CN117343138A - Flower gum oligopeptide and application thereof in treating fatty liver diseases - Google Patents
Flower gum oligopeptide and application thereof in treating fatty liver diseases Download PDFInfo
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- CN117343138A CN117343138A CN202311252573.9A CN202311252573A CN117343138A CN 117343138 A CN117343138 A CN 117343138A CN 202311252573 A CN202311252573 A CN 202311252573A CN 117343138 A CN117343138 A CN 117343138A
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- bacterial cellulose
- oligopeptide
- galactosylated
- spline
- astaxanthin
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- 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
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- A61K47/38—Cellulose; Derivatives thereof
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Abstract
The amino acid sequence of the spline oligopeptide comprises Tyr-Cys-Pro-Arg, and the spline oligopeptide and a drug delivery system consisting of galactosylated modified bacterial cellulose and astaxanthin have extremely high liver targeting and delivery rate, can relieve accumulation and peroxidation of hepatocyte lipid, and improve alcoholic or nonalcoholic fatty liver injury.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a spline 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 spline oligopeptide and astaxanthin and a preparation method of the compound and an effect of the compound in improving alcoholic liver injury.
Background
Alcoholism has become a health problem of great concern in the world today, and in recent years alcoholism has increased year by year and gradually become younger, resulting in an increasing incidence of alcoholic liver injury. Hepatic parenchymal cells act as the primary site of ethanol metabolism, and disturbances in the ethanol metabolic pathway will trigger the accumulation of oxidative substances in the body, especially lipid peroxides, thereby causing liver damage. The acetaldehyde content in the body is increased, oxidative stress occurs, further induced steatosis, inflammatory factors such as TNF-alpha, IL-1 and the like can further enhance liver injury, aggravate steatosis, induce abnormal hyperplasia and necrosis of cells, and further promote further pathological changes such as liver fibrosis and the like. In addition, non-alcoholic fatty liver disease (NAFLD) is a common chronic liver disease, is not related to alcohol, but is a pathological syndrome mainly characterized by intracellular fat accumulation and steatosis, and is developed into non-alcoholic steatohepatitis, cirrhosis and the like in severe cases. Currently, NAFLD has become a global health problem due to obesity, unhealthy eating habits, sedentary lifestyles, and the like.
The ghatti gum is called as ocean ginseng, and has extremely high edible and medicinal value. The Chinese medicine is considered to have the advantages of sweet and neutral flavor, and has the functions of nourishing yin and blood, stopping bleeding, tonifying kidney and stopping nocturnal emission. The ghatti peptide obtained by hydrolyzing the ghatti has strong antioxidant activity, can remove ROS in liver cells, lighten lipid accumulation and oxidative stress in the liver cells and lighten lipid peroxidation, and is expected to be used for preventing and treating ALD and NAFLD. However, due to stability, gastrointestinal physiological disorders, and physical and chemical limitations, ghrelin is difficult to deliver intact targeted to the liver and to exert efficacy. 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 the spline oligopeptides and the astaxanthin, the compound preparation can achieve good effects of protecting the liver and relieving liver injury after oral administration, and the construction of 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 also one of the research hotspots in recent years.
Disclosure of Invention
The present invention is directed to the above object, and in a first aspect, to solve the problem of fat accumulation of hepatocytes due to antioxidant activity of peptide fragments, some embodiments of the present application provide a spline oligopeptide, the amino acid sequence of which comprises Tyr-Cys-Pro-Arg.
In a second aspect, in some embodiments of the present application, there is provided a method of preparing the spline oligopeptides of the first aspect, comprising
S11, adding the gum into water with the mass volume of 10-20 times to prepare homogenate, and placing the homogenate into an enzymolysis tank.
S12, adding compound protease accounting for 2-5% of the mass of the gum into an enzymolysis tank, and heating and inactivating enzyme after enzymolysis to obtain the gum enzymolysis liquid, wherein the compound protease comprises neutral protease, trypsin, bromelain and flavourzyme, and the neutral protease comprises trypsin, bromelain and flavourzyme= (1-3): (2-4): (3-5): (2-5).
S13, centrifuging the microgel protein enzymolysis liquid to obtain clear liquid.
S14, performing membrane separation on the clear liquid, wherein the molecular weight cut-off is 3000Da.
S15, separating and purifying the membrane-passing solution by a gel chromatographic column, wherein the eluting solvent is deionized water, the eluting flow rate is 0.5-0.8 mL/min, detecting absorbance at 220nm, and collecting eluting peaks with retention time of 14-15 min.
S16, further purifying by using a chromatographic 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 5min,10 percent of B,5 to 15min,10 to 15 percent of B,15 to 25min,15 percent of B to 25 percent of B,25 to 40min,25 percent of B to 35 percent of B, the flow rate is 0.8mL/min, and the eluting peak with the retention time of 12 to 13min is collected to obtain the spline oligopeptide.
