CN117343137A - Oyster oligopeptide and compound thereof, preparation and application thereof in treating liver fibrosis diseases - Google Patents
Oyster oligopeptide and compound thereof, preparation and application thereof in treating liver fibrosis diseases Download PDFInfo
- Publication number
- CN117343137A CN117343137A CN202311252572.4A CN202311252572A CN117343137A CN 117343137 A CN117343137 A CN 117343137A CN 202311252572 A CN202311252572 A CN 202311252572A CN 117343137 A CN117343137 A CN 117343137A
- Authority
- CN
- China
- Prior art keywords
- oyster
- bacterial cellulose
- galactosylated
- oligopeptide
- astaxanthin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/12—Ketones
- A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
-
- A—HUMAN NECESSITIES
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Abstract
An oyster oligopeptide and a compound thereof, a preparation method and an application for treating hepatic fibrosis diseases belong to the technical field of biology, and the oyster oligopeptide has the amino acid sequence of Asp-Pro-Tyr-Pro-Phe-Lys, and has obvious hepatic stellate cell activity inhibition effect.
Description
Technical Field
The invention relates to the technical field of biology, in particular to oyster oligopeptide and a preparation method thereof, a cellulose carrier compound and a preparation method and application thereof, and particularly relates to liver targeting particles (carriers) for carrying oyster oligopeptide and astaxanthin and a preparation method of the compound thereof and an effect of the liver targeting particles (carriers) in improving liver fibrosis.
Background
Liver Fibrosis (LF) refers to the excessive proliferation and abnormal deposition of diffuse extracellular matrix (extracellular matrix, ECM), especially collagen, in the liver after the liver is damaged, and finally results in destruction of liver structure and pseudolobule formation, which is one of the main causes of liver cirrhosis and even liver cancer. The pathological mechanism of liver fibrosis is imbalance in synthesis and degradation of extracellular matrices such as liver tissue collagen, leading to collagen fiber deposition. Further development of liver fibrosis can lead to severe liver diseases such as cirrhosis, liver cancer, etc. Although many scientific studies have shown reversibility in early stages of liver fibrosis, this reversal process is very slow and even under the influence of complications, its reversal appears to be very confusing. Therefore, intensive studies on the pathogenesis and active search for effective drugs have profound significance for the treatment of liver fibrosis.
Oyster oligopeptide has various biological activities, such as increasing serum and testosterone levels of men, regulating blood lipid, inhibiting platelet aggregation, improving hyperglycemia symptoms, enhancing immunity, promoting metabolism, etc. Researches show that oyster oligopeptide can improve liver enzyme system of alcoholic liver disease patients, stabilize liver cell membrane structure and soften liver after long-term administration. Modern medical research also shows that it can induce fibroblast proliferation, activation and apoptosis, and has certain anti-fibrosis effect on different tissues and organs. In addition, oyster oligopeptide- κB (NF- κB)/iNOS signaling pathway can promote hepatic stellate cell apoptosis and reduce ECM component expression. However, oyster peptides are difficult to be targeted and delivered to the liver and to exert efficacy perfectly due to stability, gastrointestinal physiological disorders and limitations of physicochemical properties. On the other hand, astaxanthin (3, 3' -dihydroxy-4, 4' -diketo- β, β ' -carotene) is distributed in marine algae, fungi and crustaceans, with haematococcus pluvialis being the most desirable source of natural astaxanthin. Several studies have demonstrated that astaxanthin, as a natural strong antioxidant, is capable of reducing the expression of TGF-beta 1-induced fibrosis genes α -SMA and Col1A1 in LX-2 cells and inhibiting the activation of hepatic stellate cells, thereby inhibiting the development of hepatic fibrosis. In addition, the strong antioxidant capacity can regulate the oxidative stress of liver cells, and has important effects in relieving liver insulin resistance, nonalcoholic fatty liver and other aspects. However, the astaxanthin has the problems of low water solubility, poor stability and the like, and seriously influences the application of the astaxanthin in the treatment of liver injury.
Therefore, in order to improve the bioavailability of oyster oligopeptide and astaxanthin, the oyster oligopeptide and astaxanthin can achieve good effects of protecting the liver and relieving hepatic fibrosis after being taken orally, and a proper and effective technical means capable of resisting the influence of the digestive tract environment, enhancing the permeation effect of mucus and epithelial barriers and stabilizing the targeting of hepatocytes is constructed, so that the oyster oligopeptide and astaxanthin are also one of the hot spots of research in recent years.
Disclosure of Invention
The present invention is directed to the above objects, in a first aspect, to solve the problem that peptide fragments modulate hepatic stellate cell activity, reduce the accumulation of extracellular matrix in large amounts, and thereby alleviate liver fibrosis, some embodiments of the present application provide oyster oligopeptide, the amino acid sequence of which comprises Asp-Pro-Tyr-Pro-Phe-Lys.
In a second aspect, in some embodiments of the present application, there is provided a method for preparing the oyster oligopeptide of the first aspect, comprising
S11, adding the oyster into water with 15-20 times of mass volume to prepare homogenate, and placing the homogenate into an enzymolysis tank.
S12, adding compound protease accounting for 2-5% of the oyster mass into an enzymolysis tank, and heating and inactivating enzyme after enzymolysis to obtain oyster protein enzymolysis liquid, wherein the compound protease comprises pepsin, papain and flavourzyme, wherein the pepsin comprises papain and flavourzyme= (3-5): (2-3): (2-4).
S13, centrifuging the oyster protein enzymatic hydrolysate to obtain clear liquid.
S14, performing membrane separation on the clear liquid, wherein the molecular weight cut-off is 3000Da.
S15, spray drying the membrane-passing solution to obtain oyster oligopeptide powder.
S16, adding water into oyster oligopeptide powder to dissolve, wherein the concentration is 20-30 mg/mL.
S17, separating and purifying the dissolved substances by adopting a gel chromatographic column, wherein the eluting solvent is deionized water, the eluting flow rate is 0.3-0.5 mL/min, detecting the absorbance of the solution at 220nm, and collecting the eluting peak with the retention time of 5-6 min.
S18, further purifying by adopting a reverse phase column, wherein the chromatographic conditions are as follows: the mobile phase A is trifluoroacetic acid water with the volume percentage of 0.05-0.1%, the mobile phase B is acetonitrile, and the gradient elution condition is as follows: 0 to 10min,5 percent of B,10 to 20min,5 to 15 percent of B,20 to 30min,15 percent of B to 30 percent of B,30 to 40min,30 percent of B to 40 percent of B, the flow rate is 0.8 to 1.0mL/min, and chromatographic peaks with retention time of 11 to 12 min are collected to obtain the oyster oligopeptide.
