CN117343136A - Preparation of ginseng oligopeptide and compound thereof and application of ginseng oligopeptide and compound thereof in treatment of alcoholic liver injury diseases - Google Patents
Preparation of ginseng oligopeptide and compound thereof and application of ginseng oligopeptide and compound thereof in treatment of alcoholic liver injury diseases Download PDFInfo
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- CN117343136A CN117343136A CN202311252570.5A CN202311252570A CN117343136A CN 117343136 A CN117343136 A CN 117343136A CN 202311252570 A CN202311252570 A CN 202311252570A CN 117343136 A CN117343136 A CN 117343136A
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- Prior art keywords
- ginseng
- bacterial cellulose
- galactosylated
- oligopeptide
- ginseng oligopeptide
- Prior art date
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Classifications
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
- C07K17/02—Peptides being immobilised on, or in, an organic carrier
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Abstract
The ginseng oligopeptide and the compound thereof are prepared, and are used for treating alcoholic liver injury diseases, belong to the technical field of biology, and are used for solving the problem that peptide fragments have antioxidant and immunoregulatory effects.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to ginseng 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 the ginseng oligopeptide and astaxanthin and a preparation method of the compound and an effect of the compound in improving alcoholic liver injury.
Background
The mechanism by which alcoholic liver injury occurs is not well defined, but there is increasing evidence that oxidative stress and inflammation play a key etiological role in the occurrence of alcoholic liver injury, including hepatocyte dysfunction, apoptosis, fibrosis, and the like. Excessive ethanol consumption induces accumulation of various free radicals, reactive Oxygen Species (ROS), and Reactive Nitrogen Species (RNS) in the liver, including superoxide, hydrogen peroxide, lipid peroxides, nitric oxide, and peroxynitrite. In turn, the free radicals produced in large amounts exceed the oxidative metabolic capacity of the liver, disrupt the redox balance of the liver, increase the consumption of antioxidant substances, inhibit superoxide dismutase (SOD) activity, reduce the level of antioxidant enzymes, in particular Glutathione (GSH). Thereby causing a series of pathological lesions in the liver, causing peroxidation of cell membrane lipids, oxidation of proteins and DNA, leading to hepatocyte damage, inflammation, ischemia, fibrosis, necrosis and apoptosis.
The ginseng is known as 'Baicaowang' and has the effects of reinforcing primordial qi, restoring pulse, relieving depletion, tonifying spleen, benefiting lung, promoting fluid production, soothing nerves and the like, and the rich bioactive substances mainly comprise ginseng total polysaccharide, ginsenoside and ginseng protein. The micromolecular ginseng oligopeptide obtained by hydrolyzing ginseng has strong antioxidant and immunoregulatory activities, can obviously improve oxidative stress of damaged liver cells, and regulate the rise of intestinal lipopolysaccharide level caused by alcohol intake, so that the level of inflammatory cytokines caused by NF- κB passage entering liver tissue is abnormal, and further, liver oxidation-reduction imbalance, inflammatory reaction and the like caused by the large amount of alcohol intake are inhibited. However, due to stability, gastrointestinal physiological disorders, and physical and chemical limitations, ginseng oligopeptides are difficult to be perfectly 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. A plurality of researches prove that the astaxanthin serving as a natural strong antioxidant 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 ginseng oligopeptide and the astaxanthin, the effects of protecting the liver and relieving the liver injury can be achieved 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, in a first aspect, to solve the problem that peptide fragments have antioxidant and immunoregulatory effects, some embodiments of the present application provide ginseng oligopeptide, the amino acid sequence of which includes Leu-Gly-His-Glu-Ser.
In a second aspect, in some embodiments of the present application, there is provided a method for preparing a ginseng oligopeptide of the first aspect, comprising
S11, adding water 15-20 times of the ginseng into the ginseng to prepare homogenate, placing the homogenate into an enzymolysis tank, and adding cellulase accounting for 0.5-1.0% of the ginseng into the enzymolysis tank.
S12, adding compound protease accounting for 3-5% of the mass of the ginseng into an enzymolysis tank, and heating and inactivating enzyme after enzymolysis to obtain ginseng enzymolysis liquid, wherein the compound protease comprises alkaline protease, bromelain and flavourzyme, and the alkaline protease comprises bromelain and flavourzyme= (3-5): (2-4).
S13, centrifuging the ginseng 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 an ion exchange chromatographic column, wherein the eluting solvent is deionized water, the eluting flow rate is 0.8-1.0 mL/min, detecting absorbance at 220nm, and collecting eluting peaks with retention time of 13-14 min.
