阅读说明：本技术 一种榆黄蘑多糖及其应用 (Pleurotus citrinopileatus polysaccharide and application thereof ) 是由 王�琦 苏玲 杨金涛 于 2021-09-29 设计创作，主要内容包括：本发明公开了一种榆黄蘑多糖及其应用,涉及天然产物开发利用领域。所述榆黄蘑多糖包括木糖、葡萄糖、半乳糖、葡萄糖醛酸、岩藻糖、果糖和阿拉伯糖。本发明还公开了上述榆黄蘑多糖在制备治疗癌症所致的肌肉减少症药物中的应用。本发明通过结肠癌恶病质小鼠肌肉衰减实验证实,榆黄蘑多糖可增加肌肉减少症小鼠的体重及脾重,恢复肌纤维形态、数量和密度,降低炎症反应及降低肌肉减少症标志性蛋白Atrogin-1和MuRF-1的表达,缓解肿瘤所致肌肉减少症状。本发明发现了榆黄蘑多糖的新作用,为榆黄蘑的开发利用以及治疗癌症所致的肌肉减少症提供了新的思路。(The invention discloses pleurotus citrinopileatus polysaccharide and application thereof, and relates to the field of development and utilization of natural products. The pleurotus citrinopileatus polysaccharide comprises xylose, glucose, galactose, glucuronic acid, fucose, fructose and arabinose. The invention also discloses application of the pleurotus citrinopileatus polysaccharide in preparing a medicament for treating sarcopenia caused by cancer. The colon cancer cachexia mouse muscle attenuation experiment proves that the pleurotus citrinopileatus polysaccharide can increase the weight and spleen weight of a sarcopenia mouse, recover the muscle fiber form, quantity and density, reduce inflammatory reaction, reduce the expression of sarcopenia marker proteins Atrogin-1 and MuRF-1 and relieve the sarcopenia symptoms caused by tumor. The invention discovers the new function of pleurotus citrinopileatus polysaccharide and provides a new idea for developing and utilizing pleurotus citrinopileatus and treating sarcopenia caused by cancer.)

1. Pleurotus citrinopileatus polysaccharide, which comprises xylose, glucose, galactose, glucuronic acid, fucose, fructose and arabinose.

2. The pleurotus citrinopileatus polysaccharide according to claim 1, wherein the xylose accounts for more than 50% of the pleurotus citrinopileatus polysaccharide in mol percentage.

3. The pleurotus citrinopileatus polysaccharide according to claim 1, wherein the glucose accounts for more than 15% of the pleurotus citrinopileatus polysaccharide by mole percent.

4. The pleurotus citrinopileatus polysaccharide according to claim 1, wherein the galactose accounts for more than 4% of the pleurotus citrinopileatus polysaccharide by mole percent.

5. The pleurotus citrinopileatus polysaccharide according to claim 1, wherein the glucuronic acid accounts for more than 1% of the pleurotus citrinopileatus polysaccharide in mole percent.

6. The pleurotus citrinopileatus polysaccharide according to claim 1, wherein fucose, fructose and arabinose respectively account for less than 3% of the pleurotus citrinopileatus polysaccharide in mol percentage.

7. Use of the pleurotus citrinopileatus polysaccharide as defined in any one of claims 1 to 6 in the manufacture of a medicament for the treatment of sarcopenia.

8. The use of claim 7, wherein the sarcopenia is sarcopenia due to cancer.

9. The use of claim 8, wherein the cancer is colon cancer, gastric cancer, lung cancer, liver cancer, pancreatic cancer, colorectal cancer, esophageal cancer, lymphatic cancer, or breast cancer.

10. A medicament for treating sarcopenia comprising the pleurotus citrinopileatus polysaccharide of any one of claims 1 to 6 and a pharmaceutically acceptable excipient.

Technical Field

The invention relates to the field of development and utilization of natural products, in particular to pleurotus citrinopileatus polysaccharide and application thereof.

Background

Sarcopenia occurs in 50% -80% of patients with tumors, which is mainly inflammatory reaction, involves the syndrome of multiple organs of the body, has various, destructive and irreversible effects on the body, seriously affects the life quality of the patients and even leads to death. In 2016, sarcopenia was classified as a degenerative disease by the world health organization together with a wide interest in neurological diseases such as alzheimer's disease and parkinson's disease. However, no effective drug for treating sarcopenia caused by tumor exists at present.

