CN107722131B

CN107722131B - Total ganoderma lucidum spore powder refined polysaccharide with significant auxiliary antitumor activity and preparation method and application thereof

Info

Publication number: CN107722131B
Application number: CN201710986842.2A
Authority: CN
Inventors: 帅欧; 陈地灵; 吴清平; 谢意珍; 杨柏华; 谭许朋
Original assignee: Guangdong Yuewei Edible Mushroom Technology Co Ltd
Current assignee: Guangdong Yuewei Edible Mushroom Technology Co Ltd
Priority date: 2017-10-20
Filing date: 2017-10-20
Publication date: 2020-12-29
Anticipated expiration: 2037-10-20
Also published as: CN107722131A

Abstract

The invention relates to a refined polysaccharide of total ganoderma lucidum spore powder with significant auxiliary antitumor activity, a preparation method and application thereof, and belongs to the field of extraction and separation of medicinal fungi and application and development of medicinal foods. The refined polysaccharide is mainly prepared from mannose, glucose, galactose and fucose according to a molar ratio of 1: 40-50: 4-5.5: 1.5-2.0, the purity of the heteropolysaccharide is more than 80%, and the weight average molecular weight is 160-200 KD. The preparation method comprises the following steps: dynamic extraction with hot water, sedimentation, coarse filtration and deslagging, fine filtration and clarification, ultrafiltration membrane concentration and impurity removal, spray drying or alcohol precipitation and air drying. The preparation method is green, intelligent and energy-saving, can solve the difficult problem of industrialization, has obvious scale advantages, and the finished product meets the requirements on safety, effectiveness, controllability and stability in a food and drug quality control system. The refined polysaccharide has obvious functional activity in the aspect of assisting anti-tumor, can be used for developing medicaments for preventing and treating tumors, and can be used for developing foods and health-care products in related fields.

Description

Total ganoderma lucidum spore powder refined polysaccharide with significant auxiliary antitumor activity and preparation method and application thereof

Technical Field

The invention relates to a refined polysaccharide of total ganoderma lucidum spore powder with significant auxiliary antitumor activity, a preparation method and application thereof, and belongs to the field of medicinal fungus extraction and separation technology and medicinal food application and development.

Background

Ganoderma (Ganoderma Lucidum Karst) with umbrella shape, pileus kidney shape, semicircular shape or nearly circular shape is fruiting body of Ganoderma Lucidum of Polyporaceae; has effects in invigorating qi, tranquilizing mind, relieving cough and asthma, and prolonging life; can be used for treating vertigo, insomnia, palpitation, short breath, neurasthenia, and cough with asthma due to asthenia.

The growth process of the ganoderma lucidum comprises the following steps: spore → mycelium → sporophore → release of spores. Ganoderma spore, mycelium and fruiting body are different stages of their growth, and from the Ganoderma flower (fruiting body) visible to eyes, when Ganoderma is mature, the pileus turns outwards to eject the spore outwards. Ganoderma spore flying with wind will begin to germinate once it falls onto trees with proper environment, and grow a section of short hypha slowly. After a period of nutrient enrichment, the growing and dense hyphae gradually kink and differentiate under the conditions of proper temperature, humidity, illumination and the like to form growing points which are one granule, hard, flat and visible to the naked eye, and are called as primordia of the ganoderma lucidum, and the ganoderma lucidum grows, grows and matures continuously to form the sporocarp finally. That is, the ganoderma lucidum spore powder is a "seed" similar to the ganoderma lucidum fruiting body, and the ganoderma lucidum mycelium is "transformed" into the ganoderma lucidum fruiting body by tissue clustering.

According to the research of global cancer report 2014 published by the world health organization, the number of global cancer patients and death cases is continuously increased in 2012, nearly half of newly-increased cancer cases appear in asia, most of the newly-increased cancer cases come from china, and the number of the newly-increased cancer cases in china is higher than the first number. Global cancer cases are predicted to exhibit a rapidly growing situation, from 1400 million people in 2012, increasing year by year to 1900 million people in 2025, and up to 2400 million people by 2035. 1400 cancer cases are newly increased and 820 death people are existed all over the world in 2012, wherein 307 cancer patients are newly increased and about 220 death people are caused in China, and the death people respectively account for 21.9% and 26.8% of the total amount of the whole world.

Therefore, the incidence of cancer is rising year by year, the death rate is high, and products for effectively preventing and treating malignant tumors are urgently needed. The mainstream cancer treatment means at present comprise cytotoxic drugs and radiotherapy, but the means can also lead to the continuous body failure of patients and accelerate a considerable proportion of death cases. In order to relieve the pain and burden of patients, the research on anti-tumor new drugs at home and abroad is gradually changed from the original cytotoxic anti-tumor drugs to the research on targeted non-cytotoxic anti-tumor drugs.

The search for more efficient and low-toxicity non-cytotoxic drugs or drugs with adjuvant anti-tumor effects which can play a role in enhancing efficacy and reducing toxicity by assisting a treatment means with remarkable curative effect but strong toxic and side effects is an important direction for the development of anti-tumor drugs in future. Non-cytotoxic drugs in combination with cytotoxic drugs or radiation therapy would be the best option for cancer therapy. In recent years, with the deep research on the mechanism of tumor evading immune apoptosis, the research on the immune adjuvant therapy of tumor has continuously made breakthrough progress.

The ganoderma lucidum is used as a natural medicine resource, and the pharmacological functions of the ganoderma lucidum are shown in the aspects of obviously enhancing the function of an immune system, preventing and treating tumors, reducing blood pressure, preventing the generation of cardiovascular diseases, stimulating the secretion of insulin, reducing blood sugar concentration, accelerating blood microcirculation, improving the oxygen supply capacity of blood, eliminating free radicals in vivo, resisting radiation, resisting liver injury, prolonging the service life and the like. Ganoderma lucidum has become one of the first traditional Chinese medicines entering the United states pharmacopoeia in 2010 and has gradually become a potential new favorite for developing anti-tumor drugs and immunomodulators in the heart of medical workers at home and abroad. The medicine effect material basis of the anti-tumor effect of the ganoderma lucidum medicinal materials is determined, and the ganoderma lucidum medicinal materials are developed into medicine products to be put into the market, so that the ganoderma lucidum medicinal materials have wide application prospect, remarkable social benefit and good economic value.

Disclosure of Invention

Aiming at the development requirements in the prior art and the defects in the prior art, the invention aims to provide a polysaccharide prepared from whole ganoderma lucidum spore powder and having significant auxiliary antitumor activity, and a large-scale industrial preparation method and application thereof.

