CN107722131A - A kind of total ganoderma spore powder refined polysaccharide with notable adjunct antineoplastic activity and its preparation method and application - Google Patents
A kind of total ganoderma spore powder refined polysaccharide with notable adjunct antineoplastic activity and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to a kind of total ganoderma spore powder refined polysaccharide with notable adjunct antineoplastic activity and its preparation method and application, belong to medicinal fungi extraction separation and medicine food application and development field.The refined polysaccharide is mainly by mannose, glucose, galactolipin, fucose in molar ratio 1：40~50：4~5.5：The heteroglycan of 1.5~2.0 compositions, its purity are more than 80%, 160~200KD of weight average molecular weight.The preparation method includes：Hot water Dynamic Extraction, sedimentation joint coarse filtration are removed slag, refined filtration is clarified, milipore filter concentrates impurity elimination, spray drying or alcohol precipitation and air-dried.The preparation method green, intelligence, energy-conservation, can solve industrialization problem, have notable scale advantage, and manufactured goods meet the requirement on security, validity, controllability, stability in food and medicine matter body system.The refined 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
Technical field
The present invention relates to a kind of total ganoderma spore powder refined polysaccharide with notable adjunct antineoplastic activity and its preparation side
Method and application, belong to medicinal fungi extraction and separation technology and medicine food application and development field.
Background technology
Ganoderma lucidum (scientific name：Ganoderma Lucidum Karst), profile is in umbrella, cap kidney shape, semicircle or nearly circle
Shape, it is the fructification of On Polyporaceae ganoderma lucidum；With invigorating qi for tranquilization, it is relieving cough and asthma, promote longevity the effect of；For dizziness not
Dormancy, palpitation, neurasthenia, consumptive disease cough and asthma.
The growth course of ganoderma lucidum includes：Spore → mycelium → fructification → release spore.Reishi sporule, mycelium and son
Entity is the different phase of its growth, since being said the visible ganoderma lucidum piece of eyes (i.e. fructification), after ganoderma lucidum maturation, and cap
Can be turned-out, spore is outwards launched.The Reishi sporule flown away with the wind, which is once dropped on the suitable trees of environment, to be started
Sprout, slowly grow one and save short mycelia.Through nutrition accumulation after a while, longer closeer mycelia is suitable
Under the conditions such as temperature, humidity, illumination coordinate, gradually kink is differentiated to form many, hard flat, macroscopic growing point,
Referred to as " former base " of ganoderma lucidum, then grow maturation by constantly growth, ultimately form fructification.That is, lucidum spore powder is
Similar to " seed " of ganoderma lucidum fruitbody, ganoderma lucidum mycelium is by organizing clustering " variable body " ganoderma lucidum fruitbody.
Delivered according to the World Health Organization《Global cancer report 2014》Research claim, global cancer patients in 2012 and
Death is all constantly increasing, and newly-increased cases of cancer has nearly half to appear in Asia, and wherein most is new from China, China
Increase cases of cancer and be in first.Predict that swift and violent growing trend will be presented in global cases of cancer, by 14,000,000 people of 2012, by
Year is incremented to 19,000,000 people of 2025, will be up to 24,000,000 people by 2035.The whole world in 2012 increases 14,000,000 cancers newly altogether
Example, and have 8,200,000 people death, wherein, newly-increased 3,070,000 cancer patients of China, and cause about 2,200,000 people dead, the whole world is accounted for respectively
The 21.9% of total amount and 26.8%.
As can be seen here, cancer morbidity rises year by year, and the death rate is high, is badly in need of the production of effective prevention and treatment malignant tumour
Product.The treating cancer means of main flow include cytotoxic drug and radiotherapy at present, but these means also result in simultaneously
The continuous exhaustion of patient body, and accelerate the Lethal cases of significant proportion.It is both domestic and external in order to mitigate the pain of patient and burden
Anti-cancer agent research has been increasingly turned to antitumor for the non-cytotoxicity class of targeting via original cell toxicant series antineoplastic medicament
Drug research.
The non-cytotoxicity class medicine of more high-efficiency low-toxicity is found, or assists treatment means evident in efficacy but strong toxic side effect
All it is the important directions of antineoplastic development from now on so as to play the adjunct antineoplastic drugs with function of Synergy and attenuation.Acellular
The medication combined cytotoxic drug of malicious class or radiotherapy, by as the preferred plan for the treatment of of cancer.In recent years, with to tumour
The further investigation of immune Apoptosis mechanism is escaped, the immune adjuvant therapy research of tumour also constantly makes a breakthrough.
For ganoderma lucidum class medicinal material as natural pharmaceutical resources, its pharmacological function shows as significantly increasing the machine of immune system
Can, tumour is prevented and treated, is reduced blood pressure, the generation of prevention of cardiovascular disease, the secretion of insulin is stimulated, it is dense to reduce blood glucose
Degree, accelerate blood microcirculation, improve blood oxygen ability, eliminate interior free yl, anti-radiation, to anti-liver injury, extend the life-span
Deng.Ganoderma lucidum has turned into first batch of into one of Chinese medicine of American National pharmacopeia in 2010, has been increasingly becoming medicine work both at home and abroad
The potential new lover of developing anti-tumor medicaments and immunomodulator in the minds of person.Specify the effective substance of ganoderma lucidum class medicinal material antitumor action
Basis, and developed into medical product and invest market, have broad application prospects, significant social benefit and good warp
Ji value.
The content of the invention
For the exploitation demand and the shortcomings of the prior art in existing field, the present invention is intended to provide a kind of have significantly
The total ganoderma spore powder refined polysaccharide and its scale industrialization preparation method and application of adjunct antineoplastic activity.