Pleurotus citrinopileatus (Pleurotus citrinopileatus), also known as Pleurotus citrinopileatus, belongs to the subdivision Basidiomycotina, class Hymenomycetes, order Agaricales, family Pleurotaceae, genus Pleurotus, and is naturally distributed in the northern area of the northern hemisphere temperate zone, and is also distributed in the northeast three provinces, Hebei, Hunan, Sichuan and Tibet areas of China. The pleurotus citrinopileatus sporocarp is delicious in taste, rich in nutrition, sweet in taste and warm in nature, has the effects of nourishing and strengthening body, moistening lung and promoting the production of body fluid, stopping dysentery and the like after entering spleen and lung channels, and has the biological activities of reducing blood fat, resisting oxidation, improving the immunity of the organism and the like. However, no research report on the biological activity, active ingredients and preparation method of the active ingredients for relieving sarcopenia caused by tumors is available.

Disclosure of Invention

The invention aims to provide pleurotus citrinopileatus polysaccharide and application thereof, which are used for solving the problems in the prior art and improving sarcopenia caused by tumor.

In order to achieve the purpose, the invention provides the following scheme:

according to one technical scheme of the invention, the pleurotus citrinopileatus polysaccharide comprises xylose, glucose, galactose, glucuronic acid, fucose, fructose and arabinose.

Further, the pleurotus citrinopileatus polysaccharide is beta-type furanose.

Further, the xylose accounts for more than 50% of the pleurotus citrinopileatus polysaccharide molar percentage.

Further, the glucose accounts for more than 15% of the pleurotus citrinopileatus polysaccharide in mole percentage.

Further, the galactose accounts for more than 4% of the pleurotus citrinopileatus polysaccharide molar percentage.

Further, the glucuronic acid accounts for more than 1% of the pleurotus citrinopileatus polysaccharide molar percentage.

Further, the fucose, the fructose and the arabinose respectively account for less than 3 percent of the pleurotus citrinopileatus polysaccharide molar percentage.

Further, the molecular weight of the neutral homogeneous component polysaccharide in the pleurotus citrinopileatus polysaccharide is 3.00 multiplied by 10⁵～1.00×10⁶Da, molecular weight of acidic homogeneous component polysaccharide 1.20X 10⁵～3.50×10⁵Da。

In the second technical scheme of the invention, the pleurotus citrinopileatus polysaccharide is applied to preparing the medicines for treating sarcopenia.

Further, the sarcopenia is sarcopenia caused by cancer.

Further, the cancer is colon cancer, gastric cancer, lung cancer, liver cancer, pancreatic cancer, colorectal cancer, esophageal cancer, lymph cancer or breast cancer.

In another aspect of the present invention, a drug for treating sarcopenia, comprising the pleurotus citrinopileatus polysaccharide of any one of claims 1 to 6 and a pharmaceutically acceptable excipient.

The invention discloses the following technical effects:

the colon cancer cachexia mouse muscle attenuation experiment proves that the pleurotus citrinopileatus polysaccharide can increase the weight and spleen weight of a sarcopenia mouse, recover the muscle fiber form, quantity and density, reduce inflammatory reaction, reduce the expression of sarcopenia marker proteins Atrogin-1 and MuRF-1 and relieve the sarcopenia symptoms caused by tumor.

The invention discovers the new function of pleurotus citrinopileatus polysaccharide and provides a new idea for developing and utilizing pleurotus citrinopileatus and treating sarcopenia caused by cancer.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a standard curve of glucose content in Pleurotus citrinopileatus polysaccharide prepared in example 1;

FIG. 2 is a standard curve of protein content in Pleurotus citrinopileatus polysaccharide prepared in example 1;

FIG. 3 is a standard curve of standard dextran molecular weight;

FIG. 4 is a high performance gel permeation chromatogram of Pleurotus citrinopileatus polysaccharide prepared in example 1;

FIG. 5 is HPLC chart of monosaccharide composition of Pleurotus citrinopileatus Sing polysaccharide prepared in example 1;

FIG. 6 is an infrared spectrum of Pleurotus citrinopileatus polysaccharide obtained in example 1;

