CN1294196A - Immune stimulated holoside substance of bacterial strain from wood layer hole strain and its application - Google Patents

Abstract

The present invention discloses a new fissiped wood shelf fungus Yoo. A polyose substance separated from the sporophore and mycelium of it or other wood shelf funguses has the anticancer activity caused by stimulating the cellulotoxic action of T-cells and the antibody forming power of B-cells and the activity of resisting cancer transfer. Said polyose can stimulate human immunity to prevent and cure immunopathy, such as cancer and AIDS.

Description

The immunostimulation polysaccharide material and the application thereof of the new bacterial strain that derives from the wood layer hole strain kind

The present invention relates to a kind of bacterial strain that belongs to wood layer hole strain, a kind of application of originating from polysaccharide material and this polysaccharide material of this bacterial strain.Be particularly related to a kind of bacterial strain, a kind of new polysaccharide material that originates from this bacterial strain of new wood layer hole strain kind with effective immunostimulatory activity, and in for example application aspect cancer and AIDS and the mechanism research thereof of prevention and treatment immune correlated disease.

Gill fungus, a kind of higher fungi is a heterotroph; They absorb organotrophy by microstructural mycelia from the external world.In biosphere, that they serve as is a decomposer, is simple inorganic form with the organic matter degradation of complexity and finally turns back to occurring in nature.In fungi, most typical gill fungus belongs to Basidiomycetes, and their characteristics ground forms load, and each load all has four external sporidium on its surface when the ripening stage arrives.Gill fungus can be further divided into Hymenomycetes, and it automatically discharges spore from the thalamium that exposes.The representative of Hymenomycetes is Aphyllophorales and Agaricales.Ramaria botrytis, peace Di Shi thorn lead fungi, Climancodon septentrionalis, Coriolus versicolor and canodermaapplanatum belong to the Aphyllophorales class, and oyster cap fungus, Edu mushroom, commune, Trichotoma matsutake, agaricfly (Amanits muscaria) and Shaggy Mane are all in the scope of Agaricales class.

Known in the east herbal medicine in the past: many Aphyllophoraless that belong to, the gill fungus of a kind of Basidiomycetes (Basidomycetes) to multiple disease particularly tumour therapeutic activity is arranged.In Korea S, they are handed down from age to age as herbal medicine or folk medicine, and are belonging to the gill fungus of polyporaceae, particularly phellinus linteus, are considered to a kind of invaluable medicine.Yet phellinus linteus is a kind of very rare kind at occurring in nature, so that its sporophore is difficult to obtain.And the mycelium of separation and artificial culture phellinus linteus is considered to hardly may.Thereby, although phellinus linteus is considered to a kind of tumour there is very effective therapeutical agent, its effect never is used effectively.

For the treatment cancer, several different methods comprises that chemotherapy, radiotherapy and surgical operation therapy all are conventional uses.Although these methods of treatment are effective to solid carcinoma, be not enough for conquering cancer fully.Particularly, be otiose to metastatic tumour.

Usually, use the chemotherapy and radiation of anticancer for example Zorubicin agent can bring severe side effect.In order to alleviate side effect, such carcinostatic agent may the decrement administration.Yet the therapeutic action variation of medicine in this case.What is worse, even after using the chemotherapeutic treatment cancer, metastasis of cancer can't be prevented from.In this case, conventional chemotherapy is effective to suppressing the cancer growth to a certain extent, but can not cure cancer fully.

A kind of alternative medicine is to use immunosuppressor, is known as immunotherapy.The example of representative immunosuppressor is a cytokine, interleukin II for example, tumour necrosis factor or the like.Interleukin II demonstrates therapeutic action preferably, particularly to cancer metastasis.When being used for human body, interleukin II need use with heavy dose, so that can produce severe side effect.Go out lentinan and OK-432 in the research and development aspect the new immunostimulation material recently, this new immunostimulation material is non-cytokine and demonstrates the good anti-cancer activity that has no side effect.

In nearest several years, the new technology that is called as immunochemotherapy of a kind of chemotherapy and immunotherapy combined utilization is being gathered strength always.For example, Zorubicin and immunostimulant have reduced the side effect of pharmaceutical chemicals to cancer patients's co-administered when strengthening antitumous effect.

As far as our knowledge goes, up to the present there are not the microorganism of bacterial strain of wood layer hole strain kind and the research report of hereditary property.Even what microorganism was mutually of the same race also may be very different each other in substance classes that is produced and quantitative aspects, and may demonstrate very big difference aspect culture condition, strain stability and the heritable variation frequency.When the valuable product of research fungi wood layer hole strain, first thing that needs to do is the bacterial strain that screening can produce a large amount of products and can cultivate in meat soup, in addition also has low heritable variation.Various and modification arranged with microscopic examination phellinus linteus strain morphology, therefore be difficult to identify.But, rely at microscopically and to distinguish that form determines that its kind is feasible.

Recently, new DNA analysis method has been used for population heredity and phylogenetic research.Surpass the phenotype analytical method, the DNA analysis method has that can to analyze with rate of evolution and inherited tendency be the advantage of based gene type.Wherein, a kind of restrictive fragment length polymerphism (after this being called " RFLP ") technology can infer that the base sequence sudden change is so that carry out gene type assay (Dowling etc., 1990,1990. nucleic acid II: in " restriction site analysis ": molecular system D.M.Hills and C.Moritz. edit, P250-317, Sinauer combined publication society).Because nuclear DNA is not easy too greatly to analyze, the organoid genome be normally used for DNA analysis (White and Densmore, in " separation of 1992 Mitochondrial DNAs ": the molecular genetic analysis of population: a kind of practical methods, A.R.Hoelzel. edits, P29-58, the Oxford University Press).Particularly, advantage is to have adopted Mitochondrial DNA.Usually, Mitochondrial DNA is compared big or small less with nuclear DNA, and the evolution pace of change is very fast, and has maternal instinct monoploid heredity, so Mitochondrial DNA is widely used in the genetic construction and the historical research of a colony.Particularly, the RFLP of Mitochondrial DNA is used to kind of the research (Foster etc., 1988, " by the dependency between the assay Phythophothora kind of fungi Mitochondrial DNA ", " mycology " 80:466-478) of inner sudden change.

By utilize the DNA analysis method to the bacterial strain of wood layer hole strain kind thoroughgoing and painstaking and extensive studies, the inventor finds a kind of new polysaccharide material, it is produced by a kind of wood layer hole strain bacterial strain, has effective immunostimulatory activity.

Thereby an object of the present invention is to provide a kind of bacterial strain of wood layer hole strain kind, it can produce a kind of new polysaccharide material with immunostimulatory activity.

Another object of the present invention provides a kind of new polysaccharide material, demonstrates a kind of immunostimulatory activity of effective anti-immune correlated disease.

Another object of the present invention provides described polysaccharide material as a kind of purposes of immunotherapeutic agent or in the application aspect the basic mechanism research of these diseases.

