CN107574205B - Preparation method of homoarginine mixed peptide and application of homoarginine mixed peptide in treatment of gastric cancer - Google Patents
Preparation method of homoarginine mixed peptide and application of homoarginine mixed peptide in treatment of gastric cancer Download PDFInfo
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Abstract
The invention relates to a mixed peptide with high arginine content and application thereof in treating gastric cancer. The preparation method comprises the following steps: pretreating the walnut pulp and egg white protein suspension by using ultrahigh pressure, and then performing stepwise enzymolysis on pretreated products by using alkaline protease and papain under the auxiliary action of ultrasonic waves and microwaves; separating the target mixed peptide in the clear liquid after the enzymolysis liquid is filtered by a reversed-phase high performance liquid chromatography; the mixed peptide is used as a template molecule, acrylic acid and methacrylic acid are used as functional monomers, triethylene glycol dimethacrylate is used as a cross-linking agent, isopropyl thioxanthone is used as an initiator, and ultraviolet initiation polymerization is carried out to form a surface molecularly imprinted membrane which is used for separating and enriching target mixed peptide in enzymolysis clear liquid. The content of arginine in the mixed peptide is more than 18 percent; MTT (methyl thiazolyl tetrazolium) detection shows that the homoarginine mixed peptide can effectively inhibit proliferation of human gastric cancer SGC-7901 cells. The method is helpful for reducing the product cost and accelerating the realization of industrial production.
Description
Technical Field
The invention relates to a preparation method of homoarginine mixed peptide and application thereof in gastric cancer treatment, in particular to a method for preparing mixed peptide rich in arginine by taking walnut pulp and egg white protein as raw materials and application thereof in gastric cancer treatment.
Background
Cancer is a general term for malignant tumors and is characterized by loss of normal regulatory control of body cells, hyperproliferation, and various degrees of dysdifferentiation, often invading adjacent tissues or metastasizing to distant sites. It seriously threatens the health of the whole human body and is the first cause of abnormal death. The global cancer report issued by the World Health Organization (WHO)2014 shows that the newly added cancer cases in china in 2012 are the first worldwide, and the newly added cancer cases and the death number of the stomach cancer are the first worldwide. In 2015, 16 departments such as national defense council and national improvement committee jointly issued "three-year cancer prevention and treatment plan in 2015 and 2017" clearly indicates that the 8 cancers with the highest incidence and greatest harm are important in cancer prevention and treatment in China, and gastric cancer is one of the eight cancers in China.
For malignant tumors, conventional treatment methods include surgical resection, chemotherapy, radiotherapy, and the like. These methods work to some extent to treat tumors, but once local spread or potential metastasis of cancer cells occurs, treatment often ends with failure. In addition, these traditional therapies inevitably cause different degrees of damages to normal cells and autoimmune systems of human bodies, which cause adverse reactions such as vomiting, inappetence, headache, insomnia, alopecia, ulcer, erosion, inflammation, hemogram reduction and the like, and patients often cannot receive treatment continuously because the bodies of the patients are hard to bear, thereby seriously affecting the life quality of cancer patients.
Modern scientific researches find that a plurality of natural bioactive peptides have good effects of inhibiting the proliferation and the metastasis of tumor cells, and have high selectivity and small toxic and side effects. The active peptides can achieve the purpose of treating or assisting in treating malignant tumors by inducing tumor differentiation, inhibiting tumor cell growth, inhibiting tumor angiogenesis, improving the sensitivity of tumor cells to drugs, reducing chemotherapy injury, enhancing the killing power of drugs to tumor cells, interfering the DNA synthesis of tumor cells, improving the immunity of organisms and the like. In recent years, researches have continuously proved that the increment arginine has obvious effects of inhibiting tumor cells and inducing apoptosis. Polyamine is active in metabolism in tumor cells, polyamine is essential for rapid division and proliferation of the tumor cells, and the increment of arginine can inhibit biosynthesis of polyamine by inhibiting ornithine decarboxylase activity, so that proliferation of cancer cells is inhibited; arginine generates NO after the action of nitric oxide synthase, and the NO can block the energy metabolism and DNA replication of tumor cells or cause the DNA damage of the tumor cells, thereby inhibiting the growth of the tumor cells or causing the death of the tumor cells; meanwhile, NO can inhibit the expression of cell adhesion molecules so as to prevent the adhesion of cells, and the adhesion molecules play an important role in the process that tumor cells are separated from primary tissues and transferred to other parts to form new focuses; in addition, arginine can increase the production and function of T lymphocytes, increase the production of interleukin IL-2 in vivo and the expression of a receptor thereof, regulate and activate the activity of macrophages in vivo, thereby improving the autoimmunity of tumor hosts. Foreign studies have attempted to inhibit tumor growth or induce apoptosis in cancer patients by using unbalanced amino acid therapy to interfere with the metabolism and function of tumor cells by adjusting the content of certain amino acids in the tumor host. In addition, it has been reported that hydrophilic polypeptides rich in hydrophilic amino acids such as arginine can specifically act on tumor cells in an electrostatic attraction manner, causing rapid rupture of cell membranes and leakage of cell contents, and finally causing cell death; it is also known that basic amino acids (arginine, lysine, etc.) in the molecular structure of the polypeptide have certain influence on the antitumor activity of the polypeptide. These all provide a solid theoretical basis for the anti-cancer efficacy of the homoarginine mixed peptide of the present invention.
