CN111592582B - Sea cucumber intestinal peptide and preparation method and application thereof - Google Patents
Sea cucumber intestinal peptide and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a sea cucumber intestinal peptide, a preparation method and application thereof, wherein the amino acid sequence of the sea cucumber intestinal peptide is shown as SEQ ID No. 1. In the invention, the sea cucumber intestinal peptide has a prevention and treatment effect on breast cancer and cervical cancer.
Description
Technical Field
The invention belongs to the technical field of biotechnology, and particularly relates to sea cucumber intestinal peptide and a preparation method and application thereof.
Background
The sea cucumber intestine egg has higher content of precious nutrients such as polysaccharide, vanadium, nucleic acid, arginine and the like, also contains very rich gonadal pigments (beta carotene, echinocandin, astaxanthin, zeaxanthin, canthaxanthin and some unknown pigments), sea cucumber lipid, stichopus japonicus saponin, selenium, zinc, iron and other components, and has stronger effects of nourishing yin and tonifying yang, improving the microcirculation of the digestive system, enhancing the circulation flow of cardiac muscle nutrition blood and inhibiting the growth and diffusion of tumor cells.
Under the influence of modern living environment, the body can cause the canceration of normal cells of the body under the action of some carcinogenic factors such as environmental pollution, computer radiation, free radical toxin, chemical pollution, hereditary property, endocrine imbalance, immunologic dysfunction, carcinogenic substances and the like.
Disclosure of Invention
In view of the above, the invention aims to provide a sea cucumber intestinal peptide, a preparation method and an application thereof, which have a prevention and treatment effect on breast cancer and cervical cancer.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a sea cucumber intestinal peptide, and the amino acid sequence of the sea cucumber intestinal peptide is shown in SEQ ID No. 1.
The invention also provides a preparation method of the sea cucumber intestinal peptide, which comprises the following steps:
1) mixing sea cucumber intestines with water to obtain a mixture, and treating the mixture at 50-55 ℃ for 50-70 min to obtain a treated substance;
2) adjusting the pH value of the treated matter obtained in the step 1) to 8.5, mixing the treated matter with alkaline protease, performing enzymolysis for 2.5-3.5 hours at the temperature of 55-65 ℃ to obtain an zymolyte, and adjusting the pH value of the zymolyte to 5 to obtain a regulator;
3) inactivating enzymes of the regulator obtained in the step 2) to obtain an enzyme-inactivated substance, adsorbing the enzyme-inactivated substance for 50-70 min by using active carbon at 55-60 ℃, sequentially passing through a 30000Da ceramic membrane and a 1KD organic membrane, and drying to obtain a sea cucumber intestinal peptide mixture;
4) mixing the sea cucumber intestinal peptide mixture obtained in the step 3) with water to obtain a mixed solution, separating and purifying the mixed solution by a Sephadex G-50 chromatographic column, and collecting components with retention time of 26.5min to obtain the sea cucumber intestinal peptide.
Preferably, the ratio of the mass of the sea cucumber intestines to the volume of the water in the step 1) is 1000kg to 2000L.
Preferably, the mass ratio of the treated substance in the step 2) to the alkaline protease is 100: 3.
Preferably, the water in step 1) is deionized water.
Preferably, the step 4) of separating and purifying comprises: the mobile phase is 10 percent methanol solution by volume and the flow rate is 1 ml/min.
The invention also provides the application of the sea cucumber intestine peptide in the technical scheme in preparing anti-tumor drugs.
Preferably, the tumour comprises breast cancer and/or cervical cancer.
The invention provides a sea cucumber intestinal peptide, and the amino acid sequence of the sea cucumber intestinal peptide is shown in SEQ ID No. 1. In the invention, the sea cucumber intestinal peptide has a prevention and treatment effect on breast cancer and cervical cancer.
