CN107602719A - A kind of ganoderma lucidum fruitbody refined polysaccharide with notable adjunct antineoplastic activity and its preparation method and application - Google Patents

Abstract

The present invention relates to a kind of ganoderma lucidum fruitbody refined polysaccharide with notable adjunct antineoplastic activity and its preparation method and application.The refined polysaccharide is mainly by mannose, Glucosamine, glucuronic acid, glucose, galactolipin, xylose, fucose in molar ratio 3~4：1：1.3~1.7：56~65：4~6：1.3~1.8：The heteroglycan of 2.5~3.5 compositions, absolute purity is more than 52%, 7500~8500D of weight average molecular weight, molecular weight distributing index 1.3~1.5.The preparation method includes：Hot water Dynamic Extraction, coarse filtration are removed slag, refined filtration is clarified, resin bed of connecting takes off protein decolouring, milipore filter concentrates impurity elimination, spray drying or alcohol precipitation and air-dried.This method intelligence, energy-conservation, can solve industrialization problem, have notable scale advantage, finished product security, validity, controllability, stability are good.The polysaccharide has notable functional activity in terms of adjunct antineoplastic, available for the exploitation of tumor prevention and treatment medicine, can carry out the exploitation of association area food and health products.

Description

A kind of ganoderma lucidum fruitbody refined polysaccharide and its system with notable adjunct antineoplastic activity Preparation Method and application

Technical field

The present invention relates to a kind of ganoderma lucidum fruitbody refined polysaccharide with notable adjunct antineoplastic activity and preparation method thereof And application, belong to extraction and separation technology and medicine food application and development field.

Background technology

Effect of the edible mushroom to human health is increasingly by the extensive attention of international community.Edible fungi polysaccharide is as edible One of main active of bacterium, as the study hotspot of World Health industry.Numerous studies document shows that edible mushroom is more Sugar has immunological regulation, antitumor, antiviral, anti-aging, and a variety of biologies such as anti-oxidant, reducing blood lipid, hypotensive, hypoglycemic are lived Property.At present, the state such as the Asian countries headed by China and America and Europe is to a variety of foods such as ganoderma lucidum, mushroom, Agricus blazei, grifola frondosus More systematic research is carried out with granulose, wherein ganoderma lucidum and GL-B turns into research with its unique healthcare function Focus.

Modern pharmacology and clinical practice confirm, GL-B be ganoderma lucidum strengthen the body resistance to consolidate the constitution, strengthening by means of tonics, the master to promote longevity Want effective substance.GL-B has stimulation of host non-specific resistance, immune idiosyncrasy and suppresses transplantation tumor life The characteristics such as activity are managed, immunity of organisms and hypoxia-bearing capability can be improved, there is significant medicines and health protection application value, social benefit And economic benefit.

Ganoderma lucidum fruitbody is made up of main components such as chitin, cellulose, hemicellulose, lignin, close structure and work Property polysaccharide be attached on the inside of cell membrane, and it is most of molecular complex is directly formed with protein, extraction purification difficulty is larger.Mesh Preceding Extraction method of ganoderan mainly has：Hot water extraction method, extraction, ultrasound assisted extraction method, microwave―assisted extraction, Enzyme process assisted extraction.

Although extraction recovery rate is higher, equipment is corrosion-resistant to require high, and cost is expensive, and the three-dimensional of active polysaccharide is stood Body structure easily changes, and finished product color is deep, impurity is more, is unfavorable for being received by consumer, but also fixed-end forces be present Problem, operating cost accordingly improve.It is bright that although ultrasonic extraction and microwave loss mechanisms have, recovery rate is high, extraction time is short etc. Aobvious advantage, but single treatment amount is low, and equipment pays for cost height first.Also, the thermal transition efficiency of ultrasonic extraction is low, does not meet energy-conservation It is required that noise is big, it is unfavorable for health.Microwave Extraction is substantially also in laboratory stage, because the penetration depth of microwave has Limit, is only capable of penetrating 4.5cm in pure water, is not suitable for big tank extraction, and the so-called Microwave Extraction Equipment introduced at present in production is basic Only it is to replace steam or conduction oil to provide thermal source with microwave, is not microwave extraction technology truly, still falls within heat The category of water extraction, it is impossible to produce from cell interior and destroy cell membrane, promote the effect of active ingredient release.So to current Untill ultrasound assisted extraction and microwave radiation exaraction not yet realize real industrial applications.

Enzymatic Extraction belongs to efficient cryogenic extractive technique, and the yield of Enzymatic Extraction is very high, but Enzymatic Extraction is equally with aobvious The defects of work, including：(1) dissolution rate of impurity and the dissolution rate of active principle are directly proportional, have been even more than active ingredient increase Multiple, introduce substantial amounts of impurity, be unfavorable for purifying and prepare high-purity product, therefore Enzymatic Extraction technology is less is actually applied In the kind for needing later-period purification to separate.(2) Enzymatic Extraction needs medicinal material is broken as far as possible, to allow enzyme to greatest extent Effective contact is formed with substrate, so as to improve product yield, but meets that the medicinal material broken enough of enzyme process requirement is real to production Border brings many troubles, for example blocks liquid outlet and filter medium, production is not normally carried out, such as the water content of the dregs of a decoction It is high compared with the medicine materical crude slice class dregs of a decoction, target component because extract solution is adsorbed by the dregs of a decoction and caused loss is also big, fixed-end forces cost is also entered One step increase etc..(3) common enzyme will play preferable hydrolysis result on the market at present, be required for adjusting pH value to certain enzyme Near optimum PH, such as the optimum PH of pectase, cellulase, pepsin is below 4, equally can under heating environment Cause some unknown impurity that chemical property occur to change, because impurity is unknown, the reaction product of it and soda acid may also It is unknown, effective monitoring can not be formed in produce reality, therefore certain Clinical practice risk may be brought.(4) one is entered Step ground, applicant is in production practices, it was also found that the taste and flavor of enzymatic extract is significantly worse than direct decocting, it may be possible to wind There occurs the change of essence in Acid-Base System for taste composition, such as amino acid, nucleotides, volatile oil.(5) for cost viewpoint, The enzyme that can apply in production is the direct enzyme that tentatively simple purification is just launched immediately after microbial fermentation, these Enzyme is actually the dominant complex enzyme of a certain enzyme, rather than the price that biochemistry uses is high, the single specificity of high-purity Enzyme, this complex enzyme is used in production, the same risk being decomposed with desired polysaccharide.

The deficiency for more than, the present invention provide a kind of ganoderma lucidum fruitbody refined polysaccharide with notable adjunct antineoplastic activity And its preparation method and application.

The content of the invention

The present invention reaches above-mentioned purpose by following scheme：

In the first aspect of the present invention, there is provided a kind of ganoderma lucidum fruitbody with notable adjunct antineoplastic activity is refined more Sugar, the saccharide portion of the ganoderma lucidum fruitbody refined polysaccharide is mainly by mannose, Glucosamine, glucuronic acid, grape 7 kinds of monose such as sugar, galactolipin, xylose, fucose in molar ratio 3~4：1：1.3~1.7：56~65：4~6：1.3~1.8： The heteroglycan of 2.5~3.5 compositions, ganoderma lucidum fruitbody refined polysaccharide is calculated with above-mentioned 7 kinds main absolute contents for forming monose Purity, up to more than 52%；Only containing a fraction, weight average molecular weight is 7500~8500D, molecular weight distributing index D is 1.3~ 1.5, belong to narrow ditribution；Protein content (BCA methods) in UV-VIS spectrophotometry measure ganoderma lucidum fruitbody refined polysaccharide Less than 25%, polysaccharide (Anthrone-sulfuricacid method) content is more than 65%.

Preferably, a kind of ganoderma lucidum fruitbody refined polysaccharide with notable adjunct antineoplastic activity, its saccharide portion are main Be by 7 kinds of monose such as mannose, Glucosamine, glucuronic acid, glucose, galactolipin, xylose, fucose in molar ratio 3.67：1.00：1.53：61.35：5.00：1.56：The heteroglycan of 2.92 compositions, per 100g ganoderma lucidum fruitbodies refined polysaccharide difference 2.72g containing mannose, Glucosamine 0.79g, glucuronic acid 1.39g, glucose 43.71g, galactolipin 3.62g, xylose 0.90g, fucose 1.82g, the pure of ganoderma lucidum fruitbody refined polysaccharide is calculated with above-mentioned 7 kinds main absolute contents for forming monose Spend for 54.96%；Only contain a fraction, the equal relative molecular weight (Mw) of weight is 8098D, more than the accumulative section weight of 5KD molecular weight Amount area accounts for 61.18%, and molecular weight distributing index D is 1.42, belongs to narrow ditribution；UV-VIS spectrophotometry determines ganoderma lucidum Protein content (BCA methods) 23.53% in fructification refined polysaccharide, polysaccharide (Anthrone-sulfuricacid method) content 68.08%.

Verified, above-mentioned polysaccharide has significant adjunct antineoplastic activity, and its activity is significantly larger than the polysaccharide of prior art.