The walnut meal is a walnut processing byproduct, has high protein digestibility and rich and complete amino acid content, is mainly used in the feed industry in China, and causes resource depreciation due to insufficient deep processing; the egg white protein is not only rich in 8 essential amino acids of human body, but also has the composition close to that of human protein, and the human digestion utilization rate is extremely high. According to the invention, animal and plant mixed protein from eggs and walnuts is used as a raw material for the first time, and the active mixed peptide rich in arginine is prepared by means of ultrahigh pressure-ultrasonic-microwave assisted enzymolysis, surface molecular imprinting membrane separation and purification and the like, and is applied to gastric cancer treatment, so that the nutrition of a patient is supplemented, the cancer cell proliferation is inhibited, and the living quality of the gastric cancer patient is effectively improved. The application research related to the method is not reported so far.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a preparation method of a novel homoarginine mixed peptide and application of the homoarginine mixed peptide in treatment of gastric cancer. The mixed peptide is rich in arginine, and has strong effect of inhibiting gastric cancer cell proliferation.
In order to solve the technical problems, the invention provides the following technical scheme:
a preparation method of homoarginine mixed peptide comprises the following steps:
(1): uniformly mixing the degreased and crushed walnut meal, egg white protein and water, stirring, performing ultrahigh pressure pretreatment, performing ultrasonic-microwave assisted enzymolysis, heating to inactivate enzyme after the enzymolysis is finished, and performing plate-and-frame filter pressing to collect clear liquid;
(2): after the clear liquid is lyophilized, the target peptide fragment in the lyophilized coarse powder is separated by using reverse phase high performance liquid chromatography (RP-HPLC). Adopting Everest C18(4.6 multiplied by 250mm, 5 mu m, 238EV54) as a reverse phase column, acetonitrile water solution as a mobile phase, trifluoroacetic acid as an anion pair reagent, detecting the wavelength of 214nm, washing the chromatographic column with pure acetonitrile before loading, dissolving 25mg of freeze-dried powder in the mobile phase to a constant volume of 25mL, filtering with a 0.45 mu m microporous filter membrane, injecting the sample with a volume of 20 mu L, at a column temperature of 30 ℃, under the separation conditions of acetonitrile concentration of 18% (v/v), trifluoroacetic acid concentration of 0.09% (v/v), a flow rate of 1.0mL/min, a retention time of 9.64min, 11.36min and 13.80min, collecting three elution components, and obtaining the homoarginine mixed peptide powder after freeze-drying;
preferably, the preparation method of the homoarginine mixed peptide further comprises a step of separating and enriching the homoarginine mixed peptide by a surface blotting membrane, and the steps are as follows:
(3): immersing a cover glass and a glass slide by using piranha solution (concentrated sulfuric acid and 30% hydrogen peroxide in a volume ratio of 3: 1), ultrasonically cleaning for 1.5-2.5 h, cleaning by using pure water, and drying by using nitrogen for later use; soaking the cleaned cover glass in a homoarginine mixed peptide aqueous solution to obtain a mixed peptide immobilized template; soaking the cleaned glass slide in a methanol solution of 0.5-1.5% (v/v) 3-Aminopropyltriethoxysilane (APTES), oscillating at 20-40 rpm for 15-45 min, rinsing with methanol, and drying to obtain a silanized glass slide; uniformly mixing functional monomers of Acrylic Acid (AA), methacrylic acid (MAA) and a crosslinking agent of triethylene glycol dimethacrylate (TEGDMA), and adding a photoinitiator Isopropyl Thioxanthone (ITX) to obtain a prepolymerization mixed solution; introducing nitrogen into the pre-polymerization mixed solution, paving the pre-polymerization mixed solution on the surface of a silanization glass slide, rotating the glass slide, coating a mixed peptide immobilization template on the glass slide, soaking the glass slide in 8-12% (m/v) SDS (sodium dodecyl sulfate) and 8-12% (v/v) HAc solution after ultraviolet light initiated polymerization is completed, removing a cover glass, oscillating at 80-160 rpm for 4-8 h, and oscillating and rinsing with pure water to be neutral to obtain a high-arginine mixed peptide surface imprinted membrane;
(4): immersing the surface blotting membrane of the homoarginine mixed peptide obtained in the step (3) in the clear liquid obtained in the step (1), oscillating at 20-40 rpm for 1-6 h, taking out the blotting membrane adsorbed with the target peptide, immersing the blotting membrane in 0.5-1.8 mol/L NaCl solution, and performing ultrasonic treatment at 100-300W for 10-50 min to assist elution; collecting the eluent, removing NaCl through cation exchange resin, and performing low-temperature spray drying on the permeate to obtain homoarginine mixed peptide powder; rinsing the eluted surface blotting membrane by pure water, immersing the surface blotting membrane in the new clear liquid obtained in the step (1) again, and repeating the subsequent steps for separating the homoarginine mixed peptide.