In order to achieve the above object, at first cultivate the bacterial strain of multiple wood layer hole strain kind, and separate their all DNA with the SDS-phenol processes.From total DNA, only obtain Mitochondrial DNA.With restriction enzyme BamHI, ClaI, EcoRI and PvuII digestion Mitochondrial DNA, on sepharose, carry out electrophoresis then.Dependency according to the DNA between the bacterial strain of the methods analyst wood layer hole strain of Nei and Li (1979).From these analysiss of data, found a kind of new bacterial strain, it forms the new dependency different with other bacterial strains.This bacterial strain be named as phellinus linteus Yoo and on November 17th, 1997 in Korea S's bio-science and Bioteknologisk Institut Korea S type culture preservation center preservation (preserving number KCTC 0399BP).Carried out a research with regard to the microbiological property of KCTC 0399BP bacterial strain.From the sporophore of this new bacterial strain and mycelium, separate and a kind of new polysaccharide material of purifying, and its immunostimulatory activity is experimentized.Sugared unit and composition to this immunostimulation polysaccharide material are analyzed, and then its carbohydrate components are carried out structural analysis.Similarly, cultivate the bacterial strain of other multiple wood layer hole strain kinds, and its sporophore is separated and purifying with mycelium, to obtain to have equally the polysaccharide material of immunostimulatory activity.

Carry out experimentation on animals, the mouse of implanted skin cancer cell is used this immunostimulation polysaccharide material, to measure the antitumour activity of immunostimulatory activity material.And, measure when polysaccharide material and pharmaceutical chemicals are united use in the variation aspect the antitumour activity.The research polysaccharide material is to the influence of human cancer cell growth.Injected the mouse of skin cancer cell to tail vein and used polysaccharide material, so that study its influence metastasis of cancer.The mechanism of finding the mechanism of antitumour activity of this polysaccharide material and anticancer chemicals in the present invention is different.

From the description of following embodiment, will be clear that above-mentioned and other purposes of the present invention with reference to the accompanying drawings, wherein:

Accompanying drawing 1 is the genealogical tree that shows dependency between wood layer hole strain kind bacterial strain;

Accompanying drawing 2 is the synoptic diagram that show phellinus linteus mycelium culture characteristic;

Accompanying drawing 3 is the steps from the bacterial strain purified polysaccharide material of wood layer hole strain;

Accompanying drawing 4 is gas chromatograms of immunostimulation polysaccharide material sugar unit;

Accompanying drawing 5 is HW 65F gel chromatography figure of immunostimulation polysaccharide material;

Accompanying drawing 6 is carbon NMR spectrums of immunostimulation polysaccharide material;

Accompanying drawing 7 is the synoptic diagram that show according to the antitumour activity of the anti-mouse cancer cells of administering mode polysaccharide material;

Accompanying drawing 8a shows that polysaccharide material and 0.1mg/kg Zorubicin unite the synoptic diagram of antitumour activity of the anti-mouse cancer cells of use;

Accompanying drawing 8b shows that polysaccharide material and 0.3mg/kg Zorubicin unite the synoptic diagram of antitumour activity of the anti-mouse cancer cells of use;

Accompanying drawing 9a is the synoptic diagram that shows according to treat the variation of human cancer cell with polysaccharide material and Zorubicin;

Accompanying drawing 9b is the synoptic diagram that shows according to the final weight of polysaccharide material and Zorubicin treatment human cancer cell;

Accompanying drawing 10a shows the synoptic diagram of Zorubicin to the prophylactic effect of metastasis of cancer;

Accompanying drawing 10b is that this polysaccharide material of demonstration and Zorubicin are united the synoptic diagram of use to the prophylactic effect of metastasis of cancer;

Accompanying drawing 11 is the Cytotoxic synoptic diagram that show Zorubicin and polysaccharide material;

On potato dextrose agar (PDA), cultivate respectively 13 kinds of wood layer hole strain bacterial strains that comprise new phellinus linteus Yoo KCTC 0399BP. Use SDS-phenol technology and from the mycelium of every kind of culture, separate total DNA, then further separate mitochondria DNA from total DNA. These mitochondrial DNAs can be used for the analysis of correlation between bacterial strain. Analyze for this reason, digest mitochondrial DNA with restriction enzyme, and isolate postdigestive dna fragmentation by electrophoresis at Ago-Gel. Dna fragmentation is to calculate the data of restriction enzyme fragment correlation (F) and sequence divergence value (p) in the mobile distance of gel. According to these numerical value, can find a kind of new bacterial strain with correlation, with its called after " phellinus linteus Yoo (Phellinus linteus Yoo) ", and in Korea S's bioscience and Bioteknologisk Institut Korea S type culture preservation center preservation (preserving number KCTC 0399BP). The propagation that the research of phellinus linteus Yoo KCTC 0399BP fructification microbiologic properties is helped this gill fungus.

In order to extract this material that is considered to have effective therapeutic action, cultivate and breed this gill fungus. Obtain a kind of novel substance by hot water extracting, confirm that by phenol sulfuric acid and forint-phenol technical Analysis this material is a kind of polysaccharide. In addition, can disclose the architectural feature of this polysaccharide material by the NMR technology.

After using for a kind of animal, determine the immunostimulatory activity of described polysaccharide material by counting and measurement Proliferation of lymphocytes. Other wood layer hole strain bacterial strains are used the method similar to phellinus linteus Yoo. Carry out successively strain culturing, mycelium extraction and separating substances. Isolated material also is proved has immunostimulatory activity. In addition, the polysaccharide material that obtains from the fructification of wood layer hole strain bacterial strain is proved to be has the immune stimulating activity.

Carry out zoopery to measure the anticancer activity of polysaccharide material. At first, the skin cancer cell that derives from mouse is implanted mouse, then be used alone or in combination described polysaccharide material and other chemicals to mouse. The viability of more every kind of situation mouse. Implanted the activity of the anti-human cancer of nude mice mensuration polysaccharide of human cancer cell by use. Be used alone or in combination described polysaccharide material and adriamycin for this nude mice. In addition, polysaccharide material is very effective aspect the prevention metastasis of cancer. For this reason, the intravenous injection of mouse skin cancer cell is entered the afterbody of mouse, then use polysaccharide material and adriamycin to mouse. Use sulforhodamin B method to measure previous survival mice skin cancer cell and the survival human lung carcinoma cell of processing with polysaccharide material and adriamycin alone or in combination.

The present invention may be better understood from following embodiment, and following embodiment is used to illustrate of the present invention, but should not be construed as a limitation of the present invention.In following embodiment, measure the immunostimulatory activity of isolating polysaccharide material from phellinus linteus Yoo and other wood layer hole strain bacterial strains with the splenocyte of taking from the B6C3F mouse.At first, by with this mass treatment with cell immunity of spleen 2 days and with lipopolysaccharides as positive control.By the antibody that spectrophotometric determination generated, this antibody is taken a standard of immunostimulatory activity as.Measure the proliferation function of polysaccharide material by a kind of mixed lymphocytes answer techniques to the T-cell.For detecting lymphocytic propagation, measure synthetic nuclear DNA in lymphocyte recently by using the 3H-Thymine deoxyriboside.Embodiment I: the cultivation of wood layer hole strain bacterial strain

With each wood layer hole strain kind bacterial strain of being given in the following table 1 go up at potato dextrose agar (PDA), cultivation 5-12 days down of 28 ℃ of constant temperature.In 450mLPD meat soup after the inoculation, make the growth 12 days under 28 ℃, 120rpm jolting of every kind of bacterial strain.

The kind and the source thereof of the wood layer hole strain that table l is used

Bacterial strain	The source
		Phellinus linteus WD 1222	Japan
Cracked feet wood layer hole mattress ATCC 26710	The U.S.
		Phellinus linteus KCTC 0399BP	Korea S
Phellinus linteus PL5	Korea S
		Phellinus linteus PL4	Korea S
Phellinus linteus PLM	Korea S
		Narrow lid Phellinus ATCC32233	The U.S.
????P.iohnsonianus?ATCC60051	The U.S.
		Phellinus pini ATCC12240	The U.S.
????P.tuberculosis?ATCC13271	The U.S.
		Phellinus FP-71807-S turns black	The U.S.
????P.chrysoloma?HHB-3585-Sp	The U.S.