The invention achieves the above purposes through the following scheme:

in the first aspect of the invention, a total ganoderma lucidum spore powder refined polysaccharide with significant auxiliary antitumor activity is provided, and the polysaccharide is mainly prepared from 4 monosaccharides of mannose, glucose, galactose and fucose according to a molar ratio of 1: 40-50: 4-5.5: 1.5-2.0 of heteropolysaccharide; every 100g of the total ganoderma lucidum spore powder refined polysaccharide contains 1.4-1.8 g of mannitol, 60-70 g of glucose, 7-8.5 g of galactose and 2-3 g of fucose respectively; calculating the purity of the polysaccharide refined from the total ganoderma spore powder according to the absolute content of the 4 main component monosaccharides, wherein the purity is over 80 percent; the weight average relative molecular weight (Mw) is 160-200 KD, and the cumulative weight area of the slices with the molecular weight more than 10KD accounts for more than 90%; the refined polysaccharide of the whole ganoderma lucidum spore powder mainly comprises 4 fractions, wherein the peak area ratios of the fraction 1, the fraction 2, the fraction 3 and the fraction 4 are respectively 20-25%, 30-40% and 15-25%, and the weight average relative molecular weights (Mw) of the 4 fractions are respectively 620-700 KD, 90-110 KD, 14-18 KD and 4-6 KD; the content of protein (BCA method) in the refined polysaccharide of the total ganoderma spore powder is determined to be less than 8% by an ultraviolet-visible spectrophotometry method, and the content of polysaccharide (anthrone-sulfuric acid method) is greater than 85%.

In a preferred embodiment, the total ganoderma lucidum spore powder refined polysaccharide is mainly prepared from 4 monosaccharides of mannose, glucose, galactose and fucose according to a molar ratio of 1: 45.05:4.76:1.74 of a heteropolysaccharide; every 100g of the refined polysaccharide of the whole ganoderma lucidum spore powder contains 1.63g of mannitol, 68.73g of glucose, 7.79g of galactose and 2.59g of fucose respectively; the purity of the refined polysaccharide of the total ganoderma spore powder is 80.74 percent calculated according to the absolute content of the 4 main components of monosaccharide; the weight average relative molecular weight (Mw) was 179.689kD, and the cumulative weight area of the chips with molecular weights greater than 10kD accounted for 99.11%; the refined polysaccharide of the whole ganoderma spore powder mainly comprises 4 fractions, wherein the peak area ratios of the fraction 1, the fraction 2, the fraction 3 and the fraction 4 are respectively 22.21%, 23.04%, 34.40% and 20.35%, and the weight average relative molecular weights (Mw) of the 4 fractions are respectively 677.261KD, 98.706KD, 15.713KD and 5 KD; the content of protein (BCA method) in the refined polysaccharide of the total ganoderma spore powder measured by an ultraviolet-visible spectrophotometry method is 6.28%, and the content of polysaccharide (anthrone-sulfuric acid method) is 87.12%.

At present, more than 100 types of ganoderma lucidum unimodal polysaccharides obtained by academic separation are obtained, and the unimodal polysaccharides are all derived from one ganoderma lucidum medicinal material and cannot play a synergistic effect of multiple active polysaccharides. The polysaccharide refined by the total ganoderma lucidum spore powder contains active polysaccharide which can be formed in the whole growth stage of ganoderma lucidum, and has obvious auxiliary anti-tumor activity and synergistic effect.

Furthermore, most of the current unimodal active polysaccharides only stay in the laboratory stage, and the common characteristics of the current unimodal active polysaccharides are extremely low yield, extremely low single treatment capacity and extremely high cost, and the adopted process technology has the possibility of several random mold industrialization in terms of yield design and market competitiveness.

Based on the above, in the second aspect of the present invention, there is provided a method for preparing a purified polysaccharide from whole ganoderma lucidum spore powder, which has a significant auxiliary anti-tumor activity, comprising:

(1) hot water extraction: taking a proper amount of total ganoderma spore powder residue after total ganoderma spore oil extraction, adding purified water with the weight being 10-30 times of that of the residue into a multifunctional extraction tank, introducing steam for boiling, stirring or starting a material liquid pump to perform countercurrent forced circulation, slowly adding the residue, continuously stirring or intermittently starting the material liquid pump to perform countercurrent forced circulation, and stopping steam until micro-boiling time is maintained for 1-2 hours;

(2) coarse filtration and separation: after extraction, standing, settling and cooling, extracting supernatant, coarsely filtering by using a plate-frame filter or a bag filter with the filtering precision of 0.2-2.5 mu m, optionally, repeatedly extracting residues once according to the method in the step (1) and coarsely filtering again, and combining coarse filtrate;

(3) fine filtering, clarifying and removing super-large molecular impurities: fine filtering and clarifying the crude filtrate by using a 200-800 nm tubular ceramic microfiltration membrane or a micropore folding filter element with the filtering precision of 0.1-0.22 mu m;

(4) membrane ultrafiltration concentration and removal of small molecule water soluble impurities: and concentrating the clear liquid by using an ultrafiltration membrane (the molecular weight cutoff is 3500-8000D).

(5) And (3) drying: precipitating the concentrated solution with ethanol, and drying with hot air or directly spray drying.

Preferably, the preparation method of the refined polysaccharide of the total ganoderma lucidum spore powder with the significant auxiliary anti-tumor activity comprises the following steps:

(1) dynamic extraction of polysaccharide with hot water: taking a proper amount of total ganoderma spore powder residue after total ganoderma spore oil extraction, adding purified water with the weight 10-30 times of that of the residue into a multifunctional extraction tank, introducing steam for boiling, stirring at the speed of 30-60 r/min or starting a material liquid pump to perform countercurrent forced circulation, slowly adding the residue, continuously stirring or starting the material liquid pump to perform forced circulation for 5-10 min at intervals of 10-20 min, and stopping steam until micro-boiling time is maintained for 1-2 hours;

(2) the natural sedimentation is combined with the rough filtration process to realize the separation of slag and liquid: after extraction, standing for settling, cooling to below 50 ℃, extracting supernatant, roughly filtering by using a plate-frame filter or a bag filter with the filtering precision of 0.2-2.5 mu m, optionally, repeatedly extracting residues once according to the method in the step (1) and roughly filtering again, and combining the rough filtrates;

(3) the fine filtration process realizes the clarification of the liquid medicine and removes the super-large molecular impurities precipitated by hot dissolution and cold separation: performing fine filtration and clarification on the crude filtrate by using a 200-800 nm tubular ceramic microfiltration membrane or a microporous folding filter element with the filtration precision of 0.1-0.22 mu m, wherein the operation process parameters of the tubular ceramic microfiltration membrane are 30-70 ℃, the operation pressure is 0.20-0.80 Mpa, the membrane surface flow rate is 3-5 m/s, the permeation flux is 150-400 LMH, when about 20L of residual liquid is left, about 20L of purified water is added for dilution, the clarification operation is continued until about 20L of residual liquid is left, and optionally, the dilution and clarification operation is repeated once again to ensure that all target polysaccharides pass through as much as possible and reduce the process loss;

(4) the membrane ultrafiltration process realizes the concentration of target polysaccharide and removes small molecule water-soluble impurities: concentrating the clarified liquid by using an ultrafiltration membrane, wherein the molecular weight cutoff of the ultrafiltration membrane is 3500-8000D, the operating process parameters are 30-50 ℃, the operating pressure is 0.15-0.6 Mpa, the membrane surface flow rate is 2-4.5 m/s, and the permeate flux is 6-25 LMH, adding about 40L of purified water for dilution when concentrating to about 40L, continuing concentrating to about 40L, optionally repeating the dilution and concentration steps once again to remove small-molecule water-soluble impurities to the maximum extent, and finally washing the ultrafiltration membrane by using about 20L of purified water to replace all concentrated liquid so as to reduce the process loss as much as possible;

(5) and (3) a drying process: slowly adding edible alcohol which is 3-7 times of the original volume of the concentrated solution under low-speed stirring for alcohol precipitation, standing overnight, extracting supernatant, taking out precipitate, washing with a small amount of ethanol, drying by hot air or vacuum, and finally optionally crushing and sieving to obtain the compound; or the concentrated solution can be obtained by one-step drying by adopting a spray drying method.