According to the preparation method of the flower gum oligopeptide, in the step S12, the enzymolysis temperature is 50-60 ℃, the enzyme reaction pH is 8.0-9.0, and the enzymolysis time is 4 hours.
According to the preparation method of the flower gum oligopeptide, in the step S12, after enzymolysis, the temperature is raised to 80-90 ℃ for enzyme deactivation for 10min, and thus the flower gum proteolytic liquid is obtained.
According to some embodiments of the present application, in step S13, the ghrelin enzymatic hydrolysate is centrifuged at 8000 rpm for 10min.
According to the preparation method of the spline oligopeptides of some embodiments of the present application, in step S15, separation and purification are performed by using a sephadex g10 gel chromatographic column of 20mm×100 mm.
According to some embodiments of the present application, the method for preparing a spline oligopeptides, in step S16, further purifying with a C18 chromatographic column.
According to the preparation method of the spline oligopeptides, in the step S16, the spline oligopeptides are concentrated and freeze-dried to obtain the spline oligopeptide powder.
In a third aspect, in order to solve the problem of liver-targeted delivery of a galactosylated bacterial cellulose carrier, some embodiments of the present application provide a complex for delivering a galactosylated bacterial cellulose carrier with a galactosylated bacterial cellulose carrier, and a method for preparing the complex, the complex being prepared by a method comprising
S31, dissolving the flower gum oligopeptide powder and the galactosylated bacterial cellulose carrier in water, stirring, and freeze-drying to obtain the flower gum 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 some embodiments of the present application, in step S21, bacterial cellulose is dissolved in a dimethyl sulfoxide solution containing tetrabutylammonium acetate, and stirred until no jelly is coagulated, thereby obtaining a uniform bacterial cellulose solution with a mass-volume ratio of 1-5%.
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) = (1-5): 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 method of preparing the spline oligopeptides-galactosylated bacterial cellulose carrier complex, in step S23, the removing 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 method for preparing the spline oligopeptides-galactosylated bacterial cellulose carrier complex, in step S23, the dialysis time is 24-72 hours, more preferably 72 hours.
According to the method for preparing the flower gum oligopeptide-galactosylated bacterial cellulose carrier compound in some embodiments of the present application, in step S31, the flower gum oligopeptide and the galactosylated bacterial cellulose carrier powder are dissolved in 10 times volume of ultrapure water, stirred at 90-100 ℃ for reaction for 3-6 hours, and freeze-dried, so as to obtain the flower gum oligopeptide-galactosylated bacterial cellulose carrier compound powder.
According to some embodiments of the present application, the mass ratio of the spline oligopeptides to the 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 the oligopeptides and astaxanthin, in some embodiments of the present application, there is provided a complex of the oligopeptides and astaxanthin carried by galactosylated bacterial cellulose carrier of the third aspect and a method for preparing the same, which is a method for preparing a complex of oligopeptides of the same, astaxanthin, and galactosylated bacterial cellulose carrier, the complex being prepared by a method comprising
S41, dissolving astaxanthin in an ethanol solution, and dissolving the flower gum oligopeptide-galactosylated bacterial cellulose carrier compound in water.
S42, adding the ethanol solution dissolved with astaxanthin oil into the solution of the flower gum oligopeptide-galactosylated bacterial cellulose carrier complex, heating for reaction, and evaporating to remove ethanol to obtain the flower gum 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 well.
According to some embodiments of the present application, the mass to volume ratio of astaxanthin oil to ethanol solution in step S41 is 1-10%, more preferably 5%.
According to some embodiments of the present application, the mass ratio of astaxanthin oil to the cyanine 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 the spline oligopeptides-astaxanthin-galactosylated bacterial cellulose carrier complex, in step S41, the spline oligopeptides-galactosylated bacterial cellulose carrier complex is dissolved in 10 volumes of ultrapure water.
According to the method for preparing the florin oligopeptide-astaxanthin-galactosylated bacterial cellulose carrier compound 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 florin oligopeptide-galactosylated bacterial cellulose carrier compound, the reaction is performed by heating and stirring during the dripping process, ethanol is removed by evaporation, and the florin oligopeptide-astaxanthin-galactosylated bacterial cellulose carrier compound powder is obtained by freeze drying.
According to some embodiments of the present application, 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:
according to the invention, an active peptide is obtained from the propolis by gel exclusion chromatography and separation and purification of reversed-phase high performance liquid chromatography, has antioxidant activity, reduces fat accumulation of liver cells, and improves fatty liver, wherein the amino acid sequence is as follows: tyr-Cys-Tyr-Cys-Pro-Arg, and the sequence is a novel small molecule active peptide through on-line database BIOPEP and EROP-Moscow search.
