CN105194645B - Application of mud snail polypeptide in resisting prostate cancer - Google Patents
Application of mud snail polypeptide in resisting prostate cancer Download PDFInfo
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Abstract
The invention relates to application of mud snail polypeptide in resisting prostate cancer, in particular to application of mud snail polypeptide in medicines for resisting prostate cancer DU-145 cells. A crude extract of the bullacta exarata polypeptide is obtained by a trypsin enzymolysis method, and each bullacta exarata polypeptide component is obtained by separation and purification means such as an ultrafiltration method, a gel column separation method, a reversed-phase high performance liquid chromatography technology and the like, and the proliferation inhibition of each separation component on prostate cancer DU-145 cells is respectively researched, so that the research finds that each component can obviously inhibit the proliferation of the prostate cancer DU-145 cells. The method has the advantages of low cost of raw materials, simple and mild extraction conditions, and remarkable proliferation inhibition effect of the extracted bullacta exarata polypeptide on prostate cancer DU-145 cells, and provides a theoretical basis for developing the anti-prostate cancer medicament taking the bullacta exarata polypeptide as the raw material.
Description
Technical Field
The invention relates to application of mud snail enzymolysis polypeptide, in particular to application of mud snail polypeptide in resisting prostate cancer.
Background
Mud snail (Bullacta exaarata), commonly known as "iron spitting", "yellow mud snail", "plum snail", and the like, belongs to the phylum mollusca, class gastropoda, class epigillidae, order tenosynovia, family ardiac, genus mud snail, is a species of special local sea water and brackish water of the western pacific, is composed of shells and soft bodies, contains rich components such as protein, calcium, phosphorus, iron, vitamins and the like, has rich polypeptide substances, is widely distributed in Yangtze river on both sides of the east sea and the yellow sea in China, and is a common aquatic product.
Malignant tumor is a disease which seriously threatens human health and life, the incidence rate is higher and higher, and the existing chemotherapy drugs have certain curative effect on most tumors, but the problems of poor selectivity, large toxic and side effects, drug resistance and the like are still very obvious. Therefore, the search for highly effective, low-toxicity and specific antitumor drugs is still imperative. The polypeptide has small molecular weight, no immunogenicity, high activity and small side effect, and especially has good inhibitory activity on multi-drug resistant tumor cell lines. Marine biological polypeptides are an important field of research of marine biological active substances at present, and some biologically active polypeptides are often in an inactive state in proteins, and the proteins are hydrolyzed by protease to release active peptides hidden in the proteins, so that the biologically active peptides are likely to exert more biological activity than the proteins. For example: the phycocyanin enzymolysis polypeptide has obvious inhibiting effect on the in-vitro growth of the HeLa cell strain of the human cervical carcinoma as found in Dunwei and the like; mytilus edulis polypeptide obtained by enzymolysis of Mytilus edulis such as Yangyongfang has specific proliferation inhibiting effect on prostate cancer PC-3 cells and DU-145 cells, and the inhibiting effect on DU-145 cells is stronger than the proliferation inhibiting effect on PC-3 cells; the content of polypeptide in tegillarca granosa polypeptide in the study of Yao Yongyong et al is 0.25-1.0 g.L-1Within the range, the polypeptide can obviously inhibit the proliferation of lung cancer cells A549 and Ketr-3 cells, can also inhibit the synthesis of cell proteins, and has obvious dose dependence.
Prostate cancer is a highly malignant tumor in men with a high mortality rate secondary to cancer mortality, second only to lung cancer. In recent years, with the change of life style, the aging of population and the continuous improvement of diagnosis level, the incidence rate of the prostate cancer is obviously on the rise. Prostate cancer DU-145 cells are isolated from brain metastases of prostate cancer, are poorly differentiated, are androgen-dependent prostate cancer cells, have a strong metastatic potential, and lack endogenous androgen receptor expression. The time for complete adherence of DU-145 cells is usually within 24h, and the DU-145 cells have stronger invasive and angiogenesis promoting capabilities. The research on the inhibition effect of the mudsnail enzymolysis polypeptide on multiple prostate cancer DU-145 cells has important significance on the theoretical research of prostate cancer medicaments.
