CN103272217A - Pleurotus abalones protein extract and applications thereof for resisting tumor - Google Patents
Pleurotus abalones protein extract and applications thereof for resisting tumor Download PDFInfo
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Abstract
The invention discloses a pleurotus abalones protein extract and applications thereof for resisting tumor. The pleurotus abalones protein extract is prepared by adopting the method comprising the following steps of: 1) grinding pleurotus abalones fruiting body, then leaching with water, and collecting water-soluble matters to obtain water-soluble pleurotus abalones extract; and 2) carrying out 80% saturation of ammonium sulfate precipitation to the water-soluble pleurotus abalones extract, collecting the precipitate, and dialyzing the precipitate with deionized water to obtain the pleurotus abalones protein extract. After the pleurotus abalones protein extract is used for treating human liver cancer HepG2 cell, human breast cancer MCF-7 cell and human lung adenocarcinoma A-549 cell for 72 hours, the IC50 (50% inhibiting concentration) values are respectively 6mu g/mL, 6mu g/mL and 12mu g/mL, and the inhabitation ratio can be increased along with the prolonging of time and increase of concentration; and the change of cell apoptosis morphology can be observed under a light microscope.
Description
Technical field
The present invention relates to Pleurotus abalonus protein extract and antineoplastic thereof uses.
Background technology
Pleurotus abalonus (Pleurotus abalonus) belongs to Basidiomycotina, Hymenomycetes, Agaricales, Pleurotaceae, pleurotus, and Pleurotus abalonus has another name called the Taiwan Pleurotus ostreatus, the sporophore hypertrophy, and the bacterial context densification, local flavor is like Carnis Haliotidis, and is tender and crisp tasty and refreshing, is a kind of edible fungi of high temperature fruiting.Because of bright mushroom storage tolerance, be suitable for the system jar, be the famous edible fungi in subtropical zone.
At present, mainly concentrate on optimization to Pleurotus abalonus cultivation condition and planting type for the research of Pleurotus abalonus, the different formulations compost can influence the mycelial growth rate of Pleurotus abalonus, character, nutritional labeling and the output performance of sporophore.The data of relevant Pleurotus abalonus fruiting body extract activity research is less.Wang Changrong discovers, contains multiple anti-oxidation active substance in the Pleurotus abalonus sporophore.Multiple separation and purification such as integrated use gel chromatography and liquid chromatography/mass spectrometry multiple techniques and analytical method obtain and identify alkaloid and two kinds of oxidation-resistant active ingredients of soap former times class material from Pleurotus abalonus, for Pleurotus abalonus has been established theoretical basis as a kind of exploitation and application of the Natural antioxidant.Chen Rongrong has detected the influence of extractive of Abalone mushroom to diabetic mice pancreas insulin, superoxide dismutase (SOD), insulin activity factor (PDX-1) gene expression dose, discover that each gene expression dose obviously improves, extractive of Abalone mushroom can be by improving the oxidation resistance performance hypoglycemic activity of pancreas.Present separation and purification albumen and the research that tumor cell kills and wounds still belonged to blank from the Pleurotus abalonus sporophore.
Summary of the invention
A technical problem to be solved by this invention provides the Pleurotus abalonus protein extract with anti-tumor activity.
Pleurotus abalonus protein extract provided by the present invention prepares according to the method that comprises the steps:
1) the Pleurotus abalonus sporophore is pulverized the back flooding, collected water-soluble substances and obtain the Pleurotus abalonus water solubility extract;
2) described Pleurotus abalonus water solubility extract being carried out saturation is 80% ammonium sulfate precipitation, and collecting precipitation after described precipitate with deionized water dialysis, obtains described Pleurotus abalonus protein extract.
Above-mentioned steps 1) in, describedly can be at 2-6 ℃ with flooding 10-14 hour with flooding.Described water can be deionized water.
Described Pleurotus abalonus sporophore can be fresh sporophore and also can be dry sporophore.The sporophore of described drying is that fresh Pleurotus abalonus sporophore drying under room temperature (as 20-25 ℃) is obtained.
The volume ratio of described fresh Pleurotus abalonus sporophore and water can be 1:4-6, as 1:4.