According to the preparation method of oyster oligopeptide in some embodiments of the present application, in step S12, the enzymolysis temperature is 40-50 ℃, the enzyme reaction pH is 8.0-9.0, and the enzymolysis time is 4 hours.
According to the preparation method of oyster oligopeptide in some embodiments of the present application, in step S12, after enzymolysis, the temperature is raised to 80-90 ℃ for inactivating enzyme for 10 minutes, so as to obtain oyster protein enzymolysis liquid.
According to the preparation method of oyster oligopeptide of some embodiments of the present application, in step S13, oyster protein hydrolysate is centrifuged at 8000 rpm for 10 minutes.
According to some embodiments of the present application, in step S16, oyster oligopeptide powder is dissolved in water at a concentration of 25mg/mL.
According to the preparation method of oyster oligopeptide of some embodiments of the present application, in step S17, separation and purification are performed by using a SephadexG10 gel chromatographic column of 20mm×100 mm.
According to some embodiments of the present application, the oyster oligopeptide preparation method, step S18, further purification using a C18 RP-HPLC reverse phase column.
According to some embodiments of the present application, in step S18, the oyster oligopeptide is concentrated and freeze-dried to obtain oyster oligopeptide powder.
In a third aspect, in order to address the problem of liver-targeted delivery of oyster oligopeptides, in some embodiments of the present application, oyster oligopeptide-galactosylated bacterial cellulose carrier complexes are provided, as well as methods of making the same, the complexes being made by a method of making comprising
S31, dissolving oyster oligopeptide powder and a galactosylated bacterial cellulose carrier in water, stirring, and freeze-drying to obtain an oyster oligopeptide-galactosylated bacterial cellulose carrier compound.
Wherein the galactosylated bacterial cellulose carrier is prepared based on the following manner:
s21, dissolving bacterial cellulose in dimethyl sulfoxide (DMSO) solution containing tetrabutylammonium acetate, and stirring to obtain bacterial cellulose solution.
S22, dissolving lactobionic acid in dimethyl sulfoxide (DMSO) solution containing 1-ethyl-3- (3- (dimethylamino) propyl) carbodiimide (EDC) and 1-ethyl-3- (3- (dimethylamino) propyl) carbodiimide (NHS), and stirring to obtain lactobionic acid solution.
S23, adding the bacterial cellulose solution into the lactobionic acid solution, heating and stirring, and removing impurities to obtain the galactosylated bacterial cellulose carrier.
According to the preparation method of the oyster oligopeptide-galactosylated bacterial cellulose carrier compound and the preparation method thereof in some embodiments of the application, in the step S21, bacterial cellulose is dissolved in a dimethyl sulfoxide solution containing tetrabutyl ammonium acetate, and the mixture is stirred until no jelly is coagulated, so that a uniform bacterial cellulose solution with the mass-volume ratio of 1-5% is obtained.
In step S22, lactobionic acid is dissolved in dimethyl sulfoxide (DMSO) solution containing 1-ethyl-3- (3- (dimethylamino) propyl) carbodiimide (EDC) and 1-ethyl-3- (3- (dimethylamino) propyl) carbodiimide (NHS), and the mixture is stirred for carboxyl activation for 1 to 3 hours, more preferably for 2 hours, to obtain a lactobionic acid solution with a mass-volume ratio of 1 to 10%.
Wherein, lactobionic acid: 1-ethyl-3- (3- (dimethylamino) propyl) carbodiimide (EDC): 1-ethyl-3- (3- (dimethylamino) propyl) carbodiimide (NHS) =1 to 5:1:1, preferably 1:1:1.
In the step S23, the bacterial cellulose solution is added into the lactobionic acid solution, heated and stirred, impurities are removed, and the galactosylated bacterial cellulose carrier powder is obtained by freeze drying, wherein the heating and stirring temperature is 40-50 ℃, and the heating and stirring time is 10-24 hours.
According to some embodiments of the present application, the preparation of oyster oligopeptide-galactosylated bacterial cellulose carrier complex and the preparation method thereof, in step S23, the removal of impurities comprises removing unreacted lactobionic acid, bacterial cellulose and dimethyl sulfoxide (DMSO) solution containing tetrabutylammonium acetate by dialysis with distilled water in a dialysis tube.
According to some embodiments of the present application, the preparation of oyster oligopeptide-galactosylated bacterial cellulose carrier complex and the preparation method thereof, in step S23, the dialysis time is 24-72 hours, more preferably 72 hours.
According to the preparation method of the oyster oligopeptide-galactosylated bacterial cellulose carrier compound and the preparation method thereof in some embodiments of the present application, in step S31, oyster oligopeptide and galactosylated bacterial cellulose carrier powder are dissolved in 10 times volume of ultrapure water, stirred and reacted for 3 to 6 hours at 90 to 100 ℃, and freeze-dried, so as to obtain oyster oligopeptide-galactosylated bacterial cellulose carrier compound powder.
According to some embodiments of the present application, the mass ratio of oyster oligopeptide to galactosylated bacterial fiber carrier powder in step S31 is 1-10:1, more preferably 5:1.
in a fourth aspect, in order to solve the problem of liver-targeted delivery of oyster oligopeptide and astaxanthin, in some embodiments of the present application, there is provided a complex of galactosylated bacterial cellulose carrier carrying oyster oligopeptide and astaxanthin and a preparation method thereof of the third aspect, which is a method for preparing oyster oligopeptide-astaxanthin-galactosylated bacterial cellulose carrier complex, the complex being prepared by a preparation method comprising
S41, dissolving astaxanthin in an ethanol solution, and dissolving the oyster oligopeptide-galactosylated bacterial cellulose carrier compound in water.
S42, adding the ethanol solution dissolved with astaxanthin oil into the solution of the oyster oligopeptide-galactosylated bacterial cellulose carrier complex, heating for reaction, and evaporating to remove ethanol to obtain the oyster oligopeptide-astaxanthin-galactosylated bacterial cellulose carrier complex.
According to some embodiments of the present application, in step S41, astaxanthin oil having an astaxanthin content of 10% is dissolved in an ethanol solution and stirred uniformly.
According to some embodiments of the present application, the method for preparing oyster oligopeptide-astaxanthin-galactosylated bacterial cellulose carrier complex, in step S41, has a mass-volume ratio of astaxanthin oil to ethanol solution of 1-10%, more preferably 5%.
According to some embodiments of the present application, the mass ratio of astaxanthin oil to oyster oligopeptide-galactosylated bacterial cellulose carrier complex in step S41 is 1:1-10, more preferably 1:1.
According to some embodiments of the present application, the method of preparing an oyster oligopeptide-astaxanthin-galactosylated bacterial cellulose carrier complex, in step S41, the oyster oligopeptide-galactosylated bacterial cellulose carrier complex is dissolved in 10 volumes of ultrapure water.
