CN105315337B - Sinonovacula constricta active tetrapeptide and preparation method and application thereof - Google Patents
Sinonovacula constricta active tetrapeptide and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a sinonovacula constricta active tetrapeptide, the amino acid series of which is as follows: Leu-Pro-Gly-Pro, prepared by the following steps: removing shells of fresh Sinonovacula constricta Lamarck, taking out the Sinonovacula constricta Lamarck meat, cleaning the Sinonovacula constricta Lamarck meat, mashing, homogenizing tissues, and adjusting the pH value of homogenate for later use; adding pepsin into the homogenate for enzymolysis to obtain an enzymolysis sample, performing ultrafiltration on the obtained enzymolysis sample, and respectively collecting enzymolysis liquid groups of each molecular weight section for freeze drying; and (3) carrying out reversed-phase high performance liquid chromatography purification on the enzymolysis liquid groups of each molecular weight section, collecting peak components, and carrying out amino acid sequencing to obtain the sinonovacula constricta active tetrapeptide. The maximum proliferation inhibition rate of the sinonovacula constricta active tetrapeptide on human prostatic cancer PC-3 cells can reach 99.55 percent, and a theoretical basis can be provided for the research of anti-prostatic cancer drugs. The sinonovacula active tetrapeptide is obtained by means of biological enzymolysis, has wide raw material sources and low cost, and has great significance for further researching and developing sinonovacula based medicines and providing the economic value of sinonovacula.
Description
Technical Field
The invention relates to the field of bioactive peptides, in particular to a sinonovacula constricta active tetrapeptide and a preparation method and application thereof.
Background
Sinonovacula constricta (Sinonovacula constricta) common name razor clam belongs to the class of lamprex, subclass of Isodentata, order of Venerules, family Erythridae and genus of razor clam, is distributed in China coastal area, has large yield, and is one of four cultured shellfish in China. The Sinonovacula constricta meat is delicious, cold in nature and rich in nutrition, is rich in various essential amino acids, unsaturated fatty acids and other nutrient substances required by human life activities, can tonify yin and remove pathogenic heat, is used for treating symptoms such as vexation, cold dysentery, hot dysentery, postpartum deficiency and deficiency-heat of women and has higher medicinal and edible values.
Active peptides are compounds in which two or more amino acids are linked by peptide bonds, which play important physiological roles in the human body and perform physiological functions, such as: regulating the balance of water and electrolyte in the body; promoting wound healing; repairing cells, improving cell metabolism, and preventing cancer. Bioactive peptides are generally obtained by enzymolysis of bioactive materials, and active polypeptides with various functions can be obtained by protease enzymolysis of food proteins found by Zhenghuina and the like (research progress on preparation of bioactive peptides by enzymolysis of marine proteins, Zhenghuina, Chaxina, Caoshanhong, aquatic science, 2008,27(7), 370). In the research on the proteolysis products of the sinonovacula constricta meat, the rexarsia and the like research on the enzymolysis conditions of the sinonovacula constricta meat, and the discovery shows that the extracted glycosaminoglycan has certain inhibition effect on HL-60 cells cultured in vitro, and the inhibition effect is enhanced along with the increase of the dosage (preliminary research on the extraction and separation of the sinonovacula constricta glycosaminoglycan and the in vitro antitumor activity thereof, the pharmaceutical biotechnology, 2004,11 (3): 146-; studies of Zhang Yongjin and the like find that the extracted sinonovacula constricta polypeptide can promote the development of thymus and spleen of mice and the occurrence of delayed allergic reaction, and improve the carbon clearance capability and the serum hemolysin level of the mice (immunoregulation and antioxidation of the sinonovacula constricta polypeptide, Zhang Yongjuan, Zheng Huinao, Shizhen Chinese medicine, 2011,22 (5): 1076). However, the above studies generally only relate to the study of sinonovacula constricta proteolytic polypeptide mixtures, and no further study was made on specific functional polypeptide monomers, and the present application further studies on the antitumor activity of sinonovacula constricta by extracting sinonovacula constricta active tetrapeptides.
