CN113899842B - Method for detecting tripeptide-1 copper - Google Patents
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Abstract
The invention discloses a method for detecting tripeptide-1 copper, belongs to the technical field of polypeptide separation and detection, and particularly relates to a method for detecting tripeptide-1 copper, which comprises the following steps: carrying out elution and separation on the tripeptide-1 copper solution by a chromatography column of cross-linked dextran gel, and then detecting; the tripeptide-1 copper solution is prepared by dissolving a tripeptide-1 copper sample to be detected in deionized water, the sephadex is obtained by using epoxy chloropropane as a crosslinking agent to enable oat beta-glucan derivatives to form a crosslinking structure, the oat beta-glucan derivatives at least contain modified oat beta-glucan, and the modified oat beta-glucan is prepared from 6-ethoxypyrazine-3-amine and oxidized oat beta-glucan. The tripeptide-1 copper obtained by the method has good separation and detection effects, and can be used for separating and detecting the tripeptide-1 copper.
Description
Technical Field
The invention belongs to the technical field of polypeptide separation and detection, and particularly relates to a method for detecting tripeptide-1 copper.
Background
Tripeptide-1 copper can be used as an activator and antioxidant for tissue remodeling, and is also a signal peptide, which promotes the degradation of a large number of collagen aggregates outside the scar, the synthesis of normal collagen of the skin, the generation of elastin, proteoglycan and glucosaminan, the growth rate and migration of different cell types, anti-inflammation and antioxidant reactions. The tripeptide-1 copper can increase the vitality of cells, gradually repair collagen lost in vivo under the conditions of not damaging and irritating the skin, and can be used for rapidly healing wounds, thereby achieving the purposes of removing wrinkles and resisting ageing.
In the preparation and use process of tripeptide-1 copper, it is necessary to accurately measure the content of tripeptide-1 copper, and in the content detection process, the separation effect and purity of tripeptide-1 copper have great significance, so that the application aims to provide a novel detection method comprising the tripeptide-1 copper separation treatment.
Disclosure of Invention
The invention aims to provide a method for detecting tripeptide-1 copper by using sephadex with good pressure resistance and hardness.
The technical scheme adopted by the invention for realizing the purpose is as follows:
a method for detecting tripeptide-1 copper, comprising: carrying out elution and separation on the tripeptide-1 copper solution by a chromatography column of cross-linked dextran gel, and then detecting; the tripeptide-1 copper solution is prepared by dissolving a tripeptide-1 copper sample to be detected in deionized water, the sephadex is obtained by using epoxy chloropropane as a crosslinking agent to enable oat beta-glucan derivatives to form a crosslinking structure, the oat beta-glucan derivatives at least contain modified oat beta-glucan, and the modified oat beta-glucan is prepared from 6-ethoxypyrazine-3-amine and oxidized oat beta-glucan. The cross-linked dextran has a plurality of pores, and small molecules can enter the pores; macromolecules can not enter pores and can only flow in gaps among gel particles along with a mobile phase, and are eluted firstly, so that the macromolecular composite material can be used as a molecular sieve to separate molecules with different sizes. According to the invention, 6-ethoxypyrazine-3-amine is introduced into modified oat beta-glucan, and then the modified oat beta-glucan is crosslinked by using epoxy chloropropane as a crosslinking agent to obtain the cross-linked glucan gel, so that the pressure resistance of the cross-linked glucan gel is improved, the cross-linked glucan gel has better hardness, and the recovery property and the cohesiveness of the cross-linked glucan gel are improved.
Preferably, the oat β -glucan derivative comprises oxidized oat β -glucan.
Preferably, the oat β -glucan derivative comprises hydrolyzed or unhydrolyzed oat β -glucan.
Preferably, the oat β -glucan derivative comprises a glucan graft.
Preferably, the oat β -glucan is extracted from oat bran.
Preferably, in the preparation of the sephadex, the modified oat beta-glucan is added into an alkaline solution, a PVAc solution and an ECH solution are added, the temperature is raised to 40-80 ℃, the mixture is stirred and reacts for 2-8 hours, and after the reaction is finished, the mixture is washed by toluene, ethanol, distilled water, filtered and dried to obtain the sephadex.
More preferably, in the preparation of the sephadex, the alkaline solution is a sodium hydroxide solution, and the mass fraction of sodium hydroxide in the alkaline solution is 0.4-1.2 wt%.
More preferably, the amount of oxidized oat beta glucan used in the preparation of the sephadex is 1.5-5wt% of the alkaline solution.
More preferably, in the preparation of the sephadex, the PVAc solution is a dichloroethane solution of PVAc, and the mass fraction of PVAc in the PVAc solution is 6 to 14 wt%.
More preferably, the amount of the PVAc solution used in the preparation of the sephadex is between 30 and 60wt% of the alkaline solution.
