WO2022179260A1 - 一种组合物在防治酒精性脑损伤中的应用 - Google Patents

一种组合物在防治酒精性脑损伤中的应用 Download PDF

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WO2022179260A1
WO2022179260A1 PCT/CN2021/138171 CN2021138171W WO2022179260A1 WO 2022179260 A1 WO2022179260 A1 WO 2022179260A1 CN 2021138171 W CN2021138171 W CN 2021138171W WO 2022179260 A1 WO2022179260 A1 WO 2022179260A1
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composition
parts
brain injury
preventing
sodium alginate
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PCT/CN2021/138171
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English (en)
French (fr)
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胡章
卢思彤
洪鹏志
孔松芝
赵云涛
钟赛意
李程鹏
程瑜
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广东海洋大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/01—Hydrolysed proteins; Derivatives thereof
    • A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/02—Algae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/132—Amines having two or more amino groups, e.g. spermidine, putrescine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/734—Alginic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/618—Molluscs, e.g. fresh-water molluscs, oysters, clams, squids, octopus, cuttlefish, snails or slugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/205—Polysaccharides, e.g. alginate, gums; Cyclodextrin
    • A61K9/2059—Starch, including chemically or physically modified derivatives; Amylose; Amylopectin; Dextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4841—Filling excipients; Inactive ingredients
    • A61K9/4858—Organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4841—Filling excipients; Inactive ingredients
    • A61K9/4866—Organic macromolecular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
    • A61P25/32—Alcohol-abuse
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to the technical field of biomedicine, in particular to the application of a composition in preventing and treating alcohol-induced brain injury.
  • the cerebral cortex of people who drink for a long time will be in a state of anesthesia, and the damage to the nervous system will be manifested in a series of thinking and behavioral release states, such as the patient's intellectual disturbance, impulsiveness, ataxia, incoherence, vomiting, drowsiness, etc. In severe cases, it can cause Seizures, convulsions and even death. Alcohol interferes with tissue oxidative stress defense system, causes structural changes of lipids and proteins in cell membranes, alters the fluidity of nerve cell membranes and the activity of adenosine triphosphate, hinders calcium transport, causes nerve cell dysfunction, and leads to nerve cell death.
  • alcohol can cause damage to the central and peripheral nerves, and it is especially easy to penetrate the blood-brain barrier and act on the brain, causing damage to brain nerve cells.
  • the incidence of brain diseases caused by alcohol continues to increase, but there are few researches on the mechanism of alcohol damage to the brain and the protective effect of alcohol on the brain. Therefore, there is a market to find a hangover product that can effectively prevent and treat alcohol-induced brain damage. value.
  • Chinese patent CN 201510956371.1 discloses a tea bag composition for relieving alcohol-induced liver, brain and heart damage. , chrysanthemum, reed root and papaya, which have the effect of protecting the liver and heart damage caused by alcohol.
  • Chinese patent CN 201310692332.6 discloses the application of resveratrol glycosides in the preparation of drugs for the treatment of chronic alcoholism brain damage, which is mainly composed of resveratrol glycosides and has neuroprotective effects on chronic alcoholism.
  • Chinese patent CN 201510915401.4 discloses the application of black thread silver gum extract in alcohol-induced brain injury protection medicine, which is mainly composed of black thread silver gum extract, and has a protective effect on brain damage in alcoholic rats. Most of these substances have a wide range of curative effects and can increase the activity of antioxidant enzymes in the body and reduce oxidative damage to the brain, but it is difficult to explain the protective mechanism of alcohol-induced brain damage, and the bioavailability of antioxidant active substances is low.
  • the object of the present invention is to provide the application of a composition in the prevention and treatment of alcoholic brain injury, to solve the problems existing in the above-mentioned prior art, the composition is simple in formula, good in stability, strong in antioxidant properties, and can prevent and treat alcoholic brain injury The effect is good, and it is convenient to take.
  • the present invention provides the following scheme:
  • the invention provides an application of a composition in preparing a pharmaceutical preparation for preventing and treating alcoholic brain injury.
  • the composition in parts by weight, comprises the following components: 10-30 parts of seaweed polyphenols, and 2-6 parts of sodium alginate , 30-55 copies of abalone peptides and 0.5-3 copies of spermidine.
  • seaweed polyphenol is a polyphenolic compound extracted from seaweed, the main component is phloroglucinol and its derivatives, and its hydroxyl group is an important group with antioxidant activity. Due to the ability of seaweed polyphenols to scavenge free radicals and reactive oxygen species directly or indirectly, they are considered as natural antioxidants to prevent or reduce chronic diseases. However, polyphenols have poor stability and low bioavailability in vivo.
  • Sodium alginate is a kind of biopolymer material extracted from the ocean. It has the advantages of biocompatibility, hydrophilicity and non-toxicity. It can form hydrogels through ionization under mild conditions. Alginic acid hydrogels are prepared by chemical cross-linking, enzymatic cross-linking, etc., and are widely used in controlled release systems for pharmaceutical active peptides and proteins.
  • Abalone peptide is made by enzymatic hydrolysis of abalone meat. It has a complete range of amino acids and a reasonable ratio. It is known as the "soft gold" of the ocean. Abalone is rich in nutrients such as EPA, DHA, trace elements, taurine and superoxide dismutase, and has the functions of anti-oxidation, enhancing immunity and anti-fatigue.
  • Spermidine a naturally occurring polyamine, is a safe and efficient inducer of autophagy, exists in almost all cells, and has anti-aging, anti-cancer, cardiovascular and neuroprotective effects.spermidine is conducive to assisting drug delivery, improving the water solubility, stability and membrane permeability of drug molecules, thereby further improving drug absorption in vivo.
  • seaweed polyphenol particles are prepared by blending alginate and spermidine with seaweed polyphenol with antioxidative and antibacterial functions.
  • the spermidine molecule contains three amino groups, and the two ends and the middle amino groups can be connected with sodium alginate and seaweed polyphenol respectively, forming an "umbrella" protective structure for the seaweed polyphenol and improving the stability of the product.
  • Seaweed polyphenol particles and abalone peptides rich in multiple nutrients and multiple functions form complex compositions through multiple non-covalent bonds such as hydrogen bonds, electrostatics, and ⁇ - ⁇ stacking. It has a synergistic effect on the prevention and treatment of alcohol-induced brain injury.
  • sodium alginate forms alginic acid colloid under the action of gastric acid, which encapsulates algae polyphenols and abalone peptides to avoid the loss of activity in the complex environment in the body; alginic acid is converted into ionic salt state in the intestinal tract, which lasts for a long time. Releases seaweed polyphenols and abalone peptides for long-lasting activity and improved bioavailability.
  • the seaweed polyphenols are derived from edible macroalgae, including kelp, kelp, sargassum, asparagus, hijiki, wakame or gracilaria.
  • the extraction method of the seaweed polyphenol can be carried out according to the conventional extraction method of seaweed polyphenol in the art.
  • the total phenol weight content in the seaweed polyphenol used in the present invention is not less than 30%.
  • the abalone peptide is a composite peptide prepared from abalone meat through biological enzymolysis.