The present invention reaches above-mentioned purpose by following scheme：
In the first aspect of the present invention, there is provided a kind of total ganoderma spore powder with notable adjunct antineoplastic activity is refined more
Sugar, the polysaccharide is mainly by 4 kinds of mannose, glucose, galactolipin, fucose monose in molar ratio 1：40~50：4~5.5：
The heteroglycan of 1.5~2.0 compositions；Per 100g total ganoderma spore powders refined polysaccharide respectively containing 1.4~1.8g mannitol, 60~70g
Glucose, 7~8.5g galactolipins, 2~3g fucoses；Full ganoderma lucidum is calculated with above-mentioned 4 kinds main absolute contents for forming monose
The purity of conidia powder refined polysaccharide, up to more than 80%；The equal relative molecular weight (Mw) of weight is 160~200KD, more than 10KD molecular weight
Accumulative coupon weight area account for more than 90%；Total ganoderma spore powder refined polysaccharide is mainly made up of 4 fractions, fraction 1, fraction
2nd, fraction 3, the peak area accounting of fraction 4 are respectively 20~25%, 20~25%, 30~40%, 15~25%, 4 fractions
The equal relative molecular weight (Mw) of weight is respectively 620~700KD, 90~110KD, 14~18KD, 4~6KD；UV-vis spectroscopy light
Protein content (BCA methods) in degree method measure total ganoderma spore powder refined polysaccharide is less than 8%, and polysaccharide (Anthrone-sulfuricacid method) contains
Amount is more than 85%.
In a preferred embodiment, the total ganoderma spore powder refined polysaccharide is mainly by mannose, glucose, half
4 kinds of lactose, fucose monose in molar ratio 1：45.05：4.76：The heteroglycan of 1.74 compositions；Per 100g total ganoderma spore powders essence
Polysaccharide processed mannitol containing 1.63g, 68.73g glucose, 7.79g galactolipins, 2.59g fucoses respectively；With above-mentioned 4 kinds main group
The purity of total ganoderma spore powder refined polysaccharide is calculated as 80.74% into the absolute content of monose；The equal relative molecular weight (Mw) of weight
For 179.689KD, the accumulative coupon weight area more than 10KD molecular weight accounts for 99.11%；Total ganoderma spore powder refined polysaccharide master
To be made up of 4 fractions, fraction 1, fraction 2, fraction 3, the peak area accounting of fraction 4 are respectively 22.21%, 23.04%,
34.40%th, 20.35%, the equal relative molecular weights of weight (Mw) of 4 fractions be respectively 677.261KD, 98.706KD, 15.713KD,
5KD；Protein content (BCA methods) in UV-VIS spectrophotometry measure total ganoderma spore powder refined polysaccharide is 6.28%,
Polysaccharide (Anthrone-sulfuricacid method) content is 87.12%.
At present, for the isolated unimodal polysaccharide of ganoderma lucidum class of academia more than 100 kinds, these unimodal polysaccharide derive from one
Kind ganoderma lucidum class medicinal material, it can not still play the synergistic function of various active polysaccharide.The total ganoderma spore powder of the present invention is refined more
Sugar, the active polysaccharide that the whole growth phase of ganoderma lucidum can be formed is enumerated, there is notable adjunct antineoplastic activity and Synergistic to make
With.
Further, current a variety of unimodal active polysaccharides, the overwhelming majority only rest on laboratory stage, and they are common
Feature is that yield is extremely low, single treatment amount is minimum, cost is very high, and used technology is from yield design and the market competitiveness
From, the possibility of several no scale industrializations.
Based on this, in the second aspect of the present invention, there is provided a kind of ganoderma spore with notable adjunct antineoplastic activity
The preparation method of powder refined polysaccharide, including：
(1) hot water extracts：Take the total ganoderma spore powder recrement for having completed ganoderma spore oil extract appropriate, extracted in multipotency
The purified water of 10~30 times of recrement weight is added in tank, after logical steam boils, under stirring or startup feed pump adverse current forced circulation
Be slowly added to above-mentioned recrement, continue stirring or interval starts feed pump and forces circulated in countercurrent, until maintain the micro-boiling time reach 1~
Steam off after 2 hours；
(2) coarse filtration separates：After the completion of extraction, standing sedimentation simultaneously cools down, and extracts supernatant out, with 0.2~2.5 μm of filtering accuracy
Plate filter or bag filter carry out coarse filtration, alternatively, residue by step (1) method repeat extraction it is once and thick again
Filter, merge coarse filtration liquid；
(3) refined filtration is clarified and removes supramolecular impurity：200~800nm of coarse filtration liquid tubular ceramic microfiltration membranes or mistake
The micropore folding type filter element for filtering 0.1~0.22 μm of precision carries out refined filtration clarification；
(4) film is concentrated by ultrafiltration and removes micromolecular water solubility impurity：Clarified solution milipore filter (molecular cut off be 3500~
8000D) concentrated.
(5) dry：Concentrate is drying to obtain using heated-air drying after alcohol precipitation or Direct spraying.