Above-mentioned steps 1) in, can adopt the described water-soluble substances of centrifugal collection.The centrifugal force of the described water-soluble substances of centrifugal collection can be 6000-15000g(such as 12000g), centrifugation time can be 10-20 minute (as 15 minutes).Above-mentioned steps 2) in, also can adopt the described precipitation of centrifugal collection.The centrifugal force of the described precipitation of centrifugal collection can be 6000-15000g(such as 12000g), centrifugation time can be 10-20 minute (as 15 minutes).
Above-mentioned steps 2) in, at 4 ℃ described Pleurotus abalonus water solubility extract being carried out saturation is 80% ammonium sulfate precipitation.
Above-mentioned steps 2) in, the described dialysis with deionized water adopts molecular cut off to carry out for the 3kDa semipermeable membrane.
Above-mentioned preparation method also comprises the liquid in the semipermeable membrane after the dialysis at 3000-9000g(such as 6000g), centrifugal 10-20 minute (as 15 minutes), collect supernatant, this supernatant is carried out lyophilization, be prepared into the step of Pleurotus abalonus protein extract dry powder.
Another technical problem to be solved by this invention provides the purposes of above-mentioned Pleurotus abalonus protein extract.
The purposes of above-mentioned Pleurotus abalonus protein extract provided by the present invention is following A or B:
The product of A, antitumor or tumor cell (as medicine, health product and/or food), its active component are above-mentioned Pleurotus abalonus protein extract;
B, the application of above-mentioned Pleurotus abalonus protein extract in the product (as medicine, health product and/or food) of preparation antitumor or tumor cell.
In the such use, described tumor can be entity tumor.
Described entity tumor can be hepatocarcinoma, breast carcinoma and/or pulmonary carcinoma; Described tumor cell can be hepatoma carcinoma cell, breast cancer cell and/or lung carcinoma cell.Described pulmonary carcinoma can be adenocarcinoma of lung, and described lung carcinoma cell can be lung adenocarcinoma cell.
Described hepatoma carcinoma cell can be human liver cancer cell, as the HepG2 cell; Described breast cancer cell can be human breast cancer cell, as the MCF-7 cell; Described lung carcinoma cell can be human lung adenocarcinoma cell, as the A-549 cell.
Above, described Pleurotus abalonus specifically can be Pleurotus abalonus (Pleurotus abalonus) CFCC89572.
After Pleurotus abalonus protein extract of the present invention was handled human hepatoma HepG2 cell, human breast carcinoma MCF-7 cell and human lung adenocarcinoma A-549 cell 72h, the propagation of HepG2, MCF-7, A-549 cell all obviously was suppressed, and IC
50Value is respectively 6 μ g/mL, 6 μ g/mL, and 12 μ g/mL, and suppression ratio all prolongs in time and the increase of concentration and increasing; Can observe the change of apoptosis form under the light microscopic.Illustrate that the Pleurotus abalonus protein extract all has significant inhibition proliferation function to HepG2, MCF-7, A-549 cell, can be used for preparing the product of antitumor or tumor cell.
Description of drawings
Fig. 1 is BCA protein quantification test kit production standard curve.
Fig. 2 measures the Pleurotus abalonus protein extract for mtt assay and handles apoptosis situation behind the HepG2 cell 72h.
Fig. 3 measures the Pleurotus abalonus protein extract for mtt assay and handles apoptosis situation behind the MCF-7 cell 72h.
Fig. 4 measures the Pleurotus abalonus protein extract for mtt assay and handles apoptosis situation behind the A-549 cell 72h.
Among Fig. 2-Fig. 4,0,4,8,12,16,20,24,32,40 represent 0 μ g/mL group, 4 μ g/mL group, 8 μ g/mL group, 12 μ g/mL group, 16 μ g/mL group, 20 μ g/mL group, 24 μ g/mL group, 32 μ g/mL group, 40 μ g/mL group respectively; The culture plate row from left to right of Fig. 2 below are followed successively by 0 μ g/mL group, 8 μ g/mL group, 16 μ g/mL group, 24 μ g/mL group, 32 μ g/mL group, 40 μ g/mL group; The culture plate row from left to right of Fig. 3 and Fig. 4 below are followed successively by 0 μ g/mL group, 4 μ g/mL group, 8 μ g/mL group, 12 μ g/mL group, 16 μ g/mL group, 20 μ g/mL group; Data result is represented (n=3) with average ± standard deviation, and through one factor analysis of variance, wherein * represents relatively have the result of significant difference (* P＜0.05) with 0 μ g/mL group.