Embodiment II: the separation of total DNA

Use the SDS-phenol processes and from mycelium, separate total DNA.Every kind of mycelium cultivating with 20mM EDTA washing embodiment I, and by with a filter paper (Whatman NO.1) vacuum filtration collection is stored in the dark refrigerator of maintenance-70 ℃ and carries out lyophilize.With the mycelium porphyrize after the lyophilize, then every kind of powder of 2g is added 20mL and extract damping fluid (0.2M TrisHCl, pH8.5; 0.25M NaCl; 25mM EDTA; 0.5% (w/v) SDS), add 14mL phenol and 6mL chloroform then.After slowly mixing, centrifugal this solution.Handle the gained supernatant liquor 30 minutes with 15 μ lRANase (10mg/mL) down at 37 ℃, use 15 μ l Proteinase Ks (10mg/mL) under 60 ℃, to handle again then 15 minutes.After this, the supernatant liquor that enzyme was handled and the mixture thorough mixing of a kind of isopyknic phenol, chloroform and primary isoamyl alcohol (25: 24: 1), subsequently under 4 ℃ with 12, centrifugal 30 minutes of 000X.Repeat identical centrifugal before, with the mixture thorough mixing of supernatant liquor and a kind of isopyknic chloroform and primary isoamyl alcohol (24: 1).So the supernatant liquor of gained mixes with the Virahol of 0.54 volume by vortex (voltexing), and centrifugal to obtain the DNA precipitation under above-mentioned the same terms.Should precipitate with 70% washing with alcohol, and at air drying.DNA is dissolved in the 8mLTE damping fluid, and (10mM TrisHCl, lmM EDTA pH8.0), and preserve under-20 ℃.Embodiment III: the separation of Mitochondrial DNA

Carry out super centrifugally, so that Mitochondrial DNA is separated from total DNA, this total DNA obtains from every kind of bacterial strain of embodiment II.After 8.8g cesium chloride (U.S. Sigma company is on sale) and 6 μ l NSC 334072 solution (10mg/mL) mix, under 20 ℃ with 40, the super centrifugal described total dna solution of 000Xg (8mL).

After under UV-light, further identifying two DNA bands, only reclaiming band on the Mitochondrial DNA fraction under the help of No. 21 syringe needles.Add the saturated Virahol of a kind of isopyknic CsCl to the DNA of acquisition like this fraction, after faint vortex (voltexing) mixed, with 1,200Xg was centrifugal under 4 ℃.Repeated washing step 6-7 time will be so that will remove NSC 334072 fully from mitochondria DNA fragment.By in Mitochondrial DNA, adding 70% ethanol of three times of mitochondria DNA fragment volumes and under 4 ℃, removing cesium chloride in centrifugal 10 minutes with the speed of 100Xg.Repeat the once washing step again to obtain not contain the Mitochondrial DNA of NSC 334072 and Cs.Behind air drying, Mitochondrial DNA is dissolved in TE damping fluid (pH8.0), the spectrophotometer of the commodity of selling with for example U.S. Beckman company under 260nm " DU-64 Spectrometer " by name is quantitative.Store Mitochondrial DNA down at-20 ℃.Embodiment IV: digest Mitochondrial DNA with restriction enzyme

Buy restriction enzyme used the present embodiment and damping fluid and be listed in the table below in 2 from Boehringer Mannheim company (Germany) with its recognition site.The concentration that the embodiment III obtains every kind of Mitochondrial DNA sample buffer of wood layer hole strain bacterial strain is 1 μ g/10 μ l, and they were digested 3 hours down at 37 ℃.

Table 2 restriction enzyme and recognition site thereof

Restriction enzyme	Recognition site
		????BamHI	????G↓GATCC
????ClaI	????AT↓CGAT
		????EcoRI	????G↓AATTC
????PvuII	????CAG↓CTG

Embodiment V: the electrophoresis on the sepharose

To mix with the gel that is loaded with damping fluid (0.25% bromjophenol blue, the blue or green FF of 0.25% dimethylbenzene, 30% glycerine) with the DNA sample that the restriction enzyme treatment in the embodiment IV is crossed, and went up the plane electrophoresis analyzer electrophoresis sold with U.S. BRL company 4 hours at 1% sepharose (agarose MP) that German BoehringerMannheim company sells, wherein electrophoretic damping fluid is TAE damping fluid (40mMTris-acetate, 1mM EDTA, pH8.0), pass analyser with 50V.Behind the DNA electrophoresis, sepharose is immersed ethidium bromide damping fluid (0.5 μ g/mL in the water) so that detect these DNA easily under UV-lamp.The size of the Mitochondrial DNA that will determine by electrophoresis be listed in the table below in 3.

The size of table 3 isolating Mitochondrial DNA from the kind of wood layer hole strain

Bacterial strain	The DNA size
		Phellinus linteus WD1222	????58Kb
Phellinus linteus ATCC26710	????59Kb
		Phellinus linteus KCTC0399BP	????61Kb
Phellinus linteus PL5	????61Kb
		Phellinus linteus PL4	????73Kb
Phellinus linteus PLM	????63Kb
		Narrow lid Phellinus ATCC32233	????77Kb
P.johnsonianus?ATCC60051	????76Kb
		Phellinus pini ATCC12240	????76Kb
P.tuberculosis?ATCC13271	????57Kb
		Phellinus FP-71807-S turns black	????58Kb
P.chrysoloma?HHB-3585-Sp	????60Kb

The embodiment VI: limited fragment length polymorphic (RFLP) is analyzed

Carry out the analysis of DNA dependency between the wood layer hole strain bacterial strain according to Nei and Li method.If, obtain restriction enzyme segment correlation (F) and sequence divergence value (p) when moving identical promptly being considered under the mutually the same situation of dna segment of distance of electrophoresis time shift on the embodiment V sepharose.By weighing matched group method arithmetical mean (UPGMA) clustering technique not, from the p value, analyze the dependency between phellinus linteus Yoo (KCTC 0399BP) bacterial strain and other wood layer hole strain kind bacterial strains.Based on following equation, calculate P and F value respectively:

F=2n _Xy/ (n _x+ n _y) wherein F represent the dependency between two wood layer hole strain kinds, n _XyBe common fragment number between them, n _xAnd n _yBe respectively to separate x and y institute unfolded fragment overall number.

P=-(lnF)/r wherein p is the estimated value of each nucleotide position nucleotide base displacement ratio, and r is the right number of nucleotide base for the restriction endonuclease recognition site.In table 4, list the F value of gained when handling, and together list their corresponding p values with restriction enzyme BamHI.In table 5, list the F value of gained when handling, and together list their corresponding p values with restriction enzyme ClaI.In table 6, list the F value of gained when handling, and together list their corresponding p values with limiting enzyme EcoRI.In table 7, list the F value of gained when handling, and together list their corresponding p values with restriction enzyme PvuII.Comprise common pulsating ratio in the table 8 and the arithmetical mean distance corresponding to listed p value of the upper left diagonal lines top of extending of every table from table 4-7 to the bottom right.Infer genealogical tree from these data, as shown in Figure 1, we can see very significantly: bacterial strain of the present invention is compared again different with the wood layer hole strain bacterial strain with obvious dependency.

