CN106755256A - A kind of preparation method of ground bettle polypeptide, the ground bettle polypeptide for preparing and its application - Google Patents

A kind of preparation method of ground bettle polypeptide, the ground bettle polypeptide for preparing and its application Download PDF

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CN106755256A
CN106755256A CN201611263519.4A CN201611263519A CN106755256A CN 106755256 A CN106755256 A CN 106755256A CN 201611263519 A CN201611263519 A CN 201611263519A CN 106755256 A CN106755256 A CN 106755256A
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ground bettle
polypeptide
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zymolyte
enzymolysis
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庄宝祥
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Weifang Medical University
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Abstract

The present invention relates to a kind of preparation method of polypeptide, more particularly to a kind of preparation method of ground bettle polypeptide, the method comprises the following steps：(1) pre-process：Ground bettle cleaning, homogenate；(2) primary enzymolysis：Add ficin enzymolysis；(3) fermentation process：Mixed bacteria is added to be fermented；(4) secondary enzymolysis：Add trypsin digestion；(5) hyperfiltration treatment.Preparation method in the present invention, the ground bettle peptide masses for obtaining are good, and the recovery rate of ground bettle polypeptide is high, can suppress the growth of in vitro stomach cancer cell.

Description

A kind of preparation method of ground bettle polypeptide, the ground bettle polypeptide for preparing and its Using
Technical field
The present invention relates to polypeptide extractive technique field, and in particular to a kind of preparation method of ground bettle polypeptide and its be prepared into The application of the ground bettle polypeptide for arriving.
Background technology
Ground bettle is the female adult hirudo leech of Corydiidae eupolyphagasinensis walker or polyphagaplancyi, main product Fujian, Hebei, Shaanxi, Gansu, green grass or young crops The ground such as sea, Shandong, Henan, Jiangsu, Zhejiang, Hunan, are a kind of important tradition the effect of reunion of fractured tendons and bones with dissipating blood stasis with potent drugs Chinese medicine.Its organism is contained within various active albumen, amino acid, unrighted acid, trace element, alkaloid and fat-soluble dimension The materials such as raw element, present pharmacological research shows that ground bettle has the effect such as thrombolysis, anticoagulation, antitumor, regulation blood fat.
At present, active peptide is the study hotspot of medical science and Food Science, and people are also more and more comprehensive to the understanding of polypeptide, living Property peptide be the several polypeptides matters to dozens of with specific function obtained by protein hydrolysis, with antiviral, anti-height The bioactivity such as blood pressure, anti-aging, anticancer, antibacterium, anti-cholesterol, anti-oxidant and antithrombotic.It is special due to functional peptide Function so that people have extracted several functions peptide and be used for medical and health care now to its increase in demand, and such as feature is big Legumin peptide has been used to reducing blood lipid.
At present, the research to ground bettle chemical composition is concentrated mainly on amino acid, aliphatic acid, trace element, alkaloid etc. Aspect, the research to ground bettle micromolecule polypeptide is less；Prior art discloses use papain to fresh ground bettle Carry out polypeptide extraction, and ground bettle polypeptide to extracting grinds in immunoregulation effect, blood coagulation resisting function and anti-tumor aspect Study carefully.Microbial bacterial agent produces some protease during the fermentation, can be that amino acid and small molecule are more effectively by breaks down proteins Peptide, has no that report prepares ground bettle polypeptide using microbe fermentation method at present.
The content of the invention
The technical problems to be solved by the invention are：In view of the shortcomings of the prior art, there is provided a kind of ground bettle polypeptide Preparation method.
In order to solve the above technical problems, the technical scheme is that：
A kind of preparation method of ground bettle polypeptide, the preparation method comprises the following steps：
(1) pre-process：Take fresh ground bettle to clean up, according to solid-liquid ratio 1:10~18 are placed in concentration for 2~5mol/ In the NaCl solution of L, homogenized obtains homogenised tissue liquid；
(2) primary enzymolysis：Take the homogenised tissue liquid in step (1), add 0.01~0.05 times of ground bettle weight without flower Fruit protease, adjusts pH value 4~8.5, and temperature control digests 20~40min at 50~68 DEG C, and the enzyme that goes out obtains zymolyte；
(3) fermentation process：The zymolyte in step (2) is taken, the mixed bacteria of 0.01~0.05 times of zymolyte weight is added, PH5~7.5 are adjusted, at 30~40 DEG C, ferment temperature control 12~24h, obtains fermenting mixture；
(4) secondary enzymolysis：The fermenting mixture in step (3) is taken, 0.01~0.05 times of fermenting mixture weight is added Trypsase, adjusts pH5~8, and temperature control digests 20~50min at 30~40 DEG C, and go out enzyme, and zymolyte centrifugation is removed slag, and obtains Ground bettle polypeptide is slightly guided and supported.
