WO2018228431A1 - 升麻三萜皂苷类提取物、黄肉楠碱、脱氧升麻烃的用途 - Google Patents

升麻三萜皂苷类提取物、黄肉楠碱、脱氧升麻烃的用途 Download PDF

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WO2018228431A1
WO2018228431A1 PCT/CN2018/091057 CN2018091057W WO2018228431A1 WO 2018228431 A1 WO2018228431 A1 WO 2018228431A1 CN 2018091057 W CN2018091057 W CN 2018091057W WO 2018228431 A1 WO2018228431 A1 WO 2018228431A1
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group
mice
deoxyclay
pharmaceutical composition
deoxycannacin
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PCT/CN2018/091057
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English (en)
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 EP18817966.7A priority Critical patent/EP3639835B1/en
Priority to JP2019569945A priority patent/JP6961020B2/ja
Priority to US16/621,949 priority patent/US11052099B2/en
Publication of WO2018228431A1 publication Critical patent/WO2018228431A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
    • 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
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • 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
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4741—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having oxygen as a ring hetero atom, e.g. tubocuraran derivatives, noscapine, bicuculline
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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

Definitions

  • the invention relates to the field of medicine, in particular to a composition formed by Cimicifuga saponins extract, Cimicifuga, scutellaria, deoxyclay or a mixture of sulphate and deoxyclay, in preparation for autoimmune The use of a drug or functional health supplement for a disease.
  • autoimmune hepatitis Abnormal activation of natural immune cells and adaptive immune cells is a key pathogenesis of autoimmune diseases, and typical diseases are represented by autoimmune hepatitis and rheumatoid arthritis.
  • Autoimmune hepatitis is a chronic progressive hepatic inflammatory disease mediated by autoimmune response. It is characterized by elevated levels of serum transaminases and autoantibodies.
  • the histological features are interfacial properties based on lymphocyte and plasma cell infiltration. Hepatic impairment and immune disorders caused by hepatitis.
  • Rheumatoid arthritis is a systemic disease characterized by inflammatory synovitis, characterized by multiple joints of the hand and foot facet joints, symmetry, invasive joint inflammation, often accompanied by serum rheumatoid factor and inflammatory cytokine High, can lead to joint deformity and loss of function.
  • the development of new drugs or health care products from the perspective of regulating the body's immunity is an important breakthrough in the treatment of autoimmune diseases, and the development of immunomodulatory drugs or functional health products from the traditional medical resources of the motherland has broad prospects.
  • Cimicifuga foetidae is a genus of the genus Corydalis, and the triterpenoid saponin extract is obtained by extracting the roots and shoots of the cohosh. It is generally considered to have anti-tumor, endocrine, anti-osteoporosis and other physiological functions. Activity is a kind of natural product with great development prospects.
  • One of the objects of the present invention is to provide a trichoderma saponin extract, cohosh, scutellaria, deoxycannacin (alias: 27-deoxycannabinoid) or a mixture of scutellaria and deoxyclay New uses for the composition.
  • the present invention provides a composition of Cimicifuga saponins extract, Cimicifuga, scutellaria, deoxycannacin or a mixture of sulphate and deoxyclay, in the preparation of a medicament for autoimmune diseases or Use in functional health products.
  • the autoimmune disease is autoimmune hepatitis or rheumatoid arthritis.
  • the present invention also provides a composition formed by Cimicifuga saponins extract, Cimicifuga, scutellaria, deoxycannacin or scutellaria and deoxyclay, in the preparation of a composition for inhibiting inflammatory cytokines. Use in medicines or functional health supplements.
  • the inflammatory cytokines may include 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 may include 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 are examples of 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 may include TNF- ⁇ , IFN- ⁇ , IL-6, IL-9, IL-12, IL-17A, IL-18, IP-10,
  • 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., cohos saponins extract, cohosh, scutellaria, deoxycannacin, scutellaria and deoxygenation
  • the composition formed by the cocarin can be used as a medicine or a functional health supplement for preventing, alleviating, and treating the above diseases.
  • the mass ratio of the scutellaria and the deoxyclay to the anaerobic hydrocarbon may be from 1 to 5:5 to 1; preferably from 1 to 3:3. More preferably, it can be 1:1.
  • the medicine in addition to the composition of the saponin extract, the sesame, the saponin, the deoxyclay, the sulphate, and the deoxyclay, as an active ingredient, may also be used as an active ingredient.
  • a pharmaceutically acceptable carrier is included.
  • 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 comprises, in addition to the composition of the cohosh saponin extract, the cohosh, the saponin, the deoxyclay, the sulphate, and the deoxyclay, as an active ingredient. It is also possible to add common ingredients including, but not limited to, nutrients, vitamins, minerals, fragrances, colorants, tackifiers, pH adjusters, stabilizers, preservatives, and the like.
  • 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.
  • the pharmaceutical composition provided by the present invention may be a pharmaceutical composition for an autoimmune disease, which comprises the following components as an active ingredient: Cimicifuga saponins extract, Cimicifuga, and Huangnan A composition formed from a base, deoxyclay, or sulphate and deoxyclay.
  • 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, which comprises the following components as an active ingredient: Cimicifuga saponins extract, Cimicifuga, Yellow A composition of carnitine, deoxyclay, or xanthine and deoxyclay.
  • the inflammatory cytokine may include TNF- ⁇ , IFN- ⁇ , IL-6, IL-9, IL-12, IL-17A, IL-18, IP-10, MCP-1, One or more of MCP-3, MIP-1 ⁇ , MIP-1 ⁇ , MIP-2, Eotaxin, G-CSF.
  • the mass ratio of the sulphate to the deoxycrycolic hydrocarbon may be from 1 to 5:5 to 1; preferably from 1 to 3: 3 to 1; more preferably 1:1.
