Kluyveromyces marxianus high-yield cell wall polysaccharide and application thereof in improvement of colitis

Abstract

The invention discloses kluyveromyces marxianus high-yield cell wall polysaccharide and application thereof in improvement of colitis, and belongs to the technical field of fungal polysaccharide. Experiments prove that the Kluyveromyces marxianus JSA16 separated from the traditional fermented yak milk has good adaptive capacity to environment and can produce cell wall polysaccharide at high yield. Through screening, when cane sugar is used as a carbon source, the sugar yield of the JSA16 is the highest and is 177.47 mg / g. Through analysis, the extracted cell wall polysaccharide is mainly composed of mannose and accounts for 88.1% of the total sugar content. Experiments prove that the polysaccharide can significantly improve the mouse colitis symptom induced by DSS, and has a wide application prospect. The invention provides a new method for clinical treatment of colitis.

Description

Technical Field

[0001] The present invention relates to the technical field of fungal polysaccharides, and particularly to a cell wall polysaccharide with high yield from Kluyveromyces marxianus and its application in improving colitis. Background Art

[0002] The yeast cell wall accounts for 20 - 25% of the cell dry weight. The yeast cell wall is mainly composed of glucan, mannan, protein and lipid. The main active components of yeast cell wall polysaccharides are α-mannan and β-glucan. α-Mannan and β-glucan can enhance the body's immune capacity, improve the body's intestinal environment, improve animal production performance, and identify, adhere to and eliminate pathogenic microorganisms, etc., reducing the damage of pathogenic bacteria to the body.

[0003] Kluyveromyces marxianus has become the focus of research in recent years as a biosafety yeast strain. Because it is a strain derived from fermented foods, it has characteristics such as high temperature resistance, rapid growth, gastric acid resistance, and bile salt resistance. It is not only a highly potential candidate strain for single-cell protein production, but also can metabolize and produce various enzymes, so it has broad application prospects in the food and feed industries.

[0004] Ulcerative colitis (UC) is a chronic non-specific intestinal inflammatory disease, with abdominal pain, tenesmus and hematochezia as the main clinical manifestations, and it is prone to relapse. The global prevalence and incidence of UC are increasing year by year, and its risk of canceration and death is higher than that of other colonic inflammatory diseases. In recent years, probiotics and their products have shown great potential in regulating the intestinal flora, preventing and treating intestinal inflammation. Probiotics can improve intestinal health through mechanisms such as competitive exclusion of pathogenic bacteria, enhancement of intestinal barrier function, and regulation of host immune response. However, the research on Kluyveromyces marxianus and its cell wall polysaccharides in the treatment of colitis is less. Therefore, the application research of Kluyveromyces marxianus cell wall polysaccharides in the treatment of colitis has important significance. Summary of the Invention

[0005] The purpose of the present invention is to provide a cell wall polysaccharide with high yield from Kluyveromyces marxianus and its application in improving colitis, so as to solve the problems existing in the above-mentioned prior art. The present invention discovers that Kluyveromyces marxianus JSA16 can produce cell wall polysaccharides with high yield. Through experiments, it is confirmed that this polysaccharide can significantly improve the symptoms of colitis in mice induced by DSS, and has broad application prospects. The present invention provides a new method for the clinical treatment of colitis.

[0006] To achieve the above purpose, the present invention provides the following solutions:

[0007] The present invention provides a cell wall polysaccharide with high yield from Kluyveromyces marxianus. The cell wall polysaccharide is obtained by breaking the cell wall of Kluyveromyces marxianus JSA16 after fermentation, extracting the crude polysaccharide solution with alkali, and then dialyzing the crude polysaccharide solution to retain metabolites with a molecular weight cut-off greater than 8 kDa.

[0008] The cell wall polysaccharide is composed of mannose, glucose, arabinose, galactose, and glucuronic acid.

[0009] The preservation number of Kluyveromyces marxianus JSA16 is CGMCC No. 26422.