Disclosure of Invention
The first technical problem to be solved by the present invention is to provide a sinonovacula constricta active tetrapeptide against the prior art.
The second technical problem to be solved by the present invention is to provide a method for preparing the sinonovacula constricta active tetrapeptide in view of the prior art.
The third technical problem to be solved by the present invention is to provide an application of the sinonovacula constricta active tetrapeptide in view of the prior art.
The technical scheme adopted by the invention for solving the technical problems is as follows: an active tetrapeptide of Sinonovacula constricta, the amino acid series of which is as follows: Leu-Pro-Gly-Pro.
A method for preparing the sinonovacula constricta active tetrapeptide according to claim 1, comprising the steps of:
(1) removing shells of fresh sinonovacula constricta and taking meat, cleaning and mashing, adding water according to the feed-liquid ratio of 1: 1-3 for tissue homogenization, and adjusting the pH value of homogenate to 2-3.5 for later use after homogenization;
(2) adding pepsin into the homogenate to carry out enzymolysis to obtain an enzymolysis sample, wherein the enzymolysis temperature is 30-45 ℃, the enzyme adding amount is 300-1200U/g of sinonovacula constricta meat, the enzymolysis time is 2-8 h, and enzyme deactivation is carried out at 95-100 ℃ for 10-15 min after the enzymolysis is finished;
(3) performing ultrafiltration on the enzymolysis sample by 5KD and 8KD ultrafiltration membranes, respectively collecting enzymolysis liquid groups with the molecular weight of more than 8KD, the molecular weight of 8-5KD and the molecular weight of less than 5KD, and freeze-drying;
(4) and (4) respectively carrying out reversed-phase high performance liquid chromatography purification on the enzymatic hydrolysate collected in each molecular weight section in the step (3), collecting each peak component and carrying out amino acid sequencing to obtain the sinonovacula constricta active tetrapeptide.
Preferably, the preparation method of the sinonovacula constricta active tetrapeptide comprises the following steps: (1) removing shells of fresh Sinonovacula constricta Lamarck, taking out meat, cleaning, mashing, adding water according to the feed-liquid ratio of 1:1 for tissue homogenization, and adjusting the pH value of homogenate to 3 for later use;
(2) adding pepsin into the homogenate to carry out enzymolysis to obtain an enzymolysis sample, wherein the enzymolysis temperature is 45 ℃, the enzyme adding amount is 300U/g of sinonovacula constricta meat, the enzymolysis time is 8 hours, and after the enzymolysis is finished, enzyme deactivation is carried out for 10-15 min at 95-100 ℃;
(3) ultrafiltering the enzymolysis sample with 5KD ultrafiltration membrane, collecting enzymolysis solution with molecular weight less than 5KD, and freeze drying;
(4) carrying out reversed-phase high performance liquid chromatography purification on the enzymolysis liquid collected in the step (3), collecting peak components and carrying out amino acid sequencing to obtain the sinonovacula constricta active tetrapeptide;
the conditions of the reversed phase high performance liquid chromatography are as follows: agilent C18 chromatographic column 4.6X 250mm 5um, detection wavelength 280nm, column temperature 20 ℃, sample size 100ul, gradient elution, flow rate 1.0ml/min, mobile phase A0.06% TFA, B0.05% TFA acetonitrile, elution procedure: 0 to 1 percent of B is eluted for 4 minutes, 0 to 7 percent of B is eluted for 25 minutes, 7 to 100 percent of B is eluted for 1 minute, and 100 to 100 percent of B is eluted for 5 minutes.
An application of the sinonovacula constricta active tetrapeptide in antitumor drugs.
The anti-tumor is the proliferation activity of tumor cells, and the tumor is human prostatic cancer.
Furthermore, the proliferation inhibition rate of the sinonovacula constricta active tetrapeptide on human prostatic cancer PC-3 cells is in positive correlation with the concentration and the action time, and when the concentration is 5-15 mg/ml and the action time is 24-72 h, the proliferation inhibition rate is 39.75-99.55%.
