CN114835780A

CN114835780A - Oligopeptide MEDEI separated from chilli seeds and application thereof in preventing or treating cancers

Info

Publication number: CN114835780A
Application number: CN202210744911.XA
Authority: CN
Inventors: 赵靓; 廖小军; 王凤章; 王永涛; 饶雷
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2022-06-29
Filing date: 2022-06-29
Publication date: 2022-08-02
Anticipated expiration: 2042-06-29
Also published as: CN114835780B

Abstract

The oligopeptide MEDEI separated from the chilli seeds is separated from the chilli seeds, has an anti-tumor effect, can particularly effectively inhibit the growth and metabolism of HepG2 cells, and has a good application prospect.

Description

Oligopeptide MEDEI separated from chilli seeds and application thereof in preventing or treating cancers

Technical Field

The present invention relates to the field of biology. In particular, the invention relates to oligopeptide MEDEI separated from chilli seeds and application thereof in preventing or treating cancers.

Background

Capsicum, a plant of the genus Capsicum of the family Solanaceae, is native to Mexico, introduced into China, and is now mainly grown in Sichuan, Yunnan, Guizhou, Hunan, Henan, etc. The capsicum mainly comprises capsicum pulp and capsicum seeds, wherein the capsicum seeds account for 30-60% of the dry weight of the capsicum and are main byproducts in the capsicum processing process. The chilli seeds contain rich dietary fiber, fat, protein, mineral substances, vitamin E and the like, and have excellent nutritional and economic values.

With the rapid development of the pepper processing industry in China, peppers are applied to the fields of food, medicine, chemical industry and the like, pepper products such as dried peppers, pepper sauce, pepper powder, hotpot condiment and the like in the food field are more and more, capsaicin is widely applied to medicine, and a large amount of waste pepper seeds can be generated in the pepper processing process. At present, a small part of pepper seeds in China are used as animal feed, but most of the pepper seeds are not effectively utilized and treated as waste, so that environmental pollution and resource waste are caused. The pepper seeds have high yield, low price and easy acquisition, and the cake protein has complete amino acids and moderate essential amino acid content, thereby being a high-quality protein resource. Therefore, the bioactive peptide extracted from the chilli seeds can change waste into valuable, improve the resource utilization rate, reduce pollution and meet the development target of an environment-friendly society.

At present, antitumor drugs such as fluorouracil, cisplatin, daunorubicin and the like play an important role in tumor treatment, but the safety, the effectiveness, the high price and the like of the drugs limit the clinical application of the drugs. More and more studies have shown that bioactive peptides from natural foods or animals and plants have a positive effect on human health. The physiological activity function of the bioactive peptide is mainly embodied in the aspects of antibiosis, antivirus, antioxidation, antitumor, blood sugar reduction, blood pressure reduction, cholesterol reduction, immunoregulation and the like, the bioactive peptide of animal source has the problems of high cost, safety and the like, the bioactive peptide of plant source is widely applied due to pure nature and high nutritional value, such as the bioactive peptide of soybean, the bioactive peptide of peanut, the bioactive peptide of rapeseed and the like, but the research on the bioactive peptide in the chilli seed is less.

Disclosure of Invention

The present invention aims to solve at least to some extent at least one of the technical problems of the prior art.

In one aspect of the invention, the invention features an isolated polypeptide. According to an embodiment of the invention, the isolated oligopeptide has the amino acid sequence as shown in SEQ ID NO: 1 or a functional analogue thereof, and specifically, the amino acid sequence of the isolated oligopeptide is MEDEI (Met-Glu-Asp-Glu-IlE, SEQ ID NO: 1). The oligopeptide is separated from pepper seeds and the function of the oligopeptide is researched, so that the oligopeptide has an anti-tumor effect, particularly can effectively inhibit the growth and metabolism of HepG2 cells, and has a good application prospect.

According to an embodiment of the present invention, the isolated oligopeptide is derived from capsicum seeds. The inventor extracts protein in pepper seeds and unexpectedly discovers the oligopeptide with the anti-tumor effect.

In another aspect of the invention, the invention features a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes an isolated oligopeptide as described above. The nucleic acid molecules according to the embodiments of the present invention, after introduction into recipient cells, express the aforementioned isolated oligopeptides having an antitumor effect in an environment suitable for protein expression.

In the present invention, the sequence of the nucleic acid molecule is not strictly limited as long as it encodes the isolated oligopeptide described above.

In yet another aspect of the invention, the invention features a construct. According to an embodiment of the invention, the construct comprises a nucleic acid molecule as described above. After the constructs according to the embodiments of the present invention are introduced into cells, the isolated oligopeptides described above are expressed under an environment suitable for protein expression, which helps to exert the antitumor effect of the oligopeptides.

