WO2021143392A1 - 调节免疫功能或治疗免疫相关疾病的药物 - Google Patents

调节免疫功能或治疗免疫相关疾病的药物 Download PDF

Info

Publication number
WO2021143392A1
WO2021143392A1 PCT/CN2020/134171 CN2020134171W WO2021143392A1 WO 2021143392 A1 WO2021143392 A1 WO 2021143392A1 CN 2020134171 W CN2020134171 W CN 2020134171W WO 2021143392 A1 WO2021143392 A1 WO 2021143392A1
Authority
WO
WIPO (PCT)
Prior art keywords
polysaccharide
polysaccharides
fish
fish body
body surface
Prior art date
Application number
PCT/CN2020/134171
Other languages
English (en)
French (fr)
Inventor
刘牧龙
左小峰
Original Assignee
深圳元健生命科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳元健生命科技有限公司 filed Critical 深圳元健生命科技有限公司
Publication of WO2021143392A1 publication Critical patent/WO2021143392A1/zh

Links

Images

Classifications

    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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

Definitions

  • This application relates to the field of biomedicine, in particular to methods for preparing fish body surface polysaccharides and fish body surface digestive polysaccharides and their applications.
  • Cyclophosphamide is a cytotoxic drug that suppresses the immune system. It has been used to treat a variety of diseases, such as the treatment of vasculitis, multiple sclerosis, lupus erythematosus and Wegener's granulomatosis. However, cyclophosphamide can induce immunosuppression in patients, leading to low immune function in patients and forming an immunodeficiency state.
  • Fish body surface polysaccharide is a kind of polysaccharide obtained from fish body surface. It is reported that it has anti-oxidation, scavenging oxygen free radicals and other active functions.
  • a method for preparing fish body surface polysaccharides and fish body surface digestive polysaccharides is provided, and the preparation of fish body surface polysaccharides and fish body surface digestive polysaccharides is related to the preparation of immune function regulation or immune treatment.
  • Application of medicines for diseases is provided.
  • a medicine for regulating immune function or treating immune-related diseases comprising fish body surface polysaccharide or fish body surface digestive polysaccharide and optionally at least one pharmaceutically acceptable excipient.
  • mice injected with cyclophosphamide by administering fish surface polysaccharide or its body surface digestive polysaccharide, it can restore the macrophage phagocytosis rate and B lymphocyte proliferation rate caused by cyclophosphamide in mice to a certain extent.
  • T lymphocyte proliferation rate, NK cell vitality decline has the effect of restoring the vitality of mouse macrophages, B lymphocytes, T lymphocytes, and NK cells, and can increase the serum hemolysin content of mice, indicating that Fish body surface polysaccharides and their body surface digestive polysaccharides have varying degrees of repairing/improving the immunity of mice, thereby being able to treat immune-related diseases.
  • the immune-related disease is tumor or cancer.
  • the fish is a fish that has no phosphorus or tiny scales and secretes polysaccharides on the body surface.
  • the fish body surface polysaccharide is rice eel polysaccharide, catfish polysaccharide, yellow fin fish polysaccharide, long-snout yellow fin fish polysaccharide or eel polysaccharide;
  • the fish body surface digestive polysaccharide is rice eel digestive polysaccharide, catfish Digestion of polysaccharides, yellowtail fish digestion polysaccharides, long-snout yellowtail fish digestion polysaccharides or eel digestion polysaccharides.
  • a method for preparing polysaccharides on the surface of fish comprising:
  • the precipitate is dried to obtain the polysaccharide on the surface of the fish.
  • At least one of the following (a) to (f) is included:
  • the temperature of the high temperature water is about 70°C to about 90°C;
  • the weight-volume ratio of the fish body to the high-temperature water is about 1:1 to 2;
  • the enzyme is selected from at least one of papain, neutral protease, and alkaline protease;
  • the temperature of the enzymatic hydrolysis is about 50°C to about 70°C;
  • the alcohol is ethanol
  • the concentration of ethanol after mixing the ethanol and the concentrated solution is greater than about 70%.
  • a method for preparing digested polysaccharides on the surface of fish including:
  • the filtrate is dried to obtain digested polysaccharides on the surface of fish.
  • a health food for enhancing immunity including fish body surface polysaccharide or fish body surface digestive polysaccharide.
  • the fish body surface polysaccharide or fish body surface digestive polysaccharide in the health food of the present application enhances immunity by increasing the serum hemolysin content.
  • the fish body surface polysaccharide is rice eel polysaccharide, catfish polysaccharide, yellow fin fish polysaccharide, long-snout yellow fin fish polysaccharide or eel polysaccharide; fish body surface digestive polysaccharide is rice eel digestive polysaccharide, catfish digestive polysaccharide , Yellow finfish digests polysaccharides, long-snout finfish digests polysaccharides or eels digests polysaccharides.
  • Fig. 1 is a flowchart of a method for preparing polysaccharides on the surface of fish according to an embodiment
  • Fig. 2 is a flowchart of a method for preparing digested polysaccharides on the surface of fish according to an embodiment
  • Figure 3 is a graph showing changes in body weight of polysaccharide mice after cyclophosphamide injection
  • Fig. 4 is a graph showing the results of measuring the immune organ index of polysaccharide mice
  • Figures 5A to 5F are diagrams showing the phagocytosis experiment of polysaccharide mouse macrophages
  • Figure 6A is a graph showing the results of determination of phagocytic rate of polysaccharide mouse macrophages
  • Fig. 6B is a graph showing the measurement result of the phagocytic index of polysaccharide mouse macrophages
  • Figure 7A is a graph showing the proliferation rate of T lymphocytes in polysaccharide mice.
  • Figure 7B is a graph showing the proliferation rate of B lymphocytes in polysaccharide mice.
  • Figure 8 is a graph showing the activity of NK cells in polysaccharide mice.
  • Figure 9 is a diagram showing the results of the HC50 of the serum hemolysis test in mice with digested polysaccharides
  • Figure 10 is a graph showing changes in body weight of mice digested with polysaccharides after injection of cyclophosphamide
  • Figure 11 is a graph showing the results of the immune organ index measurement of mice that digested polysaccharides
  • Figures 12A-12F are diagrams showing the phagocytosis experiment of mouse macrophages that digested polysaccharides
  • Figure 13A is a graph showing the results of measuring the phagocytic rate of macrophages in mice that digested polysaccharides
  • Figure 13B is a graph showing the results of measuring the phagocytic index of macrophages in mice that digested polysaccharides
  • Figure 14A is a graph showing the proliferation rate of T lymphocytes in mice that digest polysaccharides
  • Figure 14B is a graph showing the proliferation rate of B lymphocytes in mice that digest polysaccharides
  • Figure 15 is a graph showing the activity of NK cells in mice that digest polysaccharides
  • Fig. 16 is a graph showing the HC50 result of the serum hemolysis test of the digested polysaccharide mouse.
  • a medicine for regulating immune function or treating immune-related diseases comprising fish body surface polysaccharide or fish body surface digestive polysaccharide and optionally at least one pharmaceutically acceptable excipient.
  • the disease is a tumor, cancer, or virus-related disease.
  • the medicament of this embodiment is used to regulate immune function, especially to enhance immune function, or to treat immune-related diseases, or to enhance immune function to treat diseases.
  • the immune-related diseases of this embodiment can be induced by immunosuppressive agents, such as cyclophosphamide, and can also be caused by environmental factors.
  • the immune-related disease is a disease that can be treated by restoring macrophage viability or NK cell viability; and/or increasing serum hemolysin content, enhancing T cell or B cell proliferation and activity.
  • the medicine of this embodiment can be prepared according to a method known to those skilled in the art.
  • the drugs of this embodiment are tablets, pills, soft capsules, granules, powders, solutions, syrups, and any other suitable dosage forms.
  • the excipients of this embodiment may be excipients well known to those skilled in the art, such as binders, fillers, disintegrants, lubricants in tablets; wine, vinegar, concoctions, etc. in pills; semi-solids The base part of preparations ointments and creams; preservatives, antioxidants, correctives, fragrances, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, coloring agents, etc. in liquid preparations.
  • the drug of this embodiment can be administered orally. It can also be administered by injection, for example, via intraperitoneal, intramuscular, intraarterial, intravenous, subcutaneous, and intradermal routes.
  • fish body surface polysaccharide is a kind of polysaccharide obtained from fish body surface.
  • Fish are non-phosphorus or tiny scale fish that secrete polysaccharides on the surface.
  • Fish body surface digestion polysaccharide is a substance obtained by anaerobic culture after mixing the solution of fish body surface polysaccharide digested by the gastrointestinal tract and the intestinal bacterial liquid in vitro.
  • the polysaccharide on the surface of the fish is rice field eel polysaccharide, catfish polysaccharide, yellow caraway polysaccharide, long-snout yellow caraway polysaccharide or eel polysaccharide.
  • this application also provides the application of fish body surface polysaccharides and fish body surface digestive polysaccharides in the preparation of drugs for regulating immune function or treating immune-related diseases.
  • the inventor of the present application found that in mice injected with cyclophosphamide, by administering fish surface polysaccharides or fish surface digestive polysaccharides, the macrophage phagocytic rate and B of the mice caused by cyclophosphamide can be restored to a certain extent.
  • the proliferation rate of lymphocytes, the proliferation rate of T lymphocytes, and the decline of NK cell vitality can restore the vitality of mouse macrophages, B lymphocytes, T lymphocytes, and NK cells, and can increase the serum hemolysin content of mice.
  • the effect of the fish body surface polysaccharides and fish body surface digestive polysaccharides have varying degrees of repairing/improving the immunity of mice, thereby being able to treat immune-related diseases.
  • a method for preparing the above-mentioned fish body surface polysaccharide includes:
  • step S100 the fish body is treated with high-temperature water, and the mucus on the surface of the fish body is collected.
  • the temperature of the high-temperature water is about 70°C to about 90°C.
  • the weight-volume ratio of the fish body to the high-temperature water is about 1:1 ⁇ 2;
  • step S110 the enzyme is added to the fish body surface mucus for enzymatic hydrolysis to obtain an enzymatic hydrolysate.
  • the enzyme is selected from at least one of papain, neutral protease, and alkaline protease.
  • the weight of the enzyme is 0.05% of the weight of the fish body, and the temperature of enzymatic hydrolysis is about 50°C to about 70°C; specifically, it can be 60°C or 65°C.
  • the enzymatic hydrolysis can be kept in a water bath at 60°C for about 6 hours, heated to boiling, and kept for 20 minutes;
  • step S120 the enzymolysis solution is allowed to stand for precipitation and filtered.
  • catfish diatomaceous earth 0.2% by weight of catfish diatomaceous earth is added to the enzymolysis solution, and the mixture is stirred uniformly, and then allowed to stand at 4°C for 4 hours.
  • step S130 the filtrate is concentrated to obtain a concentrated solution.
  • Step S140 adding alcohol to the concentrated solution to obtain a precipitate.
  • the alcohol is a high concentration of 95% ethanol.
  • concentration of ethanol mixed with the concentrated solution is greater than about 70%. Specifically, a white or off-white precipitate was obtained by centrifugation at 4° C. and 10000 r/min for 10 min.
  • step S150 the precipitate is dried to obtain the polysaccharide on the surface of the fish.
  • the precipitate was washed twice with 95% ethanol, and then dried by blowing at 60° C. to obtain an off-white powder.
  • the fish body surface digestible polysaccharides are rice field eel digestive polysaccharides, catfish digestive polysaccharides, yellow catfish digestive polysaccharides, long-snout yellow catfish digestive polysaccharides or eel digestive polysaccharides.
  • a method for preparing digestible polysaccharides on the surface of fish includes:
  • step S200 the fish body surface polysaccharide is mixed with the in vitro simulated gastric juice for digestion to obtain a fish body surface polysaccharide solution after gastric juice digestion.