FIG. 7 is a graph showing the body weight of a mouse in test example 1;

FIG. 8 is a graph showing the effect of histopathological morphology of gastrocnemius muscle in a mouse in test example 1;

FIG. 9 is a graph showing the serum contents of IL-6 and TNF- α in the mouse in test example 1;

FIG. 10 is a Western blot of Atrogin-1 and MuRF-1 in Experimental example 1;

FIG. 11 is a graph showing the expression amounts of mRNA of Atrogin-1 and MuRF-1 in test example 1;

FIG. 12 is a standard curve of uronic acid content in Pleurotus citrinopileatus polysaccharide prepared in example 1.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

The room temperature in the examples of the present invention means 20 to 25 ℃ unless otherwise specified.

Example 1

Step 1, putting the pleurotus citrinopileatus sporocarp and deionized water into a wall breaking machine according to the mass ratio of 1:40, and performing wall breaking extraction for 8min under the condition of 1200W power to obtain supernatant; concentrating the supernatant under reduced pressure to 20% of the original volume at room temperature, and precipitating with 80% ethanol with 4 times of the volume of the concentrated solution to obtain Pleurotus Citrinopileatus Sing crude polysaccharide solution;

step 2, adding 1/4 volumes of Sevage solution (chloroform: n-butanol is 4: 1 in volume ratio) into the pleurotus citrinopileatus crude polysaccharide solution, fully stirring for 1h by using a magnetic stirrer (the Sevage solution is used for removing protein in the polysaccharide solution), standing for layering, collecting the upper layer polysaccharide solution, repeating the operation for 3 times until no denatured protein exists between the upper layer and the lower layer, and volatilizing an organic reagent to obtain the pleurotus citrinopileatus polysaccharide.

Example 2

Step 1, putting the pleurotus citrinopileatus sporocarp and deionized water into a beaker according to the mass ratio of 1:50, and carrying out water bath at 90 ℃ for 40min to obtain a supernatant; centrifuging, extracting the precipitate for 2 times, mixing the supernatants, concentrating under reduced pressure to 20%, and precipitating with 80% ethanol with 4 times of the concentrated solution to obtain Pleurotus Citrinopileatus Sing crude polysaccharide solution;

step 2, adding 1/4 volumes of Sevage solution (chloroform: n-butanol is 4: 1 in volume ratio) into the pleurotus citrinopileatus crude polysaccharide solution, fully stirring for 1h by using a magnetic stirrer (the Sevage solution is used for removing protein in the polysaccharide solution), standing for layering, collecting the upper layer polysaccharide solution, repeating the operation for 3 times until no denatured protein exists between the upper layer and the lower layer, and volatilizing an organic reagent to obtain the pleurotus citrinopileatus polysaccharide.

Example 3

Step 1, putting the sporocarp of pleurotus citrinopileatus and deionized water into a beaker according to the mass ratio of 1:40, performing ultrasonic treatment for 16min under the condition of 500W by using an ultrasonic machine, performing water bath for 2h at 90 ℃, concentrating the supernatant into 20% of the original supernatant under reduced pressure, and performing alcohol precipitation by using ethanol with the volume fraction of 80% and 4 times that of the concentrated solution to obtain a pleurotus citrinopileatus crude polysaccharide solution;

step 2, adding 1/4 volumes of Sevage solution (chloroform: n-butanol is 4: 1 in volume ratio) into the pleurotus citrinopileatus crude polysaccharide solution, fully stirring for 1h by using a magnetic stirrer (the Sevage solution is used for removing protein in the polysaccharide solution), standing for layering, collecting the upper layer polysaccharide solution, repeating the operation for 3 times until no denatured protein exists between the upper layer and the lower layer, and volatilizing an organic reagent to obtain the pleurotus citrinopileatus polysaccharide.