F value and the P value of the PRLP of the Mitochondrial DNA of table 4BamHI digestion

	WD 1222	ATCC 26710	KCTC 0399BP	PL5	PL4	PLM	ATCC 32233	ATCC 60051	ATCC 12240	ATCC 13271	FP- 71807-S	HHB- 3585-Sp
													WD 1222	-							0.1921	0.4142		0.3465	0.2310
ATCC 26710	NC	-	0.0257	0.2570	0.3120	0.2841	0.2310		0.2841	0.2203
													KCTC 0399BP	NC	0.8571	-		0.3120	0.2841	0.2310		0.2841	0.3358
PL5	NC	0.8571	1.0000	-	0.3120	0.2841	0.2310		0.2841	0.3358
													PL4	NC	0.1538	0.1538	0.1538	-		0.3358			0.3243		0.3120
PLM	NC	0.1818	0.1818	0.1818	NC	-						0.2841
													ATCC 32233	NC	0.2500	0.2500	0.2500	0.1333	NC	-	0.3753	0.2987	0.1735		0.3465
ATCC 60051	0.3158	NC	NC	NC	NC	NC	0.1052	-	0.4209	0.3666	0.2411	0.3567
													ATCC 12240	0.0833	0.01818	0.1818	0.1818	NC	NC	0.1666	0.0800	-	0.2915	0.3996	0.2165
ATCC 13271	NC	0.2666	0.1313	0.1333	0.1428	NC	0.3530	0.111	0.1739		0.2203
													FP-718 07-S	0.1250	NC	NC	NC	NC	NC	NC	0.2353	0.0909	0.2666	-
HHB- 3585-SP	0.1250	NC	NC	NC	0.1538	0.1818	0.1250	0.1176	0.2727	NC	NC	-

F value and the P value of the PRLP of the Mitochondrial DNA of table 5CIaI digestion

	WD 1222	ATCC 26710	KCTC 0399BP	PL5	PL4	PLM	ATCC 32233	ATCC 60051	ATCC 12240	ATCC 13271	FP-71 807-S	HHB-35 85-Sp
													WD 1222	-	0.1155	0.1351	0.1527	0.2841	0.2987	0.3243	0.0372	0.1412		0.2310	0.2507
ATCC 26710	0.5000	-	0.0196	0.0851	0.1686	0.1831	0.2088	0.1527			0.1155	0.2570
													KCTC 0399BP	0.4444	0.8888	-	0.0531	0.1831	0.1288	0.2203	0.1686			0.1351	0.2682
PL5	0.4000	0.6000	0.7272	-	0.1964	0.1412	0.3465	0.1831			0.1527	0.2841
													PL4	0.1818	0.3636	0.3333	0.3077	-	0.1047	0.2411	0.1964	0.1736	0.1256	0.1686	0.2987
PLM	0.1666	0.3333	0.4615	0.4285	0.5333	-	0.3666	0.3243	0.1831	0.1831	0.1831	0.3120
													ATCC 32233	0.1428	0.2587	0.2666	0.1250	0.2353	0.1111	-	0.2310	0.2006	0.2006	0.2088	0.2203
ATCC 60051	0.8000	0.4000	0.3636	0.3333	0.3077	0.1428	0.2500	-		0.3465	0.1682	0.2841
													ATCC 12240	0.4285	NC	NC	NC	0.3529	0.3333	0.3000	NC	-	0.2682	0.3243	0.2203
ATCC 13271	NC	NC	NC	NC	0.4706	0.3333	0.3000	0.1250	0.2000	-	0.3243
													FP- 71807-S	0.2500	0.5000	0.4444	0.4000	0.3636	0.3333	0.2857	0.2000	0.1428	0.1482	-	0.2507
HHB- 3585-Sp	0.2222	0.2222	0.2000	0.1818	0.1666	0.1538	0.2666	0.1818	0.2666	NC	0.222	-

F value and the P value of the PRLP of the Mitochondrial DNA of table 6EcoRI digestion

	WD12 22	ATCC 26710	KCTC0 399BP	PL5	PL4	PLM	ATCC 32233	ATCC 60051	ATCC 12240	ATCC 13271	FP- 71807- S	HHB- 3585- SP
													WD 222	-	0.3837	0.1921	0.2006	0.2764	0.2597	0.3302	0.0175	0.4339	0.1899	0.4071	0.2987
ATCC 26710	0.1000	-	0.1442	0.2006	0.1608	0.2597	0.3302	0.3837	0.3183	0.3054	0.4071	0.4142
													KCTC 0399BP	0.3158	0.4210	-	0.0509	0.2006	0.2507	0.2567	0.4275	0.3120	0.2310	0.2165	0.2915
PL5	0.3000	0.3000	0.7368	-	0.2764	0.2597	0.1470	0.2682	0.3183	0.2378	0.2240	0.2987
													PL4	0.1904	0.3809	0.3000	0.1904	-	0.3837	0.2682	0.2764	0.4399	0.3120	0.2987	0.3054
PLM	0.2105	0.2105	0.2222	0.2105	0.1000	-	0.3243	0.2597	0.3120	0.4142		0.1964
													ATCC 32233	0.1397	0.1379	0.2143	0.4318	0.2000	0.1428	-	0.3302	0.3662	0.1256	0.3465	0.2362
ATCC 60051	0.9000	0.1000	0.0769	0.2000	0.1904	0.2105	0.1379	-	0.4339	0.2738	0.2915	0.4142
													ATCC 12240	0.0740	0.1481	0.1538	0.1481	0.0714	0.1538	0.1111	0.0740	-	0.3465	0.2203	0.2737
ATCC 13271	0.3200	0.1600	0.2500	0.2400	0.1538	0.0833	0.4705	0.2400	0.1250	-	0.2568	0?2915
													FP- 71807-S	0.0869	0.0869	0.2727	0.2680	0.1666	NC	0.1250	0.1739	0.2666	0.2142	-	0.4339
HHB- 3585-SP	0.1666	0?0833	0.1739	0.1666	0.1600	0.3077	0.2424	0.0833	0.1935	0.1379	0.0740	-

F value and the P value of the PRLP of the Mitochondrial DNA of table 7PvuII digestion

	WD 1222	ATCC 26710	KCTC 0399BP	PL5	PL4	PLM	ATCC 32233	ATCC 60051	ATCC 12240	ATCC 13271	FP- 71807-S	HHB- 3585-SP
													WD 1222	-	0.2507				0.2682	0.2411	0.0851	0.3567	0.2987	0.3358	0.3465
ATCC 26710	0.2222	-	0.1155	0.1155	0.2987	0.2507	0.3465		0.3465		0.3243
													KCTC 0399BP	NC	0.5000	-	0.0479					0.3465		0.3243
PL5	NC	0.5000	0.7500	-					0.3465
													PL4	NC	0.1666	NC	NC	-	0.1968	0.2682			0.3358
PLM	0.2000	0.2222	NC	NC	0.3070	-	0．3567	0.2682
													ATCC32 233	0.2353	0.1250	NC	NC	0.2000	0.1176	-		0.2310		0．3996	0.4071
ATCC60 051	0.6000	NC	NC	NC	NC	0.2000	NC	-	0.567	0.2987	0.3358	0.3465
													ATCC12 240	0.1176	0.1250	0.1250	0.1250	NC	NC	0.2500	0.1176	-	0.2597	0.1010	0.1760
ATCC13 271	0.1666	NC	NC	NC	0.1333	NC	NC	0.1666	0.2105	-	0.1256	0.2507
													FP- 71807-S	0.1333	0.1428	0.1428	NC	NC	NC	0.0909	0.1333	0.5454	0.4706	-	0.2088
HHB- 3585-SP	0.1250	NC	NC	NC	NC	NC	0.0869	0.1250	0.3478	0.2222	0.2857	-