  • step S210 the digested fish body surface polysaccharide solution is added to the simulated intestinal digestive juice in vitro to obtain the fish body surface polysaccharide solution after digestion of the gastrointestinal juice.
  • step S220 the fish body surface polysaccharide solution after digestion of the gastrointestinal juice is mixed with the isolated intestinal bacterial solution, followed by anaerobic culture, sterilization, and filtration to obtain a filtrate.
  • step S230 the filtrate is dried to obtain digested polysaccharides on the surface of fish.
  • the step of adjusting the pH of the fish body surface polysaccharide solution after gastric juice digestion to 7 is included.
  • the preparation of in vitro simulated gastric juice includes:
  • gastric electrolyte solution containing 3.1g/L NaCl, 1.1g/LKCl, 0.15g/LCaCl 2 , 0.6g/L NaHCO 3 ; then add 250mL gastric electrolyte solution to 187.5mg pepsin and 165mg gastric lipase, and mix well; Adjust the pH value to 2 ⁇ 0.2 with HCl; and/or;
  • the preparation of in vitro simulated intestinal fluid includes: preparing an intestinal electrolyte solution containing 5.4g/L NaCl, 0.65g/L KCl, and 0.33g/L CaCl 2 ; mixing 70g/L pancreatin and pancreatin solution with intestinal electrolyte solution in a volume ratio of 1:1 Mix well and adjust the pH value to 7 ⁇ 0.2 with NaHCO 3.
  • a health food for enhancing immunity including fish body surface polysaccharides or fish digestive body surface polysaccharides.
  • the fish body surface polysaccharide and the fish body surface digestive polysaccharide can improve the immune function by increasing the serum hemolysin content.
  • the polysaccharides on the surface of fish are rice eel polysaccharides, catfish polysaccharides, yellow finfish polysaccharides, long-snout finfish polysaccharides or eel polysaccharides.
  • the digested polysaccharides on the fish body surface are rice eel digestive polysaccharides, catfish digestive polysaccharides, yellow minnow digestive polysaccharides, long-snout yellow minnow digestive polysaccharides or eel digestive polysaccharides.
  • the health food of this embodiment may also contain other components commonly used in foods, such as various additives.
  • the fish body surface polysaccharides used in the following Examples 1 to 4 can be prepared according to the method described in the Chinese patent (application number 201811215236.1). Or according to Qin Chuanguang et al. (Qin Chuanguang, Huang Kaixun, Xu Huibi, Study on the immune function of loach polysaccharides, Chinese Pharmaceutical Journal, Vol. 37 No. 8, August 2002).
  • Standard curve of sulfuric acid-phenol method accurately weigh 100mg of glucose reference substance, place it in a 1000mL volumetric flask, add an appropriate amount of water to dissolve, dilute to the mark, and shake well to obtain 0.1mg/mL glucose mother liquor. Pipette 0.4, 0.8, 1.2, 1.6, 2.0 mL of glucose mother liquor into 25 mL test tubes, add water to make up to 2.0 mL, add 1.0 mL of 5% phenol aqueous solution and mix well. Then quickly add 5 mL of concentrated sulfuric acid, shake well for 30 seconds after 10 minutes, and bath in a water bath at 30°C for 20 minutes.
  • Sample preparation Precisely weigh an appropriate amount of sample, place it in a 25mL volumetric flask, add an appropriate amount of water to dissolve, dilute to the mark, and shake well to obtain a sample solution. Pipette 1mL of the sample to 25mL and stopper the test tube, add water to make up to 2.0mL, add 1.0mL of 5% phenol aqueous solution and mix well. Then quickly add 5 mL of concentrated sulfuric acid, shake well for 30 seconds after 10 minutes, and bath in a water bath at 30°C for 20 minutes. Accurately draw 150 ⁇ l of glucose solution into a 96-well plate, and measure the absorbance with the reagent blank solution at a wavelength of 490nm as a reference.
  • Concentration of polysaccharide on fish body surface ⁇ 0.1707 ⁇ (A sample-A blank)+0.0007 ⁇ /1 ⁇ 2 ⁇ 25/M sampling volume ⁇ 100%.
  • gastric electrolyte solution 1.55g NaCl, 0.55g KCl, 0.075g CaCl 2 , 0.3g NaHCO 3 dissolved in 0.5L of deionized water.
  • in vitro simulated gastric juice Add 250 mL of gastric electrolyte solution to 187.5 mg of pepsin and 165 mg of gastric lipase, mix well, and adjust the pH to 2 with 1 mol/L HCl.
  • Intestinal electrolyte solution 0.54g NaCl, 0.065g KCl, 0.033g CaCl 2 dissolved in 100 mL of deionized water.
  • pancreatin Dissolve 7g pancreatin in 100mL water, centrifuge at 4800 ⁇ g for 10 minutes, and take the supernatant for use.
  • In vitro simulated intestinal fluid Add 90 mL of intestinal electrolyte solution to 90 mL of pancreatin solution, mix well, and adjust the pH to 7 with 1 mol/L NaHCO 3.
  • A is 7.5 mL (1% K 2 HPO 4 ⁇ 3H 2 O); B is 37.5 mL (0.47% KH 2 PO 4 , 1.18% NaCl, 1.2% (NH 4 ) 2 SO 4 , 0.16% CaCl 2 ⁇ H 2) O, 0.45% MgSO 4 ⁇ 7H 2 O); C is 50 mL (8% Na 2 CO 3 ); 0.5 g L-cysteine, 2 mL (25% L-ascorbic acid), 1 g beef extract, 1 g peptone. Add an appropriate amount of ultrapure water above, adjust the pH to 7.5-7.7 with 1mol/L HCl, and dilute the volume to 1000mL. The obtained medium is distributed in a tube, autoclaved at 115°C at 0.07MPa for 30min, stored at 4°C or used immediately.
  • mice Male clean grade Bclb/c mice, 18g ⁇ 2g (5w), 90 mice, 6 groups, 15 mice in each group.
  • model mice 75 randomly selected were injected intraperitoneally with cyclophosphamide at a dose of 80 mg/kg/d, and the control group was given the same volume of normal saline.
  • A Normal group: normal saline 200 ⁇ l/d, continuous gastric gavage for 28 days, once a day;
  • Model group normal saline 200 ⁇ l/d, continuous gavage for 28 days, once a day;
  • Drug group 1 dose 250mg/kg/d, continuous gavage for 28 days, once a day;
  • Drug group 2 dose 250mg/kg/d, continuous gastric gavage for 28 days, once a day;
  • Drug group 3 dose 250mg/kg/d, continuous gastric gavage for 28 days, once a day;
  • Drug group 4 dosage 250mg/kg/d, continuous gastric gavage for 28 days, once a day;
  • the medicine of the medicine 1 group adopts the rice field eel polysaccharide of example 1
  • the medicine of the medicine group 2 adopts the catfish polysaccharide of example 2
  • the medicine of the medicine group 3 adopts the yellow fish polysaccharide of example 3 and the medicine of the medicine 4 group. 4 eel polysaccharides.
  • mice were weighed, and 3 mice were randomly selected for the mouse macrophage phagocytosis experiment; 3 mice were used for the splenic lymphocyte transformation experiment; 3 mice were used for the NK cell viability test , 3 mice were taken from eyeballs and serum was taken for subsequent testing. At the same time, these 3 mice were sacrificed by cervical dislocation, and their spleens and thymus were taken and weighed; 2% SRBC 0.2mL for five consecutive days).
  • mice were weighed, and the weight change and mental state of the mice were observed.
  • drug groups 1 to 4 can all improve mouse thymus injury caused by cyclophosphamide to a certain extent, and drug groups 1 to 2 have significant differences.
  • mice were sacrificed after 30 minutes. After fixing the mice, cut the mouse epidermis gently and tear it (do not cut the peritoneum layer);
  • Figure 5A is the result of the normal group
  • Figure 5B is the result of the model group
  • Figure 5C is the result of the drug group 1
  • Figure 5D is the result of the drug group 2
  • Figure 5E is the result of the drug group 3
  • Figure 5F is the result of the drug group 4 result.
  • Drug groups 1 to 4 can restore to a certain extent the decrease in macrophage phagocytic index caused by cyclophosphamide in mice, indicating that drugs 1 to 4 have the effect of restoring the viability of macrophages in mice.
  • drug group 3 There is a significant difference;
  • Drug groups 1 to 4 all recovered to a certain extent the problem of decreased macrophage phagocytic rate caused by cyclophosphamide in mice, indicating that drugs 1 to 4 have the effect of restoring the viability of mouse macrophages, of which drug group 3 has Significant difference.
  • mice were sacrificed by cervical dislocation, soaked in 75% alcohol for 5 minutes;
  • Each sample has 3 groups (control group, LPS group, ConA group), each group has three replicates, the final concentration of LPS is 1 ⁇ g/mL, and the final concentration of ConA is 5 ⁇ g/mL;
  • mice were killed by cervical dislocation, the spleen was taken out under aseptic conditions, and washed several times with sterile HANK'S solution;
  • Drugs in groups 1 to 4 can restore to a certain extent the decreased NK cell viability caused by cyclophosphamide in mice, suggesting that drugs 1 to 4 have the effect of restoring the viability of NK cells in mice, among which drug groups 3 have significant differences.
  • mice in each group were intraperitoneally injected with 200 ⁇ l of 2% SRBC;
  • the HC50 of the model group is lower than that of the normal group, and there is a significant difference, which indicates that the antibody production ability of the model group is lower than that of the normal group;
  • the following examples 6-9 used fish body surface polysaccharide raw materials can be prepared according to the above-mentioned examples 1 to 4; or according to Qin Chuanguang et al. (Qin Chuanguang, Huang Kaixun, Xu Huibi, Study on the immune function of loach polysaccharides, Chinese Pharmaceuticals Journal, August 2002, Volume 37, Issue 8) method; it can also be scraped after killing fish at high temperature.
  • Monopterus albus polysaccharide dissolve it in 20 mL purified water, mix well with 80 mL of in vitro simulated gastric juice, and digest it in a shaker at 150r/min and 37°C. Take it out after 2h, adjust the pH of the solution to 7 with 1mol/L NaHCO 3 , then add 48 mL of in vitro simulated intestinal digestion to the Erlenmeyer flask, mix well, and digest in a shaker at 150r/min at 37°C.
  • catfish polysaccharide Take 1 part of catfish polysaccharide, dissolve it in 20mL purified water, mix well with 80mL in vitro simulated gastric juice, digest it in a shaker at 150r/min, 37°C; take it out after 2h, adjust the pH of the solution to 7 with 1mol/L NaHCO 3, Then, 48 mL of in vitro simulated intestinal digestion solution was added to the Erlenmeyer flask, mixed thoroughly, and digested in a shaker at 150r/min and 37°C.
  • mice Male clean grade Bclb/c mice, 18g ⁇ 2g (5w), 90 mice, 6 groups, 15 mice in each group.
  • model mice 75 randomly selected were injected intraperitoneally with cyclophosphamide at a dose of 80 mg/kg/d, and the control group was given the same volume of normal saline.
  • A Normal group: normal saline 200ul/d, continuous gavage for 28 days, once a day;
  • Model group normal saline 200ul/d, continuous gavage for 28 days, once a day;
  • Drug group 1 dose 250mg/kg/d, continuous gavage for 28 days, once a day;
  • Drug group 2 dose 250mg/kg/d, continuous gastric gavage for 28 days, once a day;
  • Drug group 3 dose 250mg/kg/d, continuous gastric gavage for 28 days, once a day;
  • Drug group 4 dosage 250mg/kg/d, continuous gastric gavage for 28 days, once a day;
  • mice were weighed, and 3 mice were randomly selected for the mouse macrophage phagocytosis experiment; 3 mice were used for the splenic lymphocyte transformation experiment; 3 mice were used for the NK cell viability test , 3 mice were taken from eyeballs and serum was taken for subsequent testing. At the same time, these 3 mice were sacrificed by cervical dislocation, and their spleens and thymus were taken to be weighed; 3 mice were used for the determination of serum hemolysin (injection into the intraperitoneal cavity every day for five days before administration) 2% SRBC 0.2ml for five consecutive days).
  • mice were weighed, and the weight change and mental state of the mice were observed.
  • drug groups 1 to 4 can all improve mouse thymus injury caused by cyclophosphamide to a certain extent, and drug groups 2 to 4 have significant differences.
  • Fig. 12A is the result of the normal group
  • Fig. 12B is the result of the model group
  • Fig. 12C is the result of the drug 1 group
  • Fig. 12D is the result of the drug 2 group
  • Fig. 12E is the result of the drug 3 group
  • Fig. 12F is the result of the drug 4 group result.
  • Drugs 1 to 4 can all restore to a certain extent the decrease in macrophage phagocytic index caused by cyclophosphamide in mice, suggesting that drugs 1 to 4 have the effect of restoring the viability of mouse macrophages, among which drug 1, 3 to 4 groups have significant differences;
  • Drugs in groups 1 to 4 can restore to a certain extent the decrease in the proliferation rate of T lymphocytes in mice caused by cyclophosphamide, suggesting that drugs 1 to 4 have the effect of restoring the vitality of T lymphocytes in mice, and drugs 1 to 4 Both groups have significant differences;
  • Drug groups 1 to 4 can restore to a certain extent the decreased NK cell viability caused by cyclophosphamide in mice, suggesting that drugs 1 to 4 have the effect of restoring the viability of NK cells in mice. Both drug groups 1 to 4 and the model group have Significant difference.
  • the rice field eel polysaccharides, catfish polysaccharides, yellow catfish polysaccharides, long snout polysaccharides, digested rice field eel polysaccharides, digested catfish polysaccharides, digested yellow catfish polysaccharides, and digested long snout polysaccharides all have excellent immune function enhancement effects. .

