WO2021218763A1 - 杠柳苷组合物、其制备方法及其在制备治疗自身免疫性疾病的药物中的用途 - Google Patents

杠柳苷组合物、其制备方法及其在制备治疗自身免疫性疾病的药物中的用途 Download PDF

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WO2021218763A1
WO2021218763A1 PCT/CN2021/088918 CN2021088918W WO2021218763A1 WO 2021218763 A1 WO2021218763 A1 WO 2021218763A1 CN 2021088918 W CN2021088918 W CN 2021088918W WO 2021218763 A1 WO2021218763 A1 WO 2021218763A1
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ethanol
periploside
weight
mice
group
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French (fr)
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左建平
赵维民
林泽民
陈振华
何世君
张如隽
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中国科学院上海药物研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • 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
    • 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
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection

Definitions

  • the present disclosure relates to a drug for autoimmune diseases, in particular, the present disclosure relates to a psilocybin composition, a preparation method thereof, and use in the preparation of a drug for treating autoimmune diseases.
  • IBD Inflammatory Bowel Disease
  • CD Crohn's disease
  • UC Ulcerative colitis
  • CD Crohn's disease
  • Ulcerative colitis is a chronic disease that can cause inflammation and ulcers in the colon and rectum. The etiology has not been fully elucidated. It may be related to genetic factors, autoimmune diseases, changes in intestinal flora and other factors.
  • the main symptoms of the attack include abdominal pain and diarrhea accompanied by blood in the stool, weight loss, fever, and anemia may also appear during the attack of ulcerative colitis.
  • the course of its symptoms is slow and varies in severity. The symptoms often appear intermittently, and there is often an asymptomatic period between the two episodes.
  • Complications of ulcerative colitis may include Hirschsprung disease or inflammation of the eyes, joints, or liver, and colon cancer.
  • IBD has become a global disease, and clinical intervention is mainly limited to anti-inflammatory and immunomodulatory drugs.
  • the existing treatment drugs cannot completely control the progression and recurrence of IBD, and there are many shortcomings.
  • RA Rheumatoid arthritis
  • NSAIDs nonsteroidal antiinflammatory drugs
  • DMARDs disease modifying antirheumatic drugs
  • DMARDs include two major categories: 1.
  • Synthetic anti-rheumatic drugs such as hydroxychloroquine (hydroxychloroquine), sulfasalazine (SSZ), gold compounds, methotrexate, MTX), leflunomide, Cyclophosphamide, Cyclosporin A, etc.
  • sDMARDs such as hydroxychloroquine (hydroxychloroquine), sulfasalazine (SSZ), gold compounds, methotrexate, MTX), leflunomide, Cyclophosphamide, Cyclosporin A, etc.
  • Long-term medication may cause toxic side effects such as gastrointestinal, liver and kidney damage, bone marrow suppression. 2.
  • Biological DMARDs including Anakinra (Anakinra, IL-1 receptor antagonist), Infliximab (Inflectra, TNF- ⁇ monoclonal antibody), Abatacept (Abatacept, TNF- ⁇ monoclonal antibody), rituximab (Rituximab, anti-CD20 monoclonal antibody), tocilizumab (tocilizumab, IL-6 monoclonal antibody).
  • bDMARDs are expensive, which limits their wide clinical application.
  • Glucocorticoids such as prednisone, prednisolone, methylprednisolone, etc.
  • Rheumatoid arthritis belongs to the category of "Arthralgia" in the theory of traditional Chinese medicine.
  • Traditional Chinese medicine has thousands of years of experience in the treatment of Arthralgia.
  • the dialectical treatment of RA has achieved the effect of treating both the symptoms and the root causes, with the primary treatment being the root cause, and the side effects are small.
  • Periploside A C, D, E
  • K O, Q, R
  • Periploside F a trace amount of psilocybin compound
  • Periplocoside E effective compound from Periploca sepium Bge, inhibited T cell activation in vitro and in vivo[J]. Journal of Pharmacology Experimental Therapeutics,2006,316(2):662-669. [2]Zhu Y N,Zhong X G,Feng J Q,et al.Periplocoside Einhibitsexperimentalallergicencephalomyelitisby suppressioning 12-dependent-interleukin-interpression-CCR5expression dependent CXCR3 expression in T lymphocytes[J].Journal of Pharmacology and Experimental Therapeutics,2006,318(3):1153-1162.[3]Wan J,Zhu Y N,Feng J Q,et al.Periplocoside A,a pregnane glycoside from Periploca sepium Bge,prevents concanavalin A-induced mice hepatitis through inhibiting NKT-derived inflammatory cytokine productions[J].International Immunopharmacology,2008,8(9):1248
  • Periplocoside A prevents experimental autoimmune encephalomyelitis by suppressing IL-17 production and inhibition differentiation of Th17 cells[J]. Acta Pharmacologica Sinica, 2009, 30(8) :1144.[5]Yang Y,Hu X,Cheng L,et al.Periplocoside A meliorated type II collagen-induced arthritis in mice via regulation of the balance of Th17/Treg cells[J].International Immunopharma,2017,44: 43-52. ⁇
  • periploside F The content of periploside F in Periploside F is relatively low, but it can be obtained from other high-content compounds (such as Periplcoside C) through chemical transformation. Periploside F also has a significant effect on the proliferation of T lymphocytes. Inhibition. ⁇ Reference: Wang LY, Chen ZH, Zhou Y, et al. Structural revision of periplocosides and peroxides, natural immunosuppressive agents from the general Periploca.
  • the content of skin medicinal materials is about 0.1%, and there are toxic components of cardiac glycosides and a large number of fatty acids, low-grade oligosaccharides and 4-methoxy salicylaldehyde that are similar in properties to pteroside compounds in the medicinal materials. It is difficult to prepare the effective ingredients (ie, pueraria polyglycosides) containing pentaphylloside compounds, and its preparation method has not been reported in the literature.
  • the inventors found a method for preparing a puerrainin composition by exploring the combination of various extraction processes, analyzed the composition of the puerperidin compound extracted by this method, and found that the puerperidin compound is effective in treating inflammation. Effects in sexual bowel disease. This disclosure is completed on this basis.
  • An object of the present disclosure is to provide a puerperin composition.
  • Another object of the present disclosure is to provide a preparation method of the puerperin composition.
  • Another object of the present disclosure is to provide a pharmaceutical use of the puerperin composition.
  • puerperin composition which includes the following components:
  • the sum of the masses of the components Periploside A, C, D, E, K, O, Q and R is greater than 50% of the total mass of the composition, preferably greater than 60%.
  • a method for preparing a puerperin composition including:
  • the hydrocarbon solvent is one selected from C5-C8 hydrocarbon solvents (such as cyclopentane, n-hexane, cyclohexane, cyclohexene, n-heptane, n-octane, isooctane) and petroleum ether Or multiple
  • the powder mixture is purified with ethanol-water to obtain a peperidin composition.
  • a phytoside composition in the preparation of a medicament for the treatment of autoimmune diseases.
  • the inventors of the present disclosure conducted repeated experiments on the extraction method of the effective ingredients from the root bark of Salix sylvestris, and found a particularly effective extraction process in the experiment.
  • the inventors found that the periploside C and F in the periploside composition of the specific composition extracted by the extraction process are effective against autoimmune diseases, such as Crohn’s disease and ulcerative colitis. Active inflammatory bowel disease.
  • Figure 1 shows the scoring curve of body weight and inflammatory bowel disease activity index of each group of mice during the experiment of dextran sodium sulfate (DSS) inducing inflammatory bowel disease in mice.
  • DSS dextran sodium sulfate
  • Figure 2A is a representative photograph of the colorectal tissue of each group of mice at the end of the DSS-induced inflammatory bowel disease experiment in mice.
  • Fig. 2C is a representative schematic diagram of the colon tissue at the end point of the DSS-induced enteritis model mouse experiment, which was made by the H&E staining method.
  • Figure 3A is the end point of the DSS-induced inflammatory bowel disease experiment in mice.
  • Flow cytometry is used to analyze the proportion of macrophages (anti-F4/80 antibody + anti-CD11b antibody staining double positive) in the mouse mesentery lymph nodes.
  • Figure 3B is the end point of the DSS-induced inflammatory bowel disease experiment in mice.
  • Figure 3C is a representative schematic diagram of immunohistochemical analysis of the end point of the DSS-induced inflammatory bowel disease experiment in mice, and the expression level of myeloperoxidase (MPO) in the colorectal tissue of the mouse.
  • MPO myeloperoxidase
  • FITC fluorescein isothiocyanate
  • Figure 5A is the end point of the DSS-induced inflammatory bowel disease experiment in mice.
  • Figure 6A is a curve of body weight change of mice in each group during the experiment of TNBS induced inflammatory bowel disease in mice;
  • Figure 6B is a curve of the survival rate of mice in each group during the TNBS-induced inflammatory bowel disease experiment in mice;
  • Figure 6D is the result of a photo of the length of the colon.
  • Figure 7(A) shows the body weight of each group of mice during the DSS-induced inflammatory bowel disease experiment in mice.
  • Figure 7(B) shows the inflammatory bowel disease activity index score curve of each group of mice.
  • Figure 7D is a representative photograph of the colorectal tissue of each group of mice at the end of the DSS-induced inflammatory bowel disease experiment in mice.
  • Figure 8A shows the joint clinical scores of mice in each group during the collagen-induced arthritis experiment in mice.
  • Figure 8B shows the body weight of mice in each group during the collagen-induced arthritis experiment in mice.
  • Figure 9 shows the end point of the collagen-induced arthritis experiment in mice, a representative schematic diagram of pathological cuts made by the H&E staining method.
  • Figure 10A is the end point of the collagen-induced arthritis experiment in mice, and the level of total IgG in the serum of anti-collagen-specific antibodies in the mouse serum.
  • Figure 10B is the end point of the collagen-induced arthritis experiment in mice, and the level of anti-collagen-specific antibody serum IgG2a in the mouse serum.
  • Figure 11A shows the clinical scores of the joints of rats in each group during the adjuvant-induced arthritis experiment.
  • Figure 11B shows the degree of secondary foot swelling in each group of rats during the adjuvant-induced arthritis experiment in rats.
  • Figure 11C shows the weight results of rats in each group during the adjuvant-induced arthritis experiment in rats.
  • Figure 12A is the end point of the adjuvant-induced arthritis experiment in rats, and representative photos of the secondary feet of rats in each group.
  • Figure 12B is the end point of the adjuvant-induced arthritis experiment in rats, and a representative schematic diagram of the bone erosion of each group of rats by microcomputer tomography.
  • Figure 13 is the end point of the adjuvant-induced arthritis experiment in rats, a representative schematic diagram of pathological cuts made by the H&E staining method.
  • the terms “include”, “include”, “have”, “contain” or any other similar terms are all open-ended transitional phrases, which are intended to cover non-exclusive inclusions.
  • a composition or article containing plural elements is not limited to the elements listed herein, but may also include other elements that are not explicitly listed but are generally inherent in the composition or article.
  • the term “or” refers to an inclusive “or” rather than an exclusive “or”. For example, any one of the following conditions satisfies the condition "A or B": A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), Both A and B are true (or exist).
  • a numerical value should be understood to have the accuracy of the number of significant digits of the numerical value.
  • the number 40.0 should be understood to cover the range from 39.50 to 40.49.
  • puerperin composition which includes the following components:
  • the psilocin composition containing the above-mentioned components exhibits a better therapeutic effect in the process of treating autoimmune diseases.
  • the Periploside A, C, D, E, K, O, Q, R, and the sum of the masses thereof is greater than 50% of the total mass of the composition, preferably greater than 60% .
  • a preparation method of a psilocybin composition including:
  • the hydrocarbon solvent is one selected from C5-C8 hydrocarbon solvents (such as cyclopentane, n-hexane, cyclohexane, cyclohexene, n-heptane, n-octane, isooctane) and petroleum ether Or multiple
  • the powder mixture is purified with ethanol-water to obtain a peperidin composition.
  • step (1) after the alcohol extracts are combined and concentrated, the ethanol is removed due to the concentration process, and the concentrated solution becomes a suspension.
  • step (2) the addition of low-concentration ethanol to the suspension cannot dissolve the required solids, and the addition of high-concentration ethanol can only dissolve the desired compound.
  • a pharmaceutical composition containing active ingredients can be effectively obtained at low cost, while avoiding the use of high-cost chromatographic fillers, toxic solvents and chemical synthesis processes.
  • the hydrocarbon solvent is preferably cyclohexane.
  • the preparation method includes
  • the hydrocarbon solvent is one selected from C5-C8 hydrocarbon solvents (such as cyclopentane, n-hexane, cyclohexane, cyclohexene, n-heptane, n-octane, isooctane) and petroleum ether Or multiple
  • the above powder mixture was dissolved in 70%-95% ethanol, and water was added to adjust the ethanol concentration to below 60% to obtain a suspension.
  • the suspension was filtered to obtain the first supernatant and the first precipitate.
  • the second supernatant and the second precipitate are obtained by filtration.
  • the first supernatant and the second supernatant are combined and then allowed to settle and settle to obtain the third precipitate.
  • the second precipitate and the third precipitate are combined.
  • the puerperin composition is obtained by drying and pulverizing.
  • the highly polar components can be removed at low cost; in addition, by adopting different concentrations of ethanol precipitation and hydrocarbon solvent extraction processes, a large amount of impurities other than saligenin can be removed and macropores can be reduced. The amount of sample loaded on the resin column.
  • the hydrocarbon solvent is preferably cyclohexane.
  • it provides the use of the above-mentioned psilocybin composition in the preparation of a medicament for the treatment of autoimmune diseases.
  • the autoimmune disease includes inflammatory bowel disease and rheumatoid arthritis.
  • the inflammatory bowel disease includes ulcerative colitis and Crohn's disease.
  • the drug for the treatment of inflammatory bowel disease suppresses weight loss, improves loose stools and blood in the stool, protects intestinal mucosal damage, reduces pathological damage of colon tissue, and inhibits colon tissue
  • the infiltration of inflammatory cells works by down-regulating the levels of related pro-inflammatory cytokines.
  • the drug for treating rheumatoid arthritis is to relieve the swelling of the joints, reduce the inflammation and infiltration in the joint tissues, alleviate the bone erosion of the arthritic focus, and reduce the specificity in the serum.
  • the production of autoantibodies comes into play.
  • periploside C and Periploside F in the preparation of a medicament for the treatment of inflammatory bowel disease.
  • Medicinal materials 500g fragrant and peel medicinal materials.
  • Extraction Discard the water extract after three times of water extraction, and then extract twice with alcohol. Add 4L of 70% ethanol each time to obtain 8L of alcohol extract.
  • 35% ethanol precipitation 35% ethanol precipitation is performed on 20% ethanol precipitation, and 45.85g of 35% ethanol precipitation is obtained by solid-liquid separation.
  • 70% ethanol precipitation add 2 times the volume of 95% ethanol and 4 times the volume of 70% ethanol for reconstitution at 65°C, extract the supernatant to obtain 305 mL of 70% ethanol supernatant.
  • Cyclohexane extraction 305mL 70% ethanol supernatant was added with 305mL cyclohexane for three extractions, the ethanol solution was evaporated under reduced pressure to remove residual cyclohexane to obtain a deoiled ethanol phase.
  • 95% ethanol reconstituted and impurity removal 50mL of the 95% ethanol fraction concentrate was concentrated to dryness, and 12 times the solid content of 95% ethanol was added in a 65°C water bath for reconstitution, filtered and impurity removed, and the supernatant was obtained.