The raw materials adopted by the preparation method provided by the invention are all ganoderma lucidum spore powder residues which are extracted from full ganoderma lucidum spore oil by supercritical carbon dioxide in the invention patent CN 100593410C, the full ganoderma lucidum spore powder is prepared by mixing and sterilizing ganoderma lucidum fruiting bodies and ganoderma lucidum spore powder according to a certain proportion to be used as a culture medium, inoculating an 'active polysaccharide enrichment type' special ganoderma lucidum strain to be used as a 'seed source', providing nutritional conditions and environmental factors which are favorable for the growth of mycelia, and forming the dense ganoderma lucidum mycelia which are rich in active polysaccharides. The total ganoderma lucidum spore powder integrates ganoderma lucidum mycelia, ganoderma lucidum sporocarp and ganoderma lucidum spore powder, contains active polysaccharide which can be formed in the whole growth stage of ganoderma lucidum, can overcome the application limitation of single variety of polysaccharide by taking the mixed active polysaccharide as a raw material and performing extraction, separation and purification, is favorable for playing the comprehensive pharmacological action characteristics of multiple components, multiple targets, multiple layers and multiple ways of the traditional Chinese medicine, and embodies the integral appearance and characteristics of traditional Chinese medicine diagnosis and treatment.

The inventor discovers that ganoderma lucidum spore powder existing in the extraction raw material of the preparation method has extremely fine particles of about 5-7 mu m in the research process of the preparation method, and the extraction method in the prior art is very easy to naturally settle in the extraction process to form lumps, so that the yield is influenced; therefore, the technical scheme of the invention adopts dynamic extraction, and can effectively overcome the production problem. In addition, the extremely fine spore powder has extremely high requirements on the selective application of a filter medium and a filter technology; the inventors have found that when selecting a conventional filter medium or applying a single clarification technique, spore powder very easily penetrates the filter medium into the final product, affecting the quality of the finished product; meanwhile, if a terminal filtration technology is adopted, spore powder is easy to block a filter medium, so that normal production cannot be realized. Aiming at the problem, the inventor adopts three sequential clarification processes of natural sedimentation, rough filtration and fine filtration in the preparation method, and can basically remove all fragments of the raw materials from large to small (the clear liquid has no cell fragments when being inspected under a 200-fold microscope); and the rough filtration and the fine filtration process adopt a cross flow filtration mode as far as possible, so that the industrialization problem that the spore powder blocks a filter medium to cause abnormal production can be solved.

Furthermore, the extraction raw material of the preparation method is obtained by sterilizing the mixture of the ganoderma lucidum spore powder and the fruiting body according to a certain proportion to obtain a culture medium, inoculating the ganoderma lucidum strain, and providing a proper environment to generate dense ganoderma lucidum mycelia; therefore, the raw materials of the extract contain a large amount of mycelium which is in the vigorous growth stage, and the mycelium in the vigorous growth stage contains more macromolecular inactive substances such as protein, DNA, RNA, pectin, cellulose, water-insoluble hemicellulose and the like, and the common characteristics of the mycelium in the vigorous growth stage are that the mycelium can be dissolved in hot water, can be separated out after the temperature is reduced, and is suspended in an extracting solution in a colloid form, so that the extracting solution is turbid, precipitates after long-term standing, the re-solubility of a finished product is poor, and the re-solubility is difficult to remove by a conventional process. Aiming at the problem, the preparation method specially selects a high-precision filter medium, and can achieve the purpose of removing the inactive or low-activity macromolecular impurities by adjusting related parameters to a better range, thereby obtaining a good clarification effect (the turbidity is detected by a turbidity meter, and the reading is lower than No. 0.5 standard turbidity liquid).

In addition, nutrients and mineral substances which are beneficial to the growth of mycelia are introduced into the whole ganoderma lucidum raw material in the culture process, and the whole ganoderma lucidum is inactive substances.

The preparation method adopts three-step impurity removal processes of ceramic microfiltration membranes or precise microporous folding filter elements, organic ultrafiltration membranes and alcohol precipitation, can remove both hot-melt cold-separated macromolecular impurities and small-molecule water-soluble impurities, and can also remove impurities such as alcohol-water soluble pigments, polypeptides, genetic substances and the like through an alcohol precipitation process; and clarifying and removing impurities, concentrating and removing impurities, and precipitating with ethanol and separating and removing impurities simultaneously; not only can be used for preparing the polysaccharide with absolute purity of more than 80 percent, but also can save the production time and improve the production efficiency.

Meanwhile, the preparation method adopts the technical scheme of membranes in the clarification, impurity removal and concentration processes, belongs to the low-temperature production technology, can ensure that the three-dimensional structure of the active polysaccharide is not damaged, further retains the pharmacological activity of the active polysaccharide to the maximum extent, and simultaneously obviously reduces the energy consumption.

In a third aspect of the invention, the invention provides an application of the polysaccharide refined from the whole ganoderma lucidum spore powder in the aspect of assisting anti-tumor.

In the fourth aspect of the invention, the invention provides the application of the polysaccharide refined from the ganoderma lucidum spore powder in the preparation of auxiliary anti-tumor drugs, foods and health-care foods.

The polysaccharide refined from the total ganoderma lucidum spore powder has a remarkable effect on the auxiliary anti-tumor aspect, such as the auxiliary anti-tumor activity of paclitaxel; furthermore, therapeutic and/or regulatory effects are shown which specifically reverse tumor growth and abnormalities in the main biochemical markers of the immune and blood systems caused by chemotherapy. For example, the major biochemical indicators of abnormalities in the immune and blood systems caused by chemotherapy include, but are not limited to, increased stress in the spleen, significant increase in leukocytes, altered distribution of peripheral blood lymphocyte subpopulations, decreased peripheral blood red blood cell numbers, decreased peripheral blood hemoglobin concentrations, peripheral thrombocytopenia, and the like.