Disclosure of Invention
The invention aims to solve the technical problem of providing the application of mud snail polypeptide in anti-prostate cancer drugs, in particular anti-prostate cancer DU-145 cells, on the basis of obtaining mud snail polypeptide by enzymolysis of mud snail tissues.
The technical scheme adopted by the invention for solving the technical problems is as follows: the prostate cancer DU-145 cells are separated from prostate cancer brain metastasis tumors, have low differentiation degree, are androgen-dependent prostate cancer cells, have strong metastasis potential and lack the expression of endogenous androgen receptors, wherein the specific implementation form of the medicament can be tablets, capsules, granules or pills and the like.
The mud snail polypeptide is prepared by the following steps: cleaning and shelling fresh mud snails, taking mud snail tissues, mashing, adding distilled water for homogenate, wherein the material-liquid ratio is 1 (3-4), adjusting the pH value of homogenate to 8-9 after homogenate, adding trypsin with the volume of 0.4-0.5% of the homogenate, carrying out heat preservation hydrolysis at 40-50 ℃ for 7-9 h, carrying out enzyme deactivation at 95-100 ℃ for 10-15 min after hydrolysis is finished, centrifuging hydrolysate at 4 ℃ for 15-20 min at 9500-10000 r/min, taking supernatant, concentrating and drying to obtain an enzymolysis crude extract, dissolving the enzymolysis crude extract in distilled water, and separating and purifying to obtain each mud snail polypeptide component.
When the concentration of the enzymolysis crude extract is 5-25 mg/ml, the proliferation inhibition index of DU-145 cells is 36.9-80.7%, and the proliferation inhibition index is in positive correlation with the concentration of the enzymolysis crude extract.
The separation and purification steps are ultrafiltration or ultrafiltration and gel chromatography in sequence or ultrafiltration, gel chromatography and reversed-phase high performance liquid chromatography in sequence.
Furthermore, 10kd, 5kd and 3kd ultrafiltration membranes are used in the ultrafiltration step, a component A1 with the molecular weight of 10-5kd, a component A2 with the molecular weight of 5-3kd and a component A3 with the molecular weight of less than 3kd are respectively obtained, the proliferation inhibition rate of the component A3 with the concentration of 20-25 mg/ml after the component A3 acts on DU-145 cells for 36 hours is 77-78%, and the proliferation inhibition index is in positive correlation with the concentration of the component A3.
Further eluting the component A3 in the gel chromatography to respectively obtain an H1 component, an H2 component and an H3 component, wherein the proliferation inhibition index of the component H2 with the concentration of the component H2 is in positive correlation with that of the component H2 with the concentration of 5-20 mg/ml after the component H2 acts on the DU-145 cells for 24 hours, and is 44.1-81.6%; gel chromatography conditions: sephadex G-25 gel chromatography is selected, the gel is balanced by deionized water after being filled into a column, the concentration is 50mg/mL, the sample loading is 4mL, the speed is 3mL/min, the mobile phase is ultrapure water, and 280nm ultraviolet detection is carried out.
Still further, separating the H2 component by adopting a reversed-phase high performance liquid chromatography to obtain a component F1 and a component F2, wherein after the component F1 and the component F2 which are 2.5-3 mg/ml act on the DU-145 cells for 24 hours, the proliferation inhibition rates of the component F1 and the component F2 on the DU-145 cells are 31-33.72% and 40-42.04% respectively; reverse phase high performance liquid chromatography conditions: zorbax SB-C18(4.6 × 250, 5um) is selected; the column temperature is 20 ℃; mobile phase was 1% TFA and acetonitrile; gradient elution: from the beginning to the end of 30min, the acetonitrile concentration is changed from 0 to 40 percent, and the elution speed is 1.0 mL/min; the sample injection volume is 50ul, and the ultraviolet detection wavelength is 214nm and 280nm respectively.