  • the 60% ethanol supernatant is passed through column chromatography: the 60% ethanol supernatant is passed through the B17 type macroporous resin column, followed by elution with 3BV75% ethanol, 80% ethanol, and 6BV95% ethanol, and collect 80% ethanol. Two BV elution sites and 95% of the first and second BV elution sites were combined, concentrated, dried, and subjected to 60% ethanol precipitation, and the 60% precipitate was vacuum dried to obtain 57 mg of saponins crude drug (2).
  • Two-step intermediate mixing Mix the above-mentioned Pterocarpus polyglycosides (1) and (2) to obtain a total of 1.176g of intermediates.
  • 8 palisades compounds Periploside A, C, D, E, K,
  • the cumulative content of O, Q, and R is 59.09%, of which the content of 8 esperidin is Periploside A 2.52% by weight, Periploside C 31.12% by weight, Periploside D 8.54% by weight, Periploside E 5.35% by weight, Periploside K 2.62% by weight, Periploside O 3.52% by weight, Periploside Q 3.67% by weight, and Periploside R 1.75% by weight.
  • Medicinal materials 500g fragrant and peel medicinal materials.
  • Extraction Discard the water extract after three times of water extraction, and then extract twice with alcohol. Add 4L of 70% ethanol each time to obtain 8L of alcohol extract.
  • 20% ethanol precipitation add 50g 95% ethanol to the suspension to prepare a 20% ethanol suspension, and solid-liquid separation yields 164g of 20% ethanol precipitation.
  • 35% ethanol precipitation Add 164g (the same mass as the precipitation) of 35% ethanol to the 20% ethanol precipitation, mix well and separate solid-liquid to obtain 157g of 35% ethanol precipitation.
  • 70% ethanol precipitation 157 g of 35% ethanol precipitation was added with 157 mL of 95% ethanol, and 278.5 mL of 70% ethanol was used to form a 70% ethanol suspension. The solid-liquid separation yielded 400 mL of 70% ethanol supernatant.
  • Cyclohexane extraction 400 mL of 70% ethanol supernatant was added with 400 mL of cyclohexane for three extractions, the ethanol solution was evaporated under reduced pressure to remove residual cyclohexane to obtain a deoiled ethanol phase.
  • 85% ethanol recovery use the 85% ethanol elution part as the 85% ethanol sample solution, add low-concentration ethanol to 70%, pump into the column, and use 70% ethanol, 80% ethanol, and 95% ethanol for elution. , Collect 70% ethanol sites, 80% ethanol sites, and 95% ethanol sites. The 95% parts were combined and concentrated to obtain 40 mL 95% ethanol concentrate.
  • Reconstituted with 95% ethanol to remove impurities Concentrate 40 mL of the 95% ethanol concentrate to dryness to obtain 2.2 g of solids. Add 12 times the solid content of 95% ethanol in a 65°C water bath for reconstitution, filter and remove impurities to obtain the supernatant. .
  • 60% ethanol precipitation Add 17.6g of water to the 95% ethanol reconstituted supernatant to prepare a 60% ethanol solution, heat it to 30-40°C and filter while hot to obtain the precipitate, and place it in a 60°C blast oven for drying. Obtained 1.014g of the raw material drug of Pterocarpus polyglycosides, and determined the cumulative content of 8 P.
  • periploside compounds Periploside A, C, D, E, K, O, Q, and R to be 60.33%, of which the content of 8 P.
  • periplosides They are Periploside A 2.74% by weight, Periploside C 30.33% by weight, Periploside D 9.62% by weight, Periploside E 5.27% by weight, Periploside K 3.04% by weight, Periploside O 3.89% by weight, Periploside Q 3.55% by weight , Periploside R 1.89% by weight.
  • Periploside D 10.31% by weight, Periploside E 6.34% by weight, Periploside K 3.12% by weight, Periploside O 3.45% by weight, Periploside Q 3.76% by weight, Periploside R 1.98% by weight.
  • mice 8-10 weeks old, purchased from Shanghai Slack Laboratory Animal Co., Ltd.
  • DSS dextran sulfate sodium
  • CD3, CD4, IL-17, IFN- ⁇ , CD11b, GR1, F4/80 and other flow cytometry antibodies were purchased from BD Pharmingen.
  • the cytokine detection kit ProcartaPlex Mouse Th1/Th2/Th9/Th17/Th22/Treg Cytokine 17 factor detection kit was purchased from Invitrogen. Hydroxypropyl methylcellulose (HPMC) was purchased from Sigma-Aldrich, USA.
  • HPMC Hydroxypropyl methylcellulose
  • the occult blood test kit was purchased from Nanjing Jiancheng Institute of Biological Engineering.
  • Pterocarpus polyglycosides i.e. the composition of Peratoplastin prepared in Preparation Example 3, traits: light brown powder; of which 8 Peratopins Periploside A, C, D, E, K, O, Q, R The cumulative content is 64.96%, and the content of 8 psilocins is Periploside A 4.54% by weight, Periploside C 31.46% by weight, Periploside D 10.31% by weight, Periploside E 6.34% by weight, Periploside K 3.12% by weight, Periploside O 3.45% by weight, Periploside Q 3.76% by weight, Periploside R 1.98% by weight.
  • the polyglycoside is suspended in a solvent and diluted to the required concentration before use.
  • the solvent is 0.2% (w/v) HPMC.
  • Sulfasalazine colon-soluble capsules purchased from Guangdong Qiangji Pharmaceutical Co., Ltd., 250 mg/capsule, used after being diluted with 0.2% (w/v) HPMC to the required concentration.
  • mice were randomly divided into 4 groups, a normal control group, a DSS model, a sulfasalazine treatment group, and a saponins treatment group.
  • the sulfasalazine treatment group was the positive control group.
  • the drinking water of the other groups including the DSS model, the sulfasalazine treatment group, and the saponins treatment group were all added 5% DSS to their drinking water, and the intestinal inflammation model was performed for 7 consecutive days. Induced model building.
  • the sulfasalazine treatment group received 250 mg/kg of sulfasalazine orally once a day; the salivarius polyside treatment group received 12.5 mg/kg of sulfasalazine orally once a day; normal
  • the control group and the DSS model were given an equal volume of 0.2% (w/v) HPMC orally, once a day.
  • DAI Disease activity index
  • DAI score sheet
  • mice colon tissue was quickly taken, and the length was measured from the bottom of the cecum to the bottom of the rectum, recorded and photographed.
  • mice colon tissue was fixed in formalin, paraffin-embedded sections, and H&E stained to make pathological sections.
  • MPO myeloperoxidase
  • mice colon tissue was fixed in formalin, and the paraffin-embedded sections were used to detect the expression level of MPO in the mouse colon tissue by immunohistochemistry.
  • mouse mesenteric lymph nodes were taken to prepare a single cell suspension, the cells were labeled with the corresponding flow cytometry antibody, and statistical analysis was performed.
  • mice of each group were orally given FITC-labeled dextran at a dose of 400 mg/kg 4 hours before the euthanasia of each group.
  • blood was collected from mice. After standing for 2 hours at room temperature and avoiding light, centrifuged at 6000g for 10 minutes. After the serum was diluted with PBS, the concentration of FITC-dextran was measured by fluorescence microplate reader at the excitation wavelength of 488nm and emission wavelength of 520nm to reflect the mucosa of mice. Permeability.
  • Weight loss is an important disease indication for DSS-induced inflammatory bowel disease, which can reflect the health of mice.
  • the results are shown in Figure 1A.
  • Pterocarpus polyglycosides can significantly improve the weight loss caused by DSS on the sixth day and the seventh day after treatment.
  • the Disease Activity Index is a score based on the percentage of weight loss, stool consistency, and stool bleeding in mice with inflammatory bowel disease. The total score of the three results is divided by 3 to obtain the DAI value. The evaluation is small An important indicator of clinical symptoms of mouse disease. The study found that the disease activity index of the mice in the Pterocarpus polyglycoside treatment group significantly alleviated the disease activity index of the inflammatory bowel disease mice from the fifth day of treatment. The results are shown in Figure 1B.
  • DSS induces inflammatory bowel disease and causes the colon to become shorter. It can be seen from Figure 2A and Figure 2B that the colon length of DSS model mice is significantly lower than that of the normal group of mice. The colon length of the mice in the pyridine treatment group was significantly longer than that of the DSS model mice.
  • the colon pathological staining ( Figure 2C) shows that the infiltration of inflammatory cells such as neutrophils, monocytes, and multinucleated cells in the intestinal tissue of DSS model mice was significantly increased, and some crypts were destroyed. The above-mentioned conditions in the polyglycoside treatment group and the positive drug sulfasalazine treatment group were significantly alleviated. Macrophages, neutrophils, etc.
  • MPO Myeloperoxidase
  • peroxidase is an enzyme substance containing heme, which is widely present in neutrophils and tissue macrophages.
  • the expression of MPO in the colon tissue of mice with inflammatory bowel disease was detected by immunohistochemistry. It can be seen from Figure 3C that MPO in the intestinal tissue of DSS model mice was significantly higher than that in the normal control group. The MPO content in the positive drug control group was significantly lower than that in the model group. It shows that Pterocarpus polyglycosides can reduce the degree of local inflammation in the intestinal tissue of mice with DSS-induced inflammatory bowel disease.
  • Th17 cells play an important role in the pathogenesis of autoimmune diseases with chronic inflammation as the damage mechanism, and can secrete IL-17 and other inflammatory cytokines.
  • Flow cytometry was used to detect the proportion of Th17 cells (CD3 + CD4 + IL-17 + ) in the mesenteric lymph nodes, and it was found that the proportion of Th17 cells in the psilocybin treatment group was significantly lower than that of the DSS model (see Figure 5A, Figure 5B), and the tissue was uniform.
  • the content of IL-17 in the pulp was also significantly lower than that of the model group (Figure 5C).
  • Dextran sulfate sodium Dextran sulfate sodium
  • intestinal flora imbalance macrophage dysfunction
  • abnormal proliferation of intestinal mucosal epithelial cells and destruction of intestinal mucosal barrier.
  • the experimental results show that Pterocarpus polyglycosides can significantly alleviate the disease activity index of mice with inflammatory bowel disease for 5 days; Pterocarpus polyglycosides can significantly improve the weight loss of mice with inflammatory bowel disease for 6 days.
  • the treatment can improve the pathological changes of the colon in mice with inflammatory bowel disease, reduce the infiltration of inflammatory cells in the colon tissue and reduce the proportion of macrophages and neutrophils in the mesenteric lymph nodes, and improve the permeability of the intestinal mucosa of mice with inflammatory bowel disease Sex, suggesting that Pterocarpus polyglycosides have a regulatory effect on intestinal mucosal immunity. From the above results, we can confirm that Pterocarpus polyglycosides have a significant improvement effect on inflammatory bowel disease in mice.
  • TNBS 2,4,6-trinitrobenzenesulfonic acid solution 5% aqueous solution
  • the cytokine detection kit ProcartaPlex Mouse Th1/Th2/Th9/Th17/Th22/Treg Cytokine 17 factor detection kit was purchased from Invitrogen. Hydroxypropyl methylcellulose (HPMC) was purchased from Sigma-Aldrich, USA. Anhydrous ethanol was purchased from Sinopharm Chemical Reagent Co., Ltd.
  • the traits light brown powder; of which 8 P. periploside Periploside A, C, D, E, K, O, Q, R cumulative content is 64.96%, 8 Periploside content is Periploside A 4.54% by weight, Periploside C 31.46% by weight, Periploside D 10.31% by weight, Periploside E 6.34% by weight, Periploside K 3.12% by weight, Periploside O 3.45% by weight, Periploside Q 3.76% by weight, Periploside R 1.98% by weight.
  • the polyglycoside is suspended in a solvent and diluted to the required concentration before use.
  • the solvent is 0.2% (w/v) HPMC.
  • Periploside C (periploside C), traits: white amorphous powder, purity: 99%. When Periploside C is used, it is suspended in a solvent and diluted to the required concentration before use. The solvent is 0.2% (w/v) HPMC.
  • Periploside F (periploside F), traits: colorless crystals, purity: 99%.
  • Periploside F is suspended in a solvent and diluted to the required concentration before use.
  • the solvent is 0.2% (w/v) HPMC.
  • Prednisone acetate tablets purchased from Shanghai Shangyao Xinyi Pharmaceutical Co., Ltd., 5 mg/tablet, used after being diluted with 0.2% (w/v) HPMC to the required concentration.
  • mice were randomly divided into 6 groups, normal control group, model control group, prednisone acetate control group, pterygoside 12.5 mg/kg treatment group, pterygoside treatment group 6.25 mg/kg Group, the treatment group of Pterocarpus polyglycoside 3.13mg/kg.
  • the prednisone acetate control group is the positive drug control group.
  • mice After fasting for 24 hours, the mice were anesthetized with isoflurane. Introduced into the rectum of mice about 4cm away from the anus with a transparent rubber tube, model control group, prednisone acetate 2mg/kg group, sageloside 12.5mg/kg treatment group, Periploside C 12.5mg/kg treatment group, treatment group and Periploside F The mice in the 12.5 mg/kg treatment group were slowly injected with 100 ⁇ L 2.5% (v/v) TNBS. The mice in the normal control group were injected with 50% (v/v) alcohol in the same way.
  • the prednisone acetate group was given oral prednisone acetate 2 mg/kg (prednisone acetate tablets dissolved in 0.2% (v/v) HPMC) once a day; treatment with glutinate Periploside C treatment group was orally given Periploside C 12.5 mg/kg once a day; Periploside F treatment group was orally given Periploside F 12.5 mg/kg once a day; The normal control group and the model group were given an equal volume of 0.2% (w/v) HPMC orally, once a day.
  • mice During the body weight experiment, the weight of each group of mice was monitored and recorded every day.
  • mice The death of mice during the experiment was recorded, and the survival rate curve was drawn.
  • mice colon tissue was quickly taken, and the length was measured from the bottom of the cecum to the bottom of the rectum, recorded and photographed.
  • Weight loss can reflect the health of mice and is an important disease indication for TNBS-induced inflammatory bowel disease.
  • the results are shown in Figure 6A.
  • the body weight of mice in the model control group was significantly reduced the next day after being modeled.
  • Four days after the administration of polyglycoside the body weight of the mice in this group was significantly higher than that in the model group. Higher than the normal group.
  • the body weight of the 12.5 mg/kg treatment group of Pterocarpus polyglycoside has returned to a normal level 7 days after the administration.
  • the body weight of the mice was significantly improved compared with the mice in the model group.
  • the treatments with Pterocarpus polyglycoside, Periploside C and F can significantly improve the survival rate of TNBS-induced colitis mice.
  • the length of the colon of the TNBS model control group was significantly shorter than that of the normal control group.
  • the positive drug prednisone acetate group, the 12.5mg/kg treatment group and the Periploside C 12.5mg/kg treatment group The colon length of mice in the Periploside F 12.5mg/kg treatment group was significantly higher than that of the model control group.
  • the inflammatory bowel disease model induced by trinitrobenzene sulfonic acid (TNBS) in mice is similar to the pathological changes of clinical ulcerative colitis.
  • TNBS trinitrobenzene sulfonic acid
  • the changes in fecal morphology, blood in the stool, intestinal morphology and intestinal histology changes in mice are equivalent to the clinical symptoms of patients with ulcerative colitis.
  • the TNBS-induced inflammatory bowel disease model was used to evaluate the pharmacodynamics of the therapeutic effect of puerpera polyglycoside.
  • the results show that Pterocarpus polyglycosides can significantly increase the survival rate of mice with TNBS-induced colitis, alleviate the weight loss of mice, and maintain the length of the mice's colon.
  • the above results show that it is clear that Pterocarpus polyglycoside has a significant improvement effect on inflammatory bowel disease in mice.
  • mice 8-10 weeks old, purchased from Shanghai Slack Laboratory Animal Co., Ltd.
