CN109081876A - The lentinan powder and its extraction process of prevention and/or adjuvant therapy of tumors - Google Patents
The lentinan powder and its extraction process of prevention and/or adjuvant therapy of tumors Download PDFInfo
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- CN109081876A CN109081876A CN201710445531.5A CN201710445531A CN109081876A CN 109081876 A CN109081876 A CN 109081876A CN 201710445531 A CN201710445531 A CN 201710445531A CN 109081876 A CN109081876 A CN 109081876A
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
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Abstract
The present invention provides the lentinan powder and its extraction process of prevention and/or adjuvant therapy of tumors.The present invention provides a kind of extracting methods of lentinan, and the method includes making the solution containing lentinan or thick Titian mushroom polysaccharide powder contact with the alcoholic solution of various concentration and precipitate lentinan.The present invention also provides the lentinan obtained by the method and its purposes in the drug of preparation prevention and/or treatment tumour.The present invention also provides the drug or food compositions of the lentinan obtained comprising the method.The lentinan that method of the invention obtains has excellent prevention and treatment tumor effect.
Description
Technical field
The present invention relates to new lentinan powder and its extraction processes.Specifically, the present invention relates to a kind of new tools
There are prevention and/or adjuvant therapy of tumors active new lentinan powder and its extraction process.
Background technique
In view of the high-incidence of tumour, and it is limited to prevent, treat means, and there is an urgent need to a kind of preventions and/or auxiliary currently on the market
The preparation for the treatment of tumour is helped, such as from the preparation of food, to obtain the effect of preventing and treating tumour.
Lack the product of pre- anti-cancer currently on the market.The target of existing product is mostly based on direct killing tumour cell.
But they often kill immunological effect and immunological memory cell while killing tumor cell.There are also product from
Positive immunological effect is target, reinfocing effect immunocyte, namely what is often called raising immunity.However, modern tumor immunology
Studies have shown that there is normal immunological effector cell in body, immunologic escape still can occur for tumour cell.To find out its cause, be because
There is a group inhibitive ability of immunity cell to hinder immune cells and play immunosurveillance, and releases this group of inhibitive ability of immunity cells
Negative-effect is of great significance for tumor prevention and/or adjuvant treatment.
The size of cancerous tumour has been reduced with polysaccharide such as lentinan.Research shows that lentinan is made with anticancer
With, can activating T cell, natural killer cells and macrophage, to enhance the immunity of the immune system of host.Research
Have shown that lentinan has beneficial prevention and/or therapeutic effect, including such as hepatocellular carcinoma, mammary gland for kinds cancer
Cancer, gastric cancer, colorectal cancer, cancer of pancreas etc..
Although the pharmaceutical usage of known lentinan, extracting lentinan, there are many difficulties.It is currently used for pre- preventing tumor
Lentinan extraction process be Thick many candies, and the ingredient in Thick many candies is not finely divided.
Currently known extracting method can be divided into solvent extraction or ultrasound and ultra high pressure extraction.Solvent extraction method usually makes
With hot water, lye or polyethylene glycol.Lentinan crude extract can be by being obtained with extracting in boiling water.In this method, fresh son is real
After body is extracted with hot water, suspension is filtered, and is centrifuged off insoluble matter.Crude extract is concentrated, then precipitates, then will sink
Starch is re-dissolved in alkaline solution, filtering, with the neutralization of diluted acid.In addition, lye extracting method is also known, the side
Method needs to extract the fructification of fresh or dry lentinan using such as sodium hydroxide solution.Alternatively, it is also possible to pass through ultrasound
Lentinan is extracted with superhigh pressure extracting method.Mushroom powder is immersed in distilled water first, is ultrasonically treated the solution, then
It is extracted using the hot water for extracting lentinan or with sodium hydroxide extracting method.Then it goes to remove water and precipitate.It is then that solution is cold
But and sediment is collected.
Studies have shown that the subdivision component anti-tumor activity with higher of certain lentinans, such as existing research show
The component of high molecular weight shows higher levels of anti-tumor activity.Have been found that the compound performance that ultrasound assisted extraction technique generates
Lower molecular weight and lower anti-tumor activity out.Research finds that HIGH PRESSURE TREATMENT can lead to the dissociation or change of some macromoleculars
Property.It also have been discovered that harsh extraction conditions such as highly basic, high pressure and ultrasound may cause the fracture for extracting lentinan, to drop
The molecular weight of low compound is allowed to inactivate.
Therefore, this field is badly in need of can be used in preventing and/or treatment tumour is (living with high prevention and/or treatment tumour
Property) product such as novel mushroom polysaccharide powder, and prepare the extraction process of the product.