S16, further purifying by adopting a chromatographic column, wherein the mobile phase A is trifluoroacetic acid water with the volume percentage of 0.05-0.08%, the mobile phase B is methanol, and the gradient elution condition is as follows: 0 to 10min,5 percent of B,10 to 20min,5 to 10 percent of B,20 to 25min,10 to 20 percent of B,25 to 35min,20 to 30 percent of B, the flow rate is 0.8 to 1.0mL/min, and elution peaks of 27 to 28min are collected to obtain the ginseng oligopeptide.
According to some embodiments of the present application, in step S11, the enzymatic hydrolysis temperature of the cellulase is 45-50 ℃, the enzymatic hydrolysis time is 1-2 hours, and the enzymatic reaction pH is 5.0-6.0.
According to the preparation method of ginseng oligopeptide, in the step S12, the enzymolysis temperature is 50-55 ℃, the enzyme reaction pH is 9.0-10.0, and the enzymolysis time is 3-4 h.
According to the preparation method of the ginseng oligopeptide, in the step S12, after enzymolysis, the temperature is raised to 80-90 ℃ for enzyme deactivation for 10-15 minutes, and ginseng enzymolysis liquid is obtained.
According to some embodiments of the present application, in step S13, the ginseng enzymatic hydrolysate is centrifuged at 6000-8000 rpm for 8-10 minutes.
According to some embodiments of the present application, in step S15, DEAE52 ion exchange chromatography is used for separation and purification.
According to some embodiments of the present application, the ginseng oligopeptide preparation method further comprises the step of purifying the ginseng oligopeptide by using a C18 chromatographic column in step S16.
According to some embodiments of the present application, in step S16, the ginseng oligopeptide is concentrated and freeze-dried to obtain ginseng oligopeptide powder.
In a third aspect, in order to solve the problem of liver-targeted delivery of ginseng oligopeptide, in some embodiments of the present application, a complex of galactosylated bacterial cellulose carrier for delivering ginseng oligopeptide and a preparation method thereof are provided, which are a method for preparing a complex of ginseng oligopeptide-galactosylated bacterial cellulose carrier, the complex is prepared by a preparation method comprising
S31, dissolving the ginseng oligopeptide powder and the galactosylated bacterial cellulose carrier in water, stirring, and freeze-drying to obtain the ginseng 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 method for preparing the ginseng oligopeptide-galactosylated bacterial cellulose carrier complex in 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, so as to obtain 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 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 removing impurities in step S23 includes 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 dialysis time is 24-72 hours, more preferably 72 hours, in step S23.
According to the method for preparing the ginseng oligopeptide-galactosylated bacterial cellulose carrier compound in some embodiments of the present application, in step S31, the ginseng oligopeptide and the 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 the ginseng oligopeptide-galactosylated bacterial cellulose carrier compound powder.
According to some embodiments of the present application, the mass ratio of ginseng 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 ginseng oligopeptide and astaxanthin, in some embodiments of the present application, there is provided a complex of the above-mentioned galactosylated bacterial cellulose carrier carrying ginseng oligopeptide and astaxanthin and a preparation method thereof, which is a method for preparing a complex of ginseng oligopeptide-astaxanthin-galactosylated bacterial cellulose carrier, the complex being prepared by a preparation method comprising
S41, dissolving astaxanthin in an ethanol solution, and dissolving the ginseng oligopeptide-galactosylated bacterial cellulose carrier compound in water.
S42, adding the ethanol solution dissolved with astaxanthin oil into the solution of the ginseng oligopeptide-galactosylated bacterial cellulose carrier complex, heating for reaction, and evaporating to remove ethanol to obtain the ginseng 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.
In step S41 according to some embodiments of the present application, the mass-to-volume ratio of astaxanthin oil to ethanol solution is 1-10%, more preferably 5%.
In step S41 according to some embodiments of the present application, the mass ratio of astaxanthin oil to ginseng oligopeptide-galactosylated bacterial cellulose carrier complex is between 1:1 and 10, more preferably 1:1.
In step S41 according to some embodiments of the present application, the ginseng oligopeptide-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 ginseng oligopeptide-galactosylated bacterial cellulose carrier compound, heating and stirring for reaction in the dripping process, evaporating to remove ethanol, and freeze-drying to obtain the ginseng oligopeptide-astaxanthin-galactosylated bacterial cellulose carrier compound powder.
In step S42 according to some embodiments of the present application, the stirring speed is 500-1500 r/min, the reaction temperature is 40-60 ℃, and the reaction time is 2-4 h.
