CN118255839A - Preparation and application of marine bioactive peptide and derivatives thereof - Google Patents
Preparation and application of marine bioactive peptide and derivatives thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Diabetes (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Obesity (AREA)
- Endocrinology (AREA)
- Emergency Medicine (AREA)
- Hematology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses preparation and application of marine bioactive peptide and derivatives thereof, and relates to the technical field of marine organisms. The preparation method adopts enzymolysis technology to hydrolyze sea cucumber fragments, and then prepares marine bioactive peptide with the amino acid sequence of Asp-Pro-Leu-Ala-Val-Pro through separation and purification; the prepared marine bioactive peptide has good anti-fatigue, anticancer, blood sugar reducing and immune activities, and has wide application prospect in the field of marine bio-pharmaceuticals.
Description
Technical Field
The invention belongs to the technical field of marine organisms, and particularly relates to preparation and application of a marine bioactive peptide and a derivative thereof.
Background
The marine organism diversity is about half of the global species, is a huge repository of natural molecules with biological activity, provides abundant material resources for novel compounds, and from the current research situation, the marine active peptides are mainly derived from a few marine organisms such as sponge, sea squirt, conopsis, sea anemone, starfish, seaweed, sea hare and the like, and the marine organism resources are not fully developed and utilized yet. Sea cucumber is an important marine invertebrate, the survival of which has been 5000 or more years, and is mainly distributed in the temperate zone and the tropical zone of the world, the most variety and the largest resource amount in the region of the Indian ocean and the western Pacific ocean are adopted, and modern scientific researches prove that the sea cucumber contains more than 50 nutritional ingredients beneficial to the physiological activities of human bodies, wherein the protein content is extremely high. If the sea cucumber protein is degraded into sea cucumber peptide, the sea cucumber peptide is easier to digest and absorb, and has high biological potency and no antigenicity.
The sea cucumber peptide is an active peptide extracted from sea cucumber and having a special physiological function, and can be a small peptide consisting of 2-12 amino acids or a polypeptide with larger molecular weight. The biological utilization rate is high, and the protein is easier to absorb than amino acid, and is not possessed by original protein and amino acid composed of the original protein. It is reported that sea cucumber peptide has various physiological functions of reducing blood pressure, resisting fatigue, improving immunity, resisting tumor, delaying aging and the like. However, the research on the correlation between the distribution of the sea cucumber peptide amino acid and the physiological activity of the sea cucumber peptide amino acid is less, so that the correlation between the functions and the components of the product is vague, the quality standard of the product with scientific basis is difficult to formulate, and the deeper research and the product research and development are more limited. The sea cucumber peptide is prepared by an enzymolysis technology, and the amino acid sequence and the biological activity of the sea cucumber peptide are researched, so that the sea cucumber peptide has wider prospect in the aspects of functional food, cosmetics, new medicine development and the like.
Disclosure of Invention
The invention aims to provide a preparation and application of a marine bioactive peptide and a derivative thereof, and the marine bioactive peptide prepared by the method has good anti-fatigue, anticancer, blood sugar reducing and immunity performance and has wide application prospect in the field of marine bio-pharmaceuticals.
The technical scheme adopted by the invention for achieving the purpose is as follows:
a method for preparing a marine bioactive peptide, comprising: the marine bioactive peptide is obtained by performing enzymolysis, separation and purification on sea cucumber fragments by bromelain; aucubin is also added in the enzymolysis process.
The invention provides a preparation method of marine bioactive peptide, sea cucumber is an important marine food resource, has higher nutritive value, and is a key way for realizing full utilization of sea cucumber resources by converting sea cucumber protein into sea cucumber peptide. According to the invention, sea cucumber fragments are used as raw materials, bromelain is added for hydrolysis reaction, aucubin is added in the hydrolysis process, so that the enzyme activity is further promoted, the enzymolysis efficiency is improved, and the obtained enzymolysis liquid is separated and purified, so that the prepared marine bioactive peptide has good anti-fatigue, anticancer, blood sugar reducing and immune activities, has a wide application prospect in the pharmaceutical field, and provides reference for application of the sea cucumber peptide in the fields of functional foods, medicines and the like.