[0010] The present invention also provides a method for preparing the cell wall polysaccharide, which includes the following steps:

[0011] (1) Fermenting and culturing Kluyveromyces marxianus JSA16 to obtain a fermentation broth, centrifuging to collect the thalli, and preparing a cell suspension.

[0012] (2) Breaking the cell wall of the cell suspension, centrifuging to collect the precipitate.

[0013] (3) Resuspending the precipitate, extracting with alkali, centrifuging to collect the supernatant, and obtaining a crude polysaccharide solution.

[0014] (4) Obtaining the cell wall polysaccharide by dialyzing the crude polysaccharide solution.

[0015] Optionally, the carbon source used for the fermentation culture includes mannose, sucrose, glucose, or fructose.

[0016] Preferably, the carbon source used for the fermentation culture is sucrose.

[0017] Optionally, the temperature for the alkali extraction is 100 °C and the time is 4 h.

[0018] Optionally, the dialysis is to retain metabolites with a molecular weight cut-off greater than 8 kDa.

[0019] The present invention also provides the application of the cell wall polysaccharide or Kluyveromyces marxianus JSA16 in the preparation of a drug for improving colitis. The preservation number of Kluyveromyces marxianus JSA16 is CGMCC No. 26422.

[0020] The present invention also provides a drug for improving colitis, which includes the cell wall polysaccharide or Kluyveromyces marxianus JSA16;

[0021] The preservation number of Kluyveromyces marxianus JSA16 is CGMCC No. 26422.

[0022] Optionally, the drug further includes pharmaceutically acceptable excipients.

[0023] The present invention discloses the following technical effects:

[0024] Through experimental verification, the present invention finds that Kluyveromyces marxianus JSA16 isolated from traditional fermented yak milk has good environmental adaptability and can produce high yields of cell wall polysaccharides. After screening, when sucrose is used as the carbon source, the sugar production of JSA16 is the highest, reaching 177.47 mg / g. Through analysis, the cell wall polysaccharides extracted by the present invention are mainly composed of mannose, accounting for 88.1% of the total sugar content. Through experiments, it is confirmed that this polysaccharide can significantly improve the symptoms of colitis induced by DSS in mice, and has broad application prospects. The present invention provides a new method for the clinical treatment of colitis. Brief Description of the Drawings

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0026] Figure 1 Comparison of the growth of each strain at different temperatures;

[0027] Figure 2 Comparison of the growth of each strain under the condition of pH 4.5;

[0028] Figure 3 Comparison of the sugar production of the dominant strains;

[0029] Figure 4 Comparison of the growth of JSA16 using different carbon sources as substrates;

[0030] Figure 5 Elution curve of the isolation and purification of JSA16 cell wall polysaccharides;

[0031] Figure 6 Standard curve of inflammatory factors; (a): IL-10; (b): IL-6; (c): IL-1β; (d): TNF-α;

[0032] Figure 7 Percentage of mice with different levels of bloody stools in each group;

[0033] Figure 8 Comparison of the immune indexes of mice in each group;

[0034] Figure 9 Comparison of the colon lengths of mice in each group;

[0035] Figure 10Comparison of inflammatory factors in each group of mice; (a): IL-10; (b): IL-1β; (c): IL-6; (d): TNF-α. Detailed implementation manners

[0036] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0037] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0038] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0039] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the specification of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0040] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0041] Example 1 Screening of yeast strains with good environmental adaptability

[0042] Eighteen yeast strains isolated from traditional yak milk in Qinghai, fermented pickles in Northeast China, and cellar mud for liquor brewing were respectively cultured in environments at 20°C, 37°C, and pH 4.5. The inoculation amount was 1%, and three replicate experiments were set up. After 24 hours, the absorbance of the bacterial solution was measured at 600 nm, and then excellent yeast strains that could well adapt to different environments were screened out.