The common segmental ratio of table 8 and corresponding to the arithmetical mean distance of P value

	WD 222	ATCC 26710	KCTC 0399BP	PL5	PL4	PLM	ATCC 32233	ATCC 60051	ATCC 12240	ATCC 13271	FP-71 807-S	HHB-35 85-SP
													WD 1222	-	4/53	5/53	5/55	3/60	4/54	5/78	9/59	6/82	5/68	5/62	6/65
ATCC 26710	0.2499	-	6/50	4/52	8/60	6/51	7/75	3/56	5/79	4/65	4/59	2/62
													KCTC 0399BP	0.1636	0.0762	-	21/52	6/57	6/51	7/75	3/56	5/79	4/65	6/59	3/62
PL5	0.1766	0.1645	0.0379	-	5/59	6/51	9/77	4/58	5/81	4/67	5/61	3/64
													PL4	0.2802	0.2350	0.2393	0.2616	-	7/58	8/82	4/63	4/86	8/72	4/66	4/69
PLM	0.2755	0.2444	0.2212	0.2283	0.2284	-	4/76	4/57	5/80	4/66	2/60	6/63
													ATCC 32233	0.2985	0.2791	0.2360	0.2415	0.2783	0.3490	-	5/81	10/104	14/90	5/74	8/87
ATCC 60051	0.0823	0.2682	0.2980	0.2256	0.2364	0.2840	0.3121	-	3/85	6/71	6/65	4/68
													ATCC 12240	0.3365	0.3163	0.3142	0.3163	0.3067	0.2475	0.2741	0.4038	-	6/94	12/88	12/91
ATCC13 271	0.2443	0.2628	0.2834	0.2868	0.2744	0.2986	0.1665	0.3123	0.2914	-	10/74	4/77
													FP- 71807-S	0.3301	0.2823	0.2253	0.1883	0.2336	0.1831	0.3183	0.2591	0.2613	0.2317	-	5/71
HHB- 3585-SP	0.3817	0.3324	0.2498	0.2914	0.3053	0.2641	0.3025	0?3503	0.2216	0.2711	0.2978	-

Embodiment VII: the microbiological property of phellinus linteus Yoo bacterial strain KCTC 0399BP

The microbiological property of research phellinus linteus Yoo KCTC 0399BP bacterial strain, this bacterial strain is accredited as a kind of new bacterial strain that is different from the wood layer hole strain with very big dependency again.Its sporophore, no gemma, perennial contains the wooden part of semicircle cap, the wide about 10cm of semicircle cap, thick about 4cm.When plucking, the gill fungus surface is a Vandyke brown, but as time goes by, the surface has many cracks and darkens until chocolate, and its thalamium changes aspect color simultaneously, becomes tawny by bright red.Hymenial tube is the multilayered structure that has circular aperture.The long 3-4cm of its spore, subsphaeroidal, khaki.And, many setas and the wall that size is 15-30 * 8-10 μ m are arranged.We find bacterial strain of the present invention, and almost all aspects with the phellinus linteus of following record are all identical, this phellinus linteus is documented in Imazeki, R. and Hongo, T. edit, among " the color explanation of Japanese gill fungus " p189 that Hoikusha Imazeki publishes, be named as phellinus linteus Yoo.We on November 17th, 1997 at Korea S's bio-science and Bioteknologisk Institut Korea S this novel strain of type culture preservation center preservation, the preserving number that we obtain is KCTC 0399BP.Embodiment VIII: the cultivation of phellinus linteus Yoo KCTC 0399BP

Replenish KH for the culture broth that contains 50g starch, peptone 10g and yeast extract 10g in a kind of every 1L distilled water ₂PO ₄0.8g, MgSO ₄7H ₂O0.5g and CaCl ₂0.3g, it is transferred to pH6.0, the horizontal high voltage of going forward side by side sterilization.In 3.5 liters of culture broth of 5 liters of fermentation vats, cultivate phellinus linteus YooKCTC 0399BP mycelium and descend cultivation 11 days, simultaneously, in the meat soup pond, ventilate with 2vvm at 28 ℃, and with 160rpm rotation fermentation vat.Cultivate after 5 days, obtain the highest mycelial yield (30g/l), as shown in Figure 2.From cultivate the mycelium after 5 days, the hot water extraction yield of polysaccharide material is the dry mycelium of 0.873% every weight unit.

The mycelia of table 9 phellinus linteus and the productive rate of polysaccharide

Project	Cultivate the mycelium of 5 days phellinus linteus
		Dry mycelium weight (g/L)	????30
Dry raw polysaccharide weight	????262
		Crude polysaccharides productive rate (%)	????0.873

Embodiment IX: the extraction of phellinus linteus Yoo bacterial strain mycelium culture

Mycelium culture to phellinus linteus Yoo bacterial strain extracts and purifying, to obtain thick immunostimulation material.At first, the mycelium culture that is obtained in the 280g embodiment VIII is carried out hot water extraction, concentrate then, obtain the 3g hot water extract.Adding ethanol to ultimate density in extract is 80%, places 24 hours down at 4 ℃, centrifugal then.To precipitate soluble in water, then 10 ℃ down with a cellulose tube (for example Nippon Junko Jun Yaku Seihing sells) dialysis 72 hours, per 12 hours of while was changed dialyzate again one time.Solution lyophilize with in the cellulose tube obtains the 2.45g crude extract.Embodiment X: the separation of pure immunostimulation material

Finish purifying by carrying out ion exchange chromatography and gel permeation chromatography successively.

At first, the crude extract that obtains in the embodiment IX is dissolved in 5mM sodium phosphate buffer (pH7.2), last DEAE-cellulose column is with identical damping fluid washing.As elution buffer, its concentration gradient is 0-1M with a kind of NaCl solution in same buffer.It crosses post with the velocity flow of 5mL/min, and every test tube is collected elutriant 15mL.Measure wash-out sugared content partly by a kind of phenolsulfuric acid method, measure the protein content of wash-out part by Bradford ＇ s method.Top with sacchariferous part upper prop, this post is made up of porous particle, the post of for example commercially available commodity Toyopearl HW 65F by name is further purified by the moisture elutriant of gel permeation chromatography, obtains similar pure active fraction aspect molecular weight and characteristic.Measure its sugar and proteic content according to above-mentioned identical method.So the pure immunostimulation material that obtains is named as " PL ".Embodiment XI: the immunostimulatory activity of polysaccharide material

The polysaccharide material that derives from basidiomycetes (Basidomycetes) has positive influence to propagation or activated lymphocyte, and strengthen thus to external factor and even the immunity of cancer cells, this is known.This effect of polysaccharide material is not produced by direct cytotoxicity, but immunostimulant caused.Therefore, its advantage is to produce very little side effect in vivo.The polysaccharide material of basidiomycetes (Basidomycetes) causes the enhancing of cytotoxicity and lymphocyte, B-cell and T-cell to form antibody and therefore demonstrate the antitumor immune activity just.Thus, by the propagation that forms antibody lymphocyte count and measurement T-cell is proved that indirectly this polysaccharide material has the antitumor immune activity.

In this embodiment, by utilizing the method that forms the antibody cell, measure the propagation of T-cell by the mixed lymphocytes answer techniques to the B-cell counting.In addition, in order to detect this polysaccharide material, measure synthetic nuclear DNA recently by using the 3H-thymidine to lymphopoietic effect.The results are shown in the following table 10.