According to the invention, galactose modified bacterial cellulose is used as a liver targeting delivery carrier of the spline oligopeptide and the astaxanthin, on one hand, the spline oligopeptide is connected with galactose grafted on the bacterial cellulose through Maillard reaction, on the other hand, the spline oligopeptide and the astaxanthin are wrapped through emulsification, and the spline 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 spline oligopeptide and the astaxanthin can be prolonged, the spline oligopeptide and the astaxanthin have extremely high biocompatibility and biodegradability, and the structural influence of a large number of hydroxyl groups on a core material due to the change of an extreme pH value environment is well resisted.
Meanwhile, compared with a galactose modified chitosan carrier 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 the spline oligopeptides from the hydrolytic digestion of various proteases in the gastrointestinal tract and improving the stability of astaxanthin in the gastrointestinal tract. Thus, the spline oligopeptides-astaxanthin-galactosylated bacterial cellulose particles have better flexibility and high adhesive force, can not only cope with the peristaltic movement of gastrointestinal muscle and the shear stress caused by the flow rate of gastric juice in a cavity, so as to reduce the mechanical degradation of the spline oligopeptides and the astaxanthin, but also enhance the adhesion effect of a firm adhesion layer, effectively prolong the stay time in the gastrointestinal tract to enhance the absorption, and reduce the probability of phagocytosis 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 prepared spline gel oligopeptide-astaxanthin-galactosylated bacterial cellulose protects the molecular structure of the spline gel oligopeptide and astaxanthin in the gastrointestinal digestive tract, enhances the absorption and delay the release of the spline gel oligopeptide and astaxanthin, and finally is delivered to liver cells in a precise targeting way. The spline oligopeptide (Tyr-Cys-Tyr-Cys-Pro-Arg) and the astaxanthin are synergistic, so that an oxidative stress system of liver cells is regulated, the generation of ROS is reduced, the peroxidation and denaturation of lipid are relieved, meanwhile, the spline oligopeptide regulatory factor AMPK phosphorylates protein expression and can promote the oxidation of fatty acid beta, the accumulation of lipid is reduced, the spline oligopeptide has better lipid-lowering activity, and compared with single use, the spline oligopeptide has better alcoholic liver injury treatment effect. The method is simple and easy to operate, is suitable for industrial preparation scenes, and has wide application prospects in prevention and treatment of alcoholic liver injury and nonalcoholic fatty liver disease.
Drawings
FIG. 1 shows a diagram of liver lipid accumulation.
FIG. 2 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 the embodiment, the invention provides a carrier compound and a preparation method thereof, which can reduce the influence of pH value on the florin oligopeptide and astaxanthin and the inactivation of digestive hydrolase such as pepsin, trypsin and the like 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 and deliver the florin oligopeptide and astaxanthin to damaged liver cells after the absorption, thereby relieving the oxidative stress, accumulation of lipid, peroxidation and other liver cell damage.
The carrier composite is subjected to the following process steps:
step 1, preparing active spline gel oligopeptide: adding 10-20 times of water into a sample of the gum to prepare a homogenate, placing the homogenate into an enzymolysis tank, adding 2-5% of compound protease of the gum to carry out enzymolysis for 4 hours, heating to 80-90 ℃ to inactivate enzyme for 10 minutes after the enzymolysis is finished to obtain a gum protein enzymolysis liquid, centrifuging the protein enzymolysis liquid for 10 minutes at 8000 revolutions per minute to remove granular substances, separating by a membrane separation technology, separating and purifying the membrane filtration liquid by a Sephadex G10 (20 mm multiplied by 100 mm) gel chromatographic column with a molecular weight cutoff of 3000Da, eluting with deionized water at an eluting flow rate of 0.5-0.8mL/min, detecting the absorbance at 220nm, and collecting an eluting peak with a retention time of 14-15 min; c18 chromatographic column, mobile phase A is trifluoroacetic acid water with volume percent of 0.05-0.1%, mobile phase B is acetonitrile, gradient elution condition is: 0-5 min,10% of B, 5-15 min, 10-15% of B, 15-25 min,15% of B-25% of B, 25-40 min,25% of B-35% of B, the flow rate is 0.8mL/min, collecting elution peaks with retention time of 12-13min, concentrating and freeze-drying to obtain the flower-gel oligopeptide;
Further, the source of the ghua gum in the step 1 is yellow croaker.
Furthermore, the enzymolysis in the step 1 adopts neutral protease, namely trypsin, namely bromelain, namely flavourzyme=1-3:2-4:3-5:2-5 composite protease, the enzymolysis temperature is 50-60 ℃, and the pH value of the enzymatic reaction is controlled to be 8.0-9.0.
Further, the amino acid sequence of the spline gel oligopeptide in the step 1 is as follows: tyr-Cys-Tyr-Cys-Pro-Arg.