Summary of the invention
Inventor passes through research, it was found that a kind of new lentinan powder with and preparation method thereof.It is astonishing, hair
Bright people has found that the lentinan powder has the activity prevented and/or treat tumour.Inventor also found a kind of new
Lentinan powder preparation method can be obtained by the method with the lentinan for preventing and/or treating tumor promotion
Powder.Also surprisingly, it was concluded that can be finely divided by means of the present invention to the ingredient of mushroom Thick many candies.Of the invention
Method and product can obtain the one-component for improving tumor microenvironment, have significant effect.
The present invention provides a kind of for preventing and/or treating the lentinan powder extraction process of tumour.Work of the invention
Skill is finely divided the ingredient in Thick many candies in the prior art, filters out with the component for improving tumor microenvironment, restores body
Immunologic escape occurs for anti-tumor immunity, pre- preventing tumor.
In some embodiments, the present invention provides a kind of extracting method of lentinan, the method includes make containing
The step of solution of lentinan or thick Titian mushroom polysaccharide powder contact with the alcoholic solution of various concentration and precipitate lentinan.
As starting ingredient, in the present invention can using this field lentinan slightly mention solution or slightly to mention lentinan heavy
Starch carries out, and solution or the thick Titian mushroom polysaccharide precipitation object or the thick powder that mentions of slightly mentioning can be arbitrarily known by this field
The crude extract that method obtains.
In some embodiments, the alcoholic solution for precipitating lentinan may include multiple concentration, for example including 2-
20 concentration, such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 various concentrations.At this
Wen Zhong refers to that determining alcohol refers to volume by volume concentration unless expressly stated otherwise,.In some embodiments, the various concentration
Between difference can be such as 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or it is higher.In some implementations
In scheme, the concentration difference be can be different, such as the difference between first concentration and second concentration is 10%, the
Difference between two concentration and third concentration is 5% etc..
In some embodiments, the alcoholic solution of various concentration used in method of the invention includes at least three difference
Concentration, for example, at least 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 various concentrations.Some
In embodiment, sequence preferably from high to low by concentration elder generation, then from low to high is contacted with the solution containing lentinan, with
Precipitate lentinan.
In some embodiments, final concentration (v/v) of the alcoholic solution described in method of the invention in mixed solution can
To be selected from 90%-10%, such as 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 15%, 25%,
35%, 45%, 55%, 65%, 75%, 85% and they it is any between concentration range.In some embodiments, root
According to needs, it is also an option that the concentration more higher or lower than above-mentioned concentration range.For example, the reality of three concentration of use of the invention
It applies in scheme, can be 65-80% with first final concentration of crude extract of lentinan, second concentration can be 30-
50%, third concentration can be 75-85%.In some embodiments, method of the invention can be using four different alcohol
Concentration carries out, such as can be 65-80% with first final concentration of crude extract of lentinan, and second concentration can be
45-55%, third concentration can be 25-55%, and the 4th concentration can be 75-85%.In some embodiments, this hair
Bright method can be carried out using five different determining alcohols, such as can be with first final concentration of crude extract of lentinan
65-90%, second concentration can be 45-55%, and third concentration can be 25-45%, and the 4th concentration can be 45-
55%, the 5th concentration can be 75-90%.In some embodiments, method of the invention can be using six different alcohol
Concentration carries out, such as can be 65-75% with first final concentration of crude extract of lentinan, and second concentration can be
45-55%, third concentration can be 25-35%, and the 4th concentration can be 35-45%, and the 5th concentration can be 45-
55%, the 6th concentration can be 75%-85%.In some embodiments, method of the invention can use seven differences
Determining alcohol carries out, such as can be 70-90% with first final concentration of crude extract of lentinan, and second concentration can be
50-60%, third concentration can be 20-40%, and the 4th concentration can be 40-50%, and the 5th concentration can be 50-
60%, the 6th concentration can be 60%-70%, and the 7th concentration can be 70%-90%, etc..In some embodiments
In, it, can be since second concentration of above-mentioned concentration range, i.e., by institute if having obtained the thick of lentinan mentions sediment
State lentinan slightly mention sediment respectively in order with second and third, the alcoholic solution of four or more concentration contacts.
In some embodiments, it is not particularly limited for precipitating the alcohol of lentinan.In some embodiments, excellent
The alcohol of choosing includes methanol, ethyl alcohol, isopropanol or their combination.In some embodiments, it can be used comprising additive
Alcoholic solution, such as the ethanol solution containing additive, wherein the additive can be selected from methanol, isopropanol, methyl ethyl ketone,
Methyl iso-butyl ketone (MIBK).In some embodiments it is preferred that the alcoholic solution is ethanol solution.
