KR20100032714A - Extract of sparassis crispa and its use as an anti-cancer medicine - Google Patents

Abstract

PURPOSE: A composition containing Sparassis crispa extract for treating colon cancer, liver cancer, and blood cancer is provided. CONSTITUTION: A Sparassis crispa extract is cultivated by inoculating spawn of Sparassis crispa to sterilized Cryptomeria japonica. The ITS site of nucleic acid ribosome DNA has 50.6% of GC content. The Sparassis crispa extract is isolated using n-alkan of 5-8 carbon atoms, methylene chloride, alkanol of 4 or 5 carbon atoms, or their mixture solvent. The extract is apolar fraction of Sparassis crispa.

Description

Mushroom extract and its use as anticancer agent {Extract of Sparassis crispa and Its Use as an Anti-cancer Medicine}

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

Cancer is generally a malignant one of abnormalities in the cycles of the cells that make up human tissues, and cells that do not differentiate normally and grow uncontrollably.

Cancer occurs through three stages: initiation, promotion, and progression. Cellular mutations are caused by carcinogens in the environment or food, and these cells are abnormal as they are continuously stimulated by carcinogens. It is known to continue to proliferate to form cancer tissue.

Research methods for anticancer drugs for treating cancer include a method of searching for direct cytotoxic substances on cancer cells, a method of controlling substances that regulate the immune ability of the living body, a method of searching for substances that inhibit the metastasis of cancer cells, and the like. There is a method of searching for a substance that inhibits angiogenesis.

Currently used anticancer drugs can be broadly divided into biological preparations such as enzyme preparations and vaccines, chemical synthesis medicines, and natural products. Among them, biological preparations such as enzymes and vaccines are not in a practical stage. Pharmacological effects vary depending on the type of cancer (Gillman., Et al., Maxwell Macmillan., 18, pp 1202, 1986), and it is pointed out as a problem in treating cancer because of various side effects due to toxicity (Chung). , et al., J. Wonkwang Medical Sci., 3, pp 13-34, 1987).

Recently, with the rapid development of cell culture technology, studies have been actively conducted to investigate the mechanism of cytotoxicity at the cellular level by culturing various cells and administering various toxic substances, and minimizing and treating side effects of anticancer drugs. In order to enhance the effect, the development of anticancer drugs using natural products has been continuously tried.

On the other hand, Sparassis crispas ) is a woody fungus that belongs to basidiomycetes, which grow like cabbage near the live tree roots of coniferous trees or between stumps 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 anticancer 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 a blossom mushroom extract having anticancer activity.

Another object of the present invention to provide an anticancer agent composition using the flower mushroom extract.

Other objects, specific embodiments, and the like of the present invention will be presented below.

In one aspect, the present invention relates to a blossom mushroom extract having anticancer 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 (Ha Nabiotech, 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.

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

In addition, in this specification, "anticancer activity" means the improvement activity with respect to hematological cancer, colorectal cancer, and / or liver cancer. Specifically, it may be defined as the proliferation inhibitory activity of HL-60, a blood cancer cell line, HT-29, a colon cancer cell line, and / or HepG-2, a liver cancer cell line.

In addition, in this specification, "improvement" is meant to include the improvement and treatment of the pathological symptoms of cancer, in particular hematological cancer, colon cancer and / or liver cancer, and the inhibition / delay of the onset of such pathological symptoms.

In addition, in the present specification, "extract" is used as the extraction target of the mushroom mushrooms defined above, alkanol having 5 or less carbon atoms including water, distilled water, ethanol, n- having 5 to 8 carbon atoms including n-hexanoic acid as an extraction solvent. It means what is obtained using alkane, methylene chloride, ethyl acetate, or a mixed solvent thereof. These extraction solvents will be described later, but may vary depending on what anticancer activity is intended.

Here, the extraction method may be any method such as hot water, heating, cooling, ultrasonic spinning, stirring, a method of mixing them. These extraction methods may also vary depending on what anticancer activity is intended, and one of ordinary skill in the art can select a suitable extraction method for each anticancer activity within its usual capacity.