Drawings
FIG. 1 is a two-level mass spectrum of liquid chromatography-Q activity mass spectrometry of sea cucumber intestinal peptide (YQQPIIGGALF);
FIG. 2 shows the effect of sea cucumber intestinal peptide on MCF-7 apoptosis;
FIG. 3 shows the effect of sea cucumber intestinal peptide on Hela apoptosis;
FIG. 4 is a phenotypic picture of inhibition effect of sea cucumber intestinal peptide on zebra fish human breast cancer (MCF-7) transplantation tumor;
FIG. 5 shows the inhibition effect (sum of fluorescence intensity values) of sea cucumber intestinal peptide on the transplanted tumor of zebrafish human breast cancer (MCF-7), wherein p is less than 0.001;
fig. 6 is a tumor transplanted with sea cucumber intestinal peptide against zebrafish human breast cancer (MCF-7), p <0.001 compared to the model control group.
Detailed Description
The invention provides a sea cucumber intestinal peptide, the amino acid sequence of which is shown as SEQ ID No.1 and specifically comprises the following components:
YQQPIIGGALF。
the invention also provides a preparation method of the sea cucumber intestinal peptide, which comprises the following steps:
1) mixing sea cucumber intestines with water to obtain a mixture, and treating the mixture at 50-55 ℃ for 50-70 min to obtain a treated substance;
2) adjusting the pH value of the treated matter obtained in the step 1) to 8.5, mixing the treated matter with alkaline protease, performing enzymolysis for 2.5-3.5 hours at the temperature of 55-65 ℃ to obtain an zymolyte, and adjusting the pH value of the zymolyte to 5 to obtain a regulator;
3) inactivating enzymes of the regulator obtained in the step 2) to obtain an enzyme-inactivated substance, adsorbing the enzyme-inactivated substance for 50-70 min by using active carbon at 55-60 ℃, sequentially passing through a 30000Da ceramic membrane and a 1KD organic membrane, and drying to obtain a sea cucumber intestinal peptide mixture;
4) mixing the sea cucumber intestinal peptide mixture obtained in the step 3) with water to obtain a mixed solution, separating and purifying the mixed solution by a Sephadex G-50 chromatographic column, and collecting components with retention time of 26.5min to obtain the sea cucumber intestinal peptide.
The sea cucumber intestine is mixed with water to obtain a mixture, and the mixture is treated at 50-55 ℃ for 50-70 min to obtain a treated substance.
In the present invention, the ratio of the mass of the sea cucumber intestines to the volume of water is preferably 1000kg: 2000L. In the present invention, the water is preferably deionized water.
The mixture is treated at 50-55 ℃ for 50-70 min, preferably at 50-55 ℃ for 60 min.
The pH value of the treated substance obtained by adjusting is 8.5, the treated substance is mixed with alkaline protease and then is subjected to enzymolysis for 2.5-3.5 hours at the temperature of 55-65 ℃ to obtain an zymolyte, and the pH value of the zymolyte is adjusted to be 5 to obtain an adjusted substance.
In the present invention, the pH of the treated product is preferably adjusted by using a 1M sodium hydroxide solution. In the present invention, the mass ratio of the treated product to the alkaline protease is preferably 100: 3. In the present invention, the time of the enzymolysis is preferably 3h, and the temperature of the enzymolysis is preferably 60 ℃. The pH of the substrate is preferably adjusted using a 1M hydrochloric acid solution.
The method comprises the steps of inactivating enzyme of an obtained regulator to obtain an enzyme-inactivated substance, adsorbing the enzyme-inactivated substance for 50-70 min by using active carbon at 55-60 ℃, sequentially passing through a 30000Da ceramic membrane and a 1KD organic membrane, and drying to obtain the sea cucumber intestinal peptide mixture.
In the present invention, the temperature of the enzyme deactivation is preferably 110 ℃, and the time of the enzyme deactivation is preferably 5 min. In the present invention, the drying is preferably spray drying.
The sea cucumber intestinal peptide mixture is mixed with water to obtain a mixed solution, the mixed solution is separated and purified by a Sephadex G-50 chromatographic column, and components with the retention time of 26.5min are collected to obtain the sea cucumber intestinal peptide.
In the present invention, the concentration of the sea cucumber intestine peptide mixture in the mixed solution is preferably 120 mg/ml. In the present invention, the Sephadex G-50 column is 3X 100cm in size. In the present invention, the parameters of the separation and purification are preferably: the mobile phase was 10% methanol, the flow rate was 1ml/min, and the absorbance at 214nm of the eluate was measured.