(5) hyperfiltration treatment：The ground bettle polypeptide crude extract in step (4) is taken, by ultrafiltration membrance filter, then again by gained Filtrate is retained by NF membrane, and gained trapped fluid freeze-drying is obtained into ground bettle polypeptide extract.
Used as a kind of improved technical scheme, the addition of ficin is ground bettle weight in the step (2) 0.04 times, pH during enzymolysis is 5.5, and temperature is 60 DEG C, and enzymolysis time is 35min.
Used as a kind of improved technical scheme, the mixed bacteria is bacillus subtilis, saccharomycete and lactic acid bacteria, described The mass ratio of bacillus subtilis, saccharomycete and lactic acid bacteria is 0.1~0.3:0.15~0.5:0.1~0.35.
As a kind of perferred technical scheme, the mass ratio of the bacillus subtilis, saccharomycete and lactic acid bacteria is 0.25: 0.42:0.3。
As a kind of perferred technical scheme, the addition of trypsase is fermenting mixture weight in the step (4) 0.04 times, pH is 7.8, temperature be 37 DEG C.
As a kind of improved technical scheme, the zymolyte that secondary enzymolysis are obtained in step (4), by the three of pepsin The addition of secondary enzymolysis, wherein pepsin is 0.01~0.04 times of zymolyte weight, and enzymolysis goes zymolyte centrifugation after terminating Slag, obtains ground bettle polypeptide crude extract.
Used as a kind of improved technical scheme, the molecular cut off of milipore filter is 3000 in step (5).
The present invention is investigated the ground bettle polypeptide of above method preparation answering in terms of stomach cancer isolated cells growth inhibition With.
The ground bettle glycoprotein that the preparation method of the ground bettle polypeptide that the present invention sets up is obtained grows to stomach cancer isolated cells Play inhibitory action；In order to better illustrate effect of the ground bettle polypeptide in stomach cancer isolated cells growth inhibition, the present invention is logical Cross pharmacological evaluation and result is described below：
(1) culture of tumour cell
By SGC-7901 cells in 37 DEG C, 5%CO2, it is big mould containing 10% newborn calf serum and 1.25 μ g/ml celebratings Cultivated under the RPMI1640 culture medium conditions of element, fresh culture is changed 1 time every 2-3d.
(2) effect of the trypan blu e living cells count detection ground bettle glycoprotein to cell
The stomach cancer cell of exponential phase is chosen, single cell suspension, adjustment cell concentration to 2 × 10 is made5Individual/ml, In the inoculation above-mentioned cell suspensions of 1ml to the Tissue Culture Flask of 25ml, 3ml grown cultures liquid is added, cultivate 18h, the PBS of sterilizing is washed The ground bettle polypeptide solution (to maintain nutrient solution to configure, filtration sterilization) for adding 3ml various concentrations for 3 times afterwards is washed, in putting incubator Continue to cultivate 24h and 48h.Blake bottle is taken out, in 1min is made cell dissociation to come off with 0.2mL trypsin solutions, 3ml is added immediately Grown cultures liquid terminates digesting and blowing and beating into individual cells suspension, draws 0.5ml cell suspensions in clean test tube, adds 0.1ml tongues expect dyeing liquor (0.4%, match somebody with somebody with physiological saline), standing 2min are shaken up, with blood counting chamber to undyed cytometer Number, and completed in 1min.