The immunostimulatory activity of the various materials of table 10

Material	Lymphopoiesis effect (DPM * 10 4)	T cytoactive (DPM * 10 4)	B cell antibody forms (Abs576mm)
				Phellinus linteus Yoo	????8.4±0.5	????2.5±0.1	?0.66±0.01
????LPS			?0.81±0.23
				Contrast	????1.6±0.5	?0.5±0.05	?0.00±0.00

^*The working concentration of every kind of material is 10 μ gml.

Can find out significantly that from the data of table 10 polysaccharide material that extracts from new wood layer hole strain bacterial strain of the present invention is being better than control group aspect lymphopoiesis and the T-cytoactive, ratio between two all is approximately 5 times.Polysaccharide material of the present invention has the high B-cell similar to lipopolysaccharides and forms the antibody ability.Lipopolysaccharides is very effective immunosuppressor, but since it have severe side effect in vivo and can not at will use.Embodiment XII: the sugared unit and the composition of immunostimulation polysaccharide material

The polysaccharide material that will obtain from the embodiment X carries out gas chromatographic analysis to analyze its sugared unit (accompanying drawing 4).Measure its carbohydrate and albumen composition respectively with phenolsulfuric acid method and forint-phenol processes.Under following condition, on the instrument of " HP 5890 GC " by name that Hewlett Packard sells, to analyze, this instrument is equipped with a fused silica capillary columns sp-2330 (0.32mm * 30m).At first, this post was kept 2 minutes down at 200 ℃, the speed with 4 ℃ of per minute risings heats until 250 ℃ then, keeps 2 minutes at 250 ℃ then.To maintain 260 ℃ and 250 ℃ by detector and the syringe that instrument provides respectively.With every part of polysaccharide material 2N TFA hydrolysis of extracting fraction that 2mg takes from embodiment XI gained, use NaBH ₄Reduction is also used the anhydrous acetic acid acetylize, obtains the aditol acetate of air feed analysis of hplc.The results are shown in the following table 11.

The sugared unit and the composition of table 11 immunocompetence fraction

Polyoses extract	Carbohydrate (%)	Albumen (%)	Sugar unit (mol%)
						Glucose	Seminose (Monnose)	Semi-lactosi (Gatactose)
			PL	82.7	17.3	78.6	18.0	3.4

Embodiment XI II: the structural analysis of immunostimulation polysaccharide material

The immunostimulation polysaccharide material is carried out structural analysis.

At first, confirm its purity with the 65F gel filtration chromatography.Accompanying drawing 5 is tomographic results.See that as us every kind of carbohydrate and protein ingredient present single symmetrical peak, this explanation PL is pure.

From nuclear magnetic resonance of carbon wave spectrum (accompanying drawing 6), PL is accredited as a kind of galactomannan dextran (glactomannoglucan), wherein glucose unit is connected to form a long-chain by α (1 → 4) and β (1 → 6) key, and seminose and semi-lactosi are in conjunction with the side chain as long-chain.

Show that according to the structural analysis of forming the result PL a kind ofly structurally has the very new polysaccharide of big-difference with beta-glucan.Embodiment XI V: polysaccharide material extracts from the mycelium of wood layer hole strain kind, its purifying and immunostimulatory activity thereof.

The bacterial strain of wood layer hole strain kind is comprised that tinder shape Phellinus, Bao Mu Shi Phellinus, Phellinus pini, faint yellow Phellinus and the Phellinus that turns black cultivate the mycelium that obtains them.From them, extract and the purified polysaccharide material, be used to then induce strengthen cytotoxicity and lymphocyte, B-cell and T-cell formation antibody, described in example VII A I to embodiment 11.By the propagation that forms antibody lymphocyte count and mensuration T-cell being estimated indirectly the antitumor immune activity of this polysaccharide material.The gained result is similar to the result among the embodiment XI.Embodiment XV: extraction, its purifying and the immune stimulating activity thereof of polysaccharide material from wood layer hole strain kind sporophore

With the bacterial strain of wood layer hole strain kind, comprise that tinder shape Phellinus, Bao Mu Shi Phellinus, Phellinus pini, faint yellow Phellinus and the Phellinus that turns black cultivate the sporophore that obtains them.From them, extract and the purified polysaccharide material, be used to then induce strengthen cytotoxicity and lymphocyte, B-cell and T-cell formation antibody, described in example VII A I to embodiment 11.By the propagation that forms antibody lymphocyte count and mensuration T-cell being estimated indirectly the antitumor immune activity of this polysaccharide material.The gained result is similar to the result among the embodiment XI.Embodiment XVI: according to the therapeutic activity of the anticancer cell of polysaccharide material of administration time

Measure the antitumour activity of polysaccharide material with the B16F10 skin cancer cell that derives from mouse.In having replenished RPMI 1640 substratum of 10% serum, cultivate the B16F10 skin cancer cell, and succeeding transfer culture 2 times weekly.In the peritoneal cavity of every mouse, implant the B16F10 cell of many as 100,000.30 days viability of mouse implanted in record.Die young and implant for the mouse of polysaccharide material (pretreat) and cancer cells to intraperitoneal to carry out record for the mouse of polysaccharide material (treatment back) to intraperitoneal in back 12 days to implanting back 12 implant the last week from cancer cells.The results are shown in the accompanying drawing 7, be summarized in the following table 12.We see: pretreat mouse group The average survival time power is the highest, secondly is back treatment mouse group, does not treat mouse and organizes minimum.Experimental mouse does not change aspect body weight.

The immunotherapy of table 12 polysaccharide material

Experimental group (mg/kg)	Average viability	Ratio (%)	The number of the 30th day survival mouse
				Not treatment	????20.8	????100	????0/10
Pretreat (100)	????29.0	????139	????8/10
				Treatment back (100)	????25.8	????124	????2/10

Embodiment XVII: the anticancer cell activity experimental example 1 of polysaccharide material that changes with different immunochemotherapies: the antitumour activity that changes with the polysaccharide material Combined Preparation different with Zorubicin

Use the similar implantation experimental system mensuration polysaccharide material of B16F10 skin cancer cell and the antitumour activity of Zorubicin Combined Preparation.In the peritoneal cavity of mouse, implant skin cancer cell.Zorubicin passes through peritoneal administration after tumour is implanted 12 days, and polysaccharide material passed through peritoneal administration in back 12 days to implanting from implanting the last week.After 60 days, write down the viability of mouse every day.Use the low dosage Zorubicin can not damage mouse.

The results are shown among accompanying drawing 8a and the 8b, be summarized in the following table 13.Can obviously see from data: the mouse viability of pharmaceutical treatment of no use is very low, and separately the viability with the mouse of polysaccharide material treatment is far superior to untreated mouse.In accompanying drawing 8a and accompanying drawing 8b, shown as the result of mouse when using dosage is per kilogram of body weight 0.1mg/kg Zorubicin and 0.3mg/kg Zorubicin respectively.Dosage is that the viability of the mouse of 0.3mg/kg is the viability of the mouse of 0.1mg/kg significantly better than dosage.Uniting under the situation of use with polysaccharide material, the Zorubicin dosage is that the viability of the mouse of 0.3mg/kg also is better than the viability that the Zorubicin dosage is the mouse of 0.1mg/kg far away.Quantization means, the mouse of implanting the B16F10 skin cancer cell on average survived 18.1 days.When using the polysaccharide material treatment separately, the mean survival time of mouse extends to 31 days.When independent dosage with 0.1mg/kg used Zorubicin, the mean survival time of mouse was 35.4 days.And when independent dosage with 0.3mg/kg used Zorubicin, the mean survival time of mouse was 40.1 days.If Zorubicin and polysaccharide material are united use, the mean survival time significant prolongation of mouse so was by 57.8 days under the dosage that extended to Zorubicin 0.3mg/kg in 46.9 days under the dosage of Zorubicin 0.1mg/kg.The body weight of mouse does not alleviate in the experiment.