In the second aspect of the present invention, there is provided a kind of ganoderma lucidum fruitbody refined polysaccharide with notable adjunct antineoplastic activity Preparation method, the described method comprises the following steps：

(1) hot water Dynamic Extraction Thick many candies：The ganoderma lucidum fruitbody residue for the re-dry after alcoholic extraction of learning from else's experience, adds residue weight The purified water of 10~30 times of amount, lead to steam, stirring or interval, which start feed pump, forces circulated in countercurrent, until maintaining the micro-boiling time to reach Steam off after to 2~4h；

(2) coarse filtration technique realizes slag-liquid separation：After the completion of extraction, with 0.5~5 μm of plate filter or bag of filtering accuracy Formula filter carries out coarse filtration, and alternatively, residue by step (1) method repeats extraction 1~2 time and coarse filtration again, merging coarse filtration liquid.

(3) refined filtration technique realizes that decoction is clarified and removes the supramolecular impurity of thermosol cold analysis：Coarse filtration liquid with 200~ 800nm tubular ceramic microfiltration membranes or the micropore folding type filter element of 0.1~0.22 μm of filtering accuracy carry out refined filtration clarification, obtain clear Clear liquid；

(4) resin bed absorbing process of connecting realizes de- protein decolouring：Clarified solution use series connection polyamide (such as 60~ The polyamide of 100 mesh) and the de- protein decolouring of resin anion (R.A.) (such as D392 resin anion (R.A.)s), first flow through polyamide Bed, it is rear to flow into resin anion (R.A.) bed；By dispensing gauge, per the minimum outfit 250mL polyamides of kg fructification residues and 50mL D392 resin anion (R.A.)s, polyamide resin column filling height is 1~2 meter, resin anion (R.A.) post 20~40cm of bed height, uses acetic acid Or the presetting clarified solution pH value of citric acid, to 4.5~6.5, loading adsorption flow rate is 4~10BV/h of total post bed, collects efflux；

(5) film ultrafiltration technology realizes the concentration of desired polysaccharide and removes micromolecular water solubility impurity：Efflux milipore filter Carry out being concentrated to give concentrate, the molecular cut off of milipore filter is 3500~5000D；

(6) drying process：Concentrate use alcohol precipitation after heated-air drying or vacuum drying, also can Direct spraying be drying to obtain.

In a preferred embodiment, a kind of ganoderma lucidum fruitbody refined polysaccharide with notable adjunct antineoplastic activity Preparation method, it the described method comprises the following steps：

(1) hot water Dynamic Extraction Thick many candies：The ganoderma lucidum fruitbody residue of the re-dry after edible alcohol extracts in advance is taken to fit Amount, the purified water of 10~30 times of residue weight is added, lead to steam, stirred with 10~30r/min speed or at interval of 20~40min Start feed pump 3~8min of forced circulation, the steam off after maintaining the micro-boiling time to reach 2~4h；

(2) coarse filtration technique realizes slag-liquid separation：After the completion of extraction, less than 50 DEG C are cooled to, extracts supernatant out, with filtering essence The plate filter or bag filter of 0.5~5 μm of degree carry out coarse filtration, and alternatively, residue repeats extraction 1 by step (1) method ~2 times and coarse filtration again, merge coarse filtration liquid；

(3) refined filtration technique realizes that decoction is clarified and removes the supramolecular impurity of thermosol cold analysis：Coarse filtration liquid with 200~ 800nm tubular ceramic microfiltration membranes or the micropore folding type filter element of 0.1~0.22 μm of filtering accuracy carry out refined filtration clarification, tubular type pottery The Operating parameters of porcelain microfiltration membranes are 40~60 DEG C, 0.30~0.50Mpa of operating pressure, 3~4.5m/s of crossflow velocity of temperature, When 120~300LMH of percolate flux, raffinate residue about 30L, add purified water about 30L to dilute, continue clarification activities to raffinate and remain Remaining about 30L, alternatively, repeats and once dilutes clarification activities, to ensure desired polysaccharide all by reducing technique damage as far as possible Consumption；

(4) resin bed absorbing process of connecting realizes de- protein decolouring：Clarified solution using 60~100 mesh polyamides and D392 resins are packed into post bed respectively, and are cascaded, and carry out de- albumen and decolouring simultaneously using dynamic column absorbing process, first Polyamide column is flowed through, then flows into D392 resin columns；By the dispensing gauge of ganoderma lucidum fruitbody residue, 1kg fructification residues are often handled Polyamide 250mL and the D392 resin anion (R.A.) of pre-treatment has been completed in the clarified solution subsistence level configuration of extraction gained 50mL, polyamide resin column filling height is 1~2 meter, D392 resin anion (R.A.) post 20~40cm of bed height, with acetic acid or lemon For the presetting clarified solution pH value of acid to 4.5~6.5, temperature is kept for 20~35 DEG C, clarified solution loading adsorption flow rate for total post bed 4~ 10BV/h, collect efflux；End is collected, continues the water elution 2BV with pH value 4.5~6.5, continues to collect efflux, and close And efflux, to displace de- protein decolouring liquid as far as possible, reduce the process loss of desired polysaccharide；

(5) film ultrafiltration technology realizes the concentration of desired polysaccharide and removes micromolecular water solubility impurity：Efflux milipore filter Concentrated, the molecular cut off of the milipore filter is 3500~5000D, and Operating parameters are 25~45 DEG C of temperature, operation 0.2~0.5Mpa of pressure, 2.5~4m/s of crossflow velocity, 4~20LMH of percolate flux, when being concentrated into about 30L, add purified water about 30L dilutes, and continues to be concentrated into about 30L, alternatively, repeats and once dilute concentration step, to eliminate small molecule to greatest extent Water-solubility impurity, finally rinse milipore filter with 15L purified waters and displace whole concentrates, to reduce process loss as far as possible；

(6) drying process：The alcohol that 4~9 times of amounts of original volume are slowly added under concentrate stirring at low speed carries out alcohol precipitation, stands Overnight, supernatant is pumped, precipitation, edible alcohol washing, heated-air drying or vacuum drying is taken out, finally pulverizes and sieves and produce；Or Person, concentrate directly complete drying using the step of spray drying process one.

In above-mentioned preparation method, the post footpath in step (4) according to filler demand reasonable selection or by multicolumn simultaneously Connection is realized.

In above-mentioned preparation method, the raw material in the step (1) is after ganoderma lucidum fruitbody is utilized into alcoholic extraction, to carry out Slag-liquid separation, residue are used as extraction raw material after drying.

For example, in a preparation method embodiment, by ganoderma lucidum fruitbody shave or it is appropriate crush, add 10 times of weight Edible alcohol refluxing extraction 2 times, each 2h, extract extract solution out, steam is passed through to remaining residue, and to displace alcohol as far as possible residual Stay, dry to after substantially without alcohol taste, less than 60 DEG C hot-air seasonings, obtain the ganoderma lucidum of the described advance re-dry after alcoholic extraction Fructification residue, the initiation material as preparation method of the present invention.

GL-B is the glucan with helical form spatial configuration (three-dimensional structure) being made up of three strands of monose chains, and it is vertical Body configuration is similar to RNA, DNA, is a kind of macromolecular compound.Have document report at present, purify and obtain from ganoderma lucidum fruitbody The unimodal polysaccharide obtained, after chemistry or biodegradation, the presence of amino acid is detected, inferring in its structural framing to gather Conjunction has polypeptide, protein.After the stereochemical structure of GL-B is opened and is changed into planar structure or linear structure, or ganoderma lucidum After proteoglycans that may be present, the configuration in peptide glycan activated centre change in polysaccharide, many pharmacology of GL-B are lived Property also weaken or disappear therewith, so, applicant design in the extracting and developing of GL-B, purifying, drying and other steps In, using gentle physical method, to protect its three-D space structure and activated centre configuration to greatest extent, acquisition have compared with The finished product of high pharmacological activity.

Applicant detects discovery in process exploitation, and when ganoderma lucidum fruitbody is extracted using hot water, the polysaccharide in extract solution is dense Degree is far below protein concentration (polysaccharide 0.67mg/mL, protein 1.44mg/mL), or even the polysaccharide-protein ratio of indivedual raw materials reaches Into 1:2.5.The content of Thick many candies (survey by anthrone-sulphuric acid method in the GL-B that traditional water extract-alcohol precipitation drying process is obtained It is fixed) it is only 28~35%, and protein content is up to 38%~51%.By obtained GL-B with being filtered after boiling water back dissolving, filter Slag drying is weighed, and finds not redissolving the color of thing close to ater, weight up to throws the 7~13% of GL-B, through physics and chemistry Differentiate, it is found that this does not redissolve the mixture that thing belongs to protein, macromolecular pigment and cellulose.Filtrate is concentrated to alcohol precipitation again to do After dry, continue to determine the content of Thick many candies, it is found that content is increased to 38~47% sections, protein content is reduced to 32nd~44% area Between, but still it is not reaching to the purity required by active component new drug development.As can be seen here, in production practices, want to obtain Quality preferably, GL-B of high-purity, high activity, it is necessary to pointedly carry out suspended things removing, albumen removing and pigment Removing is handled.