According to the method for preparing the oyster oligopeptide-astaxanthin-galactosylated bacterial cellulose carrier complex in some embodiments of the present application, in step S42, an ethanol solution in which astaxanthin oil is dissolved is slowly dripped into the solution of the oyster oligopeptide-galactosylated bacterial cellulose carrier complex, the reaction is performed by heating and stirring during the dripping process, ethanol is removed by evaporation, and the oyster oligopeptide-astaxanthin-galactosylated bacterial cellulose carrier complex powder is obtained by freeze drying.
According to some embodiments of the present application, the method for preparing oyster oligopeptide-astaxanthin-galactosylated bacterial cellulose carrier complex in step S42, the stirring speed is 500-1500 r/min, the reaction temperature is 40-60 ℃, and the reaction time is 2-4 hours.
The beneficial effects are that:
the invention obtains an active peptide from oyster oligopeptide by gel exclusion chromatography and separation and purification of reversed phase high performance liquid chromatography, which has the functions of regulating hepatic stellate cell activity and reducing mass accumulation of extracellular matrix of liver so as to relieve hepatic fibrosis, and the amino acid sequence is as follows: asp-Pro-Tyr-Pro-Phe-Lys was searched by on-line databases BIOPEP and EROP-Moscow, and no report was found.
According to the invention, galactose modified bacterial cellulose is used as a liver targeting delivery carrier of oyster oligopeptide and astaxanthin, on one hand, the oyster oligopeptide is connected with galactose grafted on the bacterial cellulose through Maillard reaction, on the other hand, the oyster oligopeptide and the astaxanthin are wrapped through emulsification, and the oyster oligopeptide and the astaxanthin are simultaneously coated by a unique three-dimensional network structure of the bacterial cellulose, so that the release time of the oyster oligopeptide and the astaxanthin can be prolonged, the oyster oligopeptide and the astaxanthin have extremely high biocompatibility and biodegradability, and the existence of a large number of hydroxyl groups also well resists the structural influence of extreme pH value environment changes on a core material. Meanwhile, compared with a galactose modified chitosan carrying system which is reported in many cases, the three-dimensional network structure of bacterial cellulose has extremely large porosity, and the coating effect on the core material is more beneficial to protecting oyster oligopeptide from hydrolysis and digestion effects of various proteases in gastrointestinal tracts and improving the stability of astaxanthin in the gastrointestinal tracts. Therefore, the oyster oligopeptide-astaxanthin-galactosylated bacterial cellulose particles have strong flexibility and high adhesive force, can cope with the peristaltic movement of gastrointestinal muscle and the shear stress caused by the flow rate of gastric juice in a cavity, reduce the mechanical degradation of the oyster oligopeptide and the astaxanthin, enhance the adhesion effect on a firm adhesion layer, effectively prolong the stay time in the gastrointestinal tract to enhance the absorption, and reduce the phagocytic probability of macrophages in a reticuloendothelial system after the core material enters the systemic circulation, thereby improving the efficiency of delivering to target cells.
In conclusion, the oyster oligomerization active peptide-astaxanthin-galactosylated bacterial cellulose prepared by the method provided by the invention protects the molecular structure of the oyster oligomerization active peptide and astaxanthin in the gastrointestinal digestive tract, enhances the absorption and delay the release of the oyster oligomerization active peptide and astaxanthin, and finally, the oyster oligomerization active peptide and astaxanthin are delivered to liver cells in a precise targeting way. Oyster oligomerization active peptide (Asp-Pro-Tyr-Pro-Phe-Lys) and astaxanthin are synergistic, so that the pre-activation of hepatic stellate cells is inhibited, the over-expression of ECM is reduced, the oxidative stress system of the hepatic cells is regulated, the steatosis is improved, and the oyster oligomerization active peptide has better liver fibrosis treatment effect compared with single use. The method is simple and easy to operate, is suitable for industrial preparation scenes, and has wide application prospect in prevention and treatment of hepatic fibrosis.
Drawings
FIG. 1 three-dimensional network of bacterial cellulose.
Detailed Description
The invention is further described below with reference to the drawings and examples, which should not be construed as limiting the scope of the invention as claimed.
In embodiments, the present invention provides a carrier complex and a method for preparing the same, which can reduce the influence of the pH value of oyster oligopeptide and astaxanthin and the inactivation of digestive hydrolase such as pepsin and trypsin in the gastrointestinal tract, enhance the adhesion on the mucous membrane to prolong the time in the gastrointestinal tract to improve the absorption, and directly target the delivery to the damaged liver cells for fibrosis improvement after the absorption.
The carrier composite is subjected to the following process steps:
step 1, preparation of oyster oligopeptide: adding 15-20 times of water into a dried oyster sample to prepare a homogenate, placing the homogenate into an enzymolysis tank, adding compound protease with the mass of 2-5% of that of the oyster for enzymolysis for 4 hours, heating to 80-90 ℃ for enzyme deactivation for 10 minutes after the enzymolysis is finished to obtain oyster protein enzymolysis liquid, centrifuging the protease enzymolysis liquid for 10 minutes at 8000 rpm to remove granular substances, separating by adopting a membrane separation technology, intercepting the molecular weight of 3000Da, and spray-drying the membrane-filtered liquid to obtain oyster oligopeptide powder; dissolving oyster oligopeptide powder in water with concentration of 25mg/mL, separating and purifying with SephadexG 10 (20 mm×100 mm) gel chromatographic column, eluting with deionized water at flow rate of 0.3-0.5mL/min, detecting absorbance at 220nm, and collecting eluting peak with retention time of 5-6 min; further purification was performed using a C18 RP-HPLC reverse phase column under the following chromatographic conditions: the mobile phase A is trifluoroacetic acid water with the volume percentage of 0.05-0.1%, the mobile phase B is acetonitrile, and the gradient elution condition is as follows: 0 to 10min,5 percent of B,10 to 20min,5 to 15 percent of B,20 to 30min,15 percent of B to 30 percent of B,30 to 40min,30 percent of B to 40 percent of B, the flow rate is 0.8 to 1.0mL/min, the chromatographic peak with the retention time of 11 to 12 min is collected, concentrated and freeze-dried to obtain oyster oligomerization active peptide powder.
Further, the enzymolysis in the step 1 adopts pepsin, papain, flavourzyme= (3-5), flavourzyme (2-3), compound protease (2-4), the enzymolysis temperature is 40-50 ℃, and the pH value of the enzymatic reaction is controlled to be 8.0-9.0.
Further, the amino acid sequence of the oyster low-molecular weight active peptide in the step 1 is as follows: asp-Pro-Tyr-Pro-Phe-Lys.