The beneficial effects are that:
the invention obtains an active peptide with antioxidant and immunoregulatory effects from ginseng protein by gel exclusion chromatography and separation and purification of reversed phase high performance liquid chromatography, and the amino acid sequence is as follows: leu-Gly-His-Glu-Ser, and through on-line database BIOPEP and EROP-Moscow search, the sequence is one new kind of small molecule active peptide.
According to the invention, galactose modified bacterial cellulose is used as a liver targeting delivery carrier of the ginseng oligopeptide and the astaxanthin, on one hand, the ginseng oligopeptide is connected with galactose grafted on the bacterial cellulose through Maillard reaction, on the other hand, the ginseng oligopeptide and the astaxanthin are wrapped through emulsification, and the ginseng 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 ginseng oligopeptide and the astaxanthin can be prolonged, the ginseng 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 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 ginseng oligopeptide from hydrolysis and digestion effects of various proteases in the gastrointestinal tract and improving the stability of astaxanthin in the gastrointestinal tract. Therefore, the ginseng oligopeptide-astaxanthin-galactosylated bacterial cellulose particles have better flexibility and high adhesive force, not only can 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 ginseng oligopeptide and the astaxanthin, but also can enhance the adhesion effect of a firm adhesion layer, effectively prolong the stay time in the gastrointestinal tract to enhance the absorption, and the flexibility also reduces 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 ginseng oligopeptide-astaxanthin-galactosylated bacterial cellulose prepared by the method provided by the invention protects the molecular structure of the ginseng oligopeptide and astaxanthin in the gastrointestinal digestive tract, enhances the absorption and the delayed release of the ginseng oligopeptide and astaxanthin, and finally, the ginseng oligopeptide and astaxanthin are delivered to liver cells in a precise targeting manner. The ginseng oligopeptide and the astaxanthin act synergistically to regulate an oxidative stress system of liver cells, reduce generation of ROS, relieve peroxidation and denaturation of lipid, and meanwhile, the ginseng oligopeptide (Leu-Gly-His-Glu-Ser) regulates and controls expression of LPS-TLR4-NF- κB pathway related proteins, so that liver inflammatory response is improved, and compared with single use, the ginseng oligopeptide has better liver injury treatment effect. The method is simple and easy to operate, is suitable for industrial preparation scenes, and has wide application prospect in preventing and treating alcoholic liver injury.
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 the embodiment, the invention provides a carrier compound and a preparation method thereof, which can reduce the influence of pH value and inactivation of digestive hydrolase such as pepsin, trypsin and the like on ginseng oligopeptide and astaxanthin in the gastrointestinal tract, enhance adhesion on mucous membrane to prolong the time in the gastrointestinal tract so as to improve absorption, and directly target and deliver the absorbed product to damaged liver cells after absorption, thereby relieving the damage of the liver cells such as oxidative stress, cell inflammation and the like.
The carrier composite is subjected to the following process steps:
step 1, preparing ginseng oligopeptide with high antioxidant and immunoregulatory activity: after coarse crushing ginseng, adding 15-20 times of water to prepare homogenate, adding cellulase accounting for 0.5-1.0% of the ginseng, and carrying out enzymolysis for 1-2 hours at 45-50 ℃ with the pH value of enzyme reaction controlled at 5.0-6.0; adding compound protease with the mass of 3-5% of ginseng protein into an enzymolysis tank, carrying out enzymolysis for 3-4 hours at 50-55 ℃, controlling the pH value of enzyme reaction to be 9.0-10.0, wherein the mass ratio of the compound protease is as follows: alkaline protease bromelain flavourzyme= (3-5): (2-4); heating to 80-90 ℃ after enzymolysis is finished, inactivating enzyme for 10-15 minutes to obtain ginseng enzymolysis liquid, centrifuging the ginseng enzymolysis liquid for 8-10 minutes at 6000-8000 rpm, removing granular substances, separating clear liquid by adopting a membrane with a cut-off molecular weight of 3000Da, separating and purifying membrane passing liquid by using a DEAE52 ion exchange chromatographic column, eluting with deionized water at the flow rate of 0.8-1.0mL/min, detecting absorbance at 220nm, and collecting elution peaks with the retention time of 13-14 min; adopting a C18 chromatographic column, wherein the mobile phase A is trifluoroacetic acid water with the volume percentage of 0.05-0.08%, the mobile phase B is methanol, and the gradient elution condition is as follows: 0 to 10min,5 percent of B,10 to 20min,5 to 10 percent of B,20 to 25min,10 to 20 percent of B,25 to 35min,20 to 30 percent of B, the flow rate is 0.8 to 1.0mL/min, collecting elution peaks of 27 to 28min, concentrating and freeze-drying to obtain ginseng oligopeptide;
Further, the amino acid sequence of the ginseng oligopeptide in the step 1 is as follows: leu-Gly-His-Glu-Ser.