  • DSS Sodium dextran sulfate
  • Periploside C (periploside C), traits: white amorphous powder, purity: 99%. When Periploside C is used, it is suspended in a solvent and diluted to the required concentration before use. The solvent is 0.2% (w/v) HPMC.
  • Periploside F (periploside F), traits: colorless crystals, purity: 99%.
  • Periploside F is suspended in a solvent and diluted to the required concentration before use.
  • the solvent is 0.2% (w/v) HPMC.
  • Sulfasalazine colon-soluble capsules purchased from Guangdong Qiangji Pharmaceutical Co., Ltd., 250 mg/capsule, used after being diluted with 0.2% (w/v) HPMC to the required concentration.
  • mice were randomly divided into 4 groups, a normal control group, a DSS model, a sulfasalazine treatment group, and a saponins treatment group.
  • the sulfasalazine treatment group was the positive control group.
  • the drinking water of the other groups including the DSS model, the sulfasalazine treatment group, and the saponins treatment group were all added 5% DSS to their drinking water, and the intestinal inflammation model was performed for 7 consecutive days. Induced model building.
  • the sulfasalazine treatment group received 250 mg/kg of sulfasalazine orally once a day; the treatment group received oral sulfasalazine 12.5 mg/kg once a day; Periploside The C treatment group was given Periploside C 12.5mg/kg orally once a day; the Periploside F treatment group was given Periploside F12.5mg/kg orally once a day; the normal control group and the DSS model were given 0.2% of the same volume orally. (w/v) HPMC, once a day.
  • mice The body weight of each group of mice was monitored and recorded every day; the feces of the mice were monitored with an occult blood kit and scored quantitatively. According to the disease activity index scoring table, the therapeutic effect of polyglycoside on DSS-induced inflammatory bowel disease was evaluated.
  • Weight loss is an important disease indication for DSS-induced inflammatory bowel disease, which can reflect the health of mice.
  • the results are shown in Fig. 7A, the saponins treatment group and the sulfasalazine-positive drug group can significantly improve the weight loss caused by DSS on the sixth and seventh days after treatment.
  • the weight loss of mice in the Periploside C and F treatment groups improved significantly after the seventh day after treatment.
  • the Disease Activity Index is a score based on the percentage of weight loss, stool consistency, and stool bleeding in mice with inflammatory bowel disease. The total score of the three results is divided by 3 to obtain the DAI value. The evaluation is small An important indicator of clinical symptoms of mouse disease.
  • the study found that the disease activity index of mice in the saponins treatment group and the sulfasalazine-positive drug group treatment group was significantly lower than that of the model group mice on the sixth and seventh days after treatment; Periploside C treatment group and Periploside F treatment The disease activity index of the mice in the group was significantly improved compared with the mice in the model group after the seventh day after treatment. The results are shown in Figure 7B. DSS induces inflammatory bowel disease to cause the colon to become shorter.
  • Acetic acid (glacial acetic acid) (10000218) purchased from Sinopharm Chemical Reagent Co., Ltd.; Bovine Type II Collagen (Immunization Grade Bovine Type II Collagen) purchased from Chondrex; Freund's Adjuvant Complete (CFA, Freund's complete adjuvant) (F5881 -10X10mL) purchased from SIGMA; Freund's Adjuvant, Incomplete (IFA, Freund's incomplete adjuvant) purchased from SIGMA; HRP-rabbit anti-mouse IgG (H+L) (616520), HRP-rabbit anti-mouse IgG2a (610220) ) was purchased from Invitrogen.
  • CFA Freund's Adjuvant Complete
  • IFA Freund's complete adjuvant
  • HRP-rabbit anti-mouse IgG H+L
  • 616520 HRP-rabbit anti-mouse IgG2a
  • the traits light brown powder; of which 8 P. periploside Periploside A, C, D, E, K, O, Q, R cumulative content is 64.96%, 8 Periploside content is Periploside A 4.54% by weight, Periploside C 31.46% by weight, Periploside D 10.31% by weight, Periploside E 6.34% by weight, Periploside K 3.12% by weight, Periploside O 3.45% by weight, Periploside Q 3.76% by weight, Periploside R 1.98% by weight.
  • the polyglycoside is suspended in a solvent and diluted to the required concentration before use.
  • the solvent is 0.2% (w/v) HPMC.
  • Methotrexate tablets Shanghai Pharmaceutical (Group) Co., Ltd. Xinyi Pharmaceutical Factory, 2.5mg/tablet, use 0.2% (w/v) HPMC to dilute to the required concentration before use.
  • the bovine type II collagen was dissolved in 0.1M sterile acetic acid and dissolved overnight at 4°C to prepare a solution with a collagen concentration of 10 mg/mL.
  • mice were divided into 5 groups according to their weight and foot swelling score. There are 6 experimental groups, each with 10 animals: normal control group, closed model control group, and positive drug methotrexate group ( 1 mg/kg, 1 time/day), Pterocarpus polyglycosides (12.5 mg/kg/day, 6.25 mg/kg/day; 2 times/day). All groups were given oral gavage. The administration was continued for 8 weeks to investigate the effect of oral administration of polyglycoside on the indications of arthritis in CIA mice.
  • the DBA/1 mouse serum was separated at the end of the experiment, and the serum was stored in a refrigerator at -30°C.
  • the serum level of anti-bovine type II collagen (CII) specific antibody IgG was detected by ELISA.
  • mice were fixed in formalin, paraffin-embedded sections, and H&E stained to make pathological sections.
  • the clinical score of arthritis is an important indicator reflecting the occurrence and development of arthritis lesions.
  • the experimental results show that the three dose groups (12.5mg/kg, 6.25mg/kg) administered with Pterocarpus polyglycoside can reduce the incidence index of collagen-induced arthritis mice, which is expressed in the limbs and feet of collagen-induced arthritis mice.
  • the number and severity of paw swelling was significantly reduced (Figure 8A)
  • Collagen-induced arthritis mouse model mice may lose weight as the disease progresses.
  • the weight of the mice was recorded twice a week, and the results showed that the body weight of the mice in the Pterocarpus polyglycoside treatment group increased compared with the model group ( Figure 8B).
  • Model group 3/8 animals had severe inflammation, 1/8 animals had moderate inflammation, 3/8 animals had mild inflammation, and 1/8 animals had no abnormalities. Compared with the control group, severe inflammation can be seen in the ankle joint of the model group, and the histopathological morphology showed that a large number of inflammatory cells infiltrated the synovium and the periphery of the synovium, extending to the surrounding tissues, eroding cartilage and accompanied by fibrosis.
  • methotrexate group 2/8 animals had severe inflammation, 3/8 animals had moderate inflammation, and 3/8 animals had no abnormalities.
  • Pterocarpus polyglycoside group 2/8 animals in the 12.5 mg/kg group had moderate inflammation, 1/8 animals had mild inflammation, 2/8 animals had mild inflammation, and 3/8 animals had no abnormalities; 6.25 mg/kg In the group, 1/8 animals had moderate inflammation, 1/8 animals had mild inflammation, 4/8 animals had mild inflammation, and 2/8 animals had no abnormalities.
  • CII bovine type II collagen
  • Type II bovine collagen (CII)-induced DBA/1 mouse arthritis (Collagen-induced arthritis, CIA) manifests as severe multiple joint damage, its clinical and pathological manifestations, humoral immunity, and cellular immune response Similar to human rheumatoid arthritis, it is widely used in the evaluation of the efficacy of anti-arthritis drugs and the study of the mechanism of action. In this experiment, collagen was used to induce arthritis in mice, and to observe the therapeutic effect of oral administration of collagen-induced arthritis in mice.
  • Pterocarpus polyglycosides show good therapeutic effects, which can significantly improve the clinical symptoms of collagen-induced arthritis in model mice, reduce the clinical scores of arthritis, improve the pathological damage and the production of collagen-specific antibodies, indicating that Pterocarpus polyglycosides is an adjuvant Inducing arthritis in mice has a significant therapeutic effect.
  • SD rat, male, about 150-180g purchased from Shanghai Slack Laboratory Animal Co., Ltd.
  • the traits light brown powder; of which 8 P. periploside Periploside A, C, D, E, K, O, Q, R cumulative content is 64.96%, 8 Periploside content is Periploside A 4.54% by weight, Periploside C 31.46% by weight, Periploside D 10.31% by weight, Periploside E 6.34% by weight, Periploside K 3.12% by weight, Periploside O 3.45% by weight, Periploside Q 3.76% by weight, Periploside R 1.98% by weight.
  • the polyglycoside is suspended in a solvent and diluted to the required concentration before use.
  • the solvent is 0.2% (w/v) HPMC.
  • Methotrexate tablets Shanghai Pharmaceutical (Group) Co., Ltd. Xinyi Pharmaceutical Factory, 2.5mg/tablet, use 0.2% (w/v) HPMC to dilute to the required concentration before use.
  • Inactivated tuberculosis lyophilized powder (M.TUBRCULOSIS H37 Ra) was purchased from DIFCO.
  • mice Male SD rats, except the normal control group, were divided into 5 groups according to body weight, foot swelling score and swelling degree on the 13th day after modeling. There are 6 experimental groups in total, 8 in each group: normal control group, joints Inflammation model group (solvent control), positive drug MTX group (1 mg/kg, 1 time/day), sagebrush polyglycoside (10 mg/kg/day, 5 mg/kg/day, 2.5 mg/kg/day; 2 times/day) sky).
  • normal control group joints Inflammation model group (solvent control)
  • positive drug MTX group (1 mg/kg, 1 time/day
  • sagebrush polyglycoside 10 mg/kg/day, 5 mg/kg/day, 2.5 mg/kg/day; 2 times/day) sky).
  • the swelling of the primary foot (left hind foot) in rats reflects arthritic lesions, and the sensitized side (right hind foot) has secondary autoimmune swelling.
  • the toe volume meter is used to detect the swelling degree of rat arthritis, and the calculation formula is as follows:
  • Rat joint swelling ( ⁇ L) model group/experimental treatment group rat hind foot drainage volume-unmodeled normal control group hind foot average drainage volume
  • the hind limbs of the rats were fixed in formalin, paraffin-embedded sections, and H&E stained to make pathological sections.
  • Pterocarpus polyglycosides showed a good therapeutic effect in the treatment of adjuvant-induced arthritis models in rats, which can significantly improve the clinical symptoms of arthritis in model rats and reduce the clinical scores of arthritis.
  • Normal control group 2/8 of the animals showed slight or mild inflammation, and the other animals had no obvious abnormalities.
  • Model control group Compared with the control group, all animals have mild to severe inflammation.
  • the histopathological morphology of ankle joint inflammation in the model group showed that a large number of inflammatory cells infiltrated the synovium and the periphery of the synovium, extending to the surrounding tissues, eroding cartilage and bone, and accompanied by fibrosis.
  • Pterocarpus polyglycoside group No abnormalities were seen in the 10 mg/kg treatment group; 4/8 animals in the 5 mg/kg treatment group showed mild or moderate inflammation, while the rest of the animals had no obvious abnormalities; 2.5 mg/kg treatment group 2 Moderate inflammation was seen in 8 animals, and no obvious abnormalities were seen in the remaining animals.
  • Adjuvant-induced arthritis (AA) model was created by bacteriologist Freund in the 1950s. It is also known as Freund's adjuvant arthritis. It is a classic immune inflammation model and is widely used in the treatment of Preclinical evaluation of candidate rheumatoid drugs.
  • CFA was used to induce experimental adjuvant arthritis in SD rats, and to observe the therapeutic effect of oral administration of psilocybin on adjuvant-induced arthritis in SD rats.
  • Pterocarpus polyglycosides showed good therapeutic effects in the treatment of adjuvant-induced arthritis models in rats, which can significantly improve the clinical symptoms of arthritis in model rats, reduce the clinical scores of arthritis, and reduce bone erosion and inflammatory infiltration in arthritic rats Condition.
  • Acetic acid (glacial acetic acid) (10000218) purchased from Sinopharm Chemical Reagent Co., Ltd.; Bovine Type II Collagen (Immunization Grade Bovine Type II Collagen) purchased from Chondrex; Freund's Adjuvant Complete (CFA, Freund's complete adjuvant) (F5881 -10X10mL) purchased from SIGMA; Freund's Adjuvant, Incomplete (IFA, Freund's incomplete adjuvant) purchased from SIGMA; HRP-rabbit anti-mouse IgG (H+L) (616520), HRP-rabbit anti-mouse IgG2a (610220) ) was purchased from Invitrogen.
  • CFA Freund's Adjuvant Complete
  • IFA Freund's complete adjuvant
  • HRP-rabbit anti-mouse IgG H+L
  • 616520 HRP-rabbit anti-mouse IgG2a
  • the traits light brown powder; of which 8 P. periploside Periploside A, C, D, E, K, O, Q, R cumulative content is 64.96%, 8 Periploside content is Periploside A 4.54% by weight, Periploside C 31.46% by weight, Periploside D 10.31% by weight, Periploside E 6.34% by weight, Periploside K 3.12% by weight, Periploside O 3.45% by weight, Periploside Q 3.76% by weight, Periploside R 1.98% by weight.
  • the polyglycoside is suspended in a solvent and diluted to the required concentration before use.
  • the solvent is 0.2% (w/v) HPMC.
  • Periploside C (periploside C), traits: white amorphous powder, purity: 99%. When Periploside C is used, it is suspended in a solvent and diluted to the required concentration before use. The solvent is 0.2% (w/v) HPMC.
  • Methotrexate tablets Shanghai Pharmaceutical (Group) Co., Ltd. Xinyi Pharmaceutical Factory, 2.5mg/tablet, use 0.2% (w/v) HPMC to dilute to the required concentration before use.
  • the bovine type II collagen was dissolved in 0.1M sterile acetic acid and dissolved overnight at 4°C to prepare a solution with a collagen concentration of 10 mg/mL.
  • mice were divided into 5 groups according to their weight and foot swelling score. There are 6 experimental groups, each with 10 animals: normal control group, closed model control group, and positive drug methotrexate group ( 1 mg/kg, 1 time/day), Pterocarpus polyglycosides (12.5 mg/kg/day; 2 times/day), Pseudoside C (12.5 mg/kg/day; 2 times/day). All groups were given oral gavage. The administration was continued for 10 weeks to compare the effects of oral encephaloside or encephaloside C on the indications of arthritis in CIA mice, and the hind limbs of each group of mice were photographed and recorded at the 10th week after administration.
  • mice in the psilococcus polyglycoside treatment group can reduce the incidence index of collagen-induced arthritis rats after 4 weeks of administration, and the psilocin C treatment group significantly reduces the incidence index of collagen-induced arthritis rats after 6 weeks of treatment.
  • the statistically significant drug effect was 2 weeks later ( Figure 13A).
  • Collagen-induced arthritis mouse model mice may lose weight as the disease progresses.
  • the weight of the mice was recorded once a week. The results showed that the body weight of the mice in the Pterocarpus polyglycoside treatment group was higher than that of the model group and the Palibradin C treatment group.
  • Trend Figure 13B).
  • Type II bovine collagen (CII)-induced DBA/1 mouse arthritis (Collagen-induced arthritis, CIA) manifests as severe multiple joint damage, its clinical and pathological manifestations, humoral immunity, and cellular immune response Similar to human rheumatoid arthritis, it is widely used in the evaluation of the efficacy of anti-arthritis drugs and the study of the mechanism of action.
  • collagen was used to induce arthritis in mice, and the therapeutic effects of oral administration of pteroside and pteroside C on collagen induced arthritis in mice were observed.
  • Pterocarpus polyglycosides and Pseudoside C show good therapeutic effects and can significantly reduce the clinical scores of arthritis.