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Nutrition Science (AREA)
  • Mycology (AREA)
  • Virology (AREA)
  • Communicable Diseases (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Epidemiology (AREA)
  • Oncology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

一种调节免疫功能或治疗免疫相关疾病的药物和保健食品，以及一种制备鱼类体表多糖及鱼类体表消化多糖的方法。调节免疫功能或治疗免疫相关疾病的药物包括鱼类体表多糖或鱼类体表消化多糖及任选至少一种药学上可接受的赋形剂。

Description

调节免疫功能或治疗免疫相关疾病的药物
相关申请的交叉引用
本申请要求于2020年1月13日提交中国专利局、申请号为2020100320166、发明名称为“鱼类体表多糖及鱼类体表消化多糖的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本申请涉及生物医学领域，特别是涉及制备鱼类体表多糖及鱼类体表消化多糖的方法及其应用。
技术背景
环磷酰胺(Cyclophosphamide)是一种抑制免疫系统的细胞毒性药物，已被用于治疗多种疾病，例如治疗血管炎、多发性硬化症、红斑狼疮和韦格纳肉芽肿等免疫系统疾病。但是，环磷酰胺可诱导患者的免疫抑制，导致患者的免疫功能低下，形成免疫缺陷状态。
鱼类体表多糖是从鱼类体表获得的一种多糖，据报道具有抗氧化、清除氧自由基等活性功能。
目前已有关于将多糖用于调节免疫功能或治疗免疫系统疾病的报道，但没有关于鱼类体表的多糖或消化多糖用于调节免疫功能或治疗免疫系统疾病的报道。
发明内容
根据本申请的各种实施例，提供一种制备鱼类体表多糖及鱼类体表消化多糖的方法，及鱼类体表多糖及鱼类体表消化多糖在制备调节免疫功能或治疗免疫相关疾病的药物中的应用。
一种调节免疫功能或治疗免疫相关疾病的药物，包括鱼类体表多糖或鱼类体表消化多糖及任选至少一种药学上可接受的赋形剂。
已发现，在注射环磷酰胺的小鼠，通过施用鱼类体表多糖或其体表消化多糖，能够一定程度的恢复小鼠因环磷酰胺导致的巨噬细胞吞噬率、B淋巴细胞增值率、T淋巴细胞增值率、NK细胞活力的下降问题，具有恢复小鼠巨噬细胞、B淋巴细胞、T淋巴细胞、NK细胞的活力作用，且能够提高小鼠的血清溶血素含量的作用，表明鱼类体表多糖及其体表消化多糖具有不同程度的修复/提高小鼠免疫力的作用，从而能够治疗免疫性相关疾病。
在其中一个实施例中，所述免疫相关疾病为肿瘤或癌症。
在其中一个实施例中，所述鱼类为无磷或微小鳞片的且分泌体表多糖的鱼类。
在其中一个实施例中，所述鱼类体表多糖为黄鳝多糖、鲶鱼多糖、黄鲿鱼多糖、长吻黄鲿鱼多糖或鳗鱼多糖；所述鱼类体表消化多糖为黄鳝消化多糖、鲶鱼消化多糖、黄鲿鱼消化多糖、长吻黄鲿鱼消化多糖或鳗鱼消化多糖。
一种制备鱼类体表多糖的方法，包括：
用高温水处理鱼体，收集鱼类体表层黏液；
将酶加入所述鱼类体表层黏液进行酶解，以得到酶解液；
将所述酶解液静置沉淀，并过滤；
将滤液进行浓缩，得到浓缩液；
将醇加入所述浓缩液，以得到沉淀物；及
将所述沉淀物进行干燥，得到所述鱼类体表多糖。
在其中一个实施例中，包括如下(a)～(f)的至少一项：
(a)所述高温水的温度为约70℃至约90℃；
(b)所述鱼体与所述高温水的重量体积比为约1：1～2；
(c)所述酶选自木瓜蛋白酶、中性蛋白酶、碱性蛋白酶中的至少一种；
(d)所述酶解的温度为约50℃至约70℃；
(e)所述醇为乙醇；
(f)所述乙醇与所述浓缩液混合后的乙醇的浓度大于约70％。
一种制备鱼类体表消化多糖的方法，包括：
将鱼类体表多糖与体外模拟胃液混合进行消化，得到胃液消化后的鱼类体表多糖溶液；
将消化后的鱼类体表多糖溶液加入体外模拟的肠消化液，得到胃肠液消化后的鱼类体表多糖溶液；
将胃肠液消化后的鱼类体表多糖溶液与离体肠道菌液混合，依次经过厌氧培养、灭菌、过滤，得到滤液；及
将滤液进行干燥，得到鱼类体表消化多糖。
一种增强免疫力的保健食品，包括鱼类体表多糖或鱼类体表消化多糖。
本申请的保健食品中的鱼类体表多糖或鱼类体表消化多糖通过增加血清溶血素含量来增强免疫力。
在其中一个实施例中，所述鱼类体表多糖为黄鳝多糖、鲶鱼多糖、黄鲿鱼多糖、长吻黄鲿鱼多糖或鳗鱼多糖；鱼类体表消化多糖为黄鳝消化多糖、鲶鱼消化多糖、黄鲿鱼消化多糖、长吻黄鲿鱼消化多糖或鳗鱼消化多糖。
附图说明
图1是根据一实施例的制备鱼类体表多糖的方法的流程图；
图2是根据一实施例的制备鱼类体表消化多糖的方法的流程图；
图3是多糖小鼠注射环磷酰胺后体重变化图；
图4是显示多糖小鼠免疫器官指数测定结果的图；
图5A～5F是显示多糖小鼠巨噬细胞吞噬实验的图；
图6A是多糖小鼠巨噬细胞吞噬率测定结果图；
图6B是多糖小鼠巨噬细胞吞噬指数测定结果图；
图7A是显示多糖小鼠T淋巴细胞增殖率的图；
图7B是显示多糖小鼠B淋巴细胞增殖率的图；
图8是显示多糖小鼠NK细胞活性的图；
图9是显示消化多糖小鼠血清溶血实验HC50结果的图；
图10是消化多糖小鼠注射环磷酰胺后体重变化图；
图11是显示消化多糖小鼠免疫器官指数测定结果的图；
图12A～12F是显示消化多糖小鼠巨噬细胞吞噬实验的图；
图13A是消化多糖小鼠巨噬细胞吞噬率测定结果图；
图13B是消化多糖小鼠巨噬细胞吞噬指数测定结果图；
图14A是显示消化多糖小鼠T淋巴细胞增殖率的图；
图14B是显示消化多糖小鼠B淋巴细胞增殖率的图；
图15是显示消化多糖小鼠NK细胞活性的图；
图16是显示消化多糖小鼠血清溶血实验HC50结果的图。
具体实施方式
为了便于理解本申请，下面将参照相关附图对本申请进行更全面的描述。附图中给出了本申请的较佳的实施例。但是，本申请可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本申请的公开内容的理解更加透彻全面。
除非另有定义，本文所使用的所有的技术和科学术语与属于本申请的技术领域的技术人员通常理解的含义相同。本文中在本申请的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本申请。本文所使用的术语“及/或”包括一个或多个相关的所列项目的任意的和所有的组合。
一种调节免疫功能或治疗免疫相关疾病的药物，包括鱼类体表多糖或鱼类体表消化多糖及任选至少一种药学上可接受的赋形剂。
在其中一个实施例中，所述疾病为肿瘤、癌症或病毒相关的疾病。
本实施方式的药物用于调节免疫功能，特别是增强免疫功能，或治疗免疫相关疾病，或增强免疫功能治疗疾病。本实施方式的免疫相关疾病可由免疫抑制剂诱导的，如环磷酰胺，还可以由环境因素引起的。在其中一个实施例中，所述免疫相关疾病是能通过恢复巨噬细胞活力或NK细胞活力；及/或，提高血清溶血素含量、增强T细胞或B细胞增殖及活性治疗的疾病。
本实施方式的药物可根据本领域技术人员公知的方法制备。本实施方式的药物为片剂、丸剂、软胶囊剂、颗粒剂、散剂、溶液剂、糖浆剂以及任何其他合适的剂型。本实施方式的赋形剂可以是本领域技术人员公知的赋形剂，例如片剂中的黏合剂、填充剂、崩解剂、润滑剂；丸剂中的酒、醋、药汁等；半固体制剂软膏剂、霜剂中的基质部分；液体制剂中的防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂、着色剂等。本实施方式的药物可口服施用。也可注射施用，例如经腹膜内、肌内、动脉内、静脉内、皮下、皮内等途径。
具体地，鱼类体表多糖是从鱼类体表获得的一种多糖。鱼类为无磷或微小鳞片的且分泌体 表多糖的鱼类。鱼类体表消化多糖为鱼类体表多糖经胃肠消化后的溶液与离体肠道菌液混合后的经厌氧培养得到的物质。
在其中一个实施例中，鱼类体表多糖为黄鳝多糖、鲶鱼多糖、黄鲿鱼多糖、长吻黄鲿鱼多糖或鳗鱼多糖。采用上述鱼类体表多糖或消化多糖能够有效的增强机体免疫能力，治疗免疫相关疾病。
在其中一个实施例中，本申请还提供鱼类体表多糖及鱼类体表消化多糖在制备调节免疫功能或治疗免疫相关疾病的药物中的应用。
本申请发明人发现，在注射环磷酰胺的小鼠，通过施用鱼类体表多糖或鱼类体表消化多糖，能够一定程度的恢复小鼠因环磷酰胺导致的巨噬细胞吞噬率、B淋巴细胞增值率、T淋巴细胞增值率、NK细胞活力的下降问题，具有恢复小鼠巨噬细胞、B淋巴细胞、T淋巴细胞、NK细胞的活力作用，且能够提高小鼠的血清溶血素含量的作用，表明鱼类体表多糖及鱼类体表消化多糖具有不同程度的修复/提高小鼠免疫力的作用，从而能够治疗免疫性相关疾病。
参考图1，根据一实施例的制备上述鱼类体表多糖的方法包括：
步骤S100，用高温水处理鱼体，收集鱼类体表层黏液。
具体地，该高温水的温度为约70℃至约90℃。鱼体与高温水的重量体积比为约1：1～2；
步骤S110，将酶加入鱼类体表层黏液进行酶解，以得到酶解液。