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 ginseng oligopeptide-galactosylated bacterial cellulose complex carrier: and (2) re-dissolving the ginseng oligopeptide powder prepared in the step (1) and 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 ginseng oligopeptide-galactosylated bacterial cellulose composite carrier powder.
Further, in the step 3, the mass ratio of the ginseng oligopeptide powder to the galactosylated bacterial fiber powder is 1-10:1, preferably 5:1.
according to the steps, the ginseng oligopeptide-galactosylated bacterial cellulose carrier compound is obtained, and liver targeting delivery of the ginseng oligopeptide can be realized through the galactosylated bacterial cellulose carrier. As a further aspect, the ginseng oligopeptide is the ginseng oligopeptide capable of having antioxidant and immunoregulatory effects, which is described above, and it is understood that the ginseng oligopeptide may be other ginseng oligopeptides requiring liver targeting delivery in the prior art. Besides the combination of the ginseng oligopeptide and the carrier, the ginseng oligopeptide can be further combined 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, relieving nonalcoholic fatty liver and the like. Therefore, the present invention further includes the following step 4.
Step 4, preparation of a ginseng oligopeptide-astaxanthin-galactosylated bacterial cellulose complex carrier: dissolving astaxanthin oil with astaxanthin content of 10% in ethanol solution, stirring uniformly, dissolving Ginseng radix oligopeptide-galactosylated bacterial cellulose powder in 10 times of ultra-pure water, slowly dripping the astaxanthin oil ethanol solution into the stirred Ginseng radix oligopeptide-galactosylated bacterial cellulose solution, heating for reaction, evaporating to remove ethanol, and lyophilizing to obtain Ginseng radix oligopeptide-astaxanthin-galactosylated bacterial cellulose microparticle (GP-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 ginseng oligopeptide-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 the ginseng oligopeptide and the astaxanthin, on one hand, the ginseng oligopeptide is connected with galactose grafted on the bacterial cellulose through Maillard reaction, on the other hand, the ginseng oligopeptide and the astaxanthin are wrapped through emulsification, and the ginseng 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 ginseng oligopeptide and the astaxanthin can be prolonged, the ginseng 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 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 ginseng oligopeptide from hydrolysis and digestion effects of various proteases in the gastrointestinal tract and improving the stability of astaxanthin in the gastrointestinal tract. Therefore, the ginseng oligopeptide-astaxanthin-galactosylated bacterial cellulose particles have better flexibility and high adhesive force, not only can 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 ginseng oligopeptide and the astaxanthin, but also can enhance the adhesion effect of a firm adhesion layer, effectively prolong the stay time in the gastrointestinal tract to enhance the absorption, and the flexibility also reduces 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 ginseng oligopeptide-astaxanthin-galactosylated bacterial cellulose prepared by the method provided by the invention protects the molecular structure of the ginseng oligopeptide and astaxanthin in the gastrointestinal digestive tract, enhances the absorption and the delayed release of the ginseng oligopeptide and astaxanthin, and finally, the ginseng oligopeptide and astaxanthin are delivered to liver cells in a precise targeting manner. The ginseng oligopeptide and the astaxanthin act synergistically to regulate an oxidative stress system of liver cells, reduce generation of ROS, relieve peroxidation and denaturation of lipid, and meanwhile, the ginseng oligopeptide (Leu-Gly-His-Glu-Ser) regulates and controls expression of LPS-TLR4-NF- κB pathway related proteins, so that liver inflammatory response is improved, and compared with single use, the ginseng oligopeptide has better liver injury treatment effect. The method is simple and easy to operate, is suitable for industrial preparation scenes, and has wide application prospect in preventing and treating alcoholic liver injury.