A kind of it is further preferred that preparation side of the total ganoderma spore powder refined polysaccharide with notable adjunct antineoplastic activity
Method, including：
(1) hot water Dynamic Extraction polysaccharide：Take the total ganoderma spore powder recrement for having completed ganoderma spore oil extract appropriate,
The purified water of 10~30 times of recrement weight is added in multifunctional extracting pot, after logical steam boils, with the stirring of 30~60r/min speed or
Start and be slowly added to above-mentioned recrement under feed pump adverse current forced circulation, continue stirring or start feed pump at interval of 10~20min
5~10min of forced circulation, the steam off after maintaining the micro-boiling time to reach 1~2 hour；
(2) natural subsidence joint coarse filtration technique realizes slag-liquid separation：After the completion of extraction, standing sedimentation and be cooled to 50 DEG C with
Under, extract supernatant out, coarse filtration is carried out with the plate filter or bag filter of 0.2~2.5 μm of filtering accuracy, it is alternatively, residual
Slag repeats extraction once and again coarse filtration by step (1) method, merges coarse filtration liquid；
(3) refined filtration technique realizes that decoction is clarified and removes the supramolecular impurity of thermosol cold analysis：Above-mentioned 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
The Operating parameters of ceramic micro filter film are 30~70 DEG C, 0.20~0.80Mpa of operating pressure, 3~5m/s of crossflow velocity of temperature,
When 150~400LMH of percolate flux, raffinate residue about 20L, add purified water about 20L to dilute, continue clarification activities to raffinate and remain
Remaining about 20L, alternatively, repeats and once dilutes clarification activities, to ensure desired polysaccharide all by reducing technique as far as possible
Loss；
(4) film ultrafiltration technology realizes the concentration of desired polysaccharide and removes micromolecular water solubility impurity：Above-mentioned clarified solution is used super
Filter membrane is concentrated, and ultrafiltration retaining molecular weight is 3500~8000D, and Operating parameters are 30~50 DEG C of temperature, operation pressure
0.15~0.6Mpa of power, 2~4.5m/s of crossflow velocity, 6~25LMH of percolate flux, when being concentrated into about 40L, add purified water about
40L dilutes, and continues to be concentrated into about 40L, alternatively, repeats and once dilute concentration step, to eliminate small molecule to greatest extent
Water-solubility impurity, finally rinse milipore filter with about 20L purified waters and displace whole concentrates, to reduce technique damage as far as possible
Consumption；
(5) drying process：The edible alcohol that 3~7 times of amounts of original volume are slowly added under concentrate stirring at low speed carries out alcohol precipitation,
Stand overnight, pump supernatant, take out precipitation, a small amount of ethanol washing, heated-air drying or vacuum drying, finally alternatively crushed
Sieve and produce；Or concentrate can also use the step of spray drying process one to complete to be dried to obtain.
Above-mentioned preparation method provided by the invention, the raw material sources of use are complete in patent of invention CN 100593410C
Into the total ganoderma spore powder recrement of ganoderma spore oil supercritical carbon dioxide extraction, the total ganoderma spore powder is real by ganoderma lucidum
Body and lucidum spore powder are used as culture medium after mixing sterilizing by a certain percentage, are inoculated with " active polysaccharide concentration type " exclusive lucidum strain
As " introduces a collection ", and be provided with nutritional condition and environmental factor beneficial to mycelial growth, allow its formation it is dense, rich in activity
The ganoderma lucidum mycelium of polysaccharide.The total ganoderma spore powder integrates ganoderma lucidum mycelium, ganoderma lucidum fruitbody, lucidum spore powder, includes
The active polysaccharide that ganoderma lucidum whole growth phase can be formed, using its as raw material is extracted, separation, mixed active made from purifying
Polysaccharide can overcome the application limitation of single variety polysaccharide, be advantageous to give play to Chinese tradition medicine multicomponent, Mutiple Targets, multilayer
Secondary, multipath synthesis pharmacological action feature, embody the Overall View characteristic of traditional Chinese medical science diagnosis and treatment.
Inventor is carrying out research process discovery to preparation method, present in the extraction raw material of preparation method of the invention
Lucidum spore powder, particle is superfine, about 5~7 μm, using prior art extracting method when, in extraction process be easy to nature
Sedimentation forms agglomerate, influences yield；Therefore, technical solution of the present invention uses Dynamic Extraction, can effectively overcome above-mentioned production problem.
In addition, selection application of the superfine conidia powder also to filter medium and filtering technique, it is desirable to high；Inventor, which studies, to be found, when
When selecting conventional filter medium or the single clarification technique of application, conidia powder is very easy to penetrate filter medium and enter eventually
In product, quality of finished is influenceed；If blocking filtering medium is easy to, causes nothing using end filtration technology, conidia powder simultaneously
Method normally produces.For this problem, inventor is clear successively using natural subsidence, coarse filtration, three steps of refined filtration in preparation method
Clear technique, can take off substantially from big to small all fragments of raw material (inspected under 200 power microscopes, it is several acellular broken in clarified solution
Piece presence)；And coarse filtration and refined filtration technique all employ cross-flow filtration mode as far as possible, can overcome conidia powder blocking filtering medium
Cause this industrialization problem that can not normally produce.
Further, the source of the extraction raw material of preparation method of the invention, be by a certain proportion of lucidum spore powder and
The mixture of fructification is sterilized to obtain culture medium, and is inoculated with upper lucidum strain, there is provided suitable environment is dense by its generation
Ganoderma lucidum mycelium；So containing the mycelium for being largely in growth animated period in extract raw material, and these are in animated period
Mycelium in the macromoleculars such as more protein, DNA, RNA, pectin, cellulose, water-insoluble hemicellulose to be present nonactive
Material, their common feature are to be dissolved in hot water, but are separated out after temperature reduction, are suspended in colloid form in extract solution,
Cause extract solution muddy, be long placed in precipitation, finished product solubility is poor, and is difficult to be removed by common process.For this problem,
High-precision filter medium has specially been selected in preparation method of the present invention, and by adjusting relevant parameter to preferable scope, energy
Reach and eliminate these nonactive or low activity big molecular impurity purposes, obtaining good clarifying effect, (transmissometer detects, and reads
Number is less than No. 0.5 standard turbidity solution).
