CN111700903B - Application of porphyra polysaccharide and preparation method of porphyra polysaccharide - Google Patents
Application of porphyra polysaccharide and preparation method of porphyra polysaccharide Download PDFInfo
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- CN111700903B CN111700903B CN202010790126.9A CN202010790126A CN111700903B CN 111700903 B CN111700903 B CN 111700903B CN 202010790126 A CN202010790126 A CN 202010790126A CN 111700903 B CN111700903 B CN 111700903B
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- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- 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
- A61P25/24—Antidepressants
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Materials Engineering (AREA)
- Food Science & Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Neurosurgery (AREA)
- Neurology (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Psychiatry (AREA)
- Pain & Pain Management (AREA)
- Nutrition Science (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
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- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses an application of porphyra polysaccharide, provides a new application of porphyra polysaccharide, can use the porphyra acidolysis polysaccharide as an antidepressant drug for drug production, or can be used as an antidepressant functional food for food production, and has wide application prospect. The invention also provides a preparation method of the laver polysaccharide, which is characterized in that the laver acid-hydrolyzed polysaccharide is extracted from dried laver by a water extraction, alcohol precipitation and acidolysis method, and finally the yield of the laver acid-hydrolyzed polysaccharide is more than 10%. The invention takes the laver as the raw material, has wide material obtaining range and low price, and the laver is taken as a food which can be eaten for many years, has high safety and strong acceptance. Researches show that the acidolysis polysaccharide of laver has better anti-depression effect.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to preparation and application of porphyra polysaccharide.
Background
Depression, also known as depressive disorder, is characterized primarily clinically by persistent mood swings, a major type of mood disorder. Depression mainly manifests as involuntary activity of consciousness, slow thinking, cognitive function impairment, depressed mood, even pessimistic and weary appearance, suicide behavior, suicide rate as high as 10% -17%, great harmfulness, high form and recurrence rate. Currently, the etiology and pathogenesis of depression is unknown. Related scholars have proposed various hypotheses that biological, psychological and social environmental factors are involved in the pathogenesis of depression. The medicament is the main means for clinically treating depression at present, but has the defects of poor treatment effect (the effective rate is lower than 70 percent), large side effect and the like more or less. In order to more effectively treat depression, it is desirable to find more effective treatments with fewer side effects.
Seaweed is the most abundant type of marine plants, and research on seaweed physiologically active substances has become one of hot spots in the fields of medicine and food. The laver is a general name of Porphyra of the family Rhodophytaceae of the order Rhodophyta of the class Rhodophyceae of the phylum Rhodophyta, and the main cultivated species in the coastal region is Porphyra haitanensis (Porphyra haitanensis). The Chinese oceanic medicine dictionary records that the laver has the effects of cooling and refreshing, clearing heat, softening hard masses, dissipating stagnation, inducing diuresis to alleviate edema, tonifying kidney and nourishing heart. The porphyra polysaccharide is one of main active components of the porphyra, has multiple biological activities of anticoagulation, blood sugar reduction, thrombosis resistance, tumor resistance, inflammation resistance and the like, and is widely applied to industries of medicines, foods and the like.
Until now, no relevant literature report that the porphyra polysaccharide has an antidepressant effect is found.
Disclosure of Invention
In view of the above, the present invention aims to provide an application of porphyra polysaccharide in anti-depression food and medicine, i.e., a new application of porphyra polysaccharide.
The invention also aims to provide a preparation method of the porphyra polysaccharide, which provides a material basis guarantee for the research of the new application of the porphyra polysaccharide and prepares the porphyra polysaccharide with the anti-depression effect.
In order to achieve the purpose, the technical scheme of the invention is as follows:
an application of Porphyra polysaccharide, i.e. acidolysis polysaccharide of Porphyra is applied in antidepressant drugs or functional foods.
Further, the porphyra acidolysis polysaccharide is applied to an antidepressant drug, and the drug is a single agent containing a single component of the porphyra acidolysis polysaccharide.
Further, the porphyra acidolysis polysaccharide is applied to antidepressant medicines, and the medicines are multi-component mixed compound preparations containing the porphyra acidolysis polysaccharide.
