WO2018228430A1 - 异阿魏酸、含异阿魏酸中药提取物及升麻的用途 - Google Patents

异阿魏酸、含异阿魏酸中药提取物及升麻的用途 Download PDF

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WO2018228430A1
WO2018228430A1 PCT/CN2018/091056 CN2018091056W WO2018228430A1 WO 2018228430 A1 WO2018228430 A1 WO 2018228430A1 CN 2018091056 W CN2018091056 W CN 2018091056W WO 2018228430 A1 WO2018228430 A1 WO 2018228430A1
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acid
group
isoferulic acid
cohosh
chinese medicine
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PCT/CN2018/091056
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French (fr)
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曾辉
王宪波
朱鏐娈
李蕊
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首都医科大学附属北京地坛医院
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Priority to JP2019569899A priority Critical patent/JP6912606B2/ja
Priority to US16/621,935 priority patent/US20200108032A1/en
Priority to EP18818042.6A priority patent/EP3639836B1/en
Publication of WO2018228430A1 publication Critical patent/WO2018228430A1/zh

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/185Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19Carboxylic acids, e.g. valproic acid
    • A61K31/192Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, 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
    • A23L33/00Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105Plant extracts, their artificial duplicates or their derivatives
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00Drugs for skeletal disorders
    • A61P19/02Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • A61P37/02Immunomodulators
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23VINDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00Food compositions, function of food ingredients or processes for food or foodstuffs
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30Extraction of the material
    • A61K2236/33Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/331Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/71Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal

Definitions

  • the invention relates to the field of medicine, in particular to the use of isoferulic acid, a traditional Chinese medicine extract containing isoferulic acid, and the use of Cimicifuga in the preparation of a medicament or functional health care product for autoimmune diseases.
  • Innate immune cell and T lymphocyte activation are key pathogenesis of autoimmune diseases, and typical diseases are represented by autoimmune hepatitis and rheumatoid arthritis. Usually these diseases are caused by a variety of pathogenic factors to stimulate the body, causing the body's immune disorders and disease. Developing new drugs or health supplements from the perspective of regulating the body's immunity will be a breakthrough in the treatment of this type of disease. Therefore, the development of immunomodulatory drugs or functional health products from the traditional medical resources of the motherland has broad prospects.
  • Isofuric acid is the main monomeric active ingredient in Chinese herbal medicine extracts such as Cimicifuga.
  • the chemical name is 3-hydroxy-4-methoxycinnamic acid, also known as orange acid or caffeic acid-4-methyl ether. It is C 10 H 10 O 4 and has anti-oxidation and blood sugar lowering effects.
  • One of the objects of the present invention is to provide the use of isoferulic acid, an extract of a traditional Chinese medicine containing isoferulic acid, and cohosh.
  • the present invention provides use of isoferulic acid, an extract of a traditional Chinese medicine containing isoferulic acid, and cohosh in the preparation of a medicament or a functional health supplement for use in an autoimmune disease.
  • the autoimmune disease is autoimmune hepatitis or rheumatoid arthritis.
  • the invention also provides the use of isoferulic acid, a traditional Chinese medicine extract containing isoferulic acid, and cohosh in preparing a medicament or a functional health supplement for inhibiting inflammatory cytokines, including TNF - ⁇ , IFN- ⁇ , IL-6, IL-9, IL-12, IL-17A, IL-18, IP-10, MCP-1, MCP-3, MIP-1 ⁇ , MIP-1 ⁇ , MIP-2 One or more of Eotaxin and G-CSF.
  • inflammatory cytokines including TNF - ⁇ , IFN- ⁇ , IL-6, IL-9, IL-12, IL-17A, IL-18, IP-10, MCP-1, MCP-3, MIP-1 ⁇ , MIP-1 ⁇ , MIP-2
  • Eotaxin and G-CSF One or more of Eotaxin and G-CSF.
  • the inflammatory cytokine may be involved in various diseases caused by bacterial and viral infections, such as hepatitis, pneumonia, sepsis, influenza, measles, herpes simplex, etc.; and may also involve various autoimmune diseases, such as Rheumatoid arthritis, systemic vasculitis, scleroderma, multiple encephalomyelitis, etc., isoferulic acid, extracts containing isoferulic acid, and cohosh can be used as prevention, alleviation and treatment of the above diseases. Drug or functional health supplement.
  • the isofluic acid-containing traditional Chinese medicine extract may be a cohosh decoction, which is obtained by boiling the roots and roots of the cohosh.
  • the isofluic acid-containing traditional Chinese medicine extract may also be a cohos total phenolic acid extract, which mainly contains isoferulic acid, ferulic acid, caffeic acid and the like.
  • the drug may further comprise a pharmaceutically acceptable carrier in addition to the isoferulic acid, the isofluic acid-containing Chinese herbal extract, or the cohosh as an active ingredient.
  • Pharmaceutically acceptable carriers include, but are not limited to, excipients, binders, lubricants, fillers, disintegrants, emulsifiers, stabilizers, colorants, flavoring agents, preservatives, and the like.
  • the medicament may be prepared into any of the common dosage forms of the prior art, including, but not limited to, oral dosage forms such as tablets, capsules, granules, pills, and the like, as well as parenteral dosage forms such as injections, lyophilizates, and the like.
  • the functional health care product may add common ingredients, including but not limited to nutrients, vitamins, minerals, etc., in addition to isoferulic acid, isofluic acid-containing traditional Chinese medicine extract, or cohosh as an active ingredient.
  • the functional health care product can be used alone or in combination with an existing drug or health care product.
  • Another object of the present invention is to provide a pharmaceutical composition comprising isoferric acid or an extract containing a traditional Chinese medicine of ferulic acid, or a traditional Chinese medicine composition comprising Cimicifuga in the preparation of a medicament for use in an autoimmune disease.
  • the autoimmune disease is autoimmune hepatitis or rheumatoid arthritis.
  • the isofluic acid-containing traditional Chinese medicine extract may be a cohosh decoction, which is obtained by boiling the roots and roots of the cohosh.
  • the isofluic acid-containing traditional Chinese medicine extract may also be a cohos total phenolic acid extract, which mainly contains isoferulic acid, ferulic acid, caffeic acid and the like.
  • the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier other than the active ingredient, including but not limited to excipients, binders, lubricants, fillers, disintegrating agents, emulsifiers, and stabilizing agents. Agents, colorants, flavors, preservatives, and the like.
  • the pharmaceutical composition may be administered alone or in combination with existing drugs for autoimmune diseases, which may be prepared into any of the common pharmaceutical forms of the prior art, including but not limited to tablets, capsules, granules. Oral dosage forms such as agents and pills, and non-oral dosage forms such as injections and lyophilizates.
  • the traditional Chinese medicine composition may be a compatibility medicine for cohosh and other traditional Chinese medicine ingredients, such as angelica, Bupleurum, dried tangerine peel and the like.
  • the traditional Chinese medicine composition can be administered alone or in combination with an existing drug for autoimmune diseases, and can be prepared into any common dosage form in the existing pharmacy field, including but not limited to decoction, wine, Tea, lotion, pill, powder, ointment, elixir, tablet, lozenge, and the like.
  • a third object of the present invention is to provide a pharmaceutical composition.
  • the pharmaceutical composition provided by the present invention may be a pharmaceutical composition for autoimmune diseases comprising isoferulic acid, an extract containing isoferulic acid or Chinese cohosh as an active ingredient.