The impurity source of Thick many candies product is mainly impurity protein, pigment, cellulose, inhereditary material and small-molecule substance etc. Non-active ingredient, the exterior quality of GL-B product is greatly have impact on, reduce the content of active polysaccharide so as to reduce Clinical drug effect.At present, being related to the discoloration method of GL-B mainly has organic reagent preextraction, charcoal absorption, dioxygen water oxygen The methods of change, chlorite oxidation are decolourized.It is applicant's understanding that there is the defects of certain in these methods：Oxidative decoloration method can be broken The three-dimensional active structure of polysaccharide is broken, makes its many-sided reduction evident in efficacy；Charcoal absorption belongs to traditional decolouring technology, is A kind of indifference, the technology without adsorptive selectivity, while the removal of impurity is gone, the loss of polysaccharide is also very big；In ganoderma lucidum fruitbody Pigment be water miscible macromolecular pigment, organic reagent preextraction decolorizing effect is little.

At present, in ganoderma lucidum crude polysaccharide protein removal methods, use Savag methods, this method mostly in laboratory stage Using a large amount of organic solvents and cumbersome, treating capacity is low, and manpower consume is big, is not belonging to scale industrialization technology.Producing Stage mainly uses charcoal absorption, and de- albumen while decolourizing, actual removal efficiency is low, and polysaccharide loss is big.Also have one in addition Staticly settle, high speed centrifugation the methods of, belong to time-consuming method longer, single treatment amount is extremely low, have a strong impact on production effect Rate.

Therefore, applicant thinks, at present, is essentially consisted in for the extraction purification difficult point of GL-B in efficient removal While the impurity such as albumen, pigment, retain polysaccharide as much as possible, improve polysaccharide yield, and keep the activity of polysaccharide.

On this basis, applicant's analysis finds, extraction of the prior art for GL-B, main method be divided into Under several classes：

1st, as described in patent application CN201210557813, using the extraction of ultrasonic wave associated proteins enzyme, 10KD is used again The scheme of permeate, concentration and alcohol precipitation is collected in ultrafiltration, is disadvantageous in that and is used zymyhydrolyzed protein, and it is small to form hydrolysate Molecular polypeptide and amino acid, it is still present in permeate in 10KD ultrafiltration technologies, is not sloughed really.This kind of method is present Do not decolourized targetedly and/or Deproteinated problem, prepared GL-B purity is limited and/or finished appearance product Matter is general.

2nd, as described in patent application CN201410778126, cold analysis removal of impurities is carried using enzyme, then alcohol precipitation back dissolving adds tannic acid Secondary precipitation is cleaned, supernatant activated carbon decolorizing, and vacuum drying scheme is concentrated after 30KD ultra filtering clarifyings.Through looking into, tannic acid molecule Measure as 1701, and belong to two molten thing of alcohol water, described according to its technique, the tannic acid impurity being newly introduced into can not remove in subsequent technique Totally, finished product is polluted.Patent CN201510764260 uses sodium chlorite-glacial acetic acid system oxidative decoloration, then ultrasonic extraction alcohol Heavy scheme, active polysaccharide structure is destroyed big.This kind of method introduces new impurity or condition when removal of impurities be present and acutely caused very much The problem of active polysaccharide is destructurized.

3rd, as described in patent application CN200510014592, using pre-treatment, micro-filtration, freezing separation, UF membrane, film The technical matters concentrate, be concentrated under reduced pressure, being dried under reduced pressure, this kind of technique imurity-removal needs will all unconcentrated extract solution freezings To 0 degree once, cost is very big, the difficulty of equipment, production efficiency it is low, can be sayed without any volume production advantage.In another example patent CN201310742219 combines ring current method by high speed centrifugation and removes isolating protein, using 6000~14000r/min centrifugal clarifications； If belonging to commercial processes, As-Is analysis is equipped according to current industry, the centrifuge of this rotating speed can be reached in production practices Only tube centrifuge, and the single treatment capacity of tube centrifuge maximum at present is less than 25L, if needed per 25L extract solutions 10-120min is centrifuged, treating capacity is less than 150L, from production efficiency and energy cost, no volume production advantage per hour.As specially Profit application CN201510822880 adds 15~20 times of amount distilled water to extract 2 times using raw material, centrifuging and taking supernatant, adds supernatant Liquid accumulates the technical matters of 85~95% ethanol progress subsequent treatment of 3~5 times of amounts；The ethanol of the technique is extrapolated accordingly most Low usage amount is that reagent consumption expense is much larger than output benefit, without any volume production advantage per kg medicinal material 90L.This kind of method is all deposited It is too high in cost, the problem of no industrialization prospect.

4th, as described in patent CN201510541581 applications, after being redissolved again using water extract-alcohol precipitation, received with hollow-fibre membrane Collect the trapped fluid between 50,000~80,000, finally with activated carbon decolorizing, the technical matters for filtering and being spray-dried；This technical matters mesh Mark polysaccharide molecular weight cut coverage is too narrow, and inevitable yield is very low, and carries out adsorption bleaching from activated carbon, and polysaccharide loss is big.

In another example described in patent application CN201410628578, using cellulase and compound protease, and simultaneously United microwave method extracts the technical scheme of GL-B.Inferred according to the general understanding of industry practitioner, Enzymatic Extraction Impurity how much be directly proportional to the increased multiple of yield, yield is higher, and impurity is higher, and the patent configuration activated carbon inhale Attached impurity removal process, belong to non-selective adsorption impurity removing technology, specificity removal of impurities effect is not too much high, and general labourer is removed using activated carbon The shortcomings that skill, also existence time are long, loss is big, activated carbon is difficult to filtering.

5th, as described in patent application CN201110161167, using multiplex-enzyme extraction, ultrafiltration retention 100,000~1,000,000 Compound molecular weight, the technical matters of weak-base anion-exchange resin chromatogram purification；Its polysaccharide molecular weight section blocked is not The polysaccharide enrichment section of raw material, therefore yield is not high, while also there is the general defect of Enzymatic Extraction.

As described in patent application CN201110184693, using first water extraction, then concentration of alcohol is adjusted to 20~30% Precipitation, obtains light Ganoderma lucidum polysaccharide of high molecular weight, according to the general sex experience of industry practitioner, can 20~30% second The polysaccharide molecular weight being precipitated out in alcoholic solution is at least more than million grades, and the polysaccharide section is not the polysaccharide enrichment section of raw material, therefore Yield is not high.

The theoretical yield in GL-B in above-mentioned two patent application is all relatively low.

In summary, extract by research of the applicant for GL-B structure, molecular weight etc. and in the prior art The analysis of method, it has been found that in extracting method, extraction of the hot water extraction method to polysaccharide is gentleer, survivable polysaccharide Structure, still, there is the problem of protein content is high in polysaccharide extraction liquid in such mode, for this problem, prior art Generally use enzymolysis overcomes, however, applicant passes through theory analysis and practice, it is believed that：Enzyme-extracting solution by protein digestion into Amino acid, polypeptide, but prove that the active component of GL-B is holosaccharide without definite evidence at present, educational circles's analysis is purified into Unimodal polysaccharide contains certain protein, i.e. active component is likely to be by the polysaccharide of polypeptide, protein structureization modification, enzymolysis During albumen, the fracture of peptide bond is random, can both cut off impurity protein, can also cut off the activity of activated protein glycan at any time Center, so, enzymolysis step takes chemical valence link fracture removing, has the steric configuration that may change active macromolecules thing, So as to influence the risk that activated centre plays function.

Thus, applicant carries out de- protein decolouring after hot water extraction using resin, and by the research hair to resin Existing, using single strong polymeric adsorbent, such as during Anion-adsorption resin, the loss for polysaccharide is larger, it is understood that there may be peptide gathers Sugar, the loss risk of proteoglycans constituents, are combined, Cai Nengda using the moderate polyamide of absorption affinity with resin anion (R.A.) To that can remove pure impurity protein and glycoprotein as far as possible, and can, which is tried one's best, retains the purpose of proteoglycans and peptide glycan.

Applicant further repeatedly practises discovery, and it is 1~2 meter to select polyamide resin column filling height, resin anion (R.A.) When 20~40cm of post bed height series connection resin carries out de- protein decolouring, it is larger that egg white by proportion in preferably removing molecule can be reached Pure impurity protein and glycoprotein, while retain the effect of the less proteoglycans of egg white by proportion and peptide glycan again, decolourize simultaneously Effect is also preferable.

Applicant also found that GL-B is present in ganoderma lucidum fruitbody, is by multiple differences in GL-B extraction The mixture of the polysaccharide molecule composition of molecular size range, using HPGPC methods to GL-B made from traditional aqueous extraction-alcohol precipitation technology Molecular weight distribution investigation has been carried out, it is found that GL-B is substantially different from lucid ganoderma spore powder polysaccharide and lucidum mycelium polysaccharide, its The molecular weight of polysaccharide is smaller；According to appearance time, Wo Menchu corresponding to the different molecular weight dextran reference substance of narrow ditribution Step find the overwhelming majority GL-B molecular weight appearance time be respectively positioned on dextran D5000 and D50000 appearance time it Between, i.e., molecular weight is between 5.22~48.6KD.

Based on this, when developing preparation method, particularly clarification process, purifying process, concentration process steps when, to used The aperture of material carries out planned selection all referring to above-mentioned molecular weight data, realizes most preferred effect.

In the third aspect of the present invention, there is provided what a kind of above-mentioned preparation method was prepared there is notable adjunct antineoplastic to live The ganoderma lucidum fruitbody refined polysaccharide of property, the polysaccharide and the polysaccharide properties that first aspect present invention provides are same or similar.