Specifically, the preparation method of the marine bioactive peptide comprises the following steps:
Drying sea cucumber fragments, and pulverizing for use; adding a certain amount of treated sea cucumber fragments into deionized water with pH adjusted to 7-7.4, adding bromelain and aucubin for enzymolysis, performing enzymolysis at 40-45deg.C for 3.5-4.5 hr, boiling to deactivate enzyme activity, centrifuging, collecting supernatant, adding cold ethanol at volume ratio of 1:3-5 for precipitation overnight, centrifuging, dialyzing the supernatant with dialysis bag, lyophilizing, separating and purifying to obtain marine bioactive peptide.
For the invention, the mass-volume ratio of the sea cucumber fragments to the deionized water is 1g:25-30mL; the mass ratio of the sea cucumber fragments to the bromelain is 1:0.03-0.05; the mass ratio of the sea cucumber fragments to aucubin is 1:0.02-0.03.
For the purposes of the present invention, the amino acid sequence of the abovementioned marine bioactive peptide is Asp-Pro-Leu-Ala-Val-Pro.
The invention also discloses a modification method of the marine bioactive peptide, which comprises the following steps: the modified marine bioactive peptide is prepared by chemically modifying the marine bioactive peptide by using bicyclo [2.2.2] oct-5-ene-2, 3-dicarboxylic anhydride. The invention adopts the grafting reaction of anhydride in bicyclo [2.2.2] oct-5-ene-2, 3-dicarboxylic anhydride and amino of marine bioactive peptide to chemically modify the marine bioactive peptide, and the prepared modified marine bioactive peptide has obviously enhanced bioactivity and better anti-fatigue, anticancer, blood sugar reducing and immunity performance.
Specifically, the modification method of the marine bioactive peptide comprises the following steps: weighing marine bioactive peptide, dissolving in 0.05-0.06M phosphoric acid buffer solution, slowly adding dimethyl sulfoxide solution containing bicyclo [2.2.2] oct-5-ene-2, 3-dicarboxylic anhydride 0.4-0.6M, stirring in water bath at 25-30deg.C for 2-2.5 hr under pH7-7.4, and separating and purifying by dialysis to obtain modified marine bioactive peptide.
For the invention, the mass-volume ratio of the marine bioactive peptide to the phosphate buffer solution is 6g:1-2mL; the mass ratio of the marine bioactive peptide to the bicyclo [2.2.2] oct-5-ene-2, 3-dicarboxylic anhydride is 1:1-1.3.
The invention also discloses the modified marine bioactive peptide obtained by the modification method.
The invention also discloses application of the marine bioactive peptide in preparing a medicament for treating cancer and reducing blood sugar.
The invention also discloses application of the modified marine bioactive peptide in preparing a medicament for treating cancer and reducing blood sugar.
The beneficial effects of the invention include:
The invention provides a preparation method of marine bioactive peptide, sea cucumber fragments are used as raw materials, bromelain and aucubin are adopted to carry out hydrolysis reaction, and then the obtained marine bioactive peptide is further separated and purified, so that the prepared marine bioactive peptide has good anti-fatigue, anticancer, blood sugar reducing and immunity enhancing performances; the invention also provides a modification method of the marine bioactive peptide, which adopts bicyclo [2.2.2] oct-5-ene-2, 3-dicarboxylic anhydride to carry out chemical modification on the marine bioactive peptide, and the prepared modified marine bioactive peptide has better anti-fatigue, anticancer, blood sugar reducing and immunity performance.
The invention provides a preparation method and application of a marine bioactive peptide and a derivative thereof, and the marine bioactive peptide prepared by the method has good anti-fatigue, anticancer, blood sugar reducing and immunity performance, and has wide application prospects in the field of marine biopharmaceuticals.
Drawings
FIG. 1 is a Sephadex G-50 column chromatography chromatogram;
FIG. 2 is a graph showing the results of fatigue resistance testing of the components after Sephadex G-50 chromatography purification;
FIG. 3 is a high performance liquid chromatogram of the F3 component;
FIG. 4 is an infrared spectrum of marine bioactive peptides and modified marine bioactive peptides;
FIG. 5 is a graph showing the results of fatigue resistance test of marine bioactive peptides and modified marine bioactive peptides;
FIG. 6 is a graph showing the results of the blood glucose-lowering performance test of the marine bioactive peptide and the modified marine bioactive peptide;
FIG. 7 is a graph showing the results of anticancer performance test of marine bioactive peptides and modified marine bioactive peptides;
FIG. 8 shows the results of the immunopotentiation test of the marine bioactive peptide and the modified marine bioactive peptide.
