WO2015062517A1 - 马甲子提取物及其制备方法和用途 - Google Patents

马甲子提取物及其制备方法和用途 Download PDF

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Publication number
WO2015062517A1
WO2015062517A1 PCT/CN2014/089895 CN2014089895W WO2015062517A1 WO 2015062517 A1 WO2015062517 A1 WO 2015062517A1 CN 2014089895 W CN2014089895 W CN 2014089895W WO 2015062517 A1 WO2015062517 A1 WO 2015062517A1
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Prior art keywords
extract
ethanol
mazin
preparation
solvent
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PCT/CN2014/089895
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English (en)
French (fr)
Inventor
徐超群
李东晓
袁志翔
舒光明
阮佳
詹雁
谭镭
Original Assignee
四川省中医药科学院
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Priority claimed from CN201310528511.6A external-priority patent/CN103550323B/zh
Priority claimed from CN201410019123.XA external-priority patent/CN103735653B/zh
Priority claimed from CN201410018576.0A external-priority patent/CN103751315B/zh
Priority claimed from CN201410023850.3A external-priority patent/CN103751316B/zh
Priority claimed from CN201410032900.4A external-priority patent/CN103800466B/zh
Priority claimed from CN201410030860.XA external-priority patent/CN103735654B/zh
Priority to ES14858822T priority Critical patent/ES2775598T3/es
Priority to US15/033,386 priority patent/US20160256510A1/en
Priority to KR1020167014232A priority patent/KR101891417B1/ko
Application filed by 四川省中医药科学院 filed Critical 四川省中医药科学院
Priority to EP14858822.1A priority patent/EP3064212B1/en
Priority to JP2016550924A priority patent/JP6371400B2/ja
Publication of WO2015062517A1 publication Critical patent/WO2015062517A1/zh
Priority to US16/220,229 priority patent/US10987395B2/en

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    • 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)
    • A61K36/72—Rhamnaceae (Buckthorn family), e.g. buckthorn, chewstick or umbrella-tree
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • 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
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • 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
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
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    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/14—Drugs for disorders of the endocrine system of the thyroid hormones, e.g. T3, T4
    • A61P5/16—Drugs for disorders of the endocrine system of the thyroid hormones, e.g. T3, T4 for decreasing, blocking or antagonising the activity of the thyroid hormones
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/35—Extraction with lipophilic solvents, e.g. Hexane or petrol ether
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • the invention belongs to the field of medicine, and particularly relates to a mazin extract, a preparation method thereof and use thereof.
  • Paliurus ramosissimus (Lour.) Poir is a deciduous shrub and is a common medicinal plant. It is reported that the roots and flowers of the leaves are medicinal. Sexual taste is bitter, flat, non-toxic. Can remove cold and promote blood circulation, release antipyretic, swelling, cure bruises and abdominal pain. It is included in the Dictionary of Traditional Chinese Medicine and some local medicines.
  • the inventors of the present invention found that the mazin and its extract have good anti-fibrosis, anti-fungal activity, anti-tumor activity, treatment of oral and digestive tract inflammation or (and) ulcer-related diseases, and have two-way immunomodulation. effect.
  • the first technical problem solved by the present invention is to provide a new use of mazins.
  • the vest When applied, the vest uses the whole plant or any part of the mascara; wherein the medicinal part may be any part of the root, stem, leaf, flower, fruit or a mixture thereof.
  • the novel medicinal use of mazin is in the preparation of a medicament having anti-fibrotic, antifungal activity, anti-tumor activity, treatment of oral and digestive tract inflammation or (and) ulcer-related diseases, and bidirectional immunomodulatory effects. use.
  • a second technical problem solved by the present invention is to provide a novel mazin extract.
  • the mascara can be used as an extract.
  • the mazin extract of the present invention is prepared by using a conventional extraction method by using a whole plant of the mazin or any part thereof as a raw material.
  • the main components of the mazin extract obtained by the present invention include flavonoids, anthraquinones, alkaloids, and coumarins; further including the above-mentioned flavonoids, anthraquinones, alkaloids, coumarin glycosides and monomer components thereof; Sugars and cellulose.
  • the mascara is used as a raw material medicine, and the fresh product, the lyophilized product, and the organic solvent pretreatment product are used.