[0043] The culture medium used for culturing at 20°C and 30°C is YPD medium: 20.0 g of glucose, 20.0 g of peptone, 10.0 g of yeast extract powder, and 1000 mL of distilled water. The culture medium used for culturing at pH 4.5 is YPD medium, and the pH is adjusted to 4.5.

[0044] Experimental results: Strains numbered JSA16, SLBY4, BF1, BF2, BF3, BF4, and BF5 have strong abilities in adapting to the environment. Among them, JSA16 was screened and isolated from traditional yak milk in Qinghai, SLBY4 was screened and isolated from Northeast fermented pickles, and BF1, BF2, BF3, BF4, and BF5 were screened and isolated from the cellar mud for Baijiu brewing ( Figure 1 and Figure 2 ).

[0045] Example 2 Screening of high-yield cell wall polysaccharide yeast strains

[0046] According to the screening results in Example 1, strains numbered JSA16, SLBY4, BF1, BF2, BF3, BF4, and BF5 were screened for the sugar content of cell wall polysaccharides.

[0047] The extraction method used is as follows: (1) Yeast is inoculated into the fermentation medium (YPD medium) at an inoculation amount of 1%, and fermented at 30°C and 220 rmp for 24 h. (2) The cultured fermentation broth is centrifuged at 5000 rmp for 10 min to collect the cells, and a cell suspension is prepared with distilled water at a solid-liquid ratio of 1:20. (3) The cell suspension is homogenized with a high-pressure homogenizer at 1000 bar for 10 min to break the cell wall, centrifuged at 4000 rmp for 20 min, the precipitate is collected, and washed three times with distilled water until the supernatant becomes clear. (4) The precipitate is resuspended with 40 times the volume of 2 M NaOH solution, and alkali-extracted at 100°C in a water bath for 4 h, centrifuged at 4000 rmp for 20 min, the supernatant is collected, and neutralized with HCl to pH 7.0 to obtain a crude polysaccharide solution. (5) The crude polysaccharide solution is dialyzed in running water for 3 d (the molecular weight cut-off of the dialysis bag is 8 - 14 kDa, and metabolites below 8 kDa are dialyzed out, leaving metabolites above 8 kDa), and the water is changed every 8 h to obtain a polysaccharide solution. (6) The phenol-sulfuric acid method is used to detect the sugar content of the dialysis solution, and the yeast strain with the highest sugar production is screened. (7) The polysaccharide solution is frozen at -80°C in a refrigerator 24 h in advance, and the sample is freeze-dried using a freeze dryer to obtain crude polysaccharide.

[0048] Experimental results: See Figure 3 The strain numbered JSA16 has the highest sugar production. This strain was isolated from yak yogurt and sequenced using primers ITS1 and ITS4. The sequencing results show that this yeast is Kluyveromyces marxianus.

[0049] The Kluyveromyces marxianus JSA16 was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on January 6, 2023, with the deposit number CGMCC No. 26422, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0050] Example 3 Utilization of Different Substrates by JSA16

[0051] Prepare an inorganic nitrogen source medium: 10 g of carbon source, 2.5 g of ammonium sulfate, 3 g of potassium phosphate, 0.25 g of potassium chloride, and 1000 mL of distilled water. Use the inorganic nitrogen medium without carbon source as the control group, and use the inorganic nitrogen media with six monosaccharides (mannose, arabinose, fructose, xylose, rhamnose, glucose), three oligosaccharides (sucrose, maltose, lactose), and starch as carbon sources as the experimental groups to check the utilization of different carbon sources as substrates by JSA16.

[0052] The experimental results are as Figure 4 shown. JSA16 can utilize mannose, sucrose, glucose, and fructose well, while its utilization ability for the remaining monosaccharides, oligosaccharides, and starch is poor.

[0053] Example 4 Screening of High-Yield Cell Wall Polysaccharide Substrates by JSA16

[0054] The medium used is: 65.0 g of carbon source, 20.0 g of peptone, 25.0 g of yeast extract powder, and 1000 mL of distilled water. By the utilization of substrates by JSA16, prepare media with different substrates as carbon sources to screen substrates with high sugar production. The extraction method used is the same as that in Example 2.