Preferably, in terms of mass fraction, the polysaccharide retention rate 85.82% of above-mentioned preparation method preparation, albumen removal efficiency 82.61%, pigment removal rate 87.14%.

Preferably, the ganoderma lucidum fruitbody refined polysaccharide yield that prepared by above-mentioned preparation method is about 1.86% or so.

In the fourth aspect of the present invention, the present invention provides a kind of ganoderma lucidum fruitbody refined polysaccharide in terms of adjunct antineoplastic Purposes.In the fifth aspect of the present invention, the present invention provide a kind of ganoderma lucidum fruitbody refined polysaccharide prepare adjunct antineoplastic medicine, Purposes in terms of food, health food.

The ganoderma lucidum fruitbody refined polysaccharide is in terms of adjunct antineoplastic, such as tumor animal adjunct antineoplastic functional activity Evaluation result shows that ganoderma lucidum fruitbody refined polysaccharide joint paclitaxel treatment group is shown significantly within the whole test period Suppress the function of malignant breast carcinomas tumour growth, independent Paclitaxel Chemotherapy group is significantly stronger than the effect of therapeutic alliance group, and can be special Immune system caused by the growth of different in nature reversing tumor and the independent chemotherapy of taxol and hematological system main biochemical Index for examination value It is abnormal, normal value is close to, this active polysaccharide can be applied to tumor prevention and treat the exploitation of medicine, can also be applied to correlation The exploitation of field food and health products.

Above-mentioned ganoderma lucidum fruitbody refined polysaccharide is in terms of adjunct antineoplastic and otherwise effect is including pernicious breast The BALB/c female mices of adenocarcinoma cell (4T-1) are as experimental animal model, using normal mouse gavage purified water as normally Group, using tumor-bearing mice gavage purified water as model group, using the independent administering paclitaxel of tumor-bearing mice as positive controls, with warp Ganoderma lucidum fruitbody refined polysaccharide joint administering paclitaxel carries out adjunct antineoplastic as treatment group made from technical solution of the present invention Functional activity is evaluated.Final result shows that ganoderma lucidum fruitbody refined polysaccharide combines paclitaxel treatment group within the whole test period Show the significant function of suppressing malignant breast carcinomas tumour growth, be significantly stronger than independent taxol the effect of therapeutic alliance group Chemotherapy group, and immune system and hematological system main biochemical caused by the specific reversing tumor growth of energy and the independent chemotherapy of taxol The exception of Index for examination value, as spleen stress become big, leucocyte substantially increases, lymphocyte subpopulation distribution changes, outer All blood erythrocyte quantity declines, peripheral blood hemoglobin concentration reduces etc..

Therefore, the ganoderma lucidum fruitbody refined polysaccharide of the invention under effective dose is used alone, or with other activearms The compound for dividing and/or being formed with its pharmaceutically acceptable auxiliary material, carrier is used in combination, and can be applied to tumor prevention and controls The exploitation of medicine is treated, can also be applied to the fields such as the exploitation of association area food and health products.

Compared to the prior art, the invention has the advantages that：

(1) ganoderma lucidum fruitbody for being used to extract in production at present, which is substantially, completes the fructification after dusting, and cap is extremely Lignifying, fibrosis, form class flocculence after crushing, coming off for chopped fiber is easily caused during stirring is extracted, in addition Impurity in ganoderma lucidum fruitbody Aqueous extracts is mainly protein, oxidized form pigment, gel state cellulose and hemicellulose, pectin Deng, these molecules belong to macromolecular, the characteristics of thermosol cold analysis be present, the selection application to filter medium and filtering technique, Ask higher；Select conventional filter medium or use end filtration technology, impurity is easy to blocking filtering medium, led to not Normal production；The clarification process that preparation method of the present invention is combined using coarse filtration, refined filtration, post absorption, and coarse filtration and refined filtration work Skill all employs cross-flow filtration mode and has selected high-precision filter medium as far as possible, by adjusting relevant parameter to preferable Scope, it can reach and eliminate these nonactive or low activity big molecular impurity purposes, obtain good clarifying effect (transmissometer Detection, reading are less than No. 0.5 standard turbidity solution), overcome impurity blocking filtering medium to cause this industry that can not normally produce Change problem.

(2) GL-B is the glucan with helical form spatial configuration (three-dimensional structure) being made up of three strands of monose chains, Prove that the active component of GL-B is holosaccharide without definite evidence at present.Existing document report, it is pure from ganoderma lucidum fruitbody Change the unimodal polysaccharide obtained, after chemistry or biodegradation, detect the presence of certain proportion amino acid, i.e. active component It is likely to be by the polysaccharide of polypeptide, protein structureization modification, i.e. peptide glycan, proteoglycans.When the stereochemical structure of GL-B Be opened and be changed into after planar structure or linear structure, or in GL-B proteoglycans, peptide glycan that may be present work After the original valence bond structure at property center is modified, many pharmacological activity of GL-B are also with disappearing and reduce.

2 points of risks that may be present based on more than, in the extracting and developing of GL-B, purifying, concentration produce reality, The present invention avoids venturesomely taking chemical valence link fracture and bio-enzyme degradation scheme in technological design, and such as enzyme process, oxidation must From gentle physical method, to protect its three-D space structure and activated centre configuration to greatest extent, acquisition has higher medicine Manage the finished product of activity.And both met technological process, scale requirement on producing, and can gently reach what preferable impurity removed Scheme, it is preferred with physical absorption, physics screening scheme.When physical absorption removing impurities designs, avoid using it is single adsorb by force fill out Material, such as zwitterion resin, and select it is moderate to protein adsorption power, strong filler is adsorbed to pigment, design preferable technique side Case combination, Optimizing Process Parameters, pigment can be removed to greatest extent by reaching, and preferably remove pure impurity protein and glycoprotein, and More retain the purpose of proteoglycans and more peptide glycans.

Preparation method of the present invention utilizes energy of adsorption using technical scheme associated with polyamide and D392 resin anion (R.A.)s The moderate polyamide of power utilizes the stronger D392 resin anion (R.A.)s of absorption affinity as the Deproteinated pioneer main force filler that decolourizes As the Deproteinated leakproof filler of winding up of decolourizing, by the use ratio of both resins of optimization, dress post blade diameter length ratio, adsorption temp, Adsorb pH value, adsorption flow rate, with reach removing polyanionic macromolecule pigment, pure impurity protein and egg white by proportion as much as possible compared with High glycoprotein, and retain the relatively low peptide glycan of egg white by proportion, proteoglycans and holosaccharide.

According to preparation technology of the present invention, the ganoderma lucidum fruitbody refined polysaccharide of gained is purified, had both met medicinal content requirement, Natural activity is remained to greatest extent again, reaches medicinal function activity criteria.

(3) preparation method of the present invention, adsorbed using ceramic micro filter film or precise micropore foldable filter element, post, be organic Milipore filter, the step impurity removal process of alcohol precipitation four, can remove the big molecular impurity of thermosol cold analysis, and and can is removed easily by resin adsorption Intermediate molecular weight impurity, can also remove the water-solubility impurity of small molecule；And clarify and removal of impurities, concentration and the same stepping that cleans OK；It can be made the ganoderma lucidum fruitbody refined polysaccharide of about more than 52% absolute purity, obtain heavy metal index, ash content index, miscellaneous The polyose that matter index is all significantly improved, and can save the production time, improve production efficiency.

(4) preparation method of the present invention, clarification, impurity elimination, concentration technology use membrane technology and post adsorption scheme, category In temperature production technology, can ensure that the 3-D solid structure of active polysaccharide will not destroy, and then retain its medicine to greatest extent Reason activity, while energy consumption has been significantly reduced, belong to energy-conservation manufacturing technology.

(5) preparation method of the present invention, it is to be designed from big produce reality needs, through multiple pilot-scale Above actual production verifies that whole process can realize pipeline, automation, intellectualized operation, has technique simple possible, is adapted to rule The advantages of mould industrialization, low overall operation cost, product yield is of about more than 1.8%.

(6) polysaccharide that the inventive method is prepared, both using comprehensive and strict polysaccharose substance quality control standard To monitor final product quality, also carrying out the evaluation of adjunct antineoplastic functional activity by tumor-bearing model proves its effect, obtains Refined polysaccharide has the characteristics of security, validity, controllability, stability.

In summary, preparation method of the present invention, requirement of the national industrial policies on intelligence, energy-conservation had both been met, Solve industrialization problem existing for current produce reality again simultaneously, there is significant scale industrialization advantage, and it is obtained Finished product meets the requirement on security, validity, controllability, stability in food and medicine quality management system.

Brief description of the drawings

Fig. 1 is GPC molecular weight universal calibration curves in embodiment 4.

Fig. 2 is the RID collection of illustrative plates of ganoderma lucidum fruitbody refined polysaccharide in embodiment 4.

Fig. 3 is ganoderma lucidum fruitbody refined polysaccharide graph of molecular weight distribution in embodiment 4.

Fig. 4 is ganoderma lucidum fruitbody refined polysaccharide molecular weight buildup weight distribution curve in embodiment 4.