In some embodiments, thick Titian mushroom polysaccharide solution or thick Titian be can be as the lentinan of initial component
Mushroom polysaccharide precipitation object or thick Titian mushroom polysaccharide powder.In some embodiments, for obtaining the side for slightly mentioning lentinan
Method is not particularly limited, such as can be extracted by boiling water, lye extract, polyethylene glycol extract, ultrasound, ultra high pressure extraction or it
Combined method obtain.In some embodiments, the lentinan crude extract can be obtained by extracting in boiling water.One
In a little embodiments, the lentinan crude extract can be obtained by multiple extracting in boiling water.In some embodiments, it is boiling
Acid or alkali can be added in water after extracting, such as solution of trichloroacetic acid reaction can be added, and/or aqueous slkali is added.In some realities
It applies in scheme, the lentinan of acquisition slightly mentions solution and can be filtered and/or dialyse.In some embodiments, acquisition
Lentinan, which slightly mentions solution alcohol such as ethyl alcohol (such as ethyl alcohol of final concentration of 65-90%) can be added, to be precipitated, optional
Including drying steps, lentinan crude extract is obtained.In some embodiments, above-mentioned addition alcohol such as ethyl alcohol (such as eventually it is dense
Degree is the ethyl alcohol of 65-90%) the step of being precipitated first concentration of alcohol as the method for the present invention.
In some embodiments, it can according to need and washed in method of the invention, purified, and/or dry step
Suddenly, include the steps that for example chromatography method is purified, and/or such as freeze-drying or spray drying step.
In some embodiments, the lentinan obtained by means of the present invention, such as LE58 is also provided herein.One
In a little embodiments, it has proved that the lentinan obtained by means of the present invention has higher treatment and prevention of tumour activity.?
In some embodiments, the lentinan obtained by means of the present invention is also provided herein for preventing and/or treating tumour
Purposes, including be used to prepare prevention and/or treat tumour drug in purposes.Lentinan is for preventing and/or treating
The activity of tumour is known in the art, such as it can be used for preventing and/or treating hepatocellular carcinoma, breast cancer, gastric cancer, knot directly
Intestinal cancer, cancer of pancreas etc..
In some embodiments, the composition of the lentinan comprising method acquisition of the invention is also provided herein.?
In some embodiments, the composition can be drug or food compositions, such as health care product and drinks etc..
It in some embodiments, may include such as various concentration as retouched above provided herein is a kind of kit
The alcoholic solution stated, for easily extracting lentinan.In some embodiments, the kit includes using of the invention
The specification of method.
In some embodiments, provided herein is the alcoholic solutions described above of various concentration for extracting lentinan
Purposes.
In some embodiments, the present invention has following advantages and effect compared with the lentinan that the prior art obtains
Fruit:
The present invention is used for the lentinan powder extraction process of pre- preventing tumor and adjuvant therapy of tumors, in Thick many candies at
Divide and be finely divided, using multistep extraction step, obtains target components, the component is to release immune negative regulation cell to immunological effect
The inhibition of cell, the one-component for restoring immunosurveillance is target, keeps the product obtained more aobvious in terms of improving tumor microenvironment
It writes.
Detailed description of the invention
Fig. 1: lentinan LE58 has restored the proliferation of CD4+T cell.
Fig. 2: lentinan LE58 to the internal depression effect of lung cancer.
Fig. 3: the Spleen cell proliferation effect for the lentinan that method of the invention obtains is noticeably greater than commonsense method acquisition
The stimulating effect of lentinan.
Fig. 4: the anti-tumor effect for the lentinan that method of the invention obtains is noticeably greater than the mushroom of commonsense method acquisition
Polysaccharide.
Specific embodiment
The invention will be further described with reference to the accompanying drawings and embodiments.
Embodiment 1:
Lentinan powder extraction process for pre- preventing tumor of the invention, comprising:
One, it takes 1kg mushroom (Lentinus edodes (Berk.) Sing.), addition water 10L, extracting in boiling water, the time 3 is small
When, amount to and extracts 2 times.Each extracting solution merges after filtering, and heating is concentrated into small size, dialyses 48 hours to circulating water,
It is centrifuged after dialyzed solution concentration, 30% solution of trichloroacetic acid is added in 1: 1 (v/v), reacts 4 hours under the conditions of 4 DEG C, adds 10%
NaOH solution is neutralized to PH7 or so, pricks bag and dialyses 48 hours to circulating water, is centrifuged after dialyzed solution concentration, supernatant is added 3 times
95% ethyl alcohol of volume, stands overnight.Precipitating is collected by centrifugation, in 40 DEG C of dryings, obtains Thick many candies.