  • 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 pharmaceutical composition may also be a traditional Chinese medicine composition including cohosh, for example, it may be a combination of 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.
  • Concanavalin A cohos saponin extract, cohosh, scutellaria, deoxycannacin or a combination thereof, a pharmaceutical composition, etc.
  • ConA Concanavalin A
  • the acute immune liver injury caused by concanavalin A has a protective effect, which 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 for preparation of treatment. Or a drug or health supplement that relieves autoimmune hepatitis.
  • the invention also finds through an animal model experiment of collagen induced arthritis (CIA): scutellaria, deoxycannacin, cohos saponin extract, cohosh or a combination thereof, pharmaceutical composition It can protect the multiple joint damage caused by CIA, and can significantly reduce the incidence of CIA animal model, the average index of arthritis, improve the destruction of bone structure, reduce the secretion of anti-type 2 collagen antibody and various inflammatory cytokines. It has an exact anti-articular inflammation effect and can be used to prepare medicines or health care products for treating or alleviating the symptoms of rheumatoid arthritis.
  • CIA collagen induced arthritis
  • LPS-stimulated macrophages have obvious anti-inflammatory effects such as cohoductin saponin extract, cohosh, scutellaria, deoxycannacin or a combination thereof, and a pharmaceutical composition. It can significantly inhibit the activation of macrophages and the secretion of inflammatory cytokines such as IL-12 and TNF- ⁇ . It can also significantly inhibit the activation of NK, NKT and T lymphocytes and the inflammatory cytokine IFN- ⁇ by in vitro experiments. secretion. It can also be used as an immunosuppressant for treating or ameliorating diseases such as inflammation caused by various inflammatory cytokines.
  • BMDM LPS-stimulated macrophages
  • the present invention provides a novel use of the trichoderma saponin extract, the saponin, and the deoxyclay, which has developed a drug or functional for autoimmune diseases due to its extensive immunosuppressive action.
  • the application fields of health care products can not only produce these components by means of chemical synthesis or extraction, but also directly adopt the cohosh extract or the traditional Chinese medicine containing the cohosh.
  • the preparation is simple, the raw materials are widely sourced, the cost is low, and the invention has broad scope. 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.
  • Citrus 12g was soaked in distilled water for 1 ⁇ 2h, simmered for 10 minutes after simmering, and then simmered for 1h and concentrated to 75mL. Take 75 mL of sesame decoction, 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. It is extracted with ethyl acetate in an organic solvent at room temperature for 5 times, stirred, extracted, and centrifuged to obtain a supernatant, which is then extracted with 5% sodium carbonate, and recovered under reduced pressure to obtain a triterpenoid saponin extract. Up to 75mL.
  • Test materials ConA was purchased from Sigma; xanthine and deoxyclay were purchased from ChromaDex, USA.
  • mice purchased from Beijing Huakang Biotechnology Co., Ltd.
  • Grouping 40 C57BL/6 mice, male, 18-20 g, 1 day after adaptive feeding, randomly divided into 4 groups, 10 in each group: ConA model group, triterpenoid saponin group, huangnan base group, deoxygenation Hemp hydrocarbon 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. Take 4 mg of flavonoids and deoxyclay, respectively, and dissolve them in 10 ml of sterile double distilled water to prepare a concentration of 0.4 mg/mL.
  • the ConA model group was given 250 ⁇ L of distilled water, and the triterpenoid saponin group was given 250 ⁇ L of the trichosanthin extract of the preparation example 1 at a dose of 15 mg/kg, and the baconin group was administered with 250 ⁇ L of the solution of the saponin solution at a dose of 5mg/kg, 250 ⁇ L of deoxy-cyanide hydrocarbon solution in the deoxycannabinic group, the dose was 5mg/kg, and the mortality of the animals was observed at 96h.
  • the results are shown in Fig. 1.
  • the survival rate of the ConA model group was 10%, the survival rate of mice after drug treatment was increased, and the survival rate of the triterpenoid saponin group was 70% (compared with the ConA model group, ***, p ⁇ 0.001).
  • the survival rate of the alkali group was 60% (**, p ⁇ 0.01 compared with the ConA model group), and the survival rate of the deoxycannabin group was 40% (compared with the ConA model group, **, p ⁇ 0.01) .
  • 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 the trichoderma saponin extract, scutellaria and deoxycannacin increased the survival rate of ConA acute hepatitis mice.
  • test materials and animals were in accordance with Experimental Example 1.
  • mice C57BL/6 mice, males, 18-20 g, after 1 day of adaptive feeding, were randomly divided into 5 groups: normal control group, ConA model group, triterpenoid saponin group, huangnan base group and deoxycannamic hydrocarbon group. .
  • mice were injected intravenously at a dose of 15 mg/kg ConA.
  • the doses administered in each group were the same as in Experimental Example 1.
  • Results (1) Effect of drugs on serum ALT and AST in ConA hepatitis mouse model
  • Fig. 2 The results are shown in Fig. 2.
  • the hepatocytes of the mice have extensive cell degeneration, hepatic lobule structure disorder, central hepatocyte vein dilatation, central vein and hepatic sinus congestion, and even large hepatic 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 after overnight, mix well with 2mL complete Freund's adjuvant and emulsify (ice operation), The final concentration of bovine type II collagen emulsion was 1 mg/mL, and each mouse was intradermally injected with 0.1 mL. Take 4mg of scutellaria and deoxycannacin, respectively, and add 10mL of sterile double distilled water to dissolve, and prepare a mixed solution containing 0.4mg/mL of nanali and 0.4mg/mL of deoxyclay, for yellow Group administration therapy.
  • mice were subjected to secondary immunization 21 days after the initial immunization. From the 0th day of the second immunization, the CIA model group was given 250 ⁇ L of distilled water, 250 ⁇ L of the mixed solution of the yellow sulphate and deoxycannacin in the Huangtu group, the dose was 5mg/kg, and each group was intragastrically administered once a day. 21 days.
