TW200920387A - Composition of mushroom beta-glucan for anti-cancer and preparation method thereof - Google Patents

Abstract

The present invention provides a composition for anti-cancer comprising a vector and mushroom beta-glucan, wherein the mushroom is selected from Schizophyllum commue, Agarics blaze, Cordyceps sinensis, Ganoderma lucidum, Coriolus versicolor, Anthodia camphorate, Phellinus linteus, Pleurotus citrinopileatus, Lentinula edodes, Agrocybe aegerita, Hericium erinaceus, Pleurotus eryngiig, Sparrasis crispa, Auricularia auricula, Flammulina velutipes and the combination thereof.

Description

200920387 IX. Description of the Invention: [Technical Field of the Invention] The present invention relates to a mushroom 蕈 polysaccharide composition having an anticancer effect and a preparation method thereof. [Prior Art] In recent years, many studies have found that the mycelium or fruiting body of the mushroom has a considerable amount of physiologically active ingredients, such as polysaccharide (y3-Glucan), superoxide dismutase (SOD), Three kinds of substances, such as Triterpenoids, have immunomodulatory, anti-oxidative, anti-tumor effects. Among them, polysaccharides are the most in-depth and most promising anti-tumor and immunomodulatory substances. It is known that polysaccharides such as wan-glucan are a good immune-promoting agent, which can effectively stimulate immune cells, not only enhance the specific immunity of organisms, but also enhance the non-specific immune response. Polysaccharides can protect organisms from bacterial, fungal, viral and parasitic infections and inhibit tumor growth. In recent years, the mushroom polysaccharide (mushr〇(10) is _glucan) has been paid more and more attention. The main reason is that the branching degree, molecular weight, water bathing and structural stability of the mushroom polysaccharide are much higher than that of the yeast polysaccharide. Immunization promoting ability. The physiological activity of polysaccharides with anti-tumor effect can be roughly classified into three categories according to the molecular weight of polysaccharides: (〇 molecular weight is 3, 〇〇〇~5, (10) 〇' has the function of lowering blood sugar' (B) molecular weight In the case of between 1〇〇〇〇1〇〇〇〇〇, it has an anti-inflammatory effect, ((:) molecular weight is 3〇, above, it has anti-tumor effect' and the greater the molecular weight, the better the effect. 5 110484 200920387 : Most of the body is extracted from the fruiting body, but the source of the fruiting body is limited. Recently, it is known that about 50 kinds of mites can produce extracellular polysaccharides in liquid culture, so the culture solution can be taken. Purification of polysaccharides; liquid culture is faster and simpler than culture of fruit bodies. However, the components of mushroom mites are complex, such as polysaccharides, proteins, peptides, sputum, various amino acids, alkaloids, vinegar Classes and organic acids, etc., so the extraction, strontium polysaccharides need to undergo a thorough step, such as thermal extraction, extraction, acid extraction, etc., so it is easy to leave organic solvents, amino acids and proteins, such residues may be left Right person Produces a stimulatory immune response. It is known that a few tumors of the mushroom genus are mostly multi-body or a complex of multi-breast and protein, and its anti-tumor effect is related to the dose, frequency, time and route of administration. However, the multi-enzyme containing protein will cause the body's immunity, and should cause fever, redness, ascites and other discomforts. Therefore, other substances of the mushroom towel must be removed, and only the polymer polysaccharide can be extracted and purified to eliminate the disadvantage. In order to solve the above problems, the present invention provides a novel mushroom polysaccharide composition having anticancer effect and a preparation method thereof. [Summary] There is a lack of the above-mentioned prior art, 4 The main object of the invention is to provide a polysaccharide composition of the mushroom scorpion having anti-cancer effect. & Another object of the present invention is to provide an extraction method of the polysaccharide of the mushroom oyster, which is fermented by liquid mash, via alcohol Extraction, reuse of ceramic membranes and bifurcation techniques, and deuteration, the removal of high molecular weight polysaccharides, and removal of protein and other shellfish to solve the situation that is likely to cause allergic reactions 110484 6 200920387 In order to achieve the above and other objects, the present invention provides a polysaccharide composition having an anticancer effect, comprising a mushroom polysaccharide body and a carrier. The mushroom polysaccharide may be selected from all edible mushroom and medicine. The polysaccharide of the mushroom genus. The mushroom includes, by way of example and not limitation, Schizophyllum (6〇, Brazilian mushroom (aWcs), Cordyceps sinensis (6b/·sinensis), 1 foot {Ganoderma lucidwn), pen branch { Coriolus versicolor), fill { Anthodia camphorate), mulberry { Phe111nus 1inteus, P1 eurotus ci trinopi 1 eatus, Lent inula edodes, Agrocybe sp. ^ ^ ( Agrocybe aegeri ta) ' #头,兹i Hericium erinaceus )% 观兹{ Pleurotus eryngiig ), Sparrasis crispa ^ $ (Auricularia auricula ), if M ( Flammulina Fe/") or a combination of the above . A preferred embodiment of the composition of the present invention comprises a polysaccharide body extracted from various mushroom mites. A more preferred embodiment of the composition of the present invention comprises: 20-35% of polysaccharides extracted from Schizophyllum and Ganoderma lucidum; 25-45% of polysaccharides extracted from Cordyceps sinensis, Antrodia camphorata, Yunzhi and Brassica chinensis; 20-35% is extracted from the polysaccharides of mulberry yellow, coral mushroom, shiitake mushroom, willow mushroom, monkey mushroom, eryngii mushroom, petal velvet, wood velvet and enoki mushroom. According to the present invention, the mushroom polysaccharide composition can be administered to a subject in need thereof by injection or orally. In the examples, the composition may be used alone or in combination with other drugs. 