On the other hand, as confirmed in the following Examples and Experimental Examples, 80% ethanol extract of the present invention, room temperature extract using distilled water as the extraction solvent, and 40 ℃, 60 ℃ and 80 ℃ hot water extract is a colorectal cancer cell line HT-29 Although hepG-2 has a low cell proliferation inhibitory activity, the n-hexane fraction, the methylene chloride fraction, the ethyl acetate fraction and the butanol fraction have relatively high cell proliferation inhibitory activity.

In the case of HL-60, a hematologic cancer cell line, all extracts showed their anti-proliferative activity regardless of which extraction solvent was used.

In sum, the flower extract of the present invention may be specifically defined in two aspects.

In the first embodiment, (a) the cultivated cedars were inoculated with seedlings of the cultivated mushrooms, and the cultivated mushrooms having a GC content of 50.6% in the ITS region of its nuclear ribosomal DNA were used as (b) (b) ) The extraction solvent is a nonpolar or weak polar solvent, specifically n-alkanes having 5 to 8 carbon atoms including n-hexane, methylene chloride, ethyl acetate, butanol or a mixed solvent thereof, and (c) large intestine as an activity. Cancer and / or liver cancer inhibitory activity, specifically, when defined as having proliferative inhibitory activity of the colon cancer cell line HT-29 and / or liver cancer cell line HepG-2,

In the second aspect, a) a target of extraction is cultivated by inoculating sterile cedar seedlings with seedlings of flowering mushrooms, and using the mushrooms having a GC content of 50.6% in the ITS region of its nuclear ribosomal DNA, (b) The extraction solvent may be any extraction solvent, in particular an extraction solvent with or without polarity and more specifically, an alkanol having 5 or less carbon atoms including water, distilled water, ethanol and butanol, and having 5 to 8 carbon atoms including n-hexane. n-alkane, methylene chloride, ethyl acetate, or a mixed solvent thereof is used, and (c) activity is defined as having blood cancer inhibitory activity, specifically, proliferation inhibitory activity of blood cancer cell line HL-60.

On the other hand, the following Examples and Experimental Examples show that the mushroom extract is more active as fractions in the non-polar direction regardless of colon cancer, liver cancer and blood cancer.

Therefore, it is preferable that the matsutake mushroom extract of the present invention is further fractionated in the non-polar direction in any of the above embodiments.

Examples of the fractions further fractionated in the non-polar direction are n-hexane fractions obtained by extracting the flower mushroom with a polar solvent such as water or ethanol and then mixing and fractionating the extract with a non-polar solvent such as n-hexane, or the n-hexane fraction thereof as a polar solvent. For example, a second n-hexane fraction obtained by mixing and fractionating again with water or ethanol can be exemplified. After extracting the flower cluster with a nonpolar solvent such as n-hexane or methylene chloride, the n-hexane or methylene chloride fraction obtained by mixing and fractionating the extract with a polar solvent such as water or ethanol, or the n-hexane or methylene chloride fraction A second n-hexane or methylene chloride fraction obtained by mixing and fractionating again with a polar solvent such as water or ethanol may also be exemplified as a fraction further fractionated in the nonpolar direction.

Therefore, in the present specification, the "fraction fraction fractionated further in the nonpolar direction" means a fraction of a polar or non-polar solvent when the fraction is mixed by mixing a strong solvent and a weak solvent, or a mixture of a polar solvent and a non-polar solvent. It can be said. Those skilled in the art can identify and discriminate polar strength and weakness, polar solvents, and nonpolar solvents within their usual capacity. As used herein, the meanings of the polar solvent and the nonpolar solvent and the strong polar solvent and the weak polar solvent follow the conventional meaning in the art.

In another aspect, the present invention is an extract solvent as defined above and the non-polar or weak polar extract solvent, specifically n-alkanes having 5 to 8 carbon atoms including n-hexane, methylene chloride, ethyl acetate, The present invention relates to a colorectal cancer improving agent composition comprising a cauliflower extract obtained using n-alkanol having 4 or 5 carbon atoms including butanol as an active ingredient.

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 the colorectal cancer improving agent composition of the present invention, the active ingredient is preferably a fraction further fractionated in the non-polar direction. The reason is as described above.