酶选自木瓜蛋白酶、中性蛋白酶、碱性蛋白酶中的至少一种。酶的重量为鱼体重量的0.05％，酶解的温度为约50℃至约70℃；具体地可以为60℃、65℃。更进一步地，可以在60℃水浴保温酶解约6小时，加热至沸腾，保持20分钟；
步骤S120，将所述酶解液静置沉淀，并过滤。
具体地，向酶解液加入鲶鱼重量0.2％的硅藻土，搅拌均匀后4℃静置4小时。
步骤S130，将滤液进行浓缩，得到浓缩液。
步骤S140，将醇加入浓缩液，以得到沉淀物。
醇为高浓度95％乙醇。乙醇与浓缩液混合后的浓度大于约70％。具体地，通过在4℃、10000r/min离心10min，得白色或类白色沉淀。
步骤S150，将所述沉淀物进行干燥，得到所述鱼类体表多糖。
具体地，沉淀物用95％乙醇洗涤2次，然后在60℃经鼓风干燥，得到类白色粉末。
在其中一个实施例中，所述鱼类体表消化多糖为黄鳝消化多糖、鲶鱼消化多糖、黄鲿鱼消化多糖、长吻黄鲿鱼消化多糖或鳗鱼消化多糖。
参考图2，根据一实施例的制备鱼类体表消化多糖的方法包括：
步骤S200，将鱼类体表多糖与体外模拟胃液混合进行消化，得到胃液消化后的鱼类体表多糖溶液。
步骤S210，将消化后的鱼类体表多糖溶液加入体外模拟的肠消化液，得到胃肠液消化后的鱼类体表多糖溶液。
步骤S220，将胃肠液消化后的鱼类体表多糖溶液与离体肠道菌液混合，依次经过厌氧培养、灭菌、过滤，得到滤液。
步骤S230，将滤液进行干燥，得到鱼类体表消化多糖。
在其中一个实施例中，在加入体外模拟的肠消化液之前，包括将胃液消化后的鱼类体表多糖溶液的pH调节至7的步骤。
具体地，鱼类体表多糖于150r/min、37℃的摇床中消化2h后取出，用1mol/L NaHCO 3调节溶液pH值为7，然后向锥形瓶中加入48mL的体外模拟的肠消化液，充分混合后，于150r/min、37℃的摇床中消化4h，将离体肠道菌液与体表多糖经胃肠消化后的溶液，混合均匀，置于37℃厌氧培养4h，高压蒸汽121℃灭菌20min，过滤，滤液浓缩后60℃鼓风干燥。
体外模拟胃液、肠液均采用本领域常规技术实现。在其中一个实施例中，体外模拟胃液制备包括：
配制含3.1g/LNaCl、1.1g/LKCl、0.15g/LCaCl 2、0.6g/L NaHCO 3的胃电解质溶液；再将187.5mg胃蛋白酶和165mg胃脂肪酶中加入250mL胃电解质溶液，混合均匀；用HCl调pH值至2±0.2；及/或；
体外模拟肠液的制备包括：配制含5.4g/L NaCl、0.65g/L KCl、0.33g/L CaCl 2的肠电解质溶液；将70g/L胰酶胰酶溶液与肠电解质溶液以1：1体积比混合均匀，用NaHCO 3调节pH值至7±0.2。
一种增强免疫力的保健食品，包括鱼类体表多糖或鱼类消化体表多糖。该鱼类体表多糖及鱼类体表消化多糖可通过提高血清溶血素含量来提高免疫功能。
鱼类体表多糖为黄鳝多糖、鲶鱼多糖、黄鲿鱼多糖、长吻黄鲿鱼多糖或鳗鱼多糖。
具体地，鱼类体表消化多糖为黄鳝消化多糖、鲶鱼消化多糖、黄鲿鱼消化多糖、长吻黄鲿鱼消化多糖或鳗鱼消化多糖。
本实施方式的保健食品还可包含在食品中通常使用的其他组分，例如各种添加剂等。
下面通过具体的实施例进一步说明本申请，但不用于限制本申请。
下述实施例1～4中所用鱼类体表多糖可按中国专利(申请号为201811215236.1)中所述方法制备。或按钦传光等(钦传光，黄开勋，徐辉碧，泥鳅多糖的免疫作用研究，中国药学杂志，2002年8月第37卷第8期)方法制备。
1.多糖的检测
硫酸-苯酚法标准曲线：精密称取葡萄糖对照品100mg，置于1000mL容量瓶中，加适量水溶解，稀释至刻度，摇匀，即得0.1mg/mL葡萄糖母液。分别吸取葡萄糖母液0.4、0.8、1.2、1.6、2.0mL至25mL加塞试管中，加水补足至2.0mL，加入5％苯酚水溶液1.0mL并混匀。随后快速加入5mL浓硫酸，10min后摇匀30s，30℃水浴20min。准确吸取150μl葡萄糖溶液至96孔板中，在490nm波长处以试剂空白溶液为参比，测定吸光度。以吸光度为横坐标，葡萄糖浓度为纵坐标，绘制标准曲线，得到回归方程：Y＝0.1707X+0.0007(R2＝0.9997)。
样品配制：精密称取适量样品，置于25mL容量瓶中，加适量水溶解，稀释至刻度，摇匀，即得样品溶液。吸取样品1mL至25mL加塞试管中，加水补足至2.0mL，加入5％苯酚水溶液1.0mL混匀。随后快速加入5mL浓硫酸，10min后摇匀30s，30℃水浴20min。准确吸取150μl葡萄糖溶液至96孔板中，在490nm波长处以试剂空白溶液为参比，测定吸光度。
鱼类体表多糖浓度＝{0.1707×(A样品-A空白)+0.0007}/1×2×25/M取样量×100％。
2.体外模拟胃液和模拟肠液的配制：胃电解质溶液：1.55g NaCl、0.55g KCl、0.075g CaCl 2、0.3g NaHCO 3溶于0.5L的去离子水中。体外模拟胃液：187.5mg胃蛋白酶和165mg胃脂肪酶中加入250mL胃电解质溶液，混合均匀，用1mol/L HCl调pH值至2。肠电解质溶液：0.54g NaCl、0.065g KCl、0.033g CaCl 2溶于100mL的去离子水。7g/100mL胰酶：7g胰酶溶于100mL水，4800×g离心10min，取上清液待用。体外模拟肠液：90mL胰酶溶液中加入90mL肠电解质溶液，混合均匀，用1mol/L NaHCO 3调节pH值至7。
3.厌氧培养基制备培养基组成：
A为7.5mL(1％K 2HPO 4·3H 2O)；B为37.5mL(0.47％KH 2PO 4、1.18％NaCl、1.2％(NH 4) 2SO 4、0.16％CaCl 2·H 2O、0.45％MgSO 4·7H 2O)；C为50mL(8％Na 2CO 3)；0.5g L～半胱氨酸，2mL(25％L-抗坏血酸)，1g牛肉浸膏，1g蛋白胨。以上加超纯水适量，1mol/L HCl调节pH至7.5～7.7，定容至1000mL，所得培养基分配于管，115℃下以0.07MPa高压灭菌30min，4℃下贮存或立即使用。
4.粪便采集与处理(所用器具均经高压蒸汽121℃灭菌20min)：
筛选6例健康人体(男女各3例)收集新鲜粪便1次，将所得标本尽快置于－80℃冰箱中。取1个离心管，充满氮气，装入4g粪便并加入40mL生理盐水，涡旋混合制成悬浊液，5000r/min离心10min后得上清液，作为肠菌液。取肠菌液10mL置于培养瓶中，加入30mL厌氧培养液，混匀，作为肠菌培养液(整个过程于N 2流下操作)，迅速置于厌氧培养罐中，加入1个厌氧产气袋后盖上培养罐盖，置于37℃恒温培养箱中培养24h，使肠菌培养液中的肠道菌充分成长。
实施例1 黄鳝多糖的制备
经浸养干净的黄鳝2kg，置于带盖不锈钢桶中，将80℃2倍黄鳝重量体积的热水倒入桶中，盖好盖子，搅拌1分钟，过滤，收集热水液；收集液放冷后加入黄鳝重量0.05％的木瓜蛋白酶(800μ/mg)，60℃水浴保温酶解6小时；加热至沸腾，保持20分钟；向酶解液加入黄鳝重量0.2％的硅藻土，搅拌均匀后4℃静置4小时，过滤，浓缩至200mL左右，加4倍95％乙醇，使醇浓度大于70％，混合均匀，4℃静置8小时；4℃、10000r/min离心10min，得白色或类白色沉淀，沉淀用95％乙醇洗涤2次，然后在60℃经鼓风干燥，得到类白色粉末，根据检测，黄鳝多糖含量为27.8％。
实施例2 鲶鱼多糖的制备
经浸养干净的鲶鱼2kg，置于带盖不锈钢桶中，将80℃1倍鲶鱼重量体积的热水倒入桶中，盖好盖子，搅拌2分钟，过滤，收集热水液；收集液放冷后加入鲶鱼重量0.05％的木瓜蛋白酶(800μ/mg)，60℃水浴保温酶解6小时；加热至沸腾，保持20分钟；向酶解液加入鲶鱼重量0.2％的硅藻土，搅拌均匀后4℃静置4小时，过滤，浓缩至200mL左右，加4倍95％乙醇，使醇浓度大于70％，混合均匀，4℃静置8小时；4℃、10000r/min离心10min，得浅棕色或类白色沉淀，沉淀用95％乙醇洗涤2次，然后在60℃经鼓风干燥，得到浅棕色粉末。根据检测，鲶鱼多糖含量为21.9％。
实施例3 黄鲿鱼多糖的制备
经浸养干净的黄鲿鱼2kg，置于带盖不锈钢桶中，将80℃1.5倍黄鲿鱼重量体积的热水倒入桶中，盖好盖子，搅拌1分钟，过滤，收集热水液；收集液放冷后加入黄鲿鱼重量0.05％的木瓜蛋白酶(800μ/mg)，60℃水浴保温酶解6小时；加热至沸腾，保持20分钟；向酶解液加入黄鲿鱼重量0.2％的硅藻土，搅拌均匀后4℃静置4小时，过滤，浓缩至200mL左右，加4倍95％乙醇，使醇浓度大于70％，混合均匀，4℃静置8小时；4℃、10000r/min离心10min，得白色或类白色沉淀，沉淀用95％乙醇洗涤2次，然后在60℃经鼓风干燥，得到类白色粉末。根据检测，黄鲿鱼多糖含量为19.2％。
实施例4 鳗鱼多糖的制备
