Silkworm chrysalis protein anti-tumor active peptide as well as separation and screening method and application thereof

Abstract

The invention discloses silkworm pupa protein anti-tumor active peptide as well as a separation and screening method and application thereof. Silkworm chrysalis is dried, smashed and degreased to obtain degreased silkworm chrysalis powder; the preparation method comprises the following steps: dissolving degreased silkworm chrysalis meal into a NaOH solution, stirring, extracting, centrifuging, collecting supernate, adjusting the pH value, centrifuging, collecting protein precipitate, washing the protein precipitate, and freeze-drying to obtain protein powder; adding deionized water into the protein powder, performing ultrasonic oscillation to recover room temperature, adding papain, adjusting pH, stirring for enzymolysis, boiling for quenching, cooling, centrifuging at 4 DEG C, collecting supernatant, freeze-drying to obtain polypeptide powder, separating, collecting and detecting the anti-tumor cell proliferation activity of zymolyte; under the same condition, the zymolyte with the highest antitumor activity is further purified by adopting Sephadex-G15 gel chromatography, the antitumor cell proliferation activity of each component is collected and detected, the active peptide with the best antitumor effect is obtained by screening according to the inhibition ratio, and the active peptide is identified as YASPVH according to the amino acid sequence.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to a silkworm chrysalis protein anti-tumor active peptide as well as a separation and screening method and application thereof. BACKGROUND

[0002] Silkworm chrysalis is considered as a high-quality source of animal protein, and the protein content can reach 50-60%. Silkworm chrysalis protein contains 18 kinds of amino acids, of which 8 kinds of essential amino acids for human body account for 44.1% of all amino acids, and is easy to absorb and utilize, and is regarded as ideal protein. Silkworm chrysalis has many functional properties that can be used in the food and pharmaceutical industries. Silkworm chrysalis has high nutritional value and high safety for eating, and is a natural resource treasure house for the preparation of drugs, food additives and functional foods. In recent years, a variety of bioactive compounds with potential functions and health benefits have been found in silkworm chrysalis. At present, there is no related record of screening and separating anti-tumor active peptides from silkworm chrysalis protein.

[0003] In industrial production, the preparation methods of peptides include acid-base hydrolysis method, alcohol dissolution method, enzyme hydrolysis method and ionic liquid method. Among them, the silkworm chrysalis protein extraction device cost is high by acid-base method, and the high pH will denature the protein and affect its quality, and the large amount of alkali used will pollute the environment; the alcohol dissolution method is easy to denature silkworm chrysalis protein; the ionic liquid method has the advantages of simple process, no pollution and relatively mild conditions, but the degree of hydrolysis is difficult to control; and the enzyme hydrolysis method for extracting silkworm chrysalis protein has low cost, reduces the use of strong acid and strong alkali, reduces the harm to the environment, meets the requirements of green process, and is widely used.

[0004] Anti-tumor peptide is a kind of bioactive peptide with tumor inhibitory effect. Anti-tumor peptide can kill tumor cells specifically at different stages of tumor cell growth and development by inhibiting tumor cell proliferation and destroying tumor cell membrane or tumor blood vessels. Anti-tumor peptide has many advantages in treating tumors compared with other drugs, such as small molecular weight, simple structure, high activity, high selectivity, fewer side effects, multiple administration methods and less drug resistance. Therefore, the research on extracting and purifying anti-tumor peptides from food sources has become a research hotspot in the field of medical biology at home and abroad. However, there is no related report on preparing anti-tumor peptides from silkworm chrysalis protein. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a silkworm chrysalis protein anti-tumor active peptide and a separation and screening method and application thereof. The separation and screening method of the silkworm chrysalis protein anti-tumor active peptide is simple and efficient, can track the activity of the enzyme hydrolysis product to achieve directional enzyme cutting, obtain active polypeptides with specific peptide chain length, and enable the anti-tumor activity to be efficiently realized, and avoid cost waste, thereby providing a theoretical basis for the research and development of high-efficiency and low-toxicity anti-tumor drugs.

[0006] To solve the prior art problems, the present application adopts the following technical solutions:

[0007] A silkworm chrysalis protein antitumor peptide, the amino acid sequence of the silkworm chrysalis protein antitumor peptide is YASPVH, and the silkworm chrysalis protein antitumor peptide is derived from a silkworm chrysalis.