  • a joint score was performed every 3 days from 0 days after the second immunization. Animals were scored using a joint scoring method (grade 0-4) based on the degree of joint swelling and joint swelling and deformation. 0 points: no redness and swelling; 1 point: joint red is not swollen; 2 points: mild redness and swelling of the joints; 3 points: moderate redness and swelling of the joints; 4 points: severe redness and swelling of the joints with dysfunction. Each limb was scored separately, and the sum of the limbs was the arthritis index of the mouse. The highest score is 16 points. The higher the score, the more severe the joint symptoms.
  • Figure 3 shows a picture of the swelling of the paw joints before and after the CIA model mice and the medication group on day 15.
  • the picture shows that the swelling of the metatarsophalangeal joint of CIA arthritis mice is obvious, and the joint swelling of the mice in the yellow off group is significantly reduced.
  • the results showed that the combination of scutellaria and deoxycannacin significantly improved 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 The results are shown in Table 4. There was no difference in the mean body weight of the mice between the groups. The results showed that the combination of scutellaria and deoxycannacin 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, testis were taken, and the organs were quickly weighed to evaluate the organ index.
  • 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.
  • mice were anesthetized to take serum and limbs.
  • Micro-CT technique was used to examine the effects of each 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) The effect of Micro-CT technique on the joint structure of CIA mice.
  • the results are shown in Fig. 4.
  • the structure of the ankle joint of the CIA model mice is fuzzy, osteoporosis and even severe destruction.
  • the joint structure of the yellow detachment group is almost intact, especially the bone destruction is significantly lighter than the model group.
  • bone density, bone volume, bone volume fraction, and trabecular bone number were decreased in the CIA group compared with the normal group.
  • the bone dissociation indication was significantly increased in the Huangtu group compared with the CIA model group.
  • the results show that the combination of scutellaria and deoxycannacin can effectively alleviate joint damage.
  • Results (2) Effect on serum anti-type II collagen antibody content in CIA mice.
  • Results (3) Effect on cytokine secretion in serum and joint tissues of CIA mice.
  • the results are shown in Table 8.
  • the serum cytokines IFN- ⁇ , IL-12, IL-17A, IL-6, IL-9, IL-18, and chemokine MCP-1 were compared with the CIA group.
  • the colony stimulating factor G-CSF secretion level was significantly reduced, with statistical differences; at the same time, the secretion levels of IL-18 and MIP-2 in joint tissues were also significantly reduced, with statistical differences.
  • the results showed that the combination of scutellaria and deoxycannacin can significantly reduce the secretion levels of various cytokines, chemokines and colony stimulating factors in serum and joint tissues, and has a wide inhibitory effect on inflammation.
  • * represents a statistically significant difference between the CIA group and the normal group, *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001.
  • # Representative medication group and CIA group were statistically different, #, p ⁇ 0.05; ##, p ⁇ 0.01; ###, p ⁇ 0.001.
  • Test materials and animals CD3-PerCP, NK1.1-APC antibody, Ki67-PE antibody, intracellular staining kit Intracellular Fixation & Permeabilization Buffer Set purchased from eBioscience; B220-FITC antibody was purchased from BD Pharmingen; other required reagents were Same as Experimental Example 3.
  • mice purchased from Beijing Huakang Biotechnology Co., Ltd.
  • 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 by 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.
  • Intracellular staining 1) Add 100 ⁇ L of Fixation Buffer to fix the membrane at room temperature and protect the membrane for 40 min. 2) Add 1 mL of Permeabilization Buffer for 2 times, discard the supernatant; 3) Add Ki-67-PE antibody to 1 ⁇ L of room temperature. Light 40 min; 4) Step 2); 5) Detection of Ki-67 expression in CD3 - NK1.1 + B220 - NK cells by flow cytometry BD Calibar.
  • * represents a statistically significant difference between the CIA group and the normal group, *, p ⁇ 0.05; # represents a statistically significant difference between the medication group and the CIA group, ###, p ⁇ 0.001.
  • Test materials LPS was purchased from Sigma; xanthine and deoxyclay were purchased from ChromaDex, USA; FBS was purchased from PAA, USA; DMEM high glucose medium; macrophage colony stimulating factor (M-CSF) was purchased from PeproTech; ELISA kit was purchased from BD, USA.
  • mice purchased from Beijing Huakang Biotechnology Co., Ltd.
  • mice After anesthesia of C57BL/6 mice, the mice were aseptically isolated from the double femur and tibia, and the bone marrow cells were aspirated into a sterile flow tube with a 2 mL syringe. The dropper was repeatedly beaten as a single cell suspension of bone marrow. After centrifugation, bone marrow cells were harvested, and 10 mL of complete medium containing 10% FBS, 1% glutamine, 1% double antibody (anti-penicillin, streptomycin), and 10 ng/mL M-CSF were added to inoculate 100 mm cell culture.
  • BMDM mouse bone marrow-derived macrophages
  • BMDM cells were treated with different doses of flavonoids and deoxycannacin. After 1 h, 100 ng/mL LPS was used to stimulate the cells to simulate the activation of macrophages by bacterial infection. After 4 h, the cell supernatant was collected, and cytokine secretion was detected by ELISA.
  • LPS stimulated activation of BMDM cells, which showed secretion of a large amount of inflammatory cytokines IL-12 and TNF- ⁇ . It can significantly inhibit the secretion of IL-12 in the concentration range of 2.5 ng/mL to 312.5 ng/mL, and inhibit the secretion of TNF- ⁇ in the concentration range of 0.5 ng/mL to 312.5 ng/mL. Hydrocarbons can significantly inhibit IL-12 secretion in the concentration range of 250 ng/mL to 500 ng/mL, and inhibit TNF- ⁇ secretion in the concentration range of 50 ng/mL to 500 ng/mL. The results showed that the concentration of 0.5% ng/mL to 312.5 ng/mL of lanthanin and 250 ng/mL to 500 ng/mL of deoxycannamic hydrocarbon had a significant inhibitory effect on macrophage activation.