  • the enzymatic hydrolysis method of the abalone peptide can be carried out according to the conventional method in the art.
  • the molecular weight distribution range of the abalone peptides used in the present invention is: the content of polypeptides with molecular weight >3.0kDa is 2.15%, the content of polypeptides with molecular weights of 1.0-3.0kDa is 16.59%, and the content of polypeptides with molecular weights ⁇ 1.0kDa is 81.26%.
  • the composition includes the following components: 20 parts of seaweed polyphenols, 4 parts of sodium alginate, 40 parts of abalone peptides and 1.5 parts of spermidine.
  • preparation method of the composition comprises the following steps:
  • the concentrations of the sodium alginate solution and the abalone peptide solution have an important influence on the stability and synergistic effect of the composition.
  • the mass concentration of the sodium alginate solution is 1%-3.5%, preferably 1.5%; if the mass concentration of the sodium alginate solution is too large (>3.5%), it is easy to stick together, and it is difficult to form a uniform algal mass Phenol particles have poor efficacy; if the mass concentration of the sodium alginate solution is too small ( ⁇ 1%), the effect with spermidine is weak, and the protective effect on seaweed polyphenols cannot be achieved.
  • the mass concentration of the abalone peptide solution is 18%-26%, preferably 22%; if the mass concentration of the abalone peptide solution is too large (>26%), it is susceptible to microbial infection and loses activity; The mass concentration of algae is too small ( ⁇ 18%), the synergistic effect with seaweed polyphenols is weakened, and the effect of preventing and treating alcohol-induced brain injury cannot be achieved.
  • step S1 the pH range of the solution needs to be strictly controlled.
  • the method for adjusting the pH of the present invention is a conventional method. Since the sodium alginate solution is weakly alkaline, the present invention adopts a 0.1 mol/L HCl solution for adjusting the pH. If the pH of the solution is too low ( ⁇ 6.0), the acidity will be too strong, and sodium alginate will form alginic acid colloids; if the pH of the solution is too high (>7.5), spermidine will be difficult to interact with sodium alginate and algae through electrostatic interaction. Phenols form complex "umbrella" structures. Therefore, in the present invention, the pH of the sodium alginate solution is preferably 6.0-7.5, more preferably 6.5.
  • the room temperature involved in the present invention is also referred to as normal temperature or general temperature, and is generally defined as 25°C, which can be reasonably adjusted by those skilled in the art, which is also within the protection scope of the present invention.
  • the pharmaceutical preparation also includes pharmaceutically acceptable excipients.
  • the pharmaceutical preparation is a tablet
  • the auxiliary materials include corn starch, talc and magnesium stearate
  • the mass fraction of each component in the tablet is:
  • composition is 10-20wt%, corn starch 65-75wt%, talc 11-17wt% and magnesium stearate 0.5-2wt%.
  • the pharmaceutical preparation is a capsule
  • the auxiliary materials include lactose, cornstarch and talc;
  • the mass fraction of each component in the capsule is:
  • composition is 10-30 wt %, lactose 10-20 wt %, corn starch 50-60 wt % and talc 5-15 wt %.
  • the pharmaceutical preparation is a granule
  • the auxiliary materials include corn starch, sodium hydroxymethyl cellulose and magnesium stearate;
  • the mass fraction of each component in the granules is:
  • the composition comprises 5-15 wt %, corn starch 74-84 wt %, sodium hydroxymethyl cellulose 5-15 wt % and magnesium stearate 0.5-2 wt %.
  • the present invention combines seaweed polyphenols with antioxidant and antibacterial functions and abalone peptides rich in various nutrients and multiple biological effects, and forms a complex through multiple actions such as hydrogen bonds, static electricity, and ⁇ - ⁇ stacking. ; It has been proved by animal model tests that the compound plays a multi-channel, multi-target and synergistic effect in the prevention and treatment of alcoholic brain injury.
  • the present invention utilizes the hydrogel properties of alginate to form alginate colloid in the stomach with a low pH environment, and encapsulates the algae polyphenol and abalone peptide complexes to avoid the loss of its activity in the complex environment of the body;
  • the alginic acid in the intestine is converted into an ionic salt state, and the algal polyphenols and abalone peptides are released slowly and controlled to exert a lasting active effect.
  • the raw materials of the present invention all come from natural food materials, which are green and safe; the preparation process is simple, and it is easy to industrialize production; and it is safe, non-toxic, convenient to take, has no bad smell, and has good compliance.
  • Figure 1 shows the scavenging ability of the composition for preventing and treating alcohol-induced brain injury prepared by the present invention to DPPH free radicals.
  • Figure 2 shows the scavenging ability of the composition for preventing and treating alcohol-induced brain injury prepared by the present invention to OH free radicals.
  • Figure 3 shows the antioxidant stability of the composition for preventing and treating alcohol-induced brain injury prepared by the present invention.
  • Figure 4 is the effect of the composition for preventing and treating alcohol-induced brain injury prepared by the present invention on the brain index of mice.
  • Figure 5 shows the effect of the composition for preventing and treating alcohol-induced brain injury prepared by the present invention on the swimming route of mice after removing the platform.
  • the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
  • the reagents and materials used in the following examples are commercially available.
  • the seaweed polyphenols and abalone peptides used in the present invention are prepared by conventional methods, the seaweed polyphenols can also be purchased from Shandong Jiejing Group Co., Ltd., and the abalone peptides can also be purchased from Shandong Hailongyuan Biotechnology Ltd.
  • a composition for preventing and treating alcohol-induced brain injury comprising the following components in parts by weight: 20 parts of seaweed polyphenols, 4 parts of sodium alginate, 40 parts of abalone peptides and 1.5 parts of spermidine.
  • the preparation method of the composition comprises the following steps:
  • a composition for preventing and treating alcohol-induced brain injury comprising the following components in parts by weight: 15 parts of seaweed polyphenols, 3 parts of sodium alginate, 35 parts of abalone peptides and 1 part of spermidine.
  • the preparation method of the composition comprises the following steps:
  • a composition for preventing and treating alcohol-induced brain injury comprising the following components in parts by weight: 10 parts of seaweed polyphenols, 2 parts of sodium alginate, 30 parts of abalone peptides and 0.5 parts of spermidine.
  • the preparation method of the composition comprises the following steps:
  • a composition for preventing and treating alcohol-induced brain injury comprising the following components in parts by weight: 30 parts of seaweed polyphenols, 6 parts of sodium alginate, 55 parts of abalone peptides, and 3 parts of spermidine.
  • the preparation method of the composition comprises the following steps:
  • Example 1 The only difference from Example 1 is that the seaweed polyphenols are replaced by vitamin C.
  • Example 1 The only difference from Example 1 is that spermidine is used instead of spermidine.
  • Example 1 The only difference from Example 1 is that the direct raw materials are blended, as follows:
  • a composition for preventing and treating alcohol-induced brain injury comprising the following components in parts by weight: 20 parts of seaweed polyphenols, 4 parts of sodium alginate, 40 parts of abalone peptides and 1.5 parts of spermidine.
  • the preparation method of the composition comprises the following steps:
  • composition is obtained by taking the raw materials of sodium alginate, spermidine, seaweed polyphenol and abalone peptide according to the stated weight parts, adding distilled water, suspending uniformly, and freeze-drying.