Test example 1 examination of the active ingredient of the pleurotus citrinopileatus polysaccharide prepared in example 1 and the effect of attenuating the muscle of mice with colon cancer cachexia

1. Determination of content of each component in pleurotus citrinopileatus polysaccharide

Detecting sugar content in Pleurotus Citrinopileatus Sing by phenol-sulfuric acid method, wherein standard curve of glucose content in Pleurotus Citrinopileatus Sing polysaccharide is shown in FIG. 1; detecting the protein content in Pleurotus citrinopileatus by Coomassie brilliant blue G250 method, wherein the protein content standard curve is shown in FIG. 2; the content of uronic acid in the pleurotus citrinopileatus polysaccharide is determined by using the m-hydroxybiphenyl method, and a standard curve of the content of uronic acid is shown in fig. 12, and as can be seen from fig. 1, 2 and 12, the pleurotus citrinopileatus polysaccharide prepared in example 1 has a sugar content of 51.09%, a protein content of 9.87% and an uronic acid content of 1.24%.

2. And (3) measuring the molecular weight of pleurotus citrinopileatus polysaccharide:

and (3) performing molecular weight determination on the pleurotus citrinopileatus polysaccharide by adopting a high-efficiency gel permeation chromatography, and determining the molecular weight of the sample according to the retention time of the sample, wherein the smaller the retention time is, the larger the molecular weight is. The chromatographic conditions are RID-10A parallax refraction detector and TSK-gel G-3000PWXL chromatographic column (7.8 × 300nm), the mobile phase is distilled water, the flow rate is 0.6mL/min, the column temperature is 35 ℃, and the sample injection amount is 10 μ L.

The standard curve of the molecular weight of the standard glucan is shown in FIG. 3, and the equation of the molecular weight standard curve is-0.2282 x +7.8312, R²0.9922. The gel permeation chromatogram of Pleurotus Citrinopileatus Sing polysaccharide is shown in FIG. 4, and the molecular weight of neutral homogeneous component polysaccharide in Pleurotus Citrinopileatus Sing polysaccharide is calculated to be 3.00 × 10 according to retention time and standard curve⁵～1.00×10⁶Da, molecular weight of acidic homogeneous component polysaccharide 1.20X 10⁵～3.50×10⁵Da, the ratio of the two is 18.3: 1.

3. Analysis of monosaccharide composition of pleurotus citrinopileatus polysaccharide:

the pleurotus citrinopileatus polysaccharide sample prepared in example 1 is weighed to be 5mg, added with prepared 5% TFA acid solution (trifluoroacetic acid), heated at 121 ℃ for 2 hours, blown dry by nitrogen, cleaned by methanol and blown dry, and repeated for 3 times. Dissolving in sterile water, and transferring into a clean chromatographic bottle for detection. Setting a mobile phase: phase A: ddH 2O; phase B: 200mM NaOH; and C phase: 200mM NaOH/500mM NaAC; flow rate: 0.5 mL/min.

Pleurotus citrinopileatus polysaccharide monosaccharide composition HPLC As shown in FIG. 5, it can be seen from FIG. 5 that the Pleurotus citrinopileatus polysaccharide prepared in example 1 comprises xylose, glucose, galactose, glucuronic acid, fucose, fructose, arabinose, in a molar ratio of 60.905: 26.281: 7.754: 3.803: 0.697: 0.418:0.139.

4. Pleurotus citrinopileatus polysaccharide infrared spectroscopic analysis

Weighing 1.5mg of pleurotus citrinopileatus polysaccharide prepared in example 1, fully mixing the pleurotus citrinopileatus polysaccharide with 200mg of chromatographically pure KBr, grinding the mixture by using an agate mortar, tabletting, and independently tabletting 200mg of KBr as a background. An infrared spectrometer is used at 4000-500cm^-1As a result of the infrared scanning analysis within the range, as shown in FIG. 6, it can be understood from FIG. 6 that the Pleurotus citrinopileatus polysaccharide prepared in example 1 is a β -type furanose.

5. Pleurotus citrinopileatus polysaccharide anti-colorectal cancer cachexia mouse muscle attenuation effect

5.1 animal Experimental design and treatment

5.1.1 animal groups

BALB/c mice after being adaptively fed for one week are randomly divided into a normal group, a colon cancer cachexia model group, a pleurotus citrinopileatus polysaccharide low-dose group, a pleurotus citrinopileatus polysaccharide middle-dose group, a pleurotus citrinopileatus polysaccharide high-dose group and a four-monarch-particle positive control group, and 10 mice in each group.