  • Test materials CD45-PE, CD3-FITC, NK1.1-APC antibody, Intracellular Fixation & Permeabilization Buffer Set, Cell Stimulation Cocktail were purchased from eBioscience.
  • mice purchased from Beijing Huakang Biotechnology Co., Ltd.
  • Preparation of bone marrow single cell suspension After anesthesia of C57BL/6 mice, the mice were aseptically isolated from the double femur and tibia, and the bone marrow cells were aspirated into a sterile flow tube with a 2 mL syringe, and repeatedly beaten with a dropper into bone marrow. Single cell suspension. After centrifugation, bone marrow cells were harvested and 1.5 mL of complete medium containing 10% FBS, 1% glutamine, 1% double antibody (anti-penicillin, streptomycin) was added.
  • NK cells were treated with medium concentration of 62.5 ng/mL nanali and 250 ng/mL deoxycannacin, respectively. After 1 h, Cell Stimulation Cocktail (with PMA, ionomycin) was administered. Cells and protein transport inhibitors were stimulated for 5 h, stained with CD45-PE, CD3-FITC, NK1.1-APC antibody, intracellular staining with IL-12-APC antibody, and flow detection CD45 + CD3 - NK1. The percentage of positive cells expressing IFN- ⁇ in 1 + NK cells.
  • mice purchased from Beijing Huakang Biotechnology Co., Ltd.
  • NKT cells were activated in the bone marrow, which showed a large amount of secreted inflammatory cytokine IFN- ⁇ ; compared with the PMA + ionomycin group, nanali and deoxygenated liter Hemp hydrocarbon treatment significantly reduced the expression of IFN- ⁇ and inhibited the activation of NKT cells (*, p ⁇ 0.05; **, p ⁇ 0.01).
  • CD3-PE antibody was purchased from BD, USA, CD4-FITC, CD8-PerCP antibody was purchased from eBioscience; the remaining required reagents were the same as in Experimental Example 9.
  • mice purchased from Beijing Huakang Biotechnology Co., Ltd.
  • spleen single cell suspension After anesthesia of C57BL/6 mice, the spleens of the mice were isolated, and the spleen was placed in a sterile dish containing 4 mL of PBEB, and the spleen was ground with a glass matte surface and filtered through a sterile 400 mesh filter. The spleen cells were transferred to a flow tube, centrifuged, and the supernatant was discarded. After 2 min of RBC Lysis Buffer was added for 15 min at room temperature, the supernatant was centrifuged to harvest spleen cells. Subsequently, 1.5 mL of complete medium containing 10% FBS, 1% glutamine, 1% double antibody (anti-penicillin, streptomycin) was added, and the mixture was repeatedly pipetted.
  • Dosing treatment and intracellular staining after T cell induction and activation cells were treated with medium concentration of 62.5 ng/mL of lanthanin and 250 ng/mL of deoxycannacin, and cells were treated with Cell Stimulation Cocktail (with PMA, ion) 1 h later. Cells and protein transport inhibitors stimulate cells for 5 h. The percentage of positive cells expressing IFN- ⁇ in the CD3 + CD4 + T cells was detected by flow cytometry, and the percentage of positive cells expressing IFN- ⁇ in the cells of CD3 + CD8 + T cells.
  • CD4 + T cells and CD8 + T abundantly expressed the inflammatory cytokine IFN- ⁇ under the stimulation of PMA + ionomycin; compared with the PMA + ionomycin group, nanali and deoxygenated liter
  • the application of hemp hydrocarbon alone did not reduce the expression of IFN- ⁇ , but the combination of the two had a synergistic effect and significantly reduced the expression of IFN- ⁇ (**, p ⁇ 0.01; ***, p ⁇ 0.001).
  • the trichoderma saponin extract, saponin, and deoxycannacin can significantly improve the survival rate of the mouse model of hepatitis, improve liver function, reduce liver pathological damage, and inhibit the release of inflammatory factors. Has a significant liver protection.
  • Combination of scutellaria and deoxycannacin can significantly reduce the average arthritis index of arthritis mouse model, improve bone structure, reduce bone erosion, inhibit inflammatory factor release, inhibit NK cell proliferation, and have significant immune regulation. Joint protection.
  • the extract of Cimicifuga saponins and its main components, scutellaria and deoxycannacin, also have obvious anti-inflammatory effects, which can inhibit the activation of macrophages, NK and NKT cells, and the synergistic effect of the two can also inhibit T lymphocyte activation has a wide range of immunosuppressive effects.