  • the solvent in the step B is methanol, ethanol, isopropanol, ethyl acetate or petroleum ether; preferably methanol or ethanol.
  • the extraction in step B is performed by dipping, refluxing or seepage extraction.
  • the drying in the step B is vacuum drying, freeze drying, spray drying or microwave drying.
  • step C extracting the filtrate obtained in step B with solvent b, obtaining a liquid phase, and drying, that is, obtaining;
  • the filtrate obtained in step B is concentrated or dried to obtain extract 1, extracted with solvent b, and the extract is dried to obtain.
  • the mascara is used as a raw material medicine, and the fresh product, the lyophilized product, and the organic solvent pretreatment product are used.
  • the solvent a in the step B is methanol or ethanol, isopropanol; preferably methanol or ethanol.
  • the solvent b in the step C is ethyl acetate or petroleum ether.
  • the extraction in the step B and the step C is carried out by a dipping method, a reflux method, a percolation method or an extraction method.
  • the drying in steps B and C is vacuum drying, freeze drying, spray drying or microwave drying.
  • the extract prepared by the above method if prepared according to the method 1, is named as the corresponding extract by the solvent used in the step B, such as melamine ethanol extract, mazin methanol extract, mazin isopropanol extract, vest. Ethyl acetate extract, mascot petroleum ether extract.
  • the solvent b used in the step C is named as the corresponding extract, such as the ketone petroleum ether extract, the ethyl acetate extract of the horse, and the extract 1 obtained by the method 2 step C is the method 2
  • the solvent a used in the step B is named as the corresponding extract, such as melamine ethanol extract, mazin methanol extract, and mazin isopropanol extract.
  • the solvent used in the step B of the method 1 is used in a relationship with the amount of the horse, and the solvent is added in an amount of 1 to 20 times the mass of the horse; preferably, the solvent is added in an amount of 5 to 15 times the mass of the horse. Further, the amount of solvent added is 8-10 times the mass of the horse.
  • the relationship between the solvent a and the mascara used in the step B of the second method is that the solvent a is added in an amount of 1 to 20 times the mass of the mace; preferably, the solvent a is added in a mass of 5 -15 times; further, the amount of solvent a added is 8-10 times the mass of the horse.
  • the amount of methanol or ethanol added is 1 to 20 times the mass of the horse; preferably, the amount of methanol or ethanol added is 5-15 times the mass of the horse; further, Preferably, the amount of methanol or ethanol added is 8-10 times the mass of the horse.
  • the concentration of the ethanol is 10-95%.
  • the ethanol concentration is from 50 to 95%.
  • the ethanol concentration is 95%.
  • the raw material mate must be fresh or freeze-dried before being treated with an organic solvent.
  • the dried products of the vests drying or drying did not have corresponding activities during the research process, so the fresh products of vestibules were selected as raw materials.
  • fresh products are not conducive to preservation and transportation. Therefore, the inventors discovered that the use of freeze-dried and organic solvents to pretreat the fresh vestibules is conducive to the preservation of medicinal active ingredients, and the effect is comparable to that of fresh products, which can satisfy the mascara extraction of the present invention.
  • the new use of the object is conducive to preservation and transportation.
  • the method of treating with an organic solvent is: taking the mascara with an organic solvent and drying.
  • the organic solvent is ethanol, methanol, ethyl acetate, petroleum ether, isopropanol or the like; preferably methanol or ethanol.
  • the extract of the present invention can be used orally (including buccal or sublingual), nasal, topical (including buccal, sublingual or transdermal), parenteral (including subcutaneous, intradermal, intramuscular, intra-articular, slippery). Administration by intramembranous, intrasternal, intrathecal, intralesional, intravenous or intradermal injection or infusion. These preparations can be carried out by any method known in the art of pharmacy, for example by The ingredients are prepared in combination with a carrier or excipient.
  • a fourth technical problem to be solved by the present invention is to provide a pharmaceutical preparation prepared from the mazin extract of the present invention, that is, the mascara extract of the present invention can be prepared into a plurality of routes by adding a pharmaceutically acceptable adjuvant. preparation.
  • the extract of the present invention can be prepared into a conventional oral preparation, an injection preparation, and an external preparation by a conventional method.