The specific embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is conventional method.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Pleurotus abalonus among the following embodiment (Pleurotus abalonus) CFCC89572, the public can be from China Committee for Culture Collection of Microorganisms forestry microorganism center (China Forest microorganism fungus kind preservation administrative center, China Forestry Culture Collection Center english abbreviation CFCC is called for short forestry microorganism center) obtain.
The preparation of embodiment 1, Pleurotus abalonus protein extract
1, preparation Pleurotus abalonus sporophore
Pleurotus abalonus (Pleurotus abalonus) CFCC89572 slant strains is inoculated in the one-level kind culture medium activates, 25 ℃ of constant temperature culture, treat after mycelia is covered with test tube it to be inoculated in the secondary kind culture medium, 25 ℃ of constant temperature culture chambers are cultured to mycelia and cover with, the secondary kind is inoculated in the cultivating bag that culture medium for cultivating is housed under 25 ℃ the condition and sends out bacterium, strain carries out mycelium stimulation and moves into warmhouse booth after covering with cultivating bag, the fruiting condition keeps humidity more than 90%, temperature is 15-30 ℃, collect the first damp sporophore, obtain Pleurotus abalonus (Pleurotus abalonus) CFCC89572 sporophore.
Wherein, the used culture medium of this experiment is as follows:
One-level kind culture medium: 200g Rhizoma Solani tuber osi, 20g glucose, 20g agar powder, 3g KH
2PO
4, the 10mg vitamin B
1, 5g peptone, 1.5g MgSO
4, the 1000mL distilled water, through 121 ℃, the 30min autoclaving.
Secondary kind culture medium: cotton seed hulls 80%, wheat bran 18%, Gypsum Fibrosum 1%, sugar 1%., material-water ratio is 1:1, through 121 ℃, the 30min autoclaving.
Culture medium for cultivating: cotton seed hulls 57%, corn cob 20%, wheat bran 20%, Calx 3%, material-water ratio are 1:1.Through 121 ℃, the 30min autoclaving.
2, preparation Pleurotus abalonus water solubility extract
With fresh Pleurotus abalonus (Pleurotus abalonus) the CFCC89572 sporophore of the deionized water soaking step 1 of 4 times of volumes 2 hours, utilize tissue mashing machine that mixture is carried out historrhexis to pasty state, behind 4 ℃ of lixiviates (namely leaving standstill) 12h, the centrifugal 15min of 12000g, collect supernatant solution, this supernatant solution is the Pleurotus abalonus water solubility extract.
3, preparation Pleurotus abalonus protein extract
In the Pleurotus abalonus water solubility extract of step 2, add (NH at 4 ℃
4)
2SO
4To (NH
4)
2SO
4Saturation be 80%, under 4 ℃ of conditions, left standstill 4 hours, the centrifugal 15min of 12000g, collecting precipitation is dialysed to this precipitation.Wherein, the molecular cut off of the semipermeable membrane that dialysis is adopted is 3kDa, is deposited in the 5h that dialyses in the tap water that flows in the semipermeable membrane, and 12h again dialyses in deionized water.Liquid in the semipermeable membrane after the dialysis at the centrifugal 15min of 6000g, is collected supernatant, this supernatant was placed the liquid nitrogen lyophilization 36 hours, obtain the Pleurotus abalonus protein extract, as suppressing the tumor cell proliferation medicine.
Embodiment 2, Pleurotus abalonus protein extract suppress the tumor cell proliferation experiment
1.1 for the examination cell strain
Human hepatoma HepG2 cell (available from U.S. ATCC), human breast carcinoma MCF-7 cell (available from U.S. ATCC) and human lung adenocarcinoma A-549 cell (available from U.S. ATCC).