In addition, the full ganoderma lucidum of the raw material of preparation method of the present invention introduces in incubation and is advantageous to mycelial growth
Nutrient and mineral matter, belong to inert matter, and the method that preparation method of the present invention is dialysed by using film is dense in removal of impurities
Contracting, significantly simplify technological process, improves production efficiency, and the heavy metal index of obtained finished product, ash content index, impurity
Index is all significantly improved.
The preparation method of the present invention, using ceramic micro filter film or precise micropore foldable filter element, organic milipore filter, alcohol precipitation
Three step impurity removal process, the big molecular impurity of thermosol cold analysis can be removed, and can removes the water-solubility impurity of small molecule, while also can
The impurity such as the molten pigment of alcohol water two, polypeptide, inhereditary material are removed by alcohol precipitation process；And clarify with removal of impurities, concentration with removal of impurities,
Alcohol precipitation separation is synchronous with removal of impurities to be carried out；The total ganoderma spore powder refined polysaccharide of more than 80% absolute purity, and can section can be made
The about production time, improve production efficiency.
Meanwhile preparation method of the invention, the clarification of use, impurity elimination, concentration technology use membrane technology scheme, belong to low
Warm production technology, can ensure that the 3-D solid structure of active polysaccharide will not destroy, and then retain its pharmacological activity to greatest extent,
Energy consumption has been significantly reduced it simultaneously.
In the third aspect of the present invention, the present invention provides a kind of total ganoderma spore powder refined polysaccharide in terms of adjunct antineoplastic
Purposes.
In the fourth aspect of the present invention, the present invention provides a kind of total ganoderma spore powder refined polysaccharide and is preparing adjunct antineoplastic
Purposes in terms of medicine, food, health food.
The total ganoderma spore powder refined polysaccharide is in terms of adjunct antineoplastic, such as the antitumor activity side of auxiliary taxol
Face, there is significant effect；Also, also show specific reversing tumor growth and for immune system caused by chemotherapy and blood
The treatment and/or corrective action of the abnormal aspect of liquid system Main Biochemical value.For example, for immune system caused by chemotherapy
Including but not limited to spleen stress become big, leucocyte substantially increases, periphery blood strangury extremely with hematological system Main Biochemical value
Bar subsets distribution changes, peripheral blood erythrocyte quantity declines, peripheral blood hemoglobin concentration reduces, peripheral blood blood platelet
Reduce etc..
Above-mentioned total ganoderma spore powder refined polysaccharide is in terms of adjunct antineoplastic and otherwise effect is including pernicious
It is confirmed in the experiment such as BALB/c female mices of breast cancer cell (4T-1), using normal mouse gavage purified water as normally
Group, using tumor-bearing mice gavage purified water as model group, independent Paclitaxel Chemotherapy is used as using the independent administering paclitaxel of tumor-bearing mice
Group, to combine administering paclitaxel as therapeutic alliance group through total ganoderma spore powder refined polysaccharide made from technical solution of the present invention,
Carry out adjunct antineoplastic functional activity evaluation.Final result shows that total ganoderma spore powder refined polysaccharide combines paclitaxel treatment group
Show the significant function of suppressing malignant breast carcinomas tumour growth within the whole test period, show the effect of therapeutic alliance group
Work is better than independent Paclitaxel Chemotherapy group, and can immune system caused by specific reversing tumor growth and the independent chemotherapy of taxol and
The exception of hematological system main biochemical Index for examination value, as spleen stress become big, leucocyte substantially increases, PBLC
Subgroup distribution changes, peripheral blood erythrocyte quantity declines, peripheral blood hemoglobin concentration reduces, peripheral blood decrease of platelet
Deng.
Therefore, the total ganoderma spore powder refined polysaccharide of the invention under effective dose is used alone, or active with other
Component and/or with its pharmaceutically acceptable auxiliary material, carrier formed compound be used in combination, can be applied to tumor prevention
And the exploitation for the treatment of medicine, it can also be applied to the fields such as the exploitation of association area food and health products.
Heretofore described effective dose includes but is not limited to more than 500mg/Kg target organismses, target organismses include but
It is not limited to mammal such as people.
Compared to the prior art, the beneficial effects of the present invention are：
(1) preparation method of the invention is whole only uses alcohol and water, will not bring new impurity and poisonous and harmful substance into,
Risk is remained without organic reagent, ensure that the natural quality of product, Product Green, and it is environmentally friendly.
(2) the preparation method is that being designed from big produce reality needs, more than multiple pilot-scale
Actual production verifies that whole process can realize pipeline, automation, intellectualized operation, has technique simple possible, is adapted to scale production
The advantages of industry, low overall operation cost, product yield is up to more than 1.8%.
(3) 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, both met national industrial policies on green, intelligence, energy-conservation
It is required that while solve industrialization problem existing for current produce reality again, there is significant scale industrialization advantage, and made
The finished product obtained meets the requirement in food and medicine quality management system on security, validity, controllability, stability.
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 total ganoderma spore powder refined polysaccharide in embodiment 4.
Fig. 3 is total ganoderma spore powder refined polysaccharide graph of molecular weight distribution in embodiment 4.
Fig. 4 is total ganoderma spore powder 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 total ganoderma spore powder 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：
Using the full ganoderma lucidum spore that ganoderma spore oil extract has been completed in embodiment 2 in patent of invention CN 100593410C
Sub- powder recrement 40kg is standby, and 600L purified waters are added in multifunctional extracting pot, and after logical steam boils, 60r/min stirrings are lower slowly to be added
Entering above-mentioned recrement, continue to stir and maintain micro-boiling 1h, standing sedimentation is simultaneously cooled to less than 50 DEG C, extracts supernatant out, plate-frame filtering,
Residue repeats extraction once by upper method, merges coarse filtration liquid.Coarse filtration liquid is managed with the 500nm of Jiangsu Kaimi Membrane Technology Co., Ltd.