  • oral including buccal or sublingual
  • nasal including buccal, sublingual or transdermal
  • parenteral including subcutaneous, intradermal, intramuscular, intra-articular, intra-synovial, sternal
  • Administration by intra-, intrathecal, intralesional, intravenous or intradermal injection or infusion.
  • formulations may be prepared by any method known in the art of pharmacy, for example by combining the active ingredient with carriers or excipients. For example, tablets, capsules, granules, pellets, microspheres, dropping pills, controlled release preparations, sustained release preparations or injections.
  • compositions suitable for oral administration can be presented as discrete units such as capsules, tablets, powders or granules; solutions, suspensions, oil-in-water emulsions or water-in-oil emulsions in aqueous or non-aqueous liquids.
  • Oral liquids such as solutions, syrups and elixirs may be prepared in unit dosage form.
  • a syrup can be prepared by dissolving the compound in a suitably flavored aqueous solution, and the elixirs are prepared via the use of a non-toxic vehicle.
  • Solubilizers and emulsifiers such as ethoxylated isostearyl alcohol and polyoxyethylene sorbitol ether
  • preservatives such as ethoxylated isostearyl alcohol and polyoxyethylene sorbitol ether
  • flavoring additives such as peppermint oil or natural sweeteners, or saccharin or other artificial sweeteners
  • Dosage unit formulations for oral administration may be microencapsulated, as appropriate, or may be prepared by coating or embedding particulate materials in a polymer, wax or the like for prolonged or sustained release.
  • liposome delivery systems such as small unilamellar liposomes, large unilamellar liposomes and multilamellar liposomes, which can be composed of a wide variety of phospholipids, such as cholesterol.
  • phospholipids such as cholesterol.
  • Stearylamine or phosphatidylcholine is formed.
  • compositions adapted for transdermal administration can be presented in the form of separate patches intended to remain in intimate contact with the recipient's epidermis for extended periods of time.
  • Pharmaceutical preparations suitable for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, elixirs or oils.
  • a fifth technical problem solved by the present invention is to provide a medicinal new use of the mazin extract of the present invention.
  • the mazin extract of the invention including the methanol extract of mazum, the ethanol extract of mazum, the ethyl acetate extract of mazum, the petroleum ether extract of mazyl, can be used for preparing anti-fibrotic, antifungal activity and anti-fibrosis. Tumor activity, treatment of oral and digestive tract inflammation or (and) ulcer-related diseases, and new pharmaceutical uses in drugs with two-way immunomodulatory effects.
  • a pharmaceutical composition comprising a mazin extract alone or a mascara extract as a main active ingredient is used in the above-mentioned new pharmaceutical use.
  • the use of the mazin extract of the present invention in the preparation of a medicament for antitumor activity may also be combined with other traditional Chinese medicines and western medicines for treating tumors, including radiotherapy, immunotherapy, chemotherapeutic agents for DNA damage, and interference with cell replication.
  • Chemotherapeutic agents and immunomodulatory drugs specifically topoisomerase I inhibitors, topoisomerase II inhibitors, alkylating agents, DNA chimeras, DNA intercalators and free radical generators, chemotherapeutic agents that interfere with cell replication, proteins Tyrosine kinase inhibitors, protease inhibitors, antibodies, proteins or enzyme inhibitors that bind to proteins overexpressed in cancer to downregulate cell replication.
  • Other traditional Chinese medicines and western medicines for treating tumors may be selected from the group consisting of irinotecan, topotecan, camptothecin and its analogues or metabolites, doxorubicin, etoposide, teniposide, daunorubicin, and metofa Sulfonic acid, chlorambucil, busulfan, thiotepa, ifosfamide, nitrosourea mustard, lomustine, semustine, streptozotocin, aminomethamine, methylamine Chrysalis, mitomycin C, cyclophosphamide, cisplatin, oxaliplatin, carboplatin, bleomycin, 5-fluorouracil, capecitabine, gemcitabine, fludarabi Bin, cytarabine, guanidine, thioguanine, pentastatin, hydroxyurea, paclitaxel, docetaxel and related analogues, vincris
  • the mazin extract of the present invention may also be combined with other traditional Chinese medicines and western medicines having therapeutic fibrosis.