Preferably, in the step (1), the degreased and crushed walnut pulp and egg white protein are mixed according to the mass ratio of 2.96-5: 1, the protein pulp mixture and water are uniformly mixed according to the mass-to-volume ratio of 1: 4-14, the mixture is stirred for 1.5-2.5 hours at room temperature and then placed in an ultrahigh pressure host system, the processing pressure is 200-600 MPa, and the pressure maintaining time is 10-30 min, so that an ultrahigh pressure pretreated suspension is obtained; controlling the temperature of the suspension to be 40-60 ℃, adjusting the pH value to 9-10, adding alkaline protease accounting for 2-6.5% of the mass of the suspension, uniformly stirring, simultaneously carrying out ultrasonic-microwave assisted enzymolysis with the ultrasonic power of 200-400W and the ultrasonic time of 10-20 min and the microwave power of 200-600W and the microwave time of 5-15 min, adjusting the pH value to 6-8 after carrying out enzymolysis for 1.5-2.5 h, adding papain accounting for 2-6.5% of the mass of the suspension, uniformly stirring, simultaneously carrying out ultrasonic-microwave assisted enzymolysis with the ultrasonic power of 200-400W and the ultrasonic time of 10-20 min, carrying out microwave power of 200-600W and the microwave time of 5-15 min, heating to inactivate enzyme after carrying out enzymolysis for 2-3 h, and collecting clear liquid after plate frame filter pressing.
Preferably, in the step (3), the cleaned cover glass is soaked in 2-12 g/L of the homoarginine mixed peptide aqueous solution, and after oscillation at 20-40 rpm for 4-8 h, the mixed peptide immobilized template is obtained by rinsing with pure water and drying in the air.
Preferably, in the step (3), functional monomers of Acrylic Acid (AA), methacrylic acid (MAA) and a cross-linking agent of triethylene glycol dimethacrylate (TEGDMA) are uniformly mixed according to the volume ratio of 1-4: 0.5-2.5: 4-9, and 0.2-0.8 volume of 1-4 mmol/L acetone solution of Isopropyl Thioxanthone (ITX) is added as a photoinitiator to obtain a prepolymerization mixed solution.
Preferably, in the step (3), nitrogen is introduced into the pre-polymerization mixed solution for 20-40 min, and then the pre-polymerization mixed solution is spread on the surface of a silanization glass slide fixed on a rotating machine, the rotating machine rotates at 100-400 rpm for 2-10 s, then the mixed peptide immobilized template covers the glass slide, and 365nm ultraviolet light initiates polymerization for 3-6 h.
Preferably, in the step (4), the adsorption-elution process is repeated for more than 10 times by using the homoarginine mixed peptide surface imprinted membrane.
The invention also provides the homoarginine mixed peptide which is prepared by the preparation method.
Preferably, the arginine content in the mixed peptide is more than 18%.
The invention also provides application of the homoarginine mixed peptide obtained by the preparation method in preparing health-care food, special dietary food, common food and medicines related to gastric cancer treatment.
The invention has the following beneficial effects:
from the aspect of efficacy, the homoarginine mixed peptide prepared by the invention can obviously inhibit the proliferation of human gastric cancer cells, and can delay the deterioration of gastric cancer by controlling the growth of tumors; one of the major problems facing cancer patients is nutritional impairment, the mixed peptides prepared according to the invention help to reduce the inhibition of absorption by free amino acids competing for a common absorption site during intestinal digestion, and a number of scientific studies have demonstrated that overall protein deposition is higher than the corresponding free amino acid uptake or intact protein uptake when the peptide form is used as a nitrogen source. The homoarginine mixed peptide disclosed by the invention can be used for inhibiting cancer cell proliferation while supplementing nutrition to patients, and can be used for effectively improving the life quality of gastric cancer patients.
From the safety perspective, the non-edible animal and plant hydrolyzed raw materials cannot be used as the source of the monomer amino acid as specified in GB 29922-2013 food safety national standard special medical application formula food general rule, and the high arginine mixed peptide prepared by the invention is derived from natural food materials such as walnut, egg and the like, and accords with the natural healthy nutrition concept which is advocated at present; the enzyme for hydrolysis also meets the relevant regulations in the national standard of food additive use in GB 2760-2014 food safety.
From the preparation perspective, the preparation method of the invention has mild conditions, and the related instruments are common equipment in food and medicine industry; the surface imprinted membrane has good adsorption-elution effect and high repeated utilization rate; the preparation method is beneficial to reducing the production cost, has expanded production potential and is beneficial to accelerating the realization of industrial mass production of products.
Drawings
FIG. 1 is a flow chart of the preparation of homoarginine mixed peptide of the present invention;
FIG. 2 is a high performance liquid chromatogram of the homoarginine mixed peptide of the invention;
FIG. 3 is a schematic diagram of the preparation of the homoarginine mixed peptide surface imprinted membrane of the present invention;
FIG. 4 is an atomic force microscope image of an immobilized template of homoarginine mixed peptide of the present invention;
FIG. 5 is a scanning electron microscope image of the imprinted membrane on the surface of the homoarginine mixed peptide of the present invention;
FIG. 6 shows the reusability of the imprinted membrane on the surface of homoarginine-containing peptide.
Detailed Description
The invention is further illustrated by the following specific examples, which are intended to be purely exemplary of the invention and are not intended to be limiting.