The amino acid sequence of the component F1 is shown as SEQ ID NO.1, and the amino acid sequence of the component F2 is shown as SEQ ID NO. 2.
Compared with the prior art, the invention has the advantages that: the invention relates to a prostatic cancer malignant tumor which seriously affects the health of men, and the invention obtains an enzymolysis crude extract by using trypsin to carry out enzymolysis on a bullacta exarata tissue, then obtains a plurality of components of bullacta exarata polypeptide by a separation and purification means, and researches the influence of each component on prostatic cancer DU-145 cells. The mudsnail adopted in the invention is a low-value shellfish, the raw material cost is low, the extraction method of the mudsnail polypeptide has simple process and mild reaction, and each component of the mudsnail polypeptide obtained by extraction has obvious proliferation inhibition effect on prostate cancer DU-145 cells, wherein the component H2 has IC inhibition effect on the DU-145 cells50Is 1.32mg/ml, and provides a theoretical basis for developing an anti-prostate cancer medicament taking mud snail polypeptide as a raw material.
Drawings
FIG. 1 shows the inhibition rate of crude enzymatic extract of bullacta exarata on prostate cancer DU-145 cells in example 4;
FIG. 2 is a graph showing the inhibition rate of the membrane fraction on DU-145 cells in example 4;
FIG. 3 is a graph of the A3 fraction from example 4 after Sephadex G-25 column chromatography;
FIG. 4 is a line graph showing the inhibition rate of the gel assay components on DU-145 cells in example 4;
FIG. 5 is a bar graph of the inhibition rate of the gel assay components on DU-145 cells in example 4;
FIG. 6 is a reversed phase high performance liquid chromatography spectrum of the H2 component of example 4;
FIG. 7 inhibition of DU-145 cells by fractions purified by reversed phase high performance liquid chromatography.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
Example 1: preparation of mud snail polypeptide
(1.1) raw Material treatment
Taking fresh mud snails, removing shells of the mud snails, draining, and homogenizing for later use.
(1.2) enzymolysis process of mud snail
Mashing and homogenizing bullacta exarata tissues by using a high-speed tissue mashing machine, precisely weighing homogenate, adjusting the pH value by using 0.1mol/L hydrochloric acid solution and 0.1mol/L NaOH solution, adding trypsin for enzymolysis for hours, wherein the enzymolysis condition is that the enzymolysis temperature is 45 ℃, the pH value is 8.7, the material-liquid ratio is 1:4, the enzymolysis time is 8 hours, and the enzyme adding amount is 0.48%. Inactivating enzyme at 100 deg.C for 15min, centrifuging at 4 deg.C for 15min (10000r/min), and concentrating the supernatant.
Example 2: culture and passaging of DU-145 cells
(2.1) cell recovery
Taking out the stored DU-145 cell strain from the liquid nitrogen tank, quickly putting the cell strain into a constant temperature water bath kettle at 37 ℃ for thawing, putting the cell strain into a sterile working chamber for operation after thawing, sucking the cell strain into a centrifuge tube by using a sterilized straw, adding 2mL of F12 nutrient solution or RPMI1640 nutrient solution, centrifuging at 1000rpm for 10min, removing supernatant, adding 4mL of nutrient solution, and repeatedly blowing and beating the cell to form a single cell. Then, the cells were uniformly aspirated into 2 25mL culture flasks with a pipette, and 5% CO was added2Culturing in a constant-temperature incubator at 37 ℃, and pouring off dead cells which are not attached to the wall by changing the culture solution the next day.
(2.2) cell culture
Human prostate cancer cell DU-145 was inoculated into F12 and 1640 nutrient solutions containing 10% fetal bovine serum (volume fraction) FBS and diabodies (penicillin G100IU/mL, streptomycin 100IU/mL), respectively, and placed at 37 ℃ in 5% CO2Culturing in a constant-temperature incubator, carrying out adherent growth of cells, changing the liquid every 1 day, and carrying out passage when the cells grow to be about 80% of the culture bottle. The generation was carried out at a ratio of 1: 2. Cells in the logarithmic growth phase were collected for experiments.