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 a ghatti gel oligopeptide-galactosylated bacterial cellulose complex carrier: and (2) re-dissolving the spline oligopeptide 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 the spline oligopeptide-galactosylated bacterial cellulose composite carrier powder.
Further, in the step 3, the mass ratio of the spline oligopeptide powder to the galactosylated bacterial fiber powder is 1-10:1, preferably 5:1.
according to the steps, the complex of the flower gum oligopeptide-galactosylated bacterial cellulose carrier is obtained, and liver targeting delivery of the flower gum oligopeptide can be realized through the galactosylated bacterial cellulose carrier. As a further scheme, the spline oligopeptides are the spline oligopeptides which can resist oxidation, reduce fat accumulation of liver cells and improve fatty liver, and of course, it is understood that the spline oligopeptides can be other spline oligopeptides which need liver targeting delivery in the prior art. Besides the use of the compound of the flower gum oligopeptide and the carrier, the compound of the astaxanthin can be further carried out, 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 a spline oligopeptide-astaxanthin-galactosylated bacterial cellulose complex carrier: dissolving astaxanthin oil with the astaxanthin content of 10% in an ethanol solution, uniformly stirring, dissolving the powder of the cyanine oligopeptide-galactosylated bacterial cellulose in ultrapure water with the volume of 10 times, slowly dripping the astaxanthin oil ethanol solution into the stirred cyanine oligopeptide-galactosylated bacterial cellulose solution, heating for reaction, evaporating to remove ethanol, and freeze-drying to obtain the powder of the cyanine oligopeptide-astaxanthin-galactosylated bacterial cellulose particles (FMP-AST-GBC);
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 spline oligopeptides-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.
The existing galactose-mediated targeted delivery of asialoglycoprotein receptors on the cell membrane of hepatic parenchymal cells is mostly focused on directly modifying the galactose of the drug or constructing galactose-modified liposome and polymer micelle carriers to realize 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 the spline oligopeptide and the astaxanthin, on one hand, the spline oligopeptide is connected with galactose grafted on the bacterial cellulose through Maillard reaction, on the other hand, the spline oligopeptide and the astaxanthin are wrapped through emulsification, and the spline 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 spline oligopeptide and the astaxanthin can be prolonged, the spline oligopeptide and the astaxanthin have extremely high biocompatibility and biodegradability, and the structural influence of a large number of hydroxyl groups on a core material due to the change of an extreme pH value environment is well resisted.
Meanwhile, compared with a galactose modified chitosan carrying system which is reported in many cases, the three-dimensional network structure of the bacterial cellulose has extremely large porosity, the coating effect on the core material is more beneficial to protecting the spline oligopeptides from the hydrolytic digestion of various proteases in the gastrointestinal tract, and the stability of astaxanthin in the gastrointestinal tract is greatly improved. Thus, the spline oligopeptides-astaxanthin-galactosylated bacterial cellulose particles have better flexibility and high adhesive force, can not only cope with the peristaltic movement of gastrointestinal muscle and the shear stress caused by the flow rate of gastric juice in a cavity, so as to reduce the mechanical degradation of the spline oligopeptides and the astaxanthin, but also enhance the adhesion effect of a firm adhesion layer, effectively prolong the stay time in the gastrointestinal tract to enhance the absorption, and reduce the probability of phagocytosis 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 prepared spline gel oligopeptide-astaxanthin-galactosylated bacterial cellulose protects the molecular structure of the spline gel oligopeptide and astaxanthin in the gastrointestinal digestive tract, enhances the absorption and delay the release of the spline gel oligopeptide and astaxanthin, and finally is delivered to liver cells in a precise targeting way. The spline oligopeptide (Tyr-Cys-Tyr-Cys-Pro-Arg) and the astaxanthin are synergistic, so that an oxidative stress system of liver cells is regulated, the generation of ROS is reduced, the peroxidation and denaturation of lipid are relieved, meanwhile, the spline oligopeptide regulatory factor AMPK phosphorylates protein expression and can promote the oxidation of fatty acid beta, the accumulation of lipid is reduced, the spline oligopeptide has better lipid-lowering activity, and compared with single use, the spline oligopeptide has better alcoholic liver injury treatment effect. The method is simple and easy to operate, is suitable for industrial preparation scenes, and has wide application prospects in prevention and treatment of alcoholic liver injury and nonalcoholic fatty liver disease.