The effect of the whole ganoderma lucidum spore powder refined polysaccharide on the auxiliary anti-tumor aspect and other aspects is proved in tests of BALB/c female mice and the like comprising malignant breast cancer cells (4T-1), the stomach-perfused purified water of normal mice is used as a normal group, the stomach-perfused purified water of tumor-bearing mice is used as a model group, the single paclitaxel administration of the tumor-bearing mice is used as a single paclitaxel chemotherapy group, and the whole ganoderma lucidum spore powder refined polysaccharide prepared by the technical scheme of the invention is used in combination with the paclitaxel administration to be used as a combined treatment group for the auxiliary anti-tumor functional activity evaluation. The final result shows that the whole ganoderma lucidum spore powder refined polysaccharide and paclitaxel combination treatment group shows a remarkable function of inhibiting the growth of malignant breast cancer tumors in the whole test period, the curative effect of the combination treatment group is remarkably stronger than that of a single paclitaxel chemotherapy group, and the abnormality of the tumor growth and the main biochemical inspection index values of an immune system and a blood system caused by the single paclitaxel chemotherapy, such as spleen stress enlargement, obvious increase of white blood cells, change of peripheral blood lymphocyte subgroup distribution, reduction of peripheral blood hemoglobin number, reduction of peripheral blood hemoglobin concentration, peripheral blood platelet reduction and the like, can be specifically reversed.

Therefore, the polysaccharide refined from the total ganoderma lucidum spore powder under the effective dose can be used alone or combined with other active components and/or a compound formed by pharmaceutically acceptable auxiliary materials and carriers, and can be applied to the development of medicaments for preventing and treating tumors, the development of foods and health care products in related fields and the like.

Effective dosages described herein include, but are not limited to, more than 500mg/Kg of a target organism, including, but not limited to, a mammal such as a human.

Compared with the prior art, the invention has the beneficial effects that:

(1) the preparation method only uses alcohol and water in the whole process, does not bring new impurities and toxic and harmful substances, has no risk of organic reagent residue, ensures the natural quality of the product, is green and is environment-friendly.

(2) The preparation method provided by the invention is designed based on actual needs of large-scale production, and through multiple pilot-scale or above actual production verification, pipelining, automation and intelligent operation can be realized in the whole process, so that the preparation method has the advantages of simple and feasible process, suitability for large-scale industrialization, low overall operation cost and high product yield of more than 1.8%.

(3) The polysaccharide prepared by the method not only adopts comprehensive and strict quality control standards of polysaccharide substances to monitor the quality of finished products, but also carries out auxiliary antitumor functional activity evaluation through a tumor-bearing animal model to prove the effect, and the obtained refined polysaccharide has the characteristics of safety, effectiveness, controllability and stability.

In conclusion, the preparation method disclosed by the invention not only meets the requirements of national industrial policies on green, intelligence and energy conservation, but also solves the industrialization problem existing in the current production practice, has obvious scale industrialization advantages, and the prepared finished product meets the requirements on safety, effectiveness, controllability and stability in a food and drug quality management system.

Drawings

FIG. 1 is a general calibration curve for GPC molecular weights in example 4.

FIG. 2 is the RID spectrum of the purified polysaccharide from the whole Ganoderma lucidum spore powder of example 4.

FIG. 3 is a distribution diagram of the molecular weight of polysaccharides purified from Ganoderma lucidum spore powder in example 4.

FIG. 4 is a graph showing the cumulative weight distribution of the molecular weights of polysaccharides purified from whole Ganoderma lucidum spore powder in example 4.

FIG. 5 is a UV chromatogram of derivatives from twelve mixed monosaccharide controls of example 5.

FIG. 6 is the UV chromatogram of the derivative of the polysaccharide from Ganoderma lucidum spore powder in example 5.

FIG. 7 is a technical roadmap for auxiliary evaluation of antitumor functional activity according to example 6.

FIG. 8 is a photograph comparing the tumors of each group in example 6.

FIG. 9 is a curve showing the change in tumor-bearing volume of the experimental animals in example 6.

FIG. 10 is a graph of tumor weights for each group of example 6.

FIG. 11 is a graph of tumor indices for each group in example 6.

FIG. 12 is a graph of tumor volume for each group in example 6.

FIG. 13 is a graph of spleen indices for each group in example 6.

FIG. 14 is a graph of the CD 19% of each group in example 6.

FIG. 15 is a graph of the CD 3% of each group in example 6.

FIG. 16 is a graph of the CD 4% of each group in example 6.

FIG. 17 is a graph of the CD 8% of each group in example 6.

FIG. 18 is a photograph of CD4/CD8 of each group in example 6.

Fig. 19 is a graph showing WBC (white blood cell) levels of each group in example 6.

Fig. 20 is a graph of RBC (red blood cell) levels for each group in example 6.

FIG. 21 is a graph showing HGB (hemoglobin) levels of the respective groups in example 6.

FIG. 22 is a graph showing the levels of PLT (platelets) in each group in example 6.

Detailed Description

The present invention is further illustrated by the following specific examples.

In the present invention, it is acceptable that the error of the data is within 10%.

The protein content is determined by using a BCA protein concentration determination kit (enhanced type) in the invention, unless otherwise specified.

If not specifically stated, the invention adopts an anthrone-sulfuric acid method, and the content of crude polysaccharide is detected according to the steps in the item of ganoderma lucidum (content determination) polysaccharide in the first edition of Chinese pharmacopoeia 2015; using 9 dextran narrow standards with gradient molecular weight as reference, and detecting the average molecular weight and molecular weight distribution by HPGPC (differential refractometer) with double gel columns connected in series; and (3) taking 12 monosaccharides as a reference substance, and determining the monosaccharide composition and absolute purity by adopting a PMP-HPLC derivatization method.

Example 1:

the method comprises the steps of adopting 40kg of total ganoderma spore powder residue which is extracted from the total ganoderma spore oil in embodiment 2 in patent CN 100593410C for later use, adding 600L of purified water into a multifunctional extraction tank, boiling by introducing steam, slowly adding the residue while stirring at 60r/min, continuously stirring and maintaining micro boiling for 1h, standing and settling, cooling to below 50 ℃, extracting supernatant, filtering by using a plate frame, repeatedly extracting the residue once according to the method, and combining crude filtrates. The crude filtrate was subjected to 500nm tubular ceramic microfiltration membrane (CX-30-1016-4-19 type, 4 parallel membranes having a membrane area of 0.952m²) Clarifying at 50 deg.C under 0.35Mpa, with membrane surface flow rate of 4.5m/s, permeation flux of 360LMH and residual liquid content of 20L, diluting with 20L of purified water, clarifying until residual liquid content is 20L, and repeating the above steps. The clarified solution was concentrated with PT4040C2016 type organic ultrafiltration membrane (molecular weight cut-off: 5KD, membrane area: 8.3 m) manufactured by GE, USA²) Operating temperature at 40 deg.C, operating pressure at 0.5Mpa, membrane surface flow rate at 3.5m/s, permeation liquid flux at 15LMH, concentrating to 40L, adding purified water at 40L for dilution, concentrating to 40L, repeating again, and washing the ultrafiltration membrane with 20L purified water to displace all the concentrated solution. Spray drying the concentrated solution, pulverizing, and sieving.

The obtained polysaccharide from the ganoderma spore powder is gray powder, and the yield is 1.99%.