  • Other traditional Chinese medicines and western medicines with therapeutic fibrosis are selected from: Panax notoginseng, Ligustrazine, Salvia miltiorrhiza, Acanthopanax Injection, Ligustrazine Injection, Safflower Injection, Ginkgo Flavone Glycoside, Shengmai, Danshen Injection, Double Huanglian, Xiangdan Injection, Yiqi Huoxue Granule, Kangxian Granule, Feikang Granule, Lily Gujin Pill, Cordyceps sinensis, Yangshen Pill, Astragalus, Saffron, Rehmannia, Sanqi, Gynostemma, Turmeric, Yellow Sunflower , amygdalin, tetrandrine, emodin, entecavir, lamivudine, beta-carotene, vitamin
  • the application of the mazin extract of the present invention in the preparation of a medicament having two-way immunomodulatory effects specifically refers to the application of a medicament for preparing an immune function or (and) an autoimmune disease, in particular, an immune function caused by an abnormal immune function. Or (and) an autoimmune disease.
  • the immunological function of the abnormal immune function is low, including susceptibility to cold, physical deficiency, tumor, AIDS and the like.
  • the immune dysfunction autoimmune diseases include rheumatoid arthritis, lupus erythematosus, scleroderma, hyperthyroidism, juvenile diabetes, primary platelet purpura, autoimmune hemolytic anemia, ulcerative colitis, Chronic liver disease and so on.
  • the mazin extract when used for preparing a drug for antitumor activity, the whole plant of the mazin or any part thereof may be used as a drug substance, and the leaf is preferably used as a drug substance.
  • the mazin extract can be prepared by the following method: taking the whole plant of the maz, including the root, the stem, the leaf, or any of them.
  • the fresh product or the lyophilized product of the part is pretreated with the organic solvent to pretreat the fresh meringue or the lyophilized product, and then the pretreated mascara fresh product or the lyophilized product is extracted with an organic solvent.
  • the fresh lyophilized product or the lyophilized product may be directly extracted with an organic solvent to obtain a mascara extract
  • the method of extracting with an organic solvent includes, but is not limited to, a conventional extraction method in the art such as a dipping method, a reflux method, and a percolation method.
  • the vestibule is preferably fresh or lyophilized prior to treatment with an organic vehicle.
  • the organic solvent is selected from the group consisting of methanol, ethanol, isopropanol, ethyl acetate, petroleum ether, and the like, preferably 50-95% ethanol, and more preferably 95% ethanol.
  • the jatropha extract is preferably prepared by taking a whole fresh vestibule, immersing it in 8-10 parts by weight of 95% ethanol for 1 day, then pulverizing the masculin, and then adding 6-10 parts by weight of 95% ethanol.
  • the extract is immersed for 2-3 days, the extract is collected, and the solvent is recovered under reduced pressure.
  • the obtained extract concentrate is dried to obtain a melamine ethanol extract, wherein the drying method includes, but is not limited to, drying under reduced pressure, freeze drying, spray drying, and microwave. dry.
  • the fresh emazi stems and leaves are soaked in 8 times by weight of methanol for 1 day, then the mascara is pulverized, 10 times by weight of methanol is added, and the extract is refluxed, and the extract is collected, and the methanol is recovered under reduced pressure at 60 ° C to obtain methanol.
  • the extract concentrate is dried and the mazin methanol extract is obtained.
  • the ethanol extract of mazum can also be dispersed in water, and then extracted with petroleum ether and ethyl acetate in that order, concentrated, and dried to obtain a petroleum ether extract or an ethyl acetate extract.
  • the extract concentrate is further freeze-dried to obtain an alcohol extract of the leaves of the horse.
  • the traditional Chinese medicine extract is subjected to extraction treatment by petroleum ether or ethyl acetate.
  • the traditional Chinese medicine extract was extracted successively with petroleum ether, ethyl acetate and n-butanol, and the solvent was recovered and lyophilized to obtain extracts of three polar parts, which were all dissolved with isopropyl alcohol, and then tested for tumor cells.
  • the petroleum ether fraction and the ethyl acetate fraction of the extract of the traditional Chinese medicine have good antitumor activity
  • the n-butanol extract site showed no antitumor activity
  • the ethyl acetate fraction showed better antitumor activity.
  • the main components of the mazin extract include triterpenoids, flavonoids, alkaloids and coumarins.
  • the mazin extract when used for preparing an anti-fibrotic drug, the mascara whole plant or any part thereof is used as a drug substance, and the leaf is preferably used as a drug substance.