Example 1:
step 1, 1000g of gum is added with 15 times of water by mass volume to prepare homogenate, the homogenate is placed in an enzymolysis tank, then 35g of compound protease is added, the protease is composed of neutral protease, namely trypsin, bromelain, namely flavourzyme=2:4:3:3, pH is regulated to 8.5, enzymolysis is carried out for 4 hours at 50 ℃, and then the temperature is increased to 90 ℃ for enzyme deactivation for 10 minutes, so that the gum proteolytic liquid is obtained; centrifuging at 8000 rpm for 10min, ultrafiltering at 3000Da, separating and purifying the membrane-passing solution with SephadexG 10 (20 mm×100 mm) gel chromatographic column, eluting with deionized water at flow rate of 0.6mL/min, detecting absorbance at 220nm, and collecting eluting peak with retention time of 14.57 min; c18 chromatographic column, mobile phase A is trifluoroacetic acid water with volume percent of 0.05%, mobile phase B is acetonitrile, and gradient elution conditions are: 0-5 min,10% of B, 5-15 min, 10-15% of B, 15-25 min,15% of B-25% of B, 25-40 min,25% of B-35% of B, the flow rate is 0.8mL/min, the elution peak with retention time of 12.29min is collected, concentrated and freeze-dried to obtain 7.5g of flower-gel oligopeptide;
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; taking 20g of lactobionic acid, dissolving in 1000ml of DMSO solution containing 5g EDC and 5g NHS, stirring and performing carboxyl activation for 2 hours; the bacterial cellulose solution was added to a lactobionic acid solution, heated to 50℃and stirred for 12 hours, and then dialyzed for 36 hours and then freeze-dried to obtain 45g of galactosylated bacterial cellulose powder.
And 3, dissolving 6g of the spline oligopeptides powder and 2g of the galactosylated bacterial cellulose powder in 10 times of volume of ultrapure water, stirring at 100 ℃ for reaction for 3 hours, and freeze-drying to obtain 8g of the spline oligopeptides-galactosylated bacterial cellulose composite carrier powder.
And 4, dissolving 8g of astaxanthin oil in 100ml of ethanol solution, uniformly stirring, dissolving 8g of the spline oligopeptide-galactosylated bacterial cellulose powder in 10 times of ultrapure water, slowly dripping the stirred spline oligopeptide-galactosylated bacterial cellulose solution into the astaxanthin oil ethanol solution, heating to 50 ℃ for reacting for 2 hours at the stirring speed of 1500r/min, evaporating to remove ethanol, and freeze-drying to obtain 16g of FMP-AST-GBC microparticle powder.
Example 2:
step 1, 1000g of gum is added with water with the mass volume of 20 times to prepare homogenate, the homogenate is placed in an enzymolysis tank, then 40g of compound protease is added, the protease is composed of neutral protease, namely trypsin, bromelain, namely flavourzyme=3:2:5:3, pH is regulated to 8.0, enzymolysis is carried out for 4 hours at 60 ℃, and then the temperature is increased to 90 ℃ for enzyme deactivation for 10 minutes, so that the gum proteolytic liquid is obtained; centrifuging at 8000 rpm for 10min, ultrafiltering at 3000Da, separating and purifying the membrane-passing solution with SephadexG 10 (20 mm×100 mm) gel chromatographic column, eluting with deionized water at flow rate of 0.8mL/min, detecting absorbance at 220nm, and collecting eluting peak with retention time of 14.24 min; c18 chromatographic column, mobile phase A is trifluoroacetic acid water with volume percent of 0.1%, mobile phase B is acetonitrile, and gradient elution conditions are: 0-5 min,10% of B, 5-15 min, 10-15% of B, 15-25 min,15% of B-25% of B, 25-40 min,25% of B-35% of B, the flow rate is 0.8mL/min, the elution peak with retention time of 12.71min is collected, concentrated and freeze-dried to obtain 7.2g of flower-gel oligopeptide;
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 1 hour; the bacterial cellulose solution was added to lactobionic acid solution and heated to 40℃with stirring for 12 hours, and then dialyzed for 36 hours and freeze-dried to obtain 34g of galactosylated bacterial cellulose powder.
And 3, dissolving 6g of the spline oligopeptides powder and 3g of galactosylated bacterial cellulose powder in 10 times of volume of ultrapure water again, stirring at 95 ℃ for reaction for 5 hours, and freeze-drying to obtain 9g of the spline oligopeptides-galactosylated bacterial cellulose composite carrier powder.
And 4, dissolving 3g of astaxanthin oil in 100ml of ethanol solution, uniformly stirring, dissolving 9g of the spline oligopeptide-galactosylated bacterial cellulose powder in 10 times of ultrapure water, slowly dripping the stirred spline oligopeptide-galactosylated bacterial cellulose solution into the astaxanthin oil ethanol solution, heating to 60 ℃ for reaction for 3 hours at the stirring speed of 800r/min, evaporating to remove ethanol, and freeze-drying to obtain 12g of FMP-AST-GBC microparticle powder.
Comparative example 1:
10g of a powder of a complex carrier of a spline oligopeptides-galactosylated bacterial cellulose (FMP-GBC) was obtained by the procedure of step 1 to step 3 of example 1.
