CN108148110B - Method for rapidly separating and purifying aspongopus antitumor protein component - Google Patents
Method for rapidly separating and purifying aspongopus antitumor protein component Download PDFInfo
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
- C07K1/303—Extraction; Separation; Purification by precipitation by salting out
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
Abstract
The invention provides a method for quickly separating and purifying aspongopus antitumor protein components, which realizes the quick separation and purification of aspongopus antitumor protein components through high-temperature treatment, ammonium sulfate chromatographic protein and ultrafiltration, and solves the problems that the existing method has complicated steps, high requirements on instruments, high-performance liquid chromatographs, nucleic acid protein chromatographs and the like are often needed for separation and purification, the related chromatographic columns and fillers are expensive, the purification period is long and the like. The invention belongs to the field of biotechnology.
Description
Technical Field
The invention provides a rapid separation and purification method of aspongopus antitumor protein components, and belongs to the technical field of biology.
Technical Field
Aspongopus (Aspongopus chinensis Dallas) is an insect belonging to the family Insecta, the family Hemiptera, the family Birdonidae, also known as Aristolochia nigra, Eugenia glauca, Pilus testudinis, Catharsii molossus, Piper delbrueckii, Piper palustum, etc., and belongs to the family Toyota. The Chinese medicinal composition is emphasized by the functions of regulating qi, relieving pain, warming middle-jiao and tonifying yang and the medicinal components containing anticancer, antibacterial and anticoagulant medicines, but the anticancer medicinal components are not clear. The situation that the aspongopus is known and unknown seriously hinders the process of the aspongopus which is concerned by the international academic community, is a limitation on deep development and utilization of aspongopus resources, so that a deeper research on the active anticancer ingredients of the aspongopus is urgently needed, and a basis is provided for the research and development and application of the aspongopus anticancer drugs. The former experiments show that the water-soluble component of aspongopus hemolymph has a strong inhibiting effect on gastric cancer SGC-7901 proliferation, and further research results show that the water-soluble component of hemolymph can achieve an anti-cancer effect by inducing gastric cancer SGC-7901 to die. However, the conventional method for separating and purifying the anticancer active components has complicated steps and high requirements on instruments, and often requires a high performance liquid chromatograph, a nucleic acid protein chromatograph and the like for separation and purification, and the related chromatographic columns and fillers are expensive and have long purification period.
Disclosure of Invention
The invention aims to: the method for rapidly separating and purifying the aspongopus antitumor protein component is provided, and the problems that the existing method is complicated in steps, high in instrument requirement, high-performance liquid chromatography, nucleic acid protein chromatography and the like are often needed for separation and purification, the related chromatographic columns and fillers are expensive, the purification period is long and the like are solved.
In order to solve the problems, the method for quickly separating and purifying the aspongopus antitumor protein component is adopted:
extracting aspongopus hemolymph: collecting adult aspongopus, freezing and killing in a refrigerator, extracting hemolymph liquid from abdomen of the aspongopus with a 1ml syringe, injecting into a centrifugal tube containing PMSF, centrifuging at 12000r/min for 10min, and sucking the intermediate layer component with a pipette, wherein the name is component I;
② denaturing the protein without anti-tumor activity by boiling water bath: placing the centrifuge tube filled with the component I in a water bath condition at 97 ℃ for processing for 30min, centrifuging for 10min, discarding the precipitate, and reserving the supernatant, wherein the supernatant is named as a component II;
③ ammonium sulfate chromatography protein: weighing 7.67g of ammonium sulfate solid, slowly adding weighed solid ammonium sulfate powder into the supernatant of the component II while stirring to enable the final volume to be 10ml, standing for 1h, centrifuging for 10min, collecting precipitate, re-dissolving and re-suspending by PBS (phosphate buffer solution), and naming the re-suspended solution as a component III;
fourthly, ultrafiltration: ultrafiltering the above component III with 10KDa ultrafilter tube for 20min (3000r/min, 4 deg.C), placing the liquid in the suction inner tube in 30KDa ultrafilter tube, and continuously ultrafiltering for 20min (3000r/min, 4 deg.C), and taking the liquid in the outer tube, and naming as component IV;
detecting the anti-cancer effect and the protein molecular weight of the component IV: the inhibition rate of the component IV on the growth of gastric cancer is detected by an MTT method, and the molecular weight of the component IV is detected by an SDS-PAGE method.