(2.3) cell passaging
The culture flask full of cells is taken out from the constant temperature incubator and put on a sterile operation table. During passage, firstly pouring the nutrient solution in the bottle, removing dead cells growing without adherence, and carrying out mixed digestion by using 0.25% trypsin/0.02% EDTA digestive juice, wherein the digestion time of different cells is different, and the general digestion time is 3-5 min; when the cells are observed under a microscope and the cell gaps are obviously enlarged and the cells become round and bright, the cells are completely digested, and the digestive enzyme solution is removed. Adding about 2.5mL of nutrient solution into a culture bottle, repeatedly blowing digested adherent cells to form single cells, generally transferring one bottle of cells to 2 bottles, placing the passaged cells at 37 ℃ and 5% CO2The incubator of (2) for cultivation.
(2.4) cryopreservation of cells
Observing under a microscope, when the cells grow to about 80% of the culture bottle, selecting the cells with good shapes for cryopreservation, pouring out the culture solution, adding the digestive juice to digest the cells, pouring out the digestive enzyme when the intercellular space is observed to be obvious under the microscope, adding about 3mL of nutrient solution into the culture bottle, repeatedly blowing to make the cells become single cells, sucking the single cells into a centrifugal tube, centrifuging for 10min at 1000rpm, carefully sucking to remove the culture solution, adding 1mL of fetal calf serum containing 10% DMSO, repeatedly blowing the cells uniformly by using a suction tube, and sucking the cells into a sterile cryopreservation tube. Putting into a refrigerator at 4 deg.C for 30min, putting into a liquid nitrogen bottle mouth for 2h, and freezing.
Example 3: separation and purification of enzymolysis crude extract
Firstly, separating mud snail polypeptide enzymolysis crude extract by using an ultrafiltration method, respectively selecting 10kd, 5kd and 3kd as ultrafiltration membranes, respectively intercepting three components with molecular weights of 10-5kd, 5-3kd and less than 3kd, respectively preparing the components into concentrations of 20mg/mL after freeze drying, and determining the inhibition rate of each component on prostatic cancer DU145 by using an MTT method. Selecting a component with the highest activity, selecting Sephadex G-25 gel chromatography, loading the gel into a column, balancing with deionized water at a concentration of 50mg/mL, a loading amount of 4mL and a speed of 3mL/min, and detecting with ultra-pure water at 280nm as a mobile phase. Collecting the elution peak, freeze-drying into powder, detecting the proliferation inhibition rate of DU-145 cells, and drawing a curve to obtain IC50The most active large amount was collected, lyophilized and analyzed by high performance liquid chromatography.
The freeze-dried bullacta exarata sample is dissolved in 0.06% TFA water into a 0.6ml centrifuge tube, and centrifuged at 12000rpm for 10min to obtain the supernatant. High performance liquid phase conditions: zorbax SB-C18(4.6 × 250, 5um) is selected; the column temperature is 20 ℃; mobile phase was 1% TFA and acetonitrile; gradient elution: from the beginning to the end of 30min, the acetonitrile concentration is changed from 0 to 40 percent, and the elution speed is 1.0 mL/min; the sample injection volume is 50ul, and the ultraviolet detection wavelength is 214nm and 280nm respectively.