[0008] The separation and screening method of the silkworm chrysalis protein antitumor peptide comprises the following steps:

[0009] S1: The silkworm chrysalis is dried and crushed, and defatted to obtain defatted silkworm chrysalis powder;

[0010] S2: The defatted silkworm chrysalis powder is dissolved in a NaOH solution, stirred and extracted, centrifuged to collect the supernatant, and the pH value is adjusted, and the protein precipitate is collected by centrifugation, washed, and freeze-dried to obtain protein powder;

[0011] S3: The protein powder is added to deionized water, ultrasonically shaken to restore room temperature, and papain is added, the pH value is adjusted, and the enzyme is hydrolyzed by stirring, boiled to quench, cooled, centrifuged at 4 DEG C to collect the supernatant, and freeze-dried to obtain polypeptide powder, and the amount of papain added is 6%-10% of the mass of the substrate protein powder;

[0012] S4: The polypeptide powder is separated by Sephadex-G100 column, and the antitumor cell proliferation activity of each enzyme extract is collected and detected; under the same conditions, the enzyme extract with the highest antitumor activity is further purified by Sephadex-G15 gel chromatography, and the antitumor cell proliferation activity of each component is collected and detected, and the active peptide with the best antitumor effect is screened according to the inhibition rate;

[0013] S5: The amino acid sequence of the active peptide with the best antitumor effect screened in S4 is identified by Nano-LC-MS / MS, and the amino acid sequence of the silkworm chrysalis protein antitumor peptide is YASPVH.

[0014] As an improvement, the silkworm chrysalis dried and crushed in S1 is defatted by treating with petroleum ether.

[0015] As an improvement, the ratio of the defatted powder to the NaOH aqueous solution in S2 is 1:10, and the concentration of NaOH is 0.25 mol / L.

[0016] As an improvement, the extraction is carried out by constant temperature water bath stirring at 55 DEG C for 3h, 4000xg centrifugation for 15min, and the obtained supernatant is adjusted to pH 4.5 with 1 mol / L HCl.

[0017] As an improvement, the mass concentration of the protein powder in the aqueous solution in S3 is 1%; the ultrasonic oscillation is constant oscillation at 55 DEG C for 10 min; and the pH is adjusted to 10.0 by using a 0.25 mol / L NaOH solution.

[0018] As an improvement, the enzymolysis conditions in S3 are pH 10, temperature 55 DEG C, and enzymolysis time 5 h.

[0019] As an improvement, the Sephadex-G100 column in S4 is 2.5*70 cm, and the separation and purification conditions are: elution with deionized water, elution flow rate 1.0 ml / min, and detection wavelength 280 nm; and the Sephadex-G15 gel chromatography separation and purification conditions are: elution with deionized water, elution flow rate 1.0 ml / min, and detection wavelength 280 nm.

[0020] The application of the silkworm chrysalis protein active peptide in the preparation of an antitumor drug.

[0021] As an improvement, the tumor cells include, but are not limited to, breast cancer HCC-1937, breast cancer MCF-7 cells, gastric cancer BGC-823 cells, cervical cancer HeLa cells, liver cancer HepG-2 cells, and lung cancer A549 cells.

[0022] Beneficial effects

[0023] The present application is based on the high toxicity and side effects and high cost of existing antitumor drugs, and is committed to finding a natural antitumor regulator. The present application takes silkworm chrysalis as a starting point, focuses on the process control of papain enzymolysis, and prepares active polypeptides with specific peptide chain lengths, so that the antitumor activity can be efficiently realized. The enzymolysis technology adopted by the present application is simple and efficient, can track the activity of the enzymolysis product to achieve directional enzyme cutting, avoids cost waste, and the obtained polypeptide has antitumor activity. The inhibition rate of polypeptide YASPVH with a concentration of 10 mg / mL on HCC1937 after 24 hours of action is 65%, and the inhibition activity IC50 value of the enzymolysis product on human normal liver cells is greater than 1000 mg / mL, indicating that the polypeptide YASPVH has superior tumor inhibition effect and small side effects, and provides a theoretical basis for the research and development of high-efficiency and low-toxicity antitumor drugs. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Sephadex-G15 elution diagram (left) of papain enzymolysis product and antiproliferative activity (right) of each component after 24 h of action;

[0025] Figure 2 The first mass spectrum (top) and the second mass spectrum (bottom) of component II of the papain enzymolysis product;

[0026] Figure 3 For the analysis of the content of YASPVH in component II. DETAILED DESCRIPTION

[0027] The following examples further illustrate the content of the present application, but should not be construed as limiting the present application. Modifications and substitutions of the methods, steps or conditions of the present application, which do not depart from the spirit and essence of the present application, are within the scope of the present application. If not specifically indicated, the technical means used in the examples are the conventional means well known to those skilled in the art.