Compared with the prior art, the invention has the advantages that: the invention provides a brand-new sinonovacula constricta active tetrapeptide which can better inhibit the proliferation activity of human prostate cancer cells, has the maximum proliferation inhibition rate of 98.94 percent on human prostate cancer PC-3 cells, has obvious in-vitro anti-prostate cancer effect and can provide a theoretical basis for the research of anti-prostate cancer drugs. The sinonovacula active tetrapeptide is obtained through biological enzymolysis, the source of raw materials is wide, the cost is low, the internal digestion environment of a human body is simulated, pepsin is adopted for enzymolysis, the enzymolysis mode is mild, the enzymolysis product meets the natural requirement of the human body, and the sinonovacula active tetrapeptide has great significance in further researching and developing drugs based on the sinonovacula and providing the economic value of the sinonovacula.
Drawings
FIG. 1 is a reversed-phase high performance liquid chromatogram of an enzymolysis solution with a molecular weight less than 5KD in example 1 of the invention.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
Example 1: preparation of Sinonovacula constricta active tetrapeptide
1. Pretreatment:
taking fresh sinonovacula constricta, removing shells, taking sinonovacula constricta meat, cleaning, and draining for later use.
2. Enzymolysis of gastric protein
2.1 mashing the sinonovacula constricta meat by using a high-speed tissue masher, adding distilled water for homogenizing, precisely weighing homogenate according to the feed-liquid ratio of 1: 1-3, adjusting the pH value of the homogenate by using 0.1mol/L hydrochloric acid solution and 0.1mol/L NaOH solution, adding pepsin for enzymolysis for a plurality of hours, wherein the enzymolysis conditions are as follows: the enzymolysis temperature is 30-45 ℃, the pH is 2-3.5, the enzymolysis time is 2-8 h, and the enzyme adding amount is 300-1200U/g of sinonovacula constricta Lamarck meat. Inactivating enzyme at 100 deg.C for 15min, centrifuging at 4 deg.C for 15min (10000r/min), and collecting supernatant.
2.2 pepsin enzymolysis Process optimization
2.2.1, selecting 4 levels of pepsin and selecting 5 factors of A (temperature), B (pH), C (enzyme adding amount), D (feed-liquid ratio) and E (time) for L16(45) The orthogonal experiment of (2) screening enzymolysis liquid under different enzymolysis conditions through an MTT method, wherein the enzymolysis condition of the enzymolysis liquid with the highest inhibition rate on the proliferation of the PC-3 of the prostate cancer cells is determined, and a large amount of enzymolysis is carried out. Pepsin factors and levels are shown in table 1:
TABLE 1 factors and levels of pepsin
2.2.2 prostate cancer cells PC-3 hormone independent cells (originally purchased from Shanghai cell Bank of Chinese academy, and subcultured in this laboratory) were selected in this example and cultured as follows:
(1) cell recovery
Taking out the stored PC-3 cell strain from the liquid nitrogen tank, rapidly placing into a 37 ℃ constant temperature water bath for melting, entering into a sterile working room for operation after melting, sucking the cell strain into a centrifuge tube by using a sterilized straw, adding 2mL of F12 nutrient solution or RPMI1640 nutrient solution at 1000rpmCentrifuging for 10min, removing supernatant, adding 4ml of nutrient solution, and repeatedly blowing to make cells into single cells. 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) And cell culture
Human prostate cancer cell PC-3 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 cells were passaged at a ratio of 1: 2, and cells in the logarithmic growth phase were collected for experiments.
(3) Cell passage
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.
2.2.3 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 wells carefully aspiratedAn inner culture solution. 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:
2.2.4, results of pepsin enzymatic hydrolysis are shown in Table 2, and it can be seen that the IR value is the largest in experiment No. 15, so the best conditions for pepsin enzymatic hydrolysis are: enzymolysis temperature: 45 ℃, enzymolysis pH: 3, material-liquid ratio: 1:1, time: 8h, enzyme addition: 300U/g.