Further, the compound preparation is a tablet, and the components of the tablet comprise: mixing multiple kinds of acid hydrolyzed polysaccharide of thallus Porphyrae, filler, binder, wetting agent, disintegrating agent, absorption promoter, solvent, surfactant, flavoring agent, lubricant, sweetener and pigment.
Further, the components of the tablet are as follows in parts by weight: 10-50 parts of acidolysis polysaccharide of laver, 10-40 parts of microcrystalline cellulose, 2-5 parts of stearic acid, 10-30 parts of lactose, 5-15 parts of superfine silica powder and 0.2-1 part of magnesium stearate, wherein the microcrystalline cellulose and the lactose are fillers, the stearic acid and the superfine silica powder are surfactants, and the magnesium stearate is a lubricant.
Further, the compound preparation is an oral liquid preparation, and the components of the oral liquid preparation comprise: mixing various kinds of acid hydrolyzed polysaccharides of thallus Porphyrae, buffer, antioxidant synergist, correctant, sweetener, solvent, and surfactant.
Further, the oral liquid preparation comprises the following components in parts by weight: 10-50 parts of acidolysis polysaccharide of laver, 20-50 parts of sucrose, 1-3 parts of citric acid and water, wherein the citric acid is a buffering agent, a flavoring agent and an antioxidant synergist, and the sucrose is a sweetening agent.
After the technical scheme is adopted, the application of the porphyra polysaccharide has the following beneficial effects: provides a new application of the porphyra polysaccharide, can use the porphyra acidolysis polysaccharide as an antidepressant drug for drug production, or can be used as an antidepressant functional food for food production, and has wide application prospect.
A method for preparing thallus Porphyrae polysaccharide comprises extracting dried thallus Porphyrae with water extraction, alcohol precipitation and acidolysis to obtain thallus Porphyrae polysaccharide hydrolysate with yield of above 10%.
Further, pulverizing dried thallus Porphyrae, adding ethanol, heating, filtering to remove alcohol soluble substances, washing the residue with methanol, drying, extracting the dried powder with distilled water at 80-95 deg.C for 1-3 hr, centrifuging, and concentrating the centrifuged supernatant;
adding sodium acetate and acetic acid into the concentrated supernatant to obtain sodium acetate solution, adding 42% ethanol, filtering to remove the first precipitate, and adjusting the ethanol concentration of the filtrate to 60%; then centrifuging and collecting a second precipitate, dissolving the second precipitate in distilled water, dialyzing with distilled water, and freeze-drying to obtain porphyra polysaccharide;
dissolving the laver polysaccharide obtained by freeze drying in distilled water to obtain a polysaccharide solution, sequentially adding ascorbic acid and hydrogen peroxide, stirring for reaction to obtain a laver acid hydrolysis polysaccharide solution, precipitating the solution in 80% ethanol, and washing twice with 95% ethanol to obtain the laver acid hydrolysis polysaccharide.
Further, dried laver was weighed, pulverized, added with 100 times of 85% ethanol, heated at 75 ℃ for 1 hour, filtered to remove alcohol-soluble substances, repeated 3 times, the filtered residue was washed with methanol 2 times and dried, the dried powder was extracted with 200 times of distilled water at 95 ℃ for 2 hours, and then centrifuged at 8000 × g for 20 minutes, and then the centrifuged supernatant was concentrated to 1/3 of the original volume using a rotary evaporator;
adding sodium acetate and acetic acid into the supernatant to prepare 0.5M sodium acetate solution, wherein the pH value is 5.0, then adding ethanol with the final concentration of 42%, standing for 1 hour, removing a first precipitate in the solution, adjusting the final concentration of the ethanol to be 60%, centrifuging for 30 minutes at 10000 Xg to collect a second precipitate, dissolving the second precipitate in distilled water, dialyzing with the distilled water, and freeze-drying to obtain porphyra polysaccharide;
dissolving the laver polysaccharide obtained by freeze drying in distilled water to obtain 0.5% polysaccharide solution, sequentially adding 3mM ascorbic acid and 3mM hydrogen peroxide, stirring for reaction for 2 hours to obtain laver acid hydrolysis polysaccharide solution, precipitating the solution in 80% ethanol, and washing twice with 95% ethanol to obtain laver acid hydrolysis polysaccharide.