Step 2, preparation of galactosylated bacterial cellulose carrier: dissolving bacterial cellulose in dimethyl sulfoxide (DMSO) solution containing tetrabutylammonium acetate, stirring until no jelly is coagulated, and preparing into uniform bacterial cellulose solution; dissolving lactobionic acid in dimethyl sulfoxide (DMSO) solution containing 1-ethyl-3- (3- (dimethylamino) propyl) carbodiimide EDC and 1-ethyl-3- (3- (dimethylamino) propyl) carbodiimide NHS (1:1), and stirring to activate carboxyl; adding lactobionic acid solution into bacterial cellulose solution, heating and stirring, dialyzing with distilled water in dialysis tube to remove unreacted lactobionic acid, bacterial cellulose and DMSO solvent containing tetrabutylammonium acetate, and lyophilizing to obtain galactosylated bacterial cellulose powder.
Further, the mass-volume ratio of the bacterial cellulose solution in the step 2 is 1-5%.
Further, the mass volume ratio of the lactobionic acid solution in the step 2 is 1-10%.
Further, the mass ratio of the lactobionic acid, EDC and NHS in the step 2 is 1-5: 1:1, preferably 1:1:1.
Further, the activation time in step 2 is 1 to 3 hours, preferably 2 hours.
Further, the heating and stirring temperature in the step 2 is 40-50 ℃ and the stirring time is 10-24 hours.
Further, the dialysis time in step 2 is 24 to 72 hours, preferably 72 hours.
Step 3, preparation of oyster oligomerization active peptide-galactosylated bacterial cellulose complex carrier: and (2) re-dissolving the oyster oligomerization active peptide powder prepared in the step (1) and the galactosylated bacterial cellulose powder in 10 times of ultrapure water, stirring at 90-100 ℃ for reaction for 3-6 hours, and freeze-drying to obtain oyster oligomerization active peptide-galactosylated bacterial cellulose composite carrier powder.
Further, in the step 3, the mass ratio of the oyster oligomerization active peptide powder to the galactosylated bacterial fiber powder is 1-10:1, preferably 5:1.
by the steps, the oyster oligopeptide-galactosylated bacterial cellulose carrier compound is obtained, and liver targeting delivery of the oyster oligopeptide can be realized through the galactosylated bacterial cellulose carrier. As a further aspect, the oyster oligopeptide is the oyster oligopeptide with the function of regulating hepatic stellate cell activity and reducing mass accumulation of extracellular matrix so as to relieve hepatic fibrosis, which is of course understood that the oyster oligopeptide can be other oyster oligopeptides requiring liver targeting delivery in the prior art. Besides the oyster oligopeptide and the carrier, the oyster oligopeptide can be further compounded with astaxanthin, and the astaxanthin is used as a natural strong antioxidant, and has important effects in inhibiting liver fibrosis, preventing and treating liver tumor, relieving liver insulin resistance, non-alcoholic fatty liver and the like. Therefore, the present invention further includes the following step 4.
Step 4, preparation of oyster oligomerization active peptide-astaxanthin-galactosylated bacterial cellulose complex carrier: dissolving astaxanthin oil with the astaxanthin content of 10% in ethanol solution, stirring uniformly, dissolving oyster oligomerization active peptide-galactosylated bacterial cellulose powder in ultrapure water with the volume of 10 times, slowly dripping the astaxanthin oil ethanol solution into the stirred oyster oligomerization active peptide-galactosylated bacterial cellulose solution, heating for reaction, evaporating to remove ethanol, and freeze-drying to obtain oyster oligomerization active peptide-astaxanthin-galactosylated bacterial cellulose microparticle (OP-AST-GBC) powder;
further, the mass-volume ratio of the astaxanthin oil to the ethanol solution in the step 4 is 1-10%, preferably 5%.
Further, the mass ratio of the astaxanthin oil to the oyster oligomerization active peptide-galactosylated bacterial cellulose in the step 4 is 1:1-10, preferably 1:1.
Further, in the step 4, the stirring speed is 500-1500 r/min, the reaction temperature is 40-60 ℃, and the reaction time is 2-4 hours.
Although many studies have been advanced on galactose-mediated asialoglycoprotein receptor targeted delivery systems on hepatic parenchymal cell membranes, many have focused on directly modifying the drug with galactose or constructing galactose-modified liposome or polymer micelle carriers to achieve good targeted delivery performance. However, people suffering from hyperlipidemia, obesity and other diseases may not accept lipid-based delivery systems, and the high pressure homogenization, ultrasound and the like involved in the operation of emulsion delivery systems may affect the astaxanthin structure, and thus the entrapment rate, stability, bioavailability and other properties.
According to the invention, galactose modified bacterial cellulose is used as a liver targeting delivery carrier of oyster oligopeptide and astaxanthin, on one hand, the oyster oligopeptide is connected with galactose grafted on the bacterial cellulose through Maillard reaction, on the other hand, the oyster oligopeptide and the astaxanthin are wrapped through emulsification, and the oyster oligopeptide and the astaxanthin are simultaneously coated by a unique three-dimensional network structure of the bacterial cellulose, so that the release time of the oyster oligopeptide and the astaxanthin can be prolonged, the oyster oligopeptide and the astaxanthin have extremely high biocompatibility and biodegradability, and the existence of a large number of hydroxyl groups also well resists the structural influence of extreme pH value environment changes on a core material. Meanwhile, compared with a galactose modified chitosan carrying system which is reported in many cases, the three-dimensional network structure of bacterial cellulose has extremely large porosity, and the coating effect on the core material is more beneficial to protecting oyster oligopeptide from hydrolysis and digestion effects of various proteases in gastrointestinal tracts and improving the stability of astaxanthin in the gastrointestinal tracts. Therefore, the oyster oligopeptide-astaxanthin-galactosylated bacterial cellulose particles have strong flexibility and high adhesive force, can cope with the peristaltic movement of gastrointestinal muscle and the shear stress caused by the flow rate of gastric juice in a cavity, reduce the mechanical degradation of the oyster oligopeptide and the astaxanthin, enhance the adhesion effect on a firm adhesion layer, effectively prolong the stay time in the gastrointestinal tract to enhance the absorption, and reduce the phagocytic probability of macrophages in a reticuloendothelial system after the core material enters the systemic circulation, thereby improving the efficiency of delivering to target cells.
In conclusion, the oyster oligomerization active peptide-astaxanthin-galactosylated bacterial cellulose prepared by the method provided by the invention protects the molecular structure of the oyster oligomerization active peptide and astaxanthin in the gastrointestinal digestive tract, enhances the absorption and delay the release of the oyster oligomerization active peptide and astaxanthin, and finally, the oyster oligomerization active peptide and astaxanthin are delivered to liver cells in a precise targeting way. Oyster oligomerization active peptide (Asp-Pro-Tyr-Pro-Phe-Lys) and astaxanthin are synergistic, so that the pre-activation of hepatic stellate cells is inhibited, the over-expression of ECM is reduced, the oxidative stress system of the hepatic cells is regulated, the steatosis is improved, and the oyster oligomerization active peptide has better liver fibrosis treatment effect compared with single use. The method is simple and easy to operate, is suitable for industrial preparation scenes, and has wide application prospect in prevention and treatment of hepatic fibrosis.