  • psilocybin C it has a better effect on maintaining the body weight of collagen-induced arthritis mice and exerts a significant therapeutic effect earlier, indicating that psilocybin has a better effect on the treatment of arthritis.
  • mice Female, 18-20 grams, purchased from Shanghai Slack Laboratory Animal Co., Ltd.
  • Concanavalin A was purchased from Sigma; Bacterial lipopolysaccharide (LPS) was purchased from Sigma; MTT was purchased from Sigma; 3 H-thymine nucleotide (1 ⁇ Ci/mL) was purchased from PerkinElmer ; Dimethyl sulfoxide (DMSO) was purchased from Sinopharm Chemical Reagent Co., Ltd.
  • the traits light brown powder; of which 8 P. periploside Periploside A, C, D, E, K, O, Q, R cumulative content is 64.96%, 8 Periploside content is Periploside A 4.54% by weight, Periploside C 31.46% by weight, Periploside D 10.31% by weight, Periploside E 6.34% by weight, Periploside K 3.12% by weight, Periploside O 3.45% by weight, Periploside Q 3.76% by weight, Periploside R 1.98% by weight.
  • MTT method detects the effect of compounds on the immunosuppressive activity of mouse spleen lymphocytes:
  • Mouse spleen lymphocyte suspension 5*10 5 /well was inoculated into 96-well plates, and different concentrations of compounds were added at the same time, and corresponding vehicle control (cell control) and culture medium background control (blank control) were set up, the total volume was 200 ⁇ L .
  • MTT solution with a concentration of 5 mg/mL was added 4 hours before the end of the culture.
  • the supernatant was aspirated, and 200 ⁇ L DMSO was added to each well to dissolve the purple crystals, and the OD value was measured at 570 nM of the microplate reader.
  • the formula is as follows:
  • Mouse spleen lymphocyte suspension 5*10 5 /well was inoculated into 96-well plates, and ConA (final concentration 5 ⁇ g/mL) or LPS (final concentration 10 ⁇ g/mL) and different concentrations of compounds were added to make the final volume of the culture system 200 ⁇ L;
  • ConA final concentration 5 ⁇ g/mL
  • LPS final concentration 10 ⁇ g/mL
  • Each experimental group is provided with 3 multiple holes, and a non-irritating background control and corresponding solvent control group are also set. Incubate in an incubator containing 5% CO 2 at 37°C for 48 hours.
  • the selection index (SI) is the safety range for judging the effect of the drug, and the selection index is CC 50 /IC 50. The larger the index, the greater the safety range.
  • Pterocarpus polyglycoside has good immunosuppressive activity. The SI value shows that its safety is high, the immunosuppressive activity is strong, and it has potential medicinal value (see the table below).
  • Pterocarpus polyglycosides has higher cytotoxicity and the strongest immunosuppressive activity than its 8 main active ingredients. It shows that the immunosuppressive activity of Pterocarpus polyglycosides is better than that of 8 monomer Pseudosides compounds.

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Abstract

一种杠柳苷组合物、制备方法及用途。杠柳苷组合物包括以下组分：Periploside A 0.1-49%重量份、Periploside C 0.1-49%重量份、Periploside D 0.1-49%重量份、Periploside E 0.1-20%重量份、Periploside K 0.1-20%重量份、Periploside O 0.1-20%重量份、Periploside Q 0.1-20%重量份、Periploside R 0.1-20%重量份。以杠柳多苷为活性成分的药物制剂可以保护肠道组织结构，减少肠道组织中炎症细胞浸润；并且可以缓解关节组织炎症浸润情况，缓解关节炎骨侵蚀情况。

Description

杠柳苷组合物、其制备方法及其在制备治疗自身免疫性疾病的药物中的用途 技术领域
本公开涉及一种自身免疫性疾病药物，具体而言，本公开涉及一种杠柳苷组合物、其制备方法及其在制备治疗自身免疫性疾病的药物中的用途。
背景技术
炎症性肠病(Inflammatory Bowel Disease,IBD)是一组特定的慢性、反复发作的肠道疾病的统称，主要包括克罗恩病(Crohn's disease,CD)和溃疡性结肠炎(Ulcerative colitis，UC)两种。溃疡性结肠炎是一种会引起结肠与直肠发炎与溃疡的慢性疾病，病因尚未完全阐明，可能与遗传因素、自身免疫病、肠道菌群的改变等因素相关。其发作时的主要症状包括腹痛与伴有血便的腹泻，体重减轻、发热以及贫血的症状也有可能在溃疡性结肠炎发作时出现。通常其症状发生的进程缓慢，且轻重不一，其症状表现常间歇出现，两次发作中间常伴随有一段无症状期。溃疡性结肠炎的并发症可能包括巨结肠症或眼部、关节或肝脏的炎症，以及结肠癌。目前IBD已成为一种全球性疾病，临床干预主要局限于抗炎和免疫调节药物。然而，现有治疗药物无法完全控制IBD的进展和复发，存在多种缺陷，临床治疗迫切需要开发新的防治手段。
类风湿性关节炎(Rheumatoid arthritis，RA)是一种常见的自身免疫性疾病，伴有进行性残疾，可引起全身并发症，甚至早期死亡，耗费庞大的社会和经济成本。类风湿性关节炎发病机制复杂，可能与遗传、环境等因素有关，目前仍无法治愈，治疗手段主要以控制疾病发展为主。目前临床常用的RA治疗药物有非甾体类抗炎药(Nonsteroidal Antiinflammatory Drugs，NSAIDs)、改善病情的抗风湿药(Disease Modifying Antirheumatic Drugs,DMARDs)、激素类药物、生物制剂等。非甾体类抗炎药是一类不含有甾体结构的解热镇痛抗炎药，为目前治疗RA的一线药物，它主要是通过抑制前列腺素的合成，从而达到抗炎、镇痛和缓解肿胀的作用。但NSAIDs仅能缓解RA患者的临床症状，消除或减轻关节局部的炎症反应，对疾病的活动及进展无法控制，必须加用DMARDs才能有效控制病情；另外服用NSAIDs 存在潜在的心血管和消化道出血风险。临床上将具有控制病情发展的药物统称为DMARDs。DMARDs包括两大类：1.人工合成改善病情抗风湿药(sDMARDs)，如羟氯喹(hydroxychloroquine)、柳氮磺吡啶(sulfasalazine，SSZ)、金制剂(gold compounds)、甲氨蝶呤(methotrexate，MTX)、来氟米特(leflunomide)、环磷酰胺(Cyclophosphamide)、环孢素(Cyclosporin A)等，长期用药会有胃肠道、肝肾功能损伤、骨髓抑制等毒副作用。2.生物类DMARDs(bDMARDs)，包括阿那白滞素(Anakinra，IL-1受体拮抗剂)、英夫利昔单抗(Inflectra，TNF-α单抗)、阿巴西普(Abatacept，TNF-α单抗)、利妥昔单抗(Rituximab，抗CD20单抗)、托珠单抗(tocilizumab，IL-6单抗)。bDMARDs价格昂贵，限制了其临床的广泛应用。糖皮质激素类如泼尼松、泼尼松龙、甲泼尼松龙等治疗RA疗效显著，起效快、改善关节功能，控制疾病发展，但长期用药会引起许多不良反应，且不能根治RA，停药后疾病容易复发。类风湿性关节炎在传统中医学理论中属“痹证”范畴，中医对痹证的治疗有几千年的丰富经验。在中医药理论的指导下，对RA辩证施治，起到了标本兼治的效果，且以治本为主，副作用小。
中药作为中国的传统药物，其悠久的临床使用经验和治疗效果为发现慢性复杂性疾病的治疗新策略提供了新颖的资源。萝藦科杠柳属植物杠柳(Periploca sepium Bunge)的根皮，中药称为香加皮或北五加皮，是一种临床常用中药，具有悠久的药用历史，从1977年起的各版《中国药典》均有收载，具有祛风除湿的功效，被用于治疗风湿和类风湿性关节炎。中药香加皮中存在一系列杠柳苷类化合物，其结构特征是含有原酸酯基团的孕甾烷糖苷类化合物，8种主要的杠柳苷类化合物(Periploside A、C、D、E、K、O、Q、R)和微量杠柳苷类化合物Periploside F的化学结构式如下所示。其中化合物Periploside A和C分别对类风湿性关节炎、自身免疫性肝炎和实验性多发性硬化症动物模型具有良好的治疗效果。{参考文献：[1]Zhu Y N,Zhao W M,Yang Y F,et al.Periplocoside E,an effective compound from Periploca sepium Bge,inhibited T cell activation in vitro and in vivo[J].Journal of Pharmacology and Experimental Therapeutics,2006,316(2):662-669.[2]Zhu Y N,Zhong X G,Feng J Q,et al.Periplocoside E inhibits experimental allergic encephalomyelitis by suppressing interleukin 12-dependent CCR5expression and interferon-γ-dependent CXCR3expression in T lymphocytes[J].Journal of Pharmacology and Experimental Therapeutics,2006,318(3):1153-1162.[3]Wan J,Zhu Y N,Feng J Q,et al.Periplocoside A,a pregnane glycoside from Periploca  sepium Bge,prevents concanavalin A-induced mice hepatitis through inhibiting NKT-derived inflammatory cytokine productions[J].International Immunopharmacology,2008,8(9):1248-1256.[4]Zhang J,Ni J,Chen Z,et al.Periplocoside A prevents experimental autoimmune encephalomyelitis by suppressing IL-17production and inhibits differentiation of Th17 cells[J].Acta Pharmacologica Sinica,2009,30(8):1144.[5]Yang Y,Hu X,Cheng L,et al.Periplocoside A ameliorated type II collagen-induced arthritis in mice via regulation of the balance of Th17/Treg cells[J].International Immunopharmacology,2017,44:43-52.}
杠柳苷类化合物Periploside F在香加皮中含量较低，但可从其他高含量的杠柳苷类化合物(例如Periplcoside C)通过化学转化得到，Periploside F对T淋巴细胞的增殖也具有显著的抑制作用。{参考文献：Wang LY,Chen ZH,Zhou Y,et al.Structural revision of periplocosides and periperoxides,natural immunosuppressive agents from the genus Periploca.Phytochemistry,2011,72,2230-2236.}杠柳苷类化合物在香加皮药材中的含量约为0.1％，且药材中存在强心苷类有毒成分及大量与杠柳苷类化合物性质近似的脂肪酸、低级性寡糖和4-甲氧基水杨醛等成分，富含杠柳苷类化合物的有效成分(即杠柳多苷)难以制备，其制备方法尚未见文献报道。