[0055] Experimental results: When sucrose is used as the carbon source, the sugar production of JSA16 is the highest. The results are shown in Table 1.

[0056] Table 1 Sugar Production of Different Carbon Sources

[0057]

[0058] Example 5 Ion Chromatographic Separation and Purification of Cell Wall Polysaccharides of JSA16

[0059] Fill the DEAE - agarose gel into the SepaFlash chromatographic column. After washing off the protective solution with 2 column volumes of distilled water, add a 10 mg / mL polysaccharide sample to the chromatographic column and let it stand for 10 min. After the chromatographic column is balanced, elute successively with distilled water, 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, and 1.0 M NaCl solutions at an elution rate of 1 mL / min. Collect the elution solution using centrifuge tubes, with a collection volume of 3 mL per tube. After collection, detect the polysaccharide content in each tube using the phenol - sulfuric acid method and plot the elution curve. Combine the single - peak components for subsequent characterization.

[0060] Experimental results: After DEAE - agarose gel column chromatography of the JSA16 cell wall polysaccharide, collect the eluate, and a total of 2 polysaccharide components are obtained, named MPS - 1 (eluted with distilled water) and MPS - 2 (eluted with 0.1 M NaCl). The elution curve is as Figure 5 shown. Among them, MPS - 1 is eluted by distilled water, that is, it is uncharged, and it is determined to be a neutral polysaccharide; MPS - 2 is eluted by 0.1 M NaCl solution, that is, it is negatively charged, and it is determined to be an acidic polysaccharide.

[0061] Example 6 Monosaccharide composition analysis of JSA16 cell wall polysaccharide

[0062] Use ion chromatography to analyze the monosaccharide composition of MPS. Place 2 mg of MPS and 4 mL of trifluoroacetic acid (2 M) in a container and hydrolyze at 100 °C for 5 h. After hydrolysis, dry the excess trifluoroacetic acid in an oven. Redissolve the residue in 4 mL of methanol (50%, v / v), dry it in an oven to let methanol carry away the residual trifluoroacetic acid, and repeat 3 times to obtain the polysaccharide residue. Then redissolve the residue in 5 mL of deionized water, filter it through a 0.22 - μm filter, and then analyze it using a Dionex ion chromatography ICS 5000+ instrument equipped with a Carbopac PA20 analytical column. Use fructose (Fru), fucose (Fuc), arabinose (Ara), glucose (Glu), galactose (Gal), mannose (Man), xylose (Xyl), rhamnose (Rha), galacturonic acid (Gal - UA), and glucuronic acid (Glu - UA) as standards and analyze under the same experimental conditions. Determine the constituent monosaccharides of MPS and measure the monosaccharide content by comparing the retention times with the standards.

[0063] Experimental results: After hydrolysis of the MPS - 1 and MPS - 2 components of the JSA16 cell wall polysaccharide with TFA, the monosaccharide composition is determined by ion chromatography. The molar ratios of the monosaccharides and uronic acids in the polysaccharide components are shown in Table 2. From Table 2, it can be seen that both MPS - 1 and MPS - 2 are mainly composed of mannose and a small amount of glucose.

[0064] Table 2 Monosaccharide composition

[0065]

[0066] Example 7 Effect of K. marxianus JSA16 cell wall polysaccharide on bloody stools in colitis mice