  • the autoimmune disease is autoimmune hepatitis or rheumatoid arthritis.
  • the pharmaceutical composition provided by the present invention may further be a pharmaceutical composition for inhibiting inflammatory cytokines, comprising isoferulic acid, an extract containing isoferulic acid or cohosh as an active ingredient;
  • the inflammatory Cytokines include TNF- ⁇ , IFN- ⁇ , IL-6, IL-9, IL-12, IL-17A, IL-18, IP-10, MCP-1, MCP-3, MIP-1 ⁇ , MIP-1 ⁇ One or more of MIP-2, Eotaxin, and G-CSF.
  • the isofluic acid-containing traditional Chinese medicine extract may be a cohosh decoction, which is obtained by boiling the roots and roots of the cohosh.
  • the isofluic acid-containing traditional Chinese medicine extract may also be a cohos total phenolic acid extract, which mainly contains isoferulic acid, ferulic acid, caffeic acid and the like.
  • the above pharmaceutical composition may further comprise a pharmaceutically acceptable carrier other than the active ingredient, including but not limited to excipients, binders, lubricants, fillers, disintegrants, emulsifiers, stabilizers, coloring agents. Agents, flavoring agents, preservatives, etc.
  • the pharmaceutical composition may be administered alone or in combination with existing drugs for autoimmune diseases, which may be prepared into any of the common pharmaceutical forms of the prior art, including but not limited to tablets, capsules, granules. Oral dosage forms such as agents and pills, and non-oral dosage forms such as injections and lyophilizates.
  • the invention adopts an animal model experiment of Concanavalin A (ConA) to find out that acute ferellant liver damage caused by isoferulic acid and traditional Chinese medicine extracts and pharmaceutical compositions containing isoferulic acid against concanavalin A It has a protective effect and can significantly improve the survival rate, liver function level, liver pathological damage and inflammatory cytokine secretion level of animal models of autoimmune hepatitis, indicating that it can be used to prepare drugs or health products for treating or alleviating autoimmune hepatitis. .
  • ConA Concanavalin A
  • the invention also finds through the animal model experiment of collagen induced arthritis (CIA) that isoferulic acid and traditional Chinese medicine containing isoferulic acid (such as cohosh) have protective effects on multiple joint damage caused by CIA. It can significantly reduce the incidence of CIA animal models, the average index of arthritis, improve bone structure destruction, reduce the secretion of anti-type 2 collagen antibodies and various inflammatory cytokines, indicating that it has an exact anti-articular inflammation effect, which can be used Preparation of a medicament or health care product for treating or ameliorating the symptoms of rheumatoid arthritis.
  • CIA collagen induced arthritis
  • the invention also finds that isoferulic acid and traditional Chinese medicine extracts and pharmaceutical compositions containing isoferulic acid can simultaneously inhibit various inflammatory cytokines, including TNF- ⁇ , IFN- ⁇ , IL-. 6. IL-9, IL-12, IL-17A, IL-18, IP-10, MCP-1, MCP-3, MIP-1 ⁇ , MIP-1 ⁇ , MIP-2, Eotaxin, G-CSF, etc. This can also be used to treat or ameliorate diseases such as inflammation caused by various inflammatory cytokines.
  • various inflammatory cytokines including TNF- ⁇ , IFN- ⁇ , IL-. 6.
  • This can also be used to treat or ameliorate diseases such as inflammation caused by various inflammatory cytokines.
  • the present invention provides novel uses of isoferulic acid, a traditional Chinese medicine extract containing isoferulic acid, a pharmaceutical composition, and a traditional Chinese medicine composition, and has been developed for use in autoimmune diseases due to its broad immunosuppressive action.
  • the application of drugs or functional health products can not only be produced by chemical synthesis or extraction, but also can be directly used as cohosh extract or Chinese medicine containing cohosh. Wide range, low cost, and broad market application value.
  • Fig. 1 is a graph showing the survival rate of each group of mice in Experimental Example 1.
  • Fig. 2 is a photomicrograph (HE staining) of liver tissue of each group of mouse models of Experimental Example 2.
  • Fig. 3 is a photograph of the anterior and posterior paw joints of each group of mice of Experimental Example 3.
  • Hemp was purchased from Tongrentang Qianmen Head Office. Citrus 12g was soaked in distilled water for 1 ⁇ 2h, simmered for 10 minutes, and simmered for 1h, concentrated to 75mL, converted to the concentration of Shengma decoction of about 0.16g/mL, divided into 5mL centrifuge tube, stored at -20 °C spare.
  • Cimicifuga, Angelica, Bupleurum and Chenpi were purchased from Tongrentang Qianmen Head Office. Cimicifuga 12g is compatible with Angelica 6g, Bupleurum 12g, and dried tangerine peel 12g. The boiling method was the same as above, concentrated to 75 mL, the concentration was about 0.56 g/mL, and the mixture was placed in a 5 mL centrifuge tube, and stored at -20 ° C for use in the cohosh compatibility group.
  • Phenolic acid extraction Take 75 mL of sesame water decoction and add 95% ethanol to 1000 mL, precipitate overnight, and separate the supernatant. The supernatant was distilled under reduced pressure to sufficiently remove ethanol and excess water, and concentrated to 75 mL to obtain a ethanolic extract.
  • Test materials ConA was purchased from Sigma; isoferulic acid was purchased from Tianjin Side Technology Co., Ltd.
  • mice purchased from Beijing Huakang Biotechnology Co., Ltd.
  • Group 50 C57BL/6 mice, male, 18-20 g, 1 day after adaptive feeding, randomly divided into 5 groups, 10 in each group: ConA model group, Cimicifuga group, Cimicifuga group, Cimicus Acid group and isoferulic acid group.
  • Model preparation and drug treatment Weigh 150mg of ConA powder, add 40mL of sterile PBS to dissolve, leave it at room temperature for 1-2 hours, gently stir and mix in the middle, low temperature ultrasound to promote dissolution (each time not more than 5min), pay attention to avoid More foam. After it was fully dissolved, the volume was adjusted to 50 mL, and the concentration was adjusted to 3 mg/mL, and the mixture was filtered under pressure with a 0.45 ⁇ m filter. Mice were injected intravenously at a dose of 25 mg/kg, and each mouse was injected with 100 ⁇ L.
  • the ConA model group was treated with 250 ⁇ L of distilled water; the cohosh preparation group 1 was added with 250 ⁇ L of the cohosh decoction, and the bioavailability was 7 g/kg; the asparagus group was given 250 ⁇ L of the cohosh decoction of the preparation example 1 for the crude drug.
  • the dosage is 2g/kg; 250 ⁇ L of the total phenolic acid extract of the cohosh preparation group 2, the biopharmaceutical amount is 300mg/kg; the isoferulic acid group is administered 250 ⁇ L of the aqueous ferulic acid solution, the dosage is Animal mortality was observed at 5 mg/kg for 96 h.
  • RESULTS As shown in Figure 1, the mice began to die after 4-6 hours of ConA injection, and the deaths were more concentrated within 24 hours. After that, the mortality decreased and 48-96h became stable.
  • the survival rate of the ConA model group was 20%, and the survival rate of the mice after drug treatment was increased.
  • the survival rate of the cohosh group was 90% (compared with the ConA model group, **, p ⁇ 0.01).