Figure PCTCN2021088918-appb-000001
Figure PCTCN2021088918-appb-000002
发明内容
发明目的
发明人通过摸索各种提取工艺的组合，发现了一种杠柳苷组合物的制备方法，分析了通过该方法提取的杠柳苷类化合物的组成，并发现了杠柳苷类化合物在治疗炎症性肠病中的作用效果。在此基础上完成了本公开。
本公开的一个目的是提供一种杠柳苷组合物。
本公开的另一个目的是提供一种杠柳苷组合物的制备方法。
本公开的再一个目的是提供所述杠柳苷组合物的制药用途。
根据本公开的一个方面，其提供了一种杠柳苷组合物，其包括以下组分：
Periploside A 0.1-49％重量份、
Periploside C 0.1-49％重量份、
Periploside D 0.1-49％重量份、
Periploside E 0.1-20％重量份、
Periploside K 0.1-20％重量份、
Periploside O 0.1-20％重量份、
Periploside Q 0.1-20％重量份、
Periploside R 0.1-20％重量份。
所述组分Periploside A、C、D、E、K、O、Q和R的质量之和大于所述组合物总质量的50％，优选大于60％。
根据本公开的另一个方面，其提供了一种杠柳苷组合物的制备方法，包括：
(1)提取、浓缩步骤
取萝藦科杠柳属(Periploca)植物杠柳(Periploca sepium Bunge)的根皮加水提取，弃去提取液，将药渣加40％-95％乙醇提取1-10次，将醇提液合并浓缩，得到悬浊液；
(2)柱前预处理步骤
在搅拌条件下向悬浊液加入乙醇-水混合物使乙醇含量为10％-50％，进行固液分离；将固形物溶于乙醇-水混合物使乙醇含量为60％以上，从而得到清液，将清液用烃类溶剂萃取，将萃余液作为大孔树脂柱上样液；
其中，所述烃类溶剂是选自C5-C8烃溶剂(如环戊烷、正己烷、环己烷、环己烯、正庚烷、正辛烷、异辛烷)和石油醚的一种或多种；
(3)柱层析纯化步骤
用大孔树脂填充色谱柱，将上样液上样后，依次采用60％-75％乙醇除杂质，80％-90％乙醇洗脱，再用90％-95％乙醇洗脱，收集90％-95％乙醇洗脱液，浓缩，干燥得到粉末混合物；
(4)柱后处理步骤
将粉末混合物用乙醇-水精制处理，得到杠柳苷组合物。
根据本公开的另一方面，其提供了杠柳苷组合物在制备治疗自身免疫性疾病的药物中的用途。
有益效果
为了降低杠柳苷类药物的成本，本公开的发明人通过对杠柳根皮有效成分的提取方法进行反复实验，在实验中发现了特别有效的提取工艺。
通过对所述提取工艺进行进一步分析，发明人发现通过该提取工艺提取得到的特定组成的杠柳苷组合物中的periploside C和F对自身免疫性疾病，如克罗恩病和溃疡性结肠炎等炎症性肠病有活性。
附图说明
图1为葡聚糖硫酸钠(DSS)诱导小鼠炎症性肠病实验期间各组小鼠体重及炎症性肠病疾病活动指数评分曲线。
图2A为DSS诱导小鼠炎症性肠病实验终点各组小鼠结直肠组织的代表性照片。
图2B为DSS诱导小鼠炎症性肠病实验终点，各组小鼠结肠长度统计结果。与DSS造模组比较，*，p<0.05；***，p<0.001,(n＝8)。
图2C为DSS诱导肠炎模型小鼠实验终点结肠组织，通过H&E染色法制成病理切片代表性示意图。
图3A为DSS诱导小鼠炎症性肠病实验终点，通过流式细胞术分析小鼠肠系膜淋巴结中巨噬细胞(抗F4/80抗体+抗CD11b抗体的染色双阳性)比例代表性示意图和统计结果(n＝5)；
图3B为DSS诱导小鼠炎症性肠病实验终点，通过流式细胞术分析小鼠肠系膜淋巴结内中性粒细胞(抗Gr-1抗体+抗CD11b抗体的染色双阳性)比例代表性示意图以及统计结果(n＝5)；
图3C为DSS诱导小鼠炎症性肠病实验终点，小鼠结直肠组织中髓过氧化物酶(MPO)中表达水平的免疫组化分析代表性示意图。
图4为DSS诱导小鼠炎症性肠病实验终点，小鼠血清中异硫氰酸荧光素(FITC)标记标记的葡聚糖含量统计结果(n＝6)。
图5A为DSS诱导小鼠炎症性肠病实验终点，通过流式细胞术分析小鼠肠系膜淋巴结中Th17细胞比例(CD3 +CD4 +IL-17 +)。
图5B为Th17细胞比例统计结果(n＝5)。
图5C为DSS诱导小鼠炎症性肠病实验终点，小鼠血清中白细胞介素-17(IL-17)含量水平(n＝5)。
图6A为三硝基苯磺酸(TNBS)诱导小鼠炎症性肠病实验期间，各组小鼠体重变化曲线；
图6B为TNBS诱导小鼠炎症性肠病实验期间，各组小鼠生存率变化曲线；
图6C为TNBS诱导小鼠炎症性肠病实验终点，各组小鼠结肠长度统计结果(n＝3-10)。
图6D为结肠长度照片结果。
图7(A)为DSS诱导小鼠炎症性肠病实验期间各组小鼠体重。
图7(B)为各组小鼠的炎症性肠病疾病活动指数评分曲线。
图7C为DSS诱导小鼠炎症性肠病实验终点，各组小鼠结肠长度统计结果。与DSS造模组比较，*，p<0.05；***，p<0.001,(n＝10)。
图7D为DSS诱导小鼠炎症性肠病实验终点各组小鼠结直肠组织的代表性照片。
图8A为胶原诱导小鼠关节炎实验期间各组小鼠的关节临床评分。
图8B为胶原诱导小鼠关节炎实验期间各组小鼠的体重。
图9为胶原诱导小鼠关节炎实验终点，通过H&E染色法制成病理切代表性示意图。
图10A为胶原诱导小鼠关节炎实验终点，小鼠血清中抗胶原特异性抗体血清总IgG的水平。
图10B为胶原诱导小鼠关节炎实验终点，小鼠血清中抗胶原特异性抗体血清IgG2a的水平。
图11A为佐剂诱导大鼠关节炎实验期间各组大鼠关节临床评分。
图11B为佐剂诱导大鼠关节炎实验期间各组大鼠继发足肿胀程度。
图11C为佐剂诱导大鼠关节炎实验期间各组大鼠体重结果。
图12A为佐剂诱导大鼠关节炎实验终点，各组大鼠继发足代表性照片。
图12B为佐剂诱导大鼠关节炎实验终点，各组大鼠通过微计算机断层扫描骨侵蚀情况代表性示意图。
图13为佐剂诱导大鼠关节炎实验终点，通过H&E染色法制成病理切代表性示意图。
具体实施方式
为使本领域具有普通知识的人员可了解本发明的特点及效果，以下谨就说明书及申请专利范围中提及的术语及用语进行一般性的说明及定义。除非另有指明，否则文中使用的所有技术及科学上的字词，皆具有本领域技术人员对于本发明所了解的通常意义，当有冲突情形时，应以本说明书的定义为准。
在本文中，用语“包含”、“包括”、“具有”、“含有”或其他任何类似用语均属于开放性连接词(open-ended transitional phrase)，其意欲涵盖非排他性的包括物。举例而言，含有复数要素的一组合物或制品并不仅限于本文所列出的这些要素而已，而是还可包括未明确列出但却是该组合物或制品通常固有的其他要素。除此之外，除非有相反的明确说明，否则用语“或”是指涵盖性的“或”，而不是指排他性的“或”。例如，以下任何一种情况均满足条件“A或B”：A为真(或存在)且B为伪(或不存在)、A为伪(或不存在)且B为真(或存在)、A和B均为真(或存在)。此外，在本文中，用语“包含”、“包括”、“具有”、“含有”的解读应视为已具体公开并同时涵盖“由…所组成”及“实质上由…所组成”等封闭式或半封闭式连接词。
在本文中，所有以数值范围或百分比范围形式界定的特征或条件仅是为了简洁及方便。据此，数值范围或百分比范围的描述应视为已涵盖且具体公开所有可能的次级范围及范围内的个别数值，特别是整数数值。举例而言，“1至8”的范围描述应视为已经具体公开如1至7、2至8、2至6、3至6、4至8、3至8等等所有次级范围，特别是由所有整数数值所界定的次级范围，且应视为已经具体公开范围内如1、2、3、4、5、6、7、8等个别数值。除非另有指明，否则前述解释方法适用于本发明全文的所有内容，不论范围广泛与否。
若数量或其他数值或参数是以范围、较佳范围或一系列上限与下限表示，则其应理解成是本文已特定公开了由任一对该范围的上限或较佳值与该范围 的下限或较佳值构成的所有范围，不论这些范围是否有分别公开。此外，本文中若提到数值的范围时，除非另有说明，否则该范围应包括其端点以及范围内的所有整数与分数。
在本文中，在可实现发明目的的前提下，数值应理解成具有该数值有效位数的精确度。举例来说，数字40.0则应理解成涵盖从39.50至40.49的范围。
在本文中，对于使用马库什群组(Markush group)或选项式用语以描述本发明特征或实例的情形，本领域技术人员应了解马库什群组或选项列表内所有要素的次级群组或任何个别要素亦可用于描述本发明。举例而言，若X描述成“选自于由X 1、X 2及X 3所组成的群组”，亦表示已经完全描述出X为X 1的主张与X为X 1及/或X 2的主张。再者，对于使用马库什群组或选项式用语以描述本发明的特征或实例的情况，本领域技术人员应了解马库什群组或选项列表内所有要素的次级群组或个别要素的任何组合亦可用于描述本发明。据此，举例而言，若X描述成“选自于由X 1、X 2及X 3所组成的群组”，且Y描述成“选自于由Y 1、Y 2及Y 3所组成的群组”，则表示已经完全描述出X为X 1或X 2或X 3而Y为Y 1或Y 2或Y 3的主张。
以下具体实施方式本质上仅是例示性，且并不欲限制本发明及其用途。此外，本文并不受前述现有技术或发明内容或以下具体实施方式或实施例中所描述的任何理论的限制。
根据本公开的一个实施方式，其提供了一种杠柳苷组合物，其包括以下组分：
Periploside A 0.1-49％重量份、
Periploside C 0.1-49％重量份、
Periploside D 0.1-49％重量份、
Periploside E 0.1-20％重量份、
Periploside K 0.1-20％重量份、
Periploside O 0.1-20％重量份、
Periploside Q 0.1-20％重量份、
Periploside R 0.1-20％重量份。
包含上述组分的杠柳苷组合物在治疗自身免疫性疾病过程中体现出更好的治疗效果。
根据本公开的另一个实施方式，其中，所述Periploside A、C、D、E、K、O、Q、R，且其质量之和大于所述组合物总质量的50％，优选大于60％。
根据本公开的另一个实施方式，其提供了一种杠柳苷组合物的制备方法，包括：
(1)提取、浓缩步骤
取萝藦科杠柳属(Periploca)植物杠柳(Periploca sepium Bunge)的根皮加水提取，弃去提取液，将药渣加40％-95％乙醇提取1-10次，将醇提液合并浓缩，得到悬浊液；
(2)柱前预处理步骤
在搅拌条件下向悬浊液加入乙醇-水混合物使乙醇含量为10％-50％，进行固液分离；将固形物溶于乙醇-水混合物使乙醇含量为60％以上，从而得到清液，将清液用烃类溶剂萃取，将萃余液作为大孔树脂柱上样液；
其中，所述烃类溶剂是选自C5-C8烃溶剂(如环戊烷、正己烷、环己烷、环己烯、正庚烷、正辛烷、异辛烷)和石油醚的一种或多种；
(3)柱层析纯化步骤
用大孔树脂填充色谱柱，将上样液上样后，依次采用60％-75％乙醇除杂质，80％-90％乙醇洗脱，再用90％-95％乙醇洗脱，收集90％-95％乙醇洗脱液，浓缩，干燥得到粉末混合物，
(4)柱后处理步骤
将粉末混合物用乙醇-水精制处理，得到杠柳苷组合物。
其中，在步骤(1)中，在将醇提液合并浓缩后，由于浓缩过程乙醇被除去，浓缩液变为悬浊液。在步骤(2)中，悬浊液中加入低浓度乙醇不能溶解所需的固形物，而在加入高浓度乙醇后方可溶解期望的化合物。
根据上述方法，可以以低成本有效获得包含活性成分的药物组合物，而避免使用成本较高的色谱填料、有毒溶剂和化学合成工艺。
其中，所述烃类溶剂优选是环己烷。
根据本公开的另一个实施方式，所述制备方法包括
(1)提取、浓缩步骤
取萝藦科杠柳属(Periploca)植物杠柳(Periploca sepium Bunge)的根皮加2-10倍质量的水在5-100℃下提取2-10h，提取液弃去，将药渣加2-10倍质量的40％-95％乙醇提取1-10次，将醇提液合并浓缩至原料根皮的40％至80％质量，得到悬浊液；
(2)柱前预处理步骤
在搅拌条件下向悬浊液中加入40％-95％乙醇使乙醇含量为10％-50％，搅拌后固液分离取沉淀，之后，将沉淀再次悬浮于10％-50％乙醇搅拌后固液分离取沉淀，再将沉淀溶于60％以上的乙醇，过滤得到上清液，将上清液用烃类溶剂萃取3次，弃去所述烃类溶剂层，乙醇液经减压蒸除残留的烃类溶剂，得大孔树脂柱上样液；
其中，所述烃类溶剂是选自C5-C8烃溶剂(如环戊烷、正己烷、环己烷、环己烯、正庚烷、正辛烷、异辛烷)和石油醚的一种或多种；
(3)柱层析纯化步骤
用大孔树脂填充色谱柱，上样液经吸附后，依次采用60％-75％乙醇除杂质，80％-90％乙醇洗脱，再用90％-95％乙醇作为洗脱液进行第一轮洗脱，然后收集用80％-90％乙醇洗脱的洗脱液，浓缩至60％-75％乙醇后作为上样液再次上样，再依次用60％-75％乙醇、80％-90％乙醇、90％-95％乙醇作为洗脱液进行第二轮洗脱，收集第一轮和第二轮洗脱中用95％乙醇洗脱的洗脱液，浓缩，干燥得到粉末混合物；
(4)柱后处理步骤
将上述粉末混合物用70％-95％乙醇溶解，加水调至乙醇浓度为60％以下得到悬浊液，过滤该悬浊液，得第一上清液和第一沉淀物，将第一沉淀物加醇洗涤后过滤得第二上清液和第二沉淀物，将第一上清液和第二上清液合并后静置沉降，过滤得第三沉淀，合并第二沉淀和第三沉淀，干燥、粉碎即得杠柳苷组合物。
在该制备方法中，由于先使用水提工艺，可以低成本除去高极性成分；此外，通过采用不同浓度乙醇沉淀和烃类溶剂萃取工艺，可以除去杠柳苷以外的大量杂质，减少大孔树脂柱的上样量。
其中，所述烃类溶剂优选是环己烷。
根据本公开的另一个实施方式，其提供了一种根据上述方法制备的杠柳苷组合物。
根据本公开的另一个实施方式，其提供了上述杠柳苷组合物在制备治疗自身免疫性疾病的药物中的用途。
根据本公开的另一个实施方式，其中，所述自身免疫性疾病包括炎症性肠病和类风湿性关节炎。
根据本公开的另一个实施方式，其中，所述炎症性肠病包括溃疡性结肠炎和克罗恩病。
根据本公开的另一个实施方式，其中，所述治疗炎症性肠病的药物是通过抑制体重减轻，改善便溏、便血，保护肠道粘膜损伤，减轻结肠组织的病理学损伤，抑制结肠组织中炎症细胞的浸润，下调相关促炎性细胞因子的水平来起作用的。
根据本公开的另一个实施方式，其中，所述治疗类风湿性关节炎的药物是通过缓解关节部位肿胀程度，降低关节组织中炎症浸润情况，缓解关节炎病灶骨侵蚀程度，降低血清中特异性自身抗体的产生来起作用的。
根据本公开的另一个实施方式，其提供了杠柳苷化合物Periploside C和Periploside F在制备治疗炎症性肠病的药物中的用途。
制备实施例一：杠柳多苷的制备(一)
药材：500g香加皮药材。
提取：水提三次之后弃掉水提液，然后醇提两次，每次加入4L 70％乙醇，得到8L醇提液。
浓缩：醇提液进行浓缩得到300g悬浊液。
20％乙醇沉淀：悬浊液中兑乙醇至20％，固液分离得20％乙醇沉淀56.175g。
35％乙醇沉淀：对20％乙醇沉淀进行35％乙醇沉淀，固液分离得35％乙醇沉淀45.85g。
70％乙醇沉淀：加入2倍体积量95％乙醇，及4倍体积量70％乙醇进行65℃复溶，抽取上清液，获得70％乙醇上清液305mL。