In yet another aspect of the invention, the invention features a recombinant cell. According to an embodiment of the invention, the recombinant cell comprises a nucleic acid molecule as described above or a construct as described above. Thus, the aforementioned oligopeptide is expressed under an environment suitable for protein expression, and contributes to the antitumor effect of the oligopeptide. The recombinant cells of the invention do not comprise germ cells, fertilized egg cells or embryonic cells.

In yet another aspect of the invention, a medicament or food product is provided. According to an embodiment of the invention, the medicament or food product comprises: an isolated oligopeptide, nucleic acid molecule or recombinant cell as hereinbefore described. Thus, the drug or food according to the embodiment of the present invention has an effect of preventing or treating cancer.

In a further aspect of the invention, the invention provides the use of an oligopeptide, nucleic acid molecule or recombinant cell as hereinbefore described in the manufacture of a medicament or a food product. According to an embodiment of the present invention, the medicament or food is for preventing or treating cancer.

According to an embodiment of the present invention, the cancer is breast cancer, lung cancer, nasopharyngeal cancer, liver cancer, stomach cancer, esophageal cancer, colorectal cancer, pancreatic cancer, melanoma, skin cancer, prostate cancer, cervical cancer, leukemia, thyroid cancer, lymphoma, bladder cancer, kidney cancer, uterine body cancer, ovarian cancer, gall bladder cancer, oral cancer, laryngeal cancer, bone cancer, testicular cancer, or brain cancer.

In yet another aspect of the invention, the invention provides a method for obtaining the isolated oligopeptides described above. According to an embodiment of the invention, the method comprises: and (3) processing the pepper seeds to obtain the separated oligopeptide.

According to an embodiment of the invention, the method comprises: (1) crushing the chilli seeds to obtain chilli seed meal; (2) degreasing the pepper seed meal to obtain degreased pepper seed meal; (3) extracting protein in the degreased pepper seed meal to obtain a crude protein extract; (4) carrying out enzymolysis treatment on the crude protein extract, and then inactivating enzyme to obtain an enzymolysis protein solution; (5) and separating and purifying the enzymolysis protein solution to obtain the separated oligopeptide.

In the step (1), the pepper seeds are crushed so as to better perform subsequent reaction, so that the reaction is more sufficient, and the yield and the purity of the oligopeptide are improved. In the step (2), grease in the pepper seed meal can be removed through degreasing treatment, so that the extraction rate and the purity of the protein are improved. In the step (4), the proteolysis treatment can effectively decompose the protein into small molecular peptides, which is helpful for obtaining oligopeptides with anticancer function.

According to the embodiment of the invention, in the step (1), the crushed material obtained by crushing is sieved by a sieve of 60-100 meshes to obtain a trapped material and a permeated material, and the permeated material is collected to obtain the pepper seed meal. Therefore, the pepper seed powder can permeate through the membrane, impurities can be intercepted, the protein extraction rate is improved, and the influence of the impurities is avoided.

According to the embodiment of the invention, the degreasing solvent used in the degreasing treatment is n-hexane. Thus, the fat can be effectively removed, and the use is safe.

According to an embodiment of the present invention, step (3) comprises: and mixing the degreased pepper seed meal with water, adjusting the pH value of the obtained mixed solution to 9-10 by using alkali liquor, reacting for 3-5 hours, adjusting the pH value of the reaction solution to 4-5, reacting for 1-3 hours, centrifuging the reaction solution, and collecting precipitates to obtain a crude protein extract. The pH value is firstly high because the protein in the pepper seed meal can be dissolved out under the alkaline environment, then the pH value is reduced because the protein can be settled at the isoelectric point of the protein, and the separated protein can be obtained after centrifugation.

According to an embodiment of the present invention, the crude protein extract is subjected to an ultra-high pressure treatment in advance before the enzymatic treatment in step (4). The ultra-high pressure treatment can improve the physicochemical property of protein, and the activity of the product after ultra-high pressure and enzymolysis is higher than that of the product only subjected to enzymolysis.

According to the embodiment of the invention, the pressure of the ultrahigh pressure treatment is 100-400 MPa, and the time is 20-40 minutes. Therefore, the protein is beneficial to improving the physicochemical property of the protein and improving the activity of the product obtained by enzymolysis.

According to the embodiment of the invention, the temperature of the enzymolysis treatment is 30-50 ℃, the time is 1-5 hours, the pH value is 7-10, the enzyme adopted by the enzymolysis treatment is selected from alkaline protease, preferably bacillus licheniformis, and the mass ratio of the enzyme to the crude protein is 1: 20-1: 50. Thereby, the protein is decomposed into small molecular peptide, so that the anticancer oligopeptide can be obtained.