  • the survival rate was 80% (compared with the ConA model group, **, p ⁇ 0.01), and the survival rate of the ricinoleic acid group was 60% (compared with the ConA model group, *, p ⁇ 0.05).
  • the survival rate of the Wei acid group was 60% (*, p ⁇ 0.05 compared with the ConA model group).
  • the survival rate was statistically analyzed by Kaplan-Meier, and there was a significant difference between the drug treatment groups and the model group.
  • the results showed that cohosh compensatory drugs, cohosh extract, cohos phenolic acid and isoferulic acid all improved the survival rate of ConA hepatitis mice.
  • test materials and animals were in accordance with Experimental Example 1.
  • mice C57BL/6 mice, males, 18-20 g, 1 day after adaptive feeding, were randomly divided into 6 groups: normal control group, ConA model group, Cimicifuga group, Cimicifuga group, total phenolic acid group and different arsenic groups. Wei acid group.
  • mice were injected intravenously at a dose of 15 mg/kg ConA.
  • the doses of the respective drug groups were the same as those in Experimental Example 1.
  • Results (1) Effect of drugs on serum ALT and AST in ConA hepatitis mouse model
  • hepatocytes of the mice have extensive cell degeneration, hepatic lobular structure disorder, central hepatocyte vein dilatation, central vein and hepatic sinus congestion, and even large hepatocyte necrosis.
  • * represents a statistical difference between the ConA group and the normal control group, ***, p ⁇ 0.001.
  • Test materials bovine type II collagen acetate solution, complete Freund's adjuvant purchased from Chondrex company; isoferulic acid was purchased from Tianjin side technology Co., Ltd.
  • mice purchased from Beijing Huakang Biotechnology Co., Ltd.
  • Model preparation and administration Prepare 2mg/ml bovine type II collagen acetic acid solution, take 2ml of bovine type II collagen acetic acid solution overnight, mix well with 2ml complete Freund's adjuvant and emulsify (ice operation), The final concentration of bovine type II collagen emulsion is 1 mg/ml. Each mouse was intradermally injected with 0.1 ml. 21 days after the initial immunization, the second immunization was carried out for 21 days.
  • the CIA model group was given 250 ⁇ L of distilled water, and the Cimicifuga group was given 250 ⁇ L of the cohosh decoction in the preparation example 1, and the dose was 2 g/ Kg; isofuric acid group 250 ⁇ L of aqueous solution of isoferulic acid, the dose is 5mg/kg, once every 3 days, and kept for 21 days.
  • CIA mice developed redness and mild swelling at 3 days after the second immunization.
  • the mean arthritis index was 0.57.
  • the arthritis index gradually increased.
  • the average arthritis index of the isoferulic acid group and the asparagus group were 4.43 and 5.71, respectively, which significantly reduced the average arthritis index, which was significantly different from the CIA model group.
  • Figure 3 shows the picture of swelling of the paw joints in the normal group, CIA model mice and the medication group at 15 days after the second immunization.
  • the picture shows that the swelling of the metatarsophalangeal joint of CIA arthritis mice is obvious, and the joint swelling of mice in the isoferulic acid and Cimicifuga groups is significantly reduced.
  • the results showed that isoferulic acid and cohosh could improve joint redness and dysfunction in mice with CIA arthritis.
  • Model preparation and drug delivery treatment same as Experimental Example 3. On the 21st day of mouse secondary immunization, weighed.
  • mice As shown in Table 5, there was no difference in the mean body weight of the mice between the groups. The results showed that isoferulic acid and cohosh did not affect the body weight of CIA arthritis mice.
  • Model preparation and drug delivery treatment same as Experimental Example 3.
  • the anesthesia was sacrificed, and the brain, heart, liver, spleen, lung, kidney, and testes were taken. Place in a 6-well plate. Each mouse was placed in a hole and quickly weighed to evaluate the organ index.
  • results As shown in Table 6, the organs taken were represented by the spleen of the immune organs.
  • the results showed that isoferulic acid and cohosh did not affect the average spleen index of CIA mice.
  • Anti II collagen antibody ELISA kit was purchased from Chondrex Corporation, Cytokine & Chemokine 36-Plex Mouse ProcartaPlex TM Panel 1A cytokines and chemokines ThermoFisher kit was purchased from the company.
  • Model preparation and drug administration treatment The doses of the respective drug groups were the same as those in Experimental Example 3. 21 days after the second immunization, the mice were anesthetized.
  • Micro-CT technique was used to examine the effects of each drug group on the joint structure of CIA mice. Three mice in each group were treated with a broken neck, and the right hind limb was taken immediately and fixed in 4% neutral formalin. After 2-3 days of fixation, a micro-CT scan was performed using a live animal imaging system. Extraction site: Data of the area above the sacral scale of the right hind limb of the mouse was extracted. Take 3 sets of data each. Size selection: above the sacral scale plate (0.25 * 0.25 * 0.25); extraction bone measurement indications: bone density, bone volume, bone volume fraction and trabecular bone number.
  • Results (1) Micro-CT technique was used to detect the effects of drugs in each group on the joint structure of CIA mice.
  • the CIA model mouse has an ankle joint structure that is fuzzy, osteoporosis or even severely damaged.
  • the joint structure of the drug group has different degrees of damage, the degree is significantly lighter than the model group, especially the bone destruction is significantly lighter than the model.
  • the bone erosion of the cohosh group was the least, and the isoferulic acid group was the second.
  • the bone mineral density, bone volume, bone volume fraction, and trabecular bone number of the CIA group were lower than those of the normal group.
  • the isoferulic acid and the cohosh group were compared with the CIA model group. The indications have risen markedly. The results show that isoferulic acid and cohosh can effectively inhibit and alleviate joint damage.
  • Results (2) Effect of drugs on serum anti-type II collagen antibody content in CIA mice.
  • the secretion of anti-type II collagen antibody was significantly increased in the CIA group compared with the normal group, with statistical difference (***, p ⁇ 0.0001).
  • the isoferulic acid group and the Cimicifuga group can significantly reduce the secretion level of anti-type II collagen antibody in serum (###, p ⁇ 0.0001).
  • Results (3) The effect of drugs on the secretion of cytokines in serum and joint tissues of CIA mice was examined.
  • the secretion levels of MCP-3, MIP-1 ⁇ , MIP-1 ⁇ , Eotaxin and colony-stimulating factor G-CSF were significantly decreased, and there was statistical difference.
  • the secretion of IL-18, IL-9 and MIP-2 in joint tissues was observed. The level was also significantly reduced, with statistical differences.
  • the results showed that the isoferulic acid group can significantly reduce the secretion levels of various inflammatory cytokines in serum and joint tissues.
  • CD48PE and CD11b FITC antibodies were purchased from BD Pharmingen; RBC Lysis Buffer was purchased from Biolegend, USA; other required reagents were the same as in Experimental Example 3.
  • mice purchased from Beijing Huakang Biotechnology Co., Ltd.
  • Model preparation and drug administration treatment The doses of the respective drug groups were the same as those in Experimental Example 3. 21 days after the second immunization, the mice were anesthetized.