Example 2:

the method comprises the steps of adopting 30kg of total ganoderma spore powder residue which is extracted from the total ganoderma spore oil in embodiment 2 in patent CN 100593410C for later use, adding 700L of purified water into a multifunctional extraction tank, boiling by introducing steam, starting a material liquid pump to perform countercurrent forced circulation, slowly adding the residue, starting the material liquid pump to perform forced circulation every 10min for 5min, maintaining slight boiling for 1h, standing and settling, cooling to below 50 ℃, extracting supernatant, roughly filtering by using a bag filter with the filtering precision of 0.5 mu m, repeatedly extracting residues once according to the method, and combining rough filtrate. The crude filtrate is fine-filtered by a pp microporous folding filter element with the filtering precision of 0.1 mu m. The fine filtrate was concentrated with PT4040C2016 type organic ultrafiltration membrane (molecular weight cut-off 5KD, membrane area 8.3 m) from GE²) Operating temperature at 45 deg.C, operating pressure at 0.45Mpa, membrane surface flow rate at 4.0m/s, permeation liquid flux at 17LMH, concentrating to 40L, adding purified water at 40L for dilution, concentrating to 40L, repeating again, and washing the ultrafiltration membrane with 20L purified water to displace all the concentrated solution. Slowly adding edible alcohol 5 times the original volume of the concentrated solution under stirring, sealing, standing overnight, separating precipitate, hot air drying, pulverizing, and sieving.

The obtained polysaccharide refined from the ganoderma lucidum spore powder is offwhite powder, and the yield is 1.83%.

Example 3:

ultraviolet-visible spectrophotometry for determining protein and polysaccharide content of polysaccharide refined from Ganoderma spore powder

Protein content determination: the BCA Protein concentration determination Kit (Enhanced BCA Protein Assay Kit) is purchased from Shanghai Bin Yuntian biotechnology limited company and detected according to the steps of the detailed specification.

And (3) polysaccharide content determination: the detection is carried out by adopting an anthrone-sulfuric acid method according to the steps in the ganoderma lucidum item [ content determination ] polysaccharide of the first edition of Chinese pharmacopoeia 2015.

The protein content and polysaccharide content of the refined polysaccharide of the total ganoderma spore powder prepared in the example 1 are respectively measured according to the method, and the protein content and the polysaccharide content of the refined polysaccharide of the total ganoderma spore powder are respectively 6.28% and 87.12%.

Example 4:

determination of molecular weight and molecular weight distribution of polysaccharide refined from total ganoderma spore powder

Preparation of control solutions: taking dried constant weight sigma series dextran and anhydrous glucose reference substance about 50mg, precisely weighing, and adding 0.71% Na₂SO₄Dissolving the water solution, fixing the volume to a 5mL measuring flask, sealing the flask, and shaking up to obtain the product.

Preparation of a test solution: taking about 100mg of the total ganoderma lucidum spore powder refined polysaccharide prepared in the embodiment, precisely weighing, and using a small amount of 0.71% Na with the temperature of 50-60 DEG C₂SO₄The aqueous solution was dissolved and transferred to a 10mL measuring flask, and the flask was allowed to stand at room temperature (25 ℃ C.), 0.71% Na₂SO₄The water solution is fixed to the scale, the mixture is densely shaken up, about 5mL of the solution is taken, the mixture is centrifuged for 10min at 4000r/min at 25 ℃, the supernatant is taken and filtered through a 0.45 mu m microporous membrane, and about 3mL of the secondary filtrate is collected for standby.

Chromatographic conditions are as follows: an Agilent 1200 high performance liquid phase system with refractive index detector parameters set to control analog output, optical component temperature 35 deg.C, zero point compensation 5%, attenuation 500 x 10³Automatically returning to 0 and positive polarity before analysis; two TSK-GEL (7.8 × 300mm,10 μ) chromatographic columns are connected in series, the series sequence is G5000PWXL → G4000PWXL, the column temperature is 35 ℃; with 0.71% Na₂SO₄Performing mobile phase isocratic elution on the aqueous solution (ultrasonic degassing in advance) at the flow rate of 0.5 mL/min; the sample volume was 20. mu.L, and the analysis time was 1 h.

Data processing: and (3) introducing the original data of each reference substance solution and the whole ganoderma lucidum spore powder refined polysaccharide solution into special processing software for Shanghai thousand spectrum GPC gel in an AIA format, and analyzing.

The results obtained are shown in tables 1 to 2 and fig. 1 to 4.

TABLE 1 molecular weight of dextran control and Peak time in chromatogram

TABLE 2 molecular weights and distributions of polysaccharides purified from whole Ganoderma lucidum spore powder

As can be seen from the results of tables 1 to 2, and FIGS. 1 to 4, the weight average relative molecular weight (Mw) of the polysaccharides purified from Ganoderma lucidum spore powder prepared in example 1 was 179.689kD, and the cumulative weight area of the cut pieces having a molecular weight of more than 10kD accounted for 99.11%; the refined polysaccharide of the whole ganoderma spore powder mainly comprises 4 fractions, wherein the peak area ratios of the fraction 1, the fraction 2, the fraction 3 and the fraction 4 are respectively 22.21 percent, 23.04 percent, 34.40 percent and 20.35 percent, and the weight average relative molecular weights (Mw) are respectively 677.261KD, 98.706KD, 15.713KD and 5 KD.

Example 5:

PMP-HPLC derivatization method for determining purity and monosaccharide composition of polysaccharide refined from total ganoderma lucidum spore powder

Preparation of control stock solutions: taking monosaccharide reference substances such as mannose, glucosamine hydrochloride, ribose, rhamnose, glucuronic acid, galacturonic acid, galactosamine hydrochloride, glucose, galactose, xylose, arabinose, fucose and the like, precisely weighing the monosaccharide reference substances, dissolving the monosaccharide reference substances in deionized water, fixing the volume to a 25mL measuring flask, sealing, shaking up to prepare a mixed reference substance storage solution for later use.

Preparation of a stock solution of the test article: taking about 100mg of the total ganoderma lucidum spore powder refined polysaccharide prepared in the example 1, precisely weighing, dissolving with a small amount of deionized water at 50-60 ℃, transferring into a 10mL measuring flask, placing to room temperature (25 ℃), fixing the volume of the deionized water to the scale, sealing, shaking up, taking about 5mL of the solution, centrifuging at 4000r/min at 25 ℃ for 10min, taking the supernatant, passing through a 0.45 mu m microporous filter membrane, collecting about 3mL of the continuous filtrate, and preparing 10.005mg/mL of a sample stock solution for later use.

Preparation of derivatization reaction reagent: preparing a plurality of methanol solutions of 0.5mol/L PMP reagent, shaking up, sealing, shading and storing at low temperature; preparing 4mol/L trifluoroacetic acid aqueous solution, 0.6mol/L sodium hydroxide aqueous solution and 0.6mol/L hydrochloric acid aqueous solution respectively, sealing, and shaking up for later use.