In another aspect, the present invention is the extract of the above-mentioned blossom mushroom as a target for extraction and non-polar or weak polarity, specifically n- alkanes having 5 to 8 carbon atoms, including m-hexane, methylene chloride, ethyl acetate It relates to a liver cancer improver composition comprising a matsutake mushroom extract obtained using alkanol having 4 or 5 carbon atoms including butanol as an active ingredient.

Liver cancer improver composition of the present invention is also preferred if the active ingredient is a fraction further fractionated in the non-polar direction. The reason is as described above.

In another aspect, the present invention is to extract the flower mushroom as defined above, and has 5 to 8 carbon atoms of n-alkanol having up to 5 carbon atoms, including n-hexane, water, distilled water, ethanol and butanol The present invention relates to a hematological cancer improving agent composition comprising a flower mushroom extract obtained by using any extraction solvent such as n-alkane, methylene chloride, ethyl acetate, or a mixed solvent thereof.

Here too, the extract is preferably a fraction fractionated in the non-polar direction. The reason is as described above.

Meanwhile, the anticancer agent composition of the present invention (that is, the colorectal cancer improver composition, the liver cancer improver composition or the hematological cancer improver composition; referred to below) is an anticancer agent intended to use the above-described flower mushroom extract as 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 activity, and 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. An "effective amount" as used herein refers to an amount of an effective ingredient that can induce improvement, treatment of colon cancer, liver cancer or hematologic cancer, or inhibition / 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.

The anticancer agent composition of the present invention may include known compounds, natural extracts, etc., which are known to have anticancer activity, so as to increase and enhance anticancer activity, in addition to the above-described flower mushroom extract as an active ingredient thereof.

Such compounds, such as natural extracts, such as double-substituted pyrazolobenzodiazepines (WO 2006/040036) having a colorectal cancer improving activity, seaweed extracts disclosed by Korean Patent No. 844366, and docetaxel (WO 2001/15675) having a liver cancer improving activity, And allylthiopyridazine derivatives (Korean Patent No. 446103).

Subjects to which the anticancer agent compositions of the present invention can be applied (prescribed) are mammals and humans, particularly humans.

The anticancer agent 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 less toxic) than the applicable (prescribed) subject 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 (e.g. stearic acid, magnesium stearate, etc.), calcium sulfate, vegetable oils (e.g. peanut oil, cottonseed oil, sesame oil, olive oil, etc.), polyols (e.g. propylene glycol, glycerin, etc.), alginic acid, emulsifiers (e.g. TWEENS), wetting agents (e.g. 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 anticancer activity and the use of the extract as an anticancer 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 logs from the bag, lay the vinyl on the top plate in advance, spread the sponge (1cm thick) on the top plate, arrange the single trees at 15cm intervals, and water them 2-3 times a day for two 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.

<Example> Preparation of extracts of pine mushroom cultivated cedar wood

Example 1 Preparation of Extract by Hot Water Extraction

Hot water extraction was performed using a shaking thermostat, and extracted with distilled water corresponding to 20 times the weight of the sample (flower mushroom of <Reference Example>; the same). 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 2 Preparation of Extract by Hot Water Extraction

Extracted in the same process as in Example 1, the extraction temperature was extracted at 60 ℃.

Example 3 Preparation of Extract by Hot Water Extraction

Extracted in the same process as in Example 1, the extraction temperature was extracted at 80 ℃.

Example 4 Preparation of Extract by Room Temperature Extraction

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

Example 5 Preparation of Extract by Room Temperature Extraction

The extraction was carried out in the same manner as in Example 4, but the extraction solvent was extracted with 80% ethanol.

Example 6 Preparation of Extract by Solvent Fraction

For the preparation of the solvent fraction extract, first, 100% ethanol was added to 438 g of cedar log cultivated pine mushroom samples, and leached. After leaching, the filtrate was filtered through Whatman No. 2, and the filtrate was concentrated under reduced pressure. It was made.