  • Preparation of bone marrow single cell suspension After anesthesia of C57BL/6 mice, the mice were isolated from the double femur and tibia. The bone marrow cells were flushed into the flow tube with a 2 mL syringe, and repeatedly bled as a single cell suspension of the bone marrow with a dropper. The 400 mesh filter was used to filter the bone marrow to the flow tube. After centrifugation, the supernatant was discarded. After adding 2 mL of RBC Lysis Buffer for 15 min at room temperature, the supernatant was centrifuged, the bone marrow cells were harvested, resuspended in 1 mL PBS, and repeatedly sucked and mixed to absorb 100 ⁇ L. Surface dyeing is carried out in a flow tube.
  • spleen single cell suspension After anesthesia of C57BL/6 mice, the spleens of the mice were isolated, the spleen was placed in a small dish to which 4 ml of PBEB had been added, the spleen was ground with a glass matte surface, and the spleen cells were filtered to a flow through a 400 mesh filter. In the tube, after centrifugation, the supernatant was discarded, and after adding 2 mL of RBC Lysis Buffer for 15 min at room temperature, the supernatant was centrifuged, and the spleen cells were harvested. Subsequently, it was resuspended by adding 1 mL of PBS, and repeatedly mixed by suction and suction, and 100 ⁇ L was taken up into a flow tube to perform surface staining.
  • Test materials and animals CD3-PerCP, NK1.1-APC antibody, Ki67-PE antibody, intracellular staining kit Intracellular Fixation & Permeabilization Buffer Set were purchased from eBioscience; CD48PE, CD11b FITC and B220-FITC antibodies were purchased from BD Pharmingen The other required reagents were the same as in Experimental Example 7.
  • mice purchased from Beijing Huakang Biotechnology Co., Ltd.
  • Model preparation and drug administration treatment The doses of the respective drug groups were the same as those in Experimental Example 3. 21 days after the second immunization, the mice were anesthetized.
  • the proliferation ability of the spleen Ki-67 expression-reactive cells was examined by flow cytometry.
  • Two tube spleen single cell suspensions were prepared for the detection of proliferation of CD48 - CD11b + positive granulocytes and CD3 - NK1.1 + B220 - NK cells, respectively, and the spleen single cell suspension was prepared as in Experimental Example 7.
  • the surface dyeing procedure was the same as in Experimental Example 7.
  • Intracellular staining After staining on the surface, intracellular staining: 1) Add 100 ⁇ L of Fixation Buffer to fix the membrane for 40 min at room temperature; 2) Wash 2 times with 1 mL Permeabilization Buffer, discard the supernatant; 3) Add 1 ⁇ L of Ki-67-PE antibody to avoid light for 40 min at room temperature; 4) Step 2); 5) Measure the percentage of intracellular expression of Ki-67 in CD48 - CD11b + positive granulocytes by using flow cytometry BD Calibar and CD3 - NK1. 1 + B220 - Percentage of Ki-67 expressed intracellularly in NK cells.
  • cohosh companion can significantly improve the survival rate of hepatitis mouse model, improve liver function, reduce liver pathological damage, inhibit inflammation Factor release with significant liver protection.