The invention also provides the application of the sea cucumber intestine peptide in the technical scheme in preparing anti-tumor drugs. In the present invention, the tumor preferably includes breast cancer and/or cervical cancer. The dosage form of the medicine is not particularly limited, and the sea cucumber intestinal peptide can be prepared into a medically acceptable dosage form.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Step one, preparation of sea cucumber intestinal peptide
Step one, taking 1000kg of sea cucumber intestines to carry out size mixing, adding 2000L of deionized water, and enabling the substrate concentration to be 50%.
And step two, uniformly stirring the sea cucumber intestine solution obtained in the step one, heating to 55 ℃, and preserving heat for 1 hour.
And step three, adjusting the pH of the solution obtained in the step two to 8.5 by using 1MNaOH, and heating to 60 ℃.
And step four, adding 3 percent of alkaline protease (30Kg) into the solution obtained in the step three, adding 1M HCl after 3 hours of enzymolysis, and adjusting the pH value to 5.0.
And step five, heating the solution in the step four to 110 ℃, and keeping for 5 min.
And step six, cooling the enzyme deactivation solution obtained in the step five to 55-60 ℃, adding activated carbon, keeping the temperature for 1 hour, passing through a 30000Da ceramic membrane, passing through a 1Kd organic membrane, and performing spray drying to obtain the sea cucumber intestinal peptide mixture.
Step two, separation and purification of sea cucumber intestinal peptide
Step one, dissolving the sea cucumber intestinal peptide mixture produced in the step one in water to prepare 120mg/mL, separating and purifying by a Sephadex G-50 chromatographic column (3 x 100cm), collecting fractions according to chromatographic peaks, concentrating the fractions, and freeze-drying the fractions into powder by a vacuum drier, wherein the mobile phase is 10% methanol, the flow rate is 1mL/min, and the absorbance of the eluate at 214nm is measured.
And step two, verifying the lead-removing effect of different components by using the zebra fish tumor model of the different components obtained in the step one, and finding that a chromatographic peak with the retention time of 26.5min has a good anti-tumor effect. The component is further separated and purified by high performance liquid chromatography, and then the structure identification is carried out.
And step three, identifying the purity and the structure: and (3) identifying the purity and the structure of the different components obtained in the step one by adopting a nano-liter liquid chromatography-Q extraction mass spectrum combined system.
Chromatographic conditions are as follows:
(1) mobile phase: phase A: 100% purified water + 0.1% formic acid; phase B: 100% acetonitrile + 0.1% formic acid;
(2) flow rate of mobile phase: 300nl/min
(3) Sample introduction amount: 1 μ L of supernatant
(4) Mobile phase gradient program:
table 1 mobile phase gradient procedure
| Time (minutes) | 0 | 2.0 | 36.0 | 38.0 | 41.0 | 42.0 | 45.0 |
| A(％) | 97 | 97 | 63 | 10 | 10 | 97 | 97 |
| B(％) | 3 | 3 | 37 | 90 | 90 | 3 | 3 |
By using nanoliter liquid chromatography-Q EXACTIVE mass spectrometry, single-chain polypeptide with the structure of YQQPIIGGALF (Tyr-Gln-Gln-Pro-Ile-Ile-Gly-Gly-Leu-Phe)1206Da is identified, namely the sea cucumber intestinal peptide. Fig. 1.