7 110484 200920387 According to the present invention, the carrier of the mushroom genus polysaccharide composition can be liquid, solid or semi-solid and can be a pharmaceutically acceptable carrier. In the preferred embodiment, the carrier is water and more preferably re-steamed water. According to the present invention, the polysaccharide composition having the anticancer effect I-fruit can be used for treating or preventing cancer. According to the present invention, the oyster polysaccharide polysaccharide touch-up group has the effect of enhancing immunological activity, including promoting sputum phagocytic cells, tumbling activity and natural killer cell cytotoxic activity. According to the present invention, the mushroom glutinous polysaccharide has an effect of promoting the production of cytokines, including TB, TNF-a and/or interferon. (INF-). The invention further provides a method for preparing a mushroom scorpion multi-salted & ± sage, comprising: (a) fermenting the mycelium of the mushroom mycelium to obtain the mushroom; the night, (8) the mushroom obtained by the step (4) The sputum solution is homogenized and allowed to settle to collect the supernatant, (6) the supernatant obtained in the step (b) is precipitated with alcohol and collected, and (d) the precipitate is dissolved in water to form an aqueous solution, and (e) The aqueous solution was dialyzed with a ceramic membrane and a separation membrane system to obtain a mushroom polysaccharide. The obtained mushroom mash polysaccharide can be applied to the composition of the present invention. According to the present invention, the mushroom used in the above method may be selected from all edible mushroom mites and medical mushroom mites, including, by way of example and not limitation, Schizophy 11 um commue, Agarics blaze. , Cordyceps sinensis, iGanoderma lucidum, Corjolus versicolor, ^nthodia camphorate, Phel 1 inus 1 inteus, singapore 8 110484 200920387 (Pleurotus citrinopi1eatus),香兹{Lentinula ei/oi/es·), 田头名I genus (sp.) 士口才卯松菇(aegeri ta ) 'Hericium erinaceus ' PJeurotus eryngiig H petals true i Sparrasis Crispa), yjv ¥ (Auricularia auricula ) 'Golden gold (·· (Flammulina or a combination of the above garlic stalks. According to the present invention, before the preparation method, the oyster mushroom is produced in a liquid culture manner to produce a large amount of high molecular polysaccharides The body is then stirred by a homogenizer to destroy the mycelium of the mushroom, and the active ingredient contained in the mycelium is released to the culture solution, and the active ingredient is completely released by standing for a long time. In the embodiment, the culture broth is homogenized and allowed to stand for 18-24 hours. In a preferred embodiment, the static culture solution is first filtered through a sieve to remove large fragments of mycelium and then extracted with alcohol. The filtrate is used to precipitate the active substance of the mushroom (polysaccharide or a complex of the polysaccharide and the protein), wherein the alcohol is preferably equal to the filtrate (1:1), and is preferably used in an alcohol concentration of 95%. In a preferred embodiment, the ethanol-extracted precipitate is dissolved in water to obtain a multi-brewed aqueous solution. Preferably, the second distilled water is used. Next, the aqueous polysaccharide solution is purified using a ceramic membrane and a separation technique to extract high. Molecular polysaccharides and removal of impurities of small molecules and peptides, proteins and other substances that are susceptible to allergic reactions 'Let the filtrate leave only the purified mushroom 蕈 polymer polysaccharide extract 0 Because the 蕈 polysaccharide can withstand high temperature and high pressure, so The purified solution of 9 110484 200920387 can be autoclaved, and the loss of white matter and some organic substances can not only achieve the sterilization effect, but also promote the activity of the protein complex, =丨In order to avoid the allergic discomfort of the mushroom polysaccharide and the egg. It is not irritating to stimulate the immune system of the animal. In the preferred embodiment, the filter membrane is provided, and the obtained mushroom is squirmed. Equipment such as 〇·22 micron (ΑΠ1) dosage form. The dosage form can be formulated into a liquid state by using a five-segment sucrose body-stretching liquid according to the present invention;: a second-axis: a shaft body composition. Mushroom bacillus liquid, or two kinds of two "square + method of mushroom bacillus liquid can be a single one can use this preparation method is a mixture of sputum sputum liquid. Each mushroom sputum liquid is also combined. After the molecular polysaccharides are mixed, the embodiments are described below. The specific embodiments of the present invention will be explained by the specific embodiments of the present invention. I. Screening and cultivation of mushroom sputum species The mushroom strains that can be used in this month, such as Schizophyllum (Blood " (3) face), Brazilian mushroom (Maze), Cordyceps sinensis