  • Example 1 The only difference from Example 1 is that oyster peptide is used instead of abalone peptide.
  • AX is the absorbance of the sample, Absorbance of blank control.
  • Vitamin C (Vc) a strong antioxidant, served as a positive control.
  • the DPPH free radical scavenging effect of the composition for preventing and treating alcohol-induced brain damage is shown in Figure 1.
  • the IC 50 of the Examples 1 to 4 groups are all close to the Vc group, showing a strong DPPH free radical scavenging effect, especially the DPPH of Example 1.
  • the free radical scavenging ability is the strongest.
  • Group 1 of Comparative Example also showed a DPPH free radical scavenging effect similar to that of Groups 1 to 4, the scavenging ability of DPPH free radicals of Groups 2 to 4 of Comparative Example was far inferior to that of Groups 1 to 4.
  • Vitamin C (Vc), a strong antioxidant, served as a positive control.
  • the hydroxyl radical scavenging effect of the composition for preventing and treating alcohol-induced brain damage is shown in Figure 2.
  • the IC 50 of the Examples 1 to 4 groups is almost close to the Vc group, showing a strong hydroxyl radical scavenging effect, especially the hydroxyl radical of Example 1.
  • the free radical scavenging ability is the strongest.
  • the comparative example 1 group also showed a hydroxyl radical scavenging effect similar to that of the examples 1 to 4, the hydroxyl radical scavenging ability of the comparative examples 2 to 4 was far inferior to that of the examples 1 to 4.
  • the groups of Examples 1-4 all showed strong free radical scavenging effects, indicating that the composition for preventing and treating alcohol-induced brain injury prepared by the present invention has strong antioxidant activity, especially the anti-oxidative activity of Example 1 group. The most oxidative activity. If the formulation or preparation method of the composition of the present invention is changed, the expected antioxidant effect cannot be achieved.
  • the total antioxidant capacity of the composition during storage was measured using the iron ion reduction/antioxidative capacity assay (FRAP method).
  • Samples are stored in brown bottles at room temperature. Samples were taken at regular intervals and tested for total antioxidant capacity.
  • Test method Take 0.02mL FeSO 4 solution, add 0.18mL FRAP solution (prepared and used now), mix well, take a water bath at 37°C for 10min, and measure the absorbance at 593nm using a UV-Vis spectrophotometer. Taking the concentration of FeSO 4 solution as the abscissa and the absorbance value as the ordinate, the FeSO 4 equivalent standard curve was established. Taking the VC solution as the positive control, the FeSO 4 equivalent was calculated according to the standard curve. The relative percent VC was calculated for each sample based on FeSO 4 equivalents.
  • mice Kunming male mice, 6-8 weeks old, body weight 18-22 grams, SPF grade, animal feeding conditions: (25 ⁇ 1)° C., relative humidity: (55 ⁇ 5)%.
  • mice 140 mice were randomly divided into 10 groups, 14 mice in each group, divided into normal saline group, model group, Example 1-4 groups and Comparative Example 1-4 groups.
  • mice in each group were given a corresponding volume of 56-degree Hongxing Erguotou Liquor (the corresponding volume of normal saline in the normal saline group) by gavage according to the gradient concentration every day.
  • Corresponding test substances were given to each group by gavage, and the normal saline group and the model group were given normal saline by gavage. Continuous gavage for 8 weeks.
  • Mice were weighed weekly to adjust alcohol and test substance doses. In the first week, the drinking amount was 2 mL/kg ⁇ BW. In order to cause persistent alcohol damage, the drinking amount was appropriately increased according to the weekly death of mice.
  • mice Alcohol-induced brain injury can cause a significant decline in learning and memory in mice.
  • the ability of mice to learn and remember spatial location and orientation was tested by finding a platform hidden underwater in a water maze. Mice were subjected to the Morris water maze test 8 weeks after alcohol injury. Mice swam in a black circular pool with a diameter of 120 cm and a height of 40 cm. The pool was filled with water and contained a movable platform with a diameter of 8 cm. The experiment was carried out for 5 days, 4 times a day, and put into water from four different starting points. The time it took for the mice to find the platform (escape latency) was recorded on video.
  • mice were dissected, the brain tissue was taken out, the blood on the surface was washed with pre-cooled normal saline, blotted dry with filter paper, and then weighed quickly, and the brain index was calculated by the formula:
  • Brain index (%) Brain weight (g) / Body weight (g) ⁇ 100%
  • the brain index test results of the composition for preventing and treating alcohol-induced brain injury are shown in FIG. 4 . After the mice were modeled, compared with the normal saline group, the brain index of the model group was significantly increased, and there was a significant difference (p ⁇ 0.01), indicating that continuous overdrinking of alcohol would cause cerebral edema in mice.
  • the brain indexes of the mice in the groups of Examples 1 to 4 were significantly reduced, with a statistical difference (p ⁇ 0.01), among which the effect of the Example 1 group was the best, and the brain index was almost close to the level of the normal saline group, indicating that
  • the composition for preventing and treating alcohol-induced brain injury prepared by the invention can effectively improve brain index, reduce edema, hyperplasia, hypertrophy and other pathological changes in brain tissue, and has a significant preventive and therapeutic effect on alcohol-induced brain injury.
  • the brain indices of the mice in the control groups 1 to 4 were reduced, there was no statistical difference. This shows that the changes in the formula or preparation method of the patented composition cannot effectively reduce the alcohol-induced brain index.
  • the brain index has no preventive effect on alcohol-induced brain damage.
  • the damage of alcohol to body cells is mainly through the induction of cellular oxidative stress. Excessive long-term drinking can lead to changes in SOD and GSH activities in the body's brain cells and further lead to cellular oxidative stress damage to the central nervous system. Therefore, it can regulate the antioxidant enzymes in vivo. activity to prevent and treat alcohol-induced brain injury.
  • Table 1 shows the effect of the composition for preventing and treating alcohol-induced brain injury on the activity of oxidase.
  • the contents of SOD and GSH-Px in the brain tissue of the mice in the model group were significantly decreased, with a statistical difference (p ⁇ 0.01), indicating that the abnormal brain oxidative stress in mice caused by continuous excessive alcohol intake is the cause of small One of the important factors of rat brain injury.
  • the contents of SOD and GSH-Px in the brain tissue of the mice in the groups of Examples 1 to 4 were significantly increased (p ⁇ 0.01), especially in the group of Example 1, indicating that the preparation of the present invention for the prevention and treatment of alcohol-induced brain injury.
  • the composition can significantly reduce the level of peroxides in the brain of mice caused by alcohol, and enhance the antioxidant capacity of brain tissue.
  • MDA is the product of free radicals acting on lipid peroxidation, and the degree of lipid peroxidation in the body can be reflected by measuring the content of MDA.
  • the MDA content in the brain tissue of the mice in Examples 1 to 4 was significantly lower than that in the model group (p ⁇ 0.01), and the MDA content in the Example 1 group was the lowest.