Detailed Description
The technical scheme of the invention is further described in detail below with reference to the specific embodiments and the attached drawings:
Example 1:
A method for preparing marine bioactive peptide, comprising the following steps:
Drying sea cucumber fragments, and pulverizing for use; adding a certain amount of treated sea cucumber fragments into deionized water with pH adjusted to 7, adding bromelain and aucubin for enzymolysis, performing enzymolysis at 40 ℃ for 3.5 hours, boiling for enzyme deactivation, centrifuging, taking supernatant, adding cold ethanol according to a volume ratio of 1:3 for precipitation overnight, centrifuging, dialyzing the obtained supernatant with a 500Da dialysis bag and a 3000Da dialysis bag, and lyophilizing, wherein the obtained enzymolysis solution is subjected to Sephadex G-50 column chromatography, and high performance liquid chromatography separation and purification to obtain marine bioactive peptide; wherein the mass volume ratio of the sea cucumber fragments to the deionized water is 1g to 25mL; the mass ratio of the sea cucumber fragments to the bromelain is 1:0.05; the mass ratio of the sea cucumber fragments to aucubin is 1:0.02.
The specific operation steps of Sephadex G-50 column chromatography and high performance liquid chromatography separation and purification are as follows:
Sephadex G-50 column chromatography: preparing the obtained enzymolysis liquid freeze-dried sample into a solution of 3mg/mL by using distilled water, separating and purifying by using a Sephadex G-50 column, measuring absorbance at a position of 220nm at a flow rate of 10mL/h by using distilled water as eluent, separating 4 peaks F1, F2, F3 and F4 in total, collecting each elution peak component as shown in figure 1, measuring the fatigue resistance of each elution peak component, collecting a component F3 with higher fatigue resistance as shown in figure 2, and freeze-drying for later use.
High performance liquid chromatography separation and purification: f3 freeze-dried sample is prepared into a sample with the concentration of 2mg/mL by using 45wt% acetonitrile water solution containing 0.8wt% trifluoroacetic acid, and the sample injection amount is 10 mu L; chromatograph: agillent1100 high performance liquid chromatograph, chromatographic column: TSK-gel G3000 PWxl column (7.8X30 cm), detection wavelength 220nm, mobile phase: phase a-ultrapure water (containing 0.8wt% trifluoroacetic acid), phase B-45 wt% acetonitrile in water (containing 0.8wt% trifluoroacetic acid), flow rate: 0.5mL/min, column temperature: 40 ℃; elution flow: 0-20min:95% A+5% B;20-25min:40% A+60% B;25-30min:100% b; eluting to obtain chromatographic peak shown in figure 3, collecting chromatographic peak A, and lyophilizing; and (3) carrying out amino acid sequence analysis on the collected component A by adopting an amino acid automatic analyzer to obtain the marine bioactive peptide with the amino acid sequence Asp-Pro-Leu-Ala-Val-Pro.
Example 2:
The preparation method of the marine bioactive peptide is the same as that of the example 1.
The marine bioactive peptide prepared by the method is modified, and specifically comprises the following steps:
Weighing marine bioactive peptide, dissolving in 0.05M phosphoric acid buffer solution, slowly adding dimethyl sulfoxide solution containing bicyclo [2.2.2] oct-5-ene-2, 3-dicarboxylic anhydride 0.6M, stirring in water bath at 25deg.C for 2 hr under pH7, and separating and purifying by dialysis to obtain modified marine bioactive peptide; wherein the mass volume ratio of the marine bioactive peptide to the phosphate buffer solution is 6g to 1mL; the mass ratio of the marine bioactive peptide to the bicyclo [2.2.2] oct-5-ene-2, 3-dicarboxylic anhydride is 1:1.
Example 3:
And (3) scanning the sample by using a Fourier transform infrared spectrometer through KBr tabletting, wherein the scanning range is 4000-500cm -1, the scanning times are 32, and the instrument resolution is 0.5cm -1.
The above-described test was performed on the marine bioactive peptide prepared in example 1 and the modified marine bioactive peptide prepared in example 2, and the results are shown in fig. 4. As can be seen from FIG. 4, there is a characteristic absorption peak of C=C bond at 1682cm -1, indicating that bicyclo [2.2.2] oct-5-ene-2, 3-dicarboxylic anhydride is involved in the formation reaction of the modified marine bioactive peptide.