Comparative example 2:
step 1, taking 50g 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; taking 20g of lactobionic acid, dissolving in 1000ml of DMSO solution containing 5g EDC and 5g NHS, stirring and performing carboxyl activation for 2 hours; the bacterial cellulose solution was added to lactobionic acid solution and heated to 40℃with stirring for 24 hours, and then dialyzed for 48 hours and freeze-dried to obtain 68g of galactosylated bacterial cellulose powder.
Step 2, taking 10g of astaxanthin oil, dissolving in 100ml of ethanol solution, uniformly stirring, taking 10g 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 20g of astaxanthin-galactosylated bacterial cellulose (AST-GBC) microparticle powder.
Comparative example 3:
step 1: 7.8g of spline oligopeptides were obtained by carrying out the procedure of step 1, 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, dissolving 6g of the spline oligopeptides powder and 2g of the galactosylated chitosan powder in 10 times of volume of ultrapure water, stirring at 90 ℃ for reaction for 6 hours, and freeze-drying to obtain 8g of the spline oligopeptides-galactosylated chitosan powder.
And 4, dissolving 8g of astaxanthin oil in 100ml of ethanol solution, uniformly stirring, dissolving 8g of the spline oligopeptide-galactosylated chitosan powder in 10 times of ultrapure water, slowly dripping the stirred spline oligopeptide-galactosylated chitosan solution into the astaxanthin oil ethanol solution, heating to 60 ℃ for reacting for 2 hours at the stirring speed of 500r/min, evaporating to remove ethanol, and freeze-drying to obtain 16g of spline oligopeptide-astaxanthin-galactosylated chitosan (FMP-AST-GC) powder.
Experiment 1: determination of the free radical scavenging Capacity of the Di-Ph oligopeptide
The spline oligopeptides (FMP) and spline oligopeptides-astaxanthin-galactosylated bacterial cellulose (FMP-AST-GBC) obtained in example 1 were weighed out to prepare a sample solution with a mass concentration of 20. Mu.g/mL. Preparing a DPPH solution with the concentration of 0.2mmol/L, and storing the solution in a dark place. 50. Mu.L of an aqueous solution of a spline oligopeptides (20. Mu.g/mL) and 150. Mu.L of a DPPH solution were added to a 96-well plate, mixed well, reacted in a dark place for 30 minutes, and absorbance A1 was measured at 517 nm. Adding water and ethanol into the sample solution to make blank, and measuring absorbance A2; absolute ethanol was added to DPPH as a control and absorbance A3 was measured. The positive control used ascorbic acid. The calculation formula of the DPPH free radical clearance is as follows: DPPH radical clearance% = [1- (A1-A2)/A3 ] ×100. The results show that the IC50 values of FMP, FMP-AST-GBC and ascorbic acid on DPPH free radical scavenging capacity are 7.60, 5.92 and 20.63 mug/mL respectively, which shows that the spline oligopeptides purified in the step 1 have remarkable antioxidant activity, and the antioxidant activity of the spline oligopeptides and astaxanthin are not destroyed and are greatly improved after being embedded by a carrier.
Experiment 2: fatty liver cell improving effect of flower gum oligopeptide
HepG2 cells were cultured in high-sugar DMEM medium containing 10% fetal bovine serum and placed in a 37℃incubator containing 5% CO 2. When the cell density in the 6-hole plate reaches 60% -70%, the cells are divided into 3 groups, and the control group is cultured by a common culture medium (containing a medium with equal concentration); culturing the model group with 0.25mmol/L palmitic acid for 24 hours, and changing the common culture medium; the flower gum oligopeptide group is cultured for 24 hours by 0.25mmol/L palmitic acid, and is interfered for 12 hours by 16 mug/mL flower gum oligopeptide solution. Adding oil red O staining solution (oil red storage solution: deionized water=3:2) for staining for 15min, washing with distilled water for several times, and observing and photographing under a microscope. Isopropanol dissolving dyeingAfter the material is put under an enzyme-labeled instrument, the absorbance value D of each group is measured at 570nm wavelength 570 . The results show D of the floridin oligopeptide group 570 (0.28.+ -. 0.08) is far below D of model group 570 (0.49.+ -. 0.06), close to D of blank group 570 (0.21.+ -. 0.08), indicating that the interference of the ghatti-gel oligopeptide solution effectively reduced fat accumulation of fatty liver cells.