Two, the Thick many candies 10g that step 1 is extracted is melted into 1L water, is added 1L ethyl alcohol (50% alcohol precipitation), 4 DEG C stood
Night；
Three, solution in step 2 is dispensed into the centrifuge tube of 50ml, is handled 5 minutes under 10000rpm centrifugation rate；
Four, it collects through the precipitating in step 3, is sub-packed in 10 pipe 50ml centrifuge tubes, is separately added into 11ml water, it is sufficiently molten
It solves, is handled 5 minutes under 10000rpm centrifugation rate；Supernatant is obtained, (totally 5 pipe) is sub-packed in 50ml centrifuge tube, is separately added into anhydrous
Ethyl alcohol (30% alcohol precipitation) sufficiently vibrates, and 10000rpm is centrifuged 5 minutes；
Five, the supernatant of step 4 is sub-packed in 50ml centrifuge tube (totally 5 pipe), is separately added into ethyl alcohol (40% alcohol precipitation), 4 DEG C quiet
It sets overnight；
Six, the alcohol precipitation supernatant of collection step five is separately added into ethyl alcohol (50% alcohol precipitation) in 50ml centrifuge tube (5 pipe),
10000rpm is centrifuged 5 minutes；
Seven, honest and upright and thrifty 200ml is added dehydrated alcohol (final concentration 80%) in 1000ml beaker on the alcohol precipitation of collection step six
Alcohol precipitation, collection are deposited in 50ml centrifuge tube, and vacuum and low temperature freeze-drying obtains the lentinan powder.It is named as
LE58。
Embodiment 2: unkehr effect of the lentinan LE58 that the present invention extracts to inhibitive ability of immunity cell
It as shown in Fig. 1, is FCM analysis figure, (a) is normal splenocytes in Fig. 1；(b) is splenocyte in Fig. 1
In CD4+T cell stimulate lower generate to be proliferated peak at canavaline (ConA)；(c) is that (marrow source inhibits immunosuppressant cell in Fig. 1
Property cell) inhibit the proliferation of CD4+T cell；(d) is the proliferation that lentinan LE58 has restored CD4+T cell in Fig. 1.
BALB/c mouse splenocyte is prepared into single cell suspension, is inoculated in 96 orifice plate of cell culture (U-shaped bottom), cell
Inoculum density is 3 × 105/ holes, while being successively separately added into ConA, marrow source inhibitory cells MSC2, lentinan LE58, ConA
Concentration is 1.5 holes μ g/, and MSC2 cell number is 9 × 103/ holes, and polysaccharide LE58 concentration is 0.2mg/ml.After culture 3 days, streaming is thin
Born of the same parents learn detection CD4+T cell proliferative conditions, the results show that CD4+T cell Proliferation is pressed down in splenocyte+ConA+MSC2 group
System, is added lentinan LE58 in the system, and CD4+T cell restores proliferation.Through the two comparative descriptions, the present invention is to immune suppression
Property cell (MSC2) processed, which has, releases its immunosuppressive effect.
Embodiment 3: internal depression effect of the lentinan LE58 that the present invention extracts to lung cancer
As shown in Fig. 2, the corresponding line of diamond shape icon is control group lung cancer tumor-bearing mice survivorship curve；Rectangle icon is corresponding
Line be lentinan LE58 group lung cancer tumor-bearing mice survivorship curve.LLC lewis lung cancer cell is inoculated in C57 mouse web portion
Subcutaneously, inoculating cell number is 1 × 106/, constructs lung carcinoma subcutaneous Transplanted tumor model, after 10 days, tumour is formed, more using mushroom
Sugared LE58 is injected intraperitoneally, and dosage is 25mg/kg weight.Through the two comparative descriptions, the present invention has one to the growth of lung cancer
Determine depression effect.
Embodiment 4:
Comparative test 1
Non- this patent technique extracts polysaccharide: taking 1kg mushroom (Lentinus edodes (Berk.) Sing.), water is added
10L, extracting in boiling water the time 3 hours, amount to and extract 2 times.Each extracting solution merges after filtering, and heating is concentrated into small size,
It dialyses 48 hours, is centrifuged after dialyzed solution concentration, 30% solution of trichloroacetic acid is added in 1: 1 (v/v), in 4 DEG C of items to circulating water
It is reacted 4 hours under part, 10%NaOH solution is added to be neutralized to PH7 or so, pricked bag and dialyse 48 hours to circulating water, dialyzed solution concentration
After be centrifuged, supernatant be added 3 times of volumes 95% ethyl alcohol, stand overnight.Precipitating is collected by centrifugation, in 40 DEG C of dryings, is used as
The common polysaccharide of control.Experimental result display control polysaccharide extracts the effect of stimulating proliferation of splenocyte not as good as this patent technique
Lentinan.
