CN105315344B

CN105315344B - Solen active hexapeptide and preparation method and application thereof

Info

Publication number: CN105315344B
Application number: CN201510324588.0A
Authority: CN
Inventors: 黄芳芳; 丁国芳; 杨最素; 余方苗
Original assignee: Zhejiang Ocean University ZJOU
Current assignee: Zhejiang Ocean University ZJOU
Priority date: 2015-06-12
Filing date: 2015-06-12
Publication date: 2021-01-29
Anticipated expiration: 2035-06-12
Also published as: CN105315344A

Abstract

The invention relates to a solen active hexapeptide, the amino acid sequence of which is as follows: Val-Pro-Trp-Asp-Thr-Asp, ESI/MS detection molecular weight 731.7 Da; the preparation method comprises the following steps: removing shells of fresh razor clams, taking out the fresh razor clams, cleaning the razor clams, mashing the fresh razor clams, homogenizing tissues, and adjusting the pH value of homogenate for later use; adding alkaline protease into the homogenate for enzymolysis to obtain an enzymolysis sample, 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 solen active hexapeptide. The solen active hexapeptide has the maximum proliferation inhibition rate of more than 90% on human prostatic cancer PC-3 cells, can provide a theoretical basis for research on anti-prostatic cancer medicines, is obtained by a biological enzymolysis mode, has wide raw material sources and low cost, and has great significance for further research and development of solen-based medicines and foods and improvement of the economic value of solen.

Description

Solen active hexapeptide and preparation method and application thereof

Technical Field

The invention relates to the field of bioactive peptides, in particular to a solen active hexapeptide, a preparation method and application thereof.

Background

The razor clam, named razor clam king, is a marine bivalve mollusk, with an elongated body and a cylindrical shape after the two shells are clasped, so it is named razor clam. The razor clam shells are smooth, yellowish brown, glossy, crisp and thin, have protruding surfaces, are parallel to the dorsal and ventral borders, are round and rectangular at the rear ends, and have obvious growth lines which are about 11 cm long. China is distributed in south and north coastal areas, the meat taste is delicious, and the meat can be eaten fresh and dried. The razor clam has sweet and salty taste and cold nature, and enters heart, liver and kidney meridians; has effects in nourishing yin, clearing away heat, relieving restlessness, and relieving alcoholism; has certain therapeutic effects on puerperal deficiency yin, dysphoria, thirst, damp-heat edema, dysentery, alcoholic intoxication, etc.

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 current research on razor clam active peptides has mainly focused on sinonovacula constricta, such as: the enzymolysis condition of sinonovacula constricta meat is researched by Raschig et al, and the glycosaminoglycan obtained by extraction has certain inhibition effect on HL-60 cells cultured in vitro, and the inhibition effect is enhanced along with the increase of dosage (preliminary research on extraction and separation of sinonovacula constricta glycosaminoglycan and in vitro antitumor activity thereof, Raschig et al, Wu hong Gong, Fa Xiu Lian, etc., 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).

The shapes of the razor clams are different from those of the razor clams, the flavor is slightly different when the razor clams are eaten, and the research on the enzymatic hydrolysis polypeptides of the razor clams is not reported at present, so that the research on the enzymatic hydrolysis polypeptides of the razor clams is significant for further researching and developing functional foods and medicines based on the enzymatic hydrolysis polypeptides of the razor clams and improving the economic value of the razor clams.

Disclosure of Invention

The first technical problem to be solved by the invention is to provide a solen active hexapeptide with anti-tumor activity aiming at the prior art.

The second technical problem to be solved by the invention is to provide a preparation method of the razor clam active hexapeptide aiming at the prior art.

The third technical problem to be solved by the invention is to provide an application of the razor clam active hexapeptide aiming at the prior art.

The technical scheme adopted by the invention for solving the first technical problem is as follows: a solen active hexapeptide has an amino acid sequence as follows: Val-Pro-Trp-Asp-Thr-Asp, ESI/MS molecular weight 731.7 Da.

The technical scheme adopted by the invention for solving the second technical problem is as follows: the preparation method of the solen active hexapeptide comprises the following steps:

(1) removing shells of fresh razor clams, taking out the fresh razor clams, cleaning the razor clams, mashing the cleaned razor clams, adding water according to the material-liquid ratio of 1: 1-3 for tissue homogenization, and adjusting the pH value of homogenate to 8-10.4 for later use;

(2) adding alkaline protease into the homogenate for enzymolysis to obtain an enzymolysis sample, wherein the enzymolysis temperature is 35-50 ℃, the enzyme adding amount is 300-1200U/g of the razor clam 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 8-5 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 solen active hexapeptide.