Example 4: method for exploring bullacta exarata polypeptide antitumor activity by MTT method
Preparing suspension from cells in logarithmic growth phase, inoculating to 96-well plate with 200 μ L per well, setting 5 parallel wells in 5% CO2And adhering the sample to the wall for 16-48h at 37 ℃, observing under an inverted microscope, discarding the culture solution, and simultaneously dissolving the samples to be detected in the culture solution at different concentrations. Then, each well was filled with 5% CO, and a control group without the sample was placed in each well2Incubation was carried out for 36h at 37 ℃ in an incubator, washed 2 times with PBS, and the culture was continued for 4h by adding a nutrient solution containing MTT. The culture was terminated and the culture medium in the wells was carefully aspirated. Adding dimethyl Acer Truncatum Bunge, shaking on a shaker at low speed for 10min, and measuring absorbance (OD value) at 490nm with enzyme-linked immunosorbent assay. The Inhibition Index (IR) of cell proliferation, i.e., the Inhibition rate of proliferation, was calculated according to the following formula:
(4.1) study of antitumor activity of crude extract of enzymatic hydrolysis of bullacta exarata
And (3) carrying out enzymolysis on the homogenized bullacta exarata by using trypsin, centrifuging, taking supernatant, carrying out freeze drying to obtain a bullacta exarata polypeptide crude extract, and observing the influence of the bullacta exarata polypeptide crude extract on the induction of the in-vitro apoptosis of the prostate cancer DU145 cell strain by adopting an MTT method. The results show that: the inhibition rate is gradually increased along with the increase of the concentration of the polypeptide, and the concentration is 5mg-1When the concentration was 20mg.mL, the growth inhibition rate was 36.9%-1When the polypeptide is used, the inhibition rate reaches 79.6%, which indicates that the polypeptide has certain anti-prostate cancer activity. As shown in table 1 and fig. 1.
TABLE 1 inhibition of DU-145 cells (x. + -.s, n. RTM. 5)
Note: p compared to the minimum cytostatic rate of each fraction<0.05; IC of mud snail polypeptide for inhibiting DU-145 cell proliferation after 36h of co-culture with cells50It was 2.425 mg/ml.
(4.2) research on anti-tumor activity of membrane separation component of mudsnail enzymolysis polypeptide
Three fractions were obtained as described in example 3, each having a molecular weight of 10-5kd (A1), 5-3kd (A2), and less than 3kd (A3), and each fraction was lyophilized and formulated to a concentration of 20mg/mL for MTT assay, and after 36h, the inhibition rates of each fraction on DU-145 cells were 49.8%, 65.7%, and 78.3%, respectively. As shown in FIG. 2, the A3 fraction showed better inhibitory effect on DU-145 cells than the other two fractions.
(4.3) study of antitumor Activity of gel chromatography component
After ultrafiltration three fractions were obtained, which was found to be the best fraction A3 from 4.2, the A3 fraction was collected in large amounts, concentrated and freeze-dried, and eluted using gel Sephadex G-25 chromatography under the experimental conditions of example 3 to obtain three fractions H1, H2 and H3, as shown in FIG. 3.
Preparing each peak component into the concentrations of 5.0mg/mL, 10.0mg/mL, 15.0mg/mL and 20.0mg/mL respectively, detecting the proliferation inhibition effect on prostate cancer DU-145 tumor cells by using an MTT method respectively, adding a sample with the prepared concentration into the cells after culturing the cells for 24h, setting a blank control group, and carrying out T test analysis on an independent sample by SPSS software to determine whether the other concentration groups have significant difference with the group, wherein P <0.05 indicates that the peak components have significant difference with the blank control group. The graphs from table 2 are shown in fig. 4 and 5, with bars for each dose group in fig. 5 being H1, H2, and H3 from left to right. The analysis of a comprehensive chart can show that the 3 components have certain inhibition effects on DU-145 tumor cells and present a dose-effect relationship, wherein the anti-tumor activity of H2 is the highest, and the inhibition rate on DU-145 can reach 81.6% at 20.0mg/mL, and the inhibition effect is significant, as shown in Table 2.