Example 1:
the preparation method of the homoarginine mixed peptide comprises the following steps:
1. mixing degreased and crushed walnut pulp with egg white protein according to a mass ratio of 4:1, uniformly mixing the protein pulp mixture with water according to a mass-volume ratio of 1:8, stirring at room temperature for 2 hours, placing in an ultrahigh pressure host system, and carrying out treatment under the pressure of 400MPa for 20min to obtain ultrahigh pressure pretreated suspension; controlling the temperature of the suspension to 50 ℃, adjusting the pH value to 9, adding alkaline protease accounting for 3.5% of the mass of the suspension, uniformly stirring, simultaneously carrying out ultrasonic-microwave assisted enzymolysis with the ultrasonic power of 300W for 12min, the microwave power of 400W and the microwave time of 8min, adjusting the pH value to 7 after 2h of enzymolysis, adding papain accounting for 3.5% of the mass of the suspension, uniformly stirring, simultaneously carrying out ultrasonic-microwave assisted enzymolysis with the ultrasonic power of 300W for 15min, the microwave power of 400W and the microwave time of 10min, heating up to inactivate enzymes after 2.5h of enzymolysis, carrying out plate and frame filter pressing, and collecting clear liquid. The protein content is 93.6%, and the peptide content is 88.5%;
2. after the clear liquid is lyophilized, the target peptide fragment in the lyophilized coarse powder is separated by using reverse phase high performance liquid chromatography (RP-HPLC). Adopting Everest C18(4.6 multiplied by 250mm, 5 mu m, 238EV54) as a reverse phase column, acetonitrile water solution as a mobile phase, trifluoroacetic acid as an anion pair reagent, detecting the wavelength of 214nm, washing the chromatographic column with pure acetonitrile before loading, dissolving 25mg of freeze-dried powder in the mobile phase, fixing the volume to 25mL, filtering with a 0.45 mu m microporous filter membrane, injecting the sample with the volume of 20 mu L, the column temperature of 30 ℃, separating conditions of acetonitrile concentration of 18% (v/v), trifluoroacetic acid concentration of 0.09% (v/v), the flow rate of 1.0mL/min, the retention time of 9.64min, 11.36min and 13.80min, collecting three elution components, obtaining the high arginine mixed peptide powder after freeze-drying, wherein the target peptide content accounts for 23.6% of the coarse powder;
3. immersing the glass slides (cover glass and glass slide) in piranha solution (concentrated sulfuric acid and 30% hydrogen peroxide in a volume ratio of 3: 1), ultrasonically cleaning for 2h, cleaning with pure water, and drying with nitrogen for later use; soaking the cleaned cover glass in 5g/L homoarginine mixed peptide aqueous solution, oscillating at 20rpm for 6h, rinsing with pure water and drying to obtain a mixed peptide immobilized template; soaking the cleaned glass slide in a methanol solution of 1% (v/v) 3-Aminopropyltriethoxysilane (APTES), oscillating at 20rpm for 30min, rinsing with methanol, and air drying to obtain a silanized glass slide; uniformly mixing functional monomers of Acrylic Acid (AA), methacrylic acid (MAA) and a cross-linking agent of triethylene glycol dimethacrylate (TEGDMA) according to a volume ratio of 2:1:7, adding 0.5 volume of acetone solution of 2.5mmol/L Isopropyl Thioxanthone (ITX) as a photoinitiator, and obtaining a prepolymerization mixed solution; introducing nitrogen into the pre-polymerization mixed solution for 30min, spreading the pre-polymerization mixed solution on the surface of a silanization glass slide fixed on a rotating machine, rotating the rotating machine at 200rpm for 4s, covering a mixed peptide immobilized template on the glass slide, and carrying out 365nm ultraviolet light initiated polymerization for 4 h; soaking the polymerized glass slide in 10% (m/v) SDS (sodium dodecyl sulfate) 10% (v/v) HAc solution, removing the cover glass, oscillating at 120rpm for 6h, and oscillating and rinsing with pure water to be neutral to obtain a homoarginine mixed peptide surface imprinted membrane;
4. immersing the imprinted membrane on the surface of the homoarginine mixed peptide obtained in the step 3 in the clear liquid obtained in the step 1, oscillating at 20rpm for 4h, taking out the imprinted membrane adsorbed with the target peptide, immersing the imprinted membrane in 1mol/L NaCl solution, performing ultrasonic treatment at 200W for 30min to assist elution, removing NaCl from the eluate through cation exchange resin, and performing low-temperature spray drying on the permeate to obtain homoarginine mixed peptide powder. The adsorption rate of the surface imprinted membrane is 80.5% (see table 1), and the arginine content of the mixed peptide obtained by spray drying is 21.3% (see table 2); rinsing the eluted surface blotting membrane with pure water, immersing the surface blotting membrane in the new clear liquid obtained in the step 1 again, repeating the subsequent steps for separating the homoarginine mixed peptide, recycling for 20 times, and ensuring that the regeneration rate is 91.1 percent (see figure 6);
5. the adsorption rate of the target peptide in the clear liquid after the surface blotting membrane is subjected to filter pressing on the plate frame is measured by adopting a high performance liquid chromatography for three times, and the calculation formula is as follows: the adsorption rate (%) — target peptide content in eluate/target peptide content in clear solution × 100%, the results are shown in table 1:
TABLE 1 adsorption rate of high arginine mixed peptide surface blotting membrane
After the high arginine mixed peptide surface imprinted membrane is repeatedly utilized for 20 times through the adsorption-elution-re-adsorption processes, the adsorption quantity is reduced from the initial 7.38mg/g to 6.72mg/g, the regeneration rate reaches 91.1 percent, and excellent reusability is shown.