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Abstract

升麻三萜皂苷类提取物、升麻、黄肉楠碱、脱氧升麻烃或者黄肉楠碱与脱氧升麻烃形成的组合物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的药物组合物和用于抑制炎性细胞因子的药物组合物。

Description

升麻三萜皂苷类提取物、黄肉楠碱、脱氧升麻烃的用途 技术领域
本发明涉及医药领域，具体涉及升麻三萜皂苷类提取物、升麻、黄肉楠碱、脱氧升麻烃或者黄肉楠碱与脱氧升麻烃形成的组合物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
天然免疫细胞和适应性免疫细胞异常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。自身免疫性肝炎是由自身免疫反应介导的慢性进行性肝脏炎症性疾病，表现为不同程度的血清转氨酶升高、自身抗体阳性，组织学特征为以淋巴细胞、浆细胞浸润为主的界面性肝炎引起的肝脏功能受损和免疫紊乱。类风湿关节炎是以炎性滑膜炎为主的系统性疾病，特征是手、足小关节的多关节、对称性、侵袭性关节炎症，经常伴有血清类风湿因子和炎性细胞因子升高，可以导致关节畸形及功能丧失。从调节机体免疫的角度开发新药或保健品是治疗自身免疫性疾病的重要突破口，而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更是具有广阔的前景。
升麻(Cimicifuga foetidae)为毛茛科升麻属植物，三萜皂苷类提取物是由升麻根茎及地上部分提取制得，一般认为其具有抗肿瘤、调节内分泌、抗骨质疏松等多种生理活性，是一类很有开发前景的天然产物。
发明内容
本发明的目的之一是提供升麻三萜皂苷类提取物、升麻、黄肉楠碱、脱氧升麻烃(别名：27-脱氧升麻烃)或者黄肉楠碱与脱氧升麻烃形成的组合物的新的用途。
本发明提供了升麻三萜皂苷类提取物、升麻、黄肉楠碱、脱氧升麻烃或者黄肉楠碱与脱氧升麻烃形成的组合物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。
本发明还提供了升麻三萜皂苷类提取物、升麻、黄肉楠碱、脱氧升麻烃或者黄肉楠碱与脱氧升麻烃形成的组合物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。
上述用途中，所述炎性细胞因子可以包括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，升麻三萜皂苷类提取物、升麻、黄肉楠碱、脱氧升麻烃、黄肉楠碱与脱氧升麻烃形成的组合物皆可作为预防、缓解、治疗上述疾病的药物或功能性保健品。
上述用途中，黄肉楠碱与脱氧升麻烃形成的组合物中，黄肉楠碱与脱氧升麻烃的质量比可以为1～5﹕5～1；优选可以为1～3﹕3～1；更优选可以为1﹕1。
上述用途中，所述药物除升麻三萜皂苷类提取物、升麻、黄肉楠碱、脱氧升麻烃、黄肉楠碱与脱氧升麻烃形成的组合物作为活性成分外，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升麻三萜皂苷类提取物、升麻、黄肉楠碱、脱氧升麻烃、黄肉楠碱与脱氧升麻烃形成的组合物作为活性成分外，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，所述药物组合物中包含以下成分作为活性成分：升麻三萜皂苷类提取物、升麻、黄肉楠碱、脱氧升麻烃或者黄肉楠碱与脱氧升麻烃形成的组合物。
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，所述药物组合物中包含以下成分作为活性成分：升麻三萜皂苷类提取物、升麻、黄肉楠碱、脱氧升麻烃或者黄肉楠碱与脱氧升麻烃形成的组合物。
上述药物组合物中，所述炎性细胞因子可以包括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。
上述药物组合物中，黄肉楠碱与脱氧升麻烃形成的组合物中，黄肉楠碱与脱氧升麻烃的质量比可以为1～5﹕5～1；优选可以为1～3﹕3～1；更优选可以为1﹕1。
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。
上述药物组合物中，药物组合物还可以为包括升麻在内的中药组合物，例如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。
本发明通过刀豆蛋白A(Concanavalin A,ConA)的动物模型实验发现：升麻三萜皂苷类提取物、升麻、黄肉楠碱、脱氧升麻烃或其组合物、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：黄肉楠碱、脱氧升麻烃、升麻三萜皂苷类提取物、升麻或其组合物、药物组合物等对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。
本发明还通过LPS刺激的巨噬细胞(BMDM)发现：升麻三萜皂苷类提取物、升麻、黄肉楠碱、脱氧升麻烃或其组合物、药物组合物等具有确切的抗炎作用，能明显抑制巨噬细胞活化和IL-12、TNF-α等炎性细胞因子的分泌，通过体外实验发现还能明显抑制NK、NKT和T淋巴细胞活化和炎性细胞因子IFN-γ的分泌。由此还可作为免疫抑制剂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，本发明提供了升麻三萜皂苷类提取物、黄肉楠碱、脱氧升麻烃新的用途，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这些成分，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 升麻三萜皂苷提取物的制备
升麻购自同仁堂前门总店。参考文献(潘瑞乐，陈迪华，斯建勇，赵晓宏，沈连钢.升麻地上部分皂苷类成分研究.药学学报.2002,37(2):117-120)进行三萜皂苷提取。
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。取75mL升麻煎液，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，减压回收后，得到升麻三萜皂苷提取物，加水定容至75mL。
实验例1 ConA肝炎小鼠模型生存率的影响实验
试验材料：ConA购自Sigma公司；黄肉楠碱和脱氧升麻烃购自美国ChromaDex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40只，雄性，18-20g，适应性饲养1天后，随机分为4组，每组10只：ConA模型组，三萜皂苷组，黄肉楠碱组，脱氧升麻烃组。
