, inflammatory disease improving agent composition, iNOS inhibitor composition, and COX-2 inhibitor composition; refers to the same as below) intends to treat the above-mentioned flower mushroom extract, which is an active ingredient, according to the use, formulation, and formulation purpose. It may be included in any amount (effective amount) as long as it can exhibit an improving activity of an inflammatory disease, a conventional effective amount will be determined within the range of 0.001% to 15% by weight based on the total weight of the composition. The term "effective amount" as used herein refers to the amount of an effective ingredient capable of inducing the improvement, treatment, or suppression / delay of the development of such pathological symptoms in a mammal, preferably a human, to which it is applied. Such effective amounts can be determined experimentally within the range of ordinary skill in the art.

Subjects to which the compositions of the invention can be applied (prescribed) are mammals and humans, in particular humans.

The composition of the present invention can be used as a pharmaceutical composition in a specific embodiment.

The pharmaceutical composition of the present invention includes an oral dosage form (tablets, suspensions, granules, emulsions, capsules, syrups, etc.), parenteral formulations including pharmaceutically acceptable carriers, excipients, etc. Sterile injectable aqueous or oily suspensions), topical formulations (solutions, creams, ointments, gels, lotions, patches) and the like.

As used herein, "pharmaceutically acceptable" means that the subject of application (prescription) does not have more toxicity (adequately low toxicity) to which the subject of application (prescription) is adaptable without inhibiting the activity of the active ingredient.

Examples of pharmaceutically acceptable carriers include lactose, glucose, sucrose, starch (such as corn starch, potato starch, etc.), cellulose, derivatives thereof (such as sodium carboxymethyl cellulose, ethylcellulose, etc.) malt, gelatin, talc, solids Lubricants (such as stearic acid, magnesium stearate, etc.), calcium sulfate, vegetable oils (such as peanut oil, cottonseed oil, sesame oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, etc.), alginic acid, emulsifiers (such as TWEENS), wetting agents ( Sodium lauryl sulfate), colorants, flavoring agents, tableting agents, stabilizers, antioxidants, preservatives, water, saline, phosphate buffer solutions and the like. Such a carrier may be used by selecting one or more appropriate ones according to the formulation of the pharmaceutical composition of the present invention.

Excipients may be selected and used according to the formulation of the pharmaceutical composition of the present invention, for example, when the pharmaceutical composition of the present invention is prepared with an aqueous suspending agent, suitable excipients are sodium carboxymethyl cellulose, methyl cellulose, hydropropylmethylcellulose And suspending agents and dispersing agents such as sodium alginate and polyvinylpyrrolidone. Suitable excipients when prepared as injections include Ringer's solution, isotonic sodium chloride, and the like.

The pharmaceutical composition of the present invention may be administered orally or parenterally and in some cases may be administered topically.

The daily dosage of the pharmaceutical composition of the present invention is usually 0.001 ~ 150 mg / kg body weight range, it can be administered once or divided into several times. However, since the dosage of the pharmaceutical composition of the present invention is determined in view of various related factors such as the route of administration, the age, sex, weight of the patient, the severity of the patient and the like, the dosage may limit the scope of the present invention in any aspect. It should not be understood as.

The composition of the present invention may be used as a food composition in another specific embodiment.

The food composition of the present invention may be prepared from gums, vitamin complexes, dietary supplements, special nutritional supplements, functional drinks, and the like.

The food composition of the present invention, in addition to containing the mushroom extract as an active ingredient, sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, maltose, fruits such as apples, lemons, citrus fruits and green tea leaves, , Flavors obtained from teas such as jujube leaves, bioactive components such as catechin, retinol, ascorbic acid and tocopherol, and minerals such as calcium, magnesium, chromium, cobalt, copper, and fluoride may also be added. Additives may be added.

As described above, according to the present invention can provide a flower mushroom extract having anti-inflammatory activity and the use of the extract as an anti-inflammatory agent.