After the technical scheme is adopted, the preparation method of the porphyra polysaccharide has the following beneficial effects: the laver is used as a raw material, the material obtaining range is wide, the price is low, and the laver is used as a food which can be eaten for many years, so that the safety is high, and the acceptability is strong. The preparation process is simple and safe, the final yield of the laver acidolysis polysaccharide is high and can reach more than 10 percent, and a material basis guarantee is provided for the research of new application of the laver polysaccharide. Research shows that the laver acid-hydrolyzed polysaccharide can relieve the anhedonia, despair and environmental fear behaviors of depressed mice, promote the level of hippocampal neurotrophic factors, and maintain the hippocampal neurogenesis and synaptogenesis, and shows that the laver acid-hydrolyzed polysaccharide has better potential for treating depression, namely the laver acid-hydrolyzed polysaccharide has better anti-depression effect.
Drawings
FIG. 1 shows the effect of acute administration of acid-hydrolyzed porphyra polysaccharide on mouse behaviours;
in the figure, (A) represents a statistical graph of Immobility time (Immobility time) of a forced swimming test;
(B) statistical plots representing Immobility time (mobility time) for tail suspension experiments;
(C) represents the number of crossings (Crossing number) of the open field experiment;
(D) representing the number of grooming times (reading number) for open field experiments;
FIG. 2 shows the effect of chronic administration of acid-hydrolyzed laver polysaccharide on mouse behavior;
in the figure, (A) represents a sugar water preference value (Sucrose preference) statistical chart of a sugar water preference test;
(B) a feeding Latency to feed (Latency to feed) histogram representing a novel inhibition feeding experiment;
(C) a Total food consumption summary (Total food consumption) histogram representing a novel inhibition feeding experiment;
FIG. 3 shows the effect of chronic administration of acid-hydrolyzed porphyra polysaccharide on the BDNF expression level in hippocampus of mice;
in the figure, (A) represents a mRNA level (Gene expression) statistical chart;
(B) represents a statistical map of Protein levels (Protein levels),
FIG. 4 shows the chronic administration of acid-hydrolyzed polysaccharides of thallus Porphyrae on neurogenesis and synaptogenesis
In the figure, (A) represents a statistical graph of the number of double-cortin (DCX positive cells) occurring in the nerve,
(B) statistical plots representing dendritic spine Density (Density of dendritic spines) of synaptogenesis.
Detailed Description
In order to further explain the technical solution of the present invention, the present invention is explained in detail by the following specific examples.
Example 1
Method for extracting acidolysis polysaccharide of laver by water extraction, alcohol precipitation and acidolysis
The invention relates to a preparation method of porphyra polysaccharide, which comprises the following steps: weighing dried thallus Porphyrae, pulverizing, adding 100 times of 85% ethanol, heating at 75 deg.C for 1 hr, filtering to remove alcohol soluble substances, and repeating for 3 times. The residue was washed 2 times with methanol and dried. The powder was extracted with 200 times distilled water at 95 ℃ for 2h, then centrifuged at 8000 Xg for 20 minutes, and the centrifuged supernatant was concentrated to 1/3 in its original volume using a rotary evaporator. Then, sodium acetate and acetic acid were added to the supernatant to prepare a 0.5M sodium acetate solution (pH 5.0), followed by addition of ethanol (v/v) at a final concentration of 42%, and after standing for 1 hour, the first precipitate in the solution was removed by filtration to adjust the filtrate to a final concentration of 60% (v/v) ethanol. Centrifuging at 10000 Xg for 30 min to collect second precipitate, dissolving the second precipitate in distilled water, dialyzing with distilled water, and freeze-drying to obtain porphyra polysaccharide. Dissolving the laver polysaccharide obtained by freeze drying in distilled water to obtain 0.5% polysaccharide solution, sequentially adding 3mM ascorbic acid and 3mM hydrogen peroxide, and stirring for reaction for 2 hr to obtain laver acid hydrolysis polysaccharide solution. Precipitating the solution in 80% ethanol, and washing with 95% ethanol twice to obtain thallus Porphyrae acid hydrolyzed polysaccharide with yield of 10.65% calculated on dry thallus Porphyrae.