Cordyceps s 1 nens 1 s), 1 {Ganoderma lucidunf), cloud t ICoriolus versicolor, AntJhodia camphorate, i Phellinus linteus, PI euro t us citrinopi leatus, Cowes { Lentinula Wa), genus (in sp.) such as pine mushroom (in rocyk aegerita, rubbing 'acne l Hericium erinaceus), 奄Baz ίο 110484 200920387 sis crispa), (Flammulina iPleurotus eryngiig·), anti-job True {Sparra i Auricularia aurjcula, gold fraction Fe/z/hpa), etc. 'but not limited to the above strains. The strain was inoculated into YM medium (YM medium containing 〇3% (w/w) yeast extract, 0.3% malt extract '5% egg (four) (Qing t (f) e), 1.0% glucose, 1.5% agar ), on a 28 χ: culture in an incubator for 5-7 days 'the mycelium is overgrown with YM solid medium. The mycelium grown on the YM agar medium was cut into small pieces and placed in a 2 ml ml of YM medium, and cultured in a constant temperature incubator for several weeks as a liquid fermentation strain. ~ Inoculate the cultured strain to a medium containing 8 ml of glucose (containing 4% glucose and 0.5% yeast extract in a liter liquid fermentation tank, and after 30 days of incubation at room temperature, metabolites are produced, Acid fermentation: The obtained culture liquid contains polysaccharides, proteins, peptides, labels, various amino acids, bioassays, g-types and organic acids. 2. Separation and purification of mushroom polysaccharides ' Collect the above-mentioned acid fermentation broth and stir it through a homogenizer to fully mix the metabolites of the sputum mycelium, and let the 〗 δ δ w '-" scale 18~24 蚪. After the lysate is precipitated, , 200 mesh and 4 mesh screen filtration 'Removal of large fragments of bacteria and 'limb limbs to obtain a filtrate. Add an equal amount of 95% alcohol to the sputum, and let stand 4: Wait for the crystal material to sink. Collect the sink with a mesh screen and dissolve the sink (4) back to the original volume (peptide product of the above filtrate) with sterile water to obtain an aqueous solution. 110484 11 200920387 Then, using ceramic membrane and separation membrane system l (as shown in Figure 1) Dialysis of the above aqueous solution for extraction A variety of molecular weights of the 簟 簟 polymer multi-breast. The separation system uses the internal circulation of the pipeline principle, so that the sterile aqueous solution containing the mushroom scorpion polysaccharide continuously circulates in the ceramic filtration membrane column 11, small molecules (such as proteins, peptides , triterpenoids, a variety of amino acids, alkaloids, esters and organic acids, etc.) thus permeate out of the pipeline to achieve the purpose of removing small molecules, collecting the bottle 12 to collect the mushroom polysaccharide solution. Finally add the sterile Water, so that the mushroom polysaccharide solution has the volume of the original filtrate. Since the mushroom polysaccharide can withstand high temperature and high pressure, the filtrate can be sterilized by autoclaving, and the protein and some organic substances are deactivated, thereby stimulating the animal. The body's immune system produces allergic discomfort. By using a 0.22 micron (μm) filter membrane, the aggregated, inactive protein and peptides remaining in the mushroom polysaccharide polysaccharide solution after sterilization are sterilized. Filtration and removal. Then, using a sterilized dispenser in a sterile room, the mushroom polysaccharide high molecular polysaccharide solution is dispensed and prepared into a liquid dosage form, that is, The mushroom 蕈 polysaccharide composition of the present invention. The mushroom glutinous polysaccharide composition of the present invention may comprise a polysaccharide extracted from one or more mushroom mites. When the composition of the present invention comprises two or more saccharomyces polysaccharides, the mushroom 蕈The culture solution may be separately prepared as a polysaccharide composition and then mixed; or the mushroom mash culture solution may be mixed first to prepare the polysaccharide composition. The composition obtained according to the embodiment includes about 20-35% extracted from the cleavage. Polysaccharide of bacteria and Ganoderma lucidum; about 25-45% polysaccharide extracted from Cordyceps sinensis, Antrodia camphorata, Yunzhi and Brazil mushroom; and about 20-35% extracted from mulberry 12 110484 200920387 The multi-body composition obtained in this example The ratio of the total amount of polysaccharides in the sucrose body of the aunt is as follows: = 16% for bacteria, 16% for Ganoderma lucidum, 14% for Cordyceps sinensis, 8% for rods, 'Z, 8% for Yunzhi, and 6 for Brazilian mushrooms. %, mulberry yellow: butterfly: 3%, garlic 3%, lycopene 3%: AO garlic 3%, petal velvet 3%, wood velvet 3% and gold: The above composition is divided into oral and Two groups of injections, one or two: =. In the case of the injection, the second method (Phen. sulfuric acidmeth〇d) was measured ± 0. 002%. ~. Discussion on the effect of mushroom polysaccharide on phagocytic activity The following examples use five-week-old (10) strain male mice (animal source: Taida Hospital Animal Breeding and Research Center), raised at temperature U soil 2C , humidity 65 ± 5%, light and dark for 12 hours (9): L = i2 · · 12). The experimental animals were subjected to the experiment of polysaccharides after a week of adaptation. The oral group was given a stomach sputum with 1 liter of the above-mentioned polysaccharide composition, and the intraperitoneal injection group was intraperitoneally injected with 5 ml of mushrooms per day. In the control group, the SI body needle #1 was used, and the control group was replaced by the linonic acid buffer. Food and drinking water are supplied normally, and the condition of the mice is regularly checked according to the animal house management method to minimize the environmental pressure and human injury suffered by Xiaobai. 110484 ]3 200920387 The experimental results of the following examples are expressed as mean soil standard deviation (Mean ± SD), and statistical analysis using SAS (Statisticai)