6. The content of each amino acid after acidolysis of the homoarginine mixed peptide is detected by an amino acid analyzer, and the result is shown in table 2:
TABLE 2 analysis of homoarginine mixed peptide amino acids
Note: tryptophan, asparagine and glutamine are destroyed after acidolysis.
Example 2:
the preparation method of the mixed peptide comprises the following steps:
1. mixing degreased and crushed walnut pulp with egg white protein according to the mass ratio of 3:1, uniformly mixing the protein pulp mixture with water according to the mass-volume ratio of 1:6, stirring at room temperature for 1.5h, placing in an ultrahigh pressure host system, and treating at 300MPa for 15min to obtain ultrahigh pressure pretreated suspension; controlling the temperature of the suspension to 50 ℃, adjusting the pH value to 9, adding alkaline protease accounting for 3% of the mass of the suspension, uniformly stirring, simultaneously carrying out ultrasonic-microwave assisted enzymolysis with the ultrasonic power of 200W for 15min, the microwave power of 300W and the microwave time of 10min, adjusting the pH value to 7 after carrying out enzymolysis for 1.5h, adding papain accounting for 3% of the mass of the suspension, uniformly stirring, simultaneously carrying out ultrasonic-microwave assisted enzymolysis with the ultrasonic power of 200W and the ultrasonic time of 18min, the microwave power of 300W and the microwave time of 12min, heating up to inactivate enzyme after carrying out enzymolysis for 2h, and collecting clear liquid after plate and frame filter pressing. The protein content is 90.3%, and the peptide content is 81.7%;
2. after the clear liquid is lyophilized, the target peptide fragment in the lyophilized coarse powder is separated by using reverse phase high performance liquid chromatography (RP-HPLC). Adopting Everest C18(4.6 multiplied by 250mm, 5 mu m, 238EV54) as a reverse phase column, acetonitrile water solution as a mobile phase, trifluoroacetic acid as an anion pair reagent, detecting the wavelength of 214nm, washing the chromatographic column with pure acetonitrile before loading, dissolving 25mg of freeze-dried powder in the mobile phase, fixing the volume to 25mL, filtering with a 0.45 mu m microporous filter membrane, injecting the sample with the volume of 20 mu L, the column temperature of 30 ℃, separating conditions of acetonitrile concentration of 18% (v/v), trifluoroacetic acid concentration of 0.09% (v/v), the flow rate of 1.0mL/min, the retention time of 9.64min, 11.36min and 13.80min, collecting three elution components, obtaining the high arginine mixed peptide powder after freeze-drying, wherein the target peptide content accounts for 19.8% of the coarse powder;
3. immersing the glass slides (cover glass and glass slide) in piranha solution (concentrated sulfuric acid and 30% hydrogen peroxide in a volume ratio of 3: 1), ultrasonically cleaning for 1.5h, cleaning with pure water, and drying with nitrogen for later use; soaking the cleaned cover glass in 3g/L of homoarginine mixed peptide aqueous solution, oscillating at 30rpm for 4 hours, rinsing with pure water and drying in the air to obtain a mixed peptide immobilized template; soaking the cleaned glass slide in 0.8% (v/v) methanol solution of 3-Aminopropyltriethoxysilane (APTES), oscillating at 30rpm for 18min, rinsing with methanol, and air drying to obtain silanized glass slide; uniformly mixing functional monomers of Acrylic Acid (AA), methacrylic acid (MAA) and a cross-linking agent of triethylene glycol dimethacrylate (TEGDMA) according to a volume ratio of 1.5:0.8:7, and adding 0.3 volume of 3mmol/L acetone solution of Isopropyl Thioxanthone (ITX) as a photoinitiator to obtain prepolymerization mixed solution; introducing nitrogen into the pre-polymerization mixed solution for 20min, spreading the pre-polymerization mixed solution on the surface of a silanization glass slide fixed on a rotating machine, rotating the rotating machine at 100rpm for 6s, covering a mixed peptide immobilized template on the glass slide, and carrying out 365nm ultraviolet light initiated polymerization for 5 h; soaking the polymerized slide in 8% (m/v) SDS (sodium dodecyl sulfate) 8% (v/v) HAc solution, removing the cover glass, oscillating at 160rpm for 4h, and oscillating and rinsing with pure water to be neutral to obtain the homoarginine mixed peptide surface imprinted membrane;
4. immersing the surface imprinted membrane of the homoarginine mixed peptide obtained in the step 3 in the clear liquid obtained in the step 1, oscillating at 30rpm for 5h, taking out the imprinted membrane adsorbed with the target peptide, immersing in 0.8mol/L NaCl solution, simultaneously performing ultrasonic treatment at 100W for 40min to assist elution, removing NaCl from the eluent through cation exchange resin, and performing low-temperature spray drying on the permeate to obtain homoarginine mixed peptide powder. The adsorption rate of the surface imprinted membrane is 71.3%, the regeneration rate of the surface imprinted membrane after 20-time recycling is 84.2%, and the arginine content of the mixed peptide obtained by spray drying is 18.9%.