Example 1:
step 1, after 1000g of ginseng is coarsely crushed, adding 15 times of water with mass volume to prepare homogenate, adding cellulase with the mass of 1.0% of the ginseng, carrying out enzymolysis for 2 hours at 45 ℃, and controlling the pH value of enzyme reaction to be 5.0; adding compound protease with 5% of ginseng protein mass, carrying out enzymolysis for 3 hours at 50 ℃, controlling the pH value of enzyme reaction at 9.0, wherein the mass ratio of the compound protease is as follows: alkaline protease: bromelain flavourzyme = 3:2:2; heating to 90 ℃ after enzymolysis is finished, inactivating enzyme for 10 minutes to obtain ginseng enzymolysis liquid, centrifuging the ginseng enzymolysis liquid for 10 minutes at 8000 revolutions per minute, removing granular substances, separating by adopting a membrane with a cut-off molecular weight of 3000Da, separating and purifying the membrane-passing liquid by using a DEAE52 ion exchange chromatographic column, eluting with deionized water at an eluting flow rate of 0.8mL/min, detecting absorbance at 220nm, and collecting an eluting peak with a retention time of 13.63 min; adopting a C18 chromatographic column, wherein the mobile phase A is trifluoroacetic acid water with the volume percentage of 0.05%, the mobile phase B is methanol, and the gradient elution condition is as follows: 0 to 10min,5 percent of B,10 to 20min,5 to 10 percent of B,20 to 25min,10 to 20 percent of B,25 to 35min,20 to 30 percent of B, the flow rate is 0.8mL/min, the elution peak of 27.75min is collected, and 5.5g of ginseng oligopeptide is obtained by freeze drying after concentration;
Step 2, 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 50℃and stirred for 12 hours, and then dialyzed for 48 hours, and then freeze-dried to obtain 25g of galactosylated bacterial cellulose powder.
And 3, dissolving 5g of ginseng oligopeptide powder and 3g of galactosylated bacterial cellulose powder in 10 times of ultrapure water, stirring at 100 ℃ for reaction for 3 hours, and freeze-drying to obtain 8g of ginseng oligopeptide-galactosylated bacterial cellulose composite carrier powder.
And 4, dissolving 8g of astaxanthin oil in 100ml of ethanol solution, uniformly stirring, dissolving 8g of ginseng oligopeptide-galactosylated bacterial cellulose powder in ultrapure water with the volume of 10 times, then slowly dripping the astaxanthin oil ethanol solution into the stirred ginseng oligopeptide-galactosylated bacterial cellulose solution, heating to 55 ℃ for reaction for 2 hours at the stirring speed of 1000r/min, evaporating to remove ethanol, and freeze-drying to obtain 16g of GP-AST-GBC microparticle powder.
Example 2:
step 1, after 1000g of ginseng is coarsely crushed, adding water with the mass volume of 20 times to prepare homogenate, adding cellulase with the mass of 0.5% of the ginseng, carrying out enzymolysis for 2 hours at 50 ℃, and controlling the pH value of enzyme reaction to be 6.0; adding 3% of compound protease by mass of ginseng protein, performing enzymolysis for 4 hours at 50 ℃, controlling the pH value of enzyme reaction at 10.0, wherein the mass ratio of the compound protease is as follows: alkaline protease: bromelain flavourzyme=5:4:4; heating to 90 ℃ after enzymolysis is finished, inactivating enzyme for 10 minutes to obtain ginseng enzymolysis liquid, centrifuging the ginseng enzymolysis liquid for 10 minutes at 8000 revolutions per minute, removing granular substances, separating by adopting a membrane with a cut-off molecular weight of 3000Da, separating and purifying the membrane-passing liquid by using a DEAE52 ion exchange chromatographic column, eluting with deionized water at an eluting flow rate of 1.0mL/min, detecting absorbance at 220nm, and collecting an eluting peak with a retention time of 13.32 minutes; adopting a C18 chromatographic column, wherein the mobile phase A is trifluoroacetic acid water with the volume percentage of 0.08%, the mobile phase B is methanol, and the gradient elution condition is as follows: 0 to 10min,5 percent of B,10 to 20min,5 to 10 percent of B,20 to 25min,10 to 20 percent of B,25 to 35min,20 to 30 percent of B, the flow rate is 1.0mL/min, collecting an elution peak for 27.46min, concentrating and freeze-drying to obtain 5.7g of ginseng 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; 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 1 hour; the bacterial cellulose solution was added to lactobionic acid solution and heated to 40℃with stirring for 18 hours, and then dialyzed for 36 hours and freeze-dried to obtain 39g of galactosylated bacterial cellulose powder.
And 3, dissolving 5g of ginseng oligopeptide powder and 5g of galactosylated bacterial cellulose powder in 10 times of volume of ultrapure water, stirring at 95 ℃ for reaction for 5 hours, and freeze-drying to obtain 10g of ginseng oligopeptide-galactosylated bacterial cellulose composite carrier powder.
And 4, dissolving 5g of astaxanthin oil in 100ml of ethanol solution, uniformly stirring, dissolving 10g of ginseng oligopeptide-galactosylated bacterial cellulose powder in ultrapure water with the volume of 10 times, then slowly dripping the astaxanthin oil ethanol solution into the stirred ginseng oligopeptide-galactosylated bacterial cellulose solution, heating to 60 ℃ for reaction for 3 hours at the stirring speed of 1000r/min, evaporating to remove ethanol, and freeze-drying to obtain 15g of GP-AST-GBC microparticle powder.