  • Both isoferulic acid and cohosh can significantly reduce the incidence of CIA arthritis mice, reduce the average index of CIA arthritis, reduce joint swelling, reduce bone erosion bone destruction, reduce the content of anti-type II collagen antibodies, and widely inhibit inflammatory
  • the release of factors, reducing the ratio of granulocytes, NK cells and proliferation, has a wide range of immunosuppressive effects.
  • isoferulic acid, cohosh extract, cohosh, etc. also have significant inflammatory cytokine inhibition.

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Abstract

异阿魏酸、含异阿魏酸的中药提取物及升麻在制备用于自身免疫性疾病的药物或功能性保健品中的用途。以及用于自身免疫性疾病或用于抑制炎性细胞因子的包含异阿魏酸或含异阿魏酸的中药提取物或升麻的药物组合物。

Description

异阿魏酸、含异阿魏酸中药提取物及升麻的用途 技术领域
本发明涉及医药领域,具体涉及异阿魏酸、含异阿魏酸中药提取物、及升麻在制备用于自身免疫性疾病的药物或功能性保健品中的用途。
背景技术
天然免疫细胞和T淋巴细胞活化是自身免疫性疾病的关键发病机制,典型疾病的代表是自身免疫性肝炎和类风湿关节炎。通常这些疾病由多种致病因素刺激机体,引发机体的免疫紊乱而发病。从调节机体免疫的角度开发新药或保健品将是治疗此类型疾病的突破口。因此,从祖国传统医药资源中开发免疫调节药物或功能性保健品具有广阔前景。
异阿魏酸是升麻等中药材提取物中的主要单体活性成分,化学名称为3-羟基-4-甲氧基肉桂酸,也称橘皮酸或咖啡酸-4-甲醚,化学式为C 10H 10O 4,具有抗氧化、降血糖的作用。
发明内容
本发明的目的之一是提供异阿魏酸、含异阿魏酸中药提取物、及升麻的用途。
本发明提供了异阿魏酸、含异阿魏酸中药提取物、及升麻在制备用于自身免疫性疾病的药物或功能性保健品中的用途。
上述用途中,所述自身免疫性疾病为自身免疫性肝炎或类风湿关节炎。
本发明还提供了异阿魏酸、含异阿魏酸中药提取物、及升麻在制备用于抑制炎性细胞因子的药物或功能性保健品中的用途,所述炎性细胞因子包括TNF-α、IFN-γ、IL-6、IL-9、IL-12、IL-17A、IL-18、IP-10、MCP-1、MCP-3、MIP-1α、MIP-1β、MIP-2、Eotaxin、G-CSF中的一种或多种。
上述用途中,所述炎性细胞因子可涉及多种细菌、病毒感染引起的疾病,如肝炎、肺炎、脓毒症、流感、麻疹、单纯疱疹等;还可涉及多种自身免疫性疾病,如类风湿性关节炎、系统性血管炎、硬皮病、多发性脑脊髓炎等,异阿魏酸、含异阿魏酸中药提取物、及升麻皆可作为预防、缓解、治疗上述疾病的药物或功能性保健品。
其中,所述含异阿魏酸中药提取物可以为升麻水煎液,其为取升麻的根茎及须根煎煮制得。
其中,所述含异阿魏酸中药提取物还可以为升麻总酚酸提取物,其中主要含有异阿魏酸、阿魏酸、咖啡酸等。
上述用途中,所述药物除异阿魏酸、含异阿魏酸中药提取物、或升麻作为活性成分外,还可包含药学上可接受的载体。药学上可接受的载体,包括但不限于赋形剂、粘合剂、润滑剂、填充剂、崩解剂、乳化剂、稳定剂、着色剂、矫味剂、防腐剂等等。所述药物可以制备成现有药学领域的任何常见剂型,包括但不限于片剂、胶囊剂、颗粒剂、丸剂等口服剂型以及注射剂、冻干剂等非口服剂型。
上述用途中,所述功能性保健品除异阿魏酸、含异阿魏酸中药提取物、或升麻作为活性成分外,还可以添加常见配料,包括但不限于营养素、维他命、矿物质、香料、着色剂、增粘剂、pH调节剂、稳定剂、防腐剂等。所述功能性保健品可以单独食用,也可以与现有的药物或保健品配合使用。
本发明的目的之二是提供包含异阿魏酸或含异阿魏酸中药提取物的药物组合物、或者包含升麻的中药组合物在制备用于自身免疫性疾病的药物中的用途。
上述用途中,所述自身免疫性疾病为自身免疫性肝炎或类风湿关节炎。
其中,所述含异阿魏酸中药提取物可以为升麻水煎液,其为取升麻的根茎及须根煎煮制得。
其中,所述含异阿魏酸中药提取物还可以为升麻总酚酸提取物,其中主要含有异阿魏酸、阿魏酸、咖啡酸等。
上述用途中,所述药物组合物还可以包含除活性成分外的药学上可接受的载体,包括但不限于赋形剂、粘合剂、润滑剂、填充剂、崩解剂、乳化剂、稳定剂、着色剂、矫味剂、防腐剂等等。所述药物组合物可以单独施用,也可以与现有的用于自身免疫性疾病的药物配合施用,其可以制备成现有药学领域的任何常见剂型,包括但不限于片剂、胶囊剂、颗粒剂、丸剂等口服剂型以及注射剂、冻干剂等非口服剂型。
上述用途中,所述中药组合物可以为升麻与其他中药成分形成的配伍药,如当归、柴胡、陈皮等。所述中药组合物可以单独施用,也可以与现有的用于自身免疫性疾病的药物配合施用,其可以制备成现有中药学领域的任何常见剂型,包括但不限于汤剂、酒剂、茶剂、露剂、丸剂、散剂、膏剂、丹剂、片剂、锭剂等等。
本发明的目的之三是提供一种药物组合物。
本发明提供的药物组合物可以为一种用于自身免疫性疾病的药物组合物,其包含异阿魏酸、含异阿魏酸中药提取物或升麻作为活性成分。
上述药物组合物中,所述自身免疫性疾病为自身免疫性肝炎或类风湿关节炎。
本发明提供的药物组合物还可以为一种用于抑制炎性细胞因子的药物组合物,其包含 异阿魏酸、含异阿魏酸中药提取物或升麻作为活性成分;所述炎性细胞因子包括TNF-α、IFN-γ、IL-6、IL-9、IL-12、IL-17A、IL-18、IP-10、MCP-1、MCP-3、MIP-1α、MIP-1β、MIP-2、Eotaxin、G-CSF中的一种或多种。
其中,所述含异阿魏酸中药提取物可以为升麻水煎液,其为取升麻的根茎及须根煎煮制得。
其中,所述含异阿魏酸中药提取物还可以为升麻总酚酸提取物,其中主要含有异阿魏酸、阿魏酸、咖啡酸等。
上述药物组合物中,还可以包含除活性成分外的药学上可接受的载体,包括但不限于赋形剂、粘合剂、润滑剂、填充剂、崩解剂、乳化剂、稳定剂、着色剂、矫味剂、防腐剂等等。所述药物组合物可以单独施用,也可以与现有的用于自身免疫性疾病的药物配合施用,其可以制备成现有药学领域的任何常见剂型,包括但不限于片剂、胶囊剂、颗粒剂、丸剂等口服剂型以及注射剂、冻干剂等非口服剂型。
本发明通过刀豆蛋白A(Concanavalin A,ConA)的动物模型实验发现:异阿魏酸及包含异阿魏酸的中药提取物、药物组合物等对刀豆蛋白A造成的急性免疫性肝损伤具有保护作用,能够明显改善自身免疫性肝炎动物模型的生存率、肝功能水平、肝脏病理损伤和炎性细胞因子分泌水平,说明其可以用来制备治疗或缓解自身免疫性肝炎的药物或保健品。
本发明还通过胶原诱导关节炎(Collagen induced arthritis,CIA)的动物模型实验发现:异阿魏酸及包含异阿魏酸的中药(如升麻)对CIA造成的多发性关节损伤具有保护作用,能够明显降低CIA动物模型的发病率、关节炎平均指数、改善骨结构破坏、降低抗二型胶原抗体和多种炎性细胞因子的分泌,说明其具有确切的抗关节炎症的作用,可以用来制备治疗或缓解类风湿性关节炎症状的药物或保健品。