[0028] The above-mentioned separation and screening method of the silkworm chrysalis protein anti-tumor peptide is carried out according to the following steps:

[0029] S1: The silkworm chrysalis is dried at 60°C, crushed, and then degreased by adding petroleum ether, with a material-to-liquid ratio of 1:4, constant temperature water bath at 50°C for 4h, centrifugation at 3000xg for 10min, and the precipitate is dried at 60°C, repeated twice.

[0030] S2: The degreased silkworm chrysalis powder is dissolved in a 0.25mol / L NaOH solution to prepare a 10% mass fraction suspension, constant temperature water bath stirring extraction at 55°C for 3h, centrifugation at 4000xg for 15min, the supernatant is collected, the obtained supernatant is adjusted to pH 4.5 with 1mol / L HCl, centrifugation at 4000xg for 15min, the precipitate is collected, and freeze-dried to obtain the crude silkworm chrysalis protein.

[0031] S3: The crude silkworm chrysalis protein is dissolved in deionized water with a concentration of 1.0% (w / v). The amount of protease added is 8% of the mass of the substrate protein. The enzyme hydrolysis conditions are as follows: alkaline protease at pH: 8, 55°C for 2h; neutral protease at pH: 10, 50°C for 5h; papain at pH: 10, 55°C for 5h; and rennet at pH: 8, 30°C for 3.5h. After the enzyme hydrolysis reaction is completed, the reaction is terminated by boiling for 10min, centrifugation at 4000xg for 15min, and finally the supernatant is collected and vacuum freeze-dried to obtain four kinds of silkworm chrysalis protein hydrolysates.

[0032] S4: The four kinds of silkworm chrysalis protein hydrolysates are respectively dissolved in deionized water, centrifuged, the supernatant is collected and filtered with a 0.22μm filter membrane, the filtrate is separated by a Sephadex-G100 column (2.5x70cm), the column is equilibrated with distilled water, the flow rate is 1.0ml / min, the detection wavelength is 280nm, the elution components corresponding to each absorption peak are collected, vacuum dried, and the anti-tumor activity is detected.

[0033] Among them, the human five cancer cell lines A549, HCT-116, HCC-1937, MCF-7, HepG-2 and HeLa purchased from Shanghai Shengong Bioengineering Co., Ltd. and human normal liver cells L02 were selected, and the anti-tumor activity of silkworm chrysalis protein anti-tumor active peptide was determined by MTT method (tetramethyl azo salt colorimetric method). All cell strains were cultured in a incubator with 5% CO2 concentration, 37°C and saturated humidity. The specific experimental steps are as follows:

[0034] First, collect the cells in the logarithmic growth phase, adjust the cell concentration by cell counter, then inoculate the cells in the 96-well plate, add 100 μL of cell suspension in the logarithmic growth phase to each well (2000 cells per well), incubate the cells in the 96-well plate in a cell incubator at 37°C and 5% CO2 overnight, after the cells are completely adherent, dilute the anti-tumor peptide YASPVH to the required maximum concentration of 10 mg / mL with 5% fetal bovine serum medium, then perform drug treatment by half dilution, set three replicate wells for each concentration and set a blank control, incubate for 24 h, then add 10 μL (5 mg / mL) of MTT to the 96-well plate, continue to incubate for 4 h, then remove the supernatant, add 100 μL of dimethyl sulfoxide (DMSO) to each well, shake well to dissolve the formazan crystals, and then detect the absorbance by enzyme labeler. Perform 3 independent repeated experiments to ensure the reliability of the experimental results, and the results are shown in Table 1.

[0035] Table 1 IC50 of different enzyme hydrolysates after 24 h 50 (mg / mL)

[0036]

[0037] Note: The data in Table 1 are the average values of three experimental results, and the subsequent numerical values represent the standard deviation.

[0038] As can be seen from the table, the papain enzyme hydrolysate has the best anti-proliferative activity on breast cancer HCC-1937 cells.

[0039] S5: The papain enzyme hydrolysate was purified by Sephadex-G15 gel chromatography, the column was equilibrated by distillation, the flow rate was 1.0 ml / min, the detection wavelength was 280 nm, the elution components corresponding to each absorption peak were collected, vacuum dried, and the anti-tumor activity of each component was detected. As shown in Figure 1 , the proliferation inhibition rate of component II on breast cancer HCC-1937 cells was as high as 65%.

[0040] S6: After vacuum freeze-drying of component II, Nano-LC-MS / MS mass spectrometry was performed for identification, and the total ion flow diagram is as shown in Figure 2As shown in the figure, one of the amino acid sequences is YASPVH (capital letters represent single-letter amino acid code), which is the silkworm chrysalis protein anti-tumor active peptide.