Formula ceramic micro filter film (CX-30-1016-4-19 types, 4 parallel connections, membrane area 0.952m2) clarified, 50 DEG C of operation temperature, grasp
Make pressure 0.35Mpa, crossflow velocity 4.5m/s, percolate flux 360LMH, during raffinate residue 20L, add purified water 20L to dilute,
Continue clarification activities to raffinate residue 20L, repeat once.The PT4040C2016 types of GE companies of clarified solution U.S. production have
Machine milipore filter concentrates (molecular cut off 5KD, membrane area 8.3m2), 40 DEG C, operating pressure 0.5Mpa of operation temperature, film surface stream
Fast 3.5m/s, percolate flux 15LMH, is concentrated into 40L, adds purified water 40L to dilute, continues to be concentrated into 40L, repeat once,
Finally milipore filter is rinsed with 20L purified waters displace whole concentrates.Concentrate, it is spray-dried, crushes, sieving, produces.
Obtained total ganoderma spore powder refined polysaccharide is grey powder, yield 1.99%.
Embodiment 2：
Using the full ganoderma lucidum spore that ganoderma spore oil extract has been completed in embodiment 2 in patent of invention CN 100593410C
Sub- powder recrement 30kg is standby, and 700L purified waters are added in multi-function extractor, after logical steam boils, starts feed pump adverse current and forces
Above-mentioned recrement is slowly added under circulation, starts feed pump forced circulation 5min at interval of 10min, after maintaining micro-boiling 1h, it is heavy to stand
Drop and be cooled to less than 50 DEG C, extract supernatant out, with 0.5 μm of bag filter coarse filtration of filtering accuracy, residue is by the repetition of upper method
Extraction once, merges coarse filtration liquid.The pp micropore folding type filter element refined filtrations of 0.1 μm of filtering accuracy of coarse filtration liquid.The refined filtration liquid U.S.
Organic milipore filter concentration (molecular cut off 5KD, the membrane area 8.3m of PT4040C2016 types of GE companies production2), operation temperature
45 DEG C, operating pressure 0.45Mpa, crossflow velocity 4.0m/s, percolate flux 17LMH, 40L is concentrated into, adds purified water 40L dilute
Release, continue to be concentrated into 40L, repeat once, finally rinsing milipore filter with 20L purified waters displaces whole concentrates.Concentrate
The edible alcohol of 5 times of amounts of original volume is slowly added under stirring, it is closed, stand overnight, precipitation and separation, heated-air drying, crush, mistake
Sieve, is produced.
Obtained total ganoderma spore powder refined polysaccharide is pale powder, yield 1.83%.
Embodiment 3：
UV-VIS spectrophotometry determines the protein and polyoses content of total ganoderma spore powder refined polysaccharide
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.
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 ".
Protein content and polyoses content in the total ganoderma spore powder refined polysaccharide that embodiment 1 is prepared respectively by
It is measured according to the above method, the protein content for obtaining total ganoderma spore powder refined polysaccharide is 6.28%, and polyoses content is
87.12%.
Embodiment 4：
The molecular weight and molecular weight distribution determination of total ganoderma spore powder 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 total ganoderma spore powder refined polysaccharide about 100mg that Example 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：By each reference substance solution and the initial data of total ganoderma spore powder refined polysaccharide solution with AIA forms
It imported into Shanghai thousand to compose in GPC gel dedicated processes softwares, is analyzed.
Obtained result such as table 1 is to table 2, shown in Fig. 1 to Fig. 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 total ganoderma spore powder refined polysaccharide of table 2
From table 1 to table 2, and Fig. 1 to Fig. 4 result can be seen that the total ganoderma spore powder that embodiment 1 is prepared and refine
The equal relative molecular weight of weight (Mw) of polysaccharide is 179.689KD, and the accumulative coupon weight area more than 10KD molecular weight accounts for
99.11%；Total ganoderma spore powder refined polysaccharide is mainly made up of 4 fractions, fraction 1, fraction 2, fraction 3, the peak area of fraction 4
Accounting is respectively 22.21%, 23.04%, 34.40%, 20.35%, the equal relative molecular weight (Mw) of weight be respectively 677.261KD,
98.706KD、 15.713KD、5KD。
Embodiment 5：
The purity and monose composition of PMP-HPLC derivatization methods measure total ganoderma spore powder 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 total ganoderma spore powder refined polysaccharide about 100mg that Example 1 is prepared,
It is accurately weighed, with a small amount of 50 DEG C~60 DEG C of deionized water dissolvings and be transferred in 10mL measuring bottles, put to room temperature (25 DEG C), go from
Sub- 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, close
Lid, the reinforcing of sealed membrane covering, it is vortexed and fully mixes, enters 70 DEG C of water-bath insulation 30min, take out rapidly, ice bath 30s terminating reactions,
Uncap, add 2.000mL 0.6mol/L aqueous hydrochloric acid solution, be transferred to after mixing in 10mL measuring bottles, be settled to deionized water
Scale, Mi Gai, fully mix, precision is drawn 7.000mL and entered in 15mL centrifuge tubes, and pipette, extract 7mL chloroforms pour derivative liquid
In, upper strata aqueous is removed into another 15mL centrifuge tubes, is continued to be rinsed 2 times with chloroform the same manner, is drawn upper strata aqueous, room temperature
Lower 12000r/min centrifuges 10min, takes supernatant appropriate, by the 3/4,1/2,1/4,1/8,1/16,1/32,1/ of mother liquid concentration
64 are diluted, and the derivatization reference substance solution of 8 gradients is made, produces.