More preferably, the sephadex is prepared by using an ECH solution comprising ECH in dichloroethane, wherein the ECH content of the ECH solution is 3 to 12 wt%.
More preferably, the ECH solution is used in an amount of 50 to 100wt% of the alkaline solution in the preparation of the sephadex.
Preferably, in the detection of the tripeptide-1 copper, the sephadex is soaked in an ethanol solution, is stirred and swelled for 12 to 48 hours, is washed by deionized water, is loaded into a chromatographic column, is loaded with the tripeptide-1 copper solution, is eluted, and is subjected to component collection and detection by an ultraviolet spectrophotometer.
More preferably, the tripeptide-1 copper solution is prepared by dissolving a tripeptide-1 copper sample to be detected in deionized water, wherein the loading amount of the tripeptide-1 copper solution is 5-20 μ L, and the content of the tripeptide-1 copper in the tripeptide-1 copper solution is 1-5 wt%.
More preferably, the mass fraction of ethanol in the ethanol solution is 60-80 wt%.
Preferably, the method for detecting tripeptide-1 copper comprises the preparation of modified oat beta-glucan.
More preferably, in the preparation of the modified oat beta-glucan, the oxidized oat beta-glucan is added into an ethanol solution, stirred and dissolved at the temperature of 60-80 ℃, 6-ethoxypyrazine-3-amine solution is added, stirred and reacted for 6-24 hours, the reaction is cooled to room temperature after the reaction is finished, and the modified oat beta-glucan is obtained by suction filtration, washing with absolute ethyl alcohol and drying.
Still more preferably, in the preparation of the modified oat beta-glucan, the mass fraction of ethanol in the ethanol solution is 30-60 wt%.
Still more preferably, the amount of oxidized oat beta-glucan added in the preparation of the modified oat beta-glucan is 1-4wt% of the ethanol solution.
Still more preferably, in the preparation of the modified oat beta-glucan, the solvent of the 6-ethoxypyrazine-3-amine solution is ethanol, and the content of the 6-ethoxypyrazine-3-amine in the 6-ethoxypyrazine-3-amine solution is 6-24 wt%.
Still more preferably, in the preparation of the modified oat beta-glucan, the 6-ethoxypyrazin-3-amine solution is used in an amount of 7-21wt% of the ethanol solution.
Preferably, the method for detecting tripeptide-1 copper comprises the preparation of oxidized oat beta-glucan.
More preferably, in the preparation of the oxidized oat beta-glucan, the hydrolyzed oat beta-glucan is added into an ethanol solution, stirred and dissolved at the temperature of 60-80 ℃, added with a sodium periodate solution, oxidized for 4-12h in a dark place, added with ethylene glycol to stop the reaction, cooled to room temperature, filtered by suction, and dried to obtain the oxidized oat beta-glucan.
Still more preferably, in the preparation of the oxidized oat beta-glucan, the mass fraction of ethanol in the ethanol solution is 30-60 wt%.
Still more preferably, the amount of hydrolyzed oat β -glucan used in the preparation of oxidized oat β -glucan is 1-3wt% of the ethanol solution.
Still more preferably, in the preparation of the oxidized oat beta-glucan, the mass fraction of sodium periodate in the sodium periodate solution is 20-60 wt%.
Still more preferably, in the preparation of the oxidized oat beta-glucan, the sodium periodate solution is used in an amount of 2-6wt% of the ethanol solution.
Still more preferably, in the preparation of the oxidized oat beta-glucan, the amount of ethylene glycol used is 4-12wt% of the ethanol solution.
Still more preferably, in the preparation of the oxidized oat beta-glucan, the amount of ethanol used in the ethanol precipitation is more than 200wt% of the solution to be precipitated.
Preferably, the method for detecting tripeptide-1 copper comprises the steps of extracting oat beta-glucan and preparing hydrolyzed oat beta-glucan.
More preferably, in the extraction of oat beta-glucan, oat bran is ground, the obtained oat bran powder is added into an ethanol solution, the mixture is refluxed for 1 to 5 hours at the temperature of 80 to 90 ℃, alcohol extraction supernatant is removed by centrifugation, and the oat bran alcohol treatment product is obtained by ethanol washing and drying; adding into distilled water, extracting at 50-60 deg.C under stirring for 0.5-3 hr, centrifuging, and collecting supernatant to obtain oat bran water extractive solution; adding calcium chloride, adding high temperature resistant alpha-amylase at 65-85 deg.C for enzymolysis for 20-60min, adjusting pH to 7-9, adding trypsin at 30-40 deg.C for enzymolysis for 2-6h, centrifuging, collecting supernatant, adjusting pH to neutral, adding ethanol for precipitation, centrifuging, collecting precipitate, adding distilled water, and freeze drying to obtain oat beta-dextran.
