CN103285046A - Auricularia polytricha protein extract and anti-tumor application thereof - Google Patents
Auricularia polytricha protein extract and anti-tumor application thereof Download PDFInfo
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Abstract
The invention discloses an auricularia polytricha protein extract and anti-tumor application thereof. The application is application of the auricularia polytricha protein extract in preparation of a product for restricting tumor cell proliferation. The auricularia polytricha protein extract is prepared according to the method comprising the following steps of: 1) extracting by water after crushing auricularia polytricha sporocarp, collecting a water-soluble matter to obtain an auricularia polytricha water-soluble extract; 2) carrying out ammonium sulfate precipitation of which the saturation level is 80% on the auricularia polytricha water-soluble extract, collecting sediments, and obtaining the auricularia polytricha protein extract after dialyzing the sediments by deionized water. Multiplication of HepG2, MCF-7 and A-549 cells is obviously restrained after the human liver cancer HepG2 cell, the human breast cancer MCF-7 cell and the pulmonary adenocarcinoma A-549 cell are processed by the auricularia polytricha protein extract for 72 hours; the IC50 values are 45 mug/mL, 20 mug/mL, and 25 mug/mL respectively.
Description
Technical field
The present invention relates to Auricularia polytricha (Mout) Sacc. protein extract and antineoplastic thereof uses.
Background technology
Auricularia polytricha (Mout) Sacc. is Auriculariale (Auriculariales), Auriculariaceae (Auriculariaceae), and the fungus of the Auricularia polytricha (Mout) Sacc. kind (A.polytricha) in the Auricularia (Auricularia) is important edible fungi, also is used as medicine.Rich in proteins, multi mineral prime element and vitamin, the Auricularia polysaccharide that sporophore back side tapetum is abundant has higher anti-tumor activity.With the Auricularia polytricha (Mout) Sacc. cultivation technique, the research of Polysaccharide from Auricularia Polytricha Sacc extraction process and polysaccharide function is more in present research.Dialogue dorsal body setae Auricularia mycelium deep layer fermenting process such as Zheng Lixue have carried out more detailed research, serve as to investigate index with mycelium dry mass concentration, and culture medium and the process conditions of dialogue dorsal body setae Auricularia liquid submerged fermentation are optimized.For improving quality and the quality of Auricularia polytricha (Mout) Sacc. product, Ma Jing etc. have summed up Auricularia polytricha (Mout) Sacc. and have efficiently cultivated key technology, and detailed elaboration has been carried out in the requirement of Auricularia polytricha (Mout) Sacc. growing environment and cultivation technique.Tao Hong etc. have studied the liquid culture condition of Auricularia polytricha (Mout) Sacc., have determined that the appropriate media prescription is glucose, and experiment in cultivation shows that the strain preparation time can effectively shorten.Auricularia polytricha (Mout) Sacc. active component function research aspect, Zhang Weimin etc. have studied Polysaccharide from Auricularia Polytricha Sacc and have had the effect that induced platelet is assembled, and the extraction process of Auricularia polytricha (Mout) Sacc. water solublity crude polysaccharides is optimized.And at present, the research of separation and purification protein extract extracorporeal anti-tumor still belongs to blank from the Auricularia polytricha (Mout) Sacc. sporophore.
Summary of the invention
A technical problem to be solved by this invention provides the Auricularia polytricha (Mout) Sacc. protein extract with anti-tumor activity.
Auricularia polytricha (Mout) Sacc. protein extract provided by the present invention prepares according to the method that comprises the steps:
1) the Auricularia polytricha (Mout) Sacc. sporophore is pulverized the back flooding, collected water-soluble substances and obtain the Auricularia polytricha (Mout) Sacc. water solubility extract;
2) described Auricularia polytricha (Mout) Sacc. water solubility extract being carried out saturation is 80% ammonium sulfate precipitation, and collecting precipitation after described precipitate with deionized water dialysis, obtains described Auricularia polytricha (Mout) Sacc. 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 Auricularia polytricha (Mout) Sacc. sporophore can be fresh sporophore and also can be dry sporophore.The sporophore of described drying is that fresh Auricularia polytricha (Mout) Sacc. sporophore drying under room temperature (as 20-25 ℃) is obtained.
The volume ratio of described fresh Auricularia polytricha (Mout) Sacc. sporophore and water can be 1:3-5, 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).Also but 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 Auricularia polytricha (Mout) Sacc. 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 Auricularia polytricha (Mout) Sacc. protein extract dry powder.
Another technical problem to be solved by this invention provides the purposes of above-mentioned Auricularia polytricha (Mout) Sacc. protein extract.
The purposes of above-mentioned Auricularia polytricha (Mout) Sacc. 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 Auricularia polytricha (Mout) Sacc. protein extract;
B, the application of above-mentioned Auricularia polytricha (Mout) Sacc. 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 Auricularia polytricha (Mout) Sacc. specifically can be No. 3, Auricularia polytricha (Mout) Sacc. (Auricularia polytricha (Mont.) Sacc.).