  • the mazin extract can be prepared by the following method: taking the whole plant of the maz, including the root, the stem, the leaf, or any of them.
  • the fresh product or the lyophilized product of the part is pretreated with the organic solvent to pretreat the fresh meringue or the lyophilized product, and then the pretreated mascara fresh product or the lyophilized product is extracted with an organic solvent.
  • the fresh lyophilized product or the lyophilized product may be directly extracted with an organic solvent to obtain a mascara extract
  • the method of extracting with an organic solvent includes, but is not limited to, a conventional extraction method in the art such as a dipping method, a reflux method, and a percolation method.
  • the vestibule is preferably fresh or lyophilized prior to treatment with an organic vehicle.
  • the organic solvent is selected from the group consisting of methanol, 10-100% ethanol, preferably 95% ethanol.
  • the jatropha extract is preferably prepared by taking a whole fresh vestibule, immersing it in 8-10 parts by weight of 95% ethanol for 1 day, then pulverizing the masculin, and then adding 6-10 parts by weight of 95% ethanol.
  • the extract is immersed for 2-3 days, the extract is collected, and the solvent is recovered under reduced pressure.
  • the obtained extract concentrate is dried to obtain a melamine ethanol extract, wherein the drying method includes, but is not limited to, drying under reduced pressure, freeze drying, spray drying, and microwave. dry.
  • the fresh emazi stems and leaves are soaked in 8 times by weight of methanol for 1 day, then the mascara is pulverized, 10 times by weight of methanol is added, and the extract is refluxed, and the extract is collected, and the methanol is recovered under reduced pressure at 60 ° C to obtain methanol.
  • the extract concentrate is dried and the mazin methanol extract is obtained.
  • the ethanol extract of mazum can also be dispersed in water, and then extracted with petroleum ether and ethyl acetate in that order, concentrated, and dried to obtain a petroleum ether extract or an ethyl acetate extract.
  • the mazin extract when applied to the preparation of an antifungal drug, the whole plant of the mazin plant or any part thereof is used as a drug substance, and the leaf is preferably used as a drug substance.
  • the horse jasmine extract can be prepared by taking the whole plant or any part of the mazum, adding 1-20 times of the volume of the mazin to extract methanol or ethanol, or further extracting the methanol or ethanol extract with ethyl acetate or petroleum. Ether extraction; the extract was concentrated and dried to obtain a mazin extract.
  • the vest is preferably fresh, lyophilized or methanol or ethanol treated sample; the extraction method is impregnation, reflux or osmosis; the drying method is vacuum drying, freeze drying, spray drying or microwave dry.
  • the prepared raisin extract includes saccharides, flavonoids, alkaloids, coumarins and saccharides, flavonoids, alkaloids, coumarin glycosides and monomers.
  • the various preparations of the mazin extract include mascara extract tablets, granules, ointments, gels, filming agents, tinctures, lotions, and sprays.
  • the antifungal activity of the mazin extract and various preparations provided by the invention are used for antifungal activity.
  • the mazin extract is mazin methanol, ethanol, ethyl acetate, petroleum ether or a similar solvent extract. Samples prepared from mascara, lyophilized product, methanol (ethanol) or other organic solvents are used as raw materials to ensure the stability of the active ingredients.
  • the mazin extract when used for the preparation of a medicament for treating immunocompromised or (and) autoimmune diseases, the whole plant of the mazin or any part thereof is used as a raw material medicine, and preferably the leaf is used as a raw material medicine.
  • the preparation method of the horse jasmine extract is as follows: take the whole plant or any part of the vestibule, add 1-20 times of the volume of the mazin to extract methanol or ethanol, or further extract the methanol or ethanol extract with ethyl acetate or petroleum. Ether extraction; the extract was concentrated and dried to obtain a mazin extract.
  • the mazin extract, mascot is preferably fresh, lyophilized or pretreated sample such as methanol, ethanol, isopropanol; the extraction method comprises dipping, refluxing or osmosis; the drying method comprises drying under reduced pressure, freezing Dry, spray dried or microwave dried.
  • the mazin extract contains steroids, flavonoids, alkaloids, coumarins, polysaccharides, celluloses, and glucosides of steroids, flavonoids, alkaloids, and coumarins. And monomer.