Preparation of control derivatization solution: precisely sucking 2.000mL of mixed monosaccharide reference substance solution into a 15mL centrifuge tube, adding 2.000mL of 0.6mol/L sodium hydroxide aqueous solution, adding 4.000mL of 0.5mol/L PMP methanol solution, closing a cover, sealing and reinforcing a sealing membrane, fully mixing by vortex, preserving heat in 70 ℃ water bath for 30min, quickly taking out, stopping reaction in ice bath for 30s, opening the cover, adding 2.000mL of 0.6mol/L hydrochloric acid aqueous solution, uniformly mixing, transferring into a 10mL measuring flask, fixing the volume to a scale with deionized water, sealing the cover, fully mixing, precisely sucking 7.000mL into the 15mL centrifuge tube, sucking 7mL of chloroform by a pipette, flushing into the derivative solution, removing the upper layer of water into another 15mL centrifuge tube, continuously washing for 2 times in the same manner of chloroform, sucking the upper layer of water, centrifuging at room temperature of 12000r/min, taking a proper amount of supernatant, 3/4, 1/2 according to the concentration of mother solution, 1/4, 1/8, 1/16, 1/32 and 1/64, and preparing 8-gradient derivatization reference substance solutions.

Preparing a test product derivatization solution: precisely transferring a test article stock solution into an ampoule bottle of 0.200-5 mL, adding a trifluoroacetic acid solution of 0.200mL and 4mol/L, filling nitrogen into the ampoule bottle, rapidly sealing the ampoule bottle with an alcohol torch, putting the ampoule bottle in a 110 ℃ oven for hydrolysis for 4 hours, taking out, putting the ampoule bottle to room temperature, and knocking off the bottleneck of the ampoule bottle in a standing state; adding 0.5mL of methanol, drying under vacuum and reduced pressure below 60 ℃ or drying by a dry nitrogen blowing instrument, and repeatedly treating for three times until the hydrolysate is completely dried; precisely adding 0.200mL of deionized water and 0.200mL of 0.6mol/L of sodium hydroxide aqueous solution into the residues respectively, adding 0.400mL of 0.5mol/L of PMP methanol solution, performing low-frequency low-intensity ultrasound for 30s, blowing the residues on the wall of the bottle by using a liquid transfer gun for several times to completely and fully mix the residues, precisely sucking 0.700mL to 5mL of a centrifuge tube, closing a cover, sealing and reinforcing the sealing membrane, placing the centrifuge tube in a 70 ℃ water bath for heat preservation for 30min, quickly taking out the centrifuge tube, stopping the reaction in an ice bath for 30s, opening the cover, adding 0.200mL of 0.6mol/L of hydrochloric acid aqueous solution, adding deionized water to a constant volume of 2mL, fully mixing the components, sucking 2mL of chloroform each time by using a liquid transfer gun, flushing the chloroform into the derivative liquid, removing the upper-layer aqueous solution into another 5mL of centrifuge tube, continuously flushing the centrifuge tube for 2 times by using the same method of chloroform, sucking the upper-layer aqueous solution, centrifuging the upper-.

Chromatographic conditions are as follows: an Agilent 1200 high performance liquid chromatography system, a Waters symmetry ShieldRP18 (4.6X 250mm, 5 μm) chromatographic column, the column temperature is 30 ℃, ammonium acetate aqueous solution of 18% acetonitrile-82% 0.1mol/L is used as a mobile phase for isocratic elution, the flow rate is 1mL/min, the detection wavelength is 250nm, the detection time is 50min, and the sample injection amount is 20 μ L.

Data processing: respectively drawing standard curves of monosaccharides, and calculating the total content of 12 monosaccharides in the refined polysaccharide of the total ganoderma spore powder in the embodiment 1; respectively calculating the mass concentration of monosaccharide with large proportion in the refined polysaccharide, taking the monosaccharide with the minimum mass concentration in the target monosaccharide as a reference parameter 1, and converting the mass concentration of other monosaccharides with the mass concentration to obtain the multiple.

The results of the experiments are shown in tables 3 to 6 and fig. 5 to 6.

TABLE 3 prepared concentrations of twelve monosaccharide mixture control solutions and time to peak of the derivative chromatogram

TABLE 4 arrangement concentrations and corresponding peak areas of twelve monosaccharide mixtures as reference

TABLE 5 Standard curves for twelve monosaccharide mixtures as reference

TABLE 6 purity and monosaccharide composition of Total Ganoderma spore powder refined polysaccharide

Note: only monosaccharide not less than 0.3% by weight

From the above experimental results, it can be seen that the purified polysaccharide of total ganoderma spore powder prepared by the method of example 1 of the present application is a heteropolysaccharide mainly composed of 4 monosaccharides including mannose, glucose, galactose and fucose, the molar ratio is 1.00:45.05:4.76:1.74, each 100g of the purified polysaccharide of total ganoderma spore powder contains 1.63g of mannitol, 68.73g of glucose, 7.79g of galactose and 2.59g of fucose, and the purity of the purified polysaccharide of total ganoderma spore powder is 80.74% calculated by the absolute content of the main monosaccharides.

Example 6:

polysaccharide-assisted anti-tumor functional activity evaluation of whole ganoderma lucidum spore powder

1 Experimental procedures

1.1 reagent: the positive drug is paclitaxel produced by Hainan Quanxing pharmaceutical Co., Ltd, and the specification is 100mg/16.7 mL; the polysaccharide refined from the whole ganoderma lucidum spore powder is prepared according to the invention in the embodiment 1; yibao purified water.

1.2 tumor cells and experimental animals: mouse malignant breast cancer cells (4T-1), source: cell bank of Chinese academy of sciences. SPF-grade BALB/c mice, female, 6-8 weeks old, 14-16g in weight, purchased from Guangdong province medical laboratory animal center, and provided with license numbers: SCXK (yue) 2013-: no. 44007200042242.

1.3 preparation method of subcutaneous breast cancer transplantation model: 4T1 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum and 1% streptomycin under 5% CO₂Cultured in an incubator at 37 ℃, cells were digested with 0.25% trypsin-0.02% EDTA, passaged, and cells in logarithmic phase were used for experiments. After the cells were collected, they were washed with 1640 mediumCell viability was confirmed by 2 times of Trypan blue experiment>95% of viable cells were adjusted to 2 × l0 using 1640 medium⁵and/mL, for use. The inoculated part of the tumor-bearing group animals is disinfected and prepared into skin, 0.1mL (containing 2 x 10) of 4T1 cell suspension is sucked by a 1mL syringe⁴Individual cells) were inoculated under the mouse axilla, and when the tumor size was 2mm x 2mm, the model was judged to be successfully established. The blank control group was injected axillary subcutaneously with 0.1mL of complete medium.

1.4 animal groups: after the quarantine period is finished, randomly dividing the test into a normal group (8) and a tumor-bearing group (24) according to the body weight; inoculating 4T1 breast cancer cells in the tumor-bearing group, and establishing a mouse breast cancer model; after modeling for 24 hours, the animals in the tumor-bearing group are divided into the following animals according to a weight random block method: the model group comprises 8 paclitaxel groups in combination with a control group, paclitaxel group and refined polysaccharide of total ganoderma spore powder.