(3) sensitiveness of the mtt assay detection cell to ground bettle polypeptide
Cell concentration to 4 × 10 is adjusted with growth-promoting media4Individual/ml, connects hole to 96 orifice plates immediately, per the μ l of hole 200,96 orifice plate sides 36 holes of the circle of edge one are abandoned, and each hole adds the PBS that 200 μ l sterilize；37 DEG C, 5%CO2, 18h is placed in saturated humidity incubator to be made Cell attachment, sucks nutrient solution, and the PBS of each hole sterilizing is washed 3 times, removes the residual serum in hole, adds many of various concentrations Each 200 μ l of peptide solution (being made into maintaining liquid), cultivate 24h and 48h, if blank.After sample liquid effect, each hole respectively adds matches somebody with somebody The 5mg/ml for putting, the MTT solution of 25 μ l continues to cultivate 4h, topples over each boreliquid, and 150 μ l, 37 DEG C the two of pre-temperature are added per hole Methyl sulfoxide (DMSO), is put into ELIASA and makees Dual Wavelength Absorbance detection, Detection wavelength 570nm, reference wavelength after concussion 10min 630nm。
(4) experimental result
Trypan blu e living cells count detection and analysis result shows：Stomach cancer cell acts on 24h and 48h in ground bettle polypeptide Afterwards, obvious inhibitory action is presented, most cells downright bad symptom occur, there is significant difference, p ＜ compared with negative control group 0.05.Its experiment the results are shown in Table 1.
Inhibitory action of the ground bettle polypeptide of table 1 to stomach cancer cell
Ground bettle peptide concentration (mg/L) 24h inhibiting rates (%) 48h inhibiting rates (%)
0 0 0
0.083 1.46 2.97
0.820 6.23 20.35
9.500 12.46 32.46
36.00 48.79 58.54
Mtt assay testing result shows：Stomach cancer cell is more sensitive to ground bettle polypeptide solution, and effect 48h is to be presented brighter Aobvious inhibitory action, there is significant difference, p ＜ 0.05 compared with negative control group.Its experiment the results are shown in Table 2.
MTT detection of the ground bettle polypeptide of table 2 to stomach cancer cell effect 24h and 48h
Ground bettle peptide concentration (mg/L) 24h inhibiting rates (%) 48h inhibiting rates (%)
0 0 0
0.086 2.68 9.32
0.760 6.14 14.17
5.683 28.46 38.37
9.780 42.20 58.62
After employing above-mentioned technical proposal, the beneficial effects of the invention are as follows：
(1) present invention is soaked using Nacl solution to fresh ground bettle, homogenized is then carried out, because protein is readily soluble In the salting liquid of low concentration, the dissolution of protein is promoted；
(2) present invention first by ficin realize in ground bettle homogenised tissue liquid protein molecular it is abundant Degraded, then filters out effective mixed bacteria, and the synergy of mixed bacteria during the fermentation by many experiments It is not fully exerted, realizes the abundant degraded to ground bettle tissue, because the metabolism of microorganism can be to some polypeptide point Son is modified, and is made between small peptide, transposing occurs between small peptide and amino acid and resets, and obtained active peptide is without bitter taste and different Taste, and then improve the quality of polypeptide；Finally again by the enzymolysis of trypsase, the recovery rate of micromolecule polypeptide is improve；
The present invention effectively realizes the abundant drop to Eupolyphaga protein component using the technique of enzymolysis-fermentation-enzymolysis Solution, improves the quality of ground bettle polypeptide, and also improve the recovery rate of ground bettle polypeptide.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to Limit the present invention.
Embodiment 1
A kind of preparation method of ground bettle polypeptide, the preparation method comprises the following steps：
(1) pre-process：Take fresh ground bettle to clean up, according to solid-liquid ratio 1:10 are placed in the Nacl that concentration is 2mol/L In solution, homogenized obtains homogenised tissue liquid；
(2) primary enzymolysis：The homogenised tissue liquid in step (1) is taken, the ficin of 0.02 times of ground bettle weight is added Enzyme, adjusts pH value 4.5, and temperature control digests 25min at 50 DEG C, and the enzyme that goes out obtains zymolyte；
(3) fermentation process：The zymolyte in step (2) is taken, mixed bacteria (the withered grass bud of 0.02 times of zymolyte weight is added The mass ratio of spore bacillus, saccharomycete and lactic acid bacteria is 0.1:0.18:0.13), adjust pH5.5 temperature controls at 30 DEG C, ferment 12h, Obtain fermenting mixture；
(4) secondary enzymolysis：The fermenting mixture in step (3) is taken, the tryptose of 0.02 times of fermenting mixture weight is added Enzyme, adjusts pH5.3, and temperature control digests 20min at 30 DEG C, and go out enzyme, and zymolyte centrifugation is removed slag, and obtains ground bettle polypeptide and slightly carries Tuck in.