Example 3:
the preparation method of the mixed peptide comprises the following steps:
1. mixing degreased and crushed walnut meal particles with egg white protein according to a mass ratio of 5:1, uniformly mixing the protein meal mixture with water according to a mass-volume ratio of 1:10, stirring at room temperature for 2.5h, placing in an ultrahigh pressure host system, and treating at 500MPa for 25min to obtain an ultrahigh pressure pretreated suspension; controlling the temperature of the suspension to 50 ℃, adjusting the pH value to 9, adding alkaline protease accounting for 4% of the mass of the suspension, uniformly stirring, simultaneously carrying out ultrasonic-microwave assisted enzymolysis with the ultrasonic power of 400W and the ultrasonic time of 20min and the microwave power of 500W and the microwave time of 5min, adjusting the pH value to 7 after 2.5h of enzymolysis, adding papain accounting for 4% of the mass of the suspension, uniformly stirring, simultaneously carrying out ultrasonic-microwave assisted enzymolysis with the ultrasonic power of 400W and the ultrasonic time of 20min and the microwave power of 500W and the microwave time of 8min, heating up to inactivate enzyme after 3h of enzymolysis, carrying out plate and frame filter pressing, and collecting clear liquid. The protein content is 92.1%, and the peptide content is 84.7%;
2. after the clear liquid is lyophilized, the target peptide fragment in the lyophilized coarse powder is separated by using reverse phase high performance liquid chromatography (RP-HPLC). Adopting Everest C18(4.6 multiplied by 250mm, 5 mu m, 238EV54) as a reverse phase column, acetonitrile water solution as a mobile phase, trifluoroacetic acid as an anion pair reagent, detecting the wavelength of 214nm, washing the chromatographic column with pure acetonitrile before loading, dissolving 25mg of freeze-dried powder in the mobile phase to a constant volume of 25mL, filtering with a 0.45 mu m microporous filter membrane, injecting the sample with a volume of 20 mu L, at a column temperature of 30 ℃, under the separation conditions of acetonitrile concentration of 18% (v/v), trifluoroacetic acid concentration of 0.09% (v/v), a flow rate of 1.0mL/min, a retention time of 9.64min, 11.36min and 13.80min, collecting three elution components, obtaining the high arginine mixed peptide powder after freeze-drying, wherein the target peptide content accounts for 21.9% of the coarse powder;
3. immersing the glass slides (cover glass and glass slide) in piranha solution (concentrated sulfuric acid and 30% hydrogen peroxide in a volume ratio of 3: 1), ultrasonically cleaning for 2.5h, cleaning with pure water, and drying with nitrogen for later use; soaking the cleaned cover glass in 4g/L of homoarginine mixed peptide aqueous solution, oscillating at 40rpm for 8 hours, rinsing with pure water and drying in the air to obtain a mixed peptide immobilized template; soaking the cleaned glass slide in 1.2% (v/v) methanol solution of 3-Aminopropyltriethoxysilane (APTES), oscillating at 40rpm for 40min, rinsing with methanol, and air drying to obtain silanized glass slide; uniformly mixing functional monomers of Acrylic Acid (AA), methacrylic acid (MAA) and a cross-linking agent of triethylene glycol dimethacrylate (TEGDMA) according to a volume ratio of 3:1.5:8, adding 0.6 volume of 4mmol/L acetone solution of Isopropyl Thioxanthone (ITX) as a photoinitiator, and obtaining a prepolymerization mixed solution; introducing nitrogen into the pre-polymerization mixed solution for 40min, spreading the pre-polymerization mixed solution on the surface of a silanization glass slide fixed on a rotating machine, rotating the rotating machine at 300rpm for 3s, covering a mixed peptide immobilized template on the glass slide, and carrying out 365nm ultraviolet light initiated polymerization for 6 h; soaking the polymerized slide in 10% (m/v) SDS (sodium dodecyl sulfate) 8% (v/v) HAc solution, removing the cover glass, oscillating at 140rpm for 8h, and oscillating and rinsing with pure water to be neutral to obtain a homoarginine mixed peptide surface imprinted membrane;
4. immersing the surface imprinted membrane of the homoarginine mixed peptide obtained in the step 3 in the clear liquid obtained in the step 1, oscillating at 40rpm for 6h, taking out the imprinted membrane adsorbed with the target peptide, immersing the imprinted membrane in 1.2mol/L NaCl solution, simultaneously performing ultrasonic treatment at 300W for 20min to assist elution, removing NaCl from the eluent through cation exchange resin, and performing low-temperature spray drying on the permeate to obtain homoarginine mixed peptide powder. The adsorption rate of the surface imprinted membrane is 77.5%, the recycling rate of the surface imprinted membrane for 20 times is 88.0%, and the arginine content of the mixed peptide obtained by spray drying is 20.4%.