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。分别取4mg黄肉楠碱和脱氧升麻烃，加10ml无菌双蒸水溶解，配制成0.4mg/mL浓度。ConA模型组灌胃蒸馏水250 μL，三萜皂苷组灌胃制备例1的升麻三萜皂苷提取物250μL，剂量为15mg/kg，黄肉楠碱组灌胃黄肉楠碱溶液250μL，剂量为5mg/kg，脱氧升麻烃组灌胃脱氧升麻烃溶液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10％，药物处理后小鼠生存率升高，三萜皂苷组的生存率为70％(与ConA模型组相比，***，p<0.001)，黄肉楠碱组的生存率为60％(与ConA模型组相比，**，p<0.01)，脱氧升麻烃组的生存率为40％(与ConA模型组相比，**，p<0.01)。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，升麻三萜皂苷提取物、黄肉楠碱和脱氧升麻烃均能提高ConA急性肝炎小鼠的生存率。
实验例2 对肝脏炎症的治疗实验
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5组：正常对照组、ConA模型组、三萜皂苷组、黄肉楠碱组和脱氧升麻烃组。
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。给药各组给药剂量与实验例1一致。
(1)检测给药各组药物对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)检测给药各组药物对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)检测给药各组药物对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1所示，ConA模型组与正常组相比ALT、AST显著升高(**，p<0.01；***，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)。说明升麻三萜皂苷提取物、黄肉楠碱和脱氧升麻烃均能减轻ConA小鼠肝损伤。
表1.升麻三萜皂苷提取物、黄肉楠碱和脱氧升麻烃对ConA肝炎小鼠模型血清ALT和AST的影响(MEAN±SEM)
Figure PCTCN2018091057-appb-000001
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模型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；升麻三萜皂苷组与ConA组相比，肝细胞片状坏死灶明显减轻，可见少许白细胞浸润；黄肉楠碱组、脱氧升麻烃组与ConA组相比，肝细胞片状坏死明显减轻，白细胞浸润减少。
结果(3)：药物对ConA肝炎小鼠模型血清细胞因子的影响
结果如表2所示，ConA模型组与正常组相比，TNF-α、IL-12、MCP-1、IL-6和IFN-γ分泌水平明显升高，具有统计学差异(***，p<0.001)。三萜皂苷提取物和黄肉楠碱均能明显降低血清中炎性因子IL-12、TNF-α、MCP-1、IL-6和IFN-γ的分泌水平；脱氧升麻烃能明显降低IL-12、MCP-1、IL-6和IFN-γ的分泌水平( #，p<0.05； ##，p<0.01； ###，p<0.001)。
表2.升麻三萜皂苷提取物、黄肉楠碱和脱氧升麻烃对ConA肝炎小鼠模型血清炎性细胞因子分泌的影响(MEAN±SEM)
Figure PCTCN2018091057-appb-000002
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 黄肉楠碱和脱氧升麻烃联用对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3组：正常组和CIA模型组，n＝5；黄肉楠碱联合脱氧升麻烃组(下称：黄脱组)，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。分别取4mg黄肉楠碱和脱氧升麻烃，加10mL无菌双蒸水溶解，配制成含0.4mg/mL黄肉楠碱和0.4mg/mL脱氧升麻烃的混合溶液，用于黄脱组给药治疗。小鼠初次免疫21天后进行二次免疫。从二次免疫0天开始，CIA模型组灌胃蒸馏水250μL，黄脱组灌胃黄肉楠碱和脱氧升麻烃的混合溶液250μL，剂量为5mg/kg，各组每天灌胃一次，连续饲养21天。
从二次免疫0天开始，每3天进行一次关节评分。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3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，至第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7.86±0.96。黄脱组关节炎平均指数为3.71±1.17，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3 黄肉楠碱和脱氧升麻烃联用对CIA关节炎小鼠关节炎指数的影响(MEAN±SEM)
Figure PCTCN2018091057-appb-000003
注：*代表CIA组与正常组有统计学差异，**，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显示了第15天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，黄脱组小鼠关节肿胀明显减轻。结果表明，黄肉楠碱和脱氧升麻烃联用能明显改善CIA关节炎小鼠的关节红肿和功能障碍。
实验例4 黄肉楠碱和脱氧升麻烃联用对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4所示，各组之间的小鼠体重均值没有差异。结果说明，黄肉楠碱和脱氧升麻烃联用不影响CIA关节炎小鼠体重。
表4.黄肉楠碱和脱氧升麻烃联用对CIA关节炎小鼠体重的影响(MEAN±SEM)
Figure PCTCN2018091057-appb-000004
实验例5 黄肉楠碱和脱氧升麻烃联用对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，快速称重，评价脏器指数。
结果如表5所示，黄脱组与CIA组相比，平均脏器指数没有变化。结果说明，黄肉楠碱和脱氧升麻烃联用不影响CIA小鼠脏器指数。
表5.黄肉楠碱和脱氧升麻烃对CIA关节炎小鼠脏器指数的影响(MEAN±SEM,单位mg)
Figure PCTCN2018091057-appb-000005
实验例6 黄肉楠碱和脱氧升麻烃联用对CIA关节炎小鼠的治疗效果
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：各组给药剂量与实验例3一致。二次免疫21天后，麻醉小鼠取血清和四肢。
(1)Micro-CT技术检测各组对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)检测各组对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)检测各组对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技术检测对CIA小鼠关节结构的影响。