Preferably, the preparation method of the solen active hexapeptide comprises the following steps: (1) removing shells of the fresh razor clams, taking out the fresh razor clams, cleaning, mashing, adding water according to the feed-liquid ratio of 1:1 for tissue homogenization, and adjusting the pH value of homogenate to 9.6 for later use;

(2) adding alkaline protease into the homogenate for enzymolysis to obtain an enzymolysis sample, wherein the enzymolysis temperature is 50 ℃, the enzyme adding amount is 300U/g of razor clam meat, the enzymolysis time is 8 hours, and enzyme deactivation is carried out at 95-100 ℃ for 10-15 min 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 4;

(5) purifying the elution peak 4 collected in the step (4) by reversed-phase high performance liquid chromatography, collecting peak components and performing amino acid sequencing to obtain the solen active hexapeptide;

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 razor clam active hexapeptide in preparing antineoplastic medicine and food.

The anti-tumor is the proliferation activity of tumor cells, and the tumor is human prostatic cancer.

Furthermore, the proliferation inhibition rate of the solen active hexapeptide 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 advantages that: the invention provides a brand new Solen active hexapeptide which can better inhibit the proliferation activity of human prostate cancer cells, has the maximum proliferation inhibition rate of human prostate cancer PC-3 cells higher than 90 percent, has obvious in-vitro anti-prostate cancer effect and can provide a theoretical basis for the research of anti-prostate cancer medicaments. The solen active hexapeptide is obtained through biological enzymolysis, the raw material source is wide, the cost is low, alkaline protease is adopted for enzymolysis, the enzymolysis mode is mild, the enzymolysis product is fit for the natural needs of a human body, and the solen active hexapeptide has great significance for further researching and developing the solen-based medicine and providing the economic value of the solen.

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 4 sample of example 1 of the present invention;

FIG. 3 is a graph showing a mass spectrum (ESI/MS) of a solen active hexapeptide (Val-Pro-Trp-Asp-Thr-Asp) in example 1 of the present invention;

FIG. 4 is a graph showing the effect of solen active hexapeptide (Val-Pro-Trp-Asp-Thr-Asp) 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 razor clam active hexapeptide

1. Pretreatment:

taking fresh razor clams, removing shells, taking razor clam meat, cleaning, and draining for later use.

2. Basic proteolysis

2.1 mashing razor clam meat by using a high-speed tissue masher, adding distilled water for homogenate, precisely weighing homogenate according to the material-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 alkaline protease for enzymolysis for a plurality of hours, wherein the enzymolysis conditions are as follows: the enzymolysis temperature is 35-50 ℃, the pH value is 8-10.4, the enzymolysis time is 2-8 hours, and the enzyme adding amount is 300-1200U/g of razor clam meat. Inactivating enzyme at 100 deg.C for 15min, centrifuging at 4 deg.C for 15min (10000r/min), and collecting supernatant.

2.2 optimization of alkaline proteolysis Process

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. The alkaline protease factors and levels are shown in table 1:

TABLE 1 factors and levels of alkaline protease

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, quickly putting the cell strain into a constant-temperature water bath kettle at 37 ℃ for melting, putting the cell strain into a sterile working chamber 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, adding 4mL of nutrient solution, and repeatedly blowing and beating the cell strain 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) 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 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:

2.2.3, the results of the alkaline protease hydrolysis are shown in Table 2, and it can be seen that the IR value is the largest in experiment No. 15, and therefore the optimum conditions for the alkaline protease enzymatic hydrolysis are: enzymolysis temperature: 50 ℃, enzymolysis pH value: 9.6, ratio of material to liquid: 1:1, time: 8h, enzyme addition: 300U/g.

TABLE 2 results of orthogonal alkaline protease hydrolysis tests

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 is eluted with G-25 to obtain 6 peaks, and the peak 4 has the highest antitumor activity after the in vitro antitumor experiment screening, so that the peak 4 is collected, freeze-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; zorbax SB-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 FIG. 2, as can be seen from FIG. 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 Val-Pro-Trp-Asp-Thr-Asp; as shown in FIG. 3, the molecular weight of ESI/MS detection was 731.7Da ([ M + H ]]⁺731.7Da) to obtain the desired razor clam active hexapeptide.

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: study on anti-PC-3 cell proliferation activity of razor clam active hexapeptide

The MTT method was used to compare the effect of razor clam active hexapeptide on PC-3 cell proliferation at different concentrations and different action times using F12 medium as a control group, and the results are shown in FIG. 4, which shows that razor clam active hexapeptide can effectively inhibit PC-3 proliferation and is concentration and time dependent.

The main materials and reagents used in the above examples:

the razor clam is purchased from the Nanzhen market in Zhoushan, 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, Sartorius AG, 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

1. A solen active hexapeptide has an amino acid sequence as follows: Val-Pro-Trp-Asp-Thr-Asp, ESI/MS molecular weight 731.7 Da.

2. A method for preparing the solen active hexapeptide according to claim 1, comprising the steps of:

(1) removing shells of the fresh razor clams, taking out the fresh razor clams, cleaning, mashing, adding water according to the feed-liquid ratio of 1:1 for tissue homogenization, and adjusting the pH value of homogenate to 9.6 for later use;

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. An application of the razor clam active hexapeptide as defined in claim 1 in preparing an anti-prostate cancer drug.