Simulated in vivo digestion reaction of sea cucumber intestinal peptide (YQQPIIGGALF)
After the sea cucumber intestinal peptide (YQQPIIGGALF) is ingested by a human body, the sea cucumber intestinal peptide can be absorbed and utilized only through the gastrointestinal digestive tract; therefore, the in-vitro digestion stability determination of the sea cucumber intestinal peptide is developed, pepsin and trypsin are used for simulating gastrointestinal tract digestion, the sea cucumber intestinal peptide (1g) is injected into 3mL of 0.1mol/L phosphate buffer solution, 6mL of pepsin-guar mixed solution is added, the pH value is adjusted to 1.5 by utilizing 2mol/L HCl, and the mixture is magnetically stirred for 30min in a 37 ℃ constant-temperature water bath; adding 10mL of phosphate buffer solution into the solution, and adjusting the pH value to 6.9 by using NaOH solution; then 125. mu.L of MgCl was added separately2-Ca Cl2125 mul pancreatin, water to 50mL, shaking and incubating at 37 ℃ for 120 min. Inactivating enzyme in boiling water bath, and centrifuging the sample to obtainCentrifuging at 8000rpm for 10min, collecting supernatant, lyophilizing, and mass spectrometric analysis to determine its stability after absorption by gastrointestinal tract.
After the sea cucumber intestinal peptide (YQQPIIGGALF) is digested and enzymolyzed by pepsin-trypsin in vitro, the stability is 91.28%, which indicates that the sea cucumber intestinal peptide (YQQPIIGGALF) is not easy to be enzymolyzed and damaged under the action of the pepsin-trypsin.
Example 2
Inhibition effect of sea cucumber intestinal peptide on tumor cells
Two common human breast cancer cell lines (MCF-7 and MDA-MB-231) and a human cervical cancer cell line (HELA) in scientific research are adopted as research objects, the influence of the sea cucumber intestinal peptide (YQQPIIGGALF) obtained in the embodiment 1 on the growth inhibition of cancer cells is researched by an MTT method, and the influence of three polypeptides on cancer cell apoptosis is detected by a double-staining method through a flow cytometer; the inhibition effect of the sea cucumber intestinal peptide (YQQPIIGGALF) on cancer cells in vitro is determined.
1. Experimental materials and methods
1.1. Experimental Material
1.1.1 cell lines
The breast cancer cell lines (MCF-7 and MDA-MB-231) and the human cervical cancer cell line (HELA) used in the experiment are purchased from the center cell resource of the national biology classical collection center of Wuhan nationality.
1.1.2 Primary reagents
Cell culture medium, calf serum, fetal bovine serum, penicillin (80 ten thousand IU), streptomycin (100 ten thousand IU), trypsin, MTT, DMSO (analytical reagent), albumin peptide, W sea cucumber intestinal peptide, M sea cucumber intestinal peptide and an apoptosis kit.
1.1.3 Main Instrument
Flow cytometer, enzyme mark instrument (128-C), micropipettor, water purifier, thermostatic water bath, 4 deg.c chromatographic cabinet, negative 80 deg.c refrigerator, CO2The device comprises an incubator, an ultra-clean workbench, a table centrifuge, a low-temperature high-speed centrifuge, a constant-temperature magnetic stirrer, a constant-temperature vibrating 96-hole cell culture plate, a 12-hole cell culture plate, a microbalance and an inverted microscope.
1.2. Experimental methods
1.2.1 Sterilization
Placing various instruments and reagents to be sterilized in an autoclave, sterilizing for 40min, taking out the sterilized instruments and reagents, placing in an oven, and drying for use when the pressure in the autoclave is reduced to 0.
1.2.2 cell culture
Culturing the cancer cell strain in corresponding culture solution, adding 10% inactivated fetal calf serum, 2mmol/L L-glutamine, 100U/ml penicillin and 100U/ml streptomycin, and culturing in the presence of 5% CO2The culture was carried out in a 37 ℃ incubator.
1.2.3 cell passages
The selected cancer cells belong to adherent cells, after the culture bottle is full of the cancer cells, in a super clean bench, a liquid transfer device is used for sucking up a culture medium in the cell bottle, a suction tube sucks 2ml of PBS into the cell bottle, after the PBS is rinsed twice, the PBS in the cells is discarded, 2ml of 0.25% trypsin is added into the cell bottle, the cell bottle is shaken to ensure that the trypsin is uniformly contacted with the cells, the cell bottle is flapped, the cell bottle is placed in a 37 ℃ incubator for digestion for 1 to 2min, the cells are placed under an inverted microscope for observation, the original adherent cells are found to retract, the cell gaps are enlarged, when most of the cells fall down, 2ml of the culture medium containing 10% FBS is added, and digestion is stopped. And repeatedly blowing the bottle wall by using a liquid transfer device to ensure that the cells fall off from the bottle wall to form cell suspension. Centrifuging at 800r/min for 5min, discarding supernatant, adding new culture solution, repeatedly blowing to obtain single cell suspension, adjusting cell concentration, and collecting 3 × 105The cells were inoculated in a 25ml flask and cultured. The experiment was performed with cells selected in the logarithmic growth phase.