Comparative example 1:
10g of ginseng oligopeptide-galactosylated bacterial cellulose complex carrier (GP-GBC) powder was obtained by the procedure of step 1 to step 3 of example 1.
Comparative example 2:
step 1, taking 10g 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 4g of lactobionic acid, dissolving in 1000ml of DMSO solution containing 5g EDC and 5g NHS, stirring and activating carboxyl 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 14g of galactosylated bacterial cellulose powder.
And 2, dissolving 8g of astaxanthin oil in 100ml of ethanol solution, uniformly stirring, dissolving 8g of galactosylated bacterial cellulose powder 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: 5.2g of ginseng oligopeptide 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, dissolving 5g of ginseng oligopeptide 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 8g of ginseng oligopeptide-galactosylated chitosan powder.
And 4, dissolving 8g of astaxanthin oil in 100ml of ethanol solution, uniformly stirring, dissolving 8g of ginseng oligopeptide-galactosylated chitosan powder in ultrapure water with the volume of 10 times, then slowly dripping the astaxanthin oil ethanol solution into the stirred ginseng oligopeptide-galactosylated chitosan solution, heating to 60 ℃ for reaction for 2 hours at the stirring speed of 500r/min, evaporating to remove ethanol, and then freeze-drying to obtain 16g of ginseng oligopeptide-astaxanthin-galactosylated chitosan (GP-AST-GC) powder.
Experiment 1: determination of DPPH free radical scavenging Capacity of Ginseng radix oligopeptide (Leu-Gly-His-Glu-Ser)
The ginseng oligopeptide (GP) and the ginseng oligopeptide-astaxanthin-galactosylated bacterial cellulose (GP-AST-GBC) obtained in example 1 were weighed to prepare a sample solution having 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 ginseng oligopeptide solution (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 GP, GP-AST-GBC and ascorbic acid on DPPH free radical scavenging capacity are 9.35, 7.22 and 21.04 mug/mL respectively, which indicate that the ginseng oligopeptide purified in the step 1 has remarkable antioxidant activity, and the antioxidant activity of the ginseng oligopeptide and astaxanthin are not destroyed and are greatly improved after being embedded by a carrier.
Experiment 2: immunomodulatory Activity of Ginseng radix oligopeptide (Leu-Gly-His-Glu-Ser)
HepG2 cells were incubated with lipopolysaccharide LPS (1. Mu.g/mL) for 24 hours, with an aqueous solution of ginseng oligo-active peptide (2. Mu.g/mL) for 24 hours, and the secretion level of TNF-. Alpha.inflammatory factor was examined. The results show that the TNF-alpha level (12.57+/-1.93 pg/ml) of the HepG2 cells treated by the sea cucumber oligomerization active peptide solution is remarkably reduced compared with the HepG2 cell inflammation model group (57.35 +/-2.82 pg/ml) which is not treated by the ginseng oligomerization active peptide solution, and the normal HepG2 cells (5.89+/-0.64 pg/ml) close to the blank group show good liver cell inflammation reducing effect of the ginseng oligomerization active peptide (Leu-Gly-His-Glu-Ser).
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. The GP-AST-GBC1,2 obtained in examples 1 and 2 and the GP-GBC, AST-GBC and GP-AST-GC powder obtained in comparative example were respectively taken, 4mg of the powder was placed in a dialysis bag (MWCO 3500), 200mL of artificial gastric juice (or intestinal juice) was immersed, stirred in a constant temperature magnetic stirrer at 37℃for a system temperature, 1mL of the artificial gastric juice (or intestinal juice) was sampled at a rotational speed of 100rpm/min at 30min and at the time points of 1, 6, 12 and 24 hours, 1mL of the blank artificial gastric juice (or intestinal juice) with the same matrix was added after the sampling, the supernatant was obtained after centrifugation, and the content of the ginseng oligopeptide and astaxanthin was detected by a high performance liquid chromatograph after filtration with a 0.22 μm organic filter membrane. 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 the GP-AST-GBC1, 2, GP-GBC, AST-GBC and GP-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 at 6h and 12h, the GP-AST-GBC group has a protective effect on the ginseng peptide-astaxanthin, the ginseng peptide-galactose-bacterial cellulose and the 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 carrying system for independently carrying the ginseng peptide or the astaxanthin, and meanwhile, the stability is obviously better than that of a carrying system of galactosylated chitosan due to the three-dimensional network effect of the bacterial cellulose, so that the GP-AST-GBC system can stably exist in the environment of the artificial gastrointestinal fluid, and the ginseng oligopeptide and the astaxanthin are protected, and are favorable for being absorbed by human bodies.