After Auricularia polytricha (Mout) Sacc. 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 45 μ g/mL, 20 μ g/mL, and 25 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 Auricularia polytricha (Mout) Sacc. 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 Auricularia polytricha (Mout) Sacc. protein extract for mtt assay and handles apoptosis situation behind the HepG2 cell 72h.
Fig. 3 measures the Auricularia polytricha (Mout) Sacc. protein extract for mtt assay and handles apoptosis situation behind the MCF-7 cell 72h.
Fig. 4 measures the Auricularia polytricha (Mout) Sacc. protein extract for mtt assay and handles apoptosis situation behind the A-549 cell 72h.
Among Fig. 2-Fig. 4,0,10,20,30,40 and 50 represent 0 μ g/mL group respectively, 10 μ g/mL group, 20 μ g/mL group, 30 μ g/mL group, 40 μ g/mL group, 50 μ g/mL group; The culture plate row from left to right of below are followed successively by 0 μ g/mL group, 10 μ g/mL group, 20 μ g/mL group, 30 μ g/mL group, 40 μ g/mL group, 50 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.
No. 3, the Auricularia polytricha (Mout) Sacc. among the following embodiment (Auricularia polytricha (Mont.) Sacc.) (Zhao Shuan etc. the Auricularia polytricha (Mout) Sacc. mycelium produces the optimization of polysaccharide fermentation condition. the 37th the 4th phase of volume of food science and technology .2012) public can obtain this strain from the Beijing City Agriculture and Forestry Institute, to repeat the application's experiment.
The preparation of embodiment 1, Auricularia polytricha (Mout) Sacc. protein extract
1, preparation Auricularia polytricha (Mout) Sacc. sporophore
No. 3 slant strains of Auricularia polytricha (Mout) Sacc. (Auricularia polytricha (Mont.) Sacc.) are inoculated in the one-level kind culture medium activate, 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 20-25 ℃, collect the first damp sporophore, obtain Auricularia polytricha (Mout) Sacc. (Auricularia polytricha (Mont.) Sacc.) 3 work song entities.
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, 10mg vitamin B1,5g 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: wood flour 50%, cotton seed hulls 28%, wheat bran 20%, sugar 1%, Gypsum Fibrosum 1%, material-water ratio are 1:1-1.2.Through 121 ℃, the 30min autoclaving.
2, preparation Auricularia polytricha (Mout) Sacc. water solubility extract
With fresh Auricularia polytricha (Mout) Sacc. (Auricularia polytricha (Mont.) Sacc.) the 3 work song entities 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 Auricularia polytricha (Mout) Sacc. water solubility extract.
3, preparation Auricularia polytricha (Mout) Sacc. protein extract
In the Auricularia polytricha (Mout) Sacc. 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 Auricularia polytricha (Mout) Sacc. protein extract, as suppressing the tumor cell proliferation medicine.
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 Auricularia polytricha (Mout) Sacc. 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 is in 96 orifice plates, after treating that cell is adherent fully, select 18 porocytes to be divided into 6 groups at random, a matched group and 5 experimental grouies are respectively 0 μ g/mL group (matched group), 10 μ g/mL group, 20 μ g/mL group, 30 μ g/mL group, 40 μ g/mL group, 50 μ g/mL group, every group of three porocytes.Add 200 μ l serum-free mediums in every porocyte of 0 μ g/mL group, 10 μ g/mL organize every hole add 200 μ l Auricularia polytricha (Mout) Sacc. Protein Extraction substrate concentrations be 10 μ g/mL contain Auricularia polytricha (Mout) Sacc. protein extract culture fluid; 20 μ g/mL organize every hole add 200 μ l Auricularia polytricha (Mout) Sacc. Protein Extraction substrate concentrations be 20 μ g/mL contain Auricularia polytricha (Mout) Sacc. protein extract culture fluid; 30 μ g/mL organize every hole add 200 μ l Auricularia polytricha (Mout) Sacc. Protein Extraction substrate concentrations be 30 μ g/mL contain Auricularia polytricha (Mout) Sacc. protein extract culture fluid; 40 μ g/mL organize every hole add 200 μ l Auricularia polytricha (Mout) Sacc. Protein Extraction substrate concentrations be 40 μ g/mL contain Auricularia polytricha (Mout) Sacc. protein extract culture fluid; 50 μ g/mL organize every hole add 200 μ l Auricularia polytricha (Mout) Sacc. Protein Extraction substrate concentrations be 50 μ g/mL contain Auricularia polytricha (Mout) Sacc. 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 Auricularia polytricha (Mout) Sacc. protein extract to the IC50 value (apoptosis rate that makes cancerous cell is 50% the Auricularia polytricha (Mout) Sacc. Protein Extraction substrate concentration in the Auricularia polytricha (Mout) Sacc. 