Still more preferably, in the extraction of oat beta-glucan, the mass fraction of ethanol in the ethanol solution is 80-90 wt%.
Still more preferably, the oat bran powder is used in an amount of 15-25wt% of the ethanol solution in the extraction of oat β -glucan.
Still more preferably, in the extraction of oat beta-glucan, the oat bran alcohol treatment is used in an amount of 5-15wt% of distilled water.
More preferably, in the extraction of oat beta-glucan, the amount of calcium chloride used is 0.4-3.6wt% of the oat bran water extract.
More preferably, the amount of the high-temperature resistant alpha-amylase used in the extraction of the oat beta-glucan is 0.3-2.1wt% of the oat bran water extract.
More preferably, in the extraction of oat beta-glucan, the amount of trypsin is 0.2-1.2wt% of the oat bran water extract.
Still more preferably, in the extraction of oat beta-glucan, the amount of ethanol used in ethanol precipitation is more than 200wt% of the solution to be precipitated.
More preferably, in the preparation of the hydrolyzed oat beta-glucan, the oat beta-glucan is added into distilled water, stirred and dissolved at the temperature of 60-80 ℃ to obtain an oat beta-glucan solution, concentrated hydrochloric acid is added, hydrolysis is carried out at the temperature of 60-80 ℃ for 30-120min, then cooling to room temperature, adjusting the pH value to be neutral, ethanol precipitation is added, centrifugation is carried out, the precipitate is taken, distilled water is added, and freeze drying is carried out to obtain the hydrolyzed oat beta-glucan.
Still more preferably, the amount of oat beta-glucan used in the preparation of hydrolyzed oat beta-glucan is 0.6-1.5wt% of distilled water.
Still more preferably, the amount of concentrated hydrochloric acid used in the preparation of hydrolyzed oat β -glucan is 0.8-2.4wt% of distilled water.
Still more preferably, in the preparation of hydrolysed oat beta-glucan, the amount of ethanol used in the ethanol precipitation is more than 200wt% of the solution to be precipitated.
Preferably, dextran grafts may also be used in the preparation of the sephadex, the dextran grafts being used in an amount of 1-3.5wt% of the alkaline solution. In the process of preparing the cross-linked glucan gel, a certain glucan graft is introduced to form a cross-linked structure together with the modified oat beta-glucan, so that the finally obtained cross-linked glucan gel has more excellent performance, and the pressure resistance, the recovery performance and the cohesiveness are improved.
Preferably, the method for detecting the tripeptide-1 copper comprises the preparation of a dextran graft.
More preferably, in the preparation of the glucan graft, whey protein and oat beta-glucan are respectively added into distilled water, and after being uniformly mixed, the mixture is frozen and dried, and then reacts for more than 96 hours at the temperature of 50-60 ℃ and the humidity of 75-85 percent to obtain the glucan graft.
Even more preferably, the whey protein is used in an amount of 40-100wt% of oat beta-glucan in the preparation of the glucan graft.
The invention discloses a preparation method of modified oat beta-glucan, which comprises the following steps:
extracting oat beta-glucan from oat bran through an extraction process;
hydrolyzing and oxidizing the oat beta-glucan to obtain oxidized oat beta-glucan;
and (3) reacting the oxidized oat beta-glucan and 6-ethoxypyrazine-3-amine in an ethanol solution to obtain the modified oat beta-glucan.
The invention discloses a modified oat beta-glucan, which comprises the following components: the carbonyl group formed by oxidation on oat beta-glucan and 6-ethoxypyrazine-3-amine form Schiff base structure.
The invention discloses a cross-linked dextran gel, comprising: a sephadex comprising in its cross-linked structure the above-described modified oat β -glucan.
The invention discloses application of the modified oat beta-glucan in a method for detecting tripeptide-1 copper.
The invention adopts 6-ethoxypyrazine-3-amine to react with oxidized oat beta-glucan to generate modified oat beta-glucan, and then the modified oat beta-glucan reacts with epoxy chloropropane to generate cross-linked glucan gel, so the invention has the following beneficial effects: the pressure resistance of the cross-linked dextran gel is improved, the pressure resistance is represented by the flow pressure, and the flow pressure is 0.09-0.13 MPa; the hardness of the cross-linked dextran gel is good; the cross-linked dextran gel has excellent recovery performance, and the recovery performance is 0.5-0.8; good cohesiveness, the cohesiveness is 0.55-0.90. The sephadex gel obtained can be applied to a method for detecting tripeptide-1 copper to obtain an excellent separation detection effect, so that the invention is a method for detecting the tripeptide-1 copper by using the sephadex gel with good pressure resistance and good hardness.
Drawings
FIG. 1 is an infrared spectrum of modified oat beta-glucan;
FIG. 2 is a graph of the flow-through pressure of a sephadex gel;
FIG. 3 is a graph of the hardness of a sephadex gel;
FIG. 4 is a graph of the recovery of a sephadex gel;
FIG. 5 is a graph of cohesiveness of a sephadex;
FIG. 6 is an HPLC check chart of tripeptide-1 copper.