环己烷萃取：305mL 70％乙醇上清液加入305mL环己烷萃取三次，乙醇液经减压蒸除残留的环己烷得到脱油的乙醇相。
柱层析：萃取后醇层进行大孔树脂柱层析(填料名称A4)，装好填料用95％乙醇清洗干净后用70％乙醇平衡柱子，加入物料后，依次采用70％、85％、95％乙醇梯度洗脱，洗脱体积分别为3BV、3BV、6BV。
85％乙醇部位回收：将85％乙醇洗脱部位(共200mL)兑稀乙醇至70％，泵入柱子后，分别用70％乙醇，80％乙醇，95％乙醇进行洗脱，洗脱体积分别为3BV、3BV、6BV。收集70％乙醇洗脱部位，80％乙醇洗脱部位，95％ 乙醇洗脱部位。将这一步与上一步所有70％与80％乙醇洗脱部位合并浓缩，95％乙醇洗脱部位也合并浓缩，最终50mL 95％乙醇洗脱部位浓缩液。
95％乙醇复溶除杂：50mL 95％乙醇部位浓缩液浓缩至干，在65℃水浴下加入12倍固体量95％乙醇进行复溶，过滤除杂，得到上清液。
60％乙醇沉淀过滤：加水调乙醇至60％，抽滤，滤饼用50％乙醇润洗后，55℃真空干燥，得到1.119g杠柳多苷原料药(1)。
60％乙醇上清液过柱层析：将60％乙醇上清液过B17型大孔树脂柱，依次用3BV75％乙醇和80％乙醇、6BV95％乙醇洗脱，收集80％乙醇第2、3个BV洗脱部位和95％第1、2个BV洗脱部位，合并浓缩，干燥，进行60％乙醇沉淀，将60％沉淀真空干燥，得到57mg杠柳多苷原料药(2)。
两步中间体混合：将上述杠柳多苷原料药(1)和(2)混合，共得1.176g中间体，测定其中8个杠柳苷类化合物Periploside A、C、D、E、K、O、Q、R的累积含量为59.09％，其中8个杠柳苷的含量分别为Periploside A 2.52％重量份、Periploside C 31.12％重量份、Periploside D 8.54％重量份、Periploside E 5.35％重量份、Periploside K 2.62％重量份、Periploside O 3.52％重量份、Periploside Q 3.67％重量份、Periploside R 1.75％重量份。
制备实施例二：杠柳多苷的制备(二)
药材：500g香加皮药材。
提取：水提三次之后弃掉水提液，然后醇提两次，每次加入4L 70％乙醇，得到8L醇提液。
浓缩：醇提液进行浓缩得到255g悬浊液。
20％乙醇沉淀：向悬浊液中加入50g 95％乙醇配成20％乙醇混悬液，固液分离得20％乙醇沉淀为164g。
35％乙醇沉淀：向20％乙醇沉淀中加入164g(与沉淀同质量)35％乙醇，混合均匀后固液分离得35％乙醇沉淀157g。
70％乙醇沉淀：157g 35％乙醇沉淀中共加入95％乙醇157mL，与70％乙醇278.5mL配置成70％乙醇混悬液，固液分离得70％乙醇上清液400mL。
环己烷萃取：400mL 70％乙醇上清液加入400mL环己烷萃取三次，乙醇液经减压蒸除残留的环己烷得到脱油的乙醇相。
柱层析：乙醇相过大孔树脂柱层析(填料名称A19)，装好填料用95％乙醇清洗干净后用70％乙醇平衡，加入物料后，采用70％、85％、95％乙醇梯度洗脱，洗脱体积分别为3BV、3BV、6BV。
85％乙醇部位回收：将85％乙醇洗脱部位作为85％乙醇上样液，兑低浓度乙醇至70％，泵入柱子后，分别用70％乙醇，80％乙醇，95％乙醇进行洗脱，收集70％乙醇部位，80％乙醇部位，95％乙醇部位。将95％部位合并浓缩，获得40mL 95％乙醇浓缩液。
95％乙醇复溶除杂：40mL 95％乙醇部位浓缩液浓缩至干，得到2.2g固形物，在65℃水浴下加入12倍固体量95％乙醇进行复溶，过滤除杂，得到上清液。
60％乙醇沉淀：向95％乙醇复溶上清液中加入17.6g水配制成60％乙醇溶液，加热到30-40℃趁热过滤得到沉淀，将其置于60℃鼓风烘箱烘干，得到1.014g杠柳多苷原料药，测定其中8个杠柳苷类化合物Periploside A、C、D、E、K、O、Q、R的累积含量为60.33％，其中8个杠柳苷的含量分别为Periploside A 2.74％重量份、Periploside C 30.33％重量份、Periploside D 9.62％重量份、Periploside E 5.27％重量份、Periploside K 3.04％重量份、Periploside O 3.89％重量份、Periploside Q 3.55％重量份、Periploside R 1.89％重量份。
制备实施例三：杠柳多苷的制备(三)
除了使用中试设备，以100倍规模提取香加皮药材以外，已与制备实施例一相同的方式对1.0吨香加皮药材进行了提取和分离纯化，制备出2.1kg杠柳多苷原料药。其中，8个杠柳苷Periploside A、C、D、E、K、O、Q、R的累积含量为64.96％，8个杠柳苷的含量分别为Periploside A 4.54％重量份、Periploside C 31.46％重量份、Periploside D 10.31％重量份、Periploside E 6.34％重量份、Periploside K 3.12％重量份、Periploside O 3.45％重量份、Periploside Q 3.76％重量份、Periploside R 1.98％重量份。
制备实施例四：杠柳苷类化合物Periploside C和Periploside F的制备
取制备实施例一所得杠柳多苷原料500mg进行反相C18中压色谱分离，色谱柱尺寸20(内径)×460(长度)mm，70-100％甲醇-水梯度洗脱，洗脱馏分采用Merck C18 HPTLC检测，合并含有periploside C的组分，减压浓缩至干，得到白色粉末状Periploside C 150mg，纯度99％。
另取制备实施例一所得杠柳多苷原料500mg，溶于10mL 1％氢氧化钠的甲醇溶液，室温放置24小时，反应液减压浓缩，并用氯仿-水分配，氯仿 层水洗后浓缩，并进行反相C18中压色谱分离，色谱柱尺寸20(内径)×460(长度)mm，70-100％甲醇-水梯度洗脱，洗脱馏分采用Merck C18 HPTLC检测，合并含有periploside F的组分，减压浓缩，析出Periploside F无色结晶180mg，纯度99％。
实验实施例1：杠柳多苷对葡聚糖硫酸钠(DSS)诱导溃疡性结肠炎的药效学研究
实验材料：
Balb/c雌性小鼠，8-10周龄，购自上海斯莱克实验动物有限责任公司。
试剂：硫酸葡聚糖钠(DSS，购自美国MP Biomedicals生物医学公司)，CD3、CD4、IL-17、IFN-γ、CD11b、GR1、F4/80等流式细胞抗体购自BD Pharmingen公司。细胞因子检测试剂盒ProcartaPlex Mouse Th1/Th2/Th9/Th17/Th22/Treg Cytokine 17因子检测试剂盒购自美国英杰生命技术有限公司(Invitrogen)。羟丙基甲基纤维素(HPMC)购自美国Sigma-Aldrich公司。隐血测试盒，购自南京建成生物工程研究所。
使用制备实施例三中制备的杠柳多苷(即杠柳苷组合物)，性状：浅棕色粉末；其中8个杠柳苷Periploside A、C、D、E、K、O、Q、R的累积含量为64.96％，8个杠柳苷的含量分别为Periploside A 4.54％重量份、Periploside C 31.46％重量份、Periploside D 10.31％重量份、Periploside E 6.34％重量份、Periploside K 3.12％重量份、Periploside O 3.45％重量份、Periploside Q 3.76％重量份、Periploside R 1.98％重量份。杠柳多苷使用时混悬于溶剂中并稀释至所需浓度后使用。溶剂为0.2％(w/v)HPMC。
柳氮磺吡啶结肠溶胶囊，购自广东强基药业有限公司，250mg/胶囊，使用时用0.2％(w/v)HPMC稀释至所需浓度后使用。
实验方法：
动物分组及处理:Balb/c小鼠随机分成4组，正常对照组，DSS造模组，柳氮磺吡啶治疗组，杠柳多苷治疗组。其中柳氮磺吡啶治疗组为阳性药对照组。除正常对照组的小鼠以外，其他各组包括DSS造模组、柳氮磺吡啶治疗组、杠柳多苷治疗组的饮用水中均加入了5％DSS，连续7天进行肠道炎症模型的诱导造模。自造模当日起，柳氮磺吡啶治疗组经口给与柳氮磺吡啶250mg/kg，一日一次；杠柳多苷治疗组口服给予杠柳多苷12.5mg/kg，一日 一次；正常对照组和DSS造模组分别经口给予等体积的0.2％(w/v)HPMC，一日一次。
监测指标：
[一]疾病活动指数(Disease activity index,DAI)：实验期间每天对各组小鼠体重进行监测并记录；小鼠粪便采用隐血试剂盒监测并进行量化评分。依据疾病活动指数评分表评价杠柳多苷对DSS诱导炎症性肠病的治疗效果。
疾病活动指数＝(粪便形态评分+粪便隐血评分+体重减轻百分比评分)/3。
疾病活动指数(DAI)评分表：
Figure PCTCN2021088918-appb-000003
[二]小鼠结肠组织整体病理学评价
实验终点迅速取小鼠结肠组织，自盲肠底部至直肠底部量取长度，记录并拍照。
[三]小鼠结肠组织微观病理学检测
实验终点将小鼠结肠组织结肠固定于福尔马林中，石蜡包埋切片、H&E染色制作病理学切片。
[四]小鼠结肠组织髓过氧化物酶(MPO)检测
实验终点将小鼠结肠组织结肠固定于福尔马林中，石蜡包埋切片，通过免疫组化检测小鼠结肠组织中MPO表达水平。
[五]小鼠肠系膜淋巴结中性粒细胞、巨噬细胞和Th17细胞检测
实验终点取小鼠肠系膜淋巴结制备单细胞悬液，用相应流式细胞术抗体对细胞标记，并进行统计分析。
[六]DSS结肠炎小鼠肠黏膜通透性的检测
实验终点前，在各组小鼠安乐死4小时前口服给予各组小鼠FITC标记的葡聚糖，剂量为400mg/kg。实验终点，收集小鼠血液。常温避光静置2h 后，以6000g转速离心10分钟，血清用PBS稀释后，荧光酶标仪在488nm的激发波长和520nm的发射波长下测定FITC-葡聚糖浓度，用于反映小鼠黏膜通透性。
[七]小鼠结肠组织中细胞因子检测。
取各组小鼠相同节段结肠组织进行匀浆，离心后，以BCA试剂盒对匀浆上清进行蛋白定量。匀浆中细胞因子按照ProcartaPlex Mouse Th1/Th2/Th9/Th17/Th22/Treg Cytokine 17因子检测试剂盒说明书进行检测。
[八]统计学方法
采用GraphPad Prism 7软件分析，数据以均值±标准差表示，采用方差分析比较组间差异。当P<0.05认为存在显著性差异。
实验结果：
体重减轻是DSS诱导炎症性肠病的重要疾病指征，可以反映小鼠的健康状态。结果如图1A所示，杠柳多苷在治疗后第六天，第七天均能够显著改善DSS引起的体重降低。
疾病活动指数(DAI)是结合炎症性肠病小鼠的体重下降百分率、大便黏稠度和大便出血等三种情况进行评分，并将3项结果的总分除以3即得到DAI值，评价小鼠疾病临床症状的重要指标。研究发现，杠柳多苷治疗组小鼠疾病活动指数在治疗第五天起即显著性缓解炎症性肠病小鼠的疾病活动指数，结果见图1B。
DSS诱导炎症性肠病引起结肠变短，从图2A，图2B可以看出，DSS造模组小鼠结肠长度较正常组小鼠降低明显，杠柳多苷干预治疗组及阳性药柳氮磺吡啶治疗组小鼠结肠长度显著高于DSS造模组小鼠。另外，结肠病理染色(图2C)可以看出，DSS造模组小鼠肠组织中性粒细胞及单核细胞和多核细胞等炎性细胞浸润程度显著升高，部分隐窝破坏，而杠柳多苷治疗组、阳性药柳氮磺吡啶治疗组上述情况明显得到缓解。巨噬细胞、中性粒细胞等是重要的炎症性细胞，通过流式细胞术检测DSS诱导的炎症性肠病小鼠实验终点肠系膜淋巴结中巨噬细胞(F4/80 +和CD11b +双阳性细胞)和中性粒细胞(Gr-1 +和CD11b +双阳性细胞)的比例。结果(图3A，图3B)显示，杠柳多苷治疗组和阳性药治疗组小鼠肠系膜淋巴结中巨噬细胞、中性粒细胞比例较DSS造模组均能显著降低。髓过氧化物酶(MPO)又称过氧化物酶，是一类含有亚铁血红素的的酶类物质，广泛存在于中性粒细胞以及组织巨噬细胞 中。通过免疫组化检测炎症性肠病小鼠结肠组织中MPO表达情况，从图3C中可以看出，DSS造模组小鼠肠组织中MPO较正常对照组显著升高，杠柳多苷治疗组和阳性药对照组中MPO含量较模型组显著降低。表明杠柳多苷可以减轻DSS诱导炎症性肠病小鼠肠组织局部的炎症程度。
肠粘膜通透性增加，肠粘膜屏障功能受损，肠内细菌及毒素进一步通过受损的肠上皮屏障引起肠粘膜炎症和损伤，是诱发炎症性肠病的重要机制之一。通过异硫氰酸荧光素(FITC)标记的葡聚糖检测肠通透性实验结果显示(图4)，杠柳多苷治疗组和阳性药治疗组血清中FITC-葡聚糖含量较模型组显著降低，表明在杠柳多苷或者柳氮磺吡啶治疗能够更好的保护肠粘膜。
Th17细胞在以慢性炎症为损伤机制的自身免疫病发病中起重要作用，可以分泌IL-17等炎症性细胞因子。通过流式细胞术检测肠系膜淋巴结中Th17细胞比例(CD3 +CD4 +IL-17 +)发现，杠柳多苷治疗组Th17比例较DSS造模组显著降低(见图5A，图5B)，组织匀浆中IL-17含量也较模型组显著降低(图5C)。
实验结论：
葡聚糖硫酸钠(Dextran sulfate sodium，DSS)诱导的炎症性肠病发病机制复杂，可能与肠道菌群失调，巨噬细胞功能失调，肠道粘膜上皮细胞异常增生，肠粘膜屏障破坏等相关，是人类炎症性肠病理想的动物模型。实验结果显示，杠柳多苷治疗5天即可显著缓解炎症性肠病小鼠疾病活动指数；杠柳多苷治疗6天即可显著改善炎症性肠病小鼠体重减轻情况，杠柳多苷治疗能够改善炎症性肠病小鼠结肠病理学改变，减少结肠组织中炎性细胞浸润情况并降低肠系膜淋巴结中巨噬细胞、中性粒细胞比例，改善炎症性肠病小鼠肠粘膜的通透性，提示杠柳多苷对肠道粘膜免疫具有调节作用。由以上结果我们可以明确杠柳多苷对小鼠炎症性肠病具有明显的改善作用。
实验实施例2：杠柳多苷对三硝基苯磺酸(TNBS)用于诱导炎症性肠病的药效学研究
实验材料：
C57BL/6雌性小鼠，8-10周龄，购自上海斯莱克实验动物有限责任公司。
试剂：2,4,6-三硝基苯磺酸溶液5％水溶液(TNBS)购自美国Sigma-Aldrich公司。
细胞因子检测试剂盒ProcartaPlex Mouse Th1/Th2/Th9/Th17/Th22/Treg Cytokine 17因子检测试剂盒购自美国英杰生命技术有限公司(Invitrogen)。羟丙基甲基纤维素(HPMC)购自美国Sigma-Aldrich公司。无水乙醇购自国药集团化学试剂有限公司。
使用制备实施例三种制备的杠柳多苷，性状：浅棕色粉末；其中8个杠柳苷Periploside A、C、D、E、K、O、Q、R的累积含量为64.96％，8个杠柳苷的含量分别为Periploside A 4.54％重量份、Periploside C 31.46％重量份、Periploside D 10.31％重量份、Periploside E 6.34％重量份、Periploside K 3.12％重量份、Periploside O 3.45％重量份、Periploside Q 3.76％重量份、Periploside R 1.98％重量份。杠柳多苷使用时混悬于溶剂中并稀释至所需浓度后使用。溶剂为0.2％(w/v)HPMC。
杠柳苷C(periploside C)，性状：白色无定形粉末，纯度：99％。Periploside C使用时混悬于溶剂中并稀释至所需浓度后使用。溶剂为0.2％(w/v)HPMC。
杠柳苷F(periploside F)，性状：无色结晶，纯度：99％。Periploside F使用时混悬于溶剂中并稀释至所需浓度后使用。溶剂为0.2％(w/v)HPMC。