Fig. 5 is the ultraviolet chromatogram of derivative compound of 12 kinds of mixing monose reference substances in embodiment 5.

Fig. 6 is the ultraviolet chromatogram of derivative compound of ganoderma lucidum fruitbody refined polysaccharide in embodiment 5.

Fig. 7 is the adjunct antineoplastic functional activity assessment technique route map of embodiment 6.

Fig. 8 is that each group tumour in embodiment 6 contrasts photo.

Fig. 9 is experimental animal lotus knurl Volume Changes curve in embodiment 6.

Figure 10 is the tumor weight figure of each group in embodiment 6.

Figure 11 is the tamor index figure of each group in embodiment 6.

Figure 12 is the gross tumor volume figure of each group in embodiment 6.

Figure 13 is the index and spleen index figure of each group in embodiment 6.

Figure 14 is the CD19% figures of each group in embodiment 6.

Figure 15 is the CD3% figures of each group in embodiment 6.

Figure 16 is the CD4% figures of each group in embodiment 6.

Figure 17 is the CD8% figures of each group in embodiment 6.

Figure 18 is the CD4/CD8 figures of each group in embodiment 6.

Figure 19 is WBC (leucocyte) level view of each group in embodiment 6.

Figure 20 is RBC (red blood cell) level view of each group in embodiment 6.

Figure 21 is HGB (hemoglobin) level view of each group in embodiment 6.

Figure 22 is PLT (blood platelet) level view of each group in embodiment 6.

Embodiment

The present invention is further described below in conjunction with specific embodiment.

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

Unless otherwise instructed, the present invention is using " BCA method determination of protein concentration kits (enhanced) " measure protein Content.

Unless otherwise instructed, the present invention uses Anthrone-sulfuricacid method, by "《Chinese Pharmacopoeia》Under the one ganoderma lucidum item of version in 2015 【Assay】Step in polysaccharide " detects its Thick many candies content；Using 9 kinds of dextrans of gradient molecular weight it is narrow mark mark product as Control, the HPGPC methods (differential refraction detector) connected using double gel columns detect its mean molecule quantity and molecular weight distribution；With 12 kinds of monose determine its monose composition and absolute purity as reference substance using PMP-HPLC derivatization methods.

Embodiment 1：

By ganoderma lucidum fruitbody shave or the appropriate edible alcohol refluxing extraction 2 times, each 2h for crushing, adding 10 times of weight, Extract solution is released, be passed through steam into remaining fructification residue displaces alcohol residue as far as possible, dries to substantially without alcohol taste Afterwards, less than 60 DEG C hot-air seasonings, the fructification residue pre-processed.

Fructification residue 50kg is standby, adds 500L purified waters, and logical to be steam heated to boiling, 15r/min rotating speeds stir simultaneously Micro-boiling 3h is maintained, steam off, is cooled to less than 50 DEG C, supernatant is extracted out, carries out plate-frame filtering with 0.8 μm of filter membrane, residue is pressed Upper method repeats extraction 2 times, merges coarse filtration liquid.Coarse filtration liquid with 200nm tubular ceramics microfiltration membranes (CX-30-1016-4-19 types, 8 Parallel connection, membrane area 1.904m²) clarified, 40 DEG C, operating pressure 0.4Mpa, crossflow velocity 4m/s of operation temperature, permeate is led to During amount 300LMH, raffinate residue 30L, add purified water 30L to dilute, continue clarification activities to raffinate residue 30L, repeat once. 60~100 mesh polyamides and D392 resin anion (R.A.)s are purified in advance by product description and expansion process, are filled respectively Post, filling height respectively 1m (BV=31.4L) and 20cm (BV=6.28L), polyamide column (preceding) series connection D392 anion Post (rear), it is standby to rush real post bed with the aqueous acetic acid of pH value 5, clarified solution adjusts pH value to connecting at 5,26 DEG C with edible white vinegar It is continuous to be sent into post bed, Dynamic Adsorption removal of impurities is carried out, flow velocity is adjusted out to 200L/h, collects efflux, clarified solution has all been handled Afterwards, continue the acetic acid water elution 75L with pH value 5, continue to collect efflux.Efflux is produced with GE companies of the U.S. The organic milipore filter of PT4040C2016 types (molecular cut off 5000D, membrane area 8.3m²) concentration, 30 DEG C of operation temperature, operation Pressure 0.4Mpa, crossflow velocity 4m/s, percolate flux 18LMH, 30L is concentrated into, adds purified water 30L to dilute, continue to be concentrated into 30L, repeat once, finally rinsing milipore filter with 15L purified waters displaces whole concentrates.Concentrate Direct spraying is dried .

Obtained ganoderma lucidum fruitbody refined polysaccharide is brown powder, yield 1.86%.

Embodiment 2：

Take the ganoderma lucidum fruitbody recrement 30kg of the re-dry after edible alcohol extracts in advance standby, add 750L purified waters, Logical to be steam heated to boiling, interval 30min starts feed pump forced circulation 5min, maintains micro-boiling 2h, steam off, be cooled to 50 Below DEG C, supernatant is extracted out, with 1 μm of bag filter coarse filtration of filtering accuracy, residue repeats extraction 1 time by upper method, merges thick Filtrate.The pp micropore folding type filter element refined filtrations of 0.22 μm of filtering accuracy of coarse filtration liquid, collect clarified solution.60~100 mesh polyamide Resin and D392 resin anion (R.A.)s are purified in advance by product description and expansion process, post is filled respectively, filling height is respectively For 1.5m (BV=26.5L) and 30cm (BV=5.3L), polyamide column (preceding) series connection D392 anion columns (rear), pH value is used It is standby that 6 aqueous citric acid solution rushes real post bed, and clarified solution adjusts pH value to post bed is continuously introduced at 6,30 DEG C with citric acid, carries out Dynamic Adsorption cleans, and adjusts out flow velocity to 250L/h, collects efflux, after clarified solution has all been handled, continue with pH value 6 Aqueous citric acid solution elutes 63.6L, continues to collect efflux.The GH4040F types that efflux is produced with GE companies of the U.S. are organic super Filter membrane (molecular cut off 3500D, membrane area 8.3m²) concentration, 35 DEG C, operating pressure 0.45Mpa of operation temperature, crossflow velocity 3.5m/s, percolate flux 15LMH, is concentrated into 30L, adds purified water 30L to dilute, continues to be concentrated into 30L, repeat once, most Milipore filter is rinsed with 15L purified waters displace whole concentrates afterwards.The edible of 7 times of amounts of original volume is slowly added under concentrate stirring Alcohol, it is closed, stand overnight, precipitation and separation, heated-air drying, crush, sieving, produce.

Obtained ganoderma lucidum fruitbody refined polysaccharide is pale powder, yield 1.92%.

Embodiment 3：

Protein and polyoses content in UV-VIS spectrophotometry measure ganoderma lucidum fruitbody refined polysaccharide, calculate polysaccharide Retention rate, albumen removal efficiency, pigment removal rate

Protein content determination：Determined using BCA methods, BCA determination of protein concentration kit (enhanced) (Enhanced BCA Protein Assay Kit) purchase in the green skies Bioisystech Co., Ltd in Shanghai, be by the detection of detail specifications step Can.

Pigment detection：Directly use spectrophotometric determination 400nm absorbance A value.

Determination of polysaccharide：Determined using Anthrone-sulfuricacid method, by "《Chinese Pharmacopoeia》Under the one ganoderma lucidum item of version in 2015【Contain It is fixed to measure】Step detection in polysaccharide ".

Ganoderma lucidum fruitbody refined polysaccharide about 50mg, accurately weighed made from Example 1, is dissolved and is settled to purified water In 50mL measuring bottles, before use according to being actually needed appropriate dilution, determine respectively ganoderma lucidum fruitbody refined polysaccharide polyoses content and Protein content.

Coarse filtration liquid 1L in Example 1, it is standby；Take concentrate 100mL, by purge process it is actual be concentrated multiple add it is pure Change water dilution to go back, it is standby as refined solution；Protein concentration, the polysaccharide concentration in 2 kinds of solution, Yi Ji are determined respectively 400nm absorbance, is calculated as follows and produces.

Albumen removal efficiency (%)=100* (concentrate protein concentration-refined solution protein concentration)/concentrate protein concentration；It is more Sugared retention rate (%)=100* refined solutions polysaccharide concentration/concentrate polysaccharide concentration；

Pigment removal rate (%)=100* (concentrate absorbance-refined solution absorbance)/concentrate absorbance.

Experimental result：Through practical measurement, the protein content of ganoderma lucidum fruitbody refined polysaccharide is 23.53%, polyoses content For 68.08%；Under the conditions of the technical matters, polysaccharide retention rate 85.82%, albumen removal efficiency 82.61%, pigment removal rate 87.14%.

Embodiment 4：

The molecular weight and molecular weight distribution determination of ganoderma lucidum fruitbody refined polysaccharide

The preparation of reference substance solution：Take dry sigma series dextran and DEXTROSE ANHYDROUS reference substance to constant weight about 50mg, it is accurately weighed, add 0.71%Na respectively₂SO₄Aqueous dissolution is simultaneously settled in 5mL measuring bottles, close plug, is shaken up, is produced.