Detailed Description
The technical solution of the present invention is further described in detail below with reference to the following detailed description and the accompanying drawings:
the invention only provides a method which can process and detect tripeptide-1 copper samples, and if quantitative determination is needed, standard curve drawing can be carried out according to needs, and quantitative calculation can be carried out.
Example 1:
a method for detecting tripeptide-1 copper,
extracting oat beta-glucan: grinding oat bran, adding the obtained oat bran powder into an ethanol solution, refluxing for 3h at the temperature of 90 ℃, centrifuging to remove ethanol extraction supernatant, washing with ethanol, and drying to obtain oat bran ethanol-treated substance; adding into distilled water, extracting at 60 deg.C under stirring for 2 hr, centrifuging, and collecting supernatant to obtain oat bran water extractive solution; adding calcium chloride, adding high temperature resistant alpha-amylase at 80 deg.C for enzymolysis for 40min, adjusting pH to 8, adding trypsin at 35 deg.C for enzymolysis for 4h, centrifuging, collecting supernatant, adjusting pH to neutral, adding ethanol for precipitation, centrifuging, collecting precipitate, adding distilled water, and freeze drying to obtain oat beta-dextran. The mass fraction of ethanol in the ethanol solution is 85wt%, the using amount of oat bran powder is 20wt% of the ethanol solution, the using amount of oat bran alcohol treatment substance is 10wt% of distilled water, the using amount of calcium chloride is 2.5wt% of oat bran water extract, the using amount of high-temperature resistant alpha-amylase is 0.9wt% of oat bran water extract, and the using amount of trypsin is 1.0wt% of the oat bran water extract. The amount of ethanol used in the ethanol precipitation is 300wt% of the solution to be precipitated.
Preparation of hydrolyzed oat beta-glucan: adding oat beta-glucan into distilled water, stirring and dissolving at 70 ℃ to obtain an oat beta-glucan solution, adding concentrated hydrochloric acid, hydrolyzing at 70 ℃ for 90min, cooling to room temperature, adjusting pH to be neutral, adding ethanol for precipitation, centrifuging, taking the precipitate, adding distilled water, and freeze-drying to obtain hydrolyzed oat beta-glucan. The usage amount of the oat beta-glucan is 1.2wt% of the distilled water, and the usage amount of the concentrated hydrochloric acid is 2.0wt% of the distilled water. The amount of ethanol used in the ethanol precipitation is 300wt% of the solution to be precipitated.
Preparation of oxidized oat beta-glucan: adding the hydrolyzed oat beta-glucan into an ethanol solution, stirring and dissolving at the temperature of 70 ℃, adding a sodium periodate solution, oxidizing for 9 hours in a dark place, adding ethylene glycol to stop the reaction, cooling to room temperature, filtering, and drying to obtain the oxidized oat beta-glucan. The mass fraction of ethanol in the ethanol solution is 50wt%, the use amount of hydrolyzed oat beta-glucan is 2wt% of the ethanol solution, the mass fraction of sodium periodate in the sodium periodate solution is 40wt%, the use amount of the sodium periodate solution is 5wt% of the ethanol solution, and the use amount of ethylene glycol is 8wt% of the ethanol solution. The amount of ethanol used in the ethanol precipitation is 300wt% of the solution to be precipitated.
Preparing modified oat beta-glucan: adding oxidized oat beta-glucan into an ethanol solution, stirring and dissolving at the temperature of 70 ℃, adding a 6-ethoxypyrazine-3-amine solution, stirring and reacting for 12 hours, cooling to room temperature after the reaction is finished, performing suction filtration, washing with absolute ethanol, and drying to obtain the modified oat beta-glucan. The mass fraction of ethanol in the ethanol solution is 50wt%, the addition amount of the oat oxide beta-glucan is 3wt% of the ethanol solution, the solvent of the 6-ethoxypyrazine-3-amine solution is ethanol, the content of the 6-ethoxypyrazine-3-amine in the 6-ethoxypyrazine-3-amine solution is 18wt%, and the usage amount of the 6-ethoxypyrazine-3-amine solution is 9wt% of the ethanol solution.
Preparation of Sephadex: adding the modified oat beta-glucan into an alkaline solution, adding a PVAc solution and an ECH solution, heating to 80 ℃, stirring and reacting for 6 hours, washing with toluene, washing with ethanol, washing with distilled water, filtering, and drying to obtain the cross-linked dextran gel. The alkaline solution is sodium hydroxide solution, the mass fraction of sodium hydroxide in the alkaline solution is 0.8wt%, the usage amount of the oxidized oat beta-glucan is 2.5wt% of the alkaline solution, the PVAc solution is dichloroethane solution of PVAc, the mass fraction of PVAc in the PVAc solution is 10wt%, the usage amount of the PVAc solution is 40wt% of the alkaline solution, the ECH solution is dichloroethane solution of ECH, the mass fraction of ECH in the ECH solution is 10wt%, and the usage amount of the ECH solution is 50wt% of the alkaline solution.