1.2 experimental technique
1.2.1 cell line and cell culture
Human hepatoma HepG2 cell, human breast carcinoma MCF-7 cell and human lung adenocarcinoma A-549 cell activate according to conventional cultural method and go down to posterity.Wherein, human hepatoma HepG2 cell's go down to posterity and activation medium is that the high sugar of DMEM-+1% couple of anti-+ 10%FBS(is at the high sugared (Hyclone of DMEM-, SH30022.01B) add penicillin and streptomycin mixed liquor (penicillin 10000U/mL, streptomycin 10000 μ g/mL) and hyclone (FBS in, Hyclone, SV30087.02), making the above two final volume percentage compositions is that the volumn concentration of 1%, FBS is 10% culture fluid that obtains).Go down to posterity and the activation medium of human breast carcinoma MCF-7 cell and human lung adenocarcinoma A-549 cell is that RPMI1640+1% couple of anti-+ 10%FBS(is at RPMI1640(Invitrogen, add penicillin, streptomycin mixed liquor and hyclone (FBS) 11875-093), making the above two final volume percentage compositions is that the volumn concentration of 1%, FBS is 10% culture fluid that obtains).
1.2.2 cytoactive detects
Adopt the Pleurotus abalonus protein extract of tetrazolium bromide colorimetry (MTT) detection embodiment 1 to human hepatoma HepG2 cell, human breast carcinoma MCF-7 cell and human lung adenocarcinoma A-549 cell inhibiting activity, concrete grammar is as follows:
P8 generation (the 8th generation) cell of this experiment optional step 1.2.1 is with every hole 7 * 10
3Individual/mL cell inoculation in 96 orifice plates, treat that cell is fully adherent after, select 18 porocytes to be divided into 6 groups at random, a matched group and 5 experimental grouies, every group of three porocytes.6 groups of the human hepatoma HepG2 cell are respectively 0 μ g/mL group (matched group), 8 μ g/mL group, 16 μ g/mL group, 24 μ g/mL group, 32 μ g/mL group, 40 μ g/mL group.6 groups of human breast carcinoma MCF-7 cell and human lung adenocarcinoma A-549 cell is 0 μ g/mL group (matched group) respectively, 4 μ g/mL group, 8 μ g/mL group, 12 μ g/mL group, 16 μ g/mL group, 20 μ g/mL group.Add 200 μ L serum-free mediums in every porocyte of 0 μ g/mL group, 4 μ g/mL organize every hole add 200 μ L Pleurotus abalonus Protein Extraction substrate concentrations be 4 μ g/mL contain Pleurotus abalonus protein extract culture fluid; 8 μ g/mL organize every hole add 200 μ L Pleurotus abalonus Protein Extraction substrate concentrations be 8 μ g/mL contain Pleurotus abalonus protein extract culture fluid; 12 μ g/mL organize every hole add 200 μ L Pleurotus abalonus Protein Extraction substrate concentrations be 12 μ g/mL contain Pleurotus abalonus protein extract culture fluid; 16 μ g/mL organize every hole add 200 μ l Pleurotus abalonus Protein Extraction substrate concentrations be 16 μ g/mL contain Pleurotus abalonus protein extract culture fluid; 20 μ g/mL organize every hole add 200 μ L Pleurotus abalonus Protein Extraction substrate concentrations be 20 μ g/mL contain Pleurotus abalonus protein extract culture fluid; 24 μ g/mL organize every hole add 200 μ L Pleurotus abalonus Protein Extraction substrate concentrations be 24 μ g/mL contain Pleurotus abalonus protein extract culture fluid; 32 μ g/mL organize every hole add 200 μ L Pleurotus abalonus Protein Extraction substrate concentrations be 32 μ g/mL contain Pleurotus abalonus protein extract culture fluid; 40 μ g/mL organize every hole add 200 μ L Pleurotus abalonus Protein Extraction substrate concentrations be 40 μ g/mL contain Pleurotus abalonus protein extract culture fluid.Add culture fluid behind 37 ℃ of cultivation 72h, every hole adds 200 μ L MTT working solutions, and (the 5mg/mL MTT solution of sterilized water preparation: serum-free medium=1:9(volume ratio) lucifuge continues to cultivate 4h, the careful cell culture fluid of abandoning in the hole of inhaling, every hole adds 200 μ L DMSO(dimethyl sulfoxide), 10min is hatched in concussion, and crystal is fully melted.Surveying absorbance at 560nm wavelength place with microplate reader, is 100% with the cell survival rate of matched group, the survival rate of experiment with computing group cell: experimental group cell survival rate=OD(experimental group)/the OD(matched group) %.Each apoptosis rate=100%-that organizes cell respectively organizes the survival rate of cell.The experiment triplicate.