The powder sample was suspended in 1 L of distilled water, and then solvent fractionation was performed with the same amount of n-hexane, methylene chloride, ethyl acetate, and butanol after stirring for 24 hours, and the fractions were concentrated under reduced pressure and concentrated. Until stored at -20 ° C.

< Experimental Example > Genetic analysis of anticancer activity of S. mushroom extract and classification of S. mushroom cultivation

Experimental Example 1 Antitumor Activity of Cedar Log Cultivated Flower Mushroom

For this experiment, hepatic cancer cell line HL-60, colorectal cancer cell line HT-29, and liver cancer cell line HepG-2 derived from acute promyeloid leukemia patients were distributed from Korea Cell Line Bank (KCLB) and 100 units / mL penicillin- Using RPMI 1640 medium (GIBCO, Grand Island, NY, USA) containing streptomycin (GIBCO, Grand Island, NY, USA) and 10% fetal bovine serum (FBS; GIBCO, Grand Island, NY, USA) The cells were cultured in a 5% CO ₂ incubator and subcultured once every 3 to 4 days.

Each cell (2.0∼5.0 × 10 ⁴ / mL) was put in a well of a 96 well plate, and the sample of the extract of each example was added to a concentration of 200 µg / ml, and then cultured for 3 days, followed by 3 100 µg of-(4,5-dimethylthiazol) -2,5-diphenyltetrazolium bromide (MTT; Sigma, St. Louis, MO, USA) was added and further incubated for 4 hours. The cultured plate was centrifuged at 1,000 rpm for 10 minutes, and the medium was carefully removed. Then, 150 µl of dimethylsulfoxide (DMSO; Sigma, St. Louis, MO, USA) was added to dissolve the formazan precipitate produced by MTT reduction. Absorbance was measured at 540 nm using a reader (Bio-TEK instrumenis. Inc., Winooski Vermont, WI, USA) to obtain the average absorbance values for each sample group, and the absorbance values of the control group (when no sample was added). The degree of cell proliferation inhibition was investigated in comparison with

The results are shown in FIG. 2.

Referring to Figure 2, all the extracts were active against the hematological cancer cell line HL-60, but the colon cancer cell line HT-29 and hepatocellular cell line HepG2 were generally only active in the nonpolar or weak polar extracts. 2, A is an extract of <Example 4>, B is an extract of <Example 1>, C is an extract of <Example 2>, D is an extract of <Example 3>, E is <Example 5> Extract, F to I are each fraction of <Example 6>.

The experiment is represented by MEAN ± SD the results of three replicates.

<Experimental Example 2> Genetic Analysis for Strain Identification of Cedar Mushrooms

In the present experiment, in order to distinguish the strains of the cedar cultivated matsutake mushrooms obtained in the above reference example, the matsutake mushrooms native to Hallasan, Jeju (hereinafter referred to as "natural mushrooms"), and the oak cultivated matsutake mushrooms in Japan ("Oak matsutake mushrooms") ( Nucleotide sequences of nuclear ribosomeal DNA (nrDNA) and internal transcribed spacer (ITS) sites with Hana Biotech Co., Ltd. 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: 2) and ITS 4 (5'-TCC TCC GCT TAT TGA TAT GC-3': SEQ ID NO: 3) 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 1.8mM, 10 × reaction buffer (10mM Tris-HCl, 50mM KCl, 0.1% Triton X-100) and 2.5units of Taq DNA polymerase (Promega, USA) were added to sterile tertiary distilled water to reduce the total reaction amount to 25μl. It was set as. 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 for 10 minutes at room temperature. 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: 4) and M13 reverse primer (5'-AAC AGC TGT GAC CAT G-3'), a Cyc-labeled vector inner primer for sequencing : Use SEQ ID NO: 5) for 30 times at 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 40 seconds, and then keep at 10 ° C for 72 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, 0.6 × TBE buffer for buffer, and 700 min 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. 3, 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 mushrooms of the test subjects are shown in Figure 4, the nucleotide sequence of the ITS region of the nrDNA of the mushroom mushrooms cultivated using cedar, SEQ ID NO: 1 Shown in

Table 1 below shows the length or GC content of the ITS region of the nrDNA of the three blossom mushrooms.