经浸养干净的鳗鱼2kg，置于带盖不锈钢桶中，将80℃1倍鳗鱼重量体积的热水倒入桶中，盖好盖子，搅拌2分钟，过滤，收集热水液；收集液放冷后加入鳗鱼重量0.05％的木瓜蛋白酶(800μ/mg)，60℃水浴保温酶解6小时；加热至沸腾，保持20分钟；向酶解液加入鳗鱼重量0.2％的硅藻土，搅拌均匀后4℃静置4小时，过滤，浓缩至200mL左右，加4倍95％乙醇，使醇浓度大于70％，混合均匀，4℃静置8小时；4℃、10000r/min离心10min，得白色或类白色沉淀，沉淀用95％乙醇洗涤2次，然后在60℃经鼓风干燥，得到类白色粉末。根据检测，鳗鱼多糖含量为21.3％。
实施例5 多糖动物实验
5.1动物实验方案
5.1.1实验动物
雄性清洁级Bclb/c小鼠，18g±2g(5w)，90只，6组，每组15只。
5.1.2饲养条件
12h交替光照，充足食物和水供给。
5.1.3实验分组
5.1.3.1小鼠适应性培养一周后，造模小鼠(随机选取75只)按照80mg/kg/d剂量腹腔注射环磷酰胺，对照组给予相同体积的生理盐水。
5.1.3.2注射3天后，按照不同组给予药物/生理盐水，各组均自由进食进水。
5.1.3.3随机分成6组如下：
A：正常组：生理盐水200μl/d，连续灌胃28天，每天灌胃一次；
B：模型组：生理盐水200μl/d，连续灌胃28天，每天灌胃一次；
C：药物1组：剂量250mg/kg/d，连续灌胃28天，每天灌胃一次；
D：药物2组：剂量250mg/kg/d，连续灌胃28天，每天灌胃一次；
E：药物3组：剂量250mg/kg/d，连续灌胃28天，每天灌胃一次；
F：药物4组：剂量250mg/kg/d，连续灌胃28天，每天灌胃一次；
其中药物1组的药物采用实施例1的黄鳝多糖、药物2组的药物采用实施例2的鲶鱼多糖、药物3组的药物采用实施例3的黄鲿鱼多糖、药物4组的药物采用实施例4的鳗鱼多糖。
5.1.3.4末次给药24h后，各个小鼠称重，各个随机选取3只小鼠用于小鼠巨噬细胞吞噬实 验；3只用于脾淋巴细胞转化实验；3只用于NK细胞活力检测，3只小鼠摘取眼球取血清于后续检测，同时，此3只小鼠颈脱臼处死，取脾脏和胸腺称重；3只用于检测血清溶血素测定(给药前五天每天腹腔注射2％SRBC 0.2mL，连续五天)。
5.2小鼠体重增长变化
分别于实验注射环磷酰胺前(图中负d3)、1d(图中d1)、4d(图中d4)、9d(图中d9)、14d(图中d14)、21d(图中d21)和28d(图中d28)对小鼠进行称重，观察小鼠的体重变化和精神状态。
实验结果显示，造模小鼠注射环磷酰胺后，均出现不同程度的体重下降、活动减少的情况，停止注射后体重开始上升，状态开始好转。结果见图3。
5.3小鼠免疫器官指数测定
小鼠免疫器官指数测定结果显示于图4。**表示和正常组相比，具有显著性差异；^^表示和模型组相比，具有显著性差异。
5.3.1和正常组相比，模型组、药物1～4组对脾指数影响无差异，提示对脾脏影响有限；
5.3.2和模型组相比，药物1～4组均能一定程度的改善由环磷酰胺造成的小鼠胸腺损伤，其中药物1～2组具有显著性差异。
5.4小鼠巨噬细胞吞噬指数测定
5.4.1实验步骤
5.4.1.1用生理盐水将4％的鸡红细胞稀释成1％的鸡红细胞；
5.4.1.2每只受试小鼠腹腔注射上述鸡红细胞1mL，轻揉腹部使其分散；
5.4.1.3 30min后处死小鼠，固定小鼠后轻轻剪开小鼠表皮层并将其撕裂(切勿剪破腹膜层)；
5.4.1.4注射5mL的生理盐水并轻柔腹部使其充分分散；
5.4.1.5用10mL注射器针头刺入腹部，并吸出腹腔液；
5.4.1.6取200μl腹腔液置于载玻片上并分散，37度培养箱孵育30min；
5.4.1.7生理盐水从玻片一端滴下，使废液从另外一端流出，轻轻洗涤2min；
5.4.1.8丙酮：甲醇(1:1)固定液固定5min后晾干玻片；
5.4.1.9滴加吉姆萨染色液固定5min左右；
5.4.1.10自来水从玻片一端滴下，使废液从另外一端流出，轻轻洗涤3min；
5.4.1.11将洗涤好的片子放到片架上，放入冰箱使其自然干燥；
5.4.1.12显微镜观察玻片，随机选取视野计算100个巨噬细胞(深紫色，个体较大，形状不定，核大)，并计算其中的鸡红细胞(细胞核淡紫色，个体小，被吞噬的鸡红细胞部分可见裂解，核小(需要区分小鼠单核细胞))；
5.4.1.13分别计算吞噬率(吞噬鸡红细胞的巨噬细胞/计数的巨噬细胞*100％)和吞噬指数(被吞噬的鸡红细胞总数/计数的巨噬细胞)
5.4.1.14照片见图5A～图5E(400x)
图5A是正常组的结果，图5B是模型组的结果，图5C是药物1组的结果，图5D是药物 2组的结果，图5E是药物3组的结果，图5F是药物4组的结果。
5.4.2实验结果
实验结果见图6A～6B。**表示和正常组相比，具有显著性差异；^^表示和模型组相比，具有显著性差异。
5.4.3实验结论
5.4.3.1药物1～4组均能一定程度的恢复小鼠因环磷酰胺导致的巨噬细胞吞噬指数下降问题，表明药物1～4有恢复小鼠巨噬细胞活力的作用，其中药物3组具有显著性差异；
5.4.3.2药物1～4组均一定程度的恢复小鼠因环磷酰胺导致的巨噬细胞吞噬率下降问题，表明药物1～4有恢复小鼠巨噬细胞活力的作用，其中药物3组具有显著性差异。
5.5、小鼠脾淋巴细胞转化实验
5.5.1实验步骤
5.5.1.1颈椎脱臼法处死小鼠，75％酒精中浸泡5min；
5.5.1.2在生物安全柜中解剖小鼠，分离出脾脏；
5.5.1.3用眼科手术剪取部分脾脏(绿豆大小)，用注射器的注射器芯(橡胶部分)在200目的不锈钢筛上研磨，同时滴加pbs，收集透过不锈钢筛的细胞悬液；
5.5.1.4离心收集细胞，用完全培养基重悬细胞，计数；
5.5.1.5将细胞浓度调整成1*106个/mL，取100μl培养基加入到96孔细胞培养板中；
5.5.1.6每个样本设置3组(对照组、LPS组、ConA组)，每组三重复，LPS的终浓度为1μg/mL，ConA的终浓度为5μg/mL；
5.5.1.7 37度，5％CO 2的培养箱中培养68h；
5.5.1.8每孔加入10μl cck8试剂，继续培养4h；
5.5.1.9酶标仪450nm处测定OD值；
5.5.1.10计算细胞增殖率((1-对照组OD值/实验组OD值)*100％)。
5.5.2实验结果
实验结果见图7A～7B。**表示和正常组比较有显著性差异；^^表示和模型组比较有显著性差异。
5.5.3实验结论
5.5.3.1药物1～4组均能一定程度的恢复小鼠因环磷酰胺导致的T淋巴细胞增值率下降问题，提示药物1～4有恢复小鼠T淋巴细胞活力的作用，药物1～2组具有显著性差异；
5.5.3.2药物1～4组均能一定程度的恢复小鼠因环磷酰胺导致的B淋巴细胞增值率下降问题，提示药物1～4有恢复小鼠B淋巴细胞活力的作用。
5.6 NK细胞活力测定
5.6.1实验步骤
5.6.1.1.颈椎脱臼法处死小鼠，无菌条件下取出脾脏，用无菌HANK’S液清洗数次；
5.6.1.2.用手术剪刀剪碎脾脏(尽量剪成小块)后研磨，用HANK’S液洗涤数次，将洗涤液过200目无菌不锈钢网筛；
5.6.1.3. 1000rpm/min离心5分钟，弃上清，用完全培养液重悬细胞，台酚蓝染色计数(活细胞在95％以上)，调整细胞浓度为2*105个/mL；
5.6.1.4.将培养好的靶细胞(YAC-1细胞)，调整细胞浓度为1*107个/mL；
5.6.1.5.靶细胞和脾淋巴细胞各取50μl(数量比50:1)，加入准备好的96孔板中，混匀，37度，5％CO 2，培养4小时；(设立靶细胞自发释放组(不含效应细胞，用完全培养基代替)和靶细胞最大释放组(额外添加2.5％triton))。
5.6.1.6. 1000rpm/min，离心5min，收集上清液待测LDH含量；
5.6.1.7.取20μl待测样本，分别加入25μl基质缓冲液和5μl辅酶I，混匀，37度温育15min。
5.6.1.8.加入2,4-二硝基苯肼25μl，混匀，37度温育15min；
5.6.1.9.加入250μl 0.4M NaOH溶液，混匀，室温放置5min；
5.6.1.10.酶标仪450nm测定OD值。
5.6.2实验结果
实验结果见图8。**表示和正常组比较有显著性差异；^^表示和模型组比较有显著性差异。
5.6.3实验结论
药物1～4组均能一定程度的恢复小鼠因环磷酰胺导致的NK细胞活力下降问题，提示药物1～4有恢复小鼠NK细胞活力的作用，其中药物3组具有显著性差异。
5.7、小鼠血清溶血素测定
5.7.1实验步骤
5.7.1.1在小鼠给药结束前5天，受试小鼠每组5只腹腔注射2％的SRBC 200μl；
5.7.1.2五天后，摘除眼球取全血，室温放置1h后，2500rpm/min 10min分离血清；
5.7.1.3取血清，用SA缓冲液200x稀释至1mL；
5.7.1.4加入用SA缓冲液稀释(1：8)的补体1mL；
5.7.1.5加入10％SRBC 0.5mL，轻轻混匀，37度孵育30min；
5.7.1.6冰浴终止反应，2000rpm离心10min；
5.7.1.7取1mL上清液，加入3mL氰化高铁血红蛋白检测液(南京建成，C021～1)，同时取10％SRBC 0.25mL加入3.75mL氰化高铁血红蛋白检测液；