结果如图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，黄脱组关节结构几乎保持完整，特别是骨质破坏明显轻于模型组。如表6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，黄脱组与CIA模型组相比，骨计量指征明显上升。结果说明，黄肉楠碱和脱氧升麻烃联用可以有效缓解关节破坏。
表6.黄肉楠碱和脱氧升麻烃联用对CIA关节炎小鼠骨损伤的影响(MEAN±SEM)
Figure PCTCN2018091057-appb-000006
注：*代表CIA组与正常组有统计学差异，**，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)：对CIA小鼠血清抗Ⅱ型胶原抗体含量的影响。
结果如表7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)。黄脱组能明显降低血清中抗Ⅱ型胶原抗体的分泌水平(####，p<0.0001)。结果说明，黄肉楠碱和脱氧升麻烃联用能降低自身抗体分泌。
表7.黄肉楠碱和脱氧升麻烃联用对CIA关节炎小鼠血清抗Ⅱ型胶原抗体含量的影响(MEAN±SEM)
Figure PCTCN2018091057-appb-000007
注：*代表CIA组与正常组有统计学差异，***，p<0.001。#代表用药组与CIA组有统计学差异，###，p<0.001。
结果(3)：对CIA小鼠血清和关节组织细胞因子分泌的影响。
结果如表8所示，黄脱组与CIA组相比，血清中细胞因子IFN-γ、IL-12、IL-17A、IL-6、IL-9、IL-18、趋化因子MCP-1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和MIP-2的分泌水平也明显降低，具有统计学差异。结果说明，黄肉楠碱和脱氧升麻烃联用能明显降低血清和关节组织中多种细胞因子、趋化因子、集落刺激因子的分泌水平，具有广泛的抑制炎症的作用。
表8.黄肉楠碱和脱氧升麻烃联用对CIA关节炎小鼠血清和关节组织细胞因子的影响(MEAN±SEM)
Figure PCTCN2018091057-appb-000008
Figure PCTCN2018091057-appb-000009
注：*代表CIA组与正常组有统计学差异，*，p<0.05；**，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；###，p<0.001。
实验例7 黄肉楠碱和脱氧升麻烃联用对CIA关节炎小鼠粒细胞比例的影响
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；B220-FITC抗体购自BD Pharmingen公司；其他所需试剂均同实验例3。
模型制备和给药处理：各组给药剂量与实验例3一致。
动物：C57BL/6小鼠，购自北京华阜康生物科技股份有限公司。
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。加入抗CD3，B220，NK1.1抗体进行表面染色，4避光孵育15min后，加入2mL PBEB清洗，离心弃上清。进行胞内染色。
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CD3 -NK1.1 +B220 -NK细胞内Ki-67表达。
结果如表9所示，CIA关节炎小鼠与正常小鼠相比，脾脏中NK细胞的Ki-67 +细胞比例增强，表明NK细胞增殖活性增强；黄脱组与CIA组相比，Ki-67 +细胞比例降低，具有显著差异，表明黄肉楠碱和脱氧升麻烃联用能抑制NK细胞增殖。
表9.黄肉楠碱和脱氧升麻烃联用对CIA关节炎小鼠NK细胞增殖的影响(MEAN±SEM)
Figure PCTCN2018091057-appb-000010
注：*代表CIA组与正常组有统计学差异，*，p<0.05；#代表用药组与CIA组有统计学差异，###， p<0.001。
实验例8 黄肉楠碱和脱氧升麻烃对巨噬细胞分泌细胞因子的抑制作用实验
试验材料：LPS购自Sigma公司；黄肉楠碱和脱氧升麻烃购自美国ChromaDex公司；FBS购自美国PAA公司；DMEM高糖培养基；巨噬细胞集落刺激因子(M-CSF)购自PeproTech公司；ELISA试剂盒购自美国BD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。
小鼠骨髓源巨噬细胞(BMDM)分离培养：C57BL/6小鼠麻醉后，无菌分离小鼠双股骨和胫骨，用2mL注射器吸取无菌PBS将骨髓细胞冲入无菌流式管，用滴管反复吹打为骨髓单细胞悬液。离心后，收获骨髓细胞，加入10mL含10％FBS、1％谷氨酰胺、1％双抗(抗青霉素、链霉素)的完全培养基，以及10ng/mL M-CSF，接种于100mm细胞培养皿中培养，37℃培养。4天后换上述新鲜培养基，培养至第7天显微镜下可观察到体积大、贴壁生长、分化成熟的巨噬细胞。用细胞刮板将细胞刮下，反复吹吸混匀为单细胞悬液后，按照1×10 5个/每孔种于96孔板。
BMDM细胞诱导活化与给药处理：采用不同剂量的黄肉楠碱和脱氧升麻烃处理BMDM细胞，1h后给予100ng/mL LPS刺激细胞，模拟细菌感染对巨噬细胞的活化。4h后收集细胞上清，ELISA检测细胞因子分泌。
结果如表10所示，LPS刺激BMDM细胞活化，表现为分泌大量炎性细胞因子IL-12和TNF-α。黄肉楠碱在2.5ng/mL至312.5ng/mL浓度范围内均能显著抑制IL-12分泌，在0.5ng/mL至312.5ng/mL浓度范围内均能抑制TNF-α分泌；脱氧升麻烃在250ng/mL至500ng/mL浓度范围内能显著抑制IL-12分泌，在50ng/mL至500ng/mL浓度范围内能抑制TNF-α分泌。结果表明2.5ng/mL至312.5ng/mL浓度的黄肉楠碱和250ng/mL至500ng/mL浓度的脱氧升麻烃均对巨噬细胞活化有明显的抑制作用。
表10.黄肉楠碱和脱氧升麻烃抑制巨噬细胞分泌IL-12和TNF-α(MEAN±SEM)
Figure PCTCN2018091057-appb-000011
Figure PCTCN2018091057-appb-000012
注：*代表LPS组与空白组有统计学差异，***，p<0.001。#代表用药组与LPS组有统计学差异， #，p<0.05； ##，p<0.01； ###，p<0.001。
实验例9 黄肉楠碱和脱氧升麻烃对NK细胞表达IFN-γ的抑制作用实验
试验材料：CD45-PE、CD3-FITC、NK1.1-APC抗体、胞内染色试剂盒Intracellular Fixation&Permeabilization Buffer Set、细胞刺激剂Cell Stimulation Cocktail均购自eBioscience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。