TABLE 1

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 1, 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 is a graph showing the growth inhibitory effect on the hepatic cancer cell line HL-60, the colon cancer cell line HT-29, and the hepatocarcinoma cell line HepG-2.

3 is amplification of the ITS site of 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 4 is the ITS site of the jasmine mushrooms (WSB-03) nrDNA of the extract of the present invention cultivated using the Hallasan native blossom mushroom (WSB-01), the cultivated mushroom mushroom (WSB-02) and the cedar logs This is the result of comparing the homology of the nucleotide sequences. In Figure 4 (dot) means that it is the same as the nucleotide sequence of Hallasan native flower mushroom.

<110> Jeju Hi-tech Industry Development Institute <120> Extract of Sparassis crispa and Its Use as Anti-cancer Medicine <160> 5 <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> 19 <212> DNA <213> Artificial Sequence <220> <223> ITS-1 primer <400> 2 tccgtaggtg aacctgcgg 19 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ITS-4 primer <400> 3 tcctccgctt attgatatgc 20 <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> M13 forward primer <400> 4 gtaaaacgac ggccagt 17 <210> 5 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> M13 reverse primer <400> 5 aacagctgtg accatg 16  

Claims (17)

(a) As an extract, sterile mushrooms were inoculated with sterile seedlings inoculated and cultivated with mushrooms having a GC content of 50.6% in the ITS region of nuclear ribosomal DNA and having a nucleotide sequence of SEQ ID NO: 1. (B) Extraction solvent is n-alkane of 5 to 8 carbon atoms, methylene chloride, ethyl acetate, alkanol of 4 or 5 carbon atoms, or a mixed solvent thereof, and (c) colorectal cancer inhibitory activity And a mushroom extract having at least one of liver cancer inhibitory activity. The method of claim 1, The n-alkanes having 5 to 8 carbon atoms are n-hexane, and the alkanol is butanol extract. The method of claim 1, The colorectal cancer inhibitory activity is a growth inhibitory activity of the colon cancer cell line HT-29, and the liver cancer inhibitory activity is a hepatic cancer cell line HepG-2 growth inhibitory activity, characterized in that the mushroom extract. The method of claim 1, The extract is a mushroom extract, characterized in that the fraction fractionated in a non-polar direction. (a) As an extract, a sterile cultivated cedar seedling was inoculated with a seedling of a mushroom mushroom, whose nuclear liposomal DNA had a GC content of 50.6% and its nucleotide sequence of SEQ ID NO: 1 Mushrooms are used, and (b) the extraction solvent is water, distilled water, alkanols having 5 or less carbon atoms, n-alkanes having 5 to 8 carbon atoms, methylene chloride, ethyl acetate, or a mixed solvent thereof, and (c) active As a flowering mushroom extract having blood cancer inhibitory activity. The method of claim 5, The alkanol having 5 or less carbon atoms is methanol, ethanol or butanol, and the n-alkanes having 5 to 8 carbon atoms are n-hexane. The method of claim 5, The blood cancer inhibitory activity is a mushroom extract, characterized in that the proliferation inhibitory activity of the blood cancer cell line HL-60. The method of claim 5, The extract is a mushroom extract, characterized in that the fraction fractionated in a non-polar direction. Colon cancer improving agent composition comprising the extract of any one of claims 1 to 4 as an active ingredient. 10. The method of claim 9, The composition is a colorectal cancer improving agent composition, characterized in that the pharmaceutical composition. 10. The method of claim 9, The composition is a colon cancer improving composition, characterized in that the food composition. Liver cancer improver composition comprising the extract of any one of claims 1 to 4 as an active ingredient. The method of claim 12, The composition is liver cancer improver composition, characterized in that the pharmaceutical composition. The method of claim 12, The composition is liver cancer improver composition, characterized in that the food composition. A blood cancer improving agent composition comprising the extract of any one of claims 5 to 8 as an active ingredient. The method of claim 15, The composition is a blood cancer improver composition, characterized in that the pharmaceutical composition. The method of claim 15, The composition is a blood cancer improving agent composition, characterized in that the food composition.

Images