System, SAS InStltute Inc., USA) software for one-way variable analysis (One way A_A) and differential analysis between groups according to Duncan, s new multlple range (4), if between groups Differences of P < 0.05 were considered significant differences. Analysis of swallow cell activity: blood samples for swallow cell activity assay are less than U00~150 microliters (")), and need to be thoroughly mixed with anticoagulant (hepatin), which can be repeated due to consideration Sampling and avoiding sacrifice, the eye socket blood collection method is used twice a week. The method is as follows: after anesthesia, the sides of the neck of the mouse are pressed to block the venous return, so that the eyeball is fully externalized and the venous plexus of the eyelid is congested. At the front of the eye or at the back of the eye, a capillary tube with proper control and an anticoagulant on the inner wall is inserted into the eye from the periphery of the eyeball. The capillary is rotated to cut the venous plexus, and the blood is sucked into the capillary by the principle of a siphon, and then the blood sample is taken. Move to a microcentrifuge tube with anticoagulant to 'mix thoroughly and place on ice for later use. Analysis of phagocytic activity in blood: use phag〇_test kit (10) PEGEM, Ph followed, FRG), with single nuclear sphere The engulfing of E. coli (1)TC-calibrated h〇// with the globular ball was analyzed, and the swallowing ability was analyzed by flow cytometry. The experimental procedure was as follows: 1. Take 1 〇〇 microliter of heparin-containing And Shake and mix (v〇rtexmi-like whole blood to the appropriate 5 ml test tube, pay attention to the tube wall can not do two tubes per test body (one tube is the test tube, one tube is the know 'official), and then the ice Bath 1 〇 minutes. 110484 14 200920387 2. Add 20 μl of shake-mixed target cells (FITC labeled 昃 (3) hearts) to each tube. Place the test tube in water at 37 ° 〇 preheated h Valley U The effect of the gluten is 分钟Q minutes; the negative control group is left in the ice bath for 〇 minutes. The time and temperature should be accurately controlled, and the water bath should be preheated and capped. 3. Place the test tube on ice to stop phagocytosis, and Add 100 μl of ice-cold quenching liquid (quenching s〇luti〇n) (trypan blue) to each tube, shake and mix for 2 minutes to eliminate target cells that were not swallowed by the cells. Fluorescence interference. The principle is that the absorption spectrum of trypan blue overlaps with the divergence spectrum of FITC' and the cell membrane of phagocytic cells is intact, so it can avoid being stained by trypan blue, while protecting the swallowed E. coli from being stained. The phagocytic E. coli is stained by trypan blue and cannot be illuminated. Sahlin et al., 1983) 4. Wash the cells with 3 ml of rinsing solution and centrifuge at 25 rpm for 5 minutes to carefully remove the supernatant to avoid cell clumps and repeat the washing step once. · Add 2 ml of lysing so ut ion to each tube, dissolve and fix the blood, mix well, and apply for 20 minutes at room temperature. Centrifuge at 2 50 xg for 5 minutes at 4 C, aspirate the supernatant. 6. Night. Add '200 μl of DNA staining solution (staining so 1 ut ion) to each tube, mix it with ice and protect it from light for 10 minutes to dye white blood cells. 7. Use flow cytometry to analyze the swallowing ability of pellets and mononuclear spheres. The experimental results are shown in Figures 2 through 7. 110484 15 200920387 Swallowing activity is expressed as a percentage of cells that are swallowed by witchcraft (%). • Proportion of early nuclear or granules. After the oral administration of the agglutinated polysaccharides, the sauce was dried, and the d η Gole 2 was not. The j ^ high (32·93%) of the test group on the 5th day was the control group (13. , ~ 'has a difference (ρ &lt; 0. 05), and the previous time point (the second ratio increased by 53. 99%; the experimental group's phagocytic activity decreased on the 9th day (2 3. 6 8 %)» 0π λ , , ^彳—&quot; There are still significant differences in the 'day, ?, and group (Ρ &lt; 0.05), the experimental group, the day of swallowing, the tongue (17.2%) is still higher than the control group However, the phagocytic activity of the experimental group on the 16th, 19th, and 23rd days showed a % sputum f, and was significantly different from the control group (Ρ < 0.05). Overall, oral sputum polysaccharides The cell swallowing activity can be rapidly and significantly improved, and a significant difference can be obtained after one week of oral administration. ... The results of the injection of the mushroom polysaccharide by the intracavity are shown in Fig. 3, and the measured values of the experimental group after the 9th day are The control group had a significant difference (Ρ&lt;〇. 05), and the peak of the swallowing activity of the test group appeared on the second day (28. 〇 2%), which was 2.76 times that of the control group (10. 