骨髓单细胞悬液制备：C57BL/6小鼠麻醉后，无菌分离小鼠双股骨和胫骨，用2mL注射器吸取无菌PBS将骨髓细胞冲入无菌流式管，用滴管反复吹打为骨髓单细胞悬液。离心后，收获骨髓细胞，加入1.5mL含10％FBS、1％谷氨酰胺、1％双抗(抗青霉素、链霉素)的完全培养基。
给药处理及NK细胞诱导活化后胞内染色：分别采用中等浓度的62.5ng/mL黄肉楠碱和250ng/mL脱氧升麻烃处理细胞，1h后给予Cell Stimulation Cocktail(含PMA、离子霉素和蛋白转运抑制剂)刺激细胞5h，采用CD45-PE、CD3-FITC、NK1.1-APC抗体进行表面染色，采用IL-12-APC抗体进行细胞内染色，流式检测CD45 +CD3 —NK1.1 +的NK细胞中表达IFN-γ的阳性细胞百分比。
结果如表11所示，在PMA+离子霉素刺激下，骨髓中NK细胞被活化，表现为大量分泌炎性细胞因子IFN-γ；与PMA+离子霉素组相比，黄肉楠碱和脱氧升麻烃处理均能明显降低IFN-γ的表达，抑制NK细胞活化(*，p<0.05)。
表11.黄肉楠碱和脱氧升麻烃抑制NK细胞表达IFN-γ(MEAN±SEM)
Figure PCTCN2018091057-appb-000013
Figure PCTCN2018091057-appb-000014
注：*代表PMA+离子霉素组与空白组相比有统计学差异，***，p<0.001。#代表用药组与PMA+离子霉素组相比有统计学差异，#，p<0.05。
实验例10 黄肉楠碱和脱氧升麻烃对NKT细胞表达IFN-γ的抑制作用实验
试验材料：同实验例9。
动物：C57BL/6小鼠，购自北京华阜康生物科技股份有限公司。
骨髓单细胞悬液制备：同实验例9。
给药处理及NKT细胞诱导活化后胞内染色：同实验例9。流式检测CD45 +CD3 +NK1.1 +的NKT细胞中表达IFN-γ的阳性细胞百分比。
结果如表12所示，在PMA+离子霉素刺激下，骨髓中NKT细胞被活化，表现为大量分泌炎性细胞因子IFN-γ；与PMA+离子霉素组相比，黄肉楠碱和脱氧升麻烃处理均能明显降低IFN-γ的表达，抑制NKT细胞活化(*，p<0.05；**，p<0.01)。
表12.黄肉楠碱和脱氧升麻烃抑制NKT细胞表达IFN-γ(MEAN±SEM)
Figure PCTCN2018091057-appb-000015
注：*代表PMA+离子霉素组与空白组相比有统计学差异，**，p<0.01。#代表用药组与PMA+离子霉素组相比有统计学差异，#，p<0.05；##，p<0.01。
实验例11 黄肉楠碱和脱氧升麻烃对T淋巴细胞表达IFN-γ的抑制作用实验
试验材料：CD3-PE抗体购自美国BD公司，CD4-FITC、CD8-PerCP抗体购自eBioscience公司；其余所需试剂均同实验例9。
动物：C57BL/6小鼠，购自北京华阜康生物科技股份有限公司。
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的无菌小皿中，用载玻片磨砂面研磨脾脏，无菌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1.5mL含10％FBS、1％谷氨酰胺、1％双抗(抗青霉素、链霉素)的完全培养基，反复吹吸混匀。
给药处理及T细胞诱导活化后胞内染色：采用中等浓度的62.5ng/mL黄肉楠碱和250ng/mL脱氧升麻烃单独及共同处理细胞，1h后给予Cell Stimulation Cocktail(含PMA、离子霉素和蛋白转运抑制剂)刺激细胞5h。流式检测CD3 +CD4 +T细胞胞内表达IFN-γ的阳性细胞百分比，CD3 +CD8 +T细胞胞内表达IFN-γ的阳性细胞百分比。
结果，如表13所示，在PMA+离子霉素刺激下，CD4 +T细胞和CD8 +T大量表达炎性细胞因子IFN-γ；与PMA+离子霉素组相比，黄肉楠碱和脱氧升麻烃单独应用不能降低IFN-γ表达，但二者联用具有协同效应，能明显降低IFN-γ的表达(**，p<0.01；***，p<0.001)。
表13.黄肉楠碱和脱氧升麻烃抑制T淋巴细胞表达IFN-γ(MEAN±SEM)
Figure PCTCN2018091057-appb-000016
注：*代表PMA+离子霉素组与空白组相比有统计学差异，**，p<0.01；***，p<0.001。#代表用药组与PMA+离子霉素组相比有统计学差异，##，p<0.01；###，p<0.001。
综上所述，升麻三萜皂苷提取物、黄肉楠碱、脱氧升麻烃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。
黄肉楠碱和脱氧升麻烃联用能明显减轻关节炎小鼠模型的关节炎平均指数，改善骨结构，减轻骨侵蚀，抑制炎性因子释放，抑制NK细胞增殖，具有显著的免疫调节和关节保护作用。
此外，升麻三萜皂苷提取物以及其主要成分黄肉楠碱和脱氧升麻烃还具有明显的抗炎 作用，均能抑制巨噬细胞、NK和NKT细胞活化，二者协同作用还能抑制T淋巴细胞活化，具有广泛的免疫抑制作用。
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. 升麻三萜皂苷类提取物、升麻、黄肉楠碱、脱氧升麻烃或者黄肉楠碱与脱氧升麻烃形成的组合物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. 升麻三萜皂苷类提取物、升麻、黄肉楠碱、脱氧升麻烃或者黄肉楠碱与脱氧升麻烃形成的组合物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。
  4. 根据权利要求3所述的用途，其特征在于，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。
  5. 根据权利要求1-4任一项所述的用途，其特征在于，所述黄肉楠碱与脱氧升麻烃形成的组合物中，黄肉楠碱与脱氧升麻烃的质量比为1～5﹕5～1；优选为1～3﹕3～1；更优选为1﹕1。
  6. 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以下成分作为活性成分：升麻三萜皂苷类提取物、升麻、黄肉楠碱、脱氧升麻烃或者黄肉楠碱与脱氧升麻烃形成的组合物。
  7. 根据权利要求6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。
  8. 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以下成分作为活性成分：升麻三萜皂苷类提取物、升麻、黄肉楠碱、脱氧升麻烃或者黄肉楠碱与脱氧升麻烃形成的组合物。
  9. 根据权利要求8所述的药物组合物，其特征在于，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6-9任一项所述的药物组合物，其特征在于，所述黄肉楠碱与脱氧升麻烃形成的组合物中，黄肉楠碱与脱氧升麻烃的质量比为1～5﹕5～1；优选为1～3﹕3～1；更优选为1﹕1。
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