5.7.1.8充分混匀后，放置10min，以对照管为空白，于540nm处测定OD值；
5.7.1.9计算HC50，HC50＝样品OD值/SRBC半数溶血值OD值*稀释倍数。
5.7.2实验结果
实验结果见图9。**表示和正常组比较有显著性差异；^^表示和模型组比较有显著性差异。
5.7.3实验分析
5.7.3.1和正常组相比，模型组的HC50低于正常组，且显著性差异，提示模型组的小鼠抗体生成能力低于正常组；
5.7.3.2和正常组相比，各个药物组的HC50逐渐升高，但无差异。
5.7.3.3和模型组相比，正常组的HC50高于模型组，且显著性差异，提示模型组造模成功；
5.7.3.4和模型组相比，药物1～4组的HC50逐渐升高，提示药物1～4组不同程度的有修复 /提高小鼠免疫力的作用。
下述实施例6～9中所用鱼类体表多糖原料可按上述实施例1～4方法制备；或按钦传光等(钦传光，黄开勋，徐辉碧，泥鳅多糖的免疫作用研究，中国药学杂志，2002年8月第37卷第8期)方法制备；也可高温杀死鱼后刮取。
实施例6 黄鳝消化多糖的制备
取其中1份黄鳝多糖，20mL纯化水溶解，80mL体外模拟胃液充分混合后，于150r/min，37℃的摇床中消化。2h后取出，用1mol/L NaHCO 3调节溶液pH值为7，然后向锥形瓶中加入48mL的体外模拟的肠消化液，充分混合后，于150r/min，37℃的摇床中消化。4h后取出，将离体肠道菌液与黄鳝多糖经胃肠消化后的溶液，混合均匀，立即置于于厌氧培养罐中，加入1个厌氧产气袋后迅速盖上培养罐盖，置于37℃恒温培养箱，分别培养4h后，高压蒸汽121℃灭菌20min，过滤，滤液浓缩后60℃鼓风干燥，作为药物1组。
实施例7 鲶鱼消化多糖的制备
取其中1份鲶鱼多糖，20mL纯化水溶解，80mL体外模拟胃液充分混合后，于150r/min，37℃的摇床中消化；2h后取出，用1mol/L NaHCO 3调节溶液pH值为7，然后向锥形瓶中加入48mL的体外模拟的肠消化液，充分混合后，于150r/min，37℃的摇床中消化。4h后取出，将离体肠道菌液与鲶鱼多糖经胃肠消化后的溶液，混合均匀，立即置于于厌氧培养罐中，加入1个厌氧产气袋后迅速盖上培养罐盖，置于37℃恒温培养箱，分别培养4h后，高压蒸汽121℃灭菌20min，过滤，滤液浓缩后60℃鼓风干燥，作为药物2组。
实施例8 黄鲿鱼消化多糖的制备
取其中1份黄鲿鱼多糖，20mL纯化水溶解，80mL体外模拟胃液充分混合后，于150r/min，37℃的摇床中消化。2h后取出，用1mol/L NaHCO 3调节溶液pH值为7，然后向锥形瓶中加入48mL的体外模拟的肠消化液，充分混合后，于150r/min，37℃的摇床中消化。4h后取出，将离体肠道菌液与黄鲿鱼多糖经胃肠消化后的溶液，混合均匀，立即置于于厌氧培养罐中，加入1个厌氧产气袋后迅速盖上培养罐盖，置于37℃恒温培养箱，分别培养4h后，高压蒸汽121℃灭菌20min，过滤，滤液浓缩后60℃鼓风干燥，作为药物3组。
实施例9.鳗鱼消化多糖的制备
取其中1份鳗鱼多糖，20mL纯化水溶解，80mL体外模拟胃液充分混合后，于150r/min，37℃的摇床中消化。2h后取出，用1mol/L NaHCO 3调节溶液pH值为7，然后向锥形瓶中加入48mL的体外模拟的肠消化液，充分混合后，于150r/min，37℃的摇床中消化。4h后取出，将离体肠道菌液与鳗鱼多糖经胃肠消化后的溶液，混合均匀，立即置于于厌氧培养罐中，加入1个厌氧产气袋后迅速盖上培养罐盖，置于37℃恒温培养箱，分别培养4h后，高压蒸汽121℃灭菌20min，过滤，滤液浓缩后60℃鼓风干燥，作为药物4组。
实施例10 消化多糖动物实验
10.1动物实验方案
10.1.1实验动物
雄性清洁级Bclb/c小鼠，18g±2g(5w),90只，6组，每组15只。
10.1.2饲养条件
12h交替光照，充足食物和水供给。
10.1.3实验分组
10.1.3.1小鼠适应性培养一周后，造模小鼠(随机选取75只)按照80mg/kg/d剂量腹腔注射环磷酰胺，对照组给予相同体积的生理盐水。
10.1.3.2注射3天后，按照不同组给予药物/生理盐水，各组均自由进食进水。
10.1.3.3随机分成6组如下：
A：正常组：生理盐水200ul/d，连续灌胃28天，每天灌胃一次；
B：模型组：生理盐水200ul/d，连续灌胃28天，每天灌胃一次；
C：药物1组：剂量250mg/kg/d，连续灌胃28天，每天灌胃一次；
D：药物2组：剂量250mg/kg/d，连续灌胃28天，每天灌胃一次；
E：药物3组：剂量250mg/kg/d，连续灌胃28天，每天灌胃一次；
F：药物4组：剂量250mg/kg/d，连续灌胃28天，每天灌胃一次；
10.1.3.4末次给药24h后，各个小鼠称重，各个随机选取3只小鼠用于小鼠巨噬细胞吞噬实验；3只用于脾淋巴细胞转化实验；3只用于NK细胞活力检测，3只小鼠摘取眼球取血清于后续检测，同时，此3只小鼠颈脱臼处死，取脾脏和胸腺称重；3只用于检测血清溶血素测定(给药前五天每天腹腔注射2％SRBC 0.2ml，连续五天)。
10.2小鼠体重增长变化
分别于实验注射环磷酰胺前(图中负d3)、1d(图中d1)、4d(图中d4)、9d(图中d9)、14d(图中d14)、21d(图中d21)和28d(图中d28)对小鼠进行称重，观察小鼠的体重变化和精神状态。
实验结果显示，造模小鼠注射环磷酰胺后，均出现不同程度的体重下降、活动减少的情况，停止注射后体重开始上升，状态开始好转。结果见图10。
10.3小鼠免疫器官指数测定
小鼠免疫器官指数测定结果显示于图11。**表示和正常组相比，具有显著性差异；^^表示和模型组相比，具有显著性差异。
10.3.1和正常组相比，模型组、药物1～4组对脾指数影响无差异，提示对脾脏影响有限；
10.3.2和模型组相比，药物1～4组均能一定程度的改善由环磷酰胺造成的小鼠胸腺损伤，其中药物2～4组具有显著性差异。
10.4小鼠巨噬细胞吞噬指数测定
10.4.1照片见图12A～图12E(400x)
图12A是正常组的结果，图12B是模型组的结果，图12C是药物1组的结果，图12D是药物2组的结果，图12E是药物3组的结果，图12F是药物4组的结果。
10.4.2实验结果
实验结果见图13A～13B。**表示和正常组相比，具有显著性差异；^^表示和模型组相比， 具有显著性差异。
10.4.3实验结论
10.4.3.1药物1～4组均能一定程度的恢复小鼠因环磷酰胺导致的巨噬细胞吞噬指数下降问题，提示药物1～4有恢复小鼠巨噬细胞活力的作用，其中药物1、3～4组具有显著性差异；
10.4.3.2药物1～4组均一定程度的恢复小鼠因环磷酰胺导致的巨噬细胞吞噬率下降问题，提示药物1～4有恢复小鼠巨噬细胞活力的作用，其中药物1、3～4组具有显著性差异。
10.5、小鼠脾淋巴细胞转化实验
10.5.1实验结果
实验结果见图14A～14B。**表示和正常组比较有显著性差异；^^表示和模型组比较有显著性差异。
10.5.2实验结论
10.5.2.1药物1～4组均能一定程度的恢复小鼠因环磷酰胺导致的T淋巴细胞增值率下降问题，提示药物1～4有恢复小鼠T淋巴细胞活力的作用，药物1～4组均具有显著性差异；
10.5.2.2药物1～4组均能一定程度的恢复小鼠因环磷酰胺导致的B淋巴细胞增值率下降问题，提示药物1～4有恢复小鼠B淋巴细胞活力的作用，药物1～4组均具有显著性差异。
10.6 NK细胞活力测定
10.6.1实验结果
实验结果见图15。**表示和正常组比较有显著性差异；^^表示和模型组比较有显著性差异。
10.6.2实验结论
药物1～4组均能一定程度的恢复小鼠因环磷酰胺导致的NK细胞活力下降问题，提示药物1～4有恢复小鼠NK细胞活力的作用，药物1～4组与模型组均具有显著性差异。
10.7、小鼠血清溶血素测定
10.7.1实验结果
实验结果见图16。**表示和正常组比较有显著性差异；^^表示和模型组比较有显著性差异。
10.7.2实验分析
10.7.2.1和正常组相比，模型组的HC50低于正常组，且显著性差异，提示模型组的小鼠抗体生成能力低于正常组；
10.7.2.2和正常组相比，各个药物组的HC50逐渐升高，但无差异。
10.7.2.3和模型组相比，正常组的HC50高于模型组，且显著性差异，提示模型组造模成功；
10.7.2.4和模型组相比，各个药物组的HC50逐渐升高，且具有显著性差异，提示药物1～4组不同程度的有修复/提高小鼠免疫力的作用。
从上述结果可知，黄鳝多糖，鲶鱼多糖，黄颡鱼多糖，长吻鮠多糖，消化黄鳝多糖，消化鲶鱼多糖，消化黄颡鱼多糖，消化长吻鮠多糖均具有极好的增强免疫功能的效果。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本申请的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干变形和改进，这些都属于本申请的保护范围。因此，本申请专利的保护范围应以所附权利要求为准。