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-; 10 μ g/mL group, 20 μ g/mL group, 30 μ g/mL group, 40 μ g/mL group, the Auricularia polytricha (Mout) Sacc. protein extract culture fluid that contains of every hole adding was respectively to be respectively 10 μ g/mL, 20 μ g/mL, 30 μ g/mL to the high sugar of DMEM-+1% pair of Auricularia polytricha (Mout) Sacc. Protein Extraction substrate concentration that resists middle adding Auricularia polytricha (Mout) Sacc. protein extract mother solution to obtain during 50 μ g/mL organized, 40 μ g/mL, the liquid of 50 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-; 10 μ g/mL group, 20 μ g/mL group, 30 μ g/mL group, 40 μ g/mL group, the Auricularia polytricha (Mout) Sacc. protein extract culture fluid that every hole added during 50 μ g/mL organized is respectively to be respectively 10 μ g/mL, 20 μ g/mL, 30 μ g/mL to the Auricularia polytricha (Mout) Sacc. Protein Extraction substrate concentration that the two anti-middle adding Auricularia polytricha (Mout) Sacc. protein extract mother solutions of RPMI1640+1% obtain, 40 μ g/mL, the liquid of 50 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 Auricularia polytricha (Mout) Sacc. protein extract mother solution all is that being mixed with protein content is the Auricularia polytricha (Mout) Sacc. protein extract culture fluid solution of 500 μ g/mL with the Auricularia polytricha (Mout) Sacc. 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 Auricularia polytricha (Mout) Sacc. 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 Auricularia polytricha (Mout) Sacc. protein extract aqueous solution, this protein content is Auricularia polytricha (Mout) Sacc. Protein Extraction substrate concentration in the Auricularia polytricha (Mout) Sacc. protein extract aqueous solution.
2 experimental results and analysis
2.1 Auricularia polytricha (Mout) Sacc. protein extract anti-tumor activity testing result
The mtt assay measurement result shows, the Auricularia polytricha (Mout) Sacc. 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 Auricularia polytricha (Mout) Sacc. 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 45 μ g/mL, 20 μ g/mL, 25 μ g/mL; Can be observed the change of apoptosis form under the light microscopic.Concrete inhibition sees Table 1 and Fig. 2-4.
Table 1. Auricularia polytricha (Mout) Sacc. protein extract is to three strain cancer cell extracorporeal inhibiting rates
Claims (9)
1. the application of Auricularia polytricha (Mout) Sacc. protein extract in preparation antitumor product or antitumor cell product, described Auricularia polytricha (Mout) Sacc. protein extract prepares according to the method that comprises the steps:
1) the Auricularia polytricha (Mout) Sacc. sporophore is pulverized the back flooding, collected water-soluble substances and obtain the Auricularia polytricha (Mout) Sacc. water solubility extract;
2) described Auricularia polytricha (Mout) Sacc. water solubility extract being carried out saturation is 80% ammonium sulfate precipitation, and collecting precipitation after described precipitate with deionized water dialysis, obtains described Auricularia polytricha (Mout) Sacc. protein extract.
2. the application of Auricularia polytricha (Mout) Sacc. protein extract in preparation inhibition tumor cell proliferation product, described Auricularia polytricha (Mout) Sacc. protein extract prepares according to the method that comprises the steps:
1) the Auricularia polytricha (Mout) Sacc. sporophore is pulverized the back flooding, collected water-soluble substances and obtain the Auricularia polytricha (Mout) Sacc. water solubility extract;
2) described Auricularia polytricha (Mout) Sacc. water solubility extract being carried out saturation is 80% ammonium sulfate precipitation, and collecting precipitation after described precipitate with deionized water dialysis, obtains described Auricularia polytricha (Mout) Sacc. 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 Auricularia polytricha (Mout) Sacc. 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. Auricularia polytricha (Mout) Sacc. protein extract, according to the method preparation that comprises the steps:
1) the Auricularia polytricha (Mout) Sacc. sporophore is pulverized the back flooding, collected water-soluble substances and obtain the Auricularia polytricha (Mout) Sacc. water solubility extract;
2) described Auricularia polytricha (Mout) Sacc. water solubility extract being carried out saturation is 80% ammonium sulfate precipitation, and collecting precipitation after described precipitate with deionized water dialysis, obtains described Auricularia polytricha (Mout) Sacc. protein extract.
8. Auricularia polytricha (Mout) Sacc. protein extract according to claim 7 is characterized in that: described step 2), at 4 ℃ described Auricularia polytricha (Mout) Sacc. water solubility extract being carried out saturation is 80% ammonium sulfate precipitation.
9. according to claim 7 or 8 described Auricularia polytricha (Mout) Sacc. 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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| CN106905409A (en) * | 2017-02-28 | 2017-06-30 | 嵊州市派特普科技开发有限公司 | Auricularia polytricha protein extract preparation method and applications |
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