The preparation of need testing solution：The ganoderma lucidum fruitbody refined polysaccharide about 100mg that Example 1 is prepared, precision claim It is fixed, with a small amount of 50 DEG C~60 DEG C of 0.71%Na₂SO₄Aqueous dissolution is simultaneously transferred in 10mL measuring bottles, is put to room temperature (25 DEG C), 0.71%Na₂SO₄The aqueous solution is settled to scale, and close plug shakes up, and takes above-mentioned solution about 5mL, and 4000r/min is centrifuged at 25 DEG C 10min, take supernatant to cross 0.45 μm of miillpore filter, it is standby to collect subsequent filtrate about 3mL.

Chromatographic condition：The efficient liquid phase system of Agilent 1200, differential refraction detector parameter be arranged to control simulation output, 35 DEG C of optical component temperature, zero compensation 5%, decay 500*10³, return 0, positive polarity automatically before analysis；Two TSK-GEL (7.8*300mm, 10 μ) Coupled columns, series sequence are G5000PWXL → G4000PWXL, 35 DEG C of column temperature；With 0.71% Na₂SO₄The aqueous solution (ultrasound degassing in advance) makees mobile phase isocratic elution, flow velocity 0.5mL/min；The μ L of sample size 20, analysis time be 1h。

Data processing：The initial data of each reference substance solution and ganoderma lucidum fruitbody refined polysaccharide solution is led with AIA forms Enter to Shanghai thousand and compose in GPC gel dedicated processes softwares, analyzed.

Detailed Experimental data and it the results are shown in Table 1~2, Fig. 1~4.

The summit time in the molecular weight and chromatogram of the dextran reference substance of table 1

The molecular chain conformation situation of the ganoderma lucidum fruitbody refined polysaccharide of table 2

The summit time	Peak area	Mp	Mn	Mw	Mw10	Mw90	D	Accumulative coupon weight area	Accumulative number of sections area
										48.75	33795104	4475	5716	8098	25313	3266	1.42	(＞ 5KD) 61.18%	(＞ 5KD) 48.75

Test result indicates that：Through practical measurement, only contain according to the ganoderma lucidum fruitbody refined polysaccharide prepared by this patent method One fraction, the equal relative molecular weight (Mw) of weight is 8098D, and the accumulative coupon weight area more than 5KD molecular weight accounts for 61.18%, Molecular weight distributing index D is 1.42, belongs to narrow ditribution.

Embodiment 5：

The purity and monose composition of PMP-HPLC derivatization methods measure ganoderma lucidum fruitbody refined polysaccharide

The preparation of reference substance stock solution：Take mannose, aminoglucose hydrochloride, ribose, rhamnose, grape alditol The monose reference substance such as acid, galacturonic acid, amino-galactose hydrochloride, glucose, galactolipin, xylose, arabinose, fucose About 25mg, it is accurately weighed, with deionized water dissolving and it is settled in 25mL measuring bottles, close plug, shakes up, mixing reference substance deposit is made Solution, it is standby.

The preparation of test sample stock solution：The ganoderma lucidum fruitbody refined polysaccharide about 100mg that Example 1 is prepared, essence It is close weighed, with a small amount of 50 DEG C~60 DEG C of deionized water dissolvings and it is transferred in 10mL measuring bottles, puts to room temperature (25 DEG C), deionization Water is settled to scale, close plug, shakes up, and takes above-mentioned solution about 5mL, 4000r/min centrifugation 10min at 25 DEG C, takes supernatant mistake 0.45 μm of miillpore filter, subsequent filtrate about 3mL is collected, 10.005mg/mL test sample stock solution is made, it is standby.

The configuration of derivative reaction reagent：The methanol solution of fresh configuration 0.5mol/L PMP reagents is some, shakes up, close plug Shading Cord blood；4mol/L trifluoroacetic acid aqueous solution, 0.6mol/L sodium hydrate aqueous solution, 0.6mol/L is respectively configured Aqueous hydrochloric acid solution, close plug, shake up, it is standby.

The preparation of reference substance derivatization solution：Precision draws mixing monose reference substance solution 2.000mL and enters 15mL centrifuge tubes In, add 2.000mL 0.6mol/L sodium hydrate aqueous solution, then add 4.000mL 0.5mol/L PMP methanol solutions, lid is closed, Sealed membrane covering is reinforced, and is vortexed and is fully mixed, and is entered 70 DEG C of water-baths and is incubated 30min, rapid to take out, and ice bath 30s terminating reactions, is opened Lid, 2.000mL 0.6mol/L aqueous hydrochloric acid solution is added, is transferred to after mixing in 10mL measuring bottles, quarter is settled to deionized water Degree, Mi Gai, fully mixing, precision is drawn 7.000mL and entered in 15mL centrifuge tubes, and pipette, extract 7mL chloroforms are poured in derivative liquid, Upper strata aqueous is removed into another 15mL centrifuge tubes, continues to be rinsed 2 times with chloroform the same manner, draws upper strata aqueous, at room temperature 12000r/min centrifuges 10min, takes supernatant appropriate, enters by the 3/4 of mother liquid concentration, 1/2,1/4,1/8,1/16,1/32,1/64 Row dilution, is made the derivatization reference substance solution of 8 gradients, produces.

The preparation of test sample derivatization solution：Precision pipettes test sample stock solution 0.200mL into 5mL ampere bottles, adds Enter 0.200mL4mol/L trifluoroacetic acid solution, ampere bottle is full of nitrogen, uses alcohol blast burner tube sealing rapidly, puts in 110 DEG C of baking ovens 4h is hydrolyzed, takes out, puts to room temperature, knock-off ampoule bottle-neck under upright state；Add 0.5mL methanol, less than 60 DEG C vacuum decompressions Dry or dry type nitrogen evaporator dries up, handle three times, dried to hydrolyzate complete repeatedly；The accurate 0.200mL that adds is gone residue respectively Ionized water, 0.200mL0.6mol/L sodium hydrate aqueous solution, add 0.400mL0.5mol/L PMP methanol solutions, low frequency Low intensity ultrasound 30s simultaneously blows and beats bottle wall residue for several times with liquid-transfering gun, makes its dissolving completely and fully mixes, precision is drawn 0.700mL closes lid into 5mL centrifuge tubes, and sealed membrane covering is reinforced, and enters 70 DEG C of water-bath insulations 30min, rapid to take out, ice bath 30s Terminating reaction, uncap, add 0.200mL0.6mol/L aqueous hydrochloric acid solution, then add deionized water to be settled to 2mL, fully mix Afterwards, 2 milliliters every time of chloroform is drawn with liquid-transfering gun, poured in derivative liquid, removed upper strata aqueous into another 5mL centrifuge tubes, continue Rinsed 2 times with chloroform the same manner, draw upper strata aqueous, 12000r/min centrifuges 10min at room temperature, takes supernatant, produces.

Chromatographic condition：The highly effective liquid phase chromatographic system of Agilent 1200, Waters SymmetryShield RP18 (4.6 × 250mm, 5 μm) chromatographic column, 30 DEG C of column temperature, flowing equality is used as using 18% acetonitrile -82%0.1mol/L ammonium acetate aqueous solution Elution, flow velocity 1mL/min, Detection wavelength 250nm, detection time 50min, the μ L of sample size 20.

Data processing：The standard curve of monose is drawn respectively, calculates the total of 12 kinds of monose in ganoderma lucidum fruitbody refined polysaccharide Content；For the monose that accounting in refined polysaccharide is larger, its substance withdrawl syndrome is obtained respectively, by the amount of material in target monose The minimum monose of concentration is converted as basic parameter 1, the substance withdrawl syndrome of other monose with it, is tried to achieve multiple and is produced.

Detailed Experimental data and it the results are shown in Table 3~6, Fig. 5~6.

The configuration concentration of 30 two kinds of mixing monose reference substance solutions of table is looked for derivatization composes peak time

The configuration concentration and corresponding peak area of 40 two kinds of mixing monose reference substances of table

The standard curve of 50 two kinds of mixing monose reference substances of table

Purity and the monose composition of the ganoderma lucidum fruitbody refined polysaccharide of table 6

Note：Only calculate the monose that content is not less than 0.3%

It is above-mentioned test result indicates that：Through practical measurement, according to the ganoderma lucidum fruitbody essence prepared by this patent embodiment of the method 1 Polysaccharide processed is mainly by 7 kinds of mannose, Glucosamine, glucuronic acid, glucose, galactolipin, xylose, fucose monose groups Into heteroglycan, its mol ratio be 3.67：1：1.53：61.35：5.00：1.56：2.92, it is refined more per 100g ganoderma lucidum fruitbodies Sugar not 2.72g containing mannose, Glucosamine 0.79g, glucuronic acid 1.39g, glucose 43.71g, galactolipin 3.62g, Xylose 0.90g, fucose 1.82g, using the absolute content of principal monosaccharides come calculate the purity of ganoderma lucidum fruitbody refined polysaccharide as 54.96%.

Embodiment 6：

Ganoderma lucidum fruitbody refined polysaccharide adjunct antineoplastic functional activity is evaluated

1 experimental implementation

1.1 reagent：The taxol that positive drug produces for Hainan Quan Xing pharmaceutical Co. Ltds, specification 100mg/ 16.7mL；Ganoderma lucidum fruitbody refined polysaccharide is prepared by the embodiment of the present invention 1；Happy precious purified water.