Claims (15)

  1. 一种调节免疫功能或治疗免疫相关疾病的药物，包括鱼类体表多糖或鱼类体表消化多糖及任选至少一种药学上可接受的赋形剂。
  2. 根据权利要求1所述的药物，其中所述免疫相关疾病为肿瘤或癌症。
  3. 根据权利要求1所述的药物，其中所述免疫相关疾病是能通过恢复巨噬细胞活力或NK细胞活力；和/或，提高血清溶血素含量、增强T细胞或B细胞增殖及活性治疗的疾病。
  4. 根据权利要求1所述的药物，其中所述鱼类为无磷或微小鳞片的且分泌体表多糖的鱼类。
  5. 根据权利要求1所述的药物，其中所述鱼类体表多糖为黄鳝多糖、鲶鱼多糖、黄鲿鱼多糖、长吻黄鲿鱼多糖或鳗鱼多糖；所述鱼类体表消化多糖为黄鳝消化多糖、鲶鱼消化多糖、黄鲿鱼消化多糖、长吻黄鲿鱼消化多糖或鳗鱼消化多糖。
  6. 一种制备鱼类体表多糖的方法，包括：
    用高温水处理鱼体，收集鱼类体表层黏液；
    将酶加入所述鱼类体表层黏液进行酶解，以得到酶解液；
    将所述酶解液静置沉淀，并过滤；
    将滤液进行浓缩，得到浓缩液；
    将醇加入所述浓缩液，以得到沉淀物；及
    将所述沉淀物进行干燥，得到所述鱼类体表多糖。
  7. 根据权利要求6所述的方法，其中所述高温水的温度为约70℃至约90℃。
  8. 根据权利要求6所述的方法，其中所述鱼体与所述高温水的重量体积比为约1：1～2。
  9. 根据权利要求6所述的方法，其中所述酶选自木瓜蛋白酶、中性蛋白酶、碱性蛋白酶中的至少一种。
  10. 根据权利要求6所述的方法，其中所述酶解的温度为约50℃至约70℃。
  11. 根据权利要求6所述的方法，其中所述醇为乙醇。
  12. 根据权利要求11所述的方法，其中所述乙醇与所述浓缩液混合后的乙醇的浓度大于约70％。
  13. 一种制备鱼类体表消化多糖的方法，包括：
    将鱼类体表多糖与体外模拟胃液混合进行消化，得到胃液消化后的鱼类体表多糖溶液；
    将所述消化后的鱼类体表多糖溶液加入体外模拟的肠消化液，得到胃肠液消化后的鱼类体表多糖溶液；
    将所述胃肠液消化后的鱼类体表多糖溶液与离体肠道菌液混合，依次经过厌氧培养、灭菌、过滤，得到滤液；及
    将所述滤液进行干燥，得到所述鱼类体表消化多糖。
  14. 一种增强免疫力的保健食品，包括鱼类体表多糖或鱼类体表消化多糖。
  15. 根据权利要求14所述的保健食品，其中所述鱼类体表多糖为黄鳝多糖、鲶鱼多糖、黄鲿鱼多糖、长吻黄鲿鱼多糖或鳗鱼多糖；鱼类体表消化多糖为黄鳝消化多糖、鲶鱼消化多糖、 黄鲿鱼消化多糖、长吻黄鲿鱼消化多糖或鳗鱼消化多糖。
PCT/CN2020/134171 2020-01-13 2020-12-07 调节免疫功能或治疗免疫相关疾病的药物 WO2021143392A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010032016.6 2020-01-13
CN202010032016.6A CN111084783A (zh) 2020-01-13 2020-01-13 鱼类体表多糖及鱼类体表消化多糖的应用