TABLE 2 pepsin enzymolysis orthogonal test results
3. Ultrafiltration
Adding the enzymolysis liquid into an ultrafiltration system, performing ultrafiltration by using 8KD and 5KD ultrafiltration membranes, respectively collecting enzymolysis liquid groups with the molecular weight of more than 8KD, the molecular weight of 8-5KD and the molecular weight of less than 5KD, respectively preparing the enzymolysis liquid groups into enzymolysis solutions with the concentration of 10mg/ml after freeze drying for MTT test, after 24 hours of action, the proliferation inhibition rates of the enzymolysis liquid groups on the prostatic cancer PC-3 are respectively 34.23%, 46.57% and 67.21%, so that the proliferation inhibition effect of the enzymolysis liquid groups with the molecular weight of less than 5KD on the PC-3 is better than that of the other two components, and further purifying the enzymolysis liquid groups with the molecular weight of less than 5KD after freeze drying.
4. Purification by reversed phase high performance liquid chromatography (RT-HPLC) and detection of amino acid sequence
The freeze-dried enzymatic sample was dissolved in 0.06% TFA water in a 0.6ml centrifuge tube and centrifuged at 12000rpm for 10min to obtain the supernatant. Reversed-phase high-performance liquid phase conditions: the system comprises the following steps: agilent 1260 HPLC; ZorbaxSB-C18(4.6 × 250, 5um) is selected; the column temperature is 20 ℃; mobile phase a was 0.06% TFA, B was 0.05% TFA in acetonitrile, elution procedure: 0 to 1 percent of B is eluted for 4 minutes, 0 to 7 percent of B is eluted for 25 minutes, 7 to 100 percent of B is eluted for 1 minute, and 100 to 100 percent of B is eluted for 5 minutes; the flow rate is 1.0 ml/min; the sample amount is 100 ul; the detection wavelength was 280 nm.
The RT-HPLC result is shown in figure 1, as can be seen from figure 1, the enzymolysis solution group of the molecular weight section contains a plurality of components, each peak is collected one by one, the amino acid sequence is detected, the peak 8 is collected, the amino acid sequence is Leu-Pro-Gly-Pro, and the desired sinonovacula constricta active tetrapeptide is obtained.
The amino acid sequence detection adopts an N-terminal amino acid degradation detection method to determine: establishing a standard amino acid map: and (3) operating under a conventional condition by using a mixed amino acid standard product (PTH-AA) to generate a standard product chromatogram, correcting the retention time of the mixed amino acid standard product, and generating a standard method file. Preprocessing a sample: centrifuging the pure sample, and taking a supernatant for later use; 15ul of Polybrene (Polybrene) was applied to a Glass Fiber membrane (Glass Fiber Disk) and dried with nitrogen; and (3) pretreating the glass fiber membrane by a machine, namely, performing 5 cycles to add sufficient pure sample points on the pretreated glass fiber membrane, and drying by nitrogen. And thirdly, detecting on the computer: and sealing the glass fiber membrane added with the sample in a reactor of a protein sequencer PPSQ-31A by using a PTFE filter membrane, and setting the number of detected amino acids and other parameters.
Example 2: research on anti-PC-3 cell proliferation activity of sinonovacula constricta active tetrapeptide
The effects of sinonovacula constricta active tetrapeptides with different concentrations and different action times on the proliferation of PC-3 cells were compared by using the MTT method with the F12 medium as a control group, and the test results are shown in Table 3, which shows that the sinonovacula constricta active tetrapeptides can effectively inhibit the proliferation of PC-3 cells and are concentration-dependent and time-dependent.
TABLE 3 proliferation modulating effects of tetrapeptides on PC-3 cells: (
n＝9)
The main materials and reagents used in the above examples:
the Sinonovacula constricta Lamarck is purchased from Nanzhen market in Zhoushan of Zhejiang province; prostate cancer cell PC-3 hormone independent cells (originally purchased from Shanghai cell bank of Chinese academy of sciences and subcultured in the laboratory); fetal Bovine Serum (FBS), hangzhou sijiqing biologicals ltd; HamF12 medium, Gibco; pepsin, SIGMA, usa; penicillin, streptomycin, Shandong Shanglu anti-medicine GmbH; MTT was purchased from Sigma, USA; dimethyl sulfoxide (DMSO) was purchased from AMRESCO, usa; the other reagents are analytically pure.
