CN105085618B - Sinonovacula constricta active octapeptide and preparation method and application thereof - Google Patents
Sinonovacula constricta active octapeptide and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a sinonovacula active octapeptide, the amino acid sequence of which is as follows: Met-His-Thr-Asp-Asp-Asp-Val-Glu, ESI/MS detection molecular weight 960.10 Da; the preparation method comprises 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, performing ultrafiltration and G-25 gel separation reversed-phase high performance liquid chromatography purification on the obtained enzymolysis sample, collecting elution peak components, and performing amino acid sequencing to obtain the sinonovacula constricta active octapeptide. The maximum proliferation inhibition rate of the sinonovacula constricta active octapeptide on human prostatic cancer PC-3 cells can reach more than 90 percent, can provide theoretical basis for the research of drugs for resisting prostatic cancer, and has great significance for further researching and developing drugs and foods based on the sinonovacula constricta active octapeptide and improving the economic value of the sinonovacula constricta active octapeptide.
Description
Technical Field
The invention relates to the field of bioactive peptides, in particular to a sinonovacula constricta active octapeptide and a preparation method and application thereof.
Background
Marine shellfish is abundant in resources and many physiologically active substances, and thus is gradually widely researched and applied. Sinonovacula constricta (Sinonovacula constricta) common name razor clam belongs to the class of lamprex, subclass of anortholoea, order of Venerulea, family of Erythrinae and genus of razor clam, is distributed along the coast of China, has large yield, and is one of four cultured shellfish (Ansheng, Lijietai) in China. Scientific fish culture, 2005(1):4-6 ], the sinonovacula constricta meat tastes delicious, is rich in nutrition, is rich in various essential amino acids, unsaturated fatty acids and other nutrient substances required by human life activities [ Litaiwu, forest leaves, Suxiubanian, and the multivariate analysis of the nutrient components of different groups of sinonovacula constricta, and has the advantages of food science, 2008 (11): 548-; remacha, Trivino A, adaptive. regenerative cycle of the razor ram Solen margin us (Pulteney 1799) in Spain: effective study in three differential locations, Journal of shellfisheresearch, 2006, 25 (3): 869-876; anxian, nutritional and health analysis and evaluation of several sinonovacula constricta, reported in the ocean lake, 2005 (4): 99 to 103; multivariate analysis of nutritional ingredients of li taiwu, forest leaf, suxiuwu banian, different groups of sinonovacula constricta [ J ], food science, 2008, 29(11): 548-; the amino acid and fatty acid comparison research of different populations of sinonovacula constricta, such as forest leaf, suxiubanian, sunbei and the like, is scientific in food, 2006, 27 (12): 675-678].
The sinonovacula constricta lamarck is cold in nature, can tonify yin and remove pathogenic heat, and is used for treating symptoms such as vexation, cold dysentery, hot dysentery, postpartum consumptive disease of women, deficiency heat and the like, and has higher medicinal and edible values [ Jiangzhuangmiao, Zhengguping, Chenmusson, Zhoushan marine medicinal animal resources and folk application investigation [ J ] Chinese marine medicine, 2002, 85(1):35 ]; sujing, Sumingming 32741, a medicinal product for aquatic products to treat various diseases Shenyang: liaoning science and technology Press, 2000, 187. ]; the active ingredients of sinonovacula constricta also have high research value, such as Rao Xiao Ling et al [ Rao Xiao Ling, Wu hong Gong, Fa Xiu Lian Zi et al, extraction and separation of sinonovacula constricta glycosaminoglycan and initial research of in vitro antitumor activity thereof, pharmaceutical biotechnology, 2004, 11 (3): 146-149], the enzymolysis conditions of the sinonovacula constricta meat are researched, and 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. Zhang Yongjuan, etc. (Zhang Yongjuan, Zheng Hui Zhen, the immunoregulation and antioxidation action of Sinonovacula constricta polypeptide, Shizhen Chinese medicine, 2011, 22 (5): 1076 and 1077, the sinonovacula constricta polypeptide is extracted to promote the development of the thymus and spleen of the mouse and the generation of delayed type allergic reaction, and improve the carbon clearance capability and the serum hemolysin level of the mouse; PSD can also improve serum SOD and GSH-Px levels, and reduce serum MDA content.
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). The research on the anti-tumor aspect of the sinonovacula constricta is few, and particularly, the research on the anti-tumor aspect of polypeptide extraction by utilizing an enzymolysis technology is not reported. Therefore, the research on the sinonovacula constricta enzymatic hydrolysis polypeptide has great significance for further researching and developing functional foods and medicines based on the sinonovacula constricta enzymatic hydrolysis polypeptide and improving the economic value of the sinonovacula constricta.