1.5 dosing regimen: after 24h of molding, the tumor-bearing component groups were administered for 20 consecutive days. The polysaccharide and purified water of the whole ganoderma lucidum spore powder are both administered by intragastric administration, the administration volume is 0.2mL/10g, and the administration is carried out 1 time per day; paclitaxel is administered by intraperitoneal injection, with a volume of 0.1mL/10g, 1 time per week. The dosing regimen is detailed in tables 7, 8.

TABLE 7 summary of the respective group dosage numbers

Table 8 dosage regimen summary

2 observation index

2.1 tumor-bearing volume measurement: tumor mass growth was noted from the day of administration, and tumor mass volumes were measured using vernier calipers on

days

8, 10, 12, 14, 16, 18, and 20, respectively. The length is a, the length of the tumor body in the longest direction, the width is b, and the length of the vertical plane in the longest direction is the shortest; calculating Tumor Volume (TV) ═ 1/2 × a × b²And drawing a tumor-bearing volume change curve.

2.2 blood: stopping taking the medicine for 24 hours, and then measuring the indexes of the animal materials. First, blood samples were collected: (1) orbital bleeding (approximately 6 drops), heparin sodium anticoagulation, and peripheral blood lymphocyte populations were detected by flow cytometry. (2) Orbital bleeds (approximately 6 drops), 4% EDTA anticoagulation, sample sending, and blood testing routine.

2.3 tumor mass and spleen: (1) after blood was collected from the orbit, the mouse was sacrificed and then the tumor body was detached, and connective tissues such as proliferating blood vessels and capsule on the surface of the tumor body were carefully removed and weighed. (2) The spleen was further removed, and the weight was measured after removing the surface-adhering tissue. (3) And (3) photographing: after all the animal tumor masses are taken down, the animal tumor masses are arranged according to the sequence of groups, corresponding labels are attached, a ruler is placed on the background, and the ruler is photographed and stored as an original record.

3 data processing

All data are averaged plus or minus the standard deviation

And (4) showing. The average numbers among the groups are compared by adopting One-Way ANOVA (One-Way ANOVA), the average numbers among the groups are compared pairwise, and an LSD (least squares) method is adopted when the variance is uniform; dunnett's T3 method is used when variance is irregular. Finished by SPSS software, α is 0.05.

The technical route of the whole auxiliary antitumor functional activity evaluation experiment is detailed in figure 7.

4 results of the experiment

4.1 after the animal experiment, dissect the tumor-bearing mouse, strip the tumor tissue, its every group tumor tissue photo is shown in figure 8, the whole ganoderma spore powder refined polysaccharide combined with paclitaxel can inhibit the growth of tumor apparently, its curative effect is stronger than using paclitaxel alone.

4.2 the change of the tumor-bearing volume of the experimental animals is shown in table 9 and fig. 9, the tumor volume of the animals in the model group is rapidly increased, and the tumor volume of the animals in which the paclitaxel and the polysaccharide refined from the whole ganoderma lucidum spore powder are used singly and the increase trend of the tumor volume of the animals in which the paclitaxel is used is slower than that of the animals in the model group; compared with the model group, the tumor volume of the total ganoderma lucidum spore powder refined polysaccharide combined paclitaxel group is obviously reduced from the 14 th day, the 16 th day to the 20 th day of administration, and the difference has statistical significance (P is less than 0.01); compared with the paclitaxel alone, the tumor volume of the total ganoderma spore powder refined polysaccharide combined with the paclitaxel is obviously reduced from the 14 th day, the 16 th day to the 20 th day of administration, wherein the difference of the 14 th day has statistical significance (P is less than 0.01).

TABLE 9 tumor volume change of experimental animals (n ═ 8)

Note: in comparison with the set of models,^*p＜0.05，^**p is less than 0.01; compared with paclitaxel group, # p < 0.05, # p < 0.01.

4.3 the tumor-bearing weight and the related organ indexes of the experimental animals are shown in the table 10 and the figures 10-13, compared with the model group, the tumor weight, the tumor index, the tumor volume and the batch spleen index of the total ganoderma lucidum spore powder refined polysaccharide combined paclitaxel are all obviously reduced, and the difference has statistical significance (P is less than 0.01); compared with paclitaxel alone, the tumor weight, tumor index, tumor volume and spleen index of the total Ganoderma spore powder refined polysaccharide combined paclitaxel are all significantly reduced, wherein the difference of the spleen indexes has statistical significance (P is less than 0.05).

TABLE 10 tumor-bearing weights and related organ indices for experimental animals

Note: compared with the blank group, the delta p is less than 0.05, and the delta p is less than 0.01; in comparison with the set of models,^*p＜0.05，^**p is less than 0.01; compared with paclitaxel group, # p < 0.05, # p < 0.01.

4.4 peripheral blood lymphocyte subset flow analysis results are shown in table 11 and fig. 14-18, the total ganoderma lucidum spore powder polysaccharide and paclitaxel can remarkably reverse the trend of the reduction of the proportion of the peripheral blood lymphocyte subsets CD3, CD4 and CD8 caused by using paclitaxel alone, and the difference has statistical significance (P is less than 0.01).

TABLE 11 distribution of peripheral blood lymphocyte subsets (CD occupation of subsets)

4.5 blood routine test results are shown in Table 12 and figures 19-22, and compared with a model group and a paclitaxel single use, the total ganoderma lucidum spore powder refined polysaccharide combined paclitaxel can obviously reduce the peripheral blood leukocyte level to be close to a normal value, wherein the difference with the model group has statistical significance (P is less than 0.01); simultaneously, compared with a model group and a single paclitaxel group, the paclitaxel group combined with the whole ganoderma lucidum spore powder refined polysaccharide can obviously increase the level of platelets, wherein the difference with the model group has statistical significance (P is less than 0.01); in addition, compared with the model group and the paclitaxel alone, the combination of the whole ganoderma lucidum spore powder refined polysaccharide and the paclitaxel can slightly improve the level of red blood cells and the level of red blood protein of peripheral blood.

TABLE 12 results of routine blood tests on laboratory animals

5 conclusion

The total ganoderma lucidum spore powder refined polysaccharide and paclitaxel combined treatment group shows a remarkable function of inhibiting the growth of malignant breast cancer tumors in the whole test period, the curative effect of the combined treatment group is remarkably stronger than that of a single paclitaxel chemotherapy group, and the combined treatment group can specifically reverse the tumor growth and the abnormity of main biochemical inspection index values of an immune system and a blood system caused by the single paclitaxel chemotherapy.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and their concepts should be considered to be equivalent or modified within the technical scope of the present invention.