Hereinafter, the present invention will be described with reference to reference examples, examples and experimental examples. However, the scope of the present invention is not limited by these examples and experimental examples.

Reference Example Flowering Mushrooms Using Cedar

First, a cedar log of about 15 to 20 cm in diameter was taken and cut to about 15 cm. The cut wood (hereinafter referred to as "lumber") was immersed in a fresh yeast 5% aqueous solution for 24 hours, and then taken out and sealed in a plastic bag covered with a nonwoven fabric. Then, after sterilization for 8 hours at a temperature of 80 ~ 100 ℃ in the sterilizer, and transferred to a cooler equipped with a clean facility that can prevent contamination by microorganisms and naturally cooled for 24 to 30 hours. After cooling, the liquid spawn of the flower mushroom was inoculated at 30 ml per piece of cut wood in a clean booth. Immediately after inoculation, the cells were sealed in a plastic bag, incubated at a temperature of about 20 ° C. for 15 days, and then incubated at 25 ° C. for 70 days. Remove the incubated wood from the bag, lay the vinyl on the top plate, spread the sponge (1cm thick) on the top plate, arrange the single wood at 15cm intervals, and irrigate 2-3 times a day for 2 days. Supplemented. Indoor room temperature was maintained at around 20 ℃ and room humidity was high at around 95%. After mushroom cultivation, the room temperature and humidity were kept the same, and the light was kept bright by installing fluorescent lamps. For the air distribution, two fans were installed and ventilation was performed 2-3 times for 1 hour. It is a photograph of matsutake mushroom cultivated using the cedar wood of Figure 1.

<Examples> Manufacture of Extracts of Pine Mushroom Grown in Cedar

<Example 1> Preparation Example 1 of the mushroom extract

Put the 80% ethanol equivalent to 20 times the weight of the sample (flower mushroom of <Reference Example> below) and extract it for 24 hours with stirring, and filter it with Whatman No. 2 and filter the filtrate under reduced pressure. After concentration it was stored at -20 ℃ until the experiment.

<Example 2> Preparation Example 2 of the mushroom extract

Hot water was extracted using a shaking thermostat, and extracted with distilled water corresponding to 20 times the weight of the sample. The temperature was adjusted to 100 ° C., extracted for 24 hours, filtered through Whatman No. 2, and the filtrate was reduced and concentrated under reduced pressure and stored at −20 ° C. until the experiment.

<Example 3> Preparation Example 3 of the mushroom extract

1.2N NaOH 800ml was added to the residue remaining after the hot water extraction of <Example 2>, and extracted by stirring at 80 ° C for 8 hours. After extraction, the extract was centrifuged at 8000 rpm for 20 minutes at 4 ° C., and 4 times of volume of ethanol was added to the supernatant. The precipitate was centrifuged at 8000 rpm for 20 minutes at 4 ° C. The final precipitate was washed with 80% ethanol and dried.

<Example 4> Preparation Example 4 of the mushroom extract

250 ml of 1.2N NaOH was added to the residue remaining after the low-temperature alkali extraction of <Example 3>, followed by extraction at 120 ° C. for 8 hours. The supernatant was separated by centrifugation of the extract at 8000 rpm, 4 ° C. for 20 minutes, and ethanol was added to the supernatant solution for 4 minutes. The precipitate was centrifuged at 8000 rpm at 4 ° C. for 20 minutes. The final precipitate was washed with 80% ethanol and dried.

<Example 5> Preparation Example 5 of the mushroom extract

Hot water was extracted using a shaker and extracted with distilled water corresponding to 20 times the weight of the sample. The temperature was adjusted to 40 ° C., extracted for 24 hours, filtered through Whatman No. 2, and the filtrate was reduced and concentrated under reduced pressure and stored at −20 ° C. until the experiment.

<Example 6> Preparation Example 6 of the mushroom extract

Extracted in the same process as in Example 5, the extraction temperature was extracted to 60 ℃.

<Example 7> Preparation Example 7 of the mushroom extract

Extracted in the same process as in Example 5, the extraction temperature was extracted at 80 ℃.