16%). Compare the results of oral and decavitating &gt; main shots, The effect of the polysaccharides of the mushroom mites was significantly different from that of the injected polysaccharides of the mushroom (p&lt;0.05, the oral group was reached on the 5th day, and the intraperitoneal injection group was reached on the 9th day), and the highest value of the phagocytic activity was also observed in the oral administration. The group (32.93%) was higher than the intraperitoneal injection group ( 28.02%). The average fluorescence intensity (FI) of the swallowed cells represents the amount of phlegm per cell. The results of oral administration of the polysaccharides of the mushroom are as shown in Fig. 4. The average fluorescence intensity of the granule cells in the experimental group showed a gradual increase trend, and the i 2nd day (7.76) began to be significantly different from the control group (p&lt;〇〇5), and gradually became stable after 16 16 ] 10484 200920387 days. The highest value appears on the 16th day (before the third day (4.06) compared to the promotion ·)., the #c door is 2·44 hearts. The single-core ball of the firefly:: brother 6 shows the oral benefit The experiment of 蕈 polysaccharides and the peak on the 12th day (5.71) was the measured value of the control group at the same time / later to the end of the experiment. The experimental, heart, 12-day lag interval was significantly different. The above results: true I believe that (4) the amount of granulosa cells and mononuclear cells swallowing foreign matter. In the second image, the intraperitoneal injection of probiotic polysaccharide mice showed a significant mean value = the light intensity of the 'measured light intensity' was measured on the second day of the injection on the 5th day to 2 == 3.8, but did not reach significant difference; significant difference (2. Both the control group and the control group have π, , and the test group in the 0th day (2. 65) to the 2nd group of the second group: έ 'increased 7 2. 25 times' after the 9th day蕈 polysaccharides, Λ: and above. Figure 7 shows the average fluorescence intensity of the nucleus cells of the intraperitoneal injection cells, and the measured values of the granule-and::::test group after the 5th day are equal and =, different (ρ &lt; 〇〇 5), also in the first... : The most significant increase in boarding power, an increase of 2.97 times. The above results can be (iv) (iv) gambling and mononuclear effects. This force, and, in the week after the main shot, the significant activity of the active polysaccharide can be significantly improved - the effect of the cell sucrose on the activity of natural killer cells] 10484 17 200920387 Natural killer cells play a role in cellular immunity An important role, its increased activity can directly help remove pathological and cancerous cells in the body. This experiment was carried out using YAC-1 cells sensitive to natural killer cells, i.e., the mortality of YAC-1 cells was indicative of the cytotoxic activity of natural killer cells. Organ collection: After sterilizing the ventral surface of the experimental mice with alcohol, cut the abdominal skin, remove the spleen, liver and kidneys, wash the liver and kidney with PBS, blot dry and collect the weight and calculate the relative weight of the organs. (relative organ weight).丨, relative weight (%) = [organ weight (g) / weight (g) } χ 1 〇〇%. Spleen lymphocyte collection: The spleen was placed in a 20 mm (mm) petri dish containing appropriate amount of RPMI 1640 medium (Hy cl one), the spleen was shredded with a lock, and centrifuged at 1 rpm with Ficol Bu Hypaque 3 After 〇 minutes, the culture medium and the pale yellow liquid between Fic〇l and Hypaque were taken up, and this layer was the mouse spleen lymphocyte. Culture of YAC-1 cells · γAC-1 cells were cultured in an amount of 2 millimoles (mM) of L-glutamine and adjusted to contain 1.5 g/L sodium carbonate, 1 〇 mM HEPES 1. OmM sodium pyruvate and 90% RPMI 1640 medium containing 1%% calf serum were subcultured every 2 to 3 days. Subculture is directly diluted with fresh medium and then dispensed into new culture flasks.

Cytotoxic activity test: modified according to the method of Kiessling et al. (Kiessling et al., 1975) ’ using LIVE/DEAD

The Cell-Mediated Cytotoxicity kit (M〇lecuiar Probes), the experimental procedure is as follows: 110484 18 200920387 The YAC-1 cell concentration was adjusted to 1×10 cells/ml (cel 1/ml) with PBS. Add 10 // L of D10C18 per ml of YAC-1 cell suspension, add DIOCw to the bottom of a 15 ml centrifuge tube, add YAC-1 cell suspension and mix well. Incubate in a 37 ° C incubator for 20 minutes. After rinsing twice with PBS, the cells were dissolved in the medium and the cell concentration was adjusted to lxl06 cell/ml, and placed in a 37 ° C incubator to maintain a cell viability of 95% or more. Mouse spleen lymphocytes were adjusted to a cell suspension of 2 x 106 cells/ml in RPMI 1 640 medium (Hyclone). Twenty microliters of the stained YAC-1 cells were added to the spleen lymphocyte suspension in 200 μl, and stained with 220 μl of PI solution (4 μg/ml), and centrifuged at 10 μg for 30 seconds. And then at 37. (The culture was carried out for two hours in an incubator. The spleen lymphocyte activity was calculated by flow cytometry, the number of YAC-1 cells in the green edge light was calculated, and how many cells in the green cells were destroyed by the lymphocytes, so that the cell membrane was incomplete. The cells with reddish green cells; the spleen lymphocyte cytotoxic activity is calculated as: the number of cells with green and red fluorescence / the number of green fluorescent cells. The cytotoxic activity of natural killer cells in mice is as follows Figure and Figure 9. The results of the two oral administration of polysaccharides are shown in Figure 8, oral pRs.