[0067] 1. Establishment of colitis animal model

[0068] Thirty 6-week-old C57BL / 6 mice were placed in an animal laboratory at 22 ± 2°C with a 12-h light / dark cycle. The mice had free access to water and food. After 7 days of adaptation, they were weighed and randomly divided into 3 groups of 10 mice each. The normal group (no control group, NC) was gavaged with 0.2 mL of normal saline daily. The model group (dextran sulfate sodium control group, DSS) was gavaged with 0.2 mL of normal saline daily and the modeling was started in the 3rd week of the experiment by replacing the drinking water with 2.0% DSS and continuing to feed for 7 days. The K. marxianus JSA16 group (JSA16) was gavaged with 0.2 mL of an aqueous solution of K. marxianus JSA16 cell wall polysaccharide (a polysaccharide solution not separated and purified by ion chromatography) daily. In the 3rd week of the experiment, the drinking water was replaced with 2.0% DSS and feeding was continued for 7 days. The feed and drinking water conditions were the same for each group. During the experiment, the body weights of the mice 7 days before and after modeling, the consumption of feed and drinking water were recorded, and fresh feed was replaced daily.

[0069] 2. Detection indexes related to colitis

[0070] (1) Observation of bloody stools: The bloody stools of the mice were recorded daily after modeling.

[0071] (2) Determination of immune index: After the mice were fasted but allowed to drink water for 12 h, they were sacrificed by cervical dislocation. The abdominal cavity of the mice was opened, the spleen tissue was cut off, the surface liquid was blotted dry, and the wet weight of the spleen was weighed with a ten-thousandth electronic balance and recorded. The ratio of spleen weight (mg) to mouse body weight (g) was the immune index of the mouse.

[0072] (3) Measurement of colon length: After dissection, the part from the cecum to the anus was cut off intact and straightened without applying external force, and the length from the lower end of the cecum to the upper end of the anus was measured as the colon length.

[0073] 3. Detection of inflammatory factor indexes

[0074] (1) Sample pretreatment: Blood samples were collected by enucleating the eyeballs of the live animals. The blood was allowed to clot at room temperature for 30 min and then centrifuged at 3000 r / min for 15 min at room temperature. The serum was aspirated for the test, and care was taken not to freeze-thaw the samples repeatedly.

[0075] (2) Draw the standard curves of inflammatory factors IL-6, IL-10, IL-1β, and TNF-α and measure the samples according to the ELISA kit instructions. The standard curves are shown in Figure 6 .

[0076] 4. Experimental results

[0077] 4.1 Effect of JSA16 cell wall polysaccharide on fecal bleeding in colitis mice

[0078] The symptoms of murine colitis are mainly manifested as ulcerative bleeding in the colon, and the blood adheres to the feces or flows out. As Figure 7 shown, the fecal bleeding grade of the model group was significantly higher than that of the normal group, indicating successful modeling. The fecal bleeding condition in the JSA16 group was improved compared with that in the model group, indicating that JSA16 cell wall polysaccharide can form a protection in the intestine and alleviate the symptoms of colitis.

[0079] 4.2 Effect of JSA16 cell wall polysaccharide on immune index in colitis mice

[0080] The immune index refers to the ratio of the spleen weight (mg) to the body weight (g) of the mouse. The occurrence of inflammation in the intestine will cause the swelling of the spleen, resulting in the immune index of the spleen being higher than the normal value. As Figure 8 shown, after modeling, the immune index in the DSS group was significantly increased compared with that in the NC group (P<0.0001), proving that DSS induction led to inflammation in the spleen of model mice. The immune index in the JSA16 group was significantly decreased compared with that in the model group (P<0.0001), indicating that JSA16 cell wall polysaccharide can relieve the inflammation in colitis mice.

[0081] 4.3 Effect of JSA16 cell wall polysaccharide on colon length in colitis mice

[0082] The shortening of the colon length is a significant pathological feature of ulcerative colitis; it is one of the main manifestations of successful modeling of the colitis model. As Figure 9 shown, after modeling, the colon length in the DSS group was significantly decreased compared with that in the NC group (P<0.01), indicating successful modeling in this experiment. Compared with the model group, the colon length in the solid-state fermentation preparation group was significantly increased (P<0.05). It shows that JSA16 cell wall polysaccharide can play a role in protecting the colon.