  • the preparation of the mazin extract including a tablet, a capsule, a plaster, a granule, an injection, a suppository, an ointment, a gel, a coating agent, an elixir, a lotion, and a spray of a mazin extract Agent.
  • the use of the mazin extract for the preparation of a medicament for treating an immunocompromised disease or (and) an autoimmune disease comprising using a mazin extract alone or a medicinal extract containing mascara extract as a main active ingredient
  • Use of a medicament for the preparation of a medicament for treating an immunocompromised disease or (and) an autoimmune disease comprising using a mazin extract alone or a medicinal extract containing mascara extract as a main active ingredient
  • the mazin extract when used for the preparation of a medicament for treating inflammation of the oral cavity and the digestive tract or ulcer, the whole plant of the mazin or any part thereof is used as a raw material medicine, and preferably the leaf is used as a raw material medicine.
  • the preparation method of the horse jasmine extract is as follows: taking the fresh product of the whole plant of the vestibule, the stalk of the horse, or the whole plant of the horse, adding 1 to 20 times of the mass of the horse, or extracting the ethanol; or further extracting the methanol or the ethanol Ethyl acetate or petroleum ether extraction; the extract is concentrated and dried to obtain mazin extract; the components include quinones, flavonoids, alkaloids, coumarins, polysaccharides and celluloses, and quinones and flavonoids. Alkaloids, glucosides and monomers of coumarins.
  • the mazin extract is directly extracted from the fresh lyophilized product or the lyophilized product by using an organic solvent, or the fresh mascara is pretreated with an organic solvent;
  • the organic solvent includes methanol, ethanol, isopropanol, acetic acid.
  • the extraction method includes dipping, refluxing or osmosis;
  • the drying method comprises drying under reduced pressure, freeze drying, spray drying or microwave drying.
  • the preparation of the mazin extract includes a tablet, a capsule, a plaster, a granule, an injection, a suppository, an ointment, a gel, a coating, an elixir, a lotion, and a spray.
  • the use of the mazin extract includes the use of a medicinal extract alone or a mascara extract as a main active ingredient for the preparation of a medicament for treating oral and gastrointestinal inflammation or/and ulcers.
  • the sample was dissolved in acetic anhydride, and a few drops of concentrated sulfuric acid-acetic anhydride (1:20) was added to produce a color change of yellow ⁇ red ⁇ purple ⁇ blue, and finally faded, indicating that the triterpenoid compound was contained.
  • the sample was dropped on the filter paper, sprayed with 25% trichloroacetic acid in ethanol, heated to 100 ° C, reddish, and gradually turned purple, indicating the presence of triterpenoids.
  • the sample was dissolved in chloroform, and after adding concentrated sulfuric acid, the sulfuric acid layer showed red or blue, and the chloroform layer showed green fluorescence, indicating that the triterpenoid compound was contained.
  • the sample was dissolved in glacial acetic acid, and a few drops of acetyl chloride and a few crystals of zinc chloride were added. When heated slightly, it showed a reddish or purplish red color, indicating the presence of triterpenoids.
  • the sample was taken with a glass rod and the test solution was applied to the filter paper, blown dry, sprayed with 1% aluminum trichloride ethanol solution, and dried to observe the phenomenon. It is bright yellow under the UV lamp, indicating the presence of flavonoids.
  • test solution is drawn on the filter paper with a glass rod, dried, sprayed with an aqueous solution of sodium hydroxide or exposed to ammonia vapor.
  • ammonia vapor can change the color point of the sample to a bright yellow color, indicating that the flavonoid is contained. Class of compounds.
  • cerium nitrate 0.85 g was dissolved in 10 ml of glacial acetic acid and 40 ml of water; 2 8 g of potassium iodide was dissolved in 20 ml of water.
  • the test solution 1 and the test solution 2 are mixed in equal amounts and stored in a brown bottle as a stock solution. 1 ml of stock solution, 4 ml of glacial acetic acid and 12 ml of water were mixed before use.
  • the sample test solution was added to the above reagent, and it was a reddish brown solution. When distilled water was added thereto, a precipitate was produced, indicating that the alkaloid compound was contained.
  • the main components of the mazin extract of the present invention include flavonoids, anthraquinones, alkaloids, coumarins, and the above-mentioned flavonoids, anthraquinones, alkaloids, coumarin glycosides and their monomer components, and Contains polysaccharides and cellulose.