Experiment 3: liver targeting
Male SD rats were bred adaptively for 3-5d, randomly with groups GP-AST-GBC1 (5 mg/kg), AST-GBC (3 mg/kg) and GP-AST-GC (5 mg/kg), each group of 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 GP-AST-GBC was evaluated with targeting efficiency (Targeting efficiency, te) according to the method of Gupta. The results show that the liver targeting efficiency (54.79 + -4.10) of the GP-AST-GBC group is higher than that of the AST-GBC group (40.52 + -4.63) and the GP-AST-GC group (36.08+ -5.30), and the liver targeting is very high.
Experiment 4: improving effect of alcoholic liver injury mice
The 60 mice were randomly divided into 6 groups, namely, a blank group, a model group, a GP-AST-GBC group (5 mg/kg), a GP group (3 mg/kg), an AST group (3 mg/kg) and a GP-AST-GC group (5 mg/kg), 10 mice each. Day 1 to day 9 of the experiment: 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, for 9 consecutive days. Day 9-12: i.e. from day 9 of the experiment, the corresponding dose of drug was administered normally every morning, and after 1h, the remaining groups of mice were perfused with 40% ethanol solution (10 mL/kg) for 4 days. After the last administration of ethanol solution, the mice are killed by a cervical dislocation method without water inhibition after being fasted for 4 hours, the liver tissues are dissected and weighed, 9 times of physiological saline is added to prepare 10% liver homogenate, the 10% liver homogenate is centrifuged for 10 minutes at 3500r/min, and the supernatant is taken and split-packed. The liver tissue MDA and TNF-alpha content of each group of mice was examined.
The results are shown in the following table:
TABLE 4 liver MDA and TNF-alpha content
| Group of | MDA(U/mg) | TNF-α(pg/mg) |
| Blank space | 0.25±0.11 | 55.23±3.74 |
| Model | 0.72±0.18 | 82.05±4.60 |
| GP-AST-GBC | 0.30±0.12 | 59.77±5.10 |
| GP | 0.61±0.17 | 75.62±3.53 |
| AST | 0.49±0.15 | 68.34±5.05 |
| GP-AST-GC | 0.44±0.08 | 63.95±4.66 |
The result shows that the MDA content in the liver of the model group mice is obviously increased, the more serious oxidative stress reaction occurs in the model group mice, and the content of the pro-inflammatory cytokine TNF-alpha is also greatly increased, thereby causing the inflammatory injury of the liver cells. The GP-AST-GBC intervention treatment obviously reduces the lipid peroxidation degree in the liver, plays a good role in regulating inflammatory reaction, and has better effect than independently feeding the ginseng oligopeptide or astaxanthin and the ginseng oligopeptide-astaxanthin-galactosylated chitosan carrying system.
The experimental result shows that the ginseng oligopeptide (Leu-Gly-His-Glu-Ser) provided by the invention has good antioxidant and immunoregulatory activities, and the ginseng oligopeptide-astaxanthin-galactosylated bacterial cellulose delivery system has good improving effect on alcoholic liver injury, thus being a potential high-quality liver-protecting ingredient.
The invention discloses a preparation method of ginseng oligopeptide astaxanthin liver targeting microspheres and an effect of improving alcoholic liver injury. The ginseng oligopeptide solution is obtained from ginseng through the steps of chopping, enzymolysis, ultrafiltration and the like, is connected and carried with galactosylated modified bacterial cellulose, and finally the ginseng oligopeptide-galactosylated bacterial cellulose carrier reacts with astaxanthin oil and is freeze-dried to prepare the ginseng oligopeptide-astaxanthin-galactosylated bacterial cellulose particles with liver targeting. The prepared ginseng oligopeptide 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 oxidative stress and inflammation of liver cells, further improves alcoholic liver injury, and has extremely 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 ginseng oligopeptide, which is characterized in that the amino acid sequence of the ginseng oligopeptide comprises Leu-Gly-His-Glu-Ser.