<Example 8> Preparation Example 8 of the mushroom extract

Distilled water corresponding to 20 times the weight of the sample at room temperature was added and extracted for 24 hours while stirring. The resultant was filtered with Whatman No. 2, and the filtrate was reduced and concentrated under reduced pressure and stored at -20 ° C until the experiment. .

< Experimental Example > Functional Evaluation of Pine Mushroom Grown in Cedar Wood

Experimental Example 1 Evaluation of Anti-inflammatory Activity of Pine Cedar Cultivated Mushrooms

Experimental Example 1-1 Cells and Reagents

RAW264.7 cells, a mouse macrophage line, were distributed from Korean Cell Line Bank (KCLB) at 37 ° C and 5% CO ₂ using DMEM medium containing 100 units / mL penicillin-streptomycin and 10% fetal bovine serum (FBS). Experiments were performed while subcultured at intervals of 3-4 days in a thermostat. Lipopolysaccharide (LPS. E. coli serotype 0111: B4) was purchased from Sigma and used in the experiment.

Experimental Example 1-2 Evaluation of NO Production Inhibition Activity

RAW 264.7 cells were adjusted to 1.0 X 0 ⁵ cells / ml using DMEM medium added with 10% FBS, and then inoculated into 48 well plates, and 100 µg / ml of the extracts of <Examples 1 to 4> and LPS ( 1 μg / mL) were simultaneously treated and incubated for 24 hours. The amount of NO produced was measured in the form of NO ₂ ⁻ present in the cell culture solution using Griess reagent. Mix 100 µl of cell culture supernatant with 100 µl of Griess reagent [1% (w / v) sulfanilamide, 0.1% (w / v) naphylethylenediamine in 2.5% (v / v) phosphoric acid] for 10 minutes on 96 well plates. After absorbance was measured at 530nm, which was measured in the form of NO ₂ ^- present in the cell culture medium, and the amount of generated NO was compared with sodium nitrite (NaNO ₂ ) as standard. The results are shown in Table 1.

TABLE 1

NO production inhibitory activity

Sample inbibition (%) Concentration (100 μg / mL) Example 1 28.85 ± 4.05 Example 2 5.09 ± 1.15 Example 3 1.85 ± 2.54 Example 4 0.81 ± 1.73

Experimental results show that the cedar log cultivated pine mushroom extract has a general NO production inhibitory activity, but especially the 80% ethanol extract of <Example 1> has a high activity.

Experimental Example 1-3 Evaluation of Inflammatory Cytokine Production Inhibition Activity

RAW264.7 cells (4.0 × 10 ⁵ cells / mL) were incubated 18 hours ago and 100 μg / ml of extract of <Examples 1 to 4> and LPS (1 μg / mL) were co-treated for 24 hours. Total RNA was isolated using TRI-reagent (MRC).

The cells were homogenized by addition of TRI-reagent, and then centrifuged (15000 rpm, 15 minutes) by addition of chloroform. Equivalent amount of isopropanol was added to the supernatant and centrifuged (12000 rpm, 8 minutes) to precipitate RNA, and 75% DEPC (diethyl pyrocarbonate) -treated ethanol was added and centrifuged (10000 rpm, 5 minutes), followed by drying. DEPC was dissolved in the treated distilled water. RNA was quantified by measuring absorbance at 260 nm, and RNA having a value within the range of 1.7-1.9 absorbance at 260 nm and absorbance at 280 nm was used in the experiment. All experiments were done under RNase-free conditions.

1 μg of total RNA was mixed with oligo (dT) 18 primer, dNTP (0.5 μM), 1 unit RNase inhibitor and M-MuLV reverse transcriptase (2 U) at 70 ° C for 5 minutes, 25 ° C for 5 minutes, 37 ° C for 60 minutes, and The reaction was stopped by heating at 70 ° C. for 10 minutes.