Detection of tripeptide-1 copper: soaking the cross-linked dextran gel in ethanol solution, swelling for 24h under stirring, washing with deionized water, loading into a chromatographic column, loading tripeptide-1 copper solution, eluting, collecting components, and detecting with an ultraviolet spectrophotometer. The mass fraction of ethanol in the ethanol solution is 70 wt%. The ultraviolet detection wavelength is 220 nm. The tripeptide-1 copper solution is prepared by dissolving a tripeptide-1 copper sample to be detected in deionized water, wherein the loading amount of the tripeptide-1 copper solution is 10 mu L, and the content of the tripeptide-1 copper in the tripeptide-1 copper solution is 3 wt%.
In elution, the mobile phase A is acetonitrile; mobile phase B was a 0.10 wt% trifluoroacetic acid-water solution; the mobile phase was filtered through a 0.45 μm filter before use. The flow rate was 1.0 mL/min.
Elution procedure: 0-4min, 20% A; 4-5min, 10% A; 5-10min, 20% A.
Example 2:
a method for detecting tripeptide-1 copper,
this example is compared to example 1, except that 6-ethoxypyrazin-3-amine solution was used in an amount of 16wt% of the ethanol solution in the preparation of modified oat beta-glucan.
Example 3:
a method for detecting tripeptide-1 copper,
extracting oat beta-glucan: grinding oat bran, adding the obtained oat bran powder into an ethanol solution, refluxing for 3h at the temperature of 90 ℃, centrifuging to remove ethanol extraction supernatant, washing with ethanol, and drying to obtain oat bran ethanol-treated substance; adding into distilled water, extracting at 60 deg.C under stirring for 2 hr, centrifuging, and collecting supernatant to obtain oat bran water extractive solution; adding calcium chloride, adding high temperature resistant alpha-amylase at 80 deg.C for enzymolysis for 40min, adjusting pH to 8, adding trypsin at 35 deg.C for enzymolysis for 4h, centrifuging, collecting supernatant, adjusting pH to neutral, adding ethanol for precipitation, centrifuging, collecting precipitate, adding distilled water, and freeze drying to obtain oat beta-dextran. The mass fraction of ethanol in the ethanol solution is 85wt%, the using amount of oat bran powder is 20wt% of the ethanol solution, the using amount of oat bran alcohol treatment substance is 10wt% of distilled water, the using amount of calcium chloride is 2.5wt% of oat bran water extract, the using amount of high-temperature resistant alpha-amylase is 0.9wt% of oat bran water extract, and the using amount of trypsin is 1.0wt% of the oat bran water extract. The amount of ethanol used in the ethanol precipitation is 300wt% of the solution to be precipitated.
Preparation of hydrolyzed oat beta-glucan: adding oat beta-glucan into distilled water, stirring and dissolving at 70 ℃ to obtain an oat beta-glucan solution, adding concentrated hydrochloric acid, hydrolyzing at 70 ℃ for 90min, cooling to room temperature, adjusting pH to be neutral, adding ethanol for precipitation, centrifuging, taking the precipitate, adding distilled water, and freeze-drying to obtain hydrolyzed oat beta-glucan. The usage amount of the oat beta-glucan is 1.2wt% of the distilled water, and the usage amount of the concentrated hydrochloric acid is 2.0wt% of the distilled water. The amount of ethanol used in the ethanol precipitation is 300wt% of the solution to be precipitated.
Preparation of dextran grafts: respectively adding whey protein and oat beta-glucan into distilled water, uniformly mixing, freeze-drying, and then reacting at the humidity of 80% and the temperature of 60 ℃ for 168 hours to obtain the glucan graft. The usage amount of the whey protein is 60wt% of the oat beta-glucan.
Preparation of oxidized oat beta-glucan: adding the hydrolyzed oat beta-glucan into an ethanol solution, stirring and dissolving at the temperature of 70 ℃, adding a sodium periodate solution, oxidizing for 9 hours in a dark place, adding ethylene glycol to stop the reaction, cooling to room temperature, filtering, and drying to obtain the oxidized oat beta-glucan. The mass fraction of ethanol in the ethanol solution is 50wt%, the use amount of hydrolyzed oat beta-glucan is 2wt% of the ethanol solution, the mass fraction of sodium periodate in the sodium periodate solution is 40wt%, the use amount of the sodium periodate solution is 5wt% of the ethanol solution, and the use amount of ethylene glycol is 8wt% of the ethanol solution. The amount of ethanol used in the ethanol precipitation is 300wt% of the solution to be precipitated.