  • tumor cells in the logarithmic growth phase were used (the following tumor cell lines were used: cervical cancer cell line Hela; human liver cancer cell line SMMC-7721; human lung cancer cell line A549; human colon cancer cell line Caco-2) ; leukemia cell line K562; gastric cancer cell line MGC-803;), centrifuged at 2000 rpm for 5 min, adjusted to a cell concentration of 1 ⁇ 10 5 /ml with a corresponding medium containing 10% fetal bovine serum, The cells were seeded in 96-well culture plates.
  • the alcohol extract of fresh leaves of mazin and the leaves of the nectar after ethanol soaking and drying have good tumor inhibition effect, and other extracts do not have anti-tumor activity, and the analysis may be related to active extraction. It is related to the solubility and stability of the substance.
  • the content of the coumarin component was determined to be from 1% to 10%.
  • the ethanol extract of the leaves of the horses was extracted with petroleum ether, ethyl acetate and n-butanol in sequence, and the solvent was recovered and lyophilized to obtain extracts of three polar sites, all of which were dissolved in isopropyl alcohol.
  • tumor cells in the logarithmic growth phase were used (the following tumor cell lines were used: cervical cancer cell line Hela; human liver cancer cell line SMMC-7721; human lung cancer cell line A549; human colon cancer cell line Caco-2) ; leukemia cell line K562; gastric cancer cell line MGC-803;), centrifuged at 2000 rpm for 5 min, adjusted the concentration of the precipitated cells to 1 ⁇ 105 cells/ml cell suspension with the corresponding culture medium containing 10% fetal bovine serum, and then the cells were Inoculate in a 96-well culture plate.
  • mice Take Kunming mice, and inoculate 0.2ml (about 1 ⁇ 10 6 tumor cells) under the right anterior sac. After 24 hours of inoculation, the mice were randomly numbered, and the number of rats in the control group was 10. The three groups were divided into high-dose group, middle-dose group and low-dose group, with 10 rats in each group. Each group was intragastrically administered once a day for 14 times. The positive control group was intragastrically administered with cyclophosphamide (85 mg/kg) every day. The dose, frequency and time of administration were the same as those of the experimental group. 24 hours after the last administration, the animals were sacrificed, and the tumor pieces were completely excised and weighed, and the tumor inhibition rate was calculated. The results are shown in Table 3.
  • tumor cell strains used were: cervical cancer cell line Hela; human liver cancer cell line SMMC-7721; human lung cancer cell line A549; human colon cancer cell line Caco-2; leukemia cells) K562; gastric cancer cell line MGC-803;), centrifuged at 2000 rpm for 5 min, and the cell concentration was adjusted to 1 ⁇ 10 5 /ml cell suspension with the corresponding culture medium containing 10% fetal bovine serum, and then the cells were seeded. 96-well culture plate.
  • the galactose ethanol extract of Example 1 was sequentially extracted with petroleum ether, ethyl acetate and n-butanol, and the solvent was recovered and dried to obtain petroleum extract of mazum, ethyl acetate and n-butanol, respectively.
  • the propanol is dissolved.
  • tumor cell strains used were: cervical cancer cell line Hela; human liver cancer cell line SMMC-7721; human lung cancer cell line A549; human colon cancer cell line Caco-2; leukemia cells) K562; gastric cancer cell line MGC-803;), centrifuged at 2000 rpm for 5 min, and the cell concentration was adjusted to 1 ⁇ 105 cells/ml with a corresponding medium containing 10% fetal bovine serum, and then the cells were seeded at 96 rpm. Hole culture plate.
  • the final concentration of the extract in each well is 0.002, 0.01, 0.02, 0.04, 0.05, 0.08, 0.1, 0.2 mg / ml, and after mixing, set at 37 ° C, 5%
  • the culture solution was aspirated and washed twice with PBS, and then 20 ⁇ l of MTT phosphate buffer solution and 150 ⁇ l of the medium were added to each well, and the culture was terminated after continuing the culture for 4 hours under the same conditions.
  • S180 tumor-derived mice were inoculated 8 days in good condition.
  • the abdominal skin was disinfected and ascites was taken.
  • the sterile saline solution was suspended at 1:4 (ascites volume: normal saline volume) for use.