Example 4:
Fatigue resistance test
The experimental group is as follows: the marine bioactive peptide prepared in example 1 is N1; the modified marine bioactive peptide prepared in example 2 is N2; blank group is N0; the method comprises the steps of randomly dividing 30 KM mice into 3 groups, feeding 150mg/kg/d of gastric lavage, continuously feeding the same amount of normal saline into the stomach for 30d in a blank group, winding 5% weight lead skin on the root of the mice after the last gastric lavage administration for 1h, putting the mice into a swimming box for swimming, controlling the water temperature at (25+/-1) DEGC, controlling the water depth at 30cm, and recording the time from the beginning of swimming of the mice to the time when the mice cannot float out of the water for 5s in the water, wherein the time is the load swimming time.
The above-described test was performed on the marine bioactive peptide prepared in example 1 and the modified marine bioactive peptide prepared in example 2, and the results are shown in fig. 5. As can be seen from FIG. 5, compared with the blank group, the example 1 has significantly longer mouse-bearing swimming time, which indicates that the marine bioactive peptide extracted by the invention has good fatigue resistance; compared with example 1, the weight-bearing swimming time of example 2 is obviously improved, which shows that the obtained modified marine bioactive peptide has better fatigue resistance when bicyclo [2.2.2] oct-5-ene-2, 3-dicarboxylic anhydride acts on the marine bioactive peptide.
Example 5:
Blood glucose lowering Performance test
After 50 KM mice are fasted for 12 hours, 160mg/kg of tetraoxypyrimidine is injected into abdominal cavity for causing diabetes of the mice, after 5d free diet, the mice are fasted for 12 hours, blood is taken from veins at the tail of the mice, the blood sugar concentration of the mice in the fasting state is measured by a blood sugar meter, the blood sugar concentration is more than or equal to 10mmol/L as a model successful mouse, the model is determined as a diabetes model, and the mice conforming to the standard diabetes model are randomly divided into 3 groups as experimental groups; the experimental group is as follows: the marine bioactive peptide prepared in example 1 is N1; the modified marine bioactive peptide prepared in example 2 is N2; the diabetes model control group is N0; the mice of each experimental group were given a gastric lavage dose of 0.2g/kg/d, the mice of the diabetic model control group were given an equivalent amount of physiological saline every day, each time were given a gastric lavage for 6 weeks, after the last gastric lavage, the mice were fasted for 12 hours, blood was collected from the tail veins, and the blood glucose level of each experimental mouse was measured by a blood glucose meter.
The above-described test was performed on the marine bioactive peptide prepared in example 1 and the modified marine bioactive peptide prepared in example 2, and the results are shown in fig. 6. As can be seen from FIG. 6, the blood glucose concentration of the mice in the model control group is significantly higher than 10mmol/L, indicating that the modeling was successful. Compared with a control group, the blood sugar level of the marine bioactive peptide extracted by the invention is obviously reduced, which indicates that the marine bioactive peptide extracted by the invention has good blood sugar reducing performance; the significantly lower blood glucose levels of example 2 compared to example 1 indicate that the modified marine bioactive peptide obtained when bicyclo [2.2.2] oct-5-ene-2, 3-dicarboxylic anhydride was used on the marine bioactive peptide has better hypoglycemic properties.
Example 6:
Anticancer Performance test
The experimental group is as follows: the marine bioactive peptide prepared in example 1 is N1; the modified marine bioactive peptide prepared in example 2 is N2; the control group is N3; blank group is N0; taking MGC-803 cells in logarithmic phase, inoculating (5000 cells/hole) respectively to a transparent 96-well plate, culturing at 37 ℃ for 24 hours, discarding old culture solution, adding 200 mu L of fresh culture medium containing 20 mu g/mL of sample solution, taking PBS as a control group, and adding the same amount of fresh culture medium into a blank group; after 48 hours, the liquid medicine was taken out, washed with PBS, added with 20. Mu.L of MTT solution with a concentration of 5mg/mL and 180. Mu.L of fresh medium, cultured for 4 hours, the culture solution containing MTT was removed, and then 150. Mu.L of dimethyl sulfoxide was added thereto, followed by shaking treatment for 15 minutes, and OD was measured at 490 nm.