1.2.4 cell cryopreservation
Taking cancer cells in logarithmic growth phase, digesting and centrifuging, and obtaining the total amount of each 1 × 106Adding 1ml of the cryopreservation solution into each cell to prepare a single cell suspension, and transferring the single cell suspension into a cryopreservation tube with the volume of 1ml per tube. The cell type, the freezing time and concentration are indicated on the freezing tube in advance. During freezing, the freezing tube is preferably frozen at the speed of 1-10 ℃/min, and is placed in a refrigerator at the temperature of-20 ℃ for 24 hours after being placed at the temperature of 4 ℃ for 4 hours, and then is put into liquid nitrogen for preservation.
1.2.5 resuscitating cells
In a sterile super clean bench, 5ml of culture medium is added into a sterile cell bottle, the cell strain frozen in a refrigerator at minus 80 ℃ is taken out and placed in a water bath kettle at 37 ℃ to be continuously shaken, and the frozen stock solution is required to be completely thawed within 1 minute. Taking out the frozen tube, scrubbing and sterilizing with 75% alcohol, taking out cell suspension under aseptic condition, injecting into culture medium, and gently blowing uniformly. The cell suspension was transferred to a 1. sup. st sterile centrifuge tube, centrifuged at 1100rpm for 6min, and the supernatant discarded. The obtained cells were blown with 5ml of a medium containing 10% calf serum to obtain a cell suspension, which was placed in a cell bottle at 37 ℃ under 5% CO2Culturing in an incubator.
1.2.6 preparation of adherent cell suspensions
After culturing a sufficient number of cells, the gastric cancer cells are detached and dispersed by trypsin; serum-free medium was washed twice. Staining with orcinol blue, determining living cell rate above 95%, centrifuging, adding appropriate amount of normal saline, resuspending, and making into l × 107Cells/ml cell suspension ready for use.
1.2.7MTT detection of influence of sea cucumber intestinal peptide on tumor cell proliferation capacity in vitro
In 1983, Mosmann et al invented a technical method specifically for detecting cell survival and growth, which is now called tetrazolium salt colorimetric test (MTT). MTT is a yellow compound, but when it is acted on by cytochrome C and succinate dehydrogenase, it can produce blue-violet crystals of formazan (formazan), which are formed by the cleavage of tetrazole (tetrazolium) ring due to MTT acting on the mitochondrial respiratory chain. However, since the activity of succinate dehydrogenase in dead cells was lost, MTT could not be reduced, and crystals could not be formed. The blue-violet formazan crystals formed in the cells can be dissolved by DMSO, so that the number of living cells can be deduced by the OD value of an enzyme-linked immunoassay sample in a certain light wave. Therefore, the number of crystals of MTT formed and the number of viable cells were in a certain range and were positively correlated with each other. The method is sensitive, reliable and easy to popularize, so that the MTT detection method is widely used in the detection of the activity of bioactive factors, the detection of the toxicity of cells and the screening of anti-tumor drugs.
1.2.7.1
Taking three cancer cells growing in logarithmic phase 2X 105Cells/ml, counted and plated on sterile 96-well plates at 200. mu.l/well, 37 ℃ 5% CO2Culturing in an incubator for 24 h.
1.2.7.2
Drug group: adding 100 μ L culture solution containing polypeptide (20, 200, 600mg/L) with different concentrations, each group having 3 parallel wells; control group: adding 100 mul of culture solution, and arranging 3 parallel holes in each group; after adding the drugs, the 96-well plates are all placed at 37 ℃ and contain 5 percent CO2And culturing in an incubator with saturated humidity for certain time (24h, 48h and 72 h).