According to the embodiment of the invention, the enzyme deactivation is carried out for 1-10 min at 90-100 ℃. Thereby, to inactivate the enzyme, prevent over-reaction or prevent the enzyme from interfering with subsequent experiments.

According to the embodiment of the invention, in the step (5), the separation and purification are performed by using a chromatographic column, wherein the chromatographic column is an anion chromatographic column or a hydrophobic chromatographic column; when the chromatographic column is an anion chromatographic column, the adopted mobile phase is water and NaCl; when the chromatographic column is a hydrophobic chromatographic column, the adopted mobile phase is water and 40-60 v/v% methanol solution.

According to an embodiment of the present invention, in the step (5), the separation and purification includes: injecting the enzymolysis protein solution into an anion chromatographic column, eluting with a mobile phase, and collecting an effluent liquid within 35-45 min; injecting the effluent into a hydrophobic chromatographic column, eluting by using a mobile phase, and collecting the effluent within 75-90 min so as to obtain a purified product containing the oligopeptide.

The inventors have conducted extensive experiments to obtain the above-mentioned preferable separation and purification method, and thus, the isolated oligopeptide can be obtained.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 shows a schematic diagram of the analysis of the effect of different peptides on HepG2 cell proliferation according to one embodiment of the present invention.

Detailed Description

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1

In this example, the oligopeptide MEDEI in the pepper seeds was extracted as follows:

1) removing seeds: separating fresh pepper seed meat to obtain pepper seeds;

2) crushing: grinding the chilli seeds into chilli seed meal, and sieving the chilli seed meal with a 80-mesh sieve to obtain chilli seed powder;

3) degreasing: mixing the pepper seed meal and normal hexane according to a ratio of 1:10 (g/ml), stirring overnight for degreasing, and removing the normal hexane by adopting a suction filtration mode after degreasing is finished to obtain pepper seed meal;

4) protein extraction: dissolving the degreased pepper seed meal in water at a ratio of 1:10 (w/v, unit g/mL), adjusting the pH value of the solution to 9.5 by using NaOH solution, dissolving for 4 h, adjusting the pH value of the solution to 4.5 by using HCl, precipitating for 2 h, centrifuging the reaction solution at 8000 rpm for 20min, and collecting precipitate to obtain a crude protein extract;

5) ultra-high pressure auxiliary enzymolysis: dissolving the protein isolate in water, carrying out ultrahigh pressure treatment for 30min under the pressure of 300 MPa, and then carrying out enzymolysis treatment on a product obtained by the ultrahigh pressure treatment, wherein the enzyme is bacillus licheniformis, and the mass ratio of the enzyme to the substrate is 1:20 (w/w, unit g/g), 40 ℃, and adjusting the pH value to 8 by using 1mol/L NaOH for 3 hours;

6) enzyme deactivation: after enzymolysis is finished, inactivating enzyme for 10 minutes at 90 ℃ to obtain chilli seed zymolyte solution;

7) and (3) separating and purifying zymolyte: and (2) enabling the chilli seed zymolyte solution to pass through A DEAE anion chromatographic column, collecting eluent in A35-45 min time period by using deionized water and NaCl as mobile phases, then separating and purifying by using an ODS-A reversed-phase C18 column (A hydrophobic column), collecting eluent in A75-90 min time period by using deionized water and 50% methanol as the mobile phases. And carrying out mass spectrum identification analysis on the peptide segments in the obtained eluent to obtain a plurality of pieces of peptide sequence information.

Example 2

The peptide sequence obtained by mass spectrometric analysis of example 1 was chemically synthesized to give a synthetic peptide. The influence of each peptide on the proliferation of HepG2 cells is respectively researched, and the specific steps are as follows:

1) HepG2 cell culture: HepG2 cells were derived from ATCC cell bank, and HepG2 cells were cultured in DMEM medium containing 10% FBS in a growth environment of 5% carbon dioxide cell incubator at 37 ℃. At 25cm ² Culturing the cells in a culture flask, carrying out passage when the cells grow to the density of 70-90%, and inoculating the cells into a 96-well plate.

2) Peptide fragment treatment: after the cells in the 96-well plate are cultured for 24 hours, the original DMEM culture medium in the wells is sucked out, DMEM containing peptide segments is added into each well, the concentration of the peptide segments is 0.1, 0.3 and 0.6mM, and the culture is continued for 24 hours.

3) The MTT method is used for measuring the cell proliferation rate: mu.L of MTT was added to each well of the 96-well plate at a concentration of 5mg/mL, after 4 hours of incubation, the liquid was aspirated from each well, 150. mu.L of DMSO was added to each well, and the absorbance was measured after 20min of reaction.