  • the results show that the composition for preventing and treating alcohol-induced brain injury prepared by the present invention can enhance the antioxidative ability of mouse brain tissue by increasing the activities of SOD and GSH-Px, correct the balance disorder of oxidation and antioxidation, and reduce the damage to brain cells caused by free radicals. This protects against brain damage caused by chronic alcoholism.
  • Morris water maze test includes two parts: positioning navigation and space search.
  • the results of escape latency in positioning navigation test represent brain memory acquisition ability, and the results of space search test target quadrant stay time and the number of crossing platforms represent brain memory retention ability.
  • the experimental results in Tables 2 and 3 correspond to the results of positioning navigation and spatial search respectively.
  • mice in the saline group were more purposeful.
  • the results show that the brain memory acquisition function and the brain memory retention function of the mice were damaged when the excessive drinking continued to the 8th week of the experiment, and the composition for preventing and treating alcohol-induced brain injury prepared by the present invention has significant effects on the brain injury of mice caused by alcohol. improvement.
  • mice 20 healthy mice, weighing 18-22g, half male and half male, were randomly divided into experimental group and blank control group.
  • Test substance The composition of Example 1 was used as the test substance.
  • mice In the pre-experiment, the median lethal dose could not be measured, so the above-mentioned experimental dosage was used for the test. Based on the body weight of the mice, at a dose of 10 g/kg, the mice were gavaged once every 24 hours. The mice in the experimental group were continuously gavaged with distilled water for 2 weeks. During the period, the activity status, diet, feces, respiration, body weight and death of the mice were observed.
  • mice There was no death in the experimental group mice within 2 weeks of gavage with the test substance. Compared with the control group, there was no significant difference in body weight change, and there was no abnormality in eating and drinking. The mice were in good mental state and active. the animal
  • Example 5 A tablet for preventing and treating alcohol-induced brain injury
  • the tablet for preventing and treating alcohol-induced brain injury in terms of mass fraction, includes the following components:
  • Example 6 A tablet for preventing and treating alcohol-induced brain injury
  • the tablet for preventing and treating alcohol-induced brain injury in terms of mass fraction, includes the following components:
  • Example 7 A tablet for preventing and treating alcohol-induced brain injury
  • the tablet for preventing and treating alcohol-induced brain injury in terms of mass fraction, includes the following components:
  • Example 8 A capsule for preventing and treating alcohol-induced brain injury
  • the capsule for preventing and treating alcohol-induced brain injury includes the following components:
  • Embodiment 9 A kind of capsule for preventing and treating alcohol-induced brain injury