Polymerase chain reaction (PCR) was performed using 2 μL cDNA, 4 μM of 5 ′ and 3 ′ primers (see Table 2 below) for amplification of the respective inflammatory cytokine genes from the synthesized cDNA. mix with buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.1% Triton X-100), 250 μM dNTP, 25 mM MgCl ₂ , 1 unit Taq polymerase (Promega, USA) and dilute the whole to 25 μL It was then carried out using a Perkin-Elmer Thermal Cycler. At this time, PCR conditions were 94 ℃ / 20 seconds, 55 ~ 62 ℃ / 30 seconds, 72 ℃ / 40 seconds, 30 cycles. Products produced by PCR were subjected to electrophoresis on 1.2% agarose gel and stained with ethidium bromide. I identified a specific band.

TABLE 2

Primer sequence

division F / R Primer sequence Sequence ID TNF-α F 5'-TTGACCTCAGCGCTGAGTTG-3 ' 2 R 5'-CCTGTAGCCCACGTCGTAGC-3 ' 3 IL-1β F 5'-CAGGATGAGGACATGAGCACC-3 ' 4 R 5'-CTCTGCAGACTCAAACTCCAC-3 ' 5 IL-6 F 5'-GTACTCCAGAAGACCAGAGG-3 ' 6 R 5'-TGCTGGTGACAACCACGGCC-3 ' 7 β-Actin F 5'-GTGGGCCGCCCTAGGCACCAG-3 ' 8 R 5'-GGAGGAAGAGGATGCGGCAGT-3 ' 9

The results are shown in FIGS. 2 to 4.

Referring to Figures 2 to 4 it can be seen that the extract of each of the above examples has an inhibitory effect on the production of the inflammatory cytokines.

Experimental Example 1-4 Evaluation of iNOS and COX Expression Inhibition Activity

iNOS and COX expression inhibitory activity assessments were measured at the gene and protein levels.

Measurement at the gene level was made in the same manner as in <Experimental Example 1-3>, and the primers used are shown in Table 3 below.

[Table 3]

Primer sequence

division F / R Primer sequence Sequence ID iNOS F 5`-CCCTTCCGAAGTTTCTGGCAGCAGC-3` 10 R 5`-GGCTGTCAGAGCCTCGTGGCTTTGG-3` 11 COX-2 F 5'-CACTACATCCTGACCCACTT-3 ' 12 R 5'-ATGCTCCTGCTTGAGTATGT-3 ' 13 β-Actin F 5'-GTGGGCCGCCCTAGGCACCAG-3 ' 8 R 5'-GGAGGAAGAGGATGCGGCAGT-3 ' 9

Measurement at the protein level was confirmed by Western blot.

In <Experimental Example 1-3>, the cultured cells were directly dissolved and homogenized in Laemmli sample buffer containing 5% 2-mercaptoethanol. Heated at 70 ° C. for 10 minutes, electrophoresed in 8-12% polyacrylamid gel and adsorbed onto poly (vinylidenedifluoride) (PVDF). Reaction of PVDF membrane with blocking buffer (Tris-buffered saline-0.1% (w / v) Tween-20) (TBS-T) for 1 hour at room temperature, followed by primary antibody to iNOS (please specify where to buy) And TBS-T containing primary antibody to COX (please specify where to buy) (1: 1000-1: 3000) for 1 hour (25 ° C.) or 16 hours (4 ° C.). After washing three times with TBS-T and reacting with HRP-conjugated secondary antibody at room temperature for 30 minutes, images of each band were obtained by Enhanced Chemiluminescence (ECL) method.

Measurement results at the gene and protein levels are shown in FIGS. 5 to 8.

Referring to Figures 5 to 8, it can be seen that the extracts of the above examples inhibit the expression of iNOS and COX-2 at the gene and protein levels.