2 迥uo. 〇), , 3rd week (17. 96%) and 4th week (17 毋 right 'tongue is significantly different from the control group (P&lt;0.05). • 76 times, and 17% The cells were injected intraperitoneally]10484 19 200920387 The results of the polysaccharides of the mushroom scorpion were as shown in Fig. 9. The experimental group had the largest change in the week (7.7%) to the first week (17.75%). Increased 13〇·“%, 2nd week (22.99%), 3rd week (30·3%) and 4th week (29.91%) were significantly different from the control group injected with PBS (p&lt;〇 _ 〇 5 ). The experimental results confirmed that the rumen polysaccharide of the present invention can promote the cytotoxic activity of natural killer cells in vivo by either oral or intraperitoneal injection. Analysis of the influence of cytokine content The blood volume for cytokine (cyt〇kine) analysis requires about j ml, and the blood sample is collected by cardiac blood sampling. The method is as follows: after the anesthesia of the mouse, from the 1/3 of the back end of the chest cavity The left side of the sternum is inserted vertically into the ribs to reach the heart. After seeing the blood in the syringe, fix the direction and depth of the needle with one hand. The obtained blood sample was allowed to stand for serum deposition, and the serum was collected in a centrifuge tube at 12000 xg for ~20 minutes for cytokine content determination. Cytokine content analysis #素连#immunoassay EUSA), using ELISA kit The group (Bi〇_ce) was used to measure the content of cytokine (IFNm) in the serum. The detailed experimental procedure was as follows. Add the culture buffer (Incuibaticm Buffer) (5 〇 microliter/hole) to the well plate, blank detail 丨丨T, ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,

Conjugate) (50 microwells/,,,,, open/holes), blank groups are not added, and the average sentence is mixed, at 37. (: culture 1 s, n ±, • 5 small %. Wash 4 times. Add streptavidin 110484 20 200920387 Streptavidin-HRP Working Solution (10 0 μl / hole) 'blank group No addition; culture at room temperature for 3 〇 minutes. Wash 4 times. Add stable chromogen (stabi丨丨zed Chr〇丨n〇gen) (100 μl/hole) and incubate at room temperature in the dark for 2〇 Minutes. Add Stop Solution (1 〇〇 microliter/hole), mix evenly, measure the absorbance using a spectrophotometer (UltrospeclOO, PHARMACIA BIOTECH) (λ = 450nm), and the values must be deducted only for stability. The value of the blank group of chromogen and 彳τ stop solution. After adding the stop solution, the absorbance should be measured within 2 hours f. The absorbance and cytokine concentration are plotted using TNF-α and IFN-r standards and their absorbance values. The standard curve can be used to calculate the concentration of cytokines in the sample to be tested. The measurement results of TNF-α content are shown in Fig. 1 and Fig. 11. - Results of oral ruminant polysaccharides are shown in Fig. 1 In the experimental group, the average value of each sampling point is higher than that of the control. In the experimental group, the measured value reached the highest (28·8 pg/ml), the second week (27.4 pg/ml) and the third week (26.2 pg/ml) TNF-〇: the concentration decreased. By the fourth week, the concentration rose (27 pg/ml), and the second week (24·4 pg/ml) increased to f at the first week, which was 18.03%. The first, second, and fourth weeks, oral ruminant polysaccharide The experimental group of the body was significantly different from the control group of oral PBS (p &lt; 〇〇 5). The results of intraperitoneal injection of mushroom polysaccharides were as shown in Fig. 9, and the experimental group had a peak at 2 weeks (27.3 pg). /ml), the maximum increase rate occurs between =^ weeks and the second week (8%). The measured values of the 2nd week and the 4th week are significantly different between the experimental group and the control group (P&lt;〇. 〇5) 21 110484 200920387 The measurement results of IFN-7 content are shown in f 12 and f 13 . The results of η generation polysaccharides are shown in Fig. 12, and the experimental group has a relatively high point in the second and second weeks, * The IFN-r at week 4 was taken as: (28·1 pg/ml), and the results of the experimental group were significantly different in the i-week (obviously 2 7::: compared with the 4th week, the results of the control group were significantly different) <0. 05). (4) Cavity injection As shown in Fig. 13, the results of the polysaccharides in the experimental group gradually increased. In the third week, the concentration reached the highest concentration. 3 ί 二 Η, and decreased in 4 weeks, the maximum increase rate appeared in the 0th week. Between the brothers and sisters, 7.88%·'s measurements on the 2nd, 3rd, and 4th weeks were significantly different between the experimental group and the control group (ρ&lt;〇.〇5). The experimental results confirmed that the polysaccharide of the present invention can promote the increase of cytokine content in the living body whether it is administered by oral or intraperitoneal injection. Sixth, the anti-cancer effect of the mushroom 蕈 polysaccharide body 1 _ 覃 覃 polysaccharide tumor inhibition test by subcutaneous injection of lxl 〇 V5 〇 microliters of Lewis lung cancer cells (Lewls lung carcin (10) a ceUs) into c57BL/6jNarl small On the back of the mouse. After the mouse was colonized for 24 hours, the mushroom sputum polysaccharide was administered. The oral group was intragastrically administered with 1 ml of gastric tube per day, and the intraperitoneal injection group was intraperitoneally injected with 0.05 ml of polysaccharide injection per day. When the experimental mice were sacrificed, the tumor weight was taken and the tumor inhibition rate was calculated. ^ The viability of the mice before sacrifice was best in the oral polysaccharide group, followed by the intraperitoneal injection group, and the control group was the worst. Observing the appearance of the tumor, the tumor in the control group was the largest, 