TABLE 2 inhibition of proliferation of DU-145 cells by each component
(n＝3)
Note: ﹡ P <0.05 compared to the minimal cytostatic rate of each fraction
(4.4) anti-tumor Activity study of reverse phase high Performance liquid chromatography purified fractions
4.3 experiments show that the anti-tumor activity of the H2 component is the best, the H2 component is further separated, and the high performance liquid chromatography is used for detecting and comparing the components at the ultraviolet wavelengths of 214nm and 280nm respectively, and the components are marked as shown in figure 6, namely F1 and F2 are obtained respectively.
The fraction H2 obtained by gel chromatography separation has the highest activity, and the inhibition rate of proliferation of DU-145 cells after 24H of action at the concentration of 3mg/mL of 2 fractions obtained by HPLC separation is 33.72% and 42.04%, respectively, and a bar graph is drawn, as shown in FIG. 7.
As can be seen from FIG. 7, the component F2 has stronger tumor cell proliferation inhibiting effect on DU-145 tumor cells than that of the component F1, the components F1 and F2 are collected and structurally detected by PPSQ-31A, the detected amino acid sequence of F1 is shown in SEQ ID NO.1, and the amino acid sequence of F2 is shown in SEQ ID NO. 2.
The experimental materials and experimental instruments used in the above examples were as follows:
experimental Material
Fresh mud snails are collected from offshore sea areas of Zhoushan in Zhejiang;
papain; trypsin (national pharmaceutical group chemical agents limited);
formaldehyde (37.0% -40.0%); chemical agents of the national drug group, ltd;
hydrochloric acid (36% -38%); the national drug group chemical reagent company Limited is of high grade purity;
sodium hydroxide; the tin-free commercial chemical industry Co., Ltd is pure;
tetramethylazodicarbonyl blue (MTT), SIGMA corporation, USA;
f12 powdered medium, SIGMA usa;
RPMI1640 medium, Gibco;
dimethyl sulfoxide (DMSO), SIGMA, usa;
acetonitrile (CAN, Fisher Scientific Co., Ltd.)
Trifluoroacetic acid, (TFA, Merck Co.)
Polybrene, (Polybrene, Shimadzu Corporation);
PTH-amino acid standard, (PTH-AA, Shimadzu Corporation);
laboratory apparatus
BSZ-40-LCD auto section collector, Shanghai Qitt Analyzer, Inc.;
Agilent1260(Agilent.USA)
ultrafiltration cups, ultrafiltration membranes, shanghai molting scientific instruments ltd;
g-25 gel molecular sieves, Beijing Asia Tai Hengxin Biotech, Inc.;
FD-1000 freeze dryer, shanghai elan instruments ltd;
CF16RXII high speed refrigerated centrifuge, HITACHI corporation, japan;
UV1100 ultraviolet spectrophotometer, shanghai meida corporation;
BSA model 124S electronic balance, Sartorious AG, germany;
VPWS-T-20L ultrapure water, Hangzhou Yongjieda purification technology, Inc.;
ZHJH-C1209C model superclean bench, Shanghai Zhicheng Analyzer manufacturing, Inc.;
microplate reader, product of Bio-Rad, USA;
forma3111 type CO2Incubator, Thermo corporation, usa;
inverted microscope, OLYMPUS corporation, japan;
PPSQ-31A, (Shimadzu Corporation, Japan);
PTFE filters, (PTFE filter, Shimadzu Corporation);
glass Fiber membranes, (Glass Fiber Disk, Shimadzu Corporation);
cell line
Human prostate cancer DU-145 cells were purchased from Shanghai cell biology banks of Chinese academy of sciences.