[0041] Example 2

[0042] The test was performed according to the method of Example 1, and the tested tumor cells were gastric cancer BGC-823 (purchased from Shanghai Shenguo Bioengineering Co., Ltd.), which was treated with the papain vehicle prepared in Example 1. The results showed that the IC50 value of BGC-823 treated for 24 hours was 8.08±0.52. 50

[0043] In summary, the enzymatic technology adopted by the present application is simple and efficient, and can track the activity of the enzymatic product to achieve directional enzyme cutting, thereby avoiding cost waste. The obtained polypeptide has anti-tumor activity, and the inhibition rate of polypeptide YASPVH with a concentration of 10 mg / mL on HCC1937 after 24 hours of action reached 65%. Moreover, the inhibition activity IC50 value of the enzymatic product on human normal liver cells was greater than 1000 mg / mL, indicating that the polypeptide YASPVH has superior tumor inhibition effect and small side effects, thereby providing a theoretical basis for the research and development of high-efficiency and low-toxicity anti-tumor drugs.

[0044] The above only describes the preferred embodiments of the present application, and any equivalent changes and modifications made within the scope of the patent application of the present application shall be included in the scope of the present application.​

Claims

1. A silkworm pupa protein anti-tumor peptide, characterized in that: The amino acid sequence of the silkworm pupa protein anti-tumor peptide is YASPVH, and the silkworm pupa protein anti-tumor peptide is derived from silkworm pupae.

2. A method for separating and screening silkworm pupa protein anti-tumor peptides according to claim 1, characterized in that: The following steps are involved: S1: drying and crushing silkworm pupae, and defatting to obtain defatted silkworm pupae powder; S2: dissolving the defatted silkworm pupa powder in a NaOH solution, stirring and extracting, collecting the supernatant after centrifugation, adjusting the pH value, collecting the protein precipitate by centrifugation, washing the protein precipitate, and freeze-drying it to obtain protein powder; S3: Add deionized water to the protein powder, ultrasonically shake and return to room temperature, then add papain, adjust the pH, stir and hydrolyze, boil and quench, cool, centrifuge at 4°C, collect the supernatant, and freeze-dry to obtain polypeptide powder. The amount of papain added is 6%-10% of the mass of the substrate protein powder; S4: Separate the peptide powder using a Sephadex-G100 column, collect and test the anti-tumor cell proliferation activity of each hydrolyzate; under the same conditions, the hydrolyzate with the highest anti-tumor activity is further purified using Sephadex-G15 gel chromatography, and the anti-tumor cell proliferation activity of each component is collected and tested, and the active peptide with the best anti-tumor effect is screened based on the inhibition rate; S5: Nano-LC-MS / MS was used to identify the amino acid sequence of the active peptide with the best anti-tumor effect screened in S4, and the amino acid sequence of the silkworm pupa protein anti-tumor peptide was confirmed to be YASPVH.

3. The separation and screening method according to claim 2, wherein The silkworm pupae described in S1 are dried, crushed and defatted, and then treated with petroleum ether.

4. The separation and screening method according to claim 2, wherein The material-liquid ratio of the defatted powder and the NaOH aqueous solution in S2 is 1:10; the concentration of NaOH is 0.25 mol / L.

5. The separation and screening method according to claim 2, wherein The extraction in S2 is performed by stirring in a constant temperature water bath at 55° C. for 3 h, centrifuging at 4000×g for 15 min, and adjusting the pH of the obtained supernatant to 4.5 with 1 mol / L HCl.

6. The separation and screening method according to claim 2, wherein The mass concentration of the protein powder in the aqueous solution in S3 is 1%; the ultrasonic oscillation is constant temperature oscillation at 55° C. for 10 minutes; and the pH is adjusted to 10.0 using a 0.25 mol / L NaOH solution.

7. The separation and screening method according to claim 2, wherein The enzymatic hydrolysis conditions in S3 are pH 10, temperature 55° C., and enzymatic hydrolysis time 5 h.

8. The separation and screening method according to claim 2, wherein The Sephadex-G100 column described in S4 is 2.5×70 cm, and the conditions for separation and purification are: elution with deionized water, elution flow rate of 1.0 ml / min, and detection wavelength of 280 nm; the conditions for separation and purification of the Sephadex-G15 gel chromatography are: elution with deionized water, elution flow rate of 1.0 ml / min, and detection wavelength of 280 nm.

9. Use of the silkworm pupa protein active peptide according to claim 1 in the preparation of anti-tumor drugs.

10. The use according to claim 9, characterized in that: The tumor cells include but are not limited to breast cancer HCC-1937 cells, breast cancer MCF-7 cells, gastric cancer BGC-823 cells, cervical cancer HeLa cells, liver cancer HepG-2 cells, and lung cancer A549 cells.

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