  • the capsule for preventing and treating alcohol-induced brain injury includes the following components:
  • Embodiment 10 A kind of capsule for preventing and treating alcohol-induced brain injury
  • the capsule for preventing and treating alcohol-induced brain injury includes the following components:
  • Example 11 A kind of granule for preventing and treating alcohol-induced brain injury
  • the capsule for preventing and treating alcohol-induced brain injury includes the following components:
  • Example 12 A kind of granule for preventing and treating alcohol-induced brain injury
  • the capsule for preventing and treating alcohol-induced brain injury includes the following components:
  • Example 13 A kind of granule for preventing and treating alcohol-induced brain injury
  • the capsule for preventing and treating alcohol-induced brain injury includes the following components:

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Abstract

公开了一种组合物在防治酒精性脑损伤中的应用，属于生物医药技术领域，提供一种组合物在制备防治酒精性脑损伤的药物制剂中的应用，该组合物以重量份计，包括以下组分：海藻多酚10-30份、海藻酸钠2-6份、鲍鱼肽30-55份和亚精胺0.5-3份；将具有抗氧化、抗菌功能的海藻多酚和富含多种营养、多重生物功效的鲍鱼肽组合，经动物试验证明，该组合物在酒精性脑损伤的防治中发挥多途径、多靶点、协同增效作用，同时具有良好的产品稳定性，生物利用度高，而且安全无毒、服用方便，无不良气味，依从性好。

Description

一种组合物在防治酒精性脑损伤中的应用 技术领域
本发明涉及生物医药技术领域，特别是涉及一种组合物在防治酒精性脑损伤中的应用。
背景技术
随着社会的进步和经济水平的快速发展，我国人民的生活水平得到了显著改善，对于酒精消费的需求越来越多，慢性酒精病成为全球公共卫生问题之一。酒精对身体器官及其各系统的影响主要通过肝脏代谢蔓延到肝脏以外，包括中枢神经系统、心血管系统、免疫系统、肾脏、肺、胃肠道、胰腺等器官。
长期喝酒的人大脑皮质会处于麻醉状态，对神经系统的损伤，表现为一系列思维及行为释放状态，如患者理智障碍、易冲动、共济失调、语无伦次、呕吐、嗜睡等，重者可引起癫痫、抽搐甚至死亡。酒精干扰组织氧化应激防御系统，引起细胞膜中类脂和蛋白质的结构变化，改变神经细胞膜的流动性和三磷酸腺苷的活性，阻碍钙的运输，引起神经细胞功能障碍，导致神经细胞死亡。作为一种亲脂性小分子毒性物质，酒精对中枢神经和周围神经均可造成损伤，尤其容易透过血脑屏障作用于脑部，造成大脑神经细胞的损伤。目前，由于酒精引起的脑疾病发生率持续增高，但酒精对脑的损伤机制和对脑的保护作用的产品却研究较少，因此，找到一种高效防治酒精性脑损伤的解酒制品具有市场价值。
现有技术中公开了多种防治酒精性脑损伤的物质，如中国专利CN 201510956371.1公开了一种缓解酒精性肝、脑和心损伤的袋泡茶组合物，主要由葛根、山楂、枳椇子、菊花、芦根和木瓜组成，具有保护酒精性肝、心损伤的作用。中国专利CN 201310692332.6公开了白藜芦醇苷在制备治疗慢性酒精中毒脑损药物中的应用，主要由白藜芦醇苷组成，具有对慢性酒精中毒的神经保护作用。中国专利CN 201510915401.4公开了黑线银胶提取物在酒精性脑损伤保护药物中的应用，主要由黑线银胶提取物组成，具有对酒精中毒大鼠脑损伤的保护作用。这些物质多数疗效广泛，能增加体内抗氧化物酶活性，降低脑的氧化损伤，但难以说明酒精性脑损伤的保护机制，且抗氧化活性物质生物利用度低。
发明内容
本发明的目的是提供一种组合物在防治酒精性脑损伤中的应用，以解决上述现有 技术存在的问题，该组合物配方简单，稳定性好，抗氧化性强，防治酒精性脑损伤效果好，而且服用方便。
为实现上述目的，本发明提供了如下方案：
本发明提供一种组合物在制备防治酒精性脑损伤的药物制剂中的应用，所述组合物以重量份计，包括以下组分：海藻多酚10～30份、海藻酸钠2～6份、鲍鱼肽30～55份和亚精胺0.5～3份。
其中，海藻多酚是从海藻中提取出来的多酚类化合物，主要成分是间苯三酚及其衍生物，其羟基是具有抗氧化活性的重要基团。由于海藻多酚具有直接或间接清除自由基和活性氧的能力，被认为是预防或减少慢性疾病的天然抗氧化剂。但多酚类物质稳定性差，体内生物利用度低。
海藻酸钠是一类从海洋中提取的生物高分子材料，具有生物相容性、亲水性、无毒等优点，可在温和条件下通过电离作用形成水凝胶，通常采用物理交联、化学交联、酶交联等方式制备海藻酸水凝胶，广泛应用于药物活性肽和蛋白质的控制释放系统。
鲍鱼肽是鲍鱼肉经过酶解制成，其氨基酸种类齐全、配比合理，被誉为海洋“软黄金”。鲍鱼中EPA、DHA、微量元素、牛磺酸及超氧化物歧化酶等营养成分丰富，具有抗氧化、增强免疫力、抗疲劳等作用。
亚精胺是一种天然存在的多胺，是一种安全、高效的细胞自噬诱导剂，几乎存在于所有细胞中，具有抗衰老、抗癌、保护心血管和神经等作用。亚精胺有利于辅助药物输送，提高药物分子的水溶解度、稳定性和膜渗透能力从而进一步提高药物在生物体内的吸收。
本发明是将海藻酸盐和亚精胺与具有抗氧化、抗菌功能的海藻多酚通过共混，制成海藻多酚颗粒。亚精胺分子含有三个氨基，两端和中间的氨基可以分别与海藻酸钠、海藻多酚相连接，对海藻多酚形成“伞”式保护结构，提高产品的稳定性。海藻多酚颗粒和富含多种营养与多重功效的鲍鱼肽通过氢键、静电、π-π堆叠等多重非共价键作用，形成复配组合物，通过多种途径、多个靶点，对酒精性脑损伤的防治产生对协同增效作用。当组合物进入胃中，海藻酸钠在胃酸的作用下形成海藻酸胶体，包裹海藻多酚和鲍鱼肽，避免在体内复杂环境下活性的丧失；在肠道中海藻酸转换为离子盐状态，持续释放海藻多酚和鲍鱼肽，发挥持久活性，提高其生物利用度。
所述海藻多酚来源于可食用大型海藻，包括海带、昆布、马尾藻、龙须菜、羊栖菜、 裙带菜或江蓠。所述海藻多酚的提取方法可按本领域海藻多酚的常规提取方法进行。优选的，本发明所采用的海藻多酚中总酚重量含量不少于30％。
所述鲍鱼肽是采用鲍鱼肉经生物酶解制备得到的复合肽。所述鲍鱼肽的酶解方法可按本领域的常规方法酶解进行。优选的，本发明所采用的鲍鱼肽的分子量分布范围为：分子量>3.0kDa多肽含量为2.15％，分子量1.0～3.0kDa多肽含量为16.59％，分子量<1.0kDa多肽含量为81.26％。
进一步地，所述组合物以重量份计，包括以下组分：海藻多酚20份、海藻酸钠4份、鲍鱼肽40份和亚精胺1.5份。
进一步地，所述组合物的制备方法，包括以下步骤：