The preparation of test sample derivatization solution：Precision pipettes test sample stock solution 0.200mL into 5mL ampere bottles, adds
Enter 0.200mL 4mol/L trifluoroacetic acid solution, ampere bottle is full of nitrogen, uses alcohol blast burner tube sealing rapidly, puts 110 DEG C of baking ovens
Middle hydrolysis 4h, take out, put to room temperature, knock-off ampoule bottle-neck under upright state；0.5mL methanol is added, less than 60 DEG C vacuum subtract
The drying of dry or dry type nitrogen evaporator is pressed dry, handles three times, is dried to hydrolyzate complete repeatedly；Residue is accurate respectively to add 0.200mL
Deionized water, 0.200mL0.6mol/L sodium hydrate aqueous solution, add 0.400mL0.5mol/L PMP methanol solutions, low frequency
Rate 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, WatersSymmetryShieldRP18 (4.6 ×
250mm, 5 μm) chromatographic column, 30 DEG C of column temperature is equal using 18% acetonitrile -82%0.1mol/L ammonium acetate aqueous solution as flowing
Degree 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, is calculated 12 in the total ganoderma spore powder refined polysaccharide of embodiment 1
The total content of kind monose；For the monose that accounting in refined polysaccharide is larger, its substance withdrawl syndrome is obtained respectively, by target monose
The minimum monose of middle substance withdrawl syndrome is converted with it, tried to achieve as basic parameter 1, the substance withdrawl syndrome of other monose
Multiple produces.
Experimental result such as table 3 is to table 6, shown in Fig. 5 to Fig. 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 total ganoderma spore powder refined polysaccharide of table 6
Note：Only calculate the monose that content is not less than 0.3%
According to above-mentioned experimental result, it is known that according to the full ganoderma lucidum spore prepared by the method for the present patent application embodiment 1
Sub- powder refined polysaccharide is the heteroglycan being mainly made up of 4 kinds of mannose, glucose, galactolipin, fucose monose, and its mol ratio is
1.00:45.05:4.76:1.74, mannitol containing 1.63g, 68.73g grapes respectively per 100g total ganoderma spore powders refined polysaccharides
Sugar, 7.79g galactolipins, 2.59g fucoses, total ganoderma spore powder refined polysaccharide is calculated with the absolute content of principal monosaccharides
Purity is 80.74%.
Embodiment 6：
Total ganoderma spore powder 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；Total ganoderma spore powder 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
Storehouse.SPF level BALB/c mouses, female, 6-8 week old, body weight 14-16g, purchased from Guangdong Medical Lab Animal Center, production is permitted
Can the number of card：SCXK (Guangdong) 2013-0002, animal verification of conformity numbering：No.44007200042242.
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 digested with 0.25% trypsase -0.02%EDTA
Cell, 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 is real
Verify real cell viability>95%, it is 2*l0 to adjust viable cell concentrations with 1640 culture medium5/ mL, it is standby.Lotus knurl group animal inoculation pvaccination
Position sterilizes preserved skin, and drawing 4T1 cell suspensions 0.1mL with 1mL syringes (contains 2*104Individual cell) it is inoculated in mouse oxter skin
Under, as tumor size 2mm*2mm, it is judged as that model is successfully established.Blank control group oxter is subcutaneously injected 0.1mL and cultivated completely
Base.
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 is inoculated with 4T1 breast cancer cells, establishes mouse breast cancer model；After modeling 24h, lotus knurl group animal presses body weight randomized blocks
It is divided into：Model group control group, taxol group, total ganoderma spore powder 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.Total ganoderma spore powder is refined more
Sugar, 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,
16th, vernier caliper measurement volume of tumor mass is used within 18,20 days.Length=a, is the length of knurl body most length direction, and wide=b is most rectangular
To vertical plane shortest length；Calculate gross tumor volume (tumor volume, TV)=1/2 × a × b2, draw lotus knurl Volume Changes
Curve.
2.2 blood：After drug withdrawal 24h, animal materials testing index.Blood sample is gathered first：(1) eye socket takes blood (about 6 drop),
Liquaemin anti-freezing, flow cytomery PBLC group.(2) eye socket takes blood (about 6 drop), 4%EDTA anti-freezings, sample
Censorship, 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 increasing in knurl body surface face
Weighed after the connective tissues such as angiogenic, envelope.(2) continue to take spleen, weighed after removing surface attachment tissue.(3) take pictures：It is all
After animal tumor mass is removed, sequenced by group order, enclose respective labels, background is placed one ruler, taken pictures, as original record
Preserve.
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 figure
Shown in 8, total ganoderma spore powder refined polysaccharide combination taxol group can significantly inhibit the growth of tumour, and its curative effect is better than alone Japanese yew
Alcohol group.
4.2 experimental animal lotus knurl Volume Changes are as shown in table 9, Fig. 9, model group animal tumor volume rapid growth, and single
It is slow compared with model group with taxol group and total ganoderma spore powder refined polysaccharide combination taxol group animal tumor volume growth trend；
Compared with model group, up to the 20th day, total ganoderma spore powder refined polysaccharide combination taxol group swelled within the 14th day, the 16th day for administration
Knurl volume significantly reduces, and difference has statistical significance (P ＜ 0.01)；Compared with alone taxol group, administration the 14th day, the
Up to the 20th day, the gross tumor volume of total ganoderma spore powder refined polysaccharide combination taxol group substantially reduced, wherein the 14th within 16 days
It difference has statistical significance (P ＜ 0.01).