After the technical scheme is adopted, the preparation method of the porphyra polysaccharide has the following beneficial effects: the laver is used as a raw material, the material obtaining range is wide, the price is low, and the laver is used as a food which can be eaten for many years, so that the safety is high, and the acceptability is strong. The preparation process is simple and safe, the final yield of the laver acidolysis polysaccharide is high and can reach more than 10 percent, and a material basis guarantee is provided for the research of new application of the laver polysaccharide. Research shows that the laver acid-hydrolyzed polysaccharide can relieve the anhedonia, despair and environmental fear behaviors of depressed mice, promote the level of hippocampal neurotrophic factors, and maintain the hippocampal neurogenesis and synaptogenesis, and shows that the laver acid-hydrolyzed polysaccharide has better potential for treating depression, namely the laver acid-hydrolyzed polysaccharide has better anti-depression effect.
Example 2
Pharmacodynamic experiment of acute administration of laver acidic polysaccharide
1. Experimental animals Male C57/BL mice were purchased from Shanghai Slek laboratory animal center, weighing 22-26g, 8 weeks old; the animals are placed in cages of 320 multiplied by 180 multiplied by 160cm, 5 animals are placed in each cage, and the mice are adapted to the environment for one week; the mice were given free access to water for feeding throughout the experiment, with an ambient temperature of 22 + -2 deg.C and a relative humidity of 55 + -5%, and were illuminated for 12 hours per day.
2. The fluoxetine as a positive drug for drug preparation is prepared into a suspension with the concentration of 20mg/10ml by using normal saline as a solvent; the acidolysis polysaccharide of thallus Porphyrae can be prepared into medicinal liquid with three dosages of 100mg/ml, 200mg/ml and 400 mg/ml.
3. Dividing the animals into 5 groups and 10 mice in each group, wherein the groups are respectively a model control group (normal saline), a positive fluoxetine group (20mg/kg) and a laver acid hydrolysis polysaccharide (100mg/kg, 200mg/kg and 400 mg/kg); the drug is administrated by stomach irrigation according to the weight of 10ml/kg of a mouse, and the forced swimming experiment, the tail suspension experiment and the open field experiment are respectively carried out after the drug is administrated for 1 hour.
4. The forced swimming experimental animal is separately put into a cylindrical glass container (with the height of 20cm and the diameter of 14cm) containing 10cm of clean water (with the water temperature of 25 +/-2 ℃), the animal swims for 6 minutes, and the camera records the whole process; taking out the animal, wiping the body with a towel, and putting the animal back into the cage box paved with the clean padding; cleaning the container before each animal experiment, and injecting equal-height (10cm) and equal-temperature (25 +/-2 ℃) clean water; the behavior observer is a double-blind experimenter trained and having no knowledge about animal grouping and administration conditions, and observes and records the time sum that the mouse keeps in an immobile state within 4 minutes; and judging the mouse immobility state: stop struggling, float on the surface, or simply a small degree of motion necessary to keep the head out of the water.
5. The tail-suspended experimental mice were fixedly suspended in a box (length 25 × width 25 × height 30cm) from about 1cm from the tail tip, the head was suspended for 6 minutes from the bottom of the box, and the camera recorded the whole process. The animals are taken out and put back into the cage box paved with clean padding. Before each animal experiment, the carton was cleaned and the excrement of the former animal was removed. The behavioral observer was a double blind experimenter trained and having no knowledge of the animal grouping and dosing status, and observed the sum of the time that the mice remained in an immobile state within 4 minutes after recording. And (3) judging the immobility state of the mouse: stopping struggling, static suspension, or only slight flexing of the forefoot.