杠柳苷C和F的制备方法参见制备实施例四。
醋酸泼尼松片，购自上海上药信谊药厂有限公司，5mg/片，使用时用0.2％(w/v)HPMC稀释至所需浓度后使用。
配制2.5％TNBS：无水乙醇与5％的TNBS溶液按照1:1(v/v)比例混合，现配现用。
50％酒精配制：无水乙醇与双蒸水按照1:1(v/v)比例混合，现配现用。
实验方法：
动物分组及处理:Balb/c小鼠随机分成6组，正常对照组，模型对照组，醋酸泼尼松对照组，杠柳多苷12.5mg/kg治疗组，杠柳多苷6.25mg/kg治疗组，杠柳多苷3.13mg/kg治疗组。其中醋酸泼尼松对照组为阳性药对照组。
小鼠禁食24h后，使用异氟烷进行麻醉。用透明胶管将导入小鼠距肛门约4cm直肠中，模型对照组、醋酸泼尼松2mg/kg组、杠柳多苷12.5mg/kg治疗组、Periploside C 12.5mg/kg治疗组治疗组和Periploside F 12.5mg/kg治疗组小鼠缓慢注入100μL 2.5％(v/v)TNBS。正常对照组的小鼠以同样的方法注入50％(v/v)酒精。
自造模当日起，醋酸泼尼松组经口给与醋酸泼尼松2mg/kg(醋酸泼尼松片溶于0.2％(v/v)HPMC中)，一日一次；杠柳多苷治疗组口服给予杠柳多 苷12.5mg/kg，一日一次；Periploside C治疗组口服给予Periploside C12.5mg/kg，一日一次；Periploside F治疗组口服给予Periploside F 12.5mg/kg，一日一次；正常对照组和模型组分别经口给予等体积的0.2％(w/v)HPMC，一日一次。
监测指标：
[一]小鼠体重检测。
体重实验期间每天对各组小鼠体重进行监测并记录。
[二]小鼠生存率统计。
记录实验期间小鼠死亡情况，绘制生存率曲线。
[三]小鼠结肠组织整体病理学评价。
实验终点迅速取小鼠结肠组织，自盲肠底部至直肠底部量取长度，记录并拍照。
实验结果：
体重减轻可以反映小鼠的健康状态,是TNBS诱导炎症性肠病的重要疾病指征。结果如图6A所示，在TNBS诱导炎症性肠病后，模型对照组小鼠体重造模次日即显著降低，杠柳多苷给药四天后该组小鼠体重较模型组小鼠体重显著高于正常组。杠柳多苷12.5mg/kg治疗组在给药7天后，体重已恢复到正常水平。Periploside C和F治疗7天后，小鼠体重较模型组小鼠有显著的改善。如图6B所示，杠柳多苷、Periploside C和F治疗均能显著提高TNBS诱导的结肠炎小鼠的存活率。图6C，图6D所示，TNBS模型对照组小鼠结肠长度较正常对照组明显缩短，阳性药醋酸泼尼松组，杠柳多苷12.5mg/kg治疗组、Periploside C 12.5mg/kg治疗组和Periploside F 12.5mg/kg治疗组小鼠结肠长度较模型对照组均显著升高。
实验结论：
三硝基苯磺酸(TNBS)诱导小鼠的炎症性肠病模型与临床上的溃疡性结肠炎病理改变相似。该模型中小鼠出行粪便形态改变、便血、肠道形态学改变和肠道组织学改变等于溃疡性结肠炎患者临床症状相似。本实施例采用TNBS诱导炎症性肠病模型，对杠柳多苷的治疗作用进行药效学评价。结果显示，杠柳多苷能够显著提高TNBS诱导结肠炎小鼠生存率，缓解小鼠体重减轻情况，并维持小鼠结肠长度。以上结果显示，可以明确杠柳多苷对小鼠炎症性肠病具有明显的改善作用。
实验实施例3：杠柳多苷、Periploside C和F对葡聚糖硫酸钠(DSS)诱导溃疡性结肠炎的药效学研究
实验材料：
Balb/c雌性小鼠，8-10周龄，购自上海斯莱克实验动物有限责任公司。
试剂：硫酸葡聚糖钠(DSS，购自美国MP Biomedicals生物医学公司)
使用制备实施例三中制备的杠柳多苷，性状：浅棕色粉末；其中8个杠柳苷Periploside A、C、D、E、K、O、Q、R的累积含量为64.96％，8个杠柳苷的含量分别为Periploside A 4.54％重量份、Periploside C 31.46％重量份、Periploside D 10.31％重量份、Periploside E 6.34％重量份、Periploside K 3.12％重量份、Periploside O 3.45％重量份、Periploside Q 3.76％重量份、Periploside R 1.98％重量份。杠柳多苷使用时混悬于溶剂中并稀释至所需浓度后使用。溶剂为0.2％(w/v)HPMC。
杠柳苷C(periploside C)，性状：白色无定形粉末，纯度：99％。Periploside C使用时混悬于溶剂中并稀释至所需浓度后使用。溶剂为0.2％(w/v)HPMC。
杠柳苷F(periploside F)，性状：无色结晶，纯度：99％。Periploside F使用时混悬于溶剂中并稀释至所需浓度后使用。溶剂为0.2％(w/v)HPMC。
杠柳苷C和F的制备方法参见制备实施例四。
柳氮磺吡啶结肠溶胶囊，购自广东强基药业有限公司，250mg/胶囊，使用时用0.2％(w/v)HPMC稀释至所需浓度后使用。
实验方法：
动物分组及处理:Balb/c小鼠随机分成4组，正常对照组，DSS造模组，柳氮磺吡啶治疗组，杠柳多苷治疗组。其中柳氮磺吡啶治疗组为阳性药对照组。除正常对照组的小鼠以外，其他各组包括DSS造模组、柳氮磺吡啶治疗组、杠柳多苷治疗组的饮用水中均加入了5％DSS，连续7天进行肠道炎症模型的诱导造模。自造模当日起，柳氮磺吡啶治疗组经口给与柳氮磺吡啶250mg/kg，一日一次；杠柳多苷治疗组口服给予杠柳多苷12.5mg/kg，一日一次；Periploside C治疗组口服给予Periploside C 12.5mg/kg，一日一次；Periploside F治疗组口服给予Periploside F12.5mg/kg，一日一次；正常对照组和DSS造模组分别经口给予等体积的0.2％(w/v)HPMC，一日一次。
监测指标：
实验期间每天对各组小鼠体重进行监测并记录；小鼠粪便采用隐血试剂盒监测并进行量化评分。依据疾病活动指数评分表评价杠柳多苷对DSS诱导炎症性肠病的治疗效果。
实验结果：
体重减轻是DSS诱导炎症性肠病的重要疾病指征，可以反映小鼠的健康状态。结果如图7A所示，杠柳多苷治疗组、柳氮磺吡啶阳性药组在治疗后第六天和第七天均能够显著改善DSS引起的体重降低。Periploside C和F治疗组小鼠在治疗后第七天后体重降低情况显著改善。
疾病活动指数(DAI)是结合炎症性肠病小鼠的体重下降百分率、大便黏稠度和大便出血等三种情况进行评分，并将3项结果的总分除以3即得到DAI值，评价小鼠疾病临床症状的重要指标。研究发现，杠柳多苷治疗组、柳氮磺吡啶阳性药组治疗组小鼠在治疗后第六天和第七天疾病活动指数较模型组小鼠显著降低；Periploside C治疗组和Periploside F治疗组小鼠疾病活动指数在治疗后第七天后较模型组小鼠显著改善，结果见图7B。DSS诱导炎症性肠病引起结肠变短，从图7C，图7D可以看出杠柳多苷治疗组、柳氮磺吡啶阳性药组、Periploside C治疗组和Periploside SF治疗组小鼠结肠长度较模型组小鼠均显著改善。提示杠柳多苷、Periploside C和F对DSS诱导的炎症性肠病具有良好的治疗效果。
实验实施例4：杠柳多苷对胶原诱导小鼠关节炎的药效学研究
实验材料：
DBA/1小鼠，6-8周龄，购自上海斯莱克试验动物有限责任公司。
试剂：乙酸(冰醋酸)(10000218)购自国药集团化学试剂有限公司；牛Ⅱ型胶原(Immunization Grade Bovine Type II Collagen)购自Chondrex公司；Freund’s Adjuvant Complete(CFA,弗氏完全佐剂)(F5881-10X10mL)购自SIGMA；Freund’s Adjuvant,Incomplete(IFA,弗氏不完全佐剂)购自SIGMA；HRP-rabbit anti-mouse IgG(H+L)(616520)、HRP-rabbit anti-mouse IgG2a(610220)购自Invitrogen。
使用制备实施例三种制备的杠柳多苷，性状：浅棕色粉末；其中8个杠柳苷Periploside A、C、D、E、K、O、Q、R的累积含量为64.96％，8个杠柳苷的含量分别为Periploside A 4.54％重量份、Periploside C 31.46％重量份、 Periploside D 10.31％重量份、Periploside E 6.34％重量份、Periploside K 3.12％重量份、Periploside O 3.45％重量份、Periploside Q 3.76％重量份、Periploside R 1.98％重量份。杠柳多苷使用时混悬于溶剂中并稀释至所需浓度后使用。溶剂为0.2％(w/v)HPMC。
甲氨蝶呤片，上海医药(集团)有限公司信谊制药总厂，2.5mg/片，使用时用0.2％(w/v)HPMC稀释至所需浓度后使用。
实验方法：
[一]胶原诱导小鼠关节炎模型建立
牛II型胶原溶解入0.1M的无菌醋酸，4℃溶解过夜，制成胶原浓度为10mg/mL的溶液。
将牛II型胶原溶液与等体积的CFA充分乳化，DBA/1小鼠麻醉后，于小鼠的尾根部注射25μl的牛II型胶原佐剂混合乳化液进行第1次免疫诱导；3周后以相同剂量的牛II型胶原与IFA混合乳化后，于小鼠的尾根部进行免疫强化，诱导小鼠发生多关节性关节炎。
[二]动物分组及给药
动物分组及处理:在小鼠发病后，根据其体重、足肿胀评分分成5组，共有实验组6组，每组10只：正常对照组、关模型对照组、阳性药甲氨蝶呤组(1mg/kg，1次/天)、杠柳多苷(12.5mg/kg/天，6.25mg/kg/天；2次/天)。各组均采用口服灌胃给药。连续给药8周，考察口服杠柳多苷对CIA小鼠关节炎疾病指征的影响。
[三]柳多苷治疗胶原诱导小鼠关节炎药效学评价指标
每天观察比较各实验组关节炎症状的发展情况，按0-4分五级标准评分(表3-1)来评价关节炎病变及炎症严重程度，每周评分两次。
[四]胶原诱导关节炎鼠血清中抗CII特异性抗体检测
杠柳多苷治疗8周后，实验终点分离DBA/1小鼠血清，血清于-30℃冰箱保存，ELISA法检测血清中抗牛II型胶原(CII)特异性抗体IgG的水平。
[五]小鼠关节组织病理学检测
实验终点将小鼠后肢固定于福尔马林中，石蜡包埋切片、H&E染色制作病理学切片。
实验结果：
[一]杠柳多苷降低胶原诱导关节炎小鼠临床评分
牛II型胶原诱导的DBA/1小鼠关节炎表现为严重的多发性关节损伤，其临床、病理表现，体液免疫、细胞免疫应答与人类类风关相似，广泛应用于抗关节炎药物疗效评价和作用机理的研究。
关节炎临床发病指数(Clinic Score)是反映关节炎病变发生与发展的重要指标。实验结果显示，杠柳多苷治疗给药的3个剂量组(12.5mg/kg，6.25mg/kg)能够降低胶原诱导关节炎鼠的发病指数，表现为胶原诱导关节炎鼠小鼠的四肢足爪肿胀发生数以及严重程度明显减轻(图8A)
胶原诱导关节炎鼠模型小鼠随着疾病发展，可出现体重下降。在杠柳多苷治疗周期内，对小鼠进行每周2次体重记录，结果显示杠柳多苷治疗组小鼠体重较模型组有升高趋势(图8B)。
[二]病理组织学观察结果
各组后肢踝关节病理组织学结果代表图见图9，其改变及程度如下：
正常对照组：除1例动物可见轻微炎症、出血外，其它动物未见明显异常。
模型组：3/8例动物重度炎症，1/8例动物中度炎症，3/8例动物轻微炎症，1/8例动物未见异常。与对照组相比，模型组踝关节可见重度炎症，病理组织学形态表现为滑膜及滑膜周边大量炎细胞浸润，延伸到周边组织，侵蚀软骨和伴有纤维化。
阳性药甲氨蝶呤组：2/8例动物重度炎症，3/8例动物中度炎症，3/8例动物未见异常。
杠柳多苷组：12.5mg/kg组2/8例动物中度炎症，1/8例动物轻度炎症，2/8例动物轻微炎症，3/8例动物未见异常；6.25mg/kg组1/8例动物中度炎症，1/8例动物轻度炎症，4/8例动物轻微炎症，2/8例动物未见异常。
[三]杠柳多苷降低胶原诱导关节炎鼠小鼠血清中抗CII特异性抗体含量
抗牛II型胶原(CII)抗体的产生是CIA小鼠的重要病理特征，与疾病的严重程度密切相关，可以作为药效学评价的重要指标。
我们在实验终点，分离各组小鼠血清检测抗CII特异性抗体含量，结果显示，杠柳多苷治疗能够显著降低抗CII特异性抗体总IgG(图10A)及IgG2a水平(图10B)，且表现出剂量依赖性。
实验结论：
牛II型胶原(Type II bovine collagen，CII)诱导的DBA/1小鼠关节炎(Collagen-induced arthritis，CIA)表现为严重的多发性关节损伤，其临床、 病理表现，体液免疫、细胞免疫应答与人类类风关相似，广泛应用于抗关节炎药物疗效评价和作用机理的研究。本实验应用胶原诱导小鼠关节炎，观察杠柳多苷口服给药胶原诱导小鼠关节炎的治疗作用。杠柳多苷表现出良好的治疗效果，能够显著改善模型小鼠胶原诱导关节炎的临床症状，降低关节炎临床评分，改善病理损伤及胶原特异性抗体的产生，表明杠柳多苷对佐剂诱导小鼠关节炎具有显著的治疗效果。
实验实施例5：杠柳多苷对佐剂诱导大鼠关节炎的药效学研究
实验材料：
SD大鼠，雄性，约150-180g，购自上海斯莱克试验动物有限责任公司。
使用制备实施例三种制备的杠柳多苷，性状：浅棕色粉末；其中8个杠柳苷Periploside A、C、D、E、K、O、Q、R的累积含量为64.96％，8个杠柳苷的含量分别为Periploside A 4.54％重量份、Periploside C 31.46％重量份、Periploside D 10.31％重量份、Periploside E 6.34％重量份、Periploside K 3.12％重量份、Periploside O 3.45％重量份、Periploside Q 3.76％重量份、Periploside R 1.98％重量份。杠柳多苷使用时混悬于溶剂中并稀释至所需浓度后使用。溶剂为0.2％(w/v)HPMC。
甲氨蝶呤片，上海医药(集团)有限公司信谊制药总厂，2.5mg/片，使用时用0.2％(w/v)HPMC稀释至所需浓度后使用。
灭活结核杆菌冻干粉(M.TUBRCULOSIS H37 Ra)购自DIFCO公司。
实验方法：
[一]杠柳多苷治疗佐剂诱导大鼠关节炎动物分组及给药
雄性SD大鼠，除正常对照组，其余大鼠在造模后第13天，根据体重、足肿胀评分及肿胀度分成5组，共有实验组6组，每组8只：正常对照组、关节炎模型组(溶剂对照)、阳性药MTX组(1mg/kg，1次/天)、杠柳多苷(10mg/kg/天，5mg/kg/天，2.5mg/kg/天；2次/天)。
以上6组实验动物除正常对照组外，各组大鼠于实验开始的第一天自左后足垫皮内注射含10mg/mL结合杆菌灭活菌株的佐剂0.1mL，并于造模后第13天开始灌胃给药。
[三]柳多苷治疗佐剂诱导大鼠关节炎药效学评价指标
按时间点监测各组大鼠体重；
按时间点观察比较各实验组关节炎症状的发展情况，按0-4分五级标准评分(临床发病指数)来评价关节炎病变及炎症严重程度【0分：无红肿；1分：小趾关节红肿；2分：趾关节和足趾肿胀；3分：踝关节以下的足爪肿胀；4分：包括踝关节在内的全部足爪肿胀】；
大鼠原发足(左后足)肿胀反映关节炎性病变，致敏对侧(右后足)则为继发性自身免疫性肿胀。运用足趾容积测量仪检测大鼠关节炎的肿胀程度，计算公式如下：
大鼠关节肿胀度(μL)＝模型组/实验治疗组大鼠后足排水体积－未造模正常对照组大鼠的后足平均排水体积
[四]Micro-CT扫描