Example 1:
step 1, adding 1000g of dried oyster into 15 times of water by mass volume to prepare homogenate, placing the homogenate into an enzymolysis tank, and then adding 30g of compound protease, wherein the protease comprises pepsin: papain: flavourzyme=5:2:3, adjusting the pH to 8.8, carrying out enzymolysis for 5 hours at 40 ℃, and then heating to 90 ℃ for enzyme deactivation for 10 minutes to obtain oyster protein enzymolysis liquid; centrifuging at 8000 rpm for 10min, ultrafiltering at 3000Da, and spray drying to obtain oyster oligopeptide powder 100g; dissolving oyster oligopeptide powder in water with concentration of 25mg/mL, separating and purifying with SephadexG 10 (20 mm×100 mm) gel chromatographic column, eluting with deionized water at flow rate of 0.4mL/min, detecting absorbance at 220nm, and collecting eluting peak with retention time of 5.52 min; further purification was performed using a C18 RP-HPLC reverse phase column under the following chromatographic conditions: the mobile phase A is trifluoroacetic acid water with the volume percentage of 0.1%, the mobile phase B is acetonitrile, and the gradient elution condition is as follows: 0 to 10min,5 percent of B,10 to 20min,5 to 15 percent of B,20 to 30min,15 percent of B to 30 percent of B,30 to 40min,30 percent of B to 40 percent of B, the flow rate is 0.8mL/min, the chromatographic peak with the retention time of 11.35 min is collected, concentrated and freeze-dried to obtain 8.4g of oyster oligomerization active peptide powder.
Step 2, taking 20g of bacterial cellulose, dissolving the bacterial cellulose in 1000ml of DMSO solution containing tetrabutylammonium acetate, stirring until no jelly is coagulated, and preparing uniform bacterial cellulose solution; 15g of lactobionic acid is taken and dissolved in 1000ml of DMSO solution containing 5g EDC and 5g NHS, and the mixture is stirred for carboxyl activation for 2 hours; the bacterial cellulose solution was added to lactobionic acid solution and heated to 45℃with stirring for 12 hours, and then dialyzed for 24 hours, followed by freeze-drying to obtain 34g of galactosylated bacterial cellulose powder.
And 3, re-dissolving 8g of oyster oligomerization active peptide powder and 4g of galactosylated bacterial cellulose powder in 10 times of ultra-pure water, stirring at 90 ℃ for reaction for 6 hours, and freeze-drying to obtain 12g of oyster oligomerization active peptide-galactosylated bacterial cellulose composite carrier powder.
And 4, dissolving 10g of astaxanthin oil in 100ml of ethanol solution, uniformly stirring, dissolving 10g of oyster oligomerization active peptide-galactosylated bacterial cellulose powder in ultrapure water with the volume of 10 times, then slowly dripping the astaxanthin oil ethanol solution into the stirred oyster oligomerization active peptide-galactosylated bacterial cellulose solution, heating to 60 ℃ for reaction for 2 hours at the stirring speed of 500r/min, evaporating to remove ethanol, and freeze-drying to obtain 20g of OP-AST-GBC microparticle powder.
Example 2:
step 1, adding 1000g of dried oyster into 2 times of water by mass volume to prepare homogenate, placing the homogenate into an enzymolysis tank, and then adding g of compound protease, wherein the protease comprises pepsin: papain: flavourzyme=3:1:3, adjusting the pH to 8.2, carrying out enzymolysis for 6 hours at 50 ℃, and then heating to 90 ℃ for enzyme deactivation for 10 minutes to obtain oyster protein enzymolysis liquid; centrifuging the oyster protein hydrolysate at 8000 rpm for 10min, ultrafiltering and separating at 3000Da, and spray drying to obtain oyster oligopeptide powder 105g; dissolving oyster oligopeptide powder in water with concentration of 25mg/mL, separating and purifying with SephadexG10 (20 mm×100 mm) gel chromatographic column, eluting with deionized water at flow rate of 0.5mL/min, detecting absorbance at 220nm, and collecting eluting peak with retention time of 5.78 min; further purification was performed using a C18 RP-HPLC reverse phase column under the following chromatographic conditions: the mobile phase A is trifluoroacetic acid water with the volume percentage of 0.1%, the mobile phase B is acetonitrile, and the gradient elution condition is as follows: 0 to 10min,5 percent of B,10 to 20min,5 to 15 percent of B,20 to 30min,15 percent of B to 30 percent of B,30 to 40min,30 percent of B to 40 percent of B, the flow rate is 1mL/min, the chromatographic peak with retention time of 11.59min is collected, concentrated and freeze-dried to obtain 9.2g of oyster oligomerization active peptide powder.
Step 2, taking 30g of bacterial cellulose, dissolving the bacterial cellulose in 1000ml of DMSO solution containing tetrabutyl ammonium acetate, stirring until no jelly is coagulated, and preparing uniform bacterial cellulose solution; 10g of lactobionic acid is taken and dissolved in 1000ml of DMSO solution containing 4gEDC and 4gNHS, and the mixture is stirred for carboxyl activation for 1 hour; the bacterial cellulose solution was added to lactobionic acid solution and heated to 40℃with stirring for 24 hours, and then dialyzed for 36 hours and freeze-dried to obtain 38g of galactosylated bacterial cellulose powder.
And 3, re-dissolving 9g of oyster oligopeptide powder and 3g of galactosylated bacterial cellulose powder in 10 times of volume of ultrapure water, stirring at 100 ℃ for reaction for 4 hours, and freeze-drying to obtain 12g of oyster oligopeptide-galactosylated bacterial cellulose composite carrier powder.
And 4, dissolving 10g of astaxanthin oil in 100ml of ethanol solution, uniformly stirring, dissolving 10g of oyster oligomerization active peptide-galactosylated bacterial cellulose powder in ultrapure water with the volume of 10 times, then slowly dripping the astaxanthin oil ethanol solution into the stirred oyster oligomerization active peptide-galactosylated bacterial cellulose solution, heating to 55 ℃ for reaction for 3 hours at the stirring speed of 1200r/min, evaporating to remove ethanol, and freeze-drying to obtain 20g of OP-AST-GBC microparticle powder.
Comparative example 1:
10g of oyster oligoactive peptide-galactosylated bacterial cellulose complex carrier (OP-GBC) powder was obtained by the procedure of step 1 to step 3 of example 1.