Experiment 3: in vitro simulated gastric and intestinal 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. FMP-AST-GBC1,2 obtained in examples 1 and 2 and FMP-GBC, AST-GBC, FMP-AST-GC powder 4mg obtained in comparative examples were placed in dialysis bags (MWCO 3500), immersed in 200mL of artificial gastric juice (or intestinal juice), stirred in a constant temperature magnetic stirrer at 37℃at a rotational speed of 100rpm/min, sampled at 30min and at 1, 6, 12 and 24 hours, 1mL of blank artificial gastric juice (or intestinal juice) of the same matrix was added after the sampling, the supernatant was obtained after centrifugation of the sampled samples, and the content of the spline oligopeptide and astaxanthin was detected by high performance liquid chromatography after filtration with 0.22 μm organic filter membranes. The results are shown below:
TABLE 1 Release Rate in Artificial gastric juice
TABLE 2 Release Rate in Artificial intestinal juice
According to the results of the release rates of FMP-AST-GBC1, 2, FMP-GBC, AST-GBC and FMP-AST-GC in the artificial gastrointestinal fluid, the release rates of the core materials of each group in the artificial gastrointestinal fluid are gradually increased along with the increase of time, and the release rates tend to be stable at 6h and 12h, the FMP-AST-GBC group has the protection effect on the florin oligopeptide-astaxanthin, the florin peptide-galactose-bacterial cellulose and the multiple wrapping effect of the astaxanthin-bacterial cellulose, so that the stability is greatly improved, the release rate is lower than that of a carrier system for independently carrying the florin peptide or the 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 the bacterial cellulose, so that the FMP-AST-GBC system can stably exist in the environment of the artificial gastrointestinal fluid, has the protection effect on the florin oligopeptide and the astaxanthin, and is favorable for being absorbed by human bodies.
Experiment 4: liver targeting
Male SD rats were bred adaptively for 3-5d, randomly with groups FMP-AST-GBC1, 2 (5 mg/kg), AST-GBC (3 mg/kg) and FMP-AST-GC (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 FMP-AST-GBC was evaluated with targeting efficiency (Targeting efficiency, te) according to the method of Gupta. The results show that the liver targeting efficiency (57.14+ -6.23) of FMP-AST-GBC group is higher than that of AST-GBC group (41.95+ -4.33) and FMP-AST-GC group (35.22 + -3.70), and has very high liver targeting.
Experiment 5: NAFLD mice fat accumulation improving effect
After one week of adaptive feeding, 10 mice were randomly selected and fed normal diet (blank control group), and the remaining mice were given high fat diet for free feeding for 8 weeks. Mice fed high fat diet were then randomly divided into 3 groups (12 per group) for gavage, once daily, for 6 consecutive weeks: (1) normal feed+physiological saline (model group); (2) group normal feed+fmp-AST-GBC; (3) normal feed+fmp; (4) normal feed+ast; (5) general feed+FMP-AST-GC. Mice were all sacrificed 4 hours after fasted at the end of week 14. After taking out the liver, freezing and slicing the liver by a conventional method, staining with oil red O according to the instructions of a kit, extracting the oil red of the intracellular lipid drops by isopropanol, and measuring the absorbance value at 570 nm. As shown in fig. 1, compared with the blank group, the absorbance of the lipid drop oil red of the model group is remarkably increased compared with that of the blank group, which indicates that the NAFLD model is successfully established. The FMP-AST-GBC intervention treatment obviously reduces the accumulation of lipid in the liver of NAFLD mice, approaches to the level of a blank group, and has better effect than independently feeding the spline oligopeptide or astaxanthin and the spline shellfish oligopeptide-astaxanthin-galactosylated chitosan carrier system.
The experimental result shows that the spline gel oligopeptide (Tyr-Cys-Tyr-Cys-Pro-Arg) provided by the invention has good effects of regulating oxidative stress and improving fat accumulation, and the spline gel oligopeptide-astaxanthin-galactosylated bacterial cellulose delivery system has good improvement effects on nonalcoholic fatty nature and alcoholic liver injury, so that the spline gel oligopeptide-astaxanthin-galactosylated bacterial cellulose delivery system is a potential high-quality liver-protecting component.
The invention discloses a preparation method of a spline gel oligopeptide astaxanthin liver targeting microsphere and an effect of improving alcoholic liver injury. The preparation method comprises the steps of chopping, enzymolysis, ultrafiltration and the like from the flower gum to obtain a flower gum oligopeptide solution, connecting and carrying the flower gum oligopeptide solution with galactosylated modified bacterial cellulose, and finally reacting a flower gum peptide-galactosylated bacterial cellulose carrier with astaxanthin oil and freeze-drying to obtain the liver-targeted flower gum oligopeptide-astaxanthin-galactosylated bacterial cellulose particles. The prepared ghatti peptide astaxanthin delivery 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 accumulation and peroxidation of hepatocyte lipid, further improves alcoholic or non-alcoholic fatty liver injury, 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. A spline oligopeptide, which is characterized in that the amino acid sequence of the spline oligopeptide comprises Tyr-Cys-Pro-Arg.