Among the above methods, the specific method of step i is as follows: collecting Aspongopus adults in the field, bringing the Aspongopus adults back to a laboratory, freezing the Aspongopus adults in a refrigerator to kill, breaking off the heads and the wings of the Aspongopus together, putting the rest of the Aspongopus into an inner centrifugal tube with holes in the bottom, and inserting the inner centrifugal tube into an outer centrifugal tube; after centrifugation, separating hemolymph in the insect body into an external centrifuge tube, thereby obtaining aspongopus hemolymph and the insect body; sucking the obtained hemolymph into a centrifugal tube filled with PMSF (permanent magnet synchronous motor) containing 0.5mmol/L, centrifuging at 12000r/min at 4 ℃ for 10min, sucking the hemolymph water-soluble components into a new centrifugal tube to obtain the hemolymph water-soluble components, detecting the protein content by bradford, freeze-drying, preparing the hemolymph water-soluble components into corresponding concentrations by PBS, and filtering and sterilizing for later use;
in the method, the concrete method of the step two is as follows: taking a sterile 15ml centrifuge tube, treating the centrifuge tube filled with the hemolymph water-soluble components for 30min under the condition of water bath at 97 ℃, centrifuging for 10min (12000r/min, 4 ℃), discarding the precipitate, retaining the supernatant, respectively filtering and sterilizing by using a 0.22 mu m filter membrane, and immediately using for anticancer activity detection to obtain an active component II for next separation and purification;
in the method, the concrete method of the step III is as follows: weighing 7.67g of ammonium sulfate solid, slowly adding weighed solid ammonium sulfate powder into the hemolymph supernatant while stirring to avoid the solid ammonium sulfate from forming deposition at the bottom of a beaker, keeping the final volume at 10ml, standing for 1h, filling the supernatant into an activated dialysis bag, dialyzing in an ice bath, centrifuging for 10min (12000r/min, 4 ℃), collecting the deposition, re-dissolving and re-suspending by PBS, obtaining a component III, and detecting the anticancer activity by an MTT method.
In the method, the anticancer activity of each component is detected by MTT method: (1) digesting gastric cancer SGC-7901 by using 0.25% trypsin solution, inoculating 5 × 103 cells per well on a 96-well culture plate, wherein the volume of cell suspension per well is 100 μ l, and each group comprises four wells; (2) respectively adding the purified components after the cells adhere to the wall, setting a drug-free control group and a blank zero-adjusting group, and respectively culturing for 48h at 37 ℃ and 5% carbon dioxide concentration in a carbon dioxide incubator; (3) taking out the culture plate, adding 10 mu l of 5g/LMTT solution into each hole, continuously incubating for 4h at 37 ℃, adding 100 mu l of Formazan dissolving solution into each hole, continuously culturing for 4h until crystals are fully dissolved, measuring the OD value at the position of 570nm of the wavelength, wherein the calculation formula of the inhibition rate is as follows: the inhibition ratio (%) × 1- (drug group OD value-blank OD value)/(control group OD value-blank OD value) ] × 100%.
The invention is mainly based on the method for quickly purifying the aspongopus anticancer active component, which is established by finding that the aspongopus anticancer active component has the characteristic of high temperature resistance in experiments by an applicant and exploring the molecular weight range of the protein of the aspongopus anticancer active component, the method has low requirements on instruments, only needs one centrifuge and one water bath kettle, has short purification period, and can obtain the required anticancer active component after 2 hours.
The purification steps described in the present invention include: high-temperature treatment, ammonium sulfate chromatography protein and ultrafiltration.