(5) hyperfiltration treatment：The ground bettle polypeptide crude extract in step (4) is taken, by the ultrafiltration that molecular cut off is 4000 Membrane filtration, then again retains gained filtrate by NF membrane, and gained trapped fluid freeze-drying is obtained into ground bettle polypeptide extracts Thing.
Recovery rate of ground bettle polypeptide is 11.89% under the conditions of this.
Embodiment 2
A kind of preparation method of ground bettle polypeptide, the preparation method comprises the following steps：
(1) pre-process：The fresh ground bettle 1000g for cleaning up is taken, according to solid-liquid ratio 1:12 are placed in concentration for 3mol/L Nacl solution in, homogenized obtains homogenised tissue liquid；
(2) primary enzymolysis：The homogenised tissue liquid in step (1) is taken, the ficin of 0.03 times of ground bettle weight is added Enzyme, adjusts pH value 6.2, and temperature control digests 30min at 55 DEG C, and the enzyme that goes out obtains zymolyte；
(3) fermentation process：The zymolyte in step (2) is taken, mixed bacteria (the withered grass bud of 0.03 times of zymolyte weight is added The mass ratio of spore bacillus, saccharomycete and lactic acid bacteria is 0.2:0.3:0.27) pH5.8, is adjusted, at 35 DEG C, ferment temperature control 16h, Obtain fermenting mixture；
(4) secondary enzymolysis：The fermenting mixture in step (3) is taken, the tryptose of 0.03 times of fermenting mixture weight is added Enzyme, adjusts pH5.8, and temperature control digests 30min at 32 DEG C, and go out enzyme, and zymolyte centrifugation is removed slag, and obtains ground bettle polypeptide and slightly carries Tuck in.
(5) hyperfiltration treatment：The ground bettle polypeptide crude extract in step (4) is taken, by the ultrafiltration that molecular cut off is 5000 Membrane filtration, then again retains gained filtrate by NF membrane, and gained trapped fluid freeze-drying is obtained into ground bettle polypeptide extracts Thing.
Recovery rate of ground bettle polypeptide is 12.45% under the conditions of this.
Embodiment 3
A kind of preparation method of ground bettle polypeptide, the preparation method comprises the following steps：
(1) pre-process：The fresh ground bettle 1000g for cleaning up is taken, according to solid-liquid ratio 1:15 are placed in concentration for 3.5mol/ In the Nacl solution of L, homogenized obtains homogenised tissue liquid；
(2) primary enzymolysis：The homogenised tissue liquid in step (1) is taken, the ficin of 0.04 times of ground bettle weight is added Enzyme, adjusts pH value 5.5, and temperature control digests 35min at 60 DEG C, and the enzyme that goes out obtains zymolyte；
(3) fermentation process：The zymolyte in step (2) is taken, mixed bacteria (the withered grass bud of 0.04 times of zymolyte weight is added The mass ratio of spore bacillus, saccharomycete and lactic acid bacteria is 0.25:0.42:0.3) pH7.2, is adjusted, at 37 DEG C, ferment temperature control 20h, Obtain fermenting mixture；
(4) secondary enzymolysis：The fermenting mixture in step (3) is taken, the tryptose of 0.04 times of fermenting mixture weight is added Enzyme, adjusts pH7.8, and temperature control digests 45min at 37 DEG C, and go out enzyme, and zymolyte centrifugation is removed slag, and obtains ground bettle polypeptide and slightly carries Tuck in.
(5) hyperfiltration treatment：The ground bettle polypeptide crude extract in step (4) is taken, by the ultrafiltration that molecular cut off is 3000 Membrane filtration, then again retains gained filtrate by NF membrane, and gained trapped fluid freeze-drying is obtained into ground bettle polypeptide extracts Thing.
Recovery rate of ground bettle polypeptide is 13.36% under the conditions of this.