Example 4: MTT method is adopted to evaluate the inhibition effect of the homoarginine mixed peptide on the proliferation of human gastric cancer cells
1. Apparatus and materials
METERTECH Sigma 960 microplate reader is a product of Taiwan METERTECH company; (MCO-15AC) CO2The cell culture box is a product of Sanyo corporation of Japan; 1300SERIES A2 Biosafety Cabinet is product of Thermo Fisher corporation of America; the TDL-50B low-speed table centrifuge is a product of Shanghai' an pavilion scientific instrument factory; the D-1 type automatic steam sterilizing pot is a product of Beijing Fanyuko Kongmao Co.
Human gastric cancer SGC-7901 cells were purchased from cell banks of Chinese academy of sciences; RPMI-1640 medium was purchased from GIBCO, USA; fetal bovine serum was purchased from Sichuan Biochemical products, Inc., Tianjin; cell proliferation and cytotoxicity detection kits (MTT) were purchased from assist in san Francisco, Inc.; homoarginine mixed peptides were prepared as in example 1.
2. Experimental methods
Preparing 5X 10 of human gastric cancer SGC-7901 cells in logarithmic growth phase4Single cell suspension at individual/mL concentration was inoculated into a 96-well plate at 100. mu.L/well and placed at 37 ℃ in 5% CO2Culturing in an incubator for 24h, and then discarding the culture solution. The homoarginine mixed peptides (30mg/mL, 35mg/mL and 35 mg/mL) prepared in example 1 were added to the control group and the experimental group, respectively40mg/mL, diluted with RPMI-1640 medium), an equal volume of RPMI-1640 medium was added to the control group. And adding MTT working solution (5mg/mL and 10 muL/hole) after 24h of culture, absorbing 100 muL of supernatant along the upper part of the culture solution after 4h, adding 100 muL of formazan dissolving solution, measuring absorbance (wavelength of 570nm) by using an enzyme labeling instrument after continuing to culture for 4h, calculating the inhibition rate of the high-arginine mixed peptide on cell proliferation, and setting 4 repeat holes in each group.
Inhibition (%) (control group A)570Experimental group A570) Control group A570×100％
3. Results of the experiment
As shown in Table 3, after 3 high arginine mixed peptides with different concentrations (30mg/mL, 35mg/mL and 40mg/mL) act on SGC-7901 cells, the growth of the SGC-7901 cells is obviously inhibited, and the inhibition rate is increased along with the increase of the concentration of the mixed peptide.
Table 3 inhibition ratio of homoarginine mixed peptides to human gastric cancer SGC-7901 cells (n-4,
)
4. conclusion of the experiment
The homoarginine mixed peptide has stronger effect of inhibiting gastric cancer cell proliferation and is dose-dependent within a certain concentration range.
It should be understood that while the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein, and any combination of the various embodiments may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.
Claims (9)
1. A preparation method of homoarginine mixed peptide is characterized by comprising the following steps:
(1): uniformly mixing the degreased and crushed walnut meal, egg white protein and water, stirring, performing ultrahigh pressure pretreatment, performing ultrasonic-microwave assisted enzymolysis, heating to inactivate enzyme after the enzymolysis is finished, and performing plate-and-frame filter pressing to collect clear liquid; wherein the ultrasonic-microwave assisted enzymolysis comprises: adding alkaline protease accounting for 2-6.5% of the mass of the suspension into the suspension subjected to the ultrahigh pressure pretreatment, uniformly stirring, simultaneously performing ultrasonic-microwave assisted enzymolysis, adjusting the pH to 6-8 after the enzymolysis, adding papain accounting for 2-6.5% of the mass of the suspension, uniformly stirring, and simultaneously performing ultrasonic-microwave assisted enzymolysis;
(2): after the clear liquid is freeze-dried, separating a target peptide segment in the freeze-dried coarse powder by using a reversed-phase high performance liquid chromatography, adopting Everest C18 of 4.6 x 250mm, 5 mu m and 238EV54 as a reversed-phase column, using acetonitrile aqueous solution as a mobile phase, using trifluoroacetic acid as an anion pair reagent, detecting the wavelength of 214nm, washing the chromatographic column by using pure acetonitrile before sampling, dissolving 25mg of freeze-dried powder by using the mobile phase to fix the volume to 25mL, filtering by using a 0.45 mu m microporous membrane, carrying out sample injection with the volume of 20 mu L and the column temperature of 30 ℃, collecting three elution components under the separation conditions of acetonitrile concentration of 18% v/v, trifluoroacetic acid concentration of 0.09% v/v, the flow rate of 1.0mL/min, the retention time of 9.64min, 11.36min and 13.80min, and obtaining high-arginine mixed peptide powder after freeze-drying;
(3): immersing a cover glass and a glass slide in piranha solution of concentrated sulfuric acid and 30% hydrogen peroxide in a volume ratio of 3:1, ultrasonically cleaning for 1.5-2.5 h, cleaning with pure water, and drying with nitrogen for later use; soaking the cleaned cover glass in a homoarginine mixed peptide aqueous solution to obtain a mixed peptide immobilized template; soaking the cleaned glass slide in 0.5-1.5% v/v 3-aminopropyltriethoxysilane methanol solution, oscillating at 20-40 rpm for 15-45 min, rinsing with methanol, and drying to obtain a silanized glass slide; uniformly mixing functional monomers of acrylic acid, methacrylic acid and a crosslinking agent of triethylene glycol dimethacrylate, and adding a photoinitiator of isopropyl thioxanthone to obtain a prepolymerization mixed solution; introducing nitrogen into the pre-polymerization mixed solution, paving the pre-polymerization mixed solution on the surface of a silanization glass slide, rotating the glass slide, coating a mixed peptide immobilization template on the glass slide, soaking the glass slide in 8-12% m/v SDS (sodium dodecyl sulfate), namely 8-12% v/v HAc solution after ultraviolet light initiated polymerization is completed, removing a cover glass, oscillating at 80-160 rpm for 4-8 h, and oscillating and rinsing pure water to be neutral to obtain a high-arginine mixed peptide surface imprinted membrane;
(4): immersing the surface blotting membrane of the homoarginine mixed peptide obtained in the step (3) in the clear liquid obtained in the step (1), oscillating at 20-40 rpm for 1-6 h, taking out the blotting membrane adsorbed with the target peptide, immersing the blotting membrane in 0.5-1.8 mol/L NaCl solution, and performing ultrasonic treatment at 100-300W for 10-50 min to assist elution; collecting the eluent, removing NaCl through cation exchange resin, and performing low-temperature spray drying on the permeate to obtain homoarginine mixed peptide powder; rinsing the eluted surface blotting membrane by pure water, immersing the surface blotting membrane in the new clear liquid obtained in the step (1) again, and repeating the subsequent steps for separating the homoarginine mixed peptide.