In general, most of the protein in the extract is inactivated and precipitated when heated to 95 ℃, but the bioactivity of part of the thermostable protein is still strong. Examples of the heat-stable purification scheme include Stathmin, Escherichia coli alkaline phosphatase and muscle phosphatase inhibitor-1, and the research shows that the aspongopus hemolymph is treated at 97 ℃ to have a large amount of protein denaturation precipitates, centrifuged at 12000r/min for 10min, collected supernatant, filtered and sterilized to detect the anticancer activity, the supernatant still has the anticancer activity, and the hemolymph treated by the same dosage has stronger inhibition effect on cancer cells than the crude extract, so that the heat treatment can be used as an effective method for enriching anticancer active substances in a large amount at the early stage of the experiment.
"salting-out" is commonly used in crude extraction steps for protein isolation and purification. The main principle is as follows: after neutral salts are added to a protein solution, the hydration layer around the protein molecules is weakened or even disappears, because the salt has a greater affinity for water molecules than the protein. Meanwhile, after the neutral salt is added into the protein solution, the ionic strength is changed, so that the charges on the surface of the protein are greatly neutralized, the solubility of the protein is further reduced, and protein molecules are aggregated and precipitated.
In the early stages of the experiment, neutral salts may influence both non-polar (hydrophobic) interactions and electrostatic interactions between proteins to be concentrated. The electrostatic charge induction on the protein surface induces conformation, so that the protein can not be fully solvated to prevent aggregation, therefore, the protein can be dissolved in a salt solution with low concentration but is rarely dissolved or not dissolved in water, and the charge effect of the protein can be improved by increasing the electrostatic force on the neutralization surface of a small amount of salt. When the salt concentration exceeds 0.2mol/L, the electrostatic force on the surface of the protein can be neutralized, and simultaneously, water molecules are taken away from the protein, and the water molecules are necessary for dissolving the protein, and the salt competes for the water molecules. When the ion concentration reaches a certain height, the surface charge is neutralized by the interaction between proteins, and saturated ammonium sulfate is used for salting out in the experiment.
Ultrafiltration is a membrane separation process that utilizes a pressure activated membrane to separate particles by molecular weight. Under the action of external pressure, the dissolved substances and substances with smaller pore size than the membrane can be used as permeate liquid to permeate the membrane, thereby achieving the purposes of solution purification, separation and concentration. The experiment adopts two ultrafiltration membranes of 10KDa and 30KDa to divide the protein solution into 3 parts and remove most impurities. In the early stage of the experiment, a practitioner tried to perform ultrafiltration with crude hemolymph extract, but found that the ultrafiltration membrane is seriously contaminated, and in order to increase the permeation flux of the membrane and ensure the normal and stable operation of the ultrafiltration membrane, the hemolymph is selected to be pretreated first, the ultrafiltration is placed after heat treatment and salting out, and high-speed centrifugation is preferably performed each time to remove denatured proteins in the intermediate process.
Compared with the prior art, the invention is mainly based on the method for quickly purifying the aspongopus anticancer active component, which is established by finding that the aspongopus anticancer active component has the characteristic of high temperature resistance in experiments and exploring the molecular weight range of the protein of the aspongopus anticancer active component, and the purification steps comprise: the method has the characteristics of low cost, simple operation and obvious effect, has low requirement on instruments, can be completed only by one centrifuge and one water bath kettle, has short purification period, can obtain the required anticancer active components after 2 hours, has obvious progress compared with the prior method, and has very important practical significance.