Experimental Example 1-5 PGE ₂ Production Inhibition Activity Evaluation

RAW264.7 cells were adjusted to 1.5X10 ₅ cells / mL using DMEM medium, and then inoculated in a 24 well plate, and cultured 18 hours before in a 5% CO ₂ incubator. Thereafter, the medium was removed, and 50 μL of the extract sample of <Examples 1 to 4> and a fresh medium containing 450 μL of LPS (1 μg / mL) prepared at 10-fold concentration (1 mg / mL) were simultaneously treated. Incubated under the same conditions as in Example 1-3>. After 24 hours, the supernatant was obtained by centrifugation (12,000 rpm, 3 min) of the culture medium to measure prostaglandin E2 (PGE2). PGE ₂ was measured using the PGE ₂ ELISA kit (R & D Systems, Inc.) and the r ² value of the standard curve for the standard was 0.99 or more.

The results are shown in FIG. 9.

Referring to Figure 9, it can be seen that the extracts of each of the above examples have the effect of inhibiting the production of PGE ₂ .

Experimental Example 2 Evaluation of Antioxidant Activity of Pine Mushrooms

Experimental Example 2-1 Screening of Antioxidant Activity Using DPPH Radical Scavenging Activity

Electron donating ability was measured by DPPH free radical scavenging method by Blois method (Blois MS. 1958. Antioxidant determinations by the use of a stable free radical.Nature 181: 1198-1200). In other words, 100 μl of the sample dissolved in methanol (1mg / mL) was dispensed into a 96 well plate, and the same amount of 0.4 mM DPPH solution was added thereto, and the absorbance was measured at 517 nm after 10 minutes at room temperature. The DPPH radical scavenging activity was calculated from the following equation and expressed as the concentration of the sample (IC ₅₀ ) which appears when the absorbance of DPPH decreased by 50%. Each sample was repeated three times and the average value was obtained.

DPPH radical scavenging activity (%) = (A _Control -A _Sample ) / A _Control x 100

A _Sample = absorbance of the reaction solution to which the sample was added

A _Control = absorbance of the reaction solution with methanol added instead of the sample

The results are shown in Table 4 below.

Experimental Example 2-2 Xanthine Screening of Antioxidant Activity by oxidase Inhibition and Superoxide Scavenging

The production of uric acid by Xanthine / xanthine oxidase was measured by increased absorbance at 290 nm and the amount of superoxide was measured by nitroblue tetrazolium (NBT) reduction method (Cheng ZJ, Kuo SC, Chan SC, Ko FN, Teng CM. 1998). Antioxidant

properties of butein isolated from Dalbergia odorifera. Biochim Biophys Acta 1392: 291-299).

   Xanthinase enzyme reaction solution was prepared by mixing 100 μl of sample (1 mg / mL) with 100 μl of 200 mM phosphate buffer (pH 7.5) (0.5 mM xanthine and 1 mM EDTA added) and adding 50 mU / ml xanthine oxidase. . The reaction solution induced the production of uric acid and measured the production. In addition, Superoxide scavenging activity was added to the reaction solution by adding 0.5 mM NBT. Xanthine oxidase inhibition and superoxide scavenging activity were calculated from the following equations, and the results were averaged by three replicates.

Xanthine oxidase Inhibitory Activity (%) = (A _Control -A _Sample ) / A _Control x 100

superoxide scavenging activity (%) = (A _Control -A _Sample ) / A _Control x 100

A _Sample = absorbance of the reaction solution to which the sample was added

A _Control = absorbance of control (absorbance of reaction solution without sample)

The results are shown in Table 4 below along with DPPH radical scavenging activity.

[Table 4]

Antioxidant activity

DPPH radical scavenging activity (%) xanthine oxidase inhibitory activity (%) Supoeroxide radical scavenging activity (%) Example 5 19.3 ± 5.3 8.1 ± 2.2 72.7 ± 1.1 Example 6 17.2 ± 4.5 11.0 ± 2.1 70.1 ± 0.3 Example 7 17.1 ± 1.5 5.1 ± 0.9 72.7 ± 0.5 Example 8 28.6 ± 1.0 9.0 ± 2.7 72.7 ± 0.5

The above results show that the cedar cultivated pine mushroom extract of the present invention has free radical scavenging activity, xanthine oxidase inhibitory activity and superoxide radical scavenging activity of DPPH. In particular, the room temperature extract of <Example 8> was the most excellent activity.