As shown in fig. 1, it can be seen that oligopeptide MEDEI has a better HepG2 cell inhibition rate than other oligopeptides, and is helpful for preventing or treating liver cancer.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

SEQUENCE LISTING

<110> university of agriculture in China

<120> oligopeptide MEDEI isolated from capsicum seed and its use in preventing or treating cancer

<130> BI3220848

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> 1

<400> 1

Met Glu Asp Glu Ile

1 5

Claims

1. An isolated oligopeptide, wherein the amino acid sequence of the isolated oligopeptide is SEQ ID NO: 1.

2. the isolated oligopeptide according to claim 1, wherein the isolated oligopeptide is derived from capsicum seeds.

3. A nucleic acid molecule encoding the isolated oligopeptide of claim 1 or 2.

4. A construct comprising the nucleic acid molecule of claim 3.

5. A recombinant cell comprising the nucleic acid molecule of claim 3 or the construct of claim 4.

6. A medicament or food product, comprising: the isolated oligopeptide of claim 1 or 2, the nucleic acid molecule of claim 3 or the recombinant cell of claim 5.

7. Use of the isolated oligopeptide according to claim 1 or 2, the nucleic acid molecule according to claim 3 or the recombinant cell according to claim 5 in the preparation of a medicament or a foodstuff for the prevention or treatment of cancer.

8. The use according to claim 7, wherein the cancer is breast cancer, lung cancer, nasopharyngeal cancer, liver cancer, stomach cancer, esophageal cancer, colorectal cancer, pancreatic cancer, melanoma, skin cancer, prostate cancer, cervical cancer, leukemia, thyroid cancer, lymphoma, bladder cancer, kidney cancer, uterine body cancer, ovarian cancer, gallbladder cancer, oral cancer, laryngeal cancer, bone cancer, testicular cancer, or brain cancer.

9. A method for obtaining an isolated oligopeptide according to claim 1 or 2, comprising:

and (3) processing the pepper seeds to obtain the separated oligopeptide.

10. The method of claim 9, comprising:

(1) crushing the chilli seeds to obtain chilli seed powder;

(2) degreasing the chilli seed powder to obtain degreased chilli seed meal;

(3) extracting protein in the degreased pepper seed meal to obtain a crude protein extract;

(4) carrying out enzymolysis treatment on the crude protein extract, and then inactivating enzyme to obtain an enzymolysis protein solution;

(5) and separating and purifying the enzymolysis protein solution to obtain the separated oligopeptide.

11. The method according to claim 10, wherein in the step (1), the crushed material obtained by crushing is sieved by a sieve of 60-100 meshes to obtain a retentate and a permeate, and the permeate is collected to obtain the pepper seed meal;

in the step (2), a degreasing solvent adopted in the degreasing treatment is n-hexane;

the step (3) comprises the following steps: mixing the degreased pepper seed meal with water, adjusting the pH value of the obtained mixed solution to 9-10 by using alkali liquor, reacting for 3-5 hours, adjusting the pH value of the reaction solution to 4-5, reacting for 1-3 hours, centrifuging the reaction solution, and collecting precipitates to obtain a crude protein extract;

before the enzymolysis treatment in the step (4), performing ultrahigh pressure treatment on the crude protein extract in advance;

the pressure of the ultrahigh pressure treatment is 100-400 MPa, and the time is 20-40 minutes;

the temperature of the enzymolysis treatment is 30-50 ℃, the time is 1-5 hours, and the pH value is 7-10;

the enzyme adopted by the enzymolysis treatment is selected from alkaline protease, and the mass ratio of the enzyme to the crude protein is 1: 20-1: 50;

the enzyme deactivation is carried out for 1-10 min at the temperature of 90-100 ℃;

in the step (5), the separation and purification are carried out by adopting a chromatographic column, wherein the chromatographic column is an anion chromatographic column and a hydrophobic chromatographic column; when an anion chromatographic column is used, the mobile phase used comprises water and NaCl; when a hydrophobic chromatographic column is adopted, the adopted mobile phase comprises water and 40-60 v/v% methanol solution.

12. The method according to claim 10 or 11, wherein the enzyme used in the enzymatic treatment is selected from the group consisting of bacillus licheniformis;

in the step (5), the separation and purification comprises:

injecting the enzymolysis protein solution into an anion chromatographic column, eluting with a mobile phase, and collecting an effluent liquid within 35-45 min;

injecting the effluent into a hydrophobic chromatographic column, eluting by using a mobile phase, and collecting the effluent within 75-90 min so as to obtain a purified product containing the oligopeptide.