Drawings
FIG. 1 shows SDS-PAGE detection of molecular weight of purified Aspongopus anti-tumor protein fraction, and band c shows purified anti-tumor protein fraction;
FIG. 2 shows MTT method for detecting inhibitory action of Aspongopus purified protein CHP on growth of gastric cancer SGC-7901 (FIG. 2-A) and BGC-823 (FIG. 2-B) for 24h,48h and 72h, wherein Control column represents proliferation rate of Control group (without CHP treatment) cells, and the rest columns represent cell proliferation rate after intervention of gastric cancer cells by CHP of 10,20,30 and 40 μ g/mL (see the description of the invention in the specification of the invention) ((**P<0.01 shows that the difference is very obvious compared with a control group), the inhibition effect of the aspongopus purified protein CHP on gastric cancer SGC-7901 cell proliferation is enhanced along with the increase of CHP concentration, namely the aspongopus purified protein CHP can achieve the anti-cancer effect by inhibiting gastric cancer SGC-7901 and BGC-823 cell proliferation;
FIG. 3 is a graph of flow cytometry analysis of the effect of the purified protein fraction of Aspongopus on gastric cancer SGC-7901 apoptosis using Annexin V-FITC/PI, wherein A is a control group (no purified protein of Aspongopus); b, C, D and E are SGC-7901 cell apoptosis rates measured by flow cytometry after intervention of stomach cancer SGC-7901 cells by 10,20,30 and 40 mu g/mL aspongopus purified protein CHP, and Annexin V-FITC/PI
FIG. 4 is a statistical analysis of the early and late apoptosis rates of SGC-7901 cells measured by the flow cytometry Annexin V-FITC/PI assays of the above groups (A, B, C, D, E) (B, C, D, E)**P<0.01 indicates that the difference is very significant compared with the control group), it can be seen that the induction effect of the aspongopus purified protein CHP on the gastric cancer SGC-7901 cell apoptosis is enhanced along with the increase of the CHP concentration, namely the aspongopus purified protein CHP can kill the cancer cells by inducing the gastric cancer SGC-7901 cell apoptosis.
Detailed Description
In order that the present invention may be more clearly understood, further details of the invention are set forth below.
Example (b):
referring to fig. 1 to 4, this example provides a method for rapidly separating and purifying an anti-tumor protein component of aspongopus, comprising:
extracting aspongopus hemolymph: collecting Aspongopus adults in the field, bringing the Aspongopus adults back to a laboratory, freezing the Aspongopus adults in a refrigerator to kill, breaking off the heads and the wings of the Aspongopus together, putting the rest of the Aspongopus into an inner centrifugal tube with holes in the bottom, and inserting the inner centrifugal tube into an outer centrifugal tube; after centrifugation, separating hemolymph in the insect body into an external centrifuge tube, thereby obtaining aspongopus hemolymph and the insect body; sucking the obtained hemolymph into a centrifugal tube filled with PMSF (permanent magnet synchronous motor) containing 0.5mmol/L, centrifuging at 12000r/min at 4 ℃ for 10min, sucking the hemolymph water-soluble components into a new centrifugal tube to obtain the hemolymph water-soluble components, detecting the protein content by bradford, freeze-drying, preparing the hemolymph water-soluble components into corresponding concentrations by PBS (phosphate buffer solution), filtering and sterilizing for later use, and naming the hemolymph liquid as a component I;
denaturing the protein without anti-tumor activity by boiling water bath: taking a sterile 15ml centrifuge tube, treating the centrifuge tube filled with the hemolymph water-soluble components for 30min under the condition of water bath at 97 ℃, centrifuging for 10min (12000r/min, 4 ℃), discarding the precipitate, retaining the supernatant, respectively filtering and sterilizing by using a 0.22 mu m filter membrane, and immediately using for anticancer activity detection to obtain an active component II for next separation and purification;
ammonium sulfate chromatography protein: 7.67g of ammonium sulfate solid is weighed, and the weighed solid ammonium sulfate powder is slowly added into the hemolymph supernatant liquid while stirring, so that part of irreversible protein precipitation is caused due to local increase of the concentration of the ammonium sulfate, and the accuracy of the experiment is influenced. So as to avoid solid ammonium sulfate from depositing on the bottom of the beaker. The final volume was 10ml, left to stand for 1h, and the supernatant was filled into an activated dialysis bag and dialyzed in an ice bath to sufficiently remove ammonium sulfate. Centrifuging for 10min (12000r/min, 4 ℃), collecting the precipitate, re-dissolving and re-suspending by PBS, wherein the re-suspending solution is named as a component III, and detecting the anticancer activity by an MTT method;
and (3) ultrafiltration: ultrafiltering the above component III with 10KDa ultrafilter tube for 20min (3000r/min, 4 deg.C), placing the liquid in the inner tube into 30KDa ultrafilter tube, and continuously ultrafiltering for 20min (3000r/min, 4 deg.C), and collecting the liquid in the outer tube to obtain component IV (CHP);
and (3) detecting the anti-cancer effect and the protein molecular weight of the component IV (CHP): the MTT method is adopted to detect the inhibition rate of the component IV on the growth of gastric cancer, and the SDS-PAGE method is adopted to detect the molecular weight of the component IV, and the method comprises the following steps:
(1) digesting gastric cancer SGC-7901 by using 0.25% trypsin solution, inoculating 5 × 103 cells per well on a 96-well culture plate, wherein the volume of cell suspension per well is 100 μ l, and each group comprises four wells; (2) respectively adding the purified components after the cells adhere to the wall, setting a drug-free control group and a blank zero-adjusting group, and respectively culturing for 48h at 37 ℃ and 5% carbon dioxide concentration in a carbon dioxide incubator; (3) taking out the culture plate, adding 10 mu l of 5g/LMTT solution into each hole, continuously incubating for 4h at 37 ℃, adding 100 mu l of Formazan dissolving solution into each hole, continuously culturing for 4h until crystals are fully dissolved, measuring the OD value at the position of 570nm of the wavelength, wherein the calculation formula of the inhibition rate is as follows: the inhibition ratio (%) × 1- (drug group OD value-blank OD value)/(control group OD value-blank OD value) ] × 100%.