1.2 tumour cells and experimental animal：Murine Malignant breast cancer cell (4T-1), source：Chinese Academy of Sciences's cell bank. SPF level BALB/c mouses, female, 6-8 week old, body weight 14-16g, purchased from Guangdong Medical Lab Animal Center.

The preparation method of 1.3 breast carcinoma subcutaneous transplantation models：4T1 cells are used containing 10% hyclone, 1% mycillin RPMI1640 culture mediums, in 5%CO₂37 DEG C of cultures in incubator are thin with 0.25% trypsase -0.02%EDTA digestion Born of the same parents, passage, the cell in growth period of taking the logarithm are used to test.

After collecting cell, Lip river is washed 2 times with 1640 culture medium, trypan blue experiment confirms cell viability>95%, with 1640 cultures Liquid adjustment viable cell concentrations are 2*l0⁵/ mL, it is standby.Lotus knurl group animal inoculation pvaccination position sterilizes preserved skin, and 4T1 is drawn with 1mL syringes Cell suspension 0.1mL (contains 2*10⁴Individual cell) to be inoculated in mouse oxter subcutaneous, as tumor size 2mm*2mm, it is judged as mould Type is successfully established.0.1mL complete mediums are subcutaneously injected in blank control group oxter.

1.4 animal packet：After quarantine terminates, normal group (8) and lotus knurl group (24) are randomly divided into by body weight；Lotus knurl Group inoculation 4T1 breast cancer cells, establish mouse breast cancer model；After modeling 24h, lotus knurl group animal is by body weight randomized blocks point For：Model group control group, taxol group, ganoderma lucidum fruitbody refined polysaccharide combination taxol group, every group 8.

1.5 dosage regimen：After modeling 24h, lotus knurl component group starts to be administered, continuous 20 days.Ganoderma lucidum fruitbody refined polysaccharide, The equal gastric infusion of purified water, administered volume 0.2mL/10g, 1 time a day；Taxol intraperitoneal injection, administered volume are 0.1mL/10g, 1 times a week.Dosage regimen refers to table 7, table 8.

Each group dosage numbering schedule of table 7

The dosage regimen list of table 8

2 observation index

2.1 lotus knurl cubings：Began to focus on the growth of tumor mass from the administration same day, be administered the 8th respectively, 10,12,14,16, 18th, vernier caliper measurement volume of tumor mass is used within 20 days.Length=a, it is the length of knurl body most length direction, wide=b's, most length direction Vertical plane shortest length；Calculate gross tumor volume (tumor volume, TV)=1/2 × a × b², it is bent to draw lotus knurl Volume Changes Line.

2.2 blood：After drug withdrawal 24h, animal materials testing index.Blood sample is gathered first：(1) eye socket takes blood (about 6 drop), liver Plain sodium anti-freezing, flow cytomery PBLC group.(2) eye socket takes blood (about 6 drop), and 4%EDTA anti-freezings, sample is sent Inspection, survey blood routine.

2.3 tumor mass and spleen：(1) after eye socket takes blood, knurl body is peeled off after putting to death mouse, carefully removes the hyperplasia in knurl body surface face Weighed after the connective tissues such as blood vessel, envelope.(2) continue to take spleen, weighed after removing surface attachment tissue.(3) take pictures：It is all dynamic After thing tumor mass is removed, sequenced by group order, enclose respective labels, background places one ruler, takes pictures, protected as original record Deposit.

3 data processings

All data add and subtract standard deviation with meanRepresent.The comparison of multigroup mean is using single factor test variance point Analyse (One-Way ANOVA), mean compares two-by-two between group, and LSD methods are used when variance is neat；Dunnett ' s are used during heterogeneity of variance T3 methods.Completed by SPSS softwares, α=0.05.

The technology path of whole adjunct antineoplastic functional activity evaluation experimental, refers to Fig. 7.

4 experimental results

After 4.1 zooperies terminate, tumor-bearing mice is dissected, peels off tumor tissues, it is respectively grouped tumor tissues photo and sees Fig. 8 It is shown, the results showed that：Ganoderma lucidum fruitbody refined polysaccharide combination taxol group can significantly inhibit the growth of tumour, and its curative effect is better than list With taxol group.

4.2 experimental animal lotus knurl Volume Changes are as shown in table 9, Fig. 9.

The experimental animal lotus knurl Volume Changes (n=8) of table 9

Note：Compared with model group, * p ＜ 0.05, * * p ＜ 0.01；Compared with taxol group, #p ＜ 0.05, ##p ＜ 0.01.

As a result show：Model group animal tumor volume rapid growth, and alone taxol group and ganoderma lucidum fruitbody are refined more Sugar combination taxol group animal tumor volume growth trend is slow compared with model group；Compared with model group, it is administered the 14th day, the 16th day Until the 20th day, the gross tumor volume of ganoderma lucidum fruitbody refined polysaccharide combination taxol group significantly reduces, and difference has statistics Meaning (P ＜ 0.01 or P ＜ 0.05)；Compared with alone taxol group, up to the 20th day, ganoderma lucidum was sub within the 14th day, the 16th day for administration The gross tumor volume of entity refined polysaccharide combination taxol group substantially reduces

4.3 experimental animal lotus knurl weight and related organ index are as shown in table 10, Figure 10~13.

The experimental animal lotus knurl weight of table 10 and related organ index

Note：Compared with blank group, △ p ＜ 0.05, △ △ p ＜ 0.01；Compared with model group, * p ＜ 0.05, * * p ＜ 0.01；Compared with taxol group, #p ＜ 0.05, ##p ＜ 0.01.

As a result show：Compared with model group, the tumor weight of ganoderma lucidum fruitbody refined polysaccharide combination taxol group, tumour refer to Number, gross tumor volume, batch index and spleen index are remarkably decreased, and difference has statistical significance (P ＜ 0.01 or P ＜ 0.05)；With it is alone Taxol group compares, and ganoderma lucidum fruitbody refined polysaccharide is combined tumor weight, tamor index, gross tumor volume, the spleen of taxol group Index is decreased obviously.

4.4 lymphocyte subpopulation flow cytometer showed results are as shown in table 11, Figure 14~18.

The experimental animal lymphocyte subpopulation distribution situation (accounting % of the subgroup in CD) of table 11

Note：Compared with blank group, △ p ＜ 0.05, △ △ p ＜ 0.01；Compared with model group, * p ＜ 0.05, * * p ＜ 0.01；Compared with taxol group, #p ＜ 0.05, ##p ＜ 0.01.

As a result show：Ganoderma lucidum fruitbody polysaccharide combination taxol group can significantly reverse peripheral blood caused by alone taxol group The trend that lymphocyte subgroup CD3, CD4, CD8 ratio reduce, difference have statistical significance (P ＜ 0.01).

4.5 blood routine testing results are as shown in table 12, Figure 19~22.

The experimental animal blood routine testing result of table 12

Note：Compared with blank group, △ p ＜ 0.05, △ △ p ＜ 0.01；Compared with model group, * p ＜ 0.05, * * p ＜ 0.01；Compared with taxol group, #p ＜ 0.05, ##p ＜ 0.01.

As a result show：Compared with model group and alone taxol group, ganoderma lucidum fruitbody refined polysaccharide combination taxol group energy The obvious peripheral white blood cells that reduce are horizontal, it is close to normal value, wherein having statistical significance (P ＜ with the difference of model group 0.01)；Compared with model group and alone taxol group, ganoderma lucidum fruitbody refined polysaccharide combination taxol group can significantly raised periphery Blood erythrocyte (P ＜ 0.05) and hemoglobin level, make it be close to normal value；Other ganoderma lucidum fruitbody refined polysaccharide combination is purple The rise of peripheral blood platelet levels, makes it close to normal value caused by China fir alcohol group can also reverse alone taxol.

5 conclusions

Result above shows：Ganoderma lucidum fruitbody refined polysaccharide joint paclitaxel treatment group shows within the whole test period Go out the obvious function of suppressing malignant breast carcinomas tumour growth, be significantly stronger than independent Paclitaxel Chemotherapy the effect of therapeutic alliance group Group, and immune system and hematological system main biochemical inspection caused by the specific reversing tumor growth of energy and the independent chemotherapy of taxol The exception of desired value, makes it be close to normal value, and this active polysaccharide can be applied to tumor prevention and treat the exploitation of medicine, can also answer Exploitation for association area food and health products.

It is described above, it is only the preferable specific embodiment of the present invention, but protection scope of the present invention is not limited thereto, Any one skilled in the art the invention discloses technical scope in, technique according to the invention scheme and its Design is subject to equivalent substitution or change, should all cover within the scope of the present invention.

Claims (9)

1. a kind of ganoderma lucidum fruitbody refined polysaccharide with notable adjunct antineoplastic activity, it is characterised in that ganoderma lucidum is real Body refined polysaccharide is mainly by 7 kinds of mannose, Glucosamine, glucuronic acid, glucose, galactolipin, xylose, fucose lists Sugar in molar ratio 3~4：1：1.3~1.7：56~65：4~6：1.3~1.8：The heteroglycan of 2.5~3.5 compositions, with above-mentioned 7 kinds The absolute content of main composition monose calculates the purity of ganoderma lucidum fruitbody refined polysaccharide, up to more than 52%；Only contain a fraction, Weight average molecular weight is 7500~8500D, and molecular weight distributing index D is 1.3~1.5, belongs to narrow ditribution；UV-vis spectroscopy light Protein content (BCA methods) in degree method measure ganoderma lucidum fruitbody refined polysaccharide is less than 25%, polysaccharide (Anthrone-sulfuricacid method) content More than 65%.