Comparative example 2:
step 1, taking 16.2g of bacterial cellulose, dissolving the bacterial cellulose in 1000ml of DMSO solution containing tetrabutylammonium acetate, stirring until no jelly is coagulated, and preparing uniform bacterial cellulose solution; 10g of lactobionic acid is taken and dissolved in 1000ml of DMSO solution containing 5g EDC and 5g NHS, and the mixture is stirred for carboxyl activation for 2 hours; the bacterial cellulose solution was added to a lactobionic acid solution, heated to 40℃and stirred for 24 hours, and then dialyzed for 48 hours and freeze-dried to obtain 30g of galactosylated bacterial cellulose powder.
Step 2, taking 10g of astaxanthin oil, dissolving in 100ml of ethanol solution, uniformly stirring, taking 30g of galactosylated bacterial cellulose powder, redissolving in 10 times of ultrapure water, slowly dripping the astaxanthin oil ethanol solution into the stirred galactosylated bacterial cellulose solution, heating to 50 ℃ for reaction for 3 hours at the stirring speed of 1000r/min, evaporating to remove ethanol, and freeze-drying to obtain 40g of astaxanthin-galactosylated bacterial cellulose (AST-GBC) microparticle powder.
Comparative example 3:
step 1: 9.5g of oyster low-polymerization-activity peptide powder was obtained by the procedure of step 1 of example 1.
Step 2: dissolving 20g of chitosan in 1000mL of 0.1mol/L MES buffer, regulating the pH value to 5.7 after the chitosan is completely dissolved, simultaneously adding 8g of lactobionic acid, 10g of NHS and 10g of EDC, stirring for reaction for 30min, standing at 4 ℃ for 12h after the chitosan is completely dissolved, transferring to room temperature for 12h, finally dialyzing for 3d by using a dialysis bag, taking out the dialyzate, and freeze-drying to obtain 30g of galactosylated chitosan powder.
And 3, re-dissolving 9g of oyster oligomerization active peptide powder and 3g of galactosylated chitosan powder in 10 times of volume of ultrapure water, stirring at 90 ℃ for reaction for 6 hours, and freeze-drying to obtain 12g of oyster oligomerization active peptide powder-galactosylated chitosan powder.
And 4, dissolving 10g of astaxanthin oil in 100ml of ethanol solution, uniformly stirring, dissolving 20g of oyster oligomerization active peptide-galactosylated chitosan powder in 10 times of ultrapure water, slowly dripping the astaxanthin oil ethanol solution into the stirred oyster oligomerization active peptide-galactosylated chitosan solution, heating to 60 ℃ for reaction for 2 hours at the stirring speed of 500r/min, evaporating to remove ethanol, and freeze-drying to obtain 30g of oyster oligomerization active peptide-astaxanthin-galactosylated chitosan (OP-AST-GC) powder.
Experiment 1: liver astrocyte activity modulating effect of oyster oligomerization active peptide
Culturing LX-2 cells conventionally (10% fetal bovine serum and 100U/ml penicillin, 100 μg/ml streptomycin in medium), taking LX-2 cells in logarithmic phase, digesting with pancreatin, centrifuging conventionally, and adjusting cell to 1×10 5 After the cell suspension is per mL, inoculating the cell suspension into a 96-well plate, adding oyster oligopeptide water solution with the concentration of 4 mug/mL, culturing for 24 hours at 37 ℃, washing the cells for 2 times by using PBS Buffer solution with the temperature of 4 ℃, collecting the cells and preparing the cells into 1 multiplied by 10 by using Binding Buffer solution 6 Cell/ml suspension, apoptosis was detected by flow cytometry. The result shows that compared with a blank group, the LX-2 cell inhibition rate treated by the oyster oligomerization active peptide aqueous solution reaches 35.24%, the obvious hepatic stellate cell activity inhibition effect is shown, and the activation and the starting of hepatic stellate cells are very critical in the process of occurrence and development of hepatic fibrosis.
Experiment 2: in vitro simulation of gastrointestinal fluid digestion
5mL of concentrated hydrochloric acid (37%), 5mL of Tween 80,2g of Na Cl and 3.2g of pepsin were taken, water was added to a constant volume of 1L, the pH value was adjusted to 1.2, and the mixture was filtered through a 0.22 μm organic filter membrane to prepare simulated gastric fluid before use. Taking 6.8g of monopotassium phosphate, 10g of trypsin, 5mL of Tween 80, adding water to a volume of 1L, completely dissolving by ultrasonic treatment, adjusting the pH value to 6.8 by using a sodium hydroxide solution diluted by dilute hydrogen and oxygen, and filtering by using a 0.22 mu m organic filter membrane before use to obtain simulated intestinal juice. OP-AST-GBC1,2 obtained in examples 1,2 and OP-GBC, AST-GBC, OP-AST-GC powder 4mg obtained in comparative example were respectively taken, placed in dialysis bag (MWCO 3500), immersed in 200mL of artificial gastric juice (or intestinal juice), stirred in a constant temperature magnetic stirrer at 37 ℃ for system temperature at a speed of 100rpm/min, 1mL was sampled at 30min and time points of 1, 6, 12, 24 hours, respectively, while 1mL of blank artificial gastric juice (or intestinal juice) of the same matrix was added after the sampling, the supernatant was obtained after centrifugation, and after filtration with 0.22 μm organic filter membrane, the content of oyster oligopeptide and astaxanthin was detected by high performance liquid chromatograph. The results are shown below:
TABLE 1 Release Rate in Artificial gastric juice
TABLE 2 Release Rate in Artificial intestinal juice
According to the release rate results of OP-AST-GBC1, 2, OP-GBC, AST-GBC and OP-AST-GC samples in the artificial gastrointestinal fluid, the release rate of the core materials of each group in the artificial gastrointestinal fluid is gradually increased along with the increase of time, and the release rate tends to be stable in 6h and 12h, the OP-AST-GBC group has a protective effect on oyster oligopeptide-astaxanthin, oyster peptide-galactose-bacterial cellulose and astaxanthin-bacterial cellulose due to the multiple wrapping effect, so that the stability is greatly improved, the release rate is lower than that of a carrier system for independently carrying oyster peptide or astaxanthin, and meanwhile, the stability is obviously better than that of a carrier system of galactosylated chitosan due to the three-dimensional network effect of bacterial cellulose, so that the OP-AST-GBC system can stably exist in the artificial gastrointestinal fluid environment, and has a protective effect on oyster oligopeptide and astaxanthin, and is favorable for being absorbed by human bodies.