  • 82 male Kunming mice, 18-20 g, were randomly divided into 7 groups according to body weight stratification, which were model control group (0.5% tragacanth), positive control group (cyclophosphamide, CTX), ethyl acetate.
  • the extract low-dose group, the ethyl acetate extract high-dose group, the ethanol extract group, the methanol extract group, and the petroleum ether extract group were all inoculated subcutaneously with 0.2 ml of the aforementioned suspension in the right ankle.
  • the model control group and the drug group were intragastrically administered with the test substance or suspension, once a day for 14 consecutive days; the positive control group was given CTX intraperitoneally, once every other day for 7 times.
  • the mice were sacrificed by cervical dislocation 24 h after the last administration, and the tumor pieces were weighed, and the tumor inhibition rate was calculated ((1 - mean tumor weight of the experimental group / mean tumor weight of the model control group) * 100%).
  • the results are shown in Table 6.
  • mice The experimental results showed that the ethyl acetate extract of the whole plant was extracted by 1.6g/kg, which significantly inhibited S180. Growth in mice, and the intensity of 1.6g / kg dose is similar to that of cyclophosphamide 40mg / kg every day; ethanol, methanol, petroleum ether extract 4.8g / kg can also effectively reduce tumor weight, tumor inhibition rate More than 50%, suggesting that the four types of extracts have better anti-tumor activity.
  • mice were inoculated with 8 days of healthy Ehrlich ascites tumors.
  • the abdominal skin was disinfected and ascites was taken and suspended in sterile saline for 4 ⁇ 10 6 /ml.
  • 82 male Kunming mice 18-20 g, were randomly divided into 7 groups according to body weight stratification, which were model control group (0.5% tragacanth), positive control group (cyclophosphamide, CTX), ethyl acetate.
  • the extract low-dose group, the ethyl acetate extract high-dose group, the ethanol extract group, the methanol extract group, and the petroleum ether extract group were each inoculated with 0.2 ml of the aforementioned suspension in the abdominal cavity.
  • the model control group and the drug group were intragastrically administered with the test substance or suspension, once a day, and continued until the animal dying; the positive control group was given CTX intraperitoneally, once every other day for 7 times.
  • the animal dying the cervical vertebrae were sacrificed, and the dying time was counted as the number of days of survival; after the sacrifice, the body weight was weighed, then the ascites was drained, and the weight was again weighed, and the difference was calculated as the ascites weight.
  • Table 7 The results are shown in Table 7.
  • S180 tumor-derived mice were inoculated 8 days in good condition.
  • the abdominal skin was disinfected and ascites was taken.
  • the sterile saline solution was suspended at 1:4 (ascites volume: normal saline volume) for use.
  • 84 male Kunming mice, 18-20 g were randomly divided into 7 groups according to body weight stratification, which were model control group (0.5% tragacanth), positive control group (cyclophosphamide, CTX), ethyl acetate.
  • the extract group, the paclitaxel group, the scutellaria group, the paclitaxel combination group, and the scutellarin group were all inoculated with 0.2 ml of the above suspension subcutaneously in the right ankle.
  • mice After 2 hours, the model control group and the drug group were given the test substance or suspension by intragastric or intravenous injection once a day for 14 consecutive days; the positive control group was given CTX by intraperitoneal injection once every other day for 7 times.
  • the mice were sacrificed by cervical dislocation 24 h after the last administration, and the tumor pieces were weighed and the tumor inhibition rate was calculated ((1 - mean tumor weight of the experimental group / mean tumor weight of the model control group) * 100%). The results are shown in Table 8.
  • Vanillin-glacial acetic acid 0.4ml, perchloric acid 1.6ml, mixed, diluted with ethyl acetate to the mark, heated in a constant temperature water bath at 70 ° C for 15 min, cooled to room Warm, transfer to a 10 ml volumetric flask, add ethyl acetate to dilute to the mark, shake well, measure the absorbance at 540 nm wavelength, calculate the total triterpene content in the test solution (including triterpene in terms of tartanic acid). It was calculated that the triterpenoid component in 1 g of the mazin extract contained 23 mg.
  • Sixty SD rats (200-240 g, male) were randomly divided into a blank control group (0.5% scutellaria), a model control group (0.5% scutellaria), and a positive control group (dexamethasone). 1 mg/kg), mazin extract (prepared according to the method of Example 1), low dose group (