5.1.2 treatment of cells and establishment of tumor mouse model

Normally culturing mouse CT26 colon cancer cells in DMEM medium containing 10% fetal calf serum and 1% penicillin-streptomycin double antibody, adjusting with phosphate buffer solution, collecting cells with density of 2 × 10⁸the/mL is used for in vivo modeling. The right back of 5 mice except the normal group was injected with CT26 cells 1X 10 subcutaneously⁷(dissolved in 0.1mL PBS +0.1mL of primer), tumor growth was observed and the experiment was started 7 days later.

5.1.3 animal drug administration intervention

And 7d, after the formation of tumors is observed, the mice are subjected to intragastric administration, the mice in the normal group and the mice in the model group are subjected to intragastric administration according to 0.2mL of physiological saline for each mouse, the polysaccharide is dissolved in the physiological saline for the polysaccharide administration group, the intragastric administration is carried out according to 0.2 mL/mouse, the administration doses are respectively 200, 400 and 600mg/kg, the four-monarch particles for the positive group are converted into 2250mg/kg according to the daily dose of adults, the four-monarch particles are dissolved in the physiological saline for the administration group, and the intragastric administration is carried out according to 0.2 mL/mouse, and the administration dose determined by each group is safe and has no toxic or side effect. During this period all mice had free access to food and water, and the body weight of the mice was weighed every two days, and the status of the mice was observed, and the access to food and water was recorded, and the experiment was administered for 14 days.

5.1.4 animal handling and sample Collection

All mice were weighed at 14 days and then bled from the orbit and dissected, and the collected whole blood of the mice was collected and centrifuged 2 times (3500rpm/min) in sterile RNase-free tubes at 4 ℃ for 10min each time to obtain the supernatant. And the serum was stored in an ultra-low temperature refrigerator at-80 ℃ until detection. A part of the dissected heart, lung, liver, kidney, spleen and gastrocnemius muscle was fixed in 10% neutral buffered formalin, and the other part was put into a sterile RNase-free tube and then rapidly frozen with liquid nitrogen, and stored at-80 ℃ for use. Feces were carefully collected from the large intestine of mice, stored in sterile rnase-free tubes and then rapidly frozen in liquid nitrogen, also stored at-80 ℃ for use.

As a result: after the mice are dissected, the net body, the gastrocnemius muscle and the tumor mass of the mice are compared, the body weight of the mice is shown in fig. 7, and the gastrocnemius muscle and the tumor mass are shown in table 1:

TABLE 1

As can be seen from fig. 7 and table 1, the pleurotus citrinopileatus polysaccharide can improve the body weight and gastrocnemius mass of mice with sarcopenia due to colon cancer and inhibit tumor growth.

5.2 Carbamin-eosin staining of gastrocnemius and detection of partial index

Placing the gastrocnemius tissue block fixed by paraformaldehyde into an ethanol solution according to a gradient from low concentration to high concentration for dehydration, and performing transparency by using xylene, absolute ethanol and pure xylene. And (3) putting the transparent tissue block into a mixed solution of paraffin and xylene with the same volume for 15min, and then putting the transparent tissue block into pure paraffin for 20-30min to embed the tissue block. And cutting the embedded tissue wax block into slices according to the required slice thickness, pasting the slices on a glass slide, and drying the glass slide in a thermostat. Dewaxing the slices with xylene twice, placing into gradient ethanol with high concentration to low concentration for 3-5min, placing into distilled water for 3min, dyeing with hematoxylin dye solution and eosin dye solution, washing off excessive red with 95% ethanol, and placing into anhydrous ethanol for 3-5 min. The slices are put into a mixed solution of ethanol and xylene with equal volume for 5min, then put into pure xylene for transparency twice, and finally the slices are sealed by neutral gum. The slide is placed under a microscope for observation, an Eclipse Ci-L photographing microscope is used for selecting a target area of the tissue for imaging by 200 times, the tissue is filled in the whole visual field as much as possible during imaging, and the background light of each picture is ensured to be consistent. After imaging is completed, Image-Pro Plus 6.0 analysis software is used, the number of muscle fibers in each picture and the corresponding total area of the muscle fibers are respectively counted by taking millimeters as standard units, and the area of a single muscle fiber is calculated as the total area of the muscle fibers/the number of the muscle fibers, and the density of the muscle fibers is calculated as the number of the muscle fibers/the visual field area. The pathological morphological effects (HE, × 400) of gastrocnemius muscle in mice are shown in fig. 8, and the number, area and density of muscle fibers in mice are shown in table 2:

TABLE 2

Note: in comparison with the normal group,^*P＜0.05，^**p is less than 0.01; in comparison with the set of models,^△P＜0.05，^△△P＜0.01。

from fig. 8 and table 2, it can be seen that the transverse diameter of myofibroblasts is significantly increased and the intercellular substance is decreased after pretreatment with the pleurotus citrinopileatus polysaccharide stem, which improves muscle degradation caused by cachexia and is dose-dependent. The positive control group also has an improvement effect on muscle fibers, but the effect is obviously smaller than that of the polysaccharide intervention group. As can be clearly found by combining table 2, the number of myofibers in the pleurotus citrinopileatus polysaccharide and positive drug intervention group is increased compared with that in the model group and close to that in the normal group, and the number of myofibers in the polysaccharide high dose group is even more. The beneficial effects on muscle condition after administration were found both in total muscle fiber area and muscle fiber density.

5.3 serum index detection

The content of interleukin IL-6 and tumor necrosis factor TNF-alpha in a mouse serum sample is measured by using a British kit and a double-antibody sandwich method. And diluting the standard substance according to the specification. Adding 50 mu L of standard substances with different concentrations into standard substance holes of the ELISA plate, adding 40 mu L of sample diluent and 10 mu L of samples to be detected into the sample holes, and slightly shaking and uniformly mixing. Add enzyme labeling reagent 100 u L, seal the membrane sealing plate and put at 37 degrees C temperature incubation for 60 min. Discarding liquid after incubation, filling each hole with diluted washing liquid, repeatedly washing, and finally drying. Adding 50 mul of color developing agent A and B into each hole, shaking and mixing uniformly, developing color for 15min in a dark place at 37 ℃, and adding 50 mul of stop solution to stop the reaction. The absorbance values of each well were measured sequentially at a wavelength of 450 nm.

The contents of IL-6 and TNF-alpha in the mouse serum are shown in figure 9, and it can be seen from figure 9 that compared with the normal group, the contents of IL-6 and TNF-alpha in the mouse serum of tumor cachexia of the model group are obviously increased, and compared with the model group, the contents of IL-6 and TNF-alpha in the pleurotus citrinopileatus polysaccharide dried pre-group and the positive medicine group are obviously reduced, which indicates that the inflammatory reaction of the organism caused by cachexia can be obviously improved by the administration intervention of pleurotus citrinopileatus polysaccharide.

5.4 Western blot detection of muscle tissue protein expression

5.4.1 cell treatment and Total protein extraction thereof

L6 cells were seeded in 6-well plates and after overnight cell attachment, the cells were treated according to the fractions shown in table 3. After washing the cells 2-3 times with PBS, adding appropriate RIPA lysis buffer into the culture plate, standing at low temperature for 3-5min, scraping the cells, collecting into a 1.5mL centrifuge tube, and standing at 4 ℃ for 2h to ensure complete lysis of the cells. Centrifuging at 12000rpm and 4 deg.C for 10min, and collecting supernatant to obtain total protein solution. And (3) determining the protein content by using a BCA kit, normalizing the protein concentration of the sample, boiling the sample at 100 ℃ for 15min to obtain denatured protein, subpackaging the denatured protein, and storing in a refrigerator at-20 ℃ for later use.

5.4.2 electrophoresis

Separating gel with required concentration and 5% concentrated gel are prepared according to experimental requirements, a gel maker is placed in an electrophoresis tank, sufficient electrophoresis liquid is added, and then sample electrophoresis is carried out. The sample is added into the electrophoresis well, the gel voltage is 75V, and the gel voltage is 120V. The electrophoresis is terminated as soon as bromophenol blue runs out.

5.4.3 transfer film

Filter paper and PVDF membrane were prepared, which were activated with methanol before use. A clamp for transferring the membrane, two sponge pads, filter paper and an activated PVDF membrane are placed in a box with the membrane transferring liquid. The clamps are opened and the sponge and three layers of filter paper are respectively filled. The gel was carefully peeled off and placed on filter paper, and the PVDF membrane was applied to the gel to avoid air bubbles. The film is rotated according to a certain voltage and time.