Cancer cell growth inhibition rate/% = [ (OD control-OD dosing)/(OD control-OD blank) ] ×100%
The above-described test was performed on the marine bioactive peptide prepared in example 1 and the modified marine bioactive peptide prepared in example 2, and the results are shown in fig. 7. As can be seen from fig. 7, the inhibition rate of cancer cell growth of the marine bioactive peptide prepared in example 1 is 61.2%, which indicates that the marine bioactive peptide extracted by the invention has good anticancer performance; compared with the example 1, the inhibition rate of the example 2 is obviously improved, which shows that the obtained modified marine bioactive peptide has better anticancer performance when the bicyclo [2.2.2] oct-5-ene-2, 3-dicarboxylic anhydride acts on the marine bioactive peptide.
Example 7:
immune Performance test
The experimental group is as follows: the marine bioactive peptide prepared in example 1 is N1; the modified marine bioactive peptide prepared in example 2 is N2; blank group is N0; 100. Mu.L of spleen lymphocyte suspension (5X 10 6/mL) is added to each well of a 96-well plate, then 100. Mu.L of RPMI-1640 culture medium containing 0.5mg/mL of sample solution is added, the blank group is added with equal amount of RPMI-1640 culture medium, the mixture is placed in a 37 ℃ and 5% CO 2 incubator for culture for 68 hours, then 20. Mu.L of MTT solution with the concentration of 5mg/mL is added, the mixture is uniformly mixed, the mixture is continuously cultured for 4 hours, centrifugation is carried out, 100. Mu.L of supernatant is removed from each well, 100. Mu.L of dimethyl sulfoxide is added, shaking treatment is carried out for 15 minutes, and OD value is measured at 490 nm.
Lymphocyte proliferation rate/% = [ (a 1-A0)/A1 ] ×100%
Wherein A 0 is the absorbance of the blank group; a 1 is the absorbance of the sample group.
The above-described test was performed on the marine bioactive peptide prepared in example 1 and the modified marine bioactive peptide prepared in example 2, and the results are shown in fig. 8. As can be seen from fig. 8, the lymphocyte proliferation rate of the marine bioactive peptide prepared in example 1 is 34.2%, which indicates that the marine bioactive peptide extracted by the method has good immune activity; compared with example 1, the lymphocyte proliferation rate of example 2 is obviously improved, which shows that when bicyclo [2.2.2] oct-5-ene-2, 3-dicarboxylic anhydride acts on the marine bioactive peptide, the obtained modified marine bioactive peptide has better immune activity.
Example 8:
determination of relative enzyme Activity
The experimental group is as follows: bromelain group S1; bromelain + aucubin S2; blank S0; taking 3mL of sample enzyme solution prepared by phosphate buffer (pH 7.4) and having the concentration of 0.33g/mL, preserving heat for 5min at 37 ℃, and adding 5mL of casein solution having the concentration of 7.5 mg/mL; 8mL of phosphate buffer solution is added into the blank group; performing constant temperature reaction for 30min, adding 5mL of 5wt% trichloroacetic acid solution respectively, shaking to terminate the reaction, centrifuging, collecting supernatant, and measuring absorbance at 275nm in an ultraviolet spectrospectrometer; the activity of bromelain was used as a reference.
TABLE 1 relative Activity of bromelain
| Grouping | Relative Activity/% |
| S1 | 100 |
| S2 | 112.4 |
The above tests were performed on the bromelain group, bromelain + aucubin and the results are shown in table 1. As is clear from Table 1, the relative activity of bromelain increased significantly after the addition of aucubin, indicating that the aucubin and bromelain act to promote the activity of bromelain when used together.
The conventional technology in the above embodiments is known to those skilled in the art, and thus is not described in detail herein.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (6)
1. A marine bioactive peptide has an amino acid sequence of Asp-Pro-Leu-Ala-Val-Pro.
2. A method of modifying a marine bioactive peptide of claim 1, comprising: the modified marine bioactive peptide is prepared by chemically modifying the marine bioactive peptide by using bicyclo [2.2.2] oct-5-ene-2, 3-dicarboxylic anhydride.
3. The method for modifying a marine bioactive peptide according to claim 2, wherein: the mass ratio of the marine bioactive peptide to the bicyclo [2.2.2] oct-5-ene-2, 3-dicarboxylic anhydride is 1:1-1.3.
4. A modified marine bioactive peptide obtained by the modification method according to any one of claims 2 to 3.
5. Use of the marine bioactive peptide of claim 1 in the preparation of a medicament for treating cancer and reducing blood glucose.
6. The use of the modified marine bioactive peptide of claim 4 in the preparation of a medicament for treating cancer and reducing blood glucose.
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