6. Open field experimental mice are placed in a wooden box (length 40 multiplied by width 40 multiplied by height 30cm) with a grid painted on a bottom plate, the bottom plate of the box is averagely 25 grids (length 8 multiplied by width 8cm), the box is placed for 3 minutes, and a camera records the whole process; taking out the animals and putting the animals back into the cage box paved with the clean padding; before each animal is tested, the wooden box needs to be cleaned, and the excrement of the former animal is cleaned; the behavior observer is a double-blind experimenter who is trained and does not know animal grouping and administration conditions, and the number of times of grid penetration and the number of times of erection of the mouse within 3 minutes are observed and recorded as the animal spontaneous activity indexes.
7. Statistical methods the experimental results were plotted using Graphpad Prism 7 software for statistical histogram of results expressed as mean ± standard error values; for the comparison between the two groups, adopting one-factor analysis of variance combined with Tukey's analysis after the fact; statistical difference significance is indicated by a P value less than 0.05.
8. The experimental result is shown in figure 1, and the single-factor analysis of variance shows that the acidolysis polysaccharide of the laver can obviously reduce the swimming immobility time and tail suspension immobility time of the mouse; tukey's post analysis shows that compared with a model control group, fluoxetine in a positive control group can obviously reduce immobility time, and the immobility time of mice can be obviously reduced in a medium-dose group and a high-dose group of laver acid hydrolysis polysaccharide. The single-factor analysis of variance and Tukey's post analysis show that the polysaccharide acidolysis of the laver has no significant influence on the spontaneous activities (the number of lattice penetration and the number of hair organization) of the mice.
Example 3
Pharmacodynamic experiment of chronic administration of laver acidic polysaccharide
1. The experimental animals were the same as those of example 2.
2. Pharmaceutical formulation see example 2 for pharmaceutical formulation.
3. Animal grouping and mode of administration mice were divided into 6 groups of 16 mice each, each of which was: normal control group (physiological saline), normal-positive medicine fluoxetine group (20mg/kg), normal-laver acid hydrolysis polysaccharide group (200 mg/kg); model control group (physiological saline), model-positive drug fluoxetine group (20mg/kg), model-laver acid hydrolysis polysaccharide group (200mg/kg), according to the weight of the mouse 10ml/kg of intragastric administration, once a day for 28 days, after which sugar water preference experiment and forced swimming experiment are carried out.
4. The animals of the chronic mild stress model group underwent low intensity stress for 8 weeks, and weekly stimulation included: depriving food and water, inclining the cage at 45 ℃, illuminating at intervals, polluting the mouse cage, reducing space, illuminating day and night and reducing noise. The normal group animals were kept separately and 4 weeks after the stress for 4 weeks.
5. Sugar water preference experiments mice were first acclimatized to sugar water consumption for 24 hours, and then trained to deprive sugar water for 24 hours. And then, water and grain are cut off, a sugar water preference experiment is carried out after 12h, the experiment lasts for 24h, and sugar water bottles are weighed before and after the experiment respectively. The percentage of sugar water preference was calculated as sugar water consumption/(sugar water consumption + normal water consumption) × 100% by the following calculation formula.
6. The mice fed the exotic inhibition chow for 24h and then weighed the food in the very center of the empty cage. After the mice were placed on the edge, the latency time from the placement of the mice to the first meal was calculated. The mice were regarded as eating standards by starting chewing, and no eating standards were calculated for smelling or fiddling food. The intake latency when the mouse had eaten the first food and the total amount of food that the mouse had eaten within 15min were used as the observation indices.
7. PCR experiment after the behavior experiment, the mouse is sacrificed, the brain tissue hippocampus of the mouse is taken out quickly, and then is frozen by liquid nitrogen, and after the sampling is finished, the mouse is frozen in an ultra-low temperature refrigerator at minus 80 ℃. During detection, Trizol is added to extract total RNA, and after extraction and centrifugal separation, the concentration is measured by an ultramicro nucleic acid detector. Reverse transcription was performed using total RNA as template. Finally, the cDNA obtained by reverse transcription is carried out by adopting a reverse transcription reagent, and the reaction is carried out according to the following reaction program: activating the enzyme at 95 ℃ for 30 s; pre-denaturation at 95 ℃ for 5s and repeating for 40 more cycles; extension at 56 ℃ for 30 s. Finally, GAPDH (forward 5'-GGGTGTGAACCACGAGAAAT-3'; reverse5 '-GGAAGAATGGGAGTTGCTGT-3') is used as an internal reference and qPCR amplification is adoptedBrain-derived neurotrophic factor (BDNF) (forward5 '-TTATTTCATACTTCGGTTGC-3'; reverse5 '-TGTCAGCCAGTGATGTCG-3') gene, 2 was used-△△CtThe expression relative content of mouse hippocampal BDNF is calculated by a formula.