5.4.4 immune response

The transferred membrane was blocked with 5% skimmed milk (0.5% TBST) for 1h at room temperature and placed on a shaker. After blocking, PVDF membrane was placed in primary antibody dilution (TBST solubilized 5% skim milk, phosphorylated using TBST solubilized 5% BSA), shaken gently in a shaker, and incubated overnight at 4 ℃. After removal of the PVDF membrane, it was washed three times with TBST at room temperature for 5min each. After washing, the PVDF membrane was placed in secondary antibody dilutions (3000 fold diluted with TBST), incubated at room temperature for 30min, and washed three times with TBST at room temperature, 5min each.

5.4.5 chemiluminescence

After the secondary antibody incubation is finished, taking out the PVDF membrane, mixing two reagents of ECLA and ECLB in a centrifuge tube in equal volume in a dark room, pasting a double-layer glove or other transparent films on an exposure box, putting the PVDF membrane with the protein surface facing upwards between the two films of the exposure box, adding the mixed ECL solution for full reaction, removing residual liquid after 1-2min, covering the upper film and starting exposure. The exposed film is developed and fixed with a developing and fixing agent. The exposure conditions are adjusted according to the different luminous intensities.

5.4.6 gel image analysis

The film is scanned and archived, and the Alpha software processing system analyzes the optical density value of the target zone.

Western blots of Atrogin-1 and MuRF-1 are shown in FIG. 10, and the expression amounts of Atrogin-1 and MuRF-1 proteins are shown in Table 3:

TABLE 3

It can be seen from FIG. 10 and Table 3 that the Pleurotus citrinopileatus polysaccharide prepared in example 1 can down-regulate the protein expression of the sarcopenia cancer marker genes Atrogin-1 and MuRF-1.

5.5 Real-time PCR detection of muscle tissue mRNA expression

5.5.1 tissue Total RNA extraction

Total RNA (miRNA) in gastrocnemius is extracted by guanidinium isothiocyanate-phenol-chloroform extraction. Accurately weigh 100mg of tissue and lyse the tissue with 1mL of TRIzol reagent. 0.2mL of chloroform was added thereto, the mixture was vigorously shaken, allowed to stand at room temperature for 3 minutes, and then centrifuged at 12000g at 4 ℃ for 15 minutes, whereby RNA was dissolved in the upper aqueous phase. The upper aqueous phase was transferred to a 1.5mL centrifuge tube, isopropanol was added in an amount of 0.5mL isopropanol per 1mL TRIzol, left at room temperature for 10min, centrifuged at 12000g at 4 ℃ for 10min, the supernatant removed and the bottom was cleared of white RNA. Add 75% ethanol in RNase free water, vortex mix for 20s, and centrifuge at 7500g for 5 min. Removing supernatant, standing in air for 5-10min until RNA at bottom turns colorless, adding 20ul of RNase-free water, and performing reverse transcription in 60 deg.C water bath for 15min or storing at-70 deg.C.

5.5.2 Synthesis of cDNA

In 0.2mL Ep tube, the reaction solution was prepared according to the ABI reverse transcription kit instructions, and the specific procedures are as shown in Table 4-1.

TABLE 4-1 RNA extraction protocol

5.5.3 design and Synthesis of primers

Through the comparison of the reported gene sequences on Gen Bank, the specific primers are designed for the genes by using related biological software such as Primer 5.0 and the like, and the designed Primer sequences are shown in a table 4-2.

TABLE 4-2 Gene primer design and Synthesis

5.5.4 Real-time PCR

The cDNA obtained by the above reverse transcription was subjected to q-PCR reaction to detect the gene expression level, the reaction system being shown in Table 4-3.

TABLE 4-3 qPCR reaction System

Detecting the expression quantity of target genes Atrogin-1, MuRF-1 and reference gene beta-actin in the cells, obtaining a Ct value according to a qPCR reaction curve, and carrying out relative quantification by adopting a delta Ct.

The mRNA expression levels of Atrogin-1 and MuRF-1 are shown in FIG. 11, and it can be seen from FIG. 11 that the Pleurotus citrinopileatus polysaccharide prepared in example 1 can down-regulate the mRNA expression of the sarcopenia cancer marker genes Atrogin-1 and MuRF-1.