Experimental Example 3 Genetic Analysis for Classification of Species Mushrooms

In this experiment, in order to distinguish the species of cedar cultivated matsutake mushroom obtained in the above reference example, the cultivated mushroom cultivated in Hallasan, Jeju Island (hereinafter referred to as "natural mushroom"), and the cultivated oak cultivated mushroom in Japan ("Oak blossom mushroom") ( Nucleotide sequences of nuclear ribosomeal DNA (nrDNA) and internal transcribed spacer (ITS) sites with Hana Biotech, Gyeonggi-do, Korea were analyzed and compared.

<1> experimental method

<1-1> Genomic DNA Extraction

    Genomic DNA of zinnia mushroom was extracted from 10 mg of zinnia mushroom tissue and extracted using Genomic DNA Purification Kit (Promega, USA) according to the manufacturer's method. The separated DNA was subjected to electrophoresis at 100V for 20 minutes on 1.0% agarose gel to confirm that it appeared as a single band, and the isolated DNA was quantified by measuring the absorbance at 260 nm and 280 nm with UV / VIS spectrophotometer. Only the above samples were used as template DNA for gene amplification.

<1-2> Primer Design and PCR for Gene Search

A primer for the analysis of nuclear ribosomal DNA internal transcribed spacer (ITS) sites of three cultivated mushrooms was designed by White et al. (1990), and was a universal primer used for species identification and phylogenetic analysis of mushrooms. the primers ITS 1 (5'-TCC GTA GGT GAA CCT GCG G-3 ': SEQ ID NO: 14) and ITS 4 (5'-TCC TCC GCT TAT TGA TAT GC-3': SEQ ID NO: 15) Used. In the polymerase chain reaction for amplification, the reaction mixture was template DNA 10ng, primers 20pM, dNTP (Promega, USA) 200μM, MgCl ₂ 2.5 μl of 1.8 mM, 10 X reaction buffer (10 mM Tris-HCl, 50 mM KCl, 0.1% Triton X-100) and 2.5 units of Taq DNA polymerase (Promega, USA) were added to sterilized tertiary distilled water to make the total reaction volume 25 μl. . The PCR reaction was preheated for 5 minutes at 94 ° C using MyCycler Thermal Cycler (Bio-Rad, USA), followed by 30 cycles of 20 seconds at 94 ° C, 20 seconds at 57 ° C and 1 minute at 72 ° C. Next, the target gene was amplified by storing at 72 ° C. for 10 minutes.

<1-3> Cloning and Plasmid DNA Isolation

     Each gene fragment amplified by PCR was confirmed by electrophoresis at 100V for 30 minutes on 1% agarose gel, and the identified PCR-products were cloned using TOPO TA Cloning kit (Invitrogen, USA). Ligation was performed by adding sterilized water to 0.5 μl (50ng) of PCR product, 0.4μl of TOPO vector, and 0.4μl of salt solution to make the total reaction solution 2μl and reacting at room temperature for 10 minutes. After culturing the selected white colony in LB-broth (50μg / ml) medium containing ampicillin for 8 hours, plasmid DNA was isolated using Minipreps DNA Purification System (Promega, USA). It was confirmed by electrophoresis on agarose gel.

<1-4> Sequencing

Sequencing was performed using a PRISMTM Ready Dye Terminator Cycle Sequencing Kit (Perkin-Elmer, ABI, USA). All treatments were performed according to the manufacturer's manual. M5 forward primer (5'-GTA AAA CGA CGG CCA GT-3 ': SEQ ID NO: 16) and M13 reverse primer (5'-AAC AGC TGT GAC CAT G-3'), which are Cy5-labeled vector inner primers for cyclic sequencing : Use SEQ ID NO: 17) for 30 times at 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 40 seconds, and then keep at 72 ° C for 10 minutes for strand extension and store at 4 ° C until the stop solution is added. do. Electrophoresis was performed on 7 M urea, 6% acrylamide gel, using 0.6 X TBE buffer for buffer, and 700 minutes at 1000 V using ABI 310 Prism Autosequencer (Perkin-Elmer, USA).