Claims (6)
1. A method for rapidly separating and purifying aspongopus antitumor protein components is characterized by comprising the following steps:
extracting aspongopus hemolymph: collecting adult aspongopus, freezing and killing in a refrigerator, extracting hemolymph liquid from abdomen of the aspongopus with a 1ml syringe, injecting into a centrifugal tube containing PMSF, centrifuging at 12000r/min for 10min, and sucking the intermediate layer component with a pipette, wherein the name is component I;
the specific method comprises the following steps:
collecting Aspongopus adults in the field, bringing the Aspongopus adults back to a laboratory, freezing the Aspongopus adults in a refrigerator to kill, breaking off the heads and the wings of the Aspongopus together, putting the rest of the Aspongopus into an inner centrifugal tube with holes in the bottom, and inserting the inner centrifugal tube into an outer centrifugal tube; after centrifugation, separating hemolymph in the insect body into an external centrifuge tube, thereby obtaining aspongopus hemolymph and the insect body; sucking the obtained hemolymph into a centrifugal tube filled with PMSF (permanent magnet synchronous motor) containing 0.5mmol/L, centrifuging at 12000r/min at 4 ℃ for 10min, sucking the hemolymph water-soluble components into a new centrifugal tube to obtain the hemolymph water-soluble components, detecting the protein content by bradford, freeze-drying, preparing the hemolymph water-soluble components into corresponding concentrations by PBS, and filtering and sterilizing for later use;
② denaturing the protein without anti-tumor activity by boiling water bath: placing the centrifuge tube filled with the component I in a water bath condition at 97 ℃ for processing for 30min, centrifuging for 10min, discarding the precipitate, and reserving the supernatant, wherein the supernatant is named as a component II;
③ ammonium sulfate chromatography protein: weighing 7.67g of ammonium sulfate solid, slowly adding weighed solid ammonium sulfate powder into the supernatant of the component II while stirring to enable the final volume to be 10ml, standing for 1h, centrifuging for 10min, collecting precipitate, re-dissolving and re-suspending by PBS (phosphate buffer solution), and naming the re-suspended solution as a component III;
fourthly, ultrafiltration: ultrafiltering the component III with 10KDa ultrafiltering tube for 20min, sucking the liquid in the inner tube, placing in 30KDa ultrafiltering tube, and continuously ultrafiltering for 20min, and taking the liquid in the outer tube, and naming as component IV;
detecting the anti-cancer effect and the protein molecular weight of the component IV: the inhibition rate of the component IV on the growth of gastric cancer is detected by an MTT method, and the molecular weight of the component IV is detected by an SDS-PAGE method.
2. The method for rapidly separating and purifying the aspongopus antitumor protein component according to claim 1, wherein the specific method of step (II) is as follows:
taking a sterile 15ml centrifuge tube, treating the centrifuge tube filled with the hemolymph water-soluble components for 30min under the condition of water bath at 97 ℃, centrifuging for 10min, discarding the precipitate, reserving the supernatant, filtering and sterilizing by using a 0.22 mu m filter membrane, immediately using for anticancer activity detection, and obtaining an active component II for next separation and purification.