S1.取海藻酸钠加入水中配制成海藻酸钠溶液，调节溶液pH 6.0-7.5，加入亚精胺，室温搅拌5-8小时，冰水浴处理，待溶液体系降温至5-10℃，加入海藻多酚，继续搅拌1-3小时，冷冻干燥，液氮碎化，得到海藻多酚复合粉粒；
S2.用水溶解鲍鱼肽，制成鲍鱼肽溶液，在搅拌状态下加入所述海藻多酚复合粉粒，混悬均匀，冷冻干燥，即得所述组合物。
在所述组合物的制备过程中，海藻酸钠溶液和鲍鱼肽溶液的浓度对组合物的稳定性和协同增效作用有重要影响。
进一步地，所述海藻酸钠溶液的质量浓度为1％-3.5％，优选为1.5％；若海藻酸钠溶液的质量浓度过大(＞3.5％)，容易粘团，难以形成均匀的海藻多酚颗粒，功效差；若海藻酸钠溶液的质量浓度过小(＜1％)，与亚精胺作用弱，达不到对海藻多酚的保护效果。
进一步地，所述鲍鱼肽溶液的质量浓度为18％-26％，优选为22％；若鲍鱼肽溶液的质量浓度过大(＞26％)，易受微生物感染，丧失活性；若鲍鱼肽溶液的质量浓度过小(＜18％)，与海藻多酚的协同作用减弱，达不到防治酒精性脑损伤的效果。
进一步地，在步骤S1中，需要严格控制溶液的pH范围。本发明调节pH的方法为常规方法，由于海藻酸钠溶液呈弱碱性，因此，本发明调节pH采用的为0.1mol/L的HCl溶液。若溶液的pH过小(＜6.0)，则酸性过强，海藻酸钠生成海藻酸胶体析出；若溶液的pH过大(＞7.5)，亚精胺难以通过静电作用与海藻酸钠和海藻多酚形成复合“伞”式结构。因此，本发明中，所述海藻酸钠溶液pH优选为6.0-7.5，更优选为6.5。
作为说明，本发明中所涉及的室温，也成为常温或者一般温度，一般定义为25℃， 本领域技术人员可对其进行合理调整，其也在本发明的保护范围内。
进一步地，所述药物制剂还包括药学上可接受的辅料。
进一步地，所述药物制剂为片剂，所述辅料包括玉米淀粉、滑石粉和硬脂酸镁；
所述片剂中各组分的质量分数为：
所述组合物10-20wt％、玉米淀粉65-75wt％、滑石粉11-17wt％和硬脂酸镁0.5-2wt％。
进一步地，所述药物制剂为胶囊剂，所述辅料包括乳糖、玉米淀粉和滑石粉；
所述胶囊剂中各组分的质量分数为：
所述组合物10-30wt％、乳糖10-20wt％、玉米淀粉50-60wt％和滑石粉5-15wt％。
进一步地，所述药物制剂为颗粒剂，所述辅料包括玉米淀粉、羟甲基纤维素钠和硬脂酸镁；
所述颗粒剂中各组分的质量分数为：
所述组合物5-15wt％、玉米淀粉74-84wt％、羟甲基纤维素钠5-15wt％和硬脂酸镁0.5-2wt％。
本发明公开了以下技术效果：
(1)本发明将具有抗氧化、抗菌功能的海藻多酚和富含多种营养、多重生物功效的鲍鱼肽组合，通过氢键、静电、π-π堆叠等多重作用，二者形成复合物；经动物模型试验证明，该复合物在酒精性脑伤的防治中发挥多途径、多靶点、协同增效作用。
(2)本发明利用海藻酸盐的水凝胶特性，在胃中低pH环境下形成海藻酸胶质，包裹海藻多酚和鲍鱼肽复合物，避免其在体内复杂环境中活性的丧失；在肠道中海藻酸转换为离子盐状态，缓控释放海藻多酚和鲍鱼肽，发挥持久活性效应。
(3)本发明中添加的亚精胺，利用其“三胺”特性，分别与海藻酸盐和海藻多酚相连接，对海藻多酚形成“伞”式保护结构，使得海藻多酚的稳定性、物理性状及口感均得到改善，有利于其发挥在酒精性脑损伤中的防治效果。
(4)本发明原料均来自天然食材，绿色安全；制备工艺简单，易于工业化生产；而且安全无毒、服用方便，无不良气味，依从性好。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些 附图获得其他的附图。
图1是本发明制备的防治酒精性脑损伤的组合物对DPPH自由基清除能力。
图2是本发明制备的防治酒精性脑损伤的组合物对OH自由基清除能力。
图3是本发明制备的防治酒精性脑损伤的组合物的抗氧化稳定性。
图4是本发明制备的防治酒精性脑损伤的组合物对小鼠脑指数的影响。
图5是本发明制备的防治酒精性脑损伤的组合物对小鼠撤除平台后游泳路线的影响。
具体实施方式
现详细说明本发明的多种示例性实施方式，该详细说明不应认为是对本发明的限制，而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式，并非用于限制本发明。另外，对于本发明中的数值范围，应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明，否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料，但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入，用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时，以本说明书的内容为准。
在不背离本发明的范围或精神的情况下，可对本发明说明书的具体实施方式做多种改进和变化，这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本申请说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等，均为开放性的用语，即意指包含但不限于。
除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。作为一个说明，本发明所采用海藻多酚和鲍鱼肽除采用常规方法制备，所述海藻多酚也可购自山东洁晶集团股份 有限公司，所述鲍鱼肽也可购自山东海龙元生物科技有限公司。
实施例1
一种防治酒精性脑损伤的组合物，以重量份计，包括以下组分：海藻多酚20份、海藻酸钠4份、鲍鱼肽40份和亚精胺1.5份。
所述组合物的制备方法，包括以下步骤：
S1.按所述重量份数，取海藻酸钠加入蒸馏水中配制成1.5wt％海藻酸钠溶液，调节溶液pH 6.5，加入亚精胺，室温搅拌6.5小时，冰水浴处理，待溶液体系降温至8℃，加入海藻多酚，继续搅拌2小时，冷冻干燥，液氮碎化，得到海藻多酚复合粉粒；
S2.按所述重量份数，用蒸馏水溶解鲍鱼肽，制成22wt％鲍鱼肽溶液，在搅拌状态下，加入S1所述海藻多酚复合粉粒，混悬均匀，冷冻干燥，即可得到所述组合物。
实施例2
一种防治酒精性脑损伤的组合物，以重量份计，包括以下组分：海藻多酚15份、海藻酸钠3份、鲍鱼肽35份和亚精胺1份。
所述组合物的制备方法，包括以下步骤：
S1.按所述重量份数，取海藻酸钠加入蒸馏水中配制成2.0wt％海藻酸钠溶液，调节溶液pH 7.0，加入亚精胺，室温搅拌7小时，冰水浴处理，待溶液体系降温至6℃，加入海藻多酚，继续搅拌1.5小时，冷冻干燥，液氮碎化，得到海藻多酚复合粉粒；
S2.按所述重量份数，用蒸馏水溶解鲍鱼肽，制成24wt％鲍鱼肽溶液，在搅拌状态下，加入S1所述海藻多酚复合粉粒，混悬均匀，冷冻干燥，即可得到所述组合物。
实施例3
一种防治酒精性脑损伤的组合物，以重量份计，包括以下组分：海藻多酚10份、海藻酸钠2份、鲍鱼肽30份和亚精胺0.5份。
所述组合物的制备方法，包括以下步骤：
S1.按所述重量份数，取海藻酸钠加入蒸馏水中配制成1wt％海藻酸钠溶液，调节溶液pH 6.0，加入亚精胺，室温搅拌5小时，冰水浴处理，待溶液体系降温至5℃，加入海藻多酚，继续搅拌1小时，冷冻干燥，液氮碎化，得到海藻多酚复合粉粒；
S2.按所述重量份数，用蒸馏水溶解鲍鱼肽，制成18wt％鲍鱼肽溶液，在搅拌状态下，加入S1所述海藻多酚复合粉粒，混悬均匀，冷冻干燥，即可得到所述组合物。
实施例4
一种防治酒精性脑损伤的组合物，以重量份计，包括以下组分：海藻多酚30份、海藻酸钠6份、鲍鱼肽55份、亚精胺3份。
所述组合物的制备方法，包括以下步骤：
S1.按所述重量份数，取海藻酸钠加入蒸馏水中配制成3.5wt％海藻酸钠溶液，调节溶液pH 7.5，加入亚精胺，室温搅拌8小时，冰水浴处理，待溶液体系降温至10℃，加入海藻多酚，继续搅拌3小时，冷冻干燥，液氮碎化，得到海藻多酚复合粉粒；