Disclosure of Invention
The first technical problem to be solved by the present invention is to provide an active octapeptide of Sinonovacula constricta with anti-tumor activity 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 octapeptide 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 octapeptide in view of the prior art.
The technical scheme adopted by the invention for solving the first technical problem is as follows: the sinonovacula active octapeptide is characterized in that the amino acid sequence is as follows: Met-His-Thr-Asp-Asp-Asp-Val-Glu.
Further, the peak of molecular ion detected by HR-ESI-MS of the octapeptide is M/z 960.10Da [ M + H ]]+。
The technical scheme adopted by the invention for solving the second technical problem is as follows: the method comprises the following steps:
(1) removing shells of fresh sinonovacula constricta and taking out the sinonovacula constricta, cleaning the sinonovacula constricta and mashing the sinonovacula constricta, adding water according to the feed-liquid ratio of 1: 1-3 to carry out tissue homogenization, and adjusting the pH value of homogenate to 2-3.5 for later use;
(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 30-45 ℃ for 2-8 h 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 8-5 KD and the molecular weight of less than 5KD, and freeze-drying;
(4) respectively carrying out anti-tumor activity detection on the enzymolysis liquid groups of each molecular weight section in the step (3) by an MTT method, carrying out G-25 gel separation on the enzymolysis liquid group with the highest anti-tumor activity, and collecting corresponding elution peaks;
(5) and (4) purifying the elution peaks collected in the step (4) by reversed-phase high performance liquid chromatography, collecting peak components, and performing amino acid sequencing to obtain the sinonovacula constricta active octapeptide.
Preferably, the method comprises the following steps: the preparation method of the sinonovacula constricta active octapeptide 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 enzyme deactivation is carried out for 2-8 hours at 30-45 ℃ after the enzymolysis is finished;
(3) ultrafiltering the enzymolysis sample with 5KD ultrafiltration membrane, collecting enzymolysis solution with molecular weight less than 5KD, and freeze drying;
(4) carrying out G-25 gel separation on the enzymolysis liquid collected in the step (3), and collecting an elution peak 5;
(5) and (3) purifying the elution peak 5 collected in the step (4) by reversed-phase high performance liquid chromatography, collecting peak components, and performing amino acid sequencing to obtain the sinonovacula constricta active octapeptide.
The conditions of the reversed phase high performance liquid chromatography are as follows: zorbax SB-C18 column 4.6X 250mm 5um, detection wavelength 280nm/214nm, 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 0 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 technical scheme adopted by the invention for solving the third technical problem is as follows: an application of the sinonovacula constricta active octapeptide in preparing antitumor drugs and foods.
Further, the anti-tumor is the activity of inhibiting the proliferation of tumor cells, and the tumor is human prostate cancer.
Furthermore, the proliferation inhibition rate of the sinonovacula constricta active octapeptide on human prostatic cancer PC-3 cells is in a dependent relationship with concentration and action time.
Compared with the prior art, the invention has the following advantages: the invention provides a brand new sinonovacula constricta active octapeptide, which can better inhibit the proliferative activity of human prostate cancer cells, has the maximum proliferative inhibition rate of human prostate cancer PC-3 cells higher than 90 percent, has obvious in-vitro anti-prostatic cancer effect, can provide a theoretical basis for the research of anti-prostatic cancer medicines, is obtained by a biological enzymolysis mode, has wide raw material sources and low cost, adopts pepsin for enzymolysis, has mild enzymolysis mode, and is suitable for the natural needs of human bodies, thereby having great significance for further researching and developing medicines based on sinonovacula constricta and providing the economic value of the sinonovacula constricta.
Drawings
FIG. 1 is a G-25 separation and purification diagram of an enzymatic hydrolysate sample with a molecular weight of less than 5KD in example 1 of the present invention;
FIG. 2 is a reversed-phase high performance liquid chromatogram of a peak 5 sample of example 1 of the present invention;
FIG. 3 is a mass spectrum (ESI/MS) of an active octapeptide from Sinonovacula constricta in example 1 of the present invention;
FIG. 4 is a graph showing the effect of the active octapeptide of Sinonovacula constricta on the proliferation activity of PC-3 cells in example 2 of the present invention.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
Example 1: preparation of Sinonovacula constricta active octapeptide
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 alkaline protease, selecting 4 levels to perform L according to 5 factors of A (temperature), B (pH), C (enzyme adding amount), D (feed-liquid ratio) and E (time)16(45) The orthogonal experiment of (2) screening enzymolysis liquid under different enzymolysis conditions through an MTT method under the condition of highest prostate cancer cell proliferation inhibition rate, thereby determining the optimal hydrolysis condition and carrying out a large amount of enzymolysis. 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 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, centrifuging at 1000rpm for 10min, removing supernatant, addingAdding 4ml of nutrient solution, and repeatedly blowing to make the cells become 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.