Claims

1. The total ganoderma spore powder refined polysaccharide composition with the anti-tumor activity is characterized by comprising total ganoderma spore powder refined polysaccharide and paclitaxel in a mass ratio of 500:10, wherein the total ganoderma spore powder refined polysaccharide mainly comprises 4 monosaccharides of mannose, glucose, galactose and fucose, and the molar ratio of the monosaccharides to the polysaccharides is 1: 40-50: 4-5.5: 1.5-2.0 of heteropolysaccharide; calculating the purity of the polysaccharide refined from the total ganoderma spore powder according to the absolute content of the 4 main component monosaccharides, wherein the purity is over 80 percent; the weight average relative molecular weight is 160-200 KD, and the cumulative slice weight area with the molecular weight more than 10KD accounts for more than 90%; the refined polysaccharide of the whole ganoderma lucidum spore powder mainly comprises 4 fractions, wherein the peak area ratios of the fraction 1, the fraction 2, the fraction 3 and the fraction 4 are respectively 20-25%, 30-40% and 15-25%, and the weight average relative molecular weights of the 4 fractions are respectively 620-700 KD, 90-110 KD, 14-18 KD and 4-6 KD; the protein content in the whole ganoderma lucidum spore powder refined polysaccharide measured by the BCA method is less than 8%, and the polysaccharide content measured by the anthrone-sulfuric acid method is more than 85%;

the extraction method of the whole ganoderma lucidum spore powder refined polysaccharide comprises the following steps:

(1) hot water extraction: taking a proper amount of the total ganoderma spore powder residue after the total ganoderma spore oil extraction is finished, adding purified water with the weight being 10-30 times of that of the residue into a multifunctional extraction tank, introducing steam to boil, stirring or starting a material liquid pump to perform countercurrent forced circulation, slowly adding the residue, continuously stirring or intermittently starting the material liquid pump to perform the countercurrent forced circulation, and stopping the steam until the micro-boiling time is maintained for 1-2 hours;

(2) coarse filtration and separation: after extraction, standing, settling and cooling, extracting supernatant, coarsely filtering by using a plate-frame filter or a bag filter with the filtering precision of 0.2-2.5 mu m, repeatedly extracting residues once according to the method in the step (1), coarsely filtering again, and combining coarse filtrate;

(4) membrane ultrafiltration concentration and removal of small molecule water soluble impurities: concentrating the clarified liquid by using an ultrafiltration membrane with the molecular weight cutoff of 3500-8000D;

(5) and (3) drying: precipitating the concentrated solution with ethanol, and drying with hot air or directly spray drying to obtain the polysaccharide.

2. The total ganoderma spore powder refined polysaccharide composition of claim 1, wherein the total ganoderma spore powder refined polysaccharide is mainly prepared from 4 monosaccharides of mannose, glucose, galactose and fucose according to a molar ratio of 1: 42.05: 4.76:1.74 of a heteropolysaccharide; the purity of the refined polysaccharide of the total ganoderma spore powder is 80.74 percent calculated according to the absolute content of the 4 main components of monosaccharide; the weight average relative molecular weight is 179.689KD, and the cumulative weight area of the slices with the molecular weight more than 10KD accounts for 99.11%; the refined polysaccharide of the whole ganoderma lucidum spore powder mainly comprises 4 fractions, wherein the peak area ratios of the fraction 1, the fraction 2, the fraction 3 and the fraction 4 are respectively 22.21%, 23.04%, 34.40% and 20.35%, and the weight average relative molecular weights of the 4 fractions are respectively 677.261KD, 98.706KD, 15.713KD and 5 KD; the protein content in the refined polysaccharide of the total ganoderma spore powder measured by the BCA method is 6.28%, and the polysaccharide content measured by the anthrone-sulfuric acid method is 87.12%.

3. A preparation method of a total ganoderma lucidum spore powder refined polysaccharide composition with anti-tumor activity is characterized in that the composition consists of the total ganoderma lucidum spore powder refined polysaccharide and paclitaxel in a mass ratio of 500:10, and the preparation method of the total ganoderma lucidum spore powder refined polysaccharide comprises the following steps:

(1) dynamic extraction of polysaccharide with hot water: taking a proper amount of total ganoderma spore powder residue after total ganoderma spore oil extraction, adding purified water with the weight 10-30 times of that of the residue into a multifunctional extraction tank, introducing steam for boiling, stirring at the speed of 30-60 r/min or starting a material liquid pump to perform countercurrent forced circulation, slowly adding the residue, continuously stirring or starting the material liquid pump to perform forced circulation for 5-10 min at intervals of 10-20 min, and stopping steam until the micro-boiling time is maintained for 1-2 hours;

(2) the natural sedimentation is combined with the rough filtration process to realize the separation of slag and liquid: after extraction, standing for settling, cooling to below 50 ℃, extracting supernatant, roughly filtering by using a plate-frame filter or a bag filter with the filtering precision of 0.2-2.5 mu m, repeatedly extracting residues once according to the method in the step (1), roughly filtering again, and combining rough filtrate;

(3) the fine filtration process realizes the clarification of the liquid medicine and removes the super-large molecular impurities precipitated by hot dissolution and cold separation: performing fine filtration and clarification on the crude filtrate by using a 200-800 nm tubular ceramic microfiltration membrane or a microporous folding filter element with the filtration precision of 0.1-0.22 mu m, wherein the operation process parameters of the tubular ceramic microfiltration membrane are 30-70 ℃, the operation pressure is 0.20-0.80 MPa, the membrane surface flow rate is 3-5 m/s, the permeation flux is 150-400 LMH, 20L of purified water is added for diluting when 20L of residual liquid is left, the clarification operation is continued until 20L of residual liquid is left, and the dilution and clarification operation is repeated once again to ensure that the target polysaccharide completely passes through as much as possible and reduce the process loss;

(4) the membrane ultrafiltration process realizes the concentration of target polysaccharide and removes small molecule water-soluble impurities: concentrating the clarified liquid by using an ultrafiltration membrane, wherein the cutoff molecular weight of the ultrafiltration membrane is 3500-8000D, the operating process parameters are 30-50 ℃, the operating pressure is 0.15-0.6 MPa, the membrane surface flow rate is 2-4.5 m/s, the permeate flux is 6-25 LMH, when the clarified liquid is concentrated to 40L, adding 40L of purified water for dilution, continuously concentrating to 40L, repeating the dilution and concentration steps again to remove small-molecule water-soluble impurities to the maximum extent, and finally washing the ultrafiltration membrane by using 20L of purified water to replace all concentrated liquid so as to reduce the process loss as much as possible;

(5) and (3) a drying process: slowly adding edible alcohol with the volume being 3-7 times of the original volume into the concentrated solution under low-speed stirring for alcohol precipitation, standing overnight, removing supernate, taking out precipitate, washing with a small amount of ethanol, drying by hot air or vacuum, and finally crushing and sieving to obtain the polysaccharide refined from the total ganoderma spore powder; or, the concentrated solution is dried by adopting a spray drying method in one step to obtain the polysaccharide refined from the total ganoderma lucidum spore powder.

4. The purified polysaccharide composition of whole ganoderma lucidum spore powder prepared according to the method of claim 3.

5. Use of the polysaccharide composition of whole ganoderma lucidum spore powder according to any one of claims 1 to 2 or 4 in the preparation of antitumor drugs and foods for the prevention and treatment of tumors.

6. The application of the polysaccharide composition prepared from ganoderma lucidum spore powder in preparing antitumor drugs and foods for tumor prevention and treatment according to claim 5, wherein the polysaccharide composition further comprises a pharmaceutically acceptable adjuvant or carrier.