Claims (2)
1. An application of mud snail polypeptide in preparing the medicines for treating prostatic cancer DU-145 cells,
the method is characterized in that the mud snail polypeptide is prepared by the following steps: cleaning fresh bullacta exarata, removing shells, taking bullacta exarata tissues, mashing, adding distilled water for homogenate with the material-liquid ratio of 1 (3-4), adjusting the pH value of homogenate to 8-9 after homogenate, adding 0.4-0.5% of trypsin in the mass of the homogenate, preserving heat and hydrolyzing for 7-9 h at 40-50 ℃, inactivating enzyme at 95-100 ℃ for 10-15 min after hydrolysis is finished, centrifuging hydrolysate at 4 ℃ for 15-20 min at 9500-10000 r/min, taking supernatant, concentrating and drying to obtain an enzymolysis crude extract, dissolving the enzymolysis crude extract in distilled water, and separating and purifying by ultrafiltration, gel chromatography and reversed phase high performance liquid chromatography to obtain each bullacta exarata polypeptide component,
in the ultrafiltration step, 10kd, 5kd and 3kd ultrafiltration membranes are used to respectively obtain a component A1 with the molecular weight of 10-5kd, a component A2 with the molecular weight of 5-3kd and a component A3 with the molecular weight less than 3kd, the proliferation inhibition rate of the component A3 of 20-25 mg/ml after acting on DU-145 cells for 36 hours is 77-78%, and the proliferation inhibition index is in positive correlation with the concentration of the component A3,
eluting the component A3 in the gel chromatography to respectively obtain an H1 component, an H2 component and an H3 component, wherein the proliferation inhibition index of the component H2 with the concentration of the component H2 being positively correlated with that of the component H2 with the concentration of 5-20 mg/ml after the component H2 acts on the DU-145 cells for 24 hours is 44.1-81.6%;
gel chromatography conditions: selecting Sephadex G-25 gel chromatography, loading into column, balancing with deionized water at a concentration of 50mg/mL, loading amount of 4mL and speed of 3mL/min, detecting with mobile phase ultrapure water at 280nm ultraviolet,
separating the H2 component by adopting a reversed-phase high performance liquid chromatography, and separating to obtain a component F1 and a component F2, wherein after 2.5-3 mg/ml of the component F1 and the component F2 act on the DU-145 cells for 24 hours, the proliferation inhibition rates of the component F1 and the component F2 on the DU-145 cells are respectively 31-33.72% and 40-42.04%, the amino acid sequence of the component F1 is described in SEQ ID NO.1, and the amino acid sequence of the component F2 is described in SEQ ID NO. 2;
reverse phase high performance liquid chromatography conditions: zorbax SB-C18(4.6 × 250, 5um) is selected; the column temperature is 20 ℃; mobile phase was 1% TFA and acetonitrile; gradient elution: from the beginning to the end of 30min, the acetonitrile concentration is changed from 0 to 40 percent, and the elution speed is 1.0 mL/min; the sample injection volume is 50 ul; the ultraviolet detection wavelength is 214nm and 280nm respectively.
2. The use of a bullacta exarata polypeptide as claimed in claim 1 in the preparation of a medicament for treating prostate cancer DU-145 cells, wherein: when the concentration of the enzymolysis crude extract is 5-25 mg/ml, the proliferation inhibition index of DU-145 cells is 36.9-80.7%, and the proliferation inhibition index is in positive correlation with the concentration of the enzymolysis crude extract.
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| CN101402982A (en) * | 2008-11-11 | 2009-04-08 | 华东理工大学 | Mud snail glue polysaccharide, preparation method and application thereof |
| CN103204904A (en) * | 2013-02-01 | 2013-07-17 | 浙江海洋学院 | Dasyatis akajei chondroprotein polypeptide capable of resisting prostate cancer, and preparation method and application thereof |
| CN103431292A (en) * | 2013-09-01 | 2013-12-11 | 陆思烨 | Health care noodle containing bullacta exarata polypeptides and plant extract powder |
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| CN101402982A (en) * | 2008-11-11 | 2009-04-08 | 华东理工大学 | Mud snail glue polysaccharide, preparation method and application thereof |
| CN103204904A (en) * | 2013-02-01 | 2013-07-17 | 浙江海洋学院 | Dasyatis akajei chondroprotein polypeptide capable of resisting prostate cancer, and preparation method and application thereof |
| CN103431292A (en) * | 2013-09-01 | 2013-12-11 | 陆思烨 | Health care noodle containing bullacta exarata polypeptides and plant extract powder |
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