110484 22 200920387. The intraperitoneal injection group was the second, both tumors formed obvious ellipsoids in the f part of the mouse; the tumor in the oral polysaccharide group was significantly better than the control group. The intraluminal injection group is small, forming only a round lumps at the colony. During anatomy, it was found that there were obvious blood and pus accumulation around the tumor in the control group, and the tumor infiltrated into the muscle tissue, which needs to be separated by scissors; the oral polysaccharide: the tumor is - solid ^ block, only skin and There was no infiltration into the muscles and no significant blood or pus accumulation was observed. The results of the weight-removing tumors in each group were as follows: the tumors in the control group were the heaviest (〇.46g), the intra-abdominal injection of the polysaccharide group (〇.36g), and the oral polysaccharide group (lightest (0.27g)). The oral polysaccharide group and the control group reached a significant difference (P &lt; 0.05). The tumor weight of each group was compared with the control group to calculate the tumor inhibition rate, which was 40.58% in the oral polysaccharide group and 21.01% in the intraperitoneal injection polysaccharide group. The inhibitory effect of the polysaccharide composition of the oral group on the tumor was 40. 58 21. 01 Tumor weight (g) 〇6±~〇7〇5~ 〇. 27+ 〇. 〇1 〇. 36± 〇. 〇9 2. Tumor transfer inhibition test of mushroom 蕈 polysaccharides The Lewis lung cancer cells were implanted into the hind legs of mice of C57BL/6 strain. After seven days, the cancer cells in the legs will proliferate and MA (original cancer); From the last three days, the polysaccharide composition of the mushroom mites was fed twice a day. Each group was treated with cobalt 60 for 7 days after the cancer cells were injected for 5 days, and after each group of mice was observed 2 weeks after the treatment. Leg tumors, and sacrificed small ~ gas to 110484 23 200920387 to observe the tumor particles in the lungs. The results of the experiment are shown in Table 2. The mice in the control group (no polysaccharide composition, no radiation treatment) had tumor metastasis 100%, and the average number of lung tumor particles was 12.3; no polysaccharide composition was taken. But the mice that are treated with radiation, then! Tumor metastasis occurs in the sputum, the number of lung tumors is 13.7; the mice taking the polysaccharide composition but not undergoing radiation...the tumor metastasis rate is reduced and the number of lung tumors is also reduced; taking (four) body composition, and In the case of radiotherapy, the tumor metastasis rate was significantly reduced to 12.5%, while the number of lung tumors was reduced to U5. 110484 24 200920387 Control group (not taking composition, not emitting (not taking composition, taking radiation therapy) (taking composition, no radiation therapy) (taking composition for radiation therapy)

Number of lung tumors Table 2, polysaccharides and tumors for tumor metastasis such as iTail^ ~T-7Γ7;----Inhibition effect transfer ratio The above results confirmed that the mushroom 蕈 polysaccharides of the present invention or injection method have anticancer The effect: the adult reduces the tumor metastasis by mouth. In addition, the use of this tumor growth and (such as: radiation therapy or #物治疗), can make the disease control method j more effective. The above examples are merely illustrative of the compositions and preparations of the present invention and are not intended to limit the invention. Any person skilled in the art can recite the above embodiments in accordance with (4) and the specifications of the present invention. The patent application is as follows. [Simplified illustration of the drawings] Fig. 1 illustrates the use of the present invention for purifying a mushroom knot and a separation membrane system. "匕...The medicinal scorpion of the saccharide body, Fig. 2 illustrates the small activity of the ruminant granule cells of the present invention to feed the ruminant polysaccharide of the present invention. The experimental group is the daily oral administration of the present invention 110484 25 200920387 &gt; Polysaccharide (Μ liter, control group is daily oral sulphate buffer (PBS) Gl ml. * indicates that the experimental group and the control group have significant difference, Ρ &lt; 〇 · 05 (AN0VA)., Figure 3 The mice were injected intraperitoneally with the μ 苇 polysaccharide of the present invention, and the granule cells were swallowed. The experimental group was intraperitoneally injected with (4) polysaccharide injections of 0.05 ml per day, and the control group was intraperitoneally injected with squaric acid buffer per sputum. (PBS) G. G5 $ liter. * indicates that the experimental group is significantly different from the control group, P &lt; 〇.05 (AN0VA). ί : Figure 4 illustrates the oral administration of the ruminant polysaccharide of the present invention, which is ^ The phagocytic index of the granule cells was expressed by the average fluorescence intensity (fi). The experimental group was orally administered with the rumen of the rumen polysaccharide of the present invention per day, and the control group was the daily oral (tetra) acid buffer (10) ML. * indicates the experimental group Significantly different from the control group, P &lt; 〇〇 5 (an 〇 Va). Figure 5 is a description After intraperitoneal injection of the mushroom sputum polysaccharide of the present invention, the phagocytic index of the granule cells was expressed as the average camp light intensity (7), and the L experimental group was intraperitoneally injected with the sputum sputum polysaccharide injection 〇. 〇 5 ml, Second, and for the mother's day intraperitoneal injection of sulphate buffer () 〇. ml. 