The main instruments used in the above examples:
DS-1 type high-speed tissue masher, Shanghai Biao Ben model factory; BSA model 124S electronic balance, SartoriusAG, germany; SSW type microcomputer electric heating thermostatic bath, Shanghai Boxun practice Co., Ltd medical equipment factory; ultrafiltration systems, millipore corporation, usa; PHS-250 pH meter, Shanghai Lida Instrument factory; CF16RXII high speed refrigerated centrifuge, HITACHI corporation, japan; ZHJH-C1209C model superclean bench, Shanghai Zhicheng Analyzer manufacturing, Inc.; forma3111 type CO2 incubator, Thermo corporation, usa; microplate reader, Bio-Rad, USA; LGJ-18 freeze dryer, Beijing Songyuan Huaxing science and technology development Co., Ltd; agilent 1260 high performance liquid chromatograph, Agilent technologies, Inc.; micro-oscillator, Shanghai precision instruments, Inc.
Claims (2)
1. A method for preparing a sinonovacula active tetrapeptide having an activity of resisting prostate cancer, which is characterized in that the amino acid sequence of the sinonovacula active tetrapeptide is as follows: Leu-Pro-Gly-Pro,
the preparation method comprises the following steps:
(1) removing shells of fresh Sinonovacula constricta Lamarck, taking out meat, cleaning, mashing, adding water according to the feed-liquid ratio of 1:1 for tissue homogenization, and adjusting the pH value of homogenate to 3 for later use;
(2) adding pepsin into the homogenate to carry out enzymolysis to obtain an enzymolysis sample, wherein the enzymolysis temperature is 45 ℃, the enzyme adding amount is 300U/g of sinonovacula constricta meat, the enzymolysis time is 8 hours, and after the enzymolysis is finished, enzyme deactivation is carried out for 10-15 min at 95-100 ℃;
(3) ultrafiltering the enzymolysis sample with 5KD ultrafiltration membrane, collecting enzymolysis solution with molecular weight less than 5KD, and freeze drying;
(4) carrying out reversed-phase high performance liquid chromatography purification on the enzymolysis liquid collected in the step (3), collecting peak components and carrying out amino acid sequencing to obtain the sinonovacula constricta active tetrapeptide;
the conditions of the reversed phase high performance liquid chromatography are as follows: agilent C18 chromatographic column 4.6X 250mm 5um, detection wavelength 280nm, column temperature 20 ℃, sample size 100ul, gradient elution, flow rate 1.0ml/min, mobile phase A0.06% TFA, B0.05% TFA acetonitrile, elution procedure: eluting with 0% -1% B for 4 minutes, 0% -7% B for 25 minutes, 7% -100% B for 1 minute, and 100% -100% B for 5 minutes.
2. The use of the sinonovacula constricta active tetrapeptide according to claim 1 in the preparation of an anti-prostate cancer drug.
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| CN1649899A (en) * | 2002-05-03 | 2005-08-03 | 米列姆·贝尔罗吉克公司 | Connective tissue stimulating peptides |
| KR101183004B1 (en) * | 2011-11-03 | 2012-09-18 | 윤의구 | Artificial spawning ground for sinonovacula constricta |
| CN103740792A (en) * | 2013-06-07 | 2014-04-23 | 浙江海洋学院 | Preparation method of Sinonovacula constricta polypeptide with antioxidation function and application thereof |
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| CN1649899A (en) * | 2002-05-03 | 2005-08-03 | 米列姆·贝尔罗吉克公司 | Connective tissue stimulating peptides |
| KR101183004B1 (en) * | 2011-11-03 | 2012-09-18 | 윤의구 | Artificial spawning ground for sinonovacula constricta |
| CN103740792A (en) * | 2013-06-07 | 2014-04-23 | 浙江海洋学院 | Preparation method of Sinonovacula constricta polypeptide with antioxidation function and application thereof |
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| 缢蛏多肽诱导前列腺癌DU－145 和PC－3细胞的早期凋亡的研究;李潇潇等;《时珍国医国药》;20121231;第23卷(第11期);第2773-2776页 * |
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