1.2.7.3
After the incubation had reached the appropriate time, the cells were centrifuged for 10min at 1000r/min in a desk top centrifuge, the supernatant was aspirated off, 100. mu.l of medium containing 10% MTT was added to each well, and incubation was continued for 4h at 37 ℃.
1.2.7.4
Centrifugation was carried out, the supernatant was discarded, 100. mu.l of DMSO was added to each culture well, the wavelength was set to 492nm after shaking for 10min to dissolve the formazan, the Optical Density (OD) values of each group were measured on a microplate reader, and the calibration wavelength was set to 630 nm.
1.2.7.5
The cell growth inhibition rate was calculated and IC50 was calculated using SPSS software logistic curve regression. The cell inhibition rate was (1-OD of experimental group)/OD of control group × 100%.
1.2.8. Detection of apoptosis in flow cytometry
The apoptosis kit detects the principle of apoptosis: early changes in apoptosis occur at the surface of the cell membrane, and one of these changes is the translocation of Phosphatidylserine (PS) from within the cell membrane to outside the membrane. Annexin-V is species Ca2+The binding of the protein to PS is very tight, efficient and specific depending on the phospholipid binding. Therefore, Annexin-V can be used as a probe for detecting phosphatidylserine exposed outside cells. Using Annexin-V with high affinity to PS to label it with fluorescein (such as fluorescein isothiocyanate)Fluorescein FITC, etc.). The translocation of PS to the outside of the cell membrane can occur in necrotic cells, rather than being characteristic of apoptosis. Apoptosis and cell necrosis, two different modes of cell death, differ in that the cell membrane is undamaged in the initial stage of the former, while the integrity of the cell membrane is already damaged in the early stage of the latter. Propidium Iodide (PI) is a nucleic acid dye that is impermeable to the intact cell membrane, but is capable of red staining the nucleus through the cell membrane of cells in the middle and late stages of apoptosis and dead cells. Therefore, by matching Annexin-V with PI, cells in different apoptosis stages, dead cells and the like can be distinguished.
1.2.8.1
Taking logarithmic growth cancer cells (MCF-7 and Hela), 1X 106Adding polypeptide (20, 200, 1000mg/L) at different concentrations into cells/ml, standing at 37 deg.C with 5% CO2The incubator is kept overnight and the drug is treated for 24 h.
1.2.8.2
Digesting the cells with 0.25% pancreatin, collecting the cells in each well, washing with precooled PBS for 2 times to obtain single cell suspension with cell concentration of 0.5-1 × 106/ml。
1.2.8.3
Take 100. mu.l (about 1X 10)5Cells) were placed in a flow tube, 5. mu.l each of PI and FITC-labeled Annexin-V was added, mixed well, and incubated at room temperature.
1.2.8.4
Adding 1 × Binding Buffer into each tube, mixing fully, and detecting the apoptosis condition by an up-flow cytometer.
2. Results of the experiment
2.1 Effect of sea cucumber intestinal peptide on tumor cell proliferation ability in vitro
As shown in the data of Table 2, the MTT method test result shows that the sea cucumber intestinal peptide (YQQPIIGGALF) has certain inhibition effect on the growth of cancer cells MCF-7, MDA-MB-231 and Hela cell strains, the inhibition effect strength is basically in positive correlation with the polypeptide concentration, and the inhibition effect on the growth of the cancer cells is more obvious along with the prolonging of the action time. (. P < 0.01). Wherein the sea cucumber intestinal peptide (YQQPIIGGALF) has the strongest inhibition effect on two breast cancer cell strains MCF-7 and MDA-MB-231, the sea cucumber intestinal peptide (YQQPIIGGALF) has the strongest inhibition effect on Hela cell strain, and the sea cucumber intestinal peptide (which is not separated and purified) has weaker inhibition effect on three cancer cells.
TABLE 2 inhibition of cancer cell growth by polypeptides
2.2 Effect of sea cucumber intestinal peptide on apoptosis of tumor cells
In research, PI and Annexin V are often used in combination in order to distinguish between cells at different apoptotic stages. In the results of the scatter plots, the scatter in the lower right quadrant represents cells that underwent early apoptosis, the scatter in the upper right quadrant represents cells that underwent necrosis or late apoptosis, and the lower left quadrant represents live cells.