Preparing modified oat beta-glucan: adding oxidized oat beta-glucan into an ethanol solution, stirring and dissolving at the temperature of 70 ℃, adding a 6-ethoxypyrazine-3-amine solution, stirring and reacting for 12 hours, cooling to room temperature after the reaction is finished, performing suction filtration, washing with absolute ethanol, and drying to obtain the modified oat beta-glucan. The mass fraction of ethanol in the ethanol solution is 50wt%, the addition amount of the oat oxide beta-glucan is 3wt% of the ethanol solution, the solvent of the 6-ethoxypyrazine-3-amine solution is ethanol, the content of the 6-ethoxypyrazine-3-amine in the 6-ethoxypyrazine-3-amine solution is 18wt%, and the usage amount of the 6-ethoxypyrazine-3-amine solution is 16wt% of the ethanol solution.
Preparation of Sephadex: adding the modified oat beta-glucan and the glucan graft into an alkaline solution, adding a PVAc solution and an ECH solution, heating to 80 ℃, stirring for reaction for 6 hours, washing with toluene, washing with ethanol, washing with distilled water, performing suction filtration, and drying to obtain the cross-linked glucan gel. The alkaline solution is sodium hydroxide solution, the mass fraction of sodium hydroxide in the alkaline solution is 0.8wt%, the using amount of the oxidized oat beta-glucan is 2.5wt% of the alkaline solution, the using amount of the glucan graft is 1.3wt% of the alkaline solution, the PVAc solution is dichloroethane solution of PVAc, the mass fraction of PVAc in the PVAc solution is 10wt%, the using amount of the PVAc solution is 40wt% of the alkaline solution, the ECH solution is dichloroethane solution of ECH, the mass fraction of ECH in the ECH solution is 10wt%, and the using amount of the ECH solution is 50wt% of the alkaline solution.
Detection of tripeptide-1 copper: soaking the cross-linked dextran gel in ethanol solution, swelling for 24h under stirring, washing with deionized water, loading into a chromatographic column, loading tripeptide-1 copper solution, eluting, collecting components, and detecting with an ultraviolet spectrophotometer. The mass fraction of ethanol in the ethanol solution is 70 wt%. The ultraviolet detection wavelength is 220 nm. The tripeptide-1 copper solution is prepared by dissolving a tripeptide-1 copper sample to be detected in deionized water, wherein the loading amount of the tripeptide-1 copper solution is 10 mu L, and the content of the tripeptide-1 copper in the tripeptide-1 copper solution is 3 wt%.
In elution, the mobile phase A is acetonitrile; mobile phase B was a 0.10 wt% trifluoroacetic acid-water solution; the mobile phase was filtered through a 0.45 μm filter before use. The flow rate was 1.0 mL/min.
Elution procedure: 0-4min, 20% A; 4-5min, 10% A; 5-10min, 20% A.
Example 4:
a method for detecting tripeptide-1 copper,
this example is compared to example 1, except that the dextran graft is used in an amount of 2.4wt% of the alkaline solution in the preparation of the sephadex.
Comparative example 1:
this comparative example compares to example 2 only with the difference that in the preparation of the sephadex the modified oat beta glucan was replaced by oxidized oat beta glucan.
Comparative example 2:
this comparative example compares to example 4 only with the difference that in the preparation of the sephadex the modified oat beta glucan was replaced by oxidized oat beta glucan.
Test examples
1. Modified oat beta-glucan Infrared analysis
Test samples: the resulting modified oat beta glucan was prepared in the method of example 2.
Tabletting with potassium bromide, and performing infrared spectrum analysis.
The infrared spectrogram of the modified oat beta-glucan prepared by the method in the embodiment 2 of the invention is shown in figure 1, wherein the infrared spectrogram is 3413cm-1Is positioned at 2926cm and has a hydroxyl infrared absorption peak-1Is at methyl infrared absorption peak, 2862cm-1Is at a methylene infrared absorption peak of 1708cm-1The part is carbonyl infrared absorption peak, which shows that the modified oat beta-glucan has carbon-oxygen double bond residue, 1631cm-1The infrared absorption peak at the position of carbon-carbon double bond is 1579 and 1409cm-1The infrared absorption peak of the nitrogen-containing heterocycle indicates that the modified oat beta-glucan is successfully obtained.
2. Test of pressure resistance
Test samples: the examples and comparative examples prepare the resulting sephadex gels.
Swelling the sample in distilled water at room temperature for 72h, then filling a column (diameter is 1.0cm, length is 10 cm), connecting an AKTA chromatographic system, taking the distilled water as a mobile phase, increasing the flow rate after the column is stabilized, recording the corresponding column pressure of the flow rate, and taking the pressure as the pressure resistance of the test sample when the pressure is not stabilized when the flow rate is increased to a certain value.