本发明还发现:异阿魏酸及包含异阿魏酸的中药提取物、药物组合物等能够同时对多种炎性细胞因子具有明显的抑制作用,包括TNF-α、IFN-γ、IL-6、IL-9、IL-12、IL-17A、IL-18、IP-10、MCP-1、MCP-3、MIP-1α、MIP-1β、MIP-2、Eotaxin、G-CSF等,由此还可用于治疗或改善多种炎性细胞因子引起的炎症等疾病。
总之,本发明提供了异阿魏酸、包含异阿魏酸的中药提取物、药物组合物、中药组合物等新的用途,由于其具有广泛的免疫抑制作用,开拓了用于自身免疫性疾病的药物或功能性保健品的应用领域,不仅可通过化学合成或提取的方式生产得到异阿魏酸使用,也可直接采用升麻提取物或含有升麻的中药配伍药,制备简便,原料来源广泛,成本低廉,具 有广阔的市场应用价值。
附图说明
图1为实验例1的各组小鼠生存率图表。
图2为实验例2的各组小鼠模型肝脏组织显微图片(HE染色)。
图3为实验例3的各组小鼠的前后足爪关节图片。
图4为实验例6的各组小鼠的右后肢膝关节、踝关节和趾关节Mirco-CT图像。
具体实施方式
下面通过制备例和实验例对本发明进行详细说明,以使本发明的特征和优点更清楚。但应该指出,制备例和实验例用于理解本发明的构思,本发明的范围并不仅仅局限于本文中所列出的制备例和实验例。
下述制备例和实验例中所使用的实验方法如无特殊说明,均为常规方法。所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
制备例1药剂的制备
升麻购自同仁堂前门总店。升麻12g用蒸馏水浸泡1~2h,武火煎煮10min后,改文火1h,浓缩至75mL,换算升麻水煎液的浓度约为0.16g/mL,分装入5mL离心管,-20℃保存备用。
升麻、当归、柴胡、陈皮均购自同仁堂前门总店。升麻12g配伍当归6g、柴胡12g、陈皮12g。煎煮方法同上,浓缩至75mL,浓度约为0.56g/mL,分装入5mL离心管,-20℃保存备用,用于升麻配伍组。
制备例2升麻总酚酸的提取
参考文献(黄贵平,李存玉,李红阳,支兴蕾,李贺敏,刘兰平,彭国平.升麻中酚酸类成分提取工艺优选.辽宁中医药大学学报.2014,4:50-52)方法进行升麻总酚酸提取。取75mL升麻水煎液加95%乙醇至1000mL,沉淀过夜,分离上清液。将上清液减压蒸馏充分去除乙醇及过量水,浓缩至75mL,得到升麻醇提物。在室温条件下,使用有机溶剂乙酸乙酯萃取5次,搅拌,提取,离心得上清液,再经5%碳酸钠萃取,用稀盐酸调节溶液pH值至3,等量的乙酸乙酯萃取5次,减压回收后,得到升麻总酚酸提取物,水定容至75mL。采用异阿魏酸、阿魏酸和咖啡酸标准品(均购自天津一方科技有限公司)为对照, 经HPLC方法鉴定各成分含量如表1。
表1.三种主要酚酸类成分的含量
Figure PCTCN2018091056-appb-000001
实验例1 对ConA肝炎小鼠模型生存率的影响
试验材料:ConA购自Sigma公司;异阿魏酸购自天津一方科技有限公司。
动物:C57BL/6小鼠,购自北京华阜康生物科技股份有限公司。
分组:C57BL/6小鼠50只,雄性,18-20g,适应性饲养1天后,随机分为5组,每组10只:ConA模型组,升麻配伍组,升麻组,升麻总酚酸组和异阿魏酸组。
模型制备和给药处理:称取ConA粉末150mg,加40mL无菌PBS溶解,室温放置1~2小时,中间不断轻摇混匀,低温超声促进溶解(每次不超过5min),注意避免出现过多泡沫。待其充分溶解,定容至50mL,配制成3mg/mL浓度,用0.45μm滤膜加压过滤。按25mg/kg单次静脉注射小鼠,每只小鼠注射100μL。ConA模型组灌胃蒸馏水250μL;升麻配伍组灌胃制备例1的升麻配伍水煎液250μL,生药剂量为7g/kg;升麻组灌胃制备例1的升麻水煎液250μL,生药剂量为2g/kg;升麻总酚酸组灌胃制备例2的升麻总酚酸提取液250μL,生药剂量为300mg/kg;异阿魏酸组灌胃异阿魏酸水溶液250μL,剂量为5mg/kg,观察动物96h死亡率。
结果:如图1所示,ConA注射4-6h后小鼠开始死亡,24h内死亡较集中,之后死亡率下降,48-96h趋于稳定。ConA模型组的生存率是20%,药物处理后小鼠生存率升高,升麻配伍组的生存率为90%(与ConA模型组相比,**,p<0.01),升麻组的生存率为80%(与ConA模型组相比,**,p<0.01),升麻总酚酸组的生存率为60%(与ConA模型组相比,*,p<0.05),异阿魏酸组的生存率为60%(与ConA模型组相比,*,p<0.05)。生存率经Kaplan-Meier统计学分析,各药物处理组与模型组相比均有显著差异。该结果表明,升麻配伍药、升麻提取物、升麻总酚酸及异阿魏酸均能提高ConA肝炎小鼠的生存率。
实验例2 升麻及其提取物对肝脏炎症的治疗作用
试验材料和动物与实验例1一致。
分组:C57BL/6小鼠,雄性,18-20g,适应性饲养1天后,随机分为6组:正常对照 组,ConA模型组,升麻配伍组,升麻组,总酚酸组和异阿魏酸组。
模型制备和给药处理:除正常对照组外,按ConA 15mg/kg剂量单次静脉注射小鼠。各用药组给药剂量与实验例1一致。
(1)检测各用药组药物对ConA小鼠血清ALT、AST的影响。每组8只,分别在ConA注射后10h麻醉处死,取外周血并分离血清,检测ALT、AST;
(2)检测各用药组药物对ConA小鼠肝脏病理损伤的影响。每组3只,分别在ConA注射后10h麻醉处死,取肝脏左叶,切成约5mm 3大小,放10%福尔马林液中,经脱水、透明浸蜡、包埋、切片、摊片、染色后观察;
(3)检测各用药组药物对ConA小鼠血清细胞因子分泌的影响。每组6只,在ConA注射后3h取外周血并分离血清,检测细胞因子TNF-α,IL-12,IL-6和MCP-1的分泌;在ConA注射后10h分离血清,检测细胞因子IFN-γ的分泌。
结果(1):药物对ConA肝炎小鼠模型血清ALT和AST的影响
结果如表2所示,ConA肝损伤模型组与正常组相比ALT、AST具有显著差异(***,p<0.001);各用药组ALT、AST均明显下降,与ConA模型组相比差异有统计学意义( #,p<0.05; ##,p<0.01; ###,p<0.001)。说明升麻中药配伍药、升麻提取物、升麻总酚酸及异阿魏酸都能减轻ConA小鼠肝损伤。
表2.升麻配伍药、升麻提取物、升麻总酚酸以及异阿魏酸对ConA肝炎小鼠模型血清ALT和AST的影响(MEAN±SEM)
Figure PCTCN2018091056-appb-000002
注:*代表ConA组与正常对照组有统计学差异,***,p<0.001。 #代表用药组与ConA组有统计学差异, #,p<0.05; ##,p<0.01; ###,p<0.001。
结果(2):药物对ConA肝炎小鼠模型肝脏病理损伤的影响
结果如图2所示,ConA组小鼠肝细胞广泛的细胞变性,肝小叶结构紊乱,肝细胞中央静脉扩张明显,中央静脉及肝血窦充血明显,甚至出现肝细胞大片状坏死灶,大量免疫 细胞浸润;升麻配伍药、升麻提取物、升麻总酚酸以及异阿魏酸组小鼠较ConA组相比,肝细胞变性坏死减少,肝细胞片状坏死灶明显减轻,免疫细胞浸润减少。
结果(3):对ConA肝炎小鼠模型血清细胞因子的影响
结果如表3所示,ConA组与正常组相比,天然免疫细胞分泌的TNF-α、IL-12、MCP-1、IL-6和T淋巴细胞分泌的IFN-γ明显升高,具有统计学差异(***,p<0.001)。各用药组均能明显降低血清中细胞因子IL-12、TNF-α、MCP-1、IL-6和IFN-γ的分泌水平( #,p<0.05; ##,p<0.01; ###,p<0.001)。