2. The preparation method of homoarginine mixed peptide according to claim 1, wherein in step (1), the defatted and pulverized walnut meal and egg white protein are mixed according to the mass ratio of 2.96-5: 1, the protein meal mixture and water are uniformly mixed according to the mass-to-volume ratio of 1: 4-14, the mixture is stirred for 1.5-2.5 hours at room temperature and then placed in an ultrahigh pressure host system, the treatment pressure is 200-600 MPa, and the pressure maintaining time is 10-30 min, so that an ultrahigh pressure pretreated suspension is obtained; controlling the temperature of the suspension to be 40-60 ℃, adjusting the pH value to 9-10, adding alkaline protease accounting for 2-6.5% of the mass of the suspension, uniformly stirring, simultaneously carrying out ultrasonic-microwave assisted enzymolysis with the ultrasonic power of 200-400W and the ultrasonic time of 10-20 min and the microwave power of 200-600W and the microwave time of 5-15 min, adjusting the pH value to 6-8 after carrying out enzymolysis for 1.5-2.5 h, adding papain accounting for 2-6.5% of the mass of the suspension, uniformly stirring, simultaneously carrying out ultrasonic-microwave assisted enzymolysis with the ultrasonic power of 200-400W and the ultrasonic time of 10-20 min, carrying out microwave power of 200-600W and the microwave time of 5-15 min, heating to inactivate enzyme after carrying out enzymolysis for 2-3 h, and collecting clear liquid after plate frame filter pressing.
3. The preparation method according to claim 1, wherein in the step (3), the washed cover glass is soaked in 2-12 g/L of the high arginine mixed peptide aqueous solution, and after oscillation at 20-40 rpm for 4-8 h, the mixed peptide immobilized template is obtained by rinsing with pure water and drying.
4. The preparation method according to claim 1, wherein in the step (3), the functional monomers of acrylic acid, methacrylic acid and the cross-linking agent of triethylene glycol dimethacrylate are uniformly mixed according to the volume ratio of 1-4: 0.5-2.5: 4-9, and 0.2-0.8 volume of 1-4 mmol/L acetone solution of isopropyl thioxanthone is added as a photoinitiator to obtain a prepolymerization mixed solution.
5. The preparation method according to claim 1, wherein in the step (3), the pre-polymerization mixture is introduced with nitrogen for 20-40 min and then spread on the surface of a silanized glass slide fixed on a rotating machine, the mixed peptide immobilized template is covered on the glass slide after the rotating machine rotates at 100-400 rpm for 2-10 s, and 365nm ultraviolet light initiates polymerization for 3-6 h.
6. The method according to claim 1, wherein in step (4), the adsorption-elution process is repeated more than 10 times for the homoarginine mixed peptide surface imprinted membrane.
7. An homoarginine mixed peptide produced by the production method according to any one of claims 1 to 6.
8. The homoarginine hybrid peptide of claim 7, wherein the arginine content of the hybrid peptide is greater than 18%.
9. The use of the homoarginine hybrid peptide according to claim 7 for the preparation of a medicament for the treatment of gastric cancer.
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| CN1799395A (en) * | 2005-12-30 | 2006-07-12 | 深圳职业技术学院 | Blood pressure lowering polypeptide of corn gluten powder, its separation method and application |
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| CN1799395A (en) * | 2005-12-30 | 2006-07-12 | 深圳职业技术学院 | Blood pressure lowering polypeptide of corn gluten powder, its separation method and application |
| CN101775103A (en) * | 2009-12-29 | 2010-07-14 | 湖北工业大学 | Preparation method of protein molecule engram film |
| CN102212595A (en) * | 2011-04-20 | 2011-10-12 | 江南大学 | Preparation method and application of water-soluble nano grain polysaccharide |
| CN103109971A (en) * | 2013-03-04 | 2013-05-22 | 四川省均易润泽食品有限公司 | Method for preparing homoarginine walnut peptide |
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