The results of the pressure resistance test of the sephadex prepared by the present invention are shown in fig. 2, wherein a is example 1, B is example 2, C is example 3, D is example 4, E is comparative example 1, and F is comparative example 2, wherein the flow pressure of the sephadex prepared in example 1 is 0.094MPa, the flow pressure of the sephadex prepared in example 2 is 0.102MPa, the flow pressure of the sephadex prepared in comparative example 1 is 0.079MPa, and the flow pressure of the sephadex prepared in example 2 is increased by 29.11% compared to comparative example 1, which indicates that the sephadex prepared by using the modified oat β -glucan has better pressure resistance and better use effect than that of the oxidized oat β -glucan, and thus, it can be known that the 6-ethoxypyrazine-3-amine has improved effect on the oxidized oat β -glucan The performance is a key factor for improving the pressure resistance of the sephadex; the flow pressure of the sephadex prepared in example 3 was 0.120MPa, the flow pressure of the sephadex prepared in example 4 was 0.126MPa, and the flow pressure of the sephadex prepared in example 4 was increased by 23.53% compared to example 2, indicating that the pressure resistance of the sephadex was greatly improved when the modified oat β -glucan was used together with the glucan graft to prepare the sephadex; the flow pressure of the sephadex prepared in the comparative example 2 is 0.085MPa, and the comparison among the examples 2, 4 and 1-2 shows that after the oxidized oat beta-glucan is not modified by 6-ethoxypyrazine-3-amine, the pressure resistance of the sephadex prepared by jointly using the glucan graft and the oxidized oat beta-glucan is not obviously improved, and the sephadex prepared by jointly using the modified oat beta-glucan and the glucan graft has the optimal pressure resistance.
The pressure resistance of the cross-linked dextran gel obtained by the invention is improved, the pressure resistance is represented by the flow pressure, and the flow pressure is 0.09-0.13 MPa.
3. Texture parameter testing
Test samples: the examples and comparative examples prepare the resulting sephadex gels.
Adding 0.03g of the above sample into 3mL of distilled water, stirring at 75 ℃ for 6h, freezing at-20 ℃, thawing at 25 ℃, repeatedly freezing and thawing for 20 times, and performing texture test after final thawing.
Texture test conditions: the TPA mode is adopted, the probe is SMS P/5, the trigger force of 3g, the speed before measurement is 2mm/s, the test speed is 0.5 mm/s, the speed after measurement is 2mm/s, and the penetration distance is 4 mm.
The hardness test results of the sephadex prepared by the invention are shown in figure 3, A is example 1, B is example 2, C is example 3, D is example 4, E is comparative example 1, F is comparative example 2, the hardness of the sephadex obtained is above 8.5g, and the hardness is not obviously improved.
The cross-linked dextran gel obtained by the invention has good hardness.
The results of the recoverability test of the sephadex prepared by the present invention are shown in fig. 4, wherein a is example 1, B is example 2, C is example 3, D is example 4, E is comparative example 1, F is comparative example 2, wherein the recoverability of the sephadex prepared in example 1 is 0.51, the recoverability of the sephadex prepared in example 2 is 0.54, the recoverability of the sephadex prepared in comparative example 1 is 0.32, and the recoverability of the sephadex prepared in example 2 is improved by 68.75% compared with comparative example 1, which indicates that the sephadex prepared by using the modified oat β -glucan has better recoverability and the use effect of the modified oat β -glucan is better than that of the oxidized oat β -glucan, and thus, it can be known that 6-ethoxypyrazine-3-amine modifies oat β -glucan, is a key factor for improving the recoverability of the sephadex; the recovery of the sephadex prepared in example 3 was 0.66, the recovery of the sephadex prepared in example 4 was 0.74, and the recovery of the sephadex prepared in example 4 was improved by 37.04% compared to example 2, indicating that the recovery of the sephadex was greatly improved when the modified oat β -glucan was used together with the glucan graft to prepare the sephadex; the recovery of the sephadex prepared in comparative example 2 is 0.37, and the comparison between examples 2, 4 and 1-2 shows that the recovery of the sephadex prepared by using the glucan graft and the oat oxide beta-glucan is not obviously improved after the oat oxide beta-glucan is not modified by 6-ethoxypyrazine-3-amine, but the sephadex prepared by using the modified oat beta-glucan and the glucan graft has the optimal recovery.
The cross-linked dextran gel obtained by the invention has excellent recoverability which is 0.5-0.8.