The apoptosis promoting effect of the sea cucumber intestinal peptide on MCF-7 and Hela cancer cell lines is shown in fig. 2 and fig. 3 respectively, the sea cucumber intestinal peptide (YQQPIIGGALF) and the sea cucumber intestinal peptide (which is not separated and purified) can play a certain role in promoting the apoptosis activity of tumor cells, wherein the sea cucumber intestinal peptide (YQQPIIGGALF) shows strong cancer cell apoptosis promoting activity, and the sea cucumber intestinal peptide (which is not separated and purified) is weaker in activity compared with the sea cucumber intestinal peptide. Therefore, the activity difference is caused by different YQQPIIGGALF fragment contents obtained by hydrolysis, and the result shows that the sea cucumber intestinal peptide fragment (YQQPIIGGALF) plays an anti-tumor role in promoting tumor cell apoptosis. The anti-tumor effect of the existing research on the sea cucumber intestinal peptide is mainly focused on sea cucumber saponin and polysaccharide, and the activity is mainly shown in the inhibition of the growth and the period of tumor cells.
Example 3
Effect of sea cucumber intestinal peptide on zebra fish tumor model
CM-Dil is used for marking human breast cancer (MCF-7) cells, the cells are transplanted into yolk sacs of 2dpf wild AB strain zebra fish in a microinjection mode, about 300 cells are transplanted into each tail, and a zebra fish human breast cancer (MCF-7) transplantation model is established. Compared with the anti-tumor effect of the positive medicament capecitabine, the intestinal peptide fragment (YQQPIIGGALF) of sea cucumber has good anti-tumor effect, which is superior to the anti-tumor effect of intestinal peptide (which is not separated and purified) of sea cucumber.
Evaluation of antitumor Effect
Using CM-Dil to mark human breast cancer (MCF-7) cells, transplanting the cells into yolk sacs of 2dpf wild AB strain zebra fish in a microinjection mode, and transplanting about 300 cells per tail to establish a zebra fish human breast cancer (MCF-7) transplantation model; zebra fish injected with human breast cancer (MCF-7) cells were cultured at 35 ℃ to 3 dpf.
The zebra fish with better consistency of transplanted tumor cells is selected under a microscope and randomly distributed into 6-well plates, the sea cucumber intestinal peptide (YQQPIIGGALF) with the concentrations of 27.8, 83.3 and 250 mug/mL are respectively given by a water-soluble administration method, the sea cucumber intestinal peptide (not separated and purified) as a test sample has the concentration of 80 mug/mL, the capecitabine as a positive control has the concentration of 20 mug/mL, and a model control group is simultaneously arranged, wherein 30 zebra fish per well (concentration group) has the volume of 3 mL. The zebra fish of each experimental group (concentration) is continuously cultured at 35 ℃, after 2 days, a fluorescence microscope is used for photographing to obtain a fluorescence picture of the MCF-7 transplanted tumor, Nikon NIS-Elements D3.10 advanced image processing software is used for carrying out image analysis, the fluorescence intensity of cancer cells is calculated, the inhibition effect of '3 #' on the zebra fish human breast cancer (MCF-7) cell transplanted tumor is evaluated according to the statistical significance of the fluorescence intensity, and the tumor inhibition effect is calculated according to the following formula.
Drawing a concentration effect curve according to the growth inhibition effect of the transplanted tumor; statistical analysis using analysis of variance and Dunnett's T-test, p <0.05 is a significant difference; representative experimental profiles are provided.
Specifically, the following description is provided: the wild AB line zebra fish does not generate red fluorescence, after the CM-Dil marked cells are injected into the zebra fish yolk sac, the cells can be excited to generate red fluorescence under a certain wavelength, the sum of the fluorescence intensity is in positive correlation with the number of cancer cells, and the larger the sum of the fluorescence intensity is, the larger the number of the cancer cells is.