The immunotherapy of table 13 Zorubicin and polysaccharide material combined utilization

Experimental group (mg/kg)	Average viability	Ratio (%)	The number of the 60th day survival mouse
				Not treatment	????18.1	???100.0	????????0/10
????P.S. 1(100)	????31.0	???171.3	????????0/10
				????Am 2(0.1) ?Am(0.1)+P.S.(100)	????35.4 ????46.9	???195.6 ???259.1	????????0/10 ????????4/10
????Am(0.3) ?Am(0.3)+P.S.(100)	????40.1 ????57.8	???240.9 ???319.3	????????2/10 ????????9/10

¹Polysaccharide material ²Zorubicin experimental example 2: the antitumour activity that changes with the polysaccharide material Combined Preparation different with ametycin

As described in experimental example 1, use the similar implantation experimental system mensuration polysaccharide material of B16F10 skin cancer cell and the antitumour activity of mitomycin C with C administration.In the peritoneal cavity of mouse, implant skin cancer cell.Ametycin passes through peritoneal administration after tumour is implanted 12 days, and polysaccharide material passes through peritoneal administration from implanting to die young to implantation back 12 the last week.After 60 days, every day mouse viability.Use Modulation of Low Dose Mitomycin C can not damage mouse.

Obtain the result similar, and be summarized in the following table 14 to experimental example 1.Can obviously see from data: the mouse viability of pharmaceutical treatment of no use is very low, and separately the viability with the mouse of polysaccharide material treatment is far superior to untreated mouse.Quantization means, the mouse of implanting the B16F10 skin cancer cell on average survived 18.1 days.When using the polysaccharide material treatment separately, the mean survival time of mouse extends to 31 days.When independent dosage with 0.2mg/kg used mitomycin, the mean survival time of mouse was 32.5 days.And when independent dosage with 0.6mg/kg used ametycin, the mean survival time of mouse was 38.0 days.If ametycin and polysaccharide material are united use, the mean survival time significant prolongation of mouse so was by 51.5 days under the dosage that extended to ametycin 0.6mg/kg in 43.5 days under the dosage of ametycin 0.2mg/kg.The body weight of mouse does not alleviate in the experiment.

Table 14

The immunotherapy of ametycin and polysaccharide material combined utilization

Experimental group (mg/kg)	Average viability	Ratio (%)	The number of the 60th day survival mouse
				Not treatment	????18.1	????100.0	????0/10
P.S. 1(100)	????31.0	????171.3	????0/10
				M.C 2(0.2) M.C(0.2)+P.S.(100)	????32.5 ????43.5	????179.6 ????240.3	????0/10 ????3/10
M.C(0.6) M．C(0.6)+P．S.(100)	????38.0 ????51.5	????209.9 ????284.5	????1/10 ????7/10

¹Polysaccharide material ²Ametycin experimental example 3: the antitumour activity that changes with the polysaccharide material Combined Preparation different with cis-platinum

As described in above-mentioned experimental example, use the similar implantation experimental system mensuration polysaccharide material of B16F10 skin cancer cell and the antitumour activity of cisplatin combined administration.In the peritoneal cavity of mouse, implant skin cancer cell.Cis-platinum passes through peritoneal administration after tumour is implanted 12 days, and polysaccharide material passed through peritoneal administration in back 12 days to implanting from implanting the last week.After 60 days, write down the viability of mouse every day.Use the low dosage cis-platinum can not damage mouse.

Obtain the result similar, and be summarized in the following table 15 to above-mentioned experimental example.Can obviously see from data: the mouse viability of pharmaceutical treatment of no use is very low, and separately the viability with the mouse of polysaccharide material treatment is far superior to untreated mouse.Quantization means, the mouse of implanting the B16F10 skin cancer cell on average survived 18.1 days.And using polysaccharide material when treatment separately, the mean survival time of mouse extends to 31 days.When independent dosage with 0.5mg/kg used cis-platinum, the mean survival time of mouse was 33 days.And when independent dosage with 1.5mg/kg used cis-platinum, the mean survival time of mouse was 40.5 days.If cis-platinum and polysaccharide material are united use, the mean survival time significant prolongation of mouse so was by 52.6 days under the dosage that extended to cis-platinum 1.5mg/kg in 44.0 days under the dosage of cis-platinum 0.5mg/kg.The body weight of mouse does not alleviate in the experiment.

The immunotherapy of table 15 cis-platinum and polysaccharide material combined utilization

Experimental group (mg/kg)	Average viability	Ratio (%)	The number of the 50th day survival mouse
				Not treatment	????18.1	????100.0	????0/10
P．S. 1(100)	????31.0	????171.3	????0/10
				C.P 2(0.5) C.P(0.5)+P.S.(100)	????33.0 ????44.0	????182.3 ????243.0	????0/10 ????3/10
C.P(1.5) C.P(1.5)+P.S.(100)	????40.5 ????52.6	????223.8 ????290.6	????2/10 ????7/10

¹Polysaccharide material ²Cis-platinum experimental example 4: the antitumour activity that changes with the polysaccharide material Combined Preparation different with 5 FU 5 fluorouracil

As described in above-mentioned experimental example, use the similar implantation experimental system mensuration polysaccharide material of B16F10 skin cancer cell and the antitumour activity of 5 FU 5 fluorouracil Combined Preparation.In the peritoneal cavity of mouse, implant skin cancer cell.5 FU 5 fluorouracil passes through peritoneal administration after tumour is implanted 12 days, and polysaccharide material passes through peritoneal administration from implanting to die young to implantation back 12 the last week.60 die young after, write down the viability of mouse every day.Use the low dosage 5 FU 5 fluorouracil can not damage mouse.

Obtain the result similar, and be summarized in the following table 16 to above-mentioned experimental example.Can obviously see from data: the mouse viability of pharmaceutical treatment of no use is very low, and separately the viability with the mouse of polysaccharide material treatment is far superior to untreated mouse.Quantization means, when separately using 5 FU 5 fluorouracil with the dosage of 1mg/kg, the mean survival time of mouse is 34.2 days, and when 5 FU 5 fluorouracil and polysaccharide material were united use, the mean survival time of mouse was 45.5 days.When independent use 5 FU 5 fluorouracil, the dosage of 3mg/kg can make mouse on average survive 40.2 days, and unites when using with polysaccharide material, and the dosage of 3mg/kg can make mouse on average survive 54.8 days.The body weight of mouse does not alleviate in the experiment.

Table 16

Experimental group (mg/kg)	Average viability	Ratio (%)	The number of the 60th day survival mouse
				Not treatment	????18.1	????l00.0	????0/10
????P.S 1.(100)	????31.0	????171.3	????0/10
				????5-FU 2(1) 5-FU(1)+P.S.(100)	????33.0 ????44.0	????187.8 ????251.4	????0/10 ????4/10
????5-FU(3) 5-FU(3)+P.S.(100)	????40.5 ????52.6	????222 ????302.6	????2/10 ????8/10

¹ Polysaccharide material ²5 FU 5 fluorouracil embodiment XV III: polysaccharide material is to the antitumour activity of the cancer cells that derives from human body

Use the activity that NC1-H23 (a kind of lung cancer cell line) detects the anti-people's tumour of polysaccharide material.Give the subcutaneous implantation of nude mice NC1-H23 lung carcinoma cell.Give nude mice with polysaccharide material and Zorubicin through peritonaeum in 12 days after cancer cells is implanted.The size of measuring the cancer cells agglomerate on the 19th day after cancer cells is implanted is separated cancer cells then and is weighed.Because nude mice lacks the T cell, so their immunocompetence belongs to the activity of natural killer cell and scavenger cell usually.Therefore, the immuno-potentiation that is obtained after giving polysaccharide material mediates by this two classes cell.