2. A method for producing a ginseng oligopeptide according to claim 1, which comprises
S11, adding water 15-20 times of the ginseng into the ginseng to prepare homogenate, placing the homogenate into an enzymolysis tank, and adding cellulase accounting for 0.5-1.0% of the ginseng into the enzymolysis tank;
s12, adding compound protease accounting for 3-5% of the mass of the ginseng into an enzymolysis tank, and heating and inactivating enzyme after enzymolysis to obtain ginseng enzymolysis liquid, wherein the compound protease comprises alkaline protease, bromelain and flavourzyme, and the alkaline protease comprises bromelain and flavourzyme= (3-5): (2-4);
s13, centrifuging the ginseng 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 an ion exchange chromatographic column, wherein an eluting solvent is deionized water, the eluting flow rate is 0.8-1.0 mL/min, detecting absorbance at 220nm, and collecting an eluting peak with retention time of 13-14 min;
S16, further purifying by adopting a chromatographic column, wherein the mobile phase A is trifluoroacetic acid water with the volume percentage of 0.05-0.08%, the mobile phase B is methanol, and the gradient elution condition is as follows: 0 to 10min,5 percent of B,10 to 20min,5 to 10 percent of B,20 to 25min,10 to 20 percent of B,25 to 35min,20 to 30 percent of B, the flow rate is 0.8 to 1.0mL/min, and elution peaks of 27 to 28min are collected to obtain the ginseng oligopeptide.
3. The method for producing ginseng oligopeptide according to claim 2,
preferably, in the step S11, the enzymolysis temperature of the cellulase is 45-50 ℃, the enzymolysis time is 1-2 hours, and the enzyme reaction pH is 5.0-6.0;
preferably, in the step S12, the enzymolysis temperature is 50-55 ℃, the enzyme reaction pH is 9.0-10.0, and the enzymolysis time is 3-4 hours;
preferably, in the step S12, after enzymolysis, the temperature is raised to 80-90 ℃ to inactivate enzymes for 10-15 minutes, so as to obtain ginseng enzymolysis liquid;
preferably, in the step S13, the ginseng enzymolysis liquid is centrifuged for 8-10 minutes at 6000-8000 rpm;
preferably, in step S15, separation and purification are performed using a DEAE52 ion exchange chromatography column;
preferably, in step S16, further purification is performed using a C18 chromatographic column;
preferably, in step S16, the ginseng oligopeptide is concentrated and freeze-dried to obtain ginseng oligopeptide powder.
4. A method for preparing a ginseng oligopeptide-galactosylated bacterial cellulose carrier complex is characterized by comprising the following steps of
S31, dissolving ginseng oligopeptide powder and a galactosylated bacterial cellulose carrier in water, stirring, and freeze-drying to obtain a ginseng 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 ginseng 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 a ginseng oligopeptide-galactosylated bacterial cellulose carrier complex according to claim 4,
In the step S31, dissolving ginseng oligopeptide 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 ginseng oligopeptide-galactosylated bacterial cellulose carrier composite powder;
preferably, in step S31, the mass ratio of ginseng oligopeptide to galactosylated bacterial fiber carrier powder is 1-10:1, more preferably 5:1.
7. a ginseng oligopeptide-galactosylated bacterial cellulose carrier complex prepared by the preparation method according to any one of claims 4-6.
8. A method for preparing a ginseng 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 ginseng oligopeptide-galactosylated bacterial cellulose carrier compound in water;
s42, adding the ethanol solution dissolved with astaxanthin oil into the solution of the ginseng oligopeptide-galactosylated bacterial cellulose carrier complex, heating for reaction, and evaporating to remove ethanol to obtain the ginseng oligopeptide-astaxanthin-galactosylated bacterial cellulose carrier complex.
9. The method for preparing a ginseng 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 ginseng oligopeptide-galactosylated bacterial cellulose carrier complex is 1:1-10, more preferably 1:1;
preferably, in step S41, the ginseng oligopeptide-galactosylated bacterial cellulose carrier complex is dissolved in 10 volumes of ultra pure water;
in the step S42, slowly dripping the ethanol solution dissolved with astaxanthin oil into the solution of the ginseng oligopeptide-galactosylated bacterial cellulose carrier compound, heating and stirring for reaction in the dripping process, evaporating to remove ethanol, and freeze-drying to obtain the ginseng 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 ginseng oligopeptide-astaxanthin-galactosylated bacterial cellulose carrier complex prepared by the method of any one of claims 8-9.
11. Use of a ginseng oligopeptide according to claim 1 or a ginseng oligopeptide-galactosylated bacterial cellulose carrier complex according to claim 7 or a ginseng oligopeptide-astaxanthin-galactosylated bacterial cellulose carrier complex according to claim 10 for the preparation of a medicament for the treatment of liver injury.
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| CN117925766B (en) * | 2024-03-22 | 2024-05-17 | 吉林肽谷生物工程有限责任公司 | Preparation method of ginseng peptide with protective effect on acute alcoholic liver injury |
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