2. a kind of ganoderma lucidum fruitbody refined polysaccharide with notable adjunct antineoplastic activity according to claim 1, it is special Sign is that the ganoderma lucidum fruitbody refined polysaccharide is mainly by mannose, Glucosamine, glucuronic acid, glucose, gala 7 kinds of monose such as sugar, xylose, fucose in molar ratio 3.67：1.00：1.53：61.35：5.00：1.56：2.92 composition it is miscellaneous more Sugar, per 100g ganoderma lucidum fruitbodies refined polysaccharides respectively 2.72g containing mannose, Glucosamine 0.79g, glucuronic acid 1.39g, Glucose 43.71g, galactolipin 3.62g, xylose 0.90g, fucose 1.82g, contained with above-mentioned 7 kinds main the absolute of monose that form Measure to calculate the purity of ganoderma lucidum fruitbody refined polysaccharide as 54.96%；Only contain a fraction, the equal relative molecular weight (Mw) of weight is 8098D, the accumulative coupon weight area more than 5KD molecular weight account for 61.18%, and molecular weight distributing index D is 1.42, belongs to narrow point Cloth；Protein content (BCA methods) 23.53% in UV-VIS spectrophotometry measure ganoderma lucidum fruitbody refined polysaccharide, polysaccharide (Anthrone-sulfuricacid method) content 68.08%.

A kind of 3. preparation method of the ganoderma lucidum fruitbody refined polysaccharide with notable adjunct antineoplastic activity, it is characterised in that institute The method of stating comprises the following steps：

(1) hot water Dynamic Extraction Thick many candies：The ganoderma lucidum fruitbody residue for the re-dry after alcoholic extraction of learning from else's experience, adds residue weight 10 ~30 times of purified water, lead to steam, stirring or interval, which start feed pump, forces circulated in countercurrent, until maintaining the micro-boiling time to reach 2 Steam off after~4h；

(2) coarse filtration technique realizes slag-liquid separation：After the completion of extraction, with the plate filter or pocket type mistake of 0.5~5 μm of filtering accuracy Filter carries out coarse filtration, and alternatively, residue by step (1) method repeats extraction 1~2 time and coarse filtration again, merging coarse filtration liquid；

(3) refined filtration technique realizes that decoction is clarified and removes the supramolecular impurity of thermosol cold analysis：Coarse filtration liquid is with 200~800nm's The micropore folding type filter element of 0.1~0.22 μm of tubular ceramic microfiltration membranes or filtering accuracy carries out refined filtration clarification, obtains clarified solution；

(4) resin bed absorbing process of connecting realizes de- protein decolouring：Clarified solution use series connection polyamide (such as 60~100 Purpose polyamide) and the de- protein decolouring of resin anion (R.A.) (such as D392 resin anion (R.A.)s), polyamide bed is first flowed through, Resin anion (R.A.) bed is flowed into afterwards；By dispensing gauge, per the minimum outfit 250mL polyamides of kg fructification residues and 50mL D392 resin anion (R.A.)s, polyamide resin column filling height is 1~2 meter, resin anion (R.A.) post 20~40cm of bed height, uses acetic acid Or the presetting clarified solution pH value of citric acid, to 4.5~6.5, loading adsorption flow rate is 4~10BV/h of total post bed, collects efflux；

(5) film ultrafiltration technology realizes the concentration of desired polysaccharide and removes micromolecular water solubility impurity：Efflux is carried out with milipore filter Concentrate is concentrated to give, the molecular cut off of milipore filter is 3500~5000D；

(6) drying process：Concentrate use alcohol precipitation after heated-air drying or vacuum drying, also can Direct spraying be drying to obtain.

A kind of 4. preparation of ganoderma lucidum fruitbody refined polysaccharide with notable adjunct antineoplastic activity according to claim 3 Method, it is characterised in that the described method comprises the following steps：

(1) hot water Dynamic Extraction Thick many candies：Take the ganoderma lucidum fruitbody residue of the re-dry after edible alcohol extracts in advance appropriate, add Enter the purified water of 10~30 times of residue weight, lead to steam, stirred with 10~30r/min speed or started at interval of 20~40min Feed pump 3~8min of forced circulation, the steam off after maintaining the micro-boiling time to reach 2~4h；

(2) coarse filtration technique realizes slag-liquid separation：After the completion of extraction, less than 50 DEG C are cooled to, supernatant is extracted out, uses filtering accuracy 0.5~5 μm of plate filter or bag filter carries out coarse filtration, and alternatively, residue repeats extraction 1~2 by step (1) method Secondary and coarse filtration again, merge coarse filtration liquid；

(3) refined filtration technique realizes that decoction is clarified and removes the supramolecular impurity of thermosol cold analysis：Coarse filtration liquid is with 200~800nm's The micropore folding type filter element of 0.1~0.22 μm of tubular ceramic microfiltration membranes or filtering accuracy carries out refined filtration clarification, tubular ceramic micro-filtration The Operating parameters of film are 40~60 DEG C, 0.30~0.50Mpa of operating pressure, 3~4.5m/s of crossflow velocity of temperature, permeate When 120~300LMH of flux, raffinate residue about 30L, add purified water about 30L dilute, continue clarification activities to raffinate residue about 30L, alternatively, repeat and once dilute clarification activities, to ensure desired polysaccharide all by reducing process loss as far as possible；

(4) resin bed absorbing process of connecting realizes de- protein decolouring：Clarified solution uses 60~100 mesh polyamides and D392 trees Fat is packed into post bed respectively, and is cascaded, and carries out de- albumen and decolouring simultaneously using dynamic column absorbing process, first flows through poly- Acid amides post, then flow into D392 resin columns；By the dispensing gauge of ganoderma lucidum fruitbody residue, 1kg fructifications residue extraction institute is often handled Polyamide 250mL and D392 the resin anion (R.A.) 50mL of pre-treatment, polyamides have been completed in the clarified solution subsistence level configuration obtained Polyimide resin post filling height is 1~2 meter, D392 resin anion (R.A.) posts 20~40cm of bed height, presetting clear with acetic acid or citric acid Clear liquid pH value is to 4.5~6.5, and temperature is kept for 20~35 DEG C, and clarified solution loading adsorption flow rate is 4~10BV/h of total post bed, is received Collect efflux；End is collected, continues the water elution 2BV with pH value 4.5~6.5, continues to collect efflux, and merges efflux, To displace de- protein decolouring liquid as far as possible, the process loss of desired polysaccharide is reduced；

(5) film ultrafiltration technology realizes the concentration of desired polysaccharide and removes micromolecular water solubility impurity：Efflux is carried out with milipore filter Concentration, the molecular cut off of the milipore filter are 3500~5000D, and Operating parameters are 25~45 DEG C of temperature, operating pressure 0.2~0.5Mpa, 2.5~4m/s of crossflow velocity, 4~20LMH of percolate flux, when being concentrated into about 30L, add purified water about 30L Dilution, continues to be concentrated into about 30L, alternatively, repeats and once dilute concentration step, water-soluble to eliminate small molecule to greatest extent Property impurity, finally with 15L purified waters rinse milipore filter displace whole concentrates, to reduce process loss as far as possible；

(6) drying process：The alcohol that 4~9 times of amounts of original volume are slowly added under concentrate stirring at low speed carries out alcohol precipitation, stands At night, supernatant is pumped, take out precipitation, edible alcohol washing, heated-air drying or vacuum drying, finally pulverize and sieve and produce；Or Concentrate directly completes drying using the step of spray drying process one.

A kind of 5. ganoderma lucidum fruitbody refined polysaccharide with notable adjunct antineoplastic activity according to claim 3 or 4 Preparation method, it is characterised in that the post footpath in step (4) is according to filler demand reasonable selection or in parallel real by multicolumn It is existing.

6. refined according to any described a kind of ganoderma lucidum fruitbody with notable adjunct antineoplastic activity in claim 3 to 5 The preparation method of polysaccharide, it is characterised in that the raw material in the step (1) is after ganoderma lucidum fruitbody is utilized into alcoholic extraction, to enter Row slag-liquid separation, residue are used as extraction raw material after drying.

7. the ganoderma lucidum fruitbody refined polysaccharide being prepared according to any one of claim 3 to 6.

8. preparing for tumor prevention and controlling according to any described ganoderma lucidum fruitbody refined polysaccharide in claim 1 to 2 or 7 Purposes in terms of the adjunct antineoplastic medicine for the treatment of, food, health food.

9. ganoderma lucidum fruitbody refined polysaccharide according to claim 8 is anti-swollen for tumor prevention and the auxiliary for the treatment of in preparation Purposes in terms of knurl medicine, food, health food, it is characterised in that it is used alone comprising ganoderma lucidum fruitbody refined polysaccharide, or with Other active components and/or the compound formed with its pharmaceutically acceptable auxiliary material, carrier.