表3.对ConA肝炎小鼠模型血清细胞因子分泌的影响(MEAN±SEM)
Figure PCTCN2018091056-appb-000003
注:*代表ConA组与正常对照组统计学有差异,***,p<0.001。#代表用药组与ConA组统计学有差异, #,p<0.05; ##,p<0.01; ###,p<0.001。
实验例3 异阿魏酸、升麻对CIA关节炎小鼠关节炎指数的影响
试验材料:牛Ⅱ型胶原乙酸溶液、完全弗氏佐剂购自Chondrex公司;异阿魏酸购自天津一方科技有限公司。
动物:C57BL/6小鼠,购自北京华阜康生物科技股份有限公司。
分组:C57BL/6小鼠24只,雄性,18-20g,适应性饲养1天后,随机分为4组:正常对照组和CIA模型组,n=5;升麻组和异阿魏酸组,n=7。
模型制备和给药处理:配制2mg/ml牛Ⅱ型胶原乙酸溶液,取2ml过夜后的牛Ⅱ型胶原乙酸溶液,与2ml的完全弗氏佐剂充分混合并乳化(冰上操作),制得的牛Ⅱ型胶原乳剂最终浓度为l mg/ml。每只小鼠皮内注射0.1ml。初次免疫21天后,进行二次免疫21天,从二次免疫0天开始,CIA模型组灌胃蒸馏水250μL,升麻组灌胃制备例1中的升麻水煎液250μL,生药剂量为2g/kg;异阿魏酸组灌胃异阿魏酸水溶液250μL,剂量为5mg/kg,每3天灌胃一次,连续饲养21天。
采用关节评分法对动物评分(0-4级),评定依据为关节红肿程度以及关节肿大和变 形情况。0分:无红肿;1分:关节红不肿;2分:关节轻度红肿;3分:关节中度红肿;4分:关节重度红肿伴功能障碍。每个肢体分别评分,各肢体分数总和即为该鼠的关节炎指数。最高分为16分。分数越高,关节症状越严重。
结果:如表4所示,在二次免疫后3天时,CIA小鼠出现红肿及轻度肿胀,关节炎指数均值为0.57,随着病程延长,关节炎指数逐渐升高,在15天,踝关节和趾关节高度肿胀,关节表面出现充血,后肢不能负重,关节炎平均指数最高并到达平台期,为8.43。异阿魏酸组、升麻组关节炎平均指数分别为4.43和5.71,能明显降低平均关节炎指数,与CIA模型组相比有显著差异。
表4.异阿魏酸、升麻对CIA关节炎小鼠关节炎指数的影响(MEAN±SEM)
Figure PCTCN2018091056-appb-000004
注:*代表CIA组与正常对照组有统计学差异,**,p<0.01;***,p<0.001。#代表用药组与CIA组有统计学差异,#,p<0.05;##,p<0.01。
图3显示二次免疫15天时正常组、CIA模型小鼠与用药组前后足爪关节肿胀图片。图片可见:CIA关节炎小鼠踝趾关节肿胀明显,异阿魏酸、升麻组小鼠关节肿胀明显减轻。结果表明,异阿魏酸、升麻均能改善CIA关节炎小鼠的关节红肿和功能障碍。
实验例4 异阿魏酸、升麻对CIA小鼠体重的影响
试验材料和动物与实验例3一致。
模型制备和给药处理:同实验例3。在小鼠二次免疫第21天,称重。
结果:如表5所示,各用药组之间,小鼠体重均值没有差异。结果说明,异阿魏酸、升麻不影响CIA关节炎小鼠体重。
表5.异阿魏酸、升麻对CIA关节炎小鼠体重的影响(MEAN±SEM)
Figure PCTCN2018091056-appb-000005
实验例5 异阿魏酸、升麻对CIA小鼠脏器指数的影响
试验材料和动物与实验例3一致。
模型制备和给药处理:同实验例3。在小鼠二次免疫第21天,麻醉处死,取脑、心、肝、脾、肺、肾、睾丸。放入6孔板中。每只老鼠各脏器放在一个孔内,快速称重,评价脏器指数。
结果:如表6所示,所取脏器中,以免疫器官脾脏为代表计算。CIA小鼠组与正常组相比,平均脏器指数增大,而异阿魏酸组、升麻组与CIA小鼠组相比,平均脏器指数没有变化。结果说明,异阿魏酸、升麻不影响CIA小鼠脾脏平均指数。
表6.异阿魏酸、升麻对CIA关节炎小鼠脏器指数的影响(MEAN±SEM)
Figure PCTCN2018091056-appb-000006
实验例6 异阿魏酸、升麻对CIA关节炎小鼠的治疗实验
试验材料和动物:与实验例3一致。抗Ⅱ型胶原抗体ELISA试剂盒购自Chondrex公司,Cytokine&Chemokine 36-Plex Mouse ProcartaPlex TM Panel 1A细胞因子和趋化因子检测试剂盒购自ThermoFisher公司。
模型制备和给药处理:各用药组给药剂量与实验例3一致。二次免疫21天后,麻醉小鼠。
(1)Micro-CT技术检测各用药组药物对CIA小鼠关节结构的影响。每组取3只小鼠断颈处理后,立即取右后肢,4%中性福尔马林固定。固定2-3天后,采用活体动物成像系统进行micro-CT扫描。提取部位:提取小鼠右后肢胫骨垢板之上区域的数据。各取3组数据。大小选择:胫骨垢板之上部位(0.25*0.25*0.25);提取骨计量指征:骨密度、骨体积、骨体积分数及骨小梁数量。
(2)检测各用药组药物对CIA小鼠血清抗Ⅱ型胶原抗体含量的影响。每组7只,取外周血并分离血清,Elisa检测抗Ⅱ型胶原抗体。
(3)检测各用药组药物对CIA小鼠血清和关节组织细胞因子分泌的影响。每组7只, 取外周血并分离血清,多因子检测血清中细胞因子:IFN-γ、IL-6、IL-9、IL-12、IL-17A、IP-10、MCP-1、MCP-3、MIP-1α、MIP-1β、Eotaxin、G-CSF;取右后肢膝盖、关节和爪等存于液氮速冻,匀浆后提蛋白,多因子检测组织中细胞因子:IL-9、IL-18和MIP-2的分泌。
结果(1):Micro-CT技术检测各用药组药物对CIA小鼠关节结构的影响。
如图4所示,CIA模型小鼠踝关节结构模糊,骨质疏松甚至严重破坏,用药组关节结构虽也有不同程度的破坏,但程度明显轻于模型组,特别是骨质破坏明显轻于模型组,升麻组骨侵蚀骨破坏程度最小,异阿魏酸组次之。
如表7所示,CIA组小鼠与正常组相比,骨密度、骨体积、骨体积分数及骨小梁数量均下降,异阿魏酸和升麻组与CIA模型组相比,骨计量指征明显上升。结果说明,异阿魏酸、升麻可以有效抑制和缓解关节破坏。
表7.异阿魏酸、升麻对CIA关节炎小鼠骨损伤的影响(MEAN±SEM)
Figure PCTCN2018091056-appb-000007
注:*代表CIA组与正常对照组有统计学差异,**,p<0.01;***,p<0.001。#代表用药组与CIA组有统计学差异,#,p<0.05;##,p<0.01。
结果(2):药物对CIA小鼠血清抗Ⅱ型胶原抗体含量的影响。
如表8所示,CIA组与正常组相比,抗Ⅱ型胶原抗体分泌明显升高,具有统计学差异(***,p<0.0001)。异阿魏酸组和升麻组均能明显降低血清中抗Ⅱ型胶原抗体的分泌水平(###,p<0.0001)。
表8.异阿魏酸、升麻对CIA关节炎小鼠血清抗Ⅱ型胶原抗体含量的影响(MEAN±SEM)
Figure PCTCN2018091056-appb-000008
注:*代表CIA组与正常对照组有统计学差异,***,p<0.001。#代表用药组与CIA组有统计学差异,###,p<0.001。
结果(3):检测药物对CIA小鼠血清和关节组织细胞因子分泌的影响。
如表9所示,异阿魏酸组与CIA组相比,血清中细胞因子IFN-γ、IL-12、IL-17A、IL-6、IL-9,趋化因子IP-10、MCP-1、MCP-3、MIP-1α、MIP-1β、Eotaxin以及集落刺激因子G-CSF分泌水平明显降低,具有统计学差异;同时,关节组织中IL-18、IL-9和MIP-2的分泌水平也明显降低,具有统计学差异。结果说明,异阿魏酸组均能明显降低血清和关节组织中多种炎症细胞因子的分泌水平。
表9.异阿魏酸对CIA关节炎小鼠血清和关节组织细胞因子的影响(MEAN±SEM)