在实验终点，将各组小鼠安乐死后，剪取后肢，并在福尔马林中固定。采用西门子公司Inveon micro PET/CT(Inveon MM system,Siemens Preclinical Solutions)进行扫描。按照下面参数进行高分辨率扫描：扫描厚度8.5μm，电压为80千伏，电流为500微安，以360°旋转曝光，每步曝光时长1000毫秒。大鼠后肢选用中分辨率参数进行微计算机断层扫描，扫描厚度32μm，曝光时间设定为600毫秒。
[五]大鼠关节组织病理学检测
实验终点将大鼠后肢固定于福尔马林中，石蜡包埋切片、H&E染色制作病理学切片。
实验结果：
[一]杠柳多苷降低佐剂诱导大鼠关节炎临床评分
实验结果显示，杠柳多苷对大鼠佐剂关节炎有显著的治疗作用，且呈一定浓度依赖性。杠柳多苷口服治疗对继发足的临床评分(图11A)及肿胀体积(图11)均有很好的抑制作用，且表现出浓度依赖性。同时，杠柳多苷杠柳多苷口服治疗对大鼠体重无显著影响(图11C)。
杠柳多苷在治疗佐剂诱导大鼠关节炎模型中表现出良好的治疗效果，能够显著改善模型大鼠关节炎的临床症状，降低关节炎临床评分。
[二]杠柳多苷减轻佐剂诱导关节炎大鼠骨损伤程度
我们通过实验终点各组代表性继发足照片(图12A)可以看出，杠柳多苷对佐剂诱导大鼠关节炎具有足肿胀有显著的抑制作用。通过微计算机断层扫描(Micro computed tomography，micro-CT)分析了佐剂诱导关节炎大鼠 关节炎继发足、胶原诱导关节炎(CIA)小鼠左后肢的骨质侵蚀情况，从图12B可以看出，杠柳多苷对于两种关节炎模型动物中骨损伤具有明显的保护作用。
[三]病理组织学观察结果
各组后肢踝关节病理组织学结果代表图见图13，其改变及程度如下：
正常对照组：2/8例动物可见轻微或轻度炎症，其余动物未见明显异常。
模型对照组：与对照组相比，全部动物可见轻微至重度炎症。模型组踝关节炎症病理组织学形态表现为滑膜及滑膜周边大量炎细胞浸润，延伸到周边组织，侵蚀软骨和骨，伴有纤维化。
阳性药甲氨蝶呤组：1/8例动物可见轻度炎症，其余动物未见明显异常。
杠柳多苷组：10mg/kg治疗组均未见异常；5mg/kg治疗组组4/8例动物可见轻微或中度炎症，其余动物未见明显异常；2.5mg/kg治疗组组2/8例动物可见中度炎症，其余动物未见明显异常。
实验结论：
佐剂性关节炎(adjuvant-induced arthritis,AA)模型是细菌学家Freund于20世纪50年代创立的，又称弗氏佐剂关节炎，是一种经典的免疫性炎症模型，广泛应用于治疗类风关候选药物的临床前评价。本实验应用CFA诱发SD大鼠产生实验性佐剂性关节炎，观察杠柳多苷口服给药对佐剂诱导的SD大鼠关节炎的治疗作用。杠柳多苷在治疗佐剂诱导大鼠关节炎模型中表现出良好的治疗效果，能够显著改善模型大鼠关节炎的临床症状，降低关节炎临床评分，减轻关节炎大鼠骨侵蚀及炎症浸润情况。
实验实施例6：杠柳多苷、杠柳苷C对胶原诱导小鼠关节炎的药效学研究
实验材料：
DBA/1小鼠，雄性，6-8周龄，购自上海斯莱克试验动物有限责任公司。
试剂：乙酸(冰醋酸)(10000218)购自国药集团化学试剂有限公司；牛Ⅱ型胶原(Immunization Grade Bovine Type II Collagen)购自Chondrex公司；Freund’s Adjuvant Complete(CFA,弗氏完全佐剂)(F5881-10X10mL)购自SIGMA；Freund’s Adjuvant,Incomplete(IFA,弗氏不完全佐剂)购自SIGMA；HRP-rabbit anti-mouse IgG(H+L)(616520)、HRP-rabbit anti-mouse IgG2a(610220)购自Invitrogen。
使用制备实施例三种制备的杠柳多苷，性状：浅棕色粉末；其中8个杠柳苷Periploside A、C、D、E、K、O、Q、R的累积含量为64.96％，8个杠柳苷的含量分别为Periploside A 4.54％重量份、Periploside C 31.46％重量份、Periploside D 10.31％重量份、Periploside E 6.34％重量份、Periploside K 3.12％重量份、Periploside O 3.45％重量份、Periploside Q 3.76％重量份、Periploside R 1.98％重量份。杠柳多苷使用时混悬于溶剂中并稀释至所需浓度后使用。溶剂为0.2％(w/v)HPMC。
杠柳苷C(periploside C)，性状：白色无定形粉末，纯度：99％。Periploside C使用时混悬于溶剂中并稀释至所需浓度后使用。溶剂为0.2％(w/v)HPMC。
杠柳苷C的制备方法参见制备实施例四。
甲氨蝶呤片，上海医药(集团)有限公司信谊制药总厂，2.5mg/片，使用时用0.2％(w/v)HPMC稀释至所需浓度后使用。
实验方法：
[一]胶原诱导小鼠关节炎模型建立
牛II型胶原溶解入0.1M的无菌醋酸，4℃溶解过夜，制成胶原浓度为10mg/mL的溶液。
将牛II型胶原溶液与等体积的CFA充分乳化，DBA/1小鼠麻醉后，于小鼠的尾根部注射25μL的牛II型胶原佐剂混合乳化液进行第1次免疫诱导；3周后以相同剂量的牛II型胶原与IFA混合乳化后，于小鼠的尾根部进行免疫强化，诱导小鼠发生多关节性关节炎。
[二]动物分组及给药
动物分组及处理:在小鼠发病后，根据其体重、足肿胀评分分成5组，共有实验组6组，每组10只：正常对照组、关模型对照组、阳性药甲氨蝶呤组(1mg/kg，1次/天)、杠柳多苷(12.5mg/kg/天；2次/天)、杠柳苷C(12.5mg/kg/天；2次/天)。各组均采用口服灌胃给药。连续给药10周，比较口服杠柳多苷或杠柳苷C对CIA小鼠关节炎疾病指征的影响，并于给药后第10周对各组小鼠后肢拍照记录。
[三]药品治疗胶原诱导小鼠关节炎药效学评价指标
每天观察比较各实验组关节炎症状的发展情况，按0-4分五级标准评分来评价关节炎病变及炎症严重程度，每周评分两次。
实验结果：
[一]杠柳多苷降低胶原诱导关节炎小鼠临床评分
实验结果显示，杠柳多苷治疗组小鼠在给药4周后能够降低胶原诱导关节炎鼠的发病指数，杠柳苷C治疗组在治疗6周后显著降低胶原诱导关节炎鼠的发病指数，相比杠柳多苷治疗组具有统计学上显著性的药效发挥时间晚2周(图13A)。
胶原诱导关节炎鼠模型小鼠随着疾病发展，可出现体重下降。在杠柳多苷治疗周期内，对小鼠进行每周1次体重记录，结果显示杠柳多苷治疗组小鼠体重较模型组小鼠和杠柳苷C治理组小鼠体重均有升高趋势(图13B)。
实验结论：
牛II型胶原(Type II bovine collagen，CII)诱导的DBA/1小鼠关节炎(Collagen-induced arthritis，CIA)表现为严重的多发性关节损伤，其临床、病理表现，体液免疫、细胞免疫应答与人类类风关相似，广泛应用于抗关节炎药物疗效评价和作用机理的研究。本实验应用胶原诱导小鼠关节炎，观察杠柳多苷和杠柳苷C分别口服给药胶原诱导小鼠关节炎的治疗作用。杠柳多苷和杠柳苷C表现出良好的治疗效果，能够显著降低关节炎临床评分。杠柳多苷相比杠柳苷C对于维持胶原诱导关节炎鼠体重具有更好的效果且发挥出显著性治疗效果的时间更早，表明杠柳多苷在治疗关节炎上具有更优异的效果。
实验实施例7：杠柳多苷及8个主要活性成分体外免疫抑制活性研究
实验材料：
BALB/c小鼠，雌性，18-20克，购自上海斯莱克试验动物有限责任公司。
刀豆蛋白A(ConA)购自Sigma公司；细菌脂多糖(LPS)购自Sigma公司；MTT购自Sigma公司； 3H-胸腺嘧啶核苷酸(1μCi/mL)购自珀金埃尔默公司；二甲基亚砜(DMSO)购自国药集团化学试剂有限公司。
使用制备实施例三种制备的杠柳多苷，性状：浅棕色粉末；其中8个杠柳苷Periploside A、C、D、E、K、O、Q、R的累积含量为64.96％，8个杠柳苷的含量分别为Periploside A 4.54％重量份、Periploside C 31.46％重量份、Periploside D 10.31％重量份、Periploside E 6.34％重量份、Periploside K 3.12％重量份、Periploside O 3.45％重量份、Periploside Q 3.76％重量份、Periploside R 1.98％重量份。
实验方法：
[一]MTT法检测化合物对小鼠脾脏淋巴细胞免疫抑制活性的影响：
小鼠脾脏淋巴细胞悬液5*10 5/孔接种于96孔板，同时加入不同浓度化合物，另设相应的溶媒对照(细胞对照)及培养液本底对照(空白对照)，总体积为200μL。37℃，5％CO 2培养箱中培养48小时。结束培养前4小时加入浓度为5mg/mL的MTT溶液。至培养结束，吸弃上清，每孔加入200μL DMSO溶解紫色结晶，于酶标仪570nM处测定OD值。计算细胞存活率，公式如下：
Figure PCTCN2021088918-appb-000004
[二] 3H-TdR掺入法检测化合物对小鼠脾脏T|、B淋巴细胞增殖功能的影响
小鼠脾脏淋巴细胞悬液5*10 5/孔接种于96孔板，加入ConA(终浓度5μg/mL)或LPS(终浓度10μg/mL)及不同浓度化合物，使培养体系终体积为200μL；每实验组设3个复孔，另设无刺激的本底对照、相应溶剂对照组。于37℃，含5％CO 2的培养箱中培养48小时。
培养结束前8小时每孔加入25μL  3H-胸腺嘧啶核苷酸(1μCi/mL)。培养结束后先将培养板冻存至-30℃冰箱，待测。测定时将冻融的细胞收集至玻璃纤维膜上，加入闪烁液后于Beta计数仪上读取掺入细胞DNA的 3H-胸腺嘧啶核苷酸量，以cpm(Counts per minute，每分钟计数)值代表细胞增殖的情况。计算细胞增殖抑制率，公式如下：
Figure PCTCN2021088918-appb-000005
实验结果：
杠柳多苷半数细胞致死浓度(CC 50)为0.94±0.56μg/mL,能够抑制ConA诱导的T淋巴细胞增殖,其半数有效抑制浓度(IC 50)为0.10±0.01μg/mL，与其他8个有效组分相比具有最优的抑制活性；能够抑制LPS诱导的B淋巴细胞增殖，其半数有效抑制浓度(IC 50)为0.40±0.12μg/mL，与其他8个有 效组分相比具有最优的抑制活性。选择指数(SI)为判断药物效果的安全范围，选择指数为CC 50/IC 50，指数越大表明安全范围越大。杠柳多苷具有良好的免疫抑制活性，SI值表明其安全性高，免疫抑制活性强，具有潜在的药用价值(见下表)。
Figure PCTCN2021088918-appb-000006
实验结论：
通过体外免疫抑制活性筛选模型研究发现，杠柳多苷与其所含8个主要活性成分相比，具有更高的细胞毒性，且具有最强的免疫抑制活性。表明杠柳多苷的免疫抑制活性优于8个单体杠柳苷类化合物。

Claims (11)

  1. 一种杠柳苷组合物，其包括以下组分：
    Periploside A 0.1-49％重量份、
    Periploside C 0.1-49％重量份、
    Periploside D 0.1-49％重量份、
    Periploside E 0.1-20％重量份、
    Periploside K 0.1-20％重量份、
    Periploside O 0.1-20％重量份、
    Periploside Q 0.1-20％重量份、
    Periploside R 0.1-20％重量份。
  2. 根据权利要求1所述的杠柳苷组合物，其中
    所述组分Periploside A、C、D、E、K、O、Q和R的质量之和大于所述组合物总质量的50％，优选大于60％。
  3. 一种杠柳苷组合物的制备方法，包括：
    (1)提取、浓缩步骤
    取萝藦科杠柳属Periploca植物杠柳Periploca sepium Bunge的根皮加水提取，弃去提取液，将药渣加40％-95％乙醇提取1-10次，将醇提液合并浓缩，得到悬浊液；
    (2)柱前预处理步骤
    在搅拌条件下向悬浊液加入乙醇-水混合物使乙醇含量为10％-50％，进行固液分离；将固形物溶于乙醇-水混合物使乙醇含量为60％以上，从而得到清液，将清液用烃类溶剂萃取，将萃余液作为大孔树脂柱上样液；
    其中，所述烃类溶剂是选自C5-C8烃溶剂和石油醚的一种或多种；
    (3)柱层析纯化步骤
    用大孔树脂填充色谱柱，将上样液上样后，依次采用60％-75％乙醇除杂质，80％-90％乙醇洗脱，再用90％-95％乙醇洗脱，收集90％-95％乙醇洗脱液，浓缩，干燥得到粉末混合物；
    (4)柱后处理步骤
    将粉末混合物用乙醇-水精制处理，得到杠柳苷组合物。
  4. 根据权利要求3所述的制备方法，包括
    (1)提取、浓缩步骤
    取萝藦科杠柳属Periploca植物杠柳Periploca sepium Bunge的根皮加2-10倍质量的水在5-100℃下提取2-10h，提取液弃去，将药渣加2-10倍质量的40％-95％乙醇提取1-10次，将醇提液合并浓缩至原料根皮的40％至80％质量，得到悬浊液；
    (2)柱前预处理步骤
    在搅拌条件下向悬浊液加入40％-95％乙醇使乙醇含量为10％-50％，搅拌后固液分离取沉淀，之后，将沉淀再次悬浮于10％-50％乙醇搅拌后固液分离取沉淀，再将沉淀溶于60％以上的乙醇，过滤得到上清液，将上清液用等体积烃类溶剂萃取3次，弃去所述烃类溶剂层，乙醇液经减压蒸除残留的烃类溶剂，得大孔树脂柱上样液；
    其中，所述烃类溶剂是选自环戊烷、正己烷、环己烷、环己烯、正庚烷、正辛烷、异辛烷和石油醚的一种或多种；
    (3)柱层析纯化步骤
    用大孔树脂填充色谱柱，上样液经吸附后，依次采用60％-75％乙醇除杂质，80％-90％乙醇洗脱，再用90％-95％乙醇作为洗脱液进行第一轮洗脱，然后收集用80％-90％乙醇洗脱的洗脱液，浓缩至60％-75％乙醇后作为上样液再次上样，再依次用60％-75％乙醇、80％-90％乙醇、90％-95％乙醇作为洗脱液进行第二轮洗脱，收集第一轮和第二轮洗脱中用90％-95％乙醇洗脱的洗脱液，浓缩，干燥得到粉末混合物；
    (4)柱后处理步骤
    将上述粉末混合物用70％-95％乙醇溶解，加水调至乙醇浓度为60％以下得到悬浊液，过滤该悬浊液，得第一上清液和第一沉淀物，将第一沉淀物加乙醇-水洗涤后过滤得第二上清液和第二沉淀物，将第一上清液和第二上清液合并后静置沉降，过滤得第三沉淀，合并第二沉淀和第三沉淀，干燥、粉碎即得杠柳苷组合物。
  5. 一种杠柳苷组合物，其根据权利要求3或4所述的制备方法制得。
  6. 根据权利要求1-2或5中任一项所述的杠柳苷组合物在制备治疗自身免疫性疾病的药物中的用途。
  7. 根据权利要求6所述的用途，其中，所述自身免疫性疾病包括炎症性肠病和类风湿性关节炎。
  8. 杠柳苷类化合物Periploside C和Periploside F在制备治疗炎症性肠病药物中的用途。
  9. 根据权利要求7和8所述的用途，其中，所述炎症性肠病包括溃疡性结肠炎和克罗恩病。
  10. 根据权利要求7和8所述的用途，其中，所述治疗炎症性肠病的药物是通过抑制体重减轻，改善便溏、便血，保护肠道粘膜损伤，减轻结肠组织的病理学损伤，抑制结肠组织中炎症细胞的浸润，下调相关促炎性细胞因子的水平来起作用的。
  11. 根据权利要求7所述的用途，其中，所述治疗类风湿性关节炎的药物是通过缓解关节部位肿胀程度，降低关节组织中炎症浸润情况，缓解关节炎病灶骨侵蚀程度，降低血清中特异性自身抗体的产生来起作用的。
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