S2.按所述重量份数，用蒸馏水溶解鲍鱼肽，制成26wt％鲍鱼肽溶液，在搅拌状态下，加入S1所述海藻多酚复合粉粒，混悬均匀，冷冻干燥，即可得到所述组合物。
对比例1
与实施例1的不同之处仅在于，用维生素C代替海藻多酚。
对比例2
与实施例1的不同之处仅在于，用精胺代替亚精胺。
对比例3
与实施例1的不同之处仅在于，直接原料共混，具体如下：
一种防治酒精性脑损伤的组合物，以重量份计，包括以下组分：海藻多酚20份、海藻酸钠4份、鲍鱼肽40份和亚精胺1.5份。
所述组合物的制备方法，包括以下步骤：
按所述重量份数，取海藻酸钠、亚精胺、海藻多酚和鲍鱼肽原料，加入蒸馏水，混悬均匀，冷冻干燥，即可得到所述组合物。
对比例4
与实施例1的不同之处仅在于，用牡蛎肽代替鲍鱼肽。
应用例1 防治酒精性脑损伤的组合物的抗氧化实验
1、实验方法
(1)1,1-二苯基-2-三硝基苯肼(DPPH)自由基清除能力的测定
将2.0mL不同质量浓度的样品溶液(实施例1～4组和对比例1～4组制备得到的组合物)与2mL DPPH无水乙醇溶液混合，室温下避光放置60min后，在517nm处测定溶液的吸光度。以蒸馏水代替样品溶液作空白对照，按下式(1)计算DPPH自由基的清除率，用IC 50表示清除效果。
Figure PCTCN2021138171-appb-000001
其中，A X为样品的吸光度，
Figure PCTCN2021138171-appb-000002
为空白对照的吸光度。
(2)羟自由基清除能力的测定
在同一比色管中分别加入浓度为1mL H 2O 2溶液(8.8mmol/L)、1mL FeSO 4溶液(9mmol/L)和12mL不同质量浓度的样品溶液(实施例1～4组和对比例1～4组制备得到的组合物)，再加入1mL水杨酸的乙醇溶液(9mmol/L)，摇匀，置37℃水浴30min，在波长510nm处测定吸光度；以蒸馏水替代样品溶液作空白对照。羟自由基清除率按公式(1)计算，用IC 50表示清除效果。
2、实验结果
(1)DPPH自由基清除效果
IC 50值越小，清除自由基能力越强。维生素C(Vc)是一种强抗氧化剂，作为阳性对照。防治酒精性脑损伤组合物的DPPH自由基清除效果如图1所示，实施例1～4组的IC 50都接近Vc组，表现出强的DPPH自由基清除效果，尤以实施例1的DPPH自由基清除能力最强。虽然对比例1组也表现出与实施例1～4组接近的DPPH自由基清除效果，但对比例2-4组的DPPH自由基清除能力远远不如实施例1～4组。
(2)羟自由基清除效果
维生素C(Vc)是一种强抗氧化剂，作为阳性对照。防治酒精性脑损伤组合物的羟自由基清除效果如图2所示，实施例1～4组的IC 50差不多接近Vc组，表现出强的羟自由基清除效果，尤以实施例1的羟自由基清除能力最强。虽然对比例1组也表现出与实施例1～4组接近的羟自由基清除效果，但对比例2-4组的羟自由基清除能力远远不如实施例1～4组。
综合以上两个实验结果，实施例1-4组都表现出强的自由基清除效果，说明本发明制备的防治酒精性脑损伤的组合物具有强的抗氧化活性，尤以实施例1组抗氧化活性最强。本发明组合物的配方或者制备方法改变，都达不到预期的抗氧化效果。
应用例2 防治酒精性脑损伤组合物的抗氧化稳定性实验
采用铁离子还原/抗氧化能力法(FRAP法)检测组合物存储期间的总抗氧化能力。
样品储存：样品存储于棕色瓶中，室温条件下放置。样品每隔一定时间取样，进行总抗氧化能力测试。
测试方法：取0.02mL FeSO 4溶液，加入0.18mL FRAP液(现配现用)，混匀，37℃水浴10min，采用紫外-可见分光光度计在593nm测吸光值。以FeSO 4溶液浓度为横坐 标，吸光值为纵坐标，建立FeSO 4当量标准曲线。以V C溶液为阳性对照，根据标准曲线计算出FeSO 4当量。根据FeSO 4当量计算出各样品的V C相对百分比。
防治酒精性脑损伤的组合物的抗氧化稳定性结果如图3所示，由于多酚性质的不稳定性，多酚易被氧化，长期储存中极易变质，对比例3组合物的总抗氧化能力在储存过程中几乎呈直线下降，而实施例1的组合物在相同的环境中，在5天内随着储存时间的延长，其总抗氧化能力几乎保持稳定状态。由此可见，本发明组合物的配方和制备方法，可有效提高组合物的抗氧化稳定性。
应用例3 防治酒精性脑损伤组合物的护脑功效测定
1、造模方法
实验动物：昆明雄性小鼠，6-8周龄，体重18-22克，SPF级，动物饲养条件：(25±1)℃，相对湿度：(55±5)％。
实验分组：取140只小鼠随机分为10组，每组14只，分为生理盐水组、模型组、实施例1～4组和对比例1～4组。
各组小鼠每日分别按照梯度浓度灌胃小鼠相对应体积56度红星二锅头白酒(生理盐水组灌胃对应体积的生理盐水)，灌酒1小时后按10g/kg·BW(体重)剂量给各组灌胃相应受试物，生理盐水组和模型组灌胃生理盐水。连续灌胃8周。每周给小鼠称重以调节酒及受试物剂量。第一周灌酒量为2mL/kg·BW，为了造成持续性酒精伤害，之后根据每周小鼠的死亡情况等适当增加灌酒量。
2、Morris水迷宫实验
酒精性脑损伤会造成小鼠明显的学习记忆能力下降现象。通过小鼠在水迷宫中寻找隐藏在水下的平台，来测试小鼠学习和记忆空间位置和方向的能力。小鼠在酒精损伤8周后进行Morris水迷宫测试，小鼠在直径为120厘米，高度为40厘米的黑色圆形水池中游泳，水池中充满水，内含直径8厘米的可移动平台。实验5天，每天4次，分别从四个不同的起始点放入水中。录像记录小鼠找到平台的时间(逃避潜伏期)。在第6天撤除原平台，将小鼠任选1个入水点放入水中，记录小鼠在原平台的停留时间、跨越原平台的次数和路径。实验结束第二天禁食不禁水24h后进行样本采集：
(1)小鼠解剖后，取出脑组织，用预冷的生理盐水洗去表面的血水，用滤纸吸干后快速称重，用公式计算脑指数：
脑指数(％)＝脑重量(g)/体重(g)×100％
(2)快速解剖脑组织，-80℃保存，制备成10％脑组织匀浆，按照相应试剂盒要求，严格操作，测定脑组织匀浆中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和丙二醛(MDA)的含量。
2、实验结果
(1)脑指数
若脑指数增大，则代表脏器充血，水肿增生肥大等情况；若脑指数减少，则表示脏器发生萎缩及其他退行性改变。防治酒精性脑损伤组合物的脑指数实验结果如图4所示。小鼠造模后，与生理盐水组相比，模型组脑指数显著升高，具有显著差异(p＜0.01)，表明持续过量灌酒会造成小鼠脑水肿。与模型组对比，实施例1～4组的小鼠脑指数均显著降低，具有统计学差异(p＜0.01)，其中实施例1组的效果最佳，脑指数几乎接近生理盐水组水平，说明本发明制备的防治酒精性脑损伤的组合物可有效地改善脑指数，降低脑组织出现水肿、增生、肥大等病变，对酒精性脑损伤具有显著的防治作用。与模型组对比，虽然对比例1～4组的小鼠脑指数都有降低，但无统计学差异，由此说明，本专利组合物的配方或者制备方法改变，都不能有效地降低酒精诱导的脑指数，对酒精诱导的脑损伤也无防治作用。
(2)SOD、GSH-Px和MDA结果分析