[0083] 4.4 Effect of JSA16 cell wall polysaccharide on inflammatory factors in colitis mice

[0084] IL-10 is an anti-inflammatory factor in the body, and its anti-inflammatory signal participates in maintaining intestinal homeostasis. As Figure 10 can be seen, compared with the normal group, the level of IL-10 in model group mice was significantly decreased (P<0.01); compared with the model group, the level of IL-10 in JSA16 group mice increased slightly, but there was no significant difference.

[0085] The massive release of IL-6, IL-1β, and TNF-α can cause inflammation, leading to an excessive immune response in the body, thereby causing tissue damage. Figure 10 It can be seen that compared with the normal group, the levels of IL-6, IL-1β, and TNF-α in the model group of mice increased. Compared with the model group, the contents of IL-1β (P<0.05), IL-6 (P<0.005), and TNF-α (P<0.0001) in the JSA16 group were significantly decreased. This indicates that Saccharomyces cerevisiae polysaccharide can relieve the symptoms of murine colitis by inhibiting pro-inflammatory factors.

[0086] Based on the above experimental results, the present invention discloses a cell wall polysaccharide with high yield from Kluyveromyces marxianus strain, its characterization and application in improving colitis. The Kluyveromyces marxianus JSA16 was isolated from yak yogurt, and this strain has good ability to adapt to different environments, with a polysaccharide yield of 177.47 mg / g. The polysaccharide extracted in the present invention consists of a neutral component and a weakly acidic component, and its monosaccharide composition is mainly mannose, accounting for 88.1% of the total sugar content. Moreover, this polysaccharide can improve the symptoms of bloody stool, increased spleen weight, and shortened colon length in murine colitis induced by DSS, and has broad application prospects.

[0087] The above embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A high-yield cell wall polysaccharide of Kluyveromyces marxianus, characterized in that: The cell wall polysaccharide is obtained by fermenting Kluyveromyces marxianus JSA16, breaking the cell wall and extracting with alkali to obtain a crude polysaccharide solution, and then dialyzing the crude polysaccharide solution to cut off metabolites with a molecular weight greater than 8 kDa; The cell wall polysaccharide is composed of mannose, glucose, arabinose, galactose and glucuronic acid; The deposit number of the Kluyveromyces marxianus JSA16 is CGMCC No.26422.

2. The method for preparing cell wall polysaccharide according to claim 1, characterized in that: The following steps are involved: (1) fermenting and culturing Kluyveromyces marxianus JSA16 to obtain a fermentation broth, collecting the bacterial cells after centrifugation, and preparing a bacterial suspension; (2) breaking the cell walls of the bacterial suspension and collecting the precipitate after centrifugation; (3) resuspending the precipitate, extracting with alkali, and collecting the supernatant after centrifugation to obtain a crude polysaccharide solution; (4) The crude polysaccharide solution is obtained by dialysis.

3. The preparation method according to claim 2, characterized in that: The carbon source used in the fermentation culture includes mannose, sucrose, glucose or fructose.

4. The preparation method according to claim 3, characterized in that: The carbon source used in the fermentation culture is sucrose.

5. The preparation method according to claim 2, characterized in that: The temperature of the alkali extraction is 100° C. and the time is 4 hours.

6. The preparation method according to claim 2, characterized in that: The dialysis is for the retention of metabolites with a molecular weight greater than 8 kDa.

7. Use of the cell wall polysaccharide or Kluyveromyces marxianus JSA16 according to claim 1 in the preparation of a medicament for improving colitis, characterized in that: The deposit number of the Kluyveromyces marxianus JSA16 is CGMCC No.26422.

8. A drug for improving colitis, characterized in that: The drug comprises the cell wall polysaccharide of claim 1 or Kluyveromyces marxianus JSA16; The deposit number of the Kluyveromyces marxianus JSA16 is CGMCC No.26422.

9. The drug according to claim 9, characterized in that The medicine also includes pharmaceutically acceptable excipients.

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