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.
4.3 experimental animal lotus knurl weight and related organ index are as shown in table 10, Figure 10~13, compared with model group, entirely
The lucidum spore powder refined polysaccharide combination tumor weight of taxol group, tamor index, gross tumor volume, batch index and spleen index significantly under
Drop, difference have statistical significance (P ＜ 0.01)；Compared with alone taxol group, the combination of total ganoderma spore powder refined polysaccharide is purple
The tumor weight of China fir alcohol group, tamor index, gross tumor volume, index and spleen index are decreased obviously, and the difference of wherein index and spleen index has
Statistical significance (P ＜ 0.05).
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.
For 4.4 lymphocyte subpopulation flow cytometer showed results as shown in table 11, Figure 14~18, total ganoderma spore powder is more
Lymphocyte subpopulation CD3, CD4, CD8 ratio caused by sugar combination taxol group can significantly reverse alone taxol group drop
Low trend, difference have statistical significance (P ＜ 0.01).
Experimental animal lymphocyte subpopulation distribution situation (subgroup the accounting 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.
4.5 blood routine testing results are as shown in table 12, Figure 19~22, compared with model group and alone taxol group, Quan Ling
Ganoderma lucidum spore powder refined polysaccharide combination taxol group can substantially reduce peripheral white blood cells level, be close to normal value, wherein with
The difference of model group has statistical significance (P ＜ 0.01)；Simultaneously compared with model group and alone taxol group, ganoderma spore
The significantly raised platelet levels of powder refined polysaccharide combination taxol group energy, wherein there is statistical significance (P with the difference of model group
＜ 0.01)；In addition compared with model group and alone taxol group, total ganoderma spore powder refined polysaccharide combination taxol group can also be light
The erythrocyte level and hemoglobin level of micro- lifting peripheral blood.
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.
5 conclusions
Total ganoderma spore powder refined polysaccharide joint paclitaxel treatment group shows significant suppression within the whole test period
The function of malignant breast carcinomas tumour growth processed, independent Paclitaxel Chemotherapy group is significantly stronger than the effect of therapeutic alliance group, and can be special
Immune system caused by sex reversal tumour growth and the independent chemotherapy of taxol and hematological system main biochemical Index for examination value it is different
Often, this active polysaccharide can be applied to tumor prevention and treat the exploitation of medicine, can also be applied to association area food and health products
Exploitation.
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 (8)
- A kind of 1. total ganoderma spore powder refined polysaccharide with notable adjunct antineoplastic activity, it is characterised in that the full ganoderma lucidum Conidia powder refined polysaccharide is mainly by 4 kinds of mannose, glucose, galactolipin, fucose monose in molar ratio 1：40~50：4~ 5.5：The heteroglycan of 1.5~2.0 compositions；It is smart to calculate total ganoderma spore powder with the above-mentioned 4 kinds main absolute contents for forming monose The purity of polysaccharide processed, up to more than 80%；The equal relative molecular weight (Mw) of weight is 160~200KD, and accumulative more than 10KD molecular weight cuts Sheet weight area accounts for more than 90%；Total ganoderma spore powder refined polysaccharide is mainly made up of 4 fractions, fraction 1, fraction 2, fraction 3, The peak area accounting of fraction 4 is respectively 20~25%, 20~25%, 30~40%, 15~25%, and the weight of 4 fractions is relative Molecular weight (Mw) is respectively 620~700KD, 90~110KD, 14~18KD, 4~6KD；UV-VIS spectrophotometry determines Protein content (BCA methods) in total ganoderma spore powder refined polysaccharide is less than 8%, and polysaccharide (Anthrone-sulfuricacid method) content is more than 85%.
- 2. total ganoderma spore powder refined polysaccharide according to claim 1, it is characterised in that the total ganoderma spore powder refines Polysaccharide is mainly by 4 kinds of mannose, glucose, galactolipin, fucose monose in molar ratio 1：45.05：4.76：1.74 composition Heteroglycan；Per 100g total ganoderma spore powders refined polysaccharides respectively mannitol containing 1.63g, 68.73g glucose, 7.79g galactolipins, 2.59g fucose；Using the above-mentioned 4 kinds main absolute contents for forming monose calculate the purity of total ganoderma spore powder refined polysaccharide as 80.74%；The equal relative molecular weight (Mw) of weight is 179.689KD, and the accumulative coupon weight area more than 10KD molecular weight accounts for 99.11%；Total ganoderma spore powder refined polysaccharide is mainly made up of 4 fractions, fraction 1, fraction 2, fraction 3, the peak area of fraction 4 Accounting is respectively 22.21%, 23.04%, 34.40%, 20.35%, and the equal relative molecular weight of weight (Mw) of 4 fractions is respectively 677.261KD、98.706KD、15.713KD、5KD；In UV-VIS spectrophotometry measure total ganoderma spore powder refined polysaccharide Protein content (BCA methods) be 6.28%, polysaccharide (Anthrone-sulfuricacid method) content be 87.12%.