In conclusion, the pleurotus citrinopileatus polysaccharide can relieve sarcopenia caused by colon cancer.

Test example 2

The Pleurotus citrinopileatus polysaccharide prepared in example 1 is subjected to verification of muscle attenuation effect of mice with colorectal cancer cachexia, and the test process is the same as that of test example 1, except that the tumor cells are colorectal cancer cells CT-26, and the result proves that the Pleurotus citrinopileatus polysaccharide prepared in the example can relieve the symptoms of muscle reduction caused by colorectal cancer.

Test example 3

The pleurotus citrinopileatus polysaccharide prepared in example 2 is subjected to verification of the muscle attenuation effect of a mouse with liver cancer cachexia, the experimental process is the same as that of example 1, the difference is that the tumor cell is liver cancer cell H22, and the result proves that the pleurotus citrinopileatus polysaccharide prepared in the example can relieve the muscle reduction symptom caused by liver cancer.

Test example 4

The same examination as in example 1 was performed on the pleurotus citrinopileatus polysaccharide prepared in example 2, and the results showed that the pleurotus citrinopileatus polysaccharide prepared in example 2 was prepared from xylose, glucose, galactose, glucuronic acid, fucose, fructose, arabinose in a molar ratio of 59.938: 26.485: 7.234: 5.172: 0.665: 0.396: 0.125.

Test example 5

The pleurotus citrinopileatus polysaccharide prepared in example 2 is subjected to verification of the muscle attenuation effect of the gastric cancer cachexia mice, the test process is the same as that of test example 1, the difference is that the tumor cell is the gastric cancer cell MFC, and the result proves that the pleurotus citrinopileatus polysaccharide prepared in example 2 can relieve the muscle reduction symptom caused by gastric cancer.

Test example 6

The experiment process of the pleurotus citrinopileatus polysaccharide prepared in the example 2 is the same as that of the experiment example 1, except that the tumor cell is an esophageal cancer cell AKR, and the result proves that the pleurotus citrinopileatus polysaccharide prepared in the example 2 can relieve the sarcopenia caused by esophageal cancer.

Test example 7

The pleurotus citrinopileatus polysaccharide prepared in example 2 is subjected to verification of the muscle attenuation effect of the lung cancer cachexia mice, the test process is the same as that of test example 1, the difference is that the tumor cells are lung cells lewis, and the result proves that the pleurotus citrinopileatus polysaccharide prepared in example 2 can relieve the muscle attenuation symptom caused by lung cancer.

Test example 8

The muscle attenuation effect of mice with pancreatic cancer cachexia is verified on the pleurotus citrinopileatus polysaccharide prepared in example 2, the experimental process is the same as that of experimental example 1, the difference is that the tumor cell is pancreatic cell PANC02, and the result proves that the pleurotus citrinopileatus polysaccharide prepared in example 2 can relieve the muscle attenuation symptom caused by pancreatic cancer.

Test example 9

The pleurotus citrinopileatus polysaccharide prepared in example 2 is subjected to a test for the muscle attenuation effect of a lymphoma cachexia mouse, and the test process is the same as that of test example 1, except that the tumor cell is a lymphoma cell EL4, and the result proves that the pleurotus citrinopileatus polysaccharide prepared in example 2 can relieve the muscle attenuation symptom caused by the lymphoma.

Test example 10

The pholiota nameko polysaccharide prepared in example 3 was subjected to a test for a muscle attenuation effect of a mouse with lymphoma cachexia, and the test procedure was the same as that of test example 1, except that the tumor cell was a breast cancer cell 4T1, and the result proved that the pholiota nameko polysaccharide prepared in example 2 can alleviate the symptoms of muscle attenuation caused by breast cancer.

Test example 11

The same examination as in example 1 was performed on the pleurotus citrinopileatus polysaccharide prepared in example 3, and the results showed that the pleurotus citrinopileatus polysaccharide prepared in example 3 was prepared from xylose, glucose, galactose, glucuronic acid, fucose, fructose, arabinose in a molar ratio of 56.410: 29.059: 9.235: 3.464: 0.915: 0.644: 0.157.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.