The results of the cohesiveness test of the Sephadex prepared in accordance with the present invention are shown in FIG. 5, where A is example 1, B is example 2, C is example 3, D is example 4, E is comparative example 1, F is comparative example 2, where the cohesiveness of the Sephadex prepared in example 1 is 0.61, the cohesiveness of the Sephadex prepared in example 2 is 0.67, and the cohesiveness of the Sephadex prepared in comparative example 1 is 0.43, and compared with comparative example 1, the cohesiveness of the Sephadex prepared in example 2 is improved by 55.81%, indicating that the cohesiveness of the Sephadex prepared using modified Avena β -glucan is better, the use effect of modified Avena β -glucan is better than that of oxidized Avena β -glucan, and thus, it is known that 6-ethoxypyrazine-3-amine modifies Avena β -glucan, is a key factor for improving the cohesiveness of the sephadex; the cohesiveness of the sephadex prepared in example 3 was 0.78, the cohesiveness of the sephadex prepared in example 4 was 0.83, and the cohesiveness of the sephadex prepared in example 4 was improved by 23.88% compared to example 2, indicating that the cohesiveness of the sephadex was greatly improved when the modified oat β -glucan was used together with the glucan graft to prepare the sephadex; the cohesiveness of the sephadex prepared in comparative example 2 was 0.46, and the comparison between examples 2, 4 and comparative examples 1-2 shows that the cohesiveness of the sephadex prepared by using the glucan graft together with the oxidized oat β -glucan was not significantly improved without modifying the oxidized oat β -glucan with 6-ethoxypyrazine-3-amine, whereas the sephadex prepared by using the modified oat β -glucan together with the glucan graft had the optimal cohesiveness.
The invention has good cohesiveness which is 0.55-0.90.
4. HPLC detection result of tripeptide-1 copper
The product name of Zhejiang surge peptide is blue copper victimide, and the tripeptide-1 copper is detected, and the blue copper victimide is a component which is collected after being eluted by a chromatographic column according to the detection method of the embodiment 2.
Detecting the test sample solution by adopting high performance liquid chromatography; the chromatographic conditions were as follows:
a chromatographic system: agilent 1200; a chromatographic column: a C18 chromatography column; the column temperature was 25 ℃.
The mobile phase A is acetonitrile; mobile phase B was a 0.10 wt% trifluoroacetic acid-water solution; the mobile phase was filtered through a 0.45 μm filter before use. The flow rate was 1.0 mL/min.
Elution procedure:
0-4min，20％A；4-5min，10％A；5-10min，20％A。
sample introduction amount: 2.5. mu.L.
Detection wavelength of the ultraviolet detector: 220 nm.
The HPLC chart of the blue copper peptide is shown in FIG. 6, which shows that the tripeptide-1 copper obtained after elution through the chromatographic column of the invention has very high purity.
The above embodiments are merely illustrative, and not restrictive, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the protection scope of the present invention should be defined by the claims.
Claims (6)
1. A method for detecting tripeptide-1 copper, comprising: carrying out elution and separation on the tripeptide-1 copper solution by a chromatography column of cross-linked dextran gel, and then detecting; the tripeptide-1 copper solution is prepared by dissolving a tripeptide-1 copper sample to be detected in deionized water, the sephadex is obtained by using epoxy chloropropane as a crosslinking agent to enable oat beta-glucan derivatives to form a crosslinked structure, the oat beta-glucan derivatives at least contain modified oat beta-glucan, and the modified oat beta-glucan is prepared from 6-ethoxypyrazine-3-amine and oxidized oat beta-glucan.
2. The method for detecting tripeptide-1 copper according to claim 1, wherein: the oat beta-glucan derivative contains oxidized oat beta-glucan.
3. The method for detecting tripeptide-1 copper according to claim 1, wherein: the oat beta-glucan derivative contains hydrolyzed or unhydrolyzed oat beta-glucan.
4. The method for detecting tripeptide-1 copper according to claim 1, wherein: the oat beta-glucan derivative contains glucan grafts.
5. The method for detecting tripeptide-1 copper according to claim 1, wherein: the oat beta glucan is extracted from oat bran.
6. Use of a sephadex resulting from the formation of a cross-linked structure of an oat β -glucan derivative comprising at least a modified oat β -glucan by using epichlorohydrin as a cross-linking agent in a method for detecting tripeptide-1 copper; the modified oat beta-glucan is characterized in that a Schiff base structure is formed on the oat beta-glucan by carbonyl groups formed by oxidation and 6-ethoxypyrazine-3-amine.
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Address after: 518000 floor 5, building 206 plant (Building B, Hengyu Center), No. 19, Dengliang Road, Dengliang community, Nanshan street, Nanshan District, Shenzhen, Guangdong Province Patentee after: Shenzhen Branch of Zhejiang Peptide Biology Co.,Ltd. Address before: 518000 floor 5, building 206 plant (Building B, Hengyu Center), No. 19, Dengliang Road, Dengliang community, Nanshan street, Nanshan District, Shenzhen, Guangdong Province Patentee before: Zhejiang Pai peptide biology Co.,Ltd. Shenzhen Branch |
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