Experiment 3: liver targeting
Male SD rats were bred adaptively for 3-5d, randomly divided into OP-AST-GBC1, 2 groups (5 mg/kg), AST-GBC group (3 mg/kg), and OP-AST-GC group (5 mg/kg), each group being 12. The rats were fasted for 12 hours before administration, and were respectively perfused with a stomach according to doses, and after administration, the rats were sacrificed at the time points of 0.5,1,4, 12, 24 hours (12 rats per group, 3 rats per group), and after blood was discharged, liver, lung, and kidney samples were obtained, and then 2 times of physiological saline was added, and the samples were homogenized using a tissue homogenizer. Precisely sucking 200 mu L of tissue homogenate into a 1.5mL centrifuge tube, adding 600 mu L of methanol, vortex shaking for 2min, centrifuging for 5min at 10000r/min, precisely sucking 500 mu L of supernatant, blowing nitrogen to dry, re-dissolving 200 mu L of methanol, vortex mixing for 1min, taking 20 mu L of sample, and measuring astaxanthin content by HPLC. The liver targeting of OP-AST-GBC was evaluated with targeting efficiency (Targeting efficiency, te) according to the method of Gupta. The results show that the liver targeting efficiency (56.31 + -3.78) of OP-AST-GBC group is higher than that of AST-GBC group (41.77+ -2.59) and OP-AST-GC group (36.24+ -5.22), and has very high liver targeting.
Experiment 4: improvement effect of liver fibrosis of mice
The 60C 57 mice were randomly divided into 6 groups, namely, a blank group, a model group, an OP-AST-GBC group (5 mg/kg), an OP group (3 mg/kg), an AST group (3 mg/kg) and an OP-AST-GC group (5 mg/kg), 10 animals each. Except for the blank group, mice were intraperitoneally injected with an oil solution of CCl4 (50 ul/10g body weight, CCl4: peanut oil=1:9), three times a week, once a day, for 4 weeks continuously, and the blank group was intraperitoneally injected with the same dose of peanut oil. Mice in each administration group were given the corresponding dose of drug by gavage, and normal and model groups were given equal volumes of distilled water by gavage, 1 time/d, 2W in succession. After the last administration, the mice are sacrificed by a cervical dislocation method after no water control for 4 hours, liver tissues are dissected and isolated by in vitro digestion, and the apoptosis rate of the HSC is detected by double staining of acridine orange/ethidium bromide (AO/EB). Weighing liver tissue, grinding in liquid nitrogen, adding trizol, homogenizing, standing at room temperature for 5 min, removing upper water phase, adding ethanol, mixing, standing at room temperature for 3 min, centrifuging at 2-8deg.C for 5 min, collecting supernatant, and precipitating protein with isopropanol. The mixture was allowed to stand at room temperature for 10 minutes, and the supernatant was discarded by centrifugation at 12000g for 10 minutes at 2-8 ℃. And (3) adding 95% ethanol containing 0.3M guanidine hydrochloride to wash the precipitate, vacuum pumping the protein precipitate, and separating the obtained protein sample for detecting the expression of the collagen-1 and collagen-3 proteins by using Western Blot. The results are shown below:
TABLE 3 collagen expression profiles for groups and HSC apoptosis rates
The results show that the expression of important proteins of extracellular matrix in the liver of mice in the model group is sharply increased, and the liver fibrosis phenomenon is continuously aggravated. The OP-AST-GBC intervention treatment obviously reduces the expression of ECM components such as collagen and the like, and simultaneously inhibits the activation of hepatic stellate cells, so that the liver fibrosis symptom is improved, and the effect is better than that of independently feeding oyster oligopeptide or astaxanthin and an oyster oligopeptide-astaxanthin-galactosylated chitosan carrying system.
The experimental result shows that the oyster oligomerization active peptide-astaxanthin-galactosylated bacterial cellulose delivery system provided by the invention has a good improvement effect on liver fibrosis, and is a potential high-quality liver-protecting ingredient.
The invention discloses a preparation method of oyster oligopeptide astaxanthin liver targeting microspheres and an improvement effect of liver fibrosis. The oyster oligopeptide solution is obtained from oyster through the steps of chopping, enzymolysis, ultrafiltration and the like, is connected and carried with galactosylated modified bacterial cellulose, and finally the oyster peptide-galactosylated bacterial cellulose carrier reacts with astaxanthin oil and is freeze-dried to obtain the oyster oligopeptide-astaxanthin-galactosylated bacterial cellulose particles with liver targeting. The prepared oyster peptide astaxanthin administration system has good protection effect on core active ingredients in gastrointestinal digestive tracts, has extremely high liver targeting and delivery rate after absorption, can relieve liver collagen proliferation and improve liver fibrosis, and has very wide application prospect in prevention and treatment of future liver diseases.
Finally, it should also be noted that the above list is merely a few specific embodiments of the present invention. All derivatives that may be directly derived or suggested by one of ordinary skill in the art from the present disclosure are considered to be within the scope of the present invention.
Claims (11)
1. An oyster oligopeptide, characterized in that its amino acid sequence comprises Asp-Pro-Tyr-Pro-Phe-Lys.
2. A method for preparing oyster oligopeptide according to claim 1, comprising
S11, adding water 15-20 times of the mass volume of the oyster into the oyster to prepare homogenate, and placing the homogenate into an enzymolysis tank;
s12, adding compound protease accounting for 2-5% of the oyster mass into an enzymolysis tank, and heating and inactivating enzyme after enzymolysis to obtain oyster protein enzymolysis liquid, wherein the compound protease comprises pepsin, papain and flavourzyme, wherein the pepsin comprises papain and flavourzyme= (3-5): (2-3): (2-4);
s13, centrifuging oyster protein enzymatic hydrolysate to obtain clear liquid;
s14, performing membrane separation on the clear liquid, wherein the molecular weight cut-off is 3000Da;
s15, spray drying the membrane-passing solution to obtain oyster oligopeptide powder;
s16, adding water into oyster oligopeptide powder to dissolve the oyster oligopeptide powder, wherein the concentration is 20-30 mg/mL;
s17, separating and purifying the dissolved substances by adopting a gel chromatographic column, wherein an eluting solvent is deionized water, the eluting flow rate is 0.3-0.5 mL/min, detecting the absorbance of the solution at 220nm, and collecting an eluting peak with the retention time of 5-6 min;
S18, further purifying by adopting a reverse phase column, wherein the chromatographic conditions are as follows: the mobile phase A is trifluoroacetic acid water with the volume percentage of 0.05-0.1%, the mobile phase B is acetonitrile, and the gradient elution condition is as follows: 0 to 10min,5 percent of B,10 to 20min,5 to 15 percent of B,20 to 30min,15 percent of B to 30 percent of B,30 to 40min,30 percent of B to 40 percent of B, the flow rate is 0.8 to 1.0mL/min, and chromatographic peaks with retention time of 11 to 12 min are collected to obtain the oyster oligopeptide.