<1-5> Gene Sequencing and Analysis

 In order to increase the reliability of the genetic analysis of cedar cultivated mushrooms, three clones of each PCR-product were sequenced. The base sequence determination and analysis of the gene fragments were determined by comparison with previously published base sequences, and the base sequences of each gene were compared using the Clustal W program (Thompson et al., 1994).

<2> experimental results

<2-1> ITS amplification

The result of amplifying the ITS region of the nrDNA of the three blossom mushrooms was as shown in FIG. 10, and the amplification products of the same size were confirmed in all three blossom mushrooms.

<2-2> Sequence Analysis

The results of comparing the homology of the ITS sequences of the nrDNA of the three mushroom subjects NRDNA are shown in Figure 11, and the nucleotide sequence of the ITS region of the blossom mushroom using cedar is shown in <SEQ ID NO: 1>. .

In Table 5 below, the length or GC content of the ITS region of the nrDNA of the three blossom mushrooms tested was shown.

TABLE 5

Length or GC content of ITS region of nrDNA

 Classification ITS Length ( bp ) GC content (%) Wild flower mushroom 643 51.6 Oak Blossom Mushroom 643 51.6 Cedar Blossom Mushroom 644 50.6

As can be seen in Table 5, the nucleotide sequences of the ITS region of the nrDNA of the native zinnia and oak zinnia were 643 bp in length and 51.6% in GC content, respectively. However, the cultivated mushrooms of cedars had a length of 644 bp, which was 1 bp larger than that of other strains, and the GC content was 50.6%.

Figure 1 is a photograph of the matsutake mushroom extract of the present invention cultivated using cedar logs.

2 to 4 is a result showing that the blossom mushroom extract of the present invention has an inhibitory effect on the production of inflammatory cytokines TNF-α, IL-6, IL-1β.

5 to 8 is a result showing that the mushroom extract of the present invention inhibits the expression of iNOS and COX-2 at the gene and protein levels.

9 is a result showing that the mushroom extract of the present invention has a production inhibitory effect of PGE ₂ .

10 is amplification of the ITS site of the nrDNA of the extract of the present invention cultivated using the pine mushroom (lane 1), cultivated using the oak tree (lane 2) and the cedar logs of the present invention An electrophoretic picture of one result.

Figure 11 is the ITS site of the cultivated mushroom mushroom (WSB-01), cultivated using oak tree (WSB-02) and the cultivated mushroom mushroom (WSB-03) nrDNA of the present invention cultivated using solid wood This is the result of comparing the homology of the nucleotide sequences. In FIG. 11, a dot means the same as the nucleotide sequence of the native Halla matsutake mushroom.