(4) 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 well2After incubation for 36h in an incubator at 37 ℃, the cells were washed 2 times with PBS, and then added with MTT-containing nutrient solution and incubated for 4 h. The culture was terminated and the culture medium in the wells was carefully aspirated. Adding dimethyl sulfoxideThe mixture was shaken on a shaker for 10min at low speed, and absorbance (OD) was measured at 490nm using an 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:
2.2.3, the results of orthogonal pepsin hydrolysis are shown in Table 2, and it can be seen that the IR value is the greatest in experiment No. 15, so the optimum conditions for alkaline protease enzymatic hydrolysis are: enzymolysis temperature: 50 ℃, enzymolysis pH value: 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 8-5 KD 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. G-25 gel separation
Dissolving the enzymolysis sample with molecular weight less than 5KD with distilled water, centrifuging, collecting supernatant, filtering with 0.45 μm microporous membrane to obtain filtrate, filtering 1.5ml of filtrate with Sephadex G-25[90cm × 115cm (ID) ], eluting with distilled water, balancing and eluting. 3ml of each tube was collected and detected at λ 280nm and the peak eluents collected.
The results are shown in figure 1, the enzymolysis sample with molecular weight less than 5KD passes through G-25 eluent to obtain 6 peaks, and the peak 5 has the highest antitumor activity after the screening of the in vitro antitumor experiment, so the peak 5 is collected, frozen, dried and purified by reverse phase high performance liquid chromatography (RT-HPLC).
5. 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 0 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 280nm/214 nm.
The RT-HPLC result is shown in figure 2, as can be seen from figure 2, the molecular segment has only one elution peak, the elution peak is collected and subjected to amino acid sequence detection, and the amino acid sequence is Met-His-Thr-Asp-Asp-Asp-Val-Glu; as shown in FIG. 3, the molecular weight of ESI/MS detection was 960.10Da ([ M + H ]]+960.10Da) to obtain the desired sinonovacula constricta active octapeptide.
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 octapeptide
The results of the MTT method using the F12 medium as a control group to compare the effect of sinonovacula constricta active tripeptide on PC-3 cell proliferation at different concentrations and different action times are shown in FIG. 4, which shows that the sinonovacula constricta active octapeptide can effectively inhibit PC-3 cell proliferation and is concentration-dependent and time-dependent.
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; alkaline protease, 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-250pH meter, Shanghai Lida Instrument factory; CF16RXII high speed refrigerated centrifuge, HITACHI corporation, japan; ZHJH-C1209C model superclean bench, Shanghai Zhicheng Analyzer manufacturing, Inc.; forma 3111 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 (3)
1. An active octapeptide of Sinonovacula constricta, which is characterized in that the amino acid sequence is as follows: Met-His-Thr-Asp-Asp-Asp-Val-Glu.
2. The method for producing an active octapeptide from Sinonovacula constricta according to claim 1, comprising the steps of:
(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 enzyme deactivation is carried out for 2-8 hours at 30-45 ℃ after the enzymolysis is finished;
(3) ultrafiltering the enzymolysis sample with 5KD ultrafiltration membrane, collecting enzymolysis solution with molecular weight less than 5KD, and freeze drying;
(4) carrying out G-25 gel separation on the enzymolysis liquid collected in the step (3), and collecting an elution peak 5;
(5) and (3) purifying the elution peak 5 collected in the step (4) by reversed-phase high performance liquid chromatography, collecting peak components, and performing amino acid sequencing to obtain the sinonovacula constricta active octapeptide.
The conditions of the reversed phase high performance liquid chromatography are as follows: zorbax SB-C18 column 4.6X 250mm 5um, detection wavelength 280nm/214nm, 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% -0% B for 4 minutes, 0% -7% B for 25 minutes, 7% -100% B for 1 minute, and 100% -100% B for 5 minutes.
3. The use of the sinonovacula constricta active octapeptide according to claim 1 in the preparation of an antitumor drug, wherein the sinonovacula constricta active octapeptide comprises: the anti-tumor is the proliferation activity of tumor cells, and the tumor is human prostatic cancer.
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