The result is presented among following the accompanying drawing 9a and accompanying drawing 9b, and represents with numeral in table 17 and 18.From these data, can find out significantly, the size maximum of the cancer cells of Zorubicin both of no use polysaccharide material also of no use treatment, with the cancer cells of polysaccharide material treatment secondly, and with the cancer cells minimum of Zorubicin treatment.Result after weighing also is like this.The body weight of these experimental mouse does not increase or reduces.

The immunotherapy of table 17 human cancer cell (cell size mm ³)

Experimental group	The 14th day	The 17th day	The 18th day	The 19th day
					Not treatment	???43	??105	??131	??158
?P.S 1(100mg/kg) ??Am 2(1mg/kg)	???20 ???12	???39 ???27	???40 ???34	???43 ???33

¹Polysaccharide material ²Zorubicin

Table 18

The immunotherapy of human cancer cell

Experimental group	The whole weight of cancer cells
	Do not treat P.S 1(100mg/kg) Zorubicin (1mg/kg)	259 113 95

¹Polysaccharide material embodiment XI X: polysaccharide material is to the effect of mouse cancer metastasis

In order to test the restraining effect of polysaccharide material, the B16F10 skin cancer cell is planted in the tail vein of C57BL/6 mouse cancer metastasis.Whether after plantation the 14th day, the lung of excision mouse have metastasis of cancer to take place or shift with research takes place to for which kind of degree.In 12 days behind the tumor planting, give this mouse through peritonaeum with Zorubicin, simultaneously in 12 days after the 1 thoughtful plantation before plantation with polysaccharide material through peritoneal administration.Through the B16F10 skin cancer cell generation transposition of intravenous injection, on lung, form stain to the mouse tail.The result is presented among following the accompanying drawing 10a and accompanying drawing 10b, and numeral is summarised in the table 19.Shown in accompanying drawing 10a, find nearly 272 cancer spots in the mouse group with any pharmacological agent, treat the back spot by the dosage of 1mg/kg with Zorubicin and be reduced to 197, treat the back spot by the dosage of 3mg/kg with Zorubicin and further be reduced to 172.The dosage of Zorubicin 0.1-0.3mg/kg is demonstrating the potential activity aspect the anticancer growth, very weak but the dosage of its 1-3mg/kg suppresses the effect of metastasis of cancer.After independent using polysaccharides material treatment, the reduced number to 83 of these cancer spots.In the mouse group of polysaccharide material and Zorubicin combined utilization, when the dosage of Zorubicin was 1mg/kg and 3mg/kg, the cancer spot of being found was respectively 136 and 131.Therefore polysaccharide material is more effective than Zorubicin aspect the inhibition metastasis of cancer.Table 19

Experimental group	The spot number
	Do not treat P.S 1(100mg/kg) Zorubicin (1mg/kg) Zorubicin (1mg/kg)+P.S (100mg/kg) Zorubicin (3mg/kg) Zorubicin (3mg/kg)+P.S (100mg/kg)	272 83 197 136 172 131

¹Polysaccharide material embodiment XX: polysaccharide material is to the experiment of the direct cytotoxicity of cancer cells

Antitumour activity to polysaccharide material experimentizes, and is to express by immuno-potentiation to observe it, still expresses by the direct cytotoxicity to cancer cells.The Zorubicin that with polysaccharide material and dosage is 0.3-30 μ g/kg is directly treated B16F10 mouse skin cancer cell and NC1-H23 human lung carcinoma cell.After treatment the 2nd day measure and remain cancer cells alive according to sulforhodamin B method.The result is presented in the following accompanying drawing 11, and numeral is summarised in the table 20.The cell quantity of the experimental group of any pharmaceutical chemicals treatment of no use is expressed as " 100% ", the cell quantity for the treatment of experimental group is compared with this standard.From then on can be clear that in the data that Zorubicin all shows the potential cytotoxicity to this two quasi-cancer cell, and not effect of polysaccharide material.In general, above the data presentation Zorubicin that obtained among the embodiment be to bring into play antitumour activity by cytotoxicity, and polysaccharide material is to show its antitumour activity by immuno-potentiation.

Table 20

The direct cytotoxicity of polysaccharide material

Experimental group	Zorubicin		Polysaccharide material
					B16F10	NC1-H23	B16F10	NC1-H23
	Do not treat 0.3 μ g/ml, 1 μ g/ml, 3 μ g/ml, 10 μ g/ml, 30 μ g/ml	100 56 29 9 -10 -10	100 100 71 39 7 -1	100 93 89 80 78 77	100 93 89 80 78 77

Embodiment X XI: as the prevention of AIDS and the experiment of treatment adjuvant drug (Aid)

Polysaccharide material of the present invention is experimentized, observing its feasibility, and find that it is effective as the prevention of AIDS and treatment adjuvant drug.

In general, the data presentation of embodiment, according to rflp analysis, gill fungus of the present invention is a kind of new bacterial strain that has dependency with kind wood layer hole strain, and the polysaccharide material that obtains from the mycelium of wood layer hole strain kind has antitumour activity, and it is to show by the cytotoxicity that stimulates the T cell and the antibody forming capacity of B cell.In addition, polysaccharide material of the present invention shows a kind of activity of effective anticancer transfer.As a result, this polysaccharide material that can stimulate immunity of organism is to being effectively aspect the prevention of the immune correlated disease of tumour and AIDS and treatment, and therefore it is very useful in biological medicine industry.

The present invention to be illustrated, and can to recognize that used term is in order being described, rather than be limited to this.

According to top description, can carry out many modifications and change to the present invention.Therefore can think and to implement the present invention within the scope of the appended claims, and be not limited to specific description.

Claims (7)

1. new bacterial strain, phellinus linteus Yoo KCTC 0399BP, it can produce a kind of immunostimulation polysaccharide material and have size and be the Mitochondrial DNA of 61kb.

2. new natural immunity excitor substance, it is to extract from the culture of the mycelium of wood layer hole strain kind or sporophore and purifying.

3. new natural immunity excitor substance as claimed in claim 2, wherein said material is made up of seminose, semi-lactosi and glucose.

4. a method that is used to prepare a kind of new immunostimulation polysaccharide material is characterized in that it comprises the following steps:

In the substratum that contains glucose, yeast extract and peptone, cultivate the bacterial strain of wood layer hole strain to obtain mycelium or sporophore;

From mycelium or sporophore, extract described material with hot water;

By with ethanol sedimentation, suspend in water and separate this material with dialysis tubing dialysis; And

By carrying out this material of DEAE-Mierocrystalline cellulose chromatography and gel chromatography successively.

5. the medicine acceptable composition of an antitumor immune stimulating activity is characterized in that it contains the polysaccharide material or derivatives thereof that obtains as effective constituent from the mycelium of wood layer hole strain kind or sporophore.

6. utilize the purposes of the polysaccharide material described in claim 2 or 3 as a kind of immunity anticancer agent.

7. utilize the auxiliary purposes of the polysaccharide material described in claim 2 or 3 as the treatment of the immune correlated disease of a kind of AIDS of comprising.

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