<110> Jeju Hi-tech Industry Development Institute <120> Extract of Sparassis crispa and Its Use as Anti-inflammatory          Medicine <160> 17 <170> KopatentIn 1.71 <210> 1 <211> 644 <212> DNA <213> Sparassis crispa <400> 1 tccgtaggtg aacctgcgga aggatcatta tcgagttcag aagcgaggtt gtagctggcc 60 ttctcggagg catcgtgcac gcctcgcccg tcccatatca tacctgtgaa ctttttggta 120 ggcgggtttg tgtcggcctc gaaaggggtc gaccggccct ccggccgtct ttatatacac 180 accatacgag tctttagaat gtttgtgcgt ctcgacgcat cttatatata actttcggcg 240 acggatctct tggctctcgc atcgatgaag aacgcagcga aacgcgataa gtaatgtgaa 300 ttgcagaatt cagtgaatca tcgaatcttt gaacgcacct tgcgctcctc ggtattccga 360 ggagcatgcc tgtttgagtg tcatgaaatt atcaacccct tctcctttat tggtggtggg 420 gcttggactt ggaggctttg cgggctttta aatgagtcgg ctcctctcaa atgcattagc 480 tcgaaccctt gcggatcggc catcggtgtg atataatgtc gacgtcgtgg tcgtgagcgt 540 cggatcggct tctaatggtc ccctttcgga gacggaattt gaacttgtga cctcaaatca 600 ggtaggacta cccgctgaac ttaagcatat caataagcgg agga 644 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha forward primer <400> 2 ttgacctcag cgctgagttg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha reverse primer <400> 3 cctgtagccc acgtcgtagc 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IL-1 beta forward primer <400> 4 caggatgagg acatgagcac c 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IL-1 beta reverse primer <400> 5 ctctgcagac tcaaactcca c 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-6 forward primer <400> 6 gtactccaga agaccagagg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-6 reverse primer <400> 7 tgctggtgac aaccacggcc 20 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> beta-actin forward primer <400> 8 gtgggccgcc ctaggcacca g 21 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> beta-actin reverse primer <400> 9 ggaggaagag gatgcggcag t 21 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> iNOS forward primer <400> 10 cccttccgaa gtttctggca gcagc 25 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> iNOS reverse primer <400> 11 ggctgtcaga gcctcgtggc tttgg 25 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COX-2 forward primer <400> 12 cactacatcc tgacccactt 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COX-2 reverse primer <400> 13 atgctcctgc ttgagtatgt 20 <210> 14 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ITS-1 primer <400> 14 tccgtaggtg aacctgcgg 19 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ITS-4 primer <400> 15 tcctccgctt attgatatgc 20 <210> 16 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> M13 forward primer <400> 16 gtaaaacgac ggccagt 17 <210> 17 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> M13 reverse primer <400> 17 aacagctgtg accatg 16  

Claims (14)

(a) As a subject to be extracted, sterile mushrooms were inoculated with seedlings of the cultivated mushrooms, and the mushrooms having a GC content of 50.6% in the ITS region of its nuclear ribosomal DNA were used, and (b) the extraction solvent was water. , Distilled water, ethanol or a mixed solvent thereof, and (c) as an activity, the flower mushroom extract having anti-inflammatory activity. The method of claim 1, The anti-inflammatory activity includes NO production inhibitory activity, inflammatory cytokines TNF-α, IL-6, IL-1β production inhibitory activity, iNOS (inducible NOS) inhibitory activity, COX-2 inhibitory activity and PGE ₂ production inhibitory activity Blossom mushroom extract, characterized in that one or more. The method of claim 1, The nucleotide sequence of the ITS site is a mushroom extract, characterized in that consisting of the sequence of SEQ ID NO: 1. The method of claim 1, When water is used as the extraction solvent, the extract is a mushroom extract, characterized in that it further has an antioxidant activity. The method of claim 4, wherein The antioxidant activity is Blossoming mushroom extract, characterized in that at least one of the scavenging activity of the DPPH radical, xanthine oxidase (Xanthine Oxidase) inhibitory activity and the scavenging activity of the superoxide radical. Inflammatory disease improving composition comprising an extract of any one of claims 1 to 5 as an active ingredient. The method of claim 6, The composition is an inflammatory disease improving composition, characterized in that the pharmaceutical composition. The method of claim 6, The composition is an inflammatory disease improving composition, characterized in that the food composition. INOS inhibitor composition comprising the extract of any one of claims 1 to 5 as an active ingredient. 10. The method of claim 9, INOS inhibitor composition, characterized in that the composition is a pharmaceutical composition. 10. The method of claim 9, INOS inhibitor composition, characterized in that the composition is a food composition. COX-2 inhibitor composition comprising the extract of any one of claims 1 to 5 as an active ingredient. The method of claim 12, The composition is a COX-2 inhibitor composition, characterized in that the pharmaceutical composition. The method of claim 12, The composition is a COX-2 inhibitor composition, characterized in that the food composition.

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