Test all The data SPSS12.0(SPSS Inc., USA) statistics is handled in the check of the independent sample t of statistical software.Apoptosis rate according to each group cell calculates the Pleurotus abalonus protein extract to the IC50 value (apoptosis rate that makes cancerous cell is 50% the Pleurotus abalonus Protein Extraction substrate concentration in the Pleurotus abalonus protein extract culture fluid that contains) of every kind of cancerous cell.
Wherein, human hepatoma HepG2 cell's serum-free medium is that the high sugar of DMEM-+1% pair is anti-; 8 μ g/mL group, 16 μ g/mL group, 24 μ g/mL group, 32 μ g/mL group, the Pleurotus abalonus protein extract culture fluid that contains of every hole adding was respectively to be respectively 8 μ g/mL, 16 μ g/mL, 24 μ g/mL to the high sugar of DMEM-+1% pair of Pleurotus abalonus Protein Extraction substrate concentration that resists middle adding Pleurotus abalonus protein extract mother solution to obtain during 40 μ g/mL organized, 32 μ g/mL, the liquid of 40 μ g/mL.The high sugar of DMEM-+1% pair is anti-to be that (Hyclone, adding penicillin and streptomycin mixed liquor (penicillin 10000U/mL, streptomycin 10000 μ g/mL) in SH30022.01B), to make the two final volume percentage composition be 1% serum-free medium at the high sugar of DMEM-.
The serum-free medium of human breast carcinoma MCF-7 cell and human lung adenocarcinoma A-549 cell is that RPMI1640+1% is two anti-; 4 μ g/mL group, 8 μ g/mL group, 12 μ g/mL group, 16 μ g/mL group, the Pleurotus abalonus protein extract culture fluid that contains that every hole added during 20 μ g/mL organized is respectively to be respectively 4 μ g/mL, 8 μ g/mL, 12 μ g/mL to the Pleurotus abalonus Protein Extraction substrate concentration that the two anti-middle adding Pleurotus abalonus protein extract mother solutions of RPMI1640+1% obtain, 16 μ g/mL, the liquid of 20 μ g/mL.RPMI1640+1% is two anti-to be at RPMI1640(Invitrogen, and adding penicillin and streptomycin mixed liquor (penicillin 10000U/mL, streptomycin 10000 μ g/mL) in 11875-093), to make the two final volume percentage composition be 1% serum-free medium.
Above-mentioned Pleurotus abalonus protein extract mother solution all is that being mixed with protein content is the Pleurotus abalonus protein extract aqueous solution of 100 μ g/ml with the Pleurotus abalonus protein extract of corresponding serum-free medium dissolving embodiment 1 preparation of various cells.
Wherein, the assay method of protein content is as follows in the Pleurotus abalonus protein extract aqueous solution:
1) making of standard curve: utilize standard sample bovin serum albumin (BSA) to be configured to the protein solution of variable concentrations, adopt the rich Deco skill company that steps in BCA(Beijing) protein quantification test kit production standard curve (Fig. 1).
2) adopt protein content in the BCA protein quantification kit measurement Pleurotus abalonus protein extract aqueous solution, this protein content is Pleurotus abalonus Protein Extraction substrate concentration in the Pleurotus abalonus protein extract aqueous solution.
2 experimental results and analysis
2.1 Pleurotus abalonus protein extract anti-tumor activity testing result
The mtt assay measurement result shows, the Pleurotus abalonus protein extract of embodiment 1 all has inhibitory action to three strain cancerous cell, suppression ratio all shows dose dependent, compare with matched group, along with the increase of Pleurotus abalonus Protein Extraction substrate concentration, the apoptosis rate of HepG2 cell, MCF-7 cell and A-549 cell all increases, and the IC50 value is respectively 6 μ g/mL, 6 μ g/mL, 12 μ g/mL; Can be observed the change of apoptosis form under the light microscopic.Concrete inhibition sees Table 1, table 2 and Fig. 2-4.