3. The method for preparing oyster oligopeptide according to claim 2, wherein,
preferably, in the step S12, the enzymolysis temperature is 40-50 ℃, the enzyme reaction pH is 8.0-9.0, and the enzymolysis time is 4 hours;
preferably, in the step S12, after enzymolysis, the temperature is raised to 80-90 ℃ for enzyme deactivation for 10 minutes to obtain oyster protein enzymolysis liquid;
preferably, in step S13, the oyster protein hydrolysate is centrifuged at 8000 rpm for 10 minutes;
preferably, in the step S16, oyster oligopeptide powder is dissolved in water to obtain the concentration of 25mg/mL;
preferably, in step S17, separation and purification are performed using a SephadexG 10 gel column of 20mm by 100 mm;
preferably, in step S18, further purification is performed using a C18 RP-HPLC reverse phase column;
preferably, in step S18, the oyster oligopeptide is concentrated and freeze-dried to obtain oyster oligopeptide powder.
4. A method for preparing oyster oligopeptide-galactosylated bacterial cellulose carrier complex is characterized by comprising the following steps of
S31, dissolving oyster oligopeptide powder and a galactosylated bacterial cellulose carrier in water, stirring, and freeze-drying to obtain an oyster oligopeptide-galactosylated bacterial cellulose carrier compound;
wherein the galactosylated bacterial cellulose carrier is prepared based on the following manner:
s21, dissolving bacterial cellulose in a dimethyl sulfoxide (DMSO) solution containing tetrabutylammonium acetate, and stirring to obtain a bacterial cellulose solution;
s22, dissolving lactobionic acid in dimethyl sulfoxide (DMSO) solution containing 1-ethyl-3- (3- (dimethylamino) propyl) carbodiimide (EDC) and 1-ethyl-3- (3- (dimethylamino) propyl) carbodiimide (NHS), and stirring to obtain lactobionic acid solution;
s23, adding the bacterial cellulose solution into the lactobionic acid solution, heating and stirring, and removing impurities to obtain the galactosylated bacterial cellulose carrier.
5. The method for preparing oyster oligopeptide-galactosylated bacterial cellulose carrier complex according to claim 4,
in the step S21, bacterial cellulose is dissolved in dimethyl sulfoxide solution containing tetrabutyl ammonium acetate, and stirred until no jelly is coagulated, so that uniform bacterial cellulose solution with the mass-volume ratio of 1-5% is obtained;
In step S22, lactobionic acid is dissolved in dimethyl sulfoxide (DMSO) solution containing 1-ethyl-3- (3- (dimethylamino) propyl) carbodiimide (EDC) and 1-ethyl-3- (3- (dimethylamino) propyl) carbodiimide (NHS), and is stirred for carboxyl activation for 1-3 h, more preferably 2h, to obtain lactobionic acid solution with a mass-volume ratio of 1-10%;
wherein, lactobionic acid: 1-ethyl-3- (3- (dimethylamino) propyl) carbodiimide (EDC): 1-ethyl-3- (3- (dimethylamino) propyl) carbodiimide (NHS) =1 to 5:1:1, preferably 1:1:1;
in the step S23, adding the bacterial cellulose solution into a lactobionic acid solution, heating and stirring, removing impurities, and freeze-drying to obtain galactosylated bacterial cellulose carrier powder, wherein the heating and stirring temperature is 40-50 ℃, and the heating and stirring time is 10-24 hours;
preferably, in step S23, removing impurities includes removing unreacted lactobionic acid, bacterial cellulose and dimethyl sulfoxide (DMSO) solution containing tetrabutylammonium acetate by distilled water dialysis in a dialysis tube;
preferably, in step S23, the dialysis time is 24 to 72 hours, more preferably 72 hours.
6. The method for preparing oyster oligopeptide-galactosylated bacterial cellulose carrier complex according to claim 4,
In the step S31, oyster oligopeptide and galactosylated bacterial cellulose carrier powder are dissolved in 10 times of volume of ultrapure water, stirred and reacted for 3 to 6 hours at the temperature of 90 to 100 ℃, and freeze-dried to obtain oyster oligopeptide-galactosylated bacterial cellulose carrier composite powder;
preferably, in step S31, the mass ratio of oyster oligopeptide to galactosylated bacterial fiber carrier powder is (1-10): 1, more preferably 5:1.
7. an oyster oligopeptide-galactosylated bacterial cellulose carrier complex prepared by the method of any one of claims 4-6.
8. A method for preparing oyster oligopeptide-astaxanthin-galactosylated bacterial cellulose carrier complex is characterized by comprising the following steps of
S41, dissolving astaxanthin in an ethanol solution, and dissolving the oyster oligopeptide-galactosylated bacterial cellulose carrier compound in water;
s42, adding the ethanol solution dissolved with astaxanthin oil into the solution of the oyster oligopeptide-galactosylated bacterial cellulose carrier complex, heating for reaction, and evaporating to remove ethanol to obtain the oyster oligopeptide-astaxanthin-galactosylated bacterial cellulose carrier complex.
9. The method for preparing oyster oligopeptide-astaxanthin-galactosylated bacterial cellulose carrier complex according to claim 8,
In the step S41, astaxanthin oil with the astaxanthin content of 10% is dissolved in ethanol solution and stirred uniformly;
preferably, in step S41, the mass-to-volume ratio of astaxanthin oil to ethanol solution is 1-10%, more preferably 5%;
preferably, in step S41, the mass ratio of astaxanthin oil to oyster oligopeptide-galactosylated bacterial cellulose carrier complex is 1:1-10, more preferably 1:1;
preferably, in step S41, the oyster oligopeptide-galactosylated bacterial cellulose carrier complex is dissolved in 10 volumes of ultra pure water;
in the step S42, slowly dripping an ethanol solution dissolved with astaxanthin oil into the solution of the oyster oligopeptide-galactosylated bacterial cellulose carrier compound, heating and stirring for reaction in the dripping process, evaporating to remove ethanol, and freeze-drying to obtain oyster oligopeptide-astaxanthin-galactosylated bacterial cellulose carrier compound powder;
preferably, in the step S42, the stirring speed is 500-1500 r/min, the reaction temperature is 40-60 ℃, and the reaction time is 2-4 h.
10. An oyster oligopeptide-astaxanthin-galactosylated bacterial cellulose carrier complex prepared by the method of any one of claims 8-9.
11. Use of an oyster oligopeptide according to claim 1 or an oyster oligopeptide-galactosylated bacterial cellulose carrier complex according to claim 7 or an oyster oligopeptide-astaxanthin-galactosylated bacterial cellulose carrier complex according to claim 10 in the manufacture of a medicament for the treatment of liver injury.
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