8. Western blot experiment the tissue was homogenized in RIPA buffer and centrifuged, and the protein concentration of the supernatant was determined by BCA method. An equal amount of protein was subjected to SDS-PAGE, followed by membrane transfer. After 1 hour incubation of the membrane at room temperature, BDNF antibody (1:500) was added and incubated overnight at 4 ℃, followed by secondary antibody, developed by ECL and photographed by a chemiluminescence imager and analyzed for gray scale values using Image J software.
9. After the behavioral experiment of immunofluorescence is finished, 10% chloral hydrate is injected into the abdominal cavity of a mouse, the mouse is anesthetized, physiological saline and polyformaldehyde are respectively filled into the mouse, the whole brain tissue of the mouse is taken out, the mouse is immersed into 4% paraformaldehyde for fixing for 24 hours, and the mouse is subjected to gradient dehydration in 30%, 20% and 10% sucrose for about 14 hours to sink; then OCT embedded, stored at-80 ℃ for further use. Placing brain tissue taken out at-80 deg.C at-20 deg.C for 2-3d, thawing at room temperature for 15min, cutting mouse brain tissue into 18 μm slices with a freezing microtome, adhering to glass slide, and performing immunofluorescence experiment. Firstly, fixing tissue slices in 4% paraformaldehyde for 48 hours, and then washing with 1M PBS for 3 times and 5 min/time; then placing the slices in a dark box containing 100mL citric acid, boiling in a water bath kettle heated to 95-100 ℃ for 20min, cooling at room temperature, sealing the tissues, adding fluorescent biscortin (DCX) primary antibody (1:100), incubating at 4 ℃ for 12h, adding fluorescent secondary antibody, staining the nuclei by DAPI (1:5000), and finally taking pictures by a laser confocal microscope.
10. After the Golgi staining behavioural experiment is finished, injecting 10% chloral hydrate into the abdominal cavity of the mouse, quickly taking out the complete brain tissue of the mouse, and quickly washing off blood on the surface of the brain tissue by double distilled water; soaking the tissue in a soaking solution formed by mixing the Golgi staining kit solution A and the Golgi staining kit solution B in equal volume, standing for two weeks in the dark at room temperature, and replacing the soaking solution after soaking for 6 hours; then transferring the tissue to a Golgi staining kit solution C, standing for 72 hours in a dark environment at room temperature, and replacing with a new solution C after 24 hours; the tissue was embedded in TFM, snap frozen in liquid nitrogen, and frozen in a-80 ℃ ultra-low temperature freezer. During detection, taking out brain tissue, placing the brain tissue on a cold place, and dropwise adding TFM for embedding and low-temperature freezing; the brain tissue was cut into sections of 50um thickness by a cryomicrotome, adhered to a glass slide on which the solution C was dropped, and air-dried at room temperature in the dark. The slices were immersed in a mixed solution consisting of 1 part of solution D, 1 part of solution E and 2 parts of double distilled water for 10 minutes, and the slices were dehydrated in 50%, 75%, 95% ethanol sequentially for 4min per concentration gradient. The slices were then dehydrated in anhydrous ethanol. Transparent in xylene for 3 times and 4 min/time, then sealing the cover glass with resin sealing tablet, drying in the dark, and finally taking a picture in a laser confocal microscope.
11. Statistical methods the experimental results were plotted using Graphpad Prism 7 software for statistical histogram of results expressed as mean ± standard error values; for the comparison between the two groups, two-factor analysis of variance is adopted in combination with Tukey's posterior analysis; statistical difference significance is indicated by a P value less than 0.05.
12. The experimental results are shown in fig. 2, and the two-factor analysis combined with Tukey's post hoc analysis shows: compared with a normal control group, the sugar water preference value of the mouse of the model control group is obviously reduced, the ingestion latency is obviously increased, and the laver acid hydrolysis polysaccharide can obviously reverse the abnormality.