Table 1. Pleurotus abalonus protein extract is to human hepatoma HepG2 cell's extracorporeal inhibiting rate
Table 2. Pleurotus abalonus protein extract is to human breast carcinoma MCF-7 cell and human lung adenocarcinoma A-549 cells in vitro suppression ratio
Claims (9)
1. the application of Pleurotus abalonus protein extract in preparation antitumor product or antitumor cell product, described Pleurotus abalonus protein extract prepares according to the method that comprises the steps:
1) the Pleurotus abalonus sporophore is pulverized the back flooding, collected water-soluble substances and obtain the Pleurotus abalonus water solubility extract;
2) described Pleurotus abalonus water solubility extract being carried out saturation is 80% ammonium sulfate precipitation, and collecting precipitation after described precipitate with deionized water dialysis, obtains described Pleurotus abalonus protein extract.
2. the application of Pleurotus abalonus protein extract in preparation inhibition tumor cell proliferation product, described Pleurotus abalonus protein extract prepares according to the method that comprises the steps:
1) the Pleurotus abalonus sporophore is pulverized the back flooding, collected water-soluble substances and obtain the Pleurotus abalonus water solubility extract;
2) described Pleurotus abalonus water solubility extract being carried out saturation is 80% ammonium sulfate precipitation, and collecting precipitation after described precipitate with deionized water dialysis, obtains described Pleurotus abalonus protein extract.
3. application according to claim 1 and 2 is characterized in that: described tumor is entity tumor.
4. application according to claim 3 is characterized in that: described entity tumor is hepatocarcinoma, breast carcinoma and/or pulmonary carcinoma; Described tumor cell is hepatoma carcinoma cell, breast cancer cell and/or lung carcinoma cell.
5. according to arbitrary described application among the claim 1-4, it is characterized in that: described step 2), at 4 ℃ described Pleurotus abalonus water solubility extract being carried out saturation is 80% ammonium sulfate precipitation.
6. according to arbitrary described application among the claim 1-5, it is characterized in that: the described dialysis with deionized water adopts molecular cut off to carry out for the 3kDa semipermeable membrane.
7. Pleurotus abalonus protein extract, according to the method preparation that comprises the steps:
1) the Pleurotus abalonus sporophore is pulverized the back flooding, collected water-soluble substances and obtain the Pleurotus abalonus water solubility extract;
2) described Pleurotus abalonus water solubility extract being carried out saturation is 80% ammonium sulfate precipitation, and collecting precipitation after described precipitate with deionized water dialysis, obtains described Pleurotus abalonus protein extract.
8. Pleurotus abalonus protein extract according to claim 7 is characterized in that: described step 2), at 4 ℃ described Pleurotus abalonus water solubility extract being carried out saturation is 80% ammonium sulfate precipitation.
9. according to claim 7 or 8 described Pleurotus abalonus protein extracts, it is characterized in that: the described dialysis with deionized water adopts molecular cut off to carry out for the 3kDa semipermeable membrane.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH08291078A (en) * | 1995-04-21 | 1996-11-05 | M I O:Kk | Mushroom protein for food and beverage effective in preventing and treating hypertension and hyperlipemia and having antitumor action, mushroom protein for food and beverage effective in preventing and treating obesity and having antitumor action and method for extracting these proteins |
| CN1429576A (en) * | 2001-12-31 | 2003-07-16 | 钱师良 | Edible fungus formula for treating hypertension and diabetes and its preparation method |
| CN1911253A (en) * | 2006-05-16 | 2007-02-14 | 南开大学 | Extractive of Abalone mushroom, extraction method and application thereof |
-
2013
- 2013-05-30 CN CN2013102103648A patent/CN103272217A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH08291078A (en) * | 1995-04-21 | 1996-11-05 | M I O:Kk | Mushroom protein for food and beverage effective in preventing and treating hypertension and hyperlipemia and having antitumor action, mushroom protein for food and beverage effective in preventing and treating obesity and having antitumor action and method for extracting these proteins |
| CN1429576A (en) * | 2001-12-31 | 2003-07-16 | 钱师良 | Edible fungus formula for treating hypertension and diabetes and its preparation method |
| CN1911253A (en) * | 2006-05-16 | 2007-02-14 | 南开大学 | Extractive of Abalone mushroom, extraction method and application thereof |
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