Embodiment 4
A kind of preparation method of ground bettle polypeptide, the preparation method comprises the following steps：
(1) pre-process：The fresh ground bettle 1000g for cleaning up is taken, according to solid-liquid ratio 1:18 are placed in concentration for 4.5mol/ In the Nacl solution of L, homogenized obtains homogenised tissue liquid；
(2) primary enzymolysis：The homogenised tissue liquid in step (1) is taken, the ficin of 0.05 times of ground bettle weight is added Enzyme, adjusts pH value 8.2, and temperature control digests 40min at 68 DEG C, and the enzyme that goes out obtains zymolyte；
(3) fermentation process：The zymolyte in step (2) is taken, mixed bacteria (the withered grass bud of 0.05 times of zymolyte weight is added The mass ratio of spore bacillus, saccharomycete and lactic acid bacteria is 0.3:0.5:0.32) pH7.5, is adjusted, at 40 DEG C, ferment temperature control 24h, Obtain fermenting mixture；
(4) secondary enzymolysis：The fermenting mixture in step (3) is taken, the tryptose of 0.05 times of fermenting mixture weight is added Enzyme, adjusts pH7.8, and temperature control digests 50min at 40 DEG C, and go out enzyme, then to 0.03 times of addition zymolyte weight in enzymolysis product Pepsin, adjust pH2~3, enzymolysis terminate after by enzymolysis product centrifugation remove slag, obtain ground bettle polypeptide and slightly guide and support.
(5) hyperfiltration treatment：The ground bettle polypeptide crude extract in step (4) is taken, by the ultrafiltration that molecular cut off is 4000 Membrane filtration, then again retains gained filtrate by NF membrane, and gained trapped fluid freeze-drying is obtained into ground bettle polypeptide extracts Thing.
Recovery rate of ground bettle polypeptide is 13.08% under the conditions of this.
Comparative example
Using traditional method for extracting：The fresh Eupolyph aga sinesis Walker 1000g cleaned up in step (1) is weighed, phosphate is added Cushioning liquid carries out homogenized, to centrifugation after adding papain, enzymolysis to terminate in the supernatant of gained after centrifugal filtration Supernatant is taken, ground bettle polypeptide extract is obtained by freeze-drying.
Recovery rate of ground bettle glycoprotein is 10.15% under the conditions of this.
Presently preferred embodiments of the present invention is the foregoing is only, is not intended to limit the invention, it is all in essence of the invention Any modification, equivalent and improvement made within god and principle etc., should be included within the scope of the present invention.

Claims (9)

1. a kind of preparation method of ground bettle polypeptide, it is characterised in that the preparation method comprises the following steps：
(1) pre-process：Take fresh ground bettle to clean up, according to solid-liquid ratio 1:10~18 to be placed in concentration be 2~5mol/L's In NaCl solution, homogenized obtains homogenised tissue liquid；
(2) primary enzymolysis：The homogenised tissue liquid in step (1) is taken, the fig egg of 0.01~0.05 times of ground bettle weight is added White enzyme, adjusts pH value 4~8.5, and temperature control digests 20~40min at 50~68 DEG C, and the enzyme that goes out obtains zymolyte；
(3) fermentation process：The zymolyte in step (2) is taken, the mixed bacteria of 0.01~0.05 times of zymolyte weight is added, adjusted PH5~7.5, at 30~40 DEG C, ferment temperature control 12~24h, obtains fermenting mixture；
(4) secondary enzymolysis：The fermenting mixture in step (3) is taken, the pancreas egg of 0.01~0.05 times of fermenting mixture weight is added White enzyme, adjusts pH5~8, and temperature control digests 20-50min at 30~40 DEG C, and go out enzyme, and zymolyte centrifugation is removed slag, and obtains ground beetle Worm polypeptide is slightly guided and supported.
(5) hyperfiltration treatment：The ground bettle polypeptide crude extract in step (4) is taken, by ultrafiltration membrance filter, then again by gained filtrate Retained by NF membrane, gained trapped fluid freeze-drying is obtained into ground bettle polypeptide.