Publications (1)

Publication Number Publication Date
WO2021143392A1 true WO2021143392A1 (zh) 2021-07-22

Family

ID=70399132

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/134171 WO2021143392A1 (zh) 2020-01-13 2020-12-07 调节免疫功能或治疗免疫相关疾病的药物

Country Status (2)

Country Link
CN (1) CN111084783A (zh)
WO (1) WO2021143392A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111084783A (zh) * 2020-01-13 2020-05-01 深圳市生命谷生命科技研究院 鱼类体表多糖及鱼类体表消化多糖的应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015137361A (ja) * 2014-01-24 2015-07-30 国立大学法人愛媛大学 多糖類、多糖類を含む組成物、及び免疫賦活剤
CN105017440A (zh) * 2015-08-13 2015-11-04 重庆都好生物科技有限公司 一种鳝鱼表皮多糖的提取方法
CN107594187A (zh) * 2017-09-25 2018-01-19 中国标准化研究院 鲶鱼体表抗菌肽和活性多糖混合物在制备饲料中的应用
CN109223821A (zh) * 2018-10-18 2019-01-18 深圳市生命谷生命科技研究院 泥鳅多糖作为治疗免疫缺陷的药物或保健食品的用途
CN109265577A (zh) * 2018-10-18 2019-01-25 深圳市生命谷生命科技研究院 一种泥鳅多糖的制备方法
CN110638830A (zh) * 2019-09-29 2020-01-03 深圳市生命谷生命科技研究院 消化泥鳅多糖调节免疫功能或治疗免疫相关疾病的用途
CN111084783A (zh) * 2020-01-13 2020-05-01 深圳市生命谷生命科技研究院 鱼类体表多糖及鱼类体表消化多糖的应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2018517786A (ja) * 2015-06-19 2018-07-05 グローバル ヘルス ソリューションズ リミテッド ライアビリティ カンパニー カチオン性殺生物剤を含むペトロラタム系組成物
CN107212420A (zh) * 2017-05-04 2017-09-29 浙江海洋大学 一种增强免疫能力的鲟鱼鱼鳔多肽保健食品及其制备方法
CN108250314A (zh) * 2017-12-29 2018-07-06 舟山富晟食品科技有限公司 一种鱿鱼墨活性多糖

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015137361A (ja) * 2014-01-24 2015-07-30 国立大学法人愛媛大学 多糖類、多糖類を含む組成物、及び免疫賦活剤
CN105017440A (zh) * 2015-08-13 2015-11-04 重庆都好生物科技有限公司 一种鳝鱼表皮多糖的提取方法
CN107594187A (zh) * 2017-09-25 2018-01-19 中国标准化研究院 鲶鱼体表抗菌肽和活性多糖混合物在制备饲料中的应用
CN109223821A (zh) * 2018-10-18 2019-01-18 深圳市生命谷生命科技研究院 泥鳅多糖作为治疗免疫缺陷的药物或保健食品的用途
CN109265577A (zh) * 2018-10-18 2019-01-25 深圳市生命谷生命科技研究院 一种泥鳅多糖的制备方法
CN110638830A (zh) * 2019-09-29 2020-01-03 深圳市生命谷生命科技研究院 消化泥鳅多糖调节免疫功能或治疗免疫相关疾病的用途
CN111084783A (zh) * 2020-01-13 2020-05-01 深圳市生命谷生命科技研究院 鱼类体表多糖及鱼类体表消化多糖的应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
QIN CHUANGUANG, HUANG KAIXUN, XU HUIBI: "Investigation on immunomodulating activities of polysaccharide from loach mucus", CHINESE PHARMACEUTICAL JOURNAL, ZHONGGUO YAOXUEHUI ZHUBAN CHUBAN. ZHONGGUO-YAOXUE-ZAZHI BIANJI WEIYUANHUI, CN, vol. 37, no. 8, 1 January 2002 (2002-01-01), CN, pages 588 - 591, XP055829867, ISSN: 1001-2494 *
QIN, CHUAN-GUANG; HUANG, KAI-XUN; XU, HUI-BI: "Effect of Misgurnus anguillicandatus polysaccharide on immune responses of splenocytes in mice", ACTA PHARMACOL SIN, vol. 23, no. 6, 30 June 2002 (2002-06-30), pages 534 - 538, XP009529197, ISSN: 1671-4083 *
ZHANG TING, YUE XING-JIAN: "Ｒesearch Progress of Secretion Mechanisms Components and Function of Fishes Mucus", ANHUI NONGYE KEXUE (JOURNAL OF ANHUI AGRICULTURAL SCIENCES), ANHUI SHENG NONGYE KEXUEYUAN, CN, vol. 42, no. 22, 1 January 2014 (2014-01-01), CN, pages 7445 - 7458, XP055829864, ISSN: 0517-6611, DOI: 10.13989/j.cnki.0517-6611.2014.22.057 *

Also Published As

Publication number Publication date
CN111084783A (zh) 2020-05-01

Similar Documents

Publication Publication Date Title
CN105343133B (zh) 一种治疗溃疡性结肠炎的复合益生菌、药物及其制备方法
JP4771379B2 (ja) 発酵及び培養方法、植物発酵エキス、植物発酵エキス末並びに該植物発酵エキス配合物
CN101590081A (zh) 凸腹真杆菌和两形真杆菌制剂及其应用
CN110643524B (zh) 一种具有胃肠道粘膜保护作用的复合益生菌制剂及其应用
WO2021143392A1 (zh) 调节免疫功能或治疗免疫相关疾病的药物
CN101849969A (zh) 产酪酸有益菌在制备防治重症疾病肠道屏障损伤及损伤后并发症制剂中的应用
CN112889967A (zh) 一种普洱熟茶提取物制备方法及在肠道药物保健品中的应用
CN110638830A (zh) 消化泥鳅多糖调节免疫功能或治疗免疫相关疾病的用途
CN108096443A (zh) 一种清除畜禽体内毒素、提升机体免疫力的药物、其制备方法及应用
CN103108957A (zh) 全蛋蛋白肽及其制备方法和应用
CN110192653A (zh) 一种孕产妇及儿童专用益生菌的制备方法
Kellogg Autointoxication; Or, Intestinal Toxemia
CN105343132B (zh) 治疗结肠炎的组合物、药物及其制备方法
CN108273051B (zh) 猪a型产气荚膜梭菌灭活疫苗的快速高效制备方法
Neyen Foundational Article: Mechnikov I, 1909: Intestinal Bacteriotherapy
CN110123822A (zh) 乳源低聚糖在制备用于通过缓解肠道缺氧损伤而治疗或预防nec的药物或食品中的用途
Huffnagle et al. The Probiotics Revolution: The definitive guide to safe, natural health solutions using probiotic and prebiotic foods and supplements
Li et al. Probiotics Combined with Ursodeoxycholic Acid on Cholestatic Liver Disease during Pregnancy.
WO2023061100A1 (zh) 一种脆弱拟杆菌灭活菌粉及其制备方法
YASMIN et al. Effect of honey supplementation on plasma levels of short chain fatty acids in malnurished children
TWI458438B (zh) Fermentation and culture methods, plant fermentation extracts, plant fermentation extract powders, and plant fermentation extract complexes
CN109320625A (zh) 一种当归多糖的提取方法及应用
CN117223863A (zh) 一种缓解食物过敏症状的母乳低聚糖组合物及应用
Cartwright Probiotics for Crohn's & Colitis
CN117618515A (zh) 一种用于控制体重、降低肝脂肪变的中药益生菌组合物

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20913976

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20913976

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 15.03.2023)

122 Ep: pct application non-entry in european phase

Ref document number: 20913976

Country of ref document: EP

Kind code of ref document: A1