As shown in figure 3, compared with the normal control group, the BDNF content of the hippocampus of the mouse of the model control group is obviously reduced, and the abnormality is also reversed after the administration of the acidolysis polysaccharide of the laver.
As shown in FIG. 4, CMS resulted in the inhibition of both hippocampal neurogenesis and synaptogenesis, and chronic administration of acid-hydrolyzed porphyra polysaccharide could alleviate the abnormality and enhance hippocampal neurogenesis and synaptogenesis.
Through comprehensive analysis of the 4 behavioral experiments, the porphyra acidolysis polysaccharide has a good anti-depression effect. The porphyra acidolysis polysaccharide provided by the invention is taken as a food, has high safety and small toxic and side effects, so that the porphyra acidolysis polysaccharide is taken as an antidepressant drug or an antidepressant functional food, is used for pharmacy and food production, and has good clinical and health-care application prospects. Comprehensive analysis of pharmacological experiments shows that the polysaccharide acidolysis of the laver can improve the brain-derived neurotrophic factor level of depressed animals and maintain hippocampal neurogenesis and synaptogenesis.
Example 4
Porphyra acidolysis polysaccharide tablet
The invention relates to a preparation method of a laver acid hydrolysis polysaccharide tablet, which comprises the following steps:
(1) extraction: extracting acid hydrolyzed laver polysaccharide from laver according to the extraction method described in example 1;
(2) and (3) granulating: mixing above thallus Porphyrae acid hydrolyzed polysaccharide 20g, microcrystalline cellulose 40g, stearic acid 4.2g, lactose 20g and silica gel micropowder 15g, and making into wet granule;
(3) and (3) drying: drying the prepared wet granules in a drying oven for 6 hours;
(4) tabletting: adding 0.8g of magnesium stearate, mixing uniformly, and tabletting to obtain 1000 tablets with consistent content.
Example 5
Porphyra acidolysis polysaccharide oral liquid preparation
The invention relates to a preparation method of an oral liquid preparation of laver acid hydrolysis polysaccharide, which comprises the following steps:
(1) extraction: extracting acid hydrolyzed laver polysaccharide from laver according to the extraction method described in example 1;
(2) adding a small amount of distilled water into 10g of acid-hydrolyzed laver polysaccharide, slightly heating, and continuously stirring to dissolve the acid-hydrolyzed laver polysaccharide solution;
(3) dissolving 3g of citric acid in a small amount of distilled water, adding the solution into the laver acid hydrolysis polysaccharide solution, and stirring and uniformly mixing;
(4) adding 80g of sucrose, adding distilled water to 1000ml, and stirring to dissolve the mixture into a transparent solution;
(5) filtering the transparent solution to remove insoluble impurities to obtain a transparent and uniform liquid preparation;
(6) subpackaging in sterilized 10ml ampoule bottle under aseptic condition, sealing and storing to obtain thallus Porphyrae acid hydrolyzed polysaccharide oral liquid preparation.
The above examples and figures do not limit the method and applications of the present invention, and any suitable changes or modifications thereof by one of ordinary skill in the art should be considered without departing from the scope of the present invention.
Claims (10)
1. An application of porphyra polysaccharide in preparing antidepressant drugs is characterized in that: the laver polysaccharide is laver acidolysis polysaccharide;
the preparation method of the acidolysis polysaccharide of laver comprises the following steps: extracting the dried laver by water extraction, alcohol precipitation and acidolysis to obtain laver acidolysis polysaccharide;
pulverizing dried thallus Porphyrae, adding ethanol, heating, filtering to remove alcohol soluble substances, washing residue with methanol, drying, extracting dried powder with distilled water at 80-95 deg.C for 1-3 hr, centrifuging, and concentrating the centrifuged supernatant;
adding sodium acetate and acetic acid into the concentrated supernatant to obtain sodium acetate solution, adding 42% ethanol, filtering to remove the first precipitate, and adjusting the ethanol concentration of the filtrate to 60%; then centrifuging and collecting a second precipitate, dissolving the second precipitate in distilled water, dialyzing with distilled water, and freeze-drying to obtain porphyra polysaccharide;
dissolving the laver polysaccharide obtained by freeze drying in distilled water to obtain a polysaccharide solution, sequentially adding ascorbic acid and hydrogen peroxide, stirring for reaction to obtain a laver acidolysis polysaccharide solution, precipitating the solution in 80% ethanol, and washing twice with 95% ethanol to obtain the laver acidolysis polysaccharide, wherein the final yield of the laver acidolysis polysaccharide is more than 10%.