2. the preparation method of a kind of ground bettle polypeptide according to claim 1, it is characterised in that：Nothing in the step (2) The addition of ficin is 0.04 times of ground bettle weight, and pH during enzymolysis is 5.5, and temperature is 60 DEG C, and enzymolysis time is 35min。
3. the preparation method of a kind of ground bettle polypeptide according to claim 1, it is characterised in that：The mixed bacteria is withered Careless bacillus, saccharomycete and lactic acid bacteria, the mass ratio of the bacillus subtilis, saccharomycete and lactic acid bacteria is 0.1~0.3: 0.15~0.5:0.1~0.35.
4. the preparation method of a kind of ground bettle polypeptide according to claim 3, it is characterised in that：The bacillus subtilis The mass ratio of bacterium, saccharomycete and lactic acid bacteria is 0.25:0.42:0.3.
5. the preparation method of a kind of ground bettle polypeptide according to claim 1, it is characterised in that：Pancreas in the step (4) The addition of protease is 0.04 times of fermenting mixture weight, and pH is 7.8, and temperature is 37 DEG C.
6. the preparation method of a kind of ground bettle polypeptide according to claim 1, it is characterised in that：Secondary enzyme in step (4) The zymolyte that solution is obtained, by three times of pepsin enzymolysis, wherein the addition of pepsin be zymolyte weight 0.01~ 0.04 times, enzymolysis removes slag zymolyte centrifugation after terminating, and obtains ground bettle crude extract.
7. the preparation method of a kind of ground bettle polypeptide according to claim 1, it is characterised in that：Milipore filter in step (5) Molecular cut off be 3000.
8. the ground bettle polypeptide that a kind of preparation method of ground bettle polypeptide of claim 1 is prepared.
9. application of the ground bettle polypeptide of claim 8 in terms of stomach cancer isolated cells growth inhibition.
CN201611263519.4A 2016-12-30 2016-12-30 A kind of preparation method of ground bettle polypeptide, the ground bettle polypeptide for preparing and its application Pending CN106755256A (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109305994A (en) * 2017-07-26 2019-02-05 浙江医药股份有限公司新昌制药厂 The pure powder of wood louse powder enzymatic hydrolysis, its preparation and application
CN115109135A (en) * 2022-06-23 2022-09-27 南京中医药大学 Eupolyphaga sinensis protein extract with effects of resisting liver cancer and inhibiting hepatic fibrosis and application thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101696444A (en) * 2009-10-29 2010-04-21 王中华 Polypeptide extract as well as preparation method and application thereof
CN104548058A (en) * 2013-10-24 2015-04-29 河北以岭医药研究院有限公司 Application of ground beetle zymolyte
CN105343232A (en) * 2015-11-11 2016-02-24 安徽生物肽产业研究院有限公司 Composition of effective parts of ground beetle small peptides for treating cardiovascular diseases
CN106173190A (en) * 2015-05-08 2016-12-07 上海邦成生物工程有限公司 A kind of preparation method of soybean polypeptide albumen feedstuff

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101696444A (en) * 2009-10-29 2010-04-21 王中华 Polypeptide extract as well as preparation method and application thereof
CN104548058A (en) * 2013-10-24 2015-04-29 河北以岭医药研究院有限公司 Application of ground beetle zymolyte
CN106173190A (en) * 2015-05-08 2016-12-07 上海邦成生物工程有限公司 A kind of preparation method of soybean polypeptide albumen feedstuff
CN105343232A (en) * 2015-11-11 2016-02-24 安徽生物肽产业研究院有限公司 Composition of effective parts of ground beetle small peptides for treating cardiovascular diseases

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
赵鲁杭 等: "讨论", 《分子医学实验技术》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109305994A (en) * 2017-07-26 2019-02-05 浙江医药股份有限公司新昌制药厂 The pure powder of wood louse powder enzymatic hydrolysis, its preparation and application
CN109305994B (en) * 2017-07-26 2021-09-24 浙江医药股份有限公司新昌制药厂 Pure powder of ground beetle dry powder enzymolysis, preparation and application thereof
CN115109135A (en) * 2022-06-23 2022-09-27 南京中医药大学 Eupolyphaga sinensis protein extract with effects of resisting liver cancer and inhibiting hepatic fibrosis and application thereof
CN115109135B (en) * 2022-06-23 2024-05-17 南京中医药大学 Eupolyphaga Seu Steleophaga protein extract with liver cancer resisting and liver fibrosis inhibiting effects and its application

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Application publication date: 20170531