2. A method for preparing the spline oligopeptides of claim 1, comprising
S11, adding 10-20 times of water by mass volume into the gum to prepare homogenate, and placing the homogenate into an enzymolysis tank;
s12, adding compound protease accounting for 2-5% of the mass of the gum into an enzymolysis tank, and heating and inactivating enzyme after enzymolysis to obtain gum enzymolysis liquid, wherein the compound protease comprises neutral protease, trypsin, bromelain and flavourzyme, and the neutral protease comprises trypsin, bromelain and flavourzyme= (1-3): (2-4): (3-5): (2-5);
s13, centrifuging the microgel protein enzymolysis liquid to obtain clear liquid;
s14, performing membrane separation on the clear liquid, wherein the molecular weight cut-off is 3000Da;
s15, separating and purifying the membrane-passing solution through a gel chromatographic column, wherein an eluting solvent is deionized water, the eluting flow rate is 0.5-0.8 mL/min, detecting absorbance at 220nm, and collecting an eluting peak with retention time of 14-15 min;
S16, further purifying by using a chromatographic 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 5min,10 percent of B,5 to 15min,10 to 15 percent of B,15 to 25min,15 percent of B to 25 percent of B,25 to 40min,25 percent of B to 35 percent of B, the flow rate is 0.8mL/min, and the eluting peak with the retention time of 12 to 13min is collected to obtain the spline oligopeptide.
3. The method for preparing a spline oligopeptide according to claim 2,
preferably, in the step S12, the enzymolysis temperature is 50-60 ℃, 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 10min to obtain the microgel protein enzymolysis liquid;
preferably, in the step S13, the microgel enzymolysis liquid is centrifuged for 10min at 8000 rpm;
preferably, in step S15, separation and purification are performed using a SephadexG 10 gel column of 20mm by 100 mm;
preferably, in step S16, further purification is performed using a C18 chromatographic column;
preferably, in step S16, the spline oligopeptides are concentrated and freeze-dried to obtain spline oligopeptide powder.
4. A method for preparing a complex of a flower gum oligopeptide-galactosylated bacterial cellulose carrier is characterized by comprising the following steps of
S31, dissolving the flower gum oligopeptide powder and the galactosylated bacterial cellulose carrier in water, stirring, and freeze-drying to obtain a flower gum 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 a complex of a spline oligopeptide and a galactosylated bacterial cellulose carrier 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) = (1-5): 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 a complex of a spline oligopeptide and a galactosylated bacterial cellulose carrier according to claim 4,
in the step S31, dissolving the spline oligopeptides and galactosylated bacterial cellulose carrier powder in 10 times of ultrapure water, stirring at 90-100 ℃ for reaction for 3-6 hours, and freeze-drying to obtain the spline oligopeptides-galactosylated bacterial cellulose carrier composite powder;
preferably, in step S31, the mass ratio of the spline oligopeptides to the galactosylated bacterial fiber carrier powder is (1-10): 1, more preferably 5:1.
7. A spline oligopeptide-galactosylated bacterial cellulose carrier complex prepared by the method of any one of claims 4-6.
8. A method for preparing a complex of a flower gum oligopeptide-astaxanthin-galactosylated bacterial cellulose carrier is characterized by comprising the following steps of
S41, dissolving astaxanthin in an ethanol solution, and dissolving the spline gel oligopeptide-galactosylated bacterial cellulose carrier compound in water;
s42, adding the ethanol solution dissolved with astaxanthin oil into the solution of the flower gum oligopeptide-galactosylated bacterial cellulose carrier complex, heating for reaction, and evaporating to remove ethanol to obtain the flower gum oligopeptide-astaxanthin-galactosylated bacterial cellulose carrier complex.
9. The method for preparing a complex of an oligopeptides of spline-astaxanthin-galactosylated bacterial cellulose carrier 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 the spline oligopeptides-galactosylated bacterial cellulose carrier complex is 1 (1-10), more preferably 1:1;
Preferably, in step S41, the spline oligopeptides-galactosylated bacterial cellulose carrier complex is dissolved in 10 volumes of ultrapure water;
in the step S42, slowly dripping the ethanol solution dissolved with astaxanthin oil into the solution of the flower gum oligopeptide-galactosylated bacterial cellulose carrier compound, heating and stirring for reaction in the dripping process, evaporating to remove ethanol, and freeze-drying to obtain flower gum 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. A spline oligopeptide-astaxanthin-galactosylated bacterial cellulose carrier complex prepared by the method of any one of claims 8-9.
11. Use of a spline oligopeptide according to claim 1 or a spline oligopeptide-galactosylated bacterial cellulose carrier complex according to claim 7 or a spline oligopeptide-astaxanthin-galactosylated bacterial cellulose carrier complex according to claim 10 in the preparation of a medicament for the treatment of liver injury.
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