Experimental method is MTT, measures cell Proliferation, ordinate OD450 value is higher, represents cell activity and cell number is got over
It is more, and this patent extracts lentinan in 100 μ g/ml, after effect 24 hours, Spleen cell proliferation effect is significantly greater than common fragrant
The stimulating effect of mushroom polysaccharide.
Comparative experiments 2:
Killing of regulatory T cells (Tregs) the depression effect T cell to tumour, and this patent extract lentinan can be with
The ratio of Tregs in tumor-bearing mice spleen is lowered, prompts that there is adjunct antineoplastic effect, and common lentinan has no the effect
(referring to fig. 4).
The above embodiments merely illustrate the technical concept and features of the present invention, and its object is to allow person skilled in the art
Scholar cans understand the content of the present invention and implement it accordingly, and it is not intended to limit the scope of the present invention.It is all according to the present invention
Equivalent change or modification made by Spirit Essence, should be covered by the protection scope of the present invention.
Claims (10)
1. a kind of extracting method of lentinan, the method includes making the solution containing lentinan or thick Titian mushroom polysaccharide powder
The step of contacting with the alcoholic solution of various concentration and precipitate lentinan.
2. method described in claim 1, wherein the alcoholic solution of the various concentration includes 2-20 concentration, such as 2,3,4,5,
6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 various concentrations.
3. method of any of claims 1 or 2 passes through wherein the alcoholic solution of the various concentration includes at least three various concentration
First from high to low, then sequence from low to high contacts concentration with the solution containing lentinan, to precipitate lentinan.
4. method of any of claims 1-3, wherein final concentration (v/v) packet of the alcoholic solution in mixed solution
Include 90%-10%, such as 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 15%, 25%, 35%,
45%, 55%, 65%, 75%, 85% and they it is any between concentration range.
5. the described in any item methods of claim 1-4, wherein the alcoholic solution includes methanol, ethyl alcohol, isopropanol or they
Combination, the preferably described alcoholic solution are ethanol solution.
6. the described in any item methods of claim 1-5, wherein the lentinan that slightly mentions is extracted by boiling water, lye is extracted,
Polyethylene glycol extracts, ultrasound, and ultra high pressure extraction or their combination method obtain.
7. method described in any one of claims 1-6, wherein the method includes washing, purifying and/or drying steps.
8. the described in any item methods of claim 1-7, wherein being for example freeze-dried or being spray-dried the lentinan of precipitating.
9. by the lentinan and its of the method acquisition of any one of claim 1-8 in preparation prevention and/or treatment tumour
Drug in purposes.
10. a kind of drug or food compositions, it includes the lentinans that the method by any one of claim 1-8 obtains.
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| CN201710445531.5A CN109081876A (en) | 2017-06-13 | 2017-06-13 | The lentinan powder and its extraction process of prevention and/or adjuvant therapy of tumors |
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Cited By (2)
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| CN112569336A (en) * | 2020-12-16 | 2021-03-30 | 广东省微生物研究所（广东省微生物分析检测中心） | Application of grifola frondosa glycoprotein in preparation of drugs or health foods for enhancing body immunity and preventing and/or treating tumor diseases |
| CN112608393A (en) * | 2020-12-30 | 2021-04-06 | 贺伟强 | Method for extracting stropharia rugoso-annulata polysaccharide |
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| CN103724447A (en) * | 2013-12-31 | 2014-04-16 | 中国科学院长春应用化学研究所 | Extraction and classification method of lentinan |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112569336A (en) * | 2020-12-16 | 2021-03-30 | 广东省微生物研究所（广东省微生物分析检测中心） | Application of grifola frondosa glycoprotein in preparation of drugs or health foods for enhancing body immunity and preventing and/or treating tumor diseases |
| CN112569336B (en) * | 2020-12-16 | 2023-08-11 | 广东省微生物研究所（广东省微生物分析检测中心） | Application of russula cinerea glycoprotein in preparation of medicines or health foods for enhancing organism immunity and preventing and/or treating tumor diseases |
| CN112608393A (en) * | 2020-12-30 | 2021-04-06 | 贺伟强 | Method for extracting stropharia rugoso-annulata polysaccharide |
| CN112608393B (en) * | 2020-12-30 | 2022-06-03 | 嘉兴职业技术学院 | Method for extracting stropharia rugoso-annulata polysaccharide |
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