Evaluation of antitumor Effect
The results show that: the positive control drug capecitabine has the advantages that the total fluorescence intensity value of the cell of the zebra fish human breast cancer (MCF-7) transplanted tumor is 171709 pixels under the concentration of 20 mu g/mL, p is less than 0.001 compared with a model control group (385517 pixels), the tumor inhibition effect is 55 percent, and the capecitabine has obvious inhibition effect on the growth of the zebra fish human breast cancer (MCF-7) transplanted tumor. The fluorescence intensity value sum of the cell of the zebra fish human breast cancer (MCF-7) transplanted tumor with the concentration of 80 microgram/mL of the sea cucumber intestinal peptide (without separation and purification) of the test sample is 217993 pixels, compared with the model control group, p is less than 0.001, and the tumor inhibition effect is 43 percent, which shows that the sea cucumber intestinal peptide (without separation and purification) has obvious inhibition effect on the growth of the zebra fish human breast cancer (MCF-7) transplanted tumor; the total fluorescence intensity values of the transplanted tumor cells of the zebra fish breast cancer (MCF-7) of the sea cucumber intestinal peptide (YQQPIIGGALF) at the concentrations of 27.8, 83.3 and 250 mu g/mL are 284622, 193586 and 183401 pixels respectively, compared with the model control group, the p is less than 0.001, and the tumor inhibition effect is 26%, 50% and 52% respectively. The sea cucumber intestinal peptide (YQQPIIGGALF) is suggested to have obvious inhibiting effect on the zebra fish human breast cancer (MCF-7) cell transplantation tumor under the condition of the experimental concentration, and the tumor body is obviously smaller than that of a model control group. See table 3, fig. 4, fig. 5 and fig. 6 for details.
TABLE 3 inhibition of the human breast cancer (MCF-7) transplantable tumor of zebrafish by sea cucumber intestinal peptide (n 10)
P <0.001 compared to model control group.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Zhongshidu Qing (Shandong) Biotechnology Co., Ltd
<120> sea cucumber intestinal peptide, preparation method and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Tyr Gln Gln Pro Ile Ile Gly Gly Ala Leu Phe
1 5 10
Claims (5)
1. A preparation method of sea cucumber intestinal peptide is characterized by comprising the following steps:
1) mixing sea cucumber intestines with water to obtain a mixture, and treating the mixture at 55 ℃ for 60min to obtain a treated substance;
2) adjusting the pH value of the treated matter obtained in the step 1) to 8.5, mixing the treated matter with alkaline protease, performing enzymolysis for 3 hours at the temperature of 60 ℃ to obtain an zymolyte, and adjusting the pH value of the zymolyte to 5 to obtain an adjusted matter;
3) inactivating enzymes of the regulator obtained in the step 2) to obtain an enzyme-inactivated substance, adsorbing the enzyme-inactivated substance for 50-70 min by using active carbon at 55-60 ℃, sequentially passing through a 30000Da ceramic membrane and a 1KD organic membrane, and drying to obtain a sea cucumber intestinal peptide mixture;
4) mixing the sea cucumber intestinal peptide mixture obtained in the step 3) with water to obtain a mixed solution, separating and purifying the mixed solution by a Sephadex G-50 chromatographic column, and collecting components with retention time of 26.5min to obtain sea cucumber intestinal peptide;
the amino acid sequence of the sea cucumber intestinal peptide is shown in SEQ ID No. 1;
the mass ratio of the treated substance in the step 2) to the alkaline protease is 100: 3.
2. The method according to claim 1, wherein the mass to water volume ratio of the sea cucumber intestines of step 1) is 1000kg: 2000L.
3. The method of claim 1, wherein the water of step 1) is deionized water.
4. The method according to claim 1, wherein the step 4) of separating and purifying comprises: the mobile phase is 10 percent methanol solution by volume and the flow rate is 1 ml/min.
5. The use of a sea cucumber intestinal peptide obtained by the preparation method according to any one of claims 1 to 4 for the preparation of an anti-tumor medicament;
the tumor comprises breast cancer and/or cervical cancer;
the amino acid sequence of the sea cucumber intestinal peptide is shown in SEQ ID No. 1.
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