- A kind of 3. preparation method of the total ganoderma spore powder refined polysaccharide with notable adjunct antineoplastic activity, it is characterised in that It the described method comprises the following steps：(1) hot water extracts：Take the total ganoderma spore powder recrement for having completed ganoderma spore oil extract appropriate, in multifunctional extracting pot The purified water of 10~30 times of recrement weight is added, after logical steam boils, under stirring or startup feed pump adverse current forced circulation slowly Above-mentioned recrement is added, continues stirring or interval starts feed pump and forces circulated in countercurrent, it is small until maintaining the micro-boiling time to reach 1~2 When after steam off；(2) coarse filtration separates：After the completion of extraction, standing sedimentation simultaneously cools down, and supernatant is extracted out, with 0.2~2.5 μm of plate of filtering accuracy Frame filter or bag filter carry out coarse filtration, and alternatively, residue by step (1) method repeats extraction once and coarse filtration again, Merge coarse filtration liquid；(3) refined filtration is clarified and removes supramolecular impurity：200~800nm of coarse filtration liquid tubular ceramic microfiltration membranes or filtering essence The micropore folding type filter element of 0.1~0.22 μm of degree carries out refined filtration clarification；(4) film is concentrated by ultrafiltration and removes micromolecular water solubility impurity：Clarified solution milipore filter (molecular cut off be 3500~ 8000D) concentrated.(5) dry：Concentrate is drying to obtain using heated-air drying after alcohol precipitation or Direct spraying.
- A kind of 4. system of total ganoderma spore powder refined polysaccharide with notable adjunct antineoplastic activity according to claim 3 Preparation Method, it is characterised in that the described method comprises the following steps：(1) hot water Dynamic Extraction polysaccharide：Take the total ganoderma spore powder recrement for having completed ganoderma spore oil extract appropriate, in multipotency The purified water of 10~30 times of recrement weight is added in extractor, after logical steam boils, is stirred or started with 30~60r/min speed Above-mentioned recrement is slowly added under feed pump adverse current forced circulation, continues stirring or start feed pump at interval of 10~20min to force 5~10min is circulated, the steam off after maintaining the micro-boiling time to reach 1~2 hour；(2) natural subsidence joint coarse filtration technique realizes slag-liquid separation：After the completion of extraction, standing sedimentation is simultaneously cooled to less than 50 DEG C, Extract supernatant out, carry out coarse filtration with the plate filter or bag filter of 0.2~2.5 μm of filtering accuracy, alternatively, residue is pressed Step (1) method repeats extraction once and again coarse filtration, merges coarse filtration liquid；(3) refined filtration technique realizes that decoction is clarified and removes the supramolecular impurity of thermosol cold analysis：Above-mentioned 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 30~70 DEG C, 0.20~0.80Mpa of operating pressure, 3~5m/s of crossflow velocity of temperature, thoroughly Cross liquid 150~400LMH of flux, during raffinate residue about 20L, add purified water about 20L to dilute, it is remaining to raffinate to continue clarification activities About 20L, alternatively, repeat and once dilute clarification activities, to ensure desired polysaccharide all by reducing technique damage as far as possible Consumption；(4) film ultrafiltration technology realizes the concentration of desired polysaccharide and removes micromolecular water solubility impurity：Above-mentioned clarified solution milipore filter Concentrated, ultrafiltration retaining molecular weight is 3500~8000D, and Operating parameters are 30~50 DEG C of temperature, operating pressure 0.15~0.6Mpa, 2~4.5m/s of crossflow velocity, 6~25LMH of percolate flux, when being concentrated into about 40L, add purified water about 40L Dilution, continues to be concentrated into about 40L, alternatively, repeats and once dilute concentration step, water-soluble to eliminate small molecule to greatest extent Property impurity, finally with about 20L purified waters rinse milipore filter displace whole concentrates, to reduce process loss as far as possible；(5) drying process：The edible alcohol that 3~7 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, a small amount of ethanol washing, heated-air drying or vacuum drying is taken out, finally alternatively pulverizes and sieves i.e. ；Or concentrate can also use the step of spray drying process one to complete to be dried to obtain.
- A kind of 5. total ganoderma spore powder refined polysaccharide with notable adjunct antineoplastic activity according to claim 3 or 4 Preparation method, it is characterised in that the total ganoderma spore powder recrement includes having completed in patent of invention CN 100593410C complete The total ganoderma spore powder recrement of Reishi sporule oil extract.
- 6. the total ganoderma spore powder refined polysaccharide being prepared according to any one of claim 3 to 5.
- 7. according to any described total ganoderma spore powder refined polysaccharide in claim 1 to 2 or 6 preparing for tumor prevention and Purposes in terms of the adjunct antineoplastic medicine for the treatment of, food, health food.
- 8. total ganoderma spore powder refined polysaccharide according to claim 7 resists in preparation for tumor prevention and the auxiliary for the treatment of Purposes in terms of tumour medicine, food, health food, it is characterised in that it is used alone comprising total ganoderma spore powder refined polysaccharide, Or the compound formed with other active components and/or with its pharmaceutically acceptable auxiliary material, carrier.
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| CN110218717A (en) * | 2019-05-22 | 2019-09-10 | 暨南大学 | A kind of preparation method of ganoderma lucidum polysaccharide and its protective effect to the dry inactivation of superoxide dismutase |
| CN113768141A (en) * | 2021-09-18 | 2021-12-10 | 广东粤微食用菌技术有限公司 | Anti-oxidation repairing ganoderma lucidum extract and preparation method thereof |
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| CN113768141A (en) * | 2021-09-18 | 2021-12-10 | 广东粤微食用菌技术有限公司 | Anti-oxidation repairing ganoderma lucidum extract and preparation method thereof |
| CN113768141B (en) * | 2021-09-18 | 2022-10-18 | 广东粤微食用菌技术有限公司 | Anti-oxidation repairing ganoderma lucidum extract and preparation method thereof |
| WO2023040033A1 (en) * | 2021-09-18 | 2023-03-23 | 广东粤微食用菌技术有限公司 | Anti-oxidant repair ganoderma lucidum extract and preparation method therefor |
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