2. Use according to claim 1, characterized in that: the medicine is a single agent containing a single component of the laver acid hydrolysis polysaccharide.
3. Use according to claim 1, characterized in that: the medicine is a multi-component mixed compound preparation containing laver acid hydrolysis polysaccharide.
4. A use as claimed in claim 3, wherein: the compound preparation is a tablet, and the components of the tablet comprise: a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, a flavoring agent, a lubricant, a sweetener, and a pigment.
5. Use according to claim 4, characterized in that: the tablet comprises the following components in parts by weight: 10-50 parts of acidolysis polysaccharide of laver, 10-40 parts of microcrystalline cellulose, 2-5 parts of stearic acid, 10-30 parts of lactose, 5-15 parts of aerosil and 0.2-1 part of magnesium stearate, wherein the microcrystalline cellulose and the lactose are fillers, the stearic acid is a surfactant, and the magnesium stearate is a lubricant.
6. A use as claimed in claim 3, wherein: the compound preparation is an oral liquid preparation, and the components of the oral liquid preparation comprise: buffer, antioxidant synergist, correctant, solvent, and surfactant.
7. Use according to claim 6, characterized in that: the oral liquid preparation comprises the following components in parts by weight: 10-50 parts of acidolysis polysaccharide of laver, 20-50 parts of sucrose, 1-3 parts of citric acid and water, wherein the citric acid is a buffering agent, a flavoring agent and an antioxidant synergist.
8. A use as claimed in claim 3, wherein: the compound preparation is an oral liquid preparation, and the components of the oral liquid preparation comprise: buffer, antioxidant synergist, sweetener, solvent, and surfactant.
9. Use according to claim 8, characterized in that: the oral liquid preparation comprises the following components in parts by weight: 10-50 parts of acidolysis polysaccharide of laver, 20-50 parts of sucrose, 1-3 parts of citric acid and water, wherein the citric acid is a buffering agent and an antioxidant synergist, and the sucrose is a sweetening agent.
10. AUse according to claim 1, characterized in that: weighing dried thallus Porphyrae, pulverizing, adding 100 times of 85% ethanol, heating at 75 deg.C for 1 hr, filtering to remove alcohol soluble substances, repeating for 3 times, washing the filtered residue with methanol for 2 times, drying, extracting the dried powder with 200 times of distilled water at 95 deg.C for 2 hr, and making into health food with 8000-gCentrifugation was carried out for 20 minutes, and the centrifuged supernatant was then concentrated to 1/3 in the original volume using a rotary evaporator;
adding sodium acetate and acetic acid into the supernatant to prepare 0.5M sodium acetate solution, wherein the pH value is 5.0, then adding ethanol with the final concentration of 42%, standing for 1 hour, removing a first precipitate in the solution, adjusting the final concentration of the ethanol to be 60%, centrifuging for 30 minutes at 10000 Xg to collect a second precipitate, then dissolving the second precipitate in distilled water, dialyzing with the distilled water, and freeze-drying to obtain porphyra polysaccharide;
dissolving the laver polysaccharide obtained by freeze drying in distilled water to obtain 0.5% polysaccharide solution, sequentially adding 3mM ascorbic acid and 3mM hydrogen peroxide, stirring for reaction for 2 hours to obtain laver acid hydrolysis polysaccharide solution, precipitating the solution in 80% ethanol, and washing twice with 95% ethanol to obtain laver acid hydrolysis polysaccharide.
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| 坛紫菜多糖提取及其生理活性研究;应苗苗;《中国博士学位论文全文数据库工程科技Ⅰ辑》;20190315(第3期);第B016-3页 * |
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