氺 indicates that the group and the control group are significantly different 'p &lt; 〇〇 5 (10) map. ^ The first diagram illustrates oral feeding of the present invention The anthraquinone index of the small nucleus mononuclear cells of the mushroom polysaccharide, the average fluorescence intensity (F1) indicates that the shell test group is daily oral administration of the mushroom 蕈 polysaccharide 丨 ml, and the control group is daily oral phosphate buffer. Liquid (PBS) Ol ml. * indicates that the experimental group is significantly different from the control group, p &lt; 〇 · 〇 5 (an〇va). Fig. 7 is a graph showing the phagocytic index of mononuclear cells of a small 110484 26 200920387 mouse injected intraperitoneally with the polysaccharide of the mushroom of the present invention, expressed as mean fluorescence intensity (FI). The experimental group was intraperitoneally injected with 0.05 mg of sputum sputum polysaccharide injection, and the control group was intraperitoneally injected with phosphate buffer (PBS) 0.05 ml per day. * indicates that the experimental group is significantly different from the control group, p&lt;〇.〇5 (AN0VA). Fig. 8 is a view showing the toxicity of the spleen natural killer cells of the mice which were orally fed with the mushroom stalk polysaccharide of the present invention. The experimental group was 0.1 ml of the polysaccharide of the present invention, and the control group was 0.1 ml of phosphate buffer (PBS) per mouth. * indicates that the experimental group is significantly different from the control group, P &lt; 〇. 05 (AN0VA). Fig. 9 is a view showing the toxicity of the spleen natural killer cells of the mice which were peri-injected to the mushroom stalk polysaccharide of the present invention by intraperitoneal injection. The experimental group was injected intraperitoneally with a sputum sputum polysaccharide injection 0.55 ml, and the control group was intraperitoneally injected with phosphate buffer (PBS) O. 05 ml.氺 indicates that the experimental group is significantly different from the control group, Ρ &lt;0· 05 (AN0VA). Fig. 10 is a graph showing the TNF-α content (concentration pg/ml) in blood of mice which were orally fed with the mushroom stalk polysaccharide of the present invention. 1毫升。 The test group was orally administered with 0.1% of the oral phosphate buffer (PBS). * indicates that there is a significant difference between the experimental group and the control group, P &lt; 0.05 (AN0VA). Fig. 11 is a view showing the TNF-? content (concentration pg/ml) in the blood of each week of mice which were intraperitoneally injected with the polysaccharide of the mushroom genus of the present invention. The experimental group was intraperitoneally injected with 0.05 ml of rumen polysaccharide injection, and the control group was intraperitoneally injected with phosphate buffered saline (PBS) 0.05 ml. * indicates that the experimental group was significantly different from the control group, P &lt; 0.05 (AN0VA). 27 110484 200920387 Fig. 12 is a diagram showing the IFN-γ content (concentration pg/ml) in blood of mice peri-weekly fed with the mushroom scorpion polysaccharide of the present invention. 1毫升。 The test group was orally administered with 0.1% of the oral phosphate buffer (PBS). * indicates that there is a significant difference between the experimental group and the control group, P &lt; 0.05 (AN0VA). Fig. 13 is a view showing the IFN-7 content (concentration pg/ml) in the blood of each week of mice which were intraperitoneally injected with the polysaccharide of the mushroom genus of the present invention. The experimental group was daily intraperitoneal injection of mushroom sputum polysaccharide injection 0.55 ml, the control group was daily intraperitoneal injection of phosphate buffer (PBS) O. 05 ml. * indicates that the experimental group was significantly different from the control group, P &lt; 0.05 (AN0VA). 28 110484

Claims (1)

200920387 X. Patent application scope: 1. A multi-audio composition with anti-cancer effect, including: carrier; and mushroom polysaccharide, wherein the mushroom is selected from Schizophy 1 lum com/ Nue), Agarics blaze, winter watch l % l Cordyceps sinensis, H (Ganoderma lucidum), Coriolus versicolor, Anthodia camphorate, Phe 11 inus linteus, Shan Pleurotus ci trinopi leatus 'Lent inula edodes' ' 'Agrocyhe aegen· ta ), 'He Hericium erinaceus' ' Pleurotus eryngiig~ ' ' Petal Sparrasis crispa, , ^ l Auricularia auricula, Jinnuz { Flammulina velutipes, a combination of anti-reports. The composition according to claim 1, wherein the polysaccharide comprises 20 to 35% of polysaccharides extracted from Schizophyllum and Ganoderma lucidum; 25 to 45% is extracted from Cordyceps sinensis, Antrodia camphorata, and cloud Polysaccharide of Zhizhi and Brazilian mushrooms; and 20 to 35% of polysaccharides extracted from mulberry, coral, mushroom, willow mushroom, Hericium erinaceus, Pleurotus eryngii, petal velvet, wood velvet and enoki mushroom. 3. The composition described in item 1 of the patent application may be administered to a subject in need, either by injection or orally. 4. The composition of claim 1, wherein the carrier is 29 110484 200920387 5. 6. 7. 8. Liquid, solid or semi-solid. For example, the κ, and the composition described in the patent application section, wherein the carrier is water. A method for preparing a polysaccharide body of the first aspect of the invention is as follows: (a) fermenting the mycelium of the mushroom mycelium to obtain the mushroom liquid; (b) a) Knowing the mushroom sputum liquid homogenization and standing sedimentation 'to obtain the supernatant; (c) precipitating the supernatant with alcohol and collecting the sediment; (d) using water/gluten solution 5 Xuan Shen Temple, And obtaining an aqueous solution; and (e) dialyzing the aqueous solution with a ceramic membrane and a separation membrane system to obtain a mushroom polysaccharide. X. The method of claim 6, wherein the homogenization is allowed to stand for 18 to 24 hours. Night The method of claim 6, wherein the alcohol is an alcohol equivalent to the supernatant and having a concentration of 95%. Month total 10848 30