Figure PCTCN2018091056-appb-000009
注:#代表用药组与CIA组有统计学差异,#,p<0.05;##,p<0.01;###,p<0.001。
实验例7 异阿魏酸、升麻对CIA关节炎小鼠粒细胞比例的影响
试验材料和动物:CD48PE和CD11b FITC抗体均购自BD Pharmingen公司;红细胞裂解液(RBC Lysis Buffer)购自美国Biolegend公司;其他所需试剂均同实验例3。
动物:C57BL/6小鼠,购自北京华阜康生物科技股份有限公司。
模型制备和给药处理:各用药组给药剂量与实验例3一致。二次免疫21天后,麻醉小鼠。
骨髓单细胞悬液制备:C57BL/6小鼠麻醉后,分离小鼠双股骨和胫骨,用2mL注射器吸取PBS将骨髓细胞冲入流式管,用滴管反复吹打为骨髓单细胞悬液。400目滤网过滤骨髓至流式管,离心后弃上清,加入2mL RBC Lysis Buffer室温裂解15min后,离心弃上清,收获骨髓细胞,加入1mL PBS重悬,反复吹吸混匀后吸取100μL至流式管中,进行表面染色。
脾脏单细胞悬液制备:C57BL/6小鼠麻醉后,分离小鼠脾脏,将脾脏置于已加入4ml PBEB的小皿中,用载玻片磨砂面研磨脾脏,400目滤网过滤脾细胞至流式管中,离心后弃上清,加入2mL RBC Lysis Buffer室温裂解15min后,离心弃上清,收获脾细胞。随后加入1mL PBS重悬,反复吹吸混匀后吸取100μL至流式管中,进行表面染色。
表面染色:加入抗体CD48-PE 0.5μL,CD11b-FITC 1μL避光,4°孵育15min后,加入2mL PBEB离心,弃上清。采用流式细胞仪BD Calibar检测CD48 -CD11b +阳性粒细胞占白细胞的百分比。
结果:如表10所示,骨髓和脾脏中,CIA关节炎小鼠粒细胞的比例均升高,异阿魏酸组均能降低骨髓和脾脏中粒细胞的比例;升麻组能降低脾脏中粒细胞的比例,具有统计学差异。结果说明:异阿魏酸、升麻降低CIA关节炎小鼠粒细胞的比例。
表10.异阿魏酸、升麻对CIA关节炎小鼠粒细胞比例的影响(MEAN±SEM)
Figure PCTCN2018091056-appb-000010
注:#代表用药组与CIA组有统计学差异,#,p<0.05;##,p<0.01。
实验例8 异阿魏酸、升麻对CIA关节炎小鼠粒细胞和NK细胞增殖能力的影响
试验材料和动物:CD3-PerCP、NK1.1-APC抗体、Ki67-PE抗体、胞内染色试剂盒Intracellular Fixation&Permeabilization Buffer Set购自eBioscience公司;CD48PE、CD11b FITC和B220-FITC抗体均购自BD Pharmingen公司;其他所需试剂均同实验例7。
动物:C57BL/6小鼠,购自北京华阜康生物科技股份有限公司。
模型制备和给药处理:各用药组给药剂量与实验例3一致。二次免疫21天后,麻醉小鼠。
通过流式细胞仪检测脾脏Ki-67的表达反应细胞的增殖能力。制备2管脾脏单细胞悬液,分别用于检测CD48 -CD11b +阳性粒细胞和CD3 NK1.1 +B220 NK细胞的增殖,脾脏单细胞悬液制备同实验例7。
表面染色步骤同实验例7。
胞内染色:在表面染色后,进行胞内染色:1)加入100μL固定破膜液Fixation Buffer,室温避光,固定破膜40min;2)加入1mL Permeabilization Buffer洗2遍,弃上清;3)加入Ki-67-PE抗体1μL室温避光40min;4)步骤同2);5)采用流式细胞仪BD Calibar检测CD48 -CD11b +阳性粒细胞胞内表达Ki-67的百分比和CD3 NK1.1 +B220 NK细胞胞内表达Ki-67的百分比。
结果:如表11所示,CIA关节炎小鼠与正常小鼠相比,骨髓、脾脏中粒细胞的增殖能力以及脾脏中NK细胞的增殖能力增强,异阿魏酸组的增殖能力明显减弱,具有显著差异;升麻组与CIA关节炎小鼠相比,骨髓中粒细胞和脾脏中NK细胞增殖能力明显减弱,具有显著差异。结果说明:异阿魏酸、升麻减弱CIA关节炎小鼠粒细胞和NK细胞的增殖能力。
表11.异阿魏酸、升麻对CIA关节炎小鼠粒细胞和NK细胞增殖能力的影响(MEAN±SEM)
Figure PCTCN2018091056-appb-000011
注:#代表用药组与CIA组有统计学差异,#,p<0.05;##,p<0.01。
综上所述,升麻配伍药、升麻提取物、升麻总酚酸及异阿魏酸均能明显提高肝炎小鼠 模型的生存率,改善肝功能水平,减轻肝脏病理损伤,抑制炎性因子释放,具有显著的肝脏保护作用。
异阿魏酸、升麻均能明显降低CIA关节炎小鼠的发病率,降低CIA关节炎平均指数,减轻关节肿胀,减轻骨侵蚀骨破坏,降低抗Ⅱ型胶原抗体的含量,广泛抑制炎性因子的释放,降低粒细胞、NK细胞的比例和增殖能力,具有广泛的免疫抑制作用。
此外,异阿魏酸、升麻提取物、升麻等还具有明显的炎性细胞因子抑制作用。
除非特别限定,本发明所用术语均为本领域技术人员通常理解的含义。
本发明所描述的实施方式仅出于示例性目的,并非用以限制本发明的保护范围,本领域技术人员可在本发明的范围内作出各种其他替换、改变和改进,因而,本发明不限于上述实施方式,而仅由权利要求限定。

Claims (10)

  1. 异阿魏酸、含异阿魏酸中药提取物、及升麻在制备用于自身免疫性疾病的药物或功能性保健品中的用途。
  2. 根据权利要求1所述的用途,其特征在于,所述自身免疫性疾病为自身免疫性肝炎或类风湿关节炎。
  3. 异阿魏酸、含异阿魏酸中药提取物、及升麻在制备用于抑制炎性细胞因子的药物或功能性保健品中的用途,所述炎性细胞因子包括TNF-α、IFN-γ、IL-6、IL-9、IL-12、IL-17A、IL-18、IP-10、MCP-1、MCP-3、MIP-1α、MIP-1β、MIP-2、Eotaxin、G-CSF中的一种或多种。
  4. 包含异阿魏酸或含异阿魏酸中药提取物的药物组合物、或者包含升麻的中药组合物在制备用于自身免疫性疾病的药物中的用途。
  5. 根据权利要求4所述的用途,其特征在于,所述自身免疫性疾病为自身免疫性肝炎或类风湿关节炎。
  6. 根据权利要求1-5任一项所述的用途,其特征在于,所述含异阿魏酸中药提取物为升麻水煎液或升麻总酚酸提取物。
  7. 一种用于自身免疫性疾病的药物组合物,其特征在于,所述药物组合物中包含异阿魏酸、含异阿魏酸中药提取物或升麻作为活性成分。
  8. 根据权利要求7所述的药物组合物,其特征在于,所述自身免疫性疾病为自身免疫性肝炎或类风湿关节炎。
  9. 一种用于抑制炎性细胞因子的药物组合物,其特征在于,所述药物组合物中包含异阿魏酸、含异阿魏酸中药提取物或升麻作为活性成分;所述炎性细胞因子包括TNF-α、IFN-γ、IL-6、IL-9、IL-12、IL-17A、IL-18、IP-10、MCP-1、MCP-3、MIP-1α、MIP-1β、MIP-2、Eotaxin、G-CSF中的一种或多种。
  10. 根据权利要求7-9任一项所述的药物组合物,其特征在于,所述含异阿魏酸中药提取物为升麻水煎液或升麻总酚酸提取物。
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US20200108032A1 (en) 2020-04-09
EP3639836B1 (en) 2021-09-15
EP3639836A4 (en) 2020-05-06
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