酒精对机体细胞的损害主要通过诱导细胞氧化应激，过量长期饮酒会导致机体脑细胞中SOD、GSH活性的改变并进一步导致细胞氧化应激损害中枢神经，因此可通过调节体内抗氧化物酶的活性来防治酒精性脑损伤。防治酒精性脑损伤的组合物对氧化物酶活性影响结果如表1所示。与生理盐水组相比，模型组小鼠脑组织中SOD和GSH-Px含量显著降低，具有统计学差异(p＜0.01)，表明持续过量灌酒导致的小鼠脑氧化应激异常是造成小鼠脑损伤的重要因素之一。与模型组对比，实施例1～4组小鼠脑组织中SOD和GSH-Px含量显著提高(p＜0.01)，其中实施例1组尤以为甚，说明本发明制备的防治酒精性脑损伤的组合物能显著降低酒精引起小鼠脑中过氧化物水平，增强脑组织抗氧化能力。MDA是自由基作用于脂质过氧化的产物，通过测定MDA含量可以反映体内脂质过氧化的程度。实施例1～4组小鼠脑组织中MDA含量显著低于模型组(p＜0.01)，其中实施例1组MDA含量最低。结果表明，本发明制备的防治酒精性脑损伤的组合物可通过增加SOD和GSH-Px活性，增强小鼠脑组织抗氧化能力，纠正氧化和抗氧化平衡紊乱，减轻自由基对脑细胞损伤，从而对慢性酒精中毒造成的脑损伤 起到保护作用。
表1各组小鼠脑组织中SOD、GSH-Px和MDA含量
Figure PCTCN2021138171-appb-000003
注：与生理盐水组比较， ##p<0.01；与模型组比较，*p<0.05，**p<0.01。
(3)小鼠Morris水迷宫的实验结果
Morris水迷宫实验包括定位航行和空间搜索两部分，定位航行试验逃避潜伏期结果代表脑记忆获得能力，空间搜索试验目标象限停留时间和穿越平台次数结果则代表脑记忆保持能力。表2～3实验结果分别对应定位航行结果和空间搜索结果，经过5d的Morris实验，各实验组小鼠的逃避潜伏期均逐渐缩短，与生理盐水组相比，模型组小鼠逃避潜伏期明显延长，差异具有统计学意义(p＜0.05)；与模型组相比，实施例1～4组和对比例1～4组小鼠从第2d开始，逃避潜伏期逐天减少，其中实施例1～4组显著减少(p＜0.01)；与模型组相比，实施例1～4组小鼠的撤除原平台后目标象限停留时间和穿越平台次数都有极显著性差异(p＜0.01)，其中实施例1组小鼠目标象限停留时间最长和穿越平台次数最多；此外，由撤除平台后小鼠游泳路线图5可以看出，模型组小鼠寻找原平台目的性不强，实施例1组与生理盐水组小鼠目的性较强。结果表明，持续过量灌酒到实验第8周时小鼠脑记忆获得功能和脑记忆保持功能受损，本发明制备的防治酒精性脑损伤的组合物对酒精导致的小鼠脑损伤具有显著的改善作用。
表2小鼠Morris定位航行试验结果
Figure PCTCN2021138171-appb-000004
Figure PCTCN2021138171-appb-000005
注：与生理盐水组比较， ##p<0.01；与模型组比较，*p<0.05，**p<0.01。
表3小鼠Morris空间搜索试验结果
Figure PCTCN2021138171-appb-000006
Figure PCTCN2021138171-appb-000007
注：与生理盐水组比较， ##p<0.01；与模型组比较，*p<0.05，**p<0.01。
应用例4 急性毒性实验
动物：取健康小鼠，体重18-22g，雌雄各半，共20只，随机分为实验组和空白对 照两组。
受试物：以实施例1的组合物作为受试物。
实验方法：在预实验中，未能测出半数致死量，故选用上述实验给药量进行试验。以小鼠的体重为准，按10g/kg的剂量，每24h灌胃一次，连续灌胃实验组小鼠2周,空白对照组灌胃蒸馏水，在饲喂期间均自由饮水和觅食，实验期间观察小鼠的活动状态、饮食、粪便、呼吸、体重及死亡情况。
实验结果：实验组小鼠灌胃受试物2周内无一例死亡，与对照组相比，体重变化无显著差异，进食和饮水均无异常，且小鼠精神状态良好，活泼好动。将动物
处死后，肉眼观察各主要脏器无异常现象。结果表明，本发明的防治酒精性脑损伤组合物口服应用，对动物无明显损害，未发现对机体产生的毒性反应，安全无毒。
实施例5 一种防治酒精性脑损伤的片剂
该防治酒精性脑损伤的片剂，以质量分数计，包括以下组分：
Figure PCTCN2021138171-appb-000008
实施例6 一种防治酒精性脑损伤的片剂
该防治酒精性脑损伤的片剂，以质量分数计，包括以下组分：
Figure PCTCN2021138171-appb-000009
实施例7 一种防治酒精性脑损伤的片剂
该防治酒精性脑损伤的片剂，以质量分数计，包括以下组分：
Figure PCTCN2021138171-appb-000010
实施例8 一种防治酒精性脑损伤的胶囊剂
该防治酒精性脑损伤的胶囊剂，以质量分数计，包括以下组分：
Figure PCTCN2021138171-appb-000011
实施例9 一种防治酒精性脑损伤的胶囊剂
该防治酒精性脑损伤的胶囊剂，以质量分数计，包括以下组分：
Figure PCTCN2021138171-appb-000012
实施例10 一种防治酒精性脑损伤的胶囊剂
该防治酒精性脑损伤的胶囊剂，以质量分数计，包括以下组分：
Figure PCTCN2021138171-appb-000013
实施例11 一种防治酒精性脑损伤的颗粒剂
该防治酒精性脑损伤的胶囊剂，以质量分数计，包括以下组分：
Figure PCTCN2021138171-appb-000014
实施例12 一种防治酒精性脑损伤的颗粒剂
该防治酒精性脑损伤的胶囊剂，以质量分数计，包括以下组分：
Figure PCTCN2021138171-appb-000015
实施例13 一种防治酒精性脑损伤的颗粒剂
该防治酒精性脑损伤的胶囊剂，以质量分数计，包括以下组分：
Figure PCTCN2021138171-appb-000016
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。

Claims (9)

  1. 一种组合物在制备防治酒精性脑损伤的药物制剂中的应用，所述组合物以重量份计，包括以下组分：海藻多酚10～30份、海藻酸钠2～6份、鲍鱼肽30～55份和亚精胺0.5～3份。
  2. 根据权利要求1所述的应用，其特征在于，所述组合物以重量份计，包括以下组分：海藻多酚20份、海藻酸钠4份、鲍鱼肽40份和亚精胺1.5份。
  3. 根据权利要求1或2所述的应用，其特征在于，所述组合物的制备方法，包括以下步骤：
    S1.取海藻酸钠加入水中配制成海藻酸钠溶液，调节溶液pH 6.0-7.5，加入亚精胺，室温搅拌5-8小时，冰水浴处理，待溶液体系降温至5-10℃，加入海藻多酚，继续搅拌1-3小时，冷冻干燥，液氮碎化，得到海藻多酚复合粉粒；
    S2.用水溶解鲍鱼肽，制成鲍鱼肽溶液，在搅拌状态下加入所述海藻多酚复合粉粒，混悬均匀，冷冻干燥，即得所述组合物。
  4. 根据权利要求3所述的应用，其特征在于，所述海藻酸钠溶液的质量浓度为1％-3.5％；所述鲍鱼肽溶液的质量浓度为18％-26％。
  5. 根据权利要求3所述的应用，其特征在于，所述海藻酸钠溶液pH调节至6.5。
  6. 根据权利要求1所述的应用，其特征在于，所述药物制剂还包括药学上可接受的辅料。
  7. 根据权利要求6所述的应用，其特征在于，所述药物制剂为片剂，所述辅料包括玉米淀粉、滑石粉和硬脂酸镁；
    所述片剂中各组分的质量分数为：
    所述组合物10-20wt％、玉米淀粉65-75wt％、滑石粉11-17wt％和硬脂酸镁0.5-2wt％。
  8. 根据权利要求6所述的应用，其特征在于，所述药物制剂为胶囊剂，所述辅料包括乳糖、玉米淀粉和滑石粉；
    所述胶囊剂中各组分的质量分数为：
    所述组合物10-30wt％、乳糖10-20wt％、玉米淀粉50-60wt％和滑石粉5-15wt％。
  9. 根据权利要求6所述的应用，其特征在于，所述药物制剂为颗粒剂，所述辅料包括玉米淀粉、羟甲基纤维素钠和硬脂酸镁；
    所述颗粒剂中各组分的质量分数为：
    所述组合物5-15wt％、玉米淀粉74-84wt％、羟甲基纤维素钠5-15wt％和硬脂酸镁 0.5-2wt％。
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