3. The method for rapidly separating and purifying the aspongopus antitumor protein component according to claim 1, wherein the specific method of step (c) is as follows: weighing 7.67g of ammonium sulfate solid, slowly adding weighed solid ammonium sulfate powder into the component II while stirring, avoiding the solid ammonium sulfate from forming deposition at the bottom of a beaker, keeping the final volume at 10ml, standing for 1h, filling the supernatant into an activated dialysis bag, dialyzing in an ice bath, centrifuging for 10min, collecting the precipitate, re-dissolving and re-suspending PBS, and obtaining the component III, wherein the anticancer activity is detected by an MTT method.
4. The method for rapidly separating and purifying the aspongopus antitumor protein component according to claim 1, wherein the step (v) is to detect the antitumor activity of each component by MTT method:
(1) gastric carcinoma SGC-7901 digested with 0.25% trypsin solution at 5X 10 per well3Inoculating each cell on a 96-well culture plate, wherein the volume of cell suspension in each well is 100 mu l, and each group comprises four wells;
(2) respectively adding the purified components after the cells adhere to the wall, setting a drug-free control group and a blank zero-adjusting group, and respectively culturing for 48h at 37 ℃ and 5% carbon dioxide concentration in a carbon dioxide incubator;
(3) taking out the culture plate, adding 10 mu l of 5g/LMTT solution into each hole, continuously incubating for 4h at 37 ℃, adding 100 mu l of Formazan dissolving solution into each hole, continuously culturing for 4h until crystals are fully dissolved, measuring the OD value at the position of 570nm of the wavelength, wherein the calculation formula of the inhibition rate is as follows: inhibition (%) =1- (drug OD value-blank OD value)/(control OD value-blank OD value) ] × 100%.
5. The method for rapidly separating and purifying the aspongopus antitumor protein component according to claim 1, wherein the method comprises the following steps: the specific conditions of the centrifugation in the second step and the third step are 12000r/min, 4 ℃ and 10min of centrifugation.
6. The method for rapidly separating and purifying the aspongopus antitumor protein component according to claim 1, wherein the method comprises the following steps: the specific conditions of ultrafiltration in the step (IV) are 3000r/min, 4 ℃, and centrifugation is carried out for 20 min.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102670660A (en) * | 2012-05-30 | 2012-09-19 | 贵州大学 | Processing method and application of aspongopus |
| CN106692943A (en) * | 2017-02-09 | 2017-05-24 | 赵德润 | Method for preparing aspongopus bioactive peptide |
| CN107522770A (en) * | 2017-10-25 | 2017-12-29 | 贵州大学 | Aspongopus medicinal ingredient isolation and purification method |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102670660A (en) * | 2012-05-30 | 2012-09-19 | 贵州大学 | Processing method and application of aspongopus |
| CN106692943A (en) * | 2017-02-09 | 2017-05-24 | 赵德润 | Method for preparing aspongopus bioactive peptide |
| CN107522770A (en) * | 2017-10-25 | 2017-12-29 | 贵州大学 | Aspongopus medicinal ingredient isolation and purification method |
Non-Patent Citations (4)
| Title |
|---|
| The biosynthetic products of Chinese insect medicine, Aspongopus chinensis;Luo XiaoHong等;《FITOTERAPIA》;20120630;第83卷(第4期);754-758 * |
| 不同方法处理的九香虫对人胃癌SGC-7901细胞体外作用比较及其抑癌组分分布;曹米兰等;《生物资源》;20171009;第39卷(第5期);第329-330页第1.4.5节 * |
| 九香虫血淋巴对胃癌SGC-7901细胞体外增殖的抑制作用;檀军等;《山地农业生物学报》;20131231;第32卷(第2期);第120页第1.2.2节，第122页最后1段 * |
| 基于新型加工方法的九香虫药食两用价值研究;蔡仁莲等;《遵义医学院学报》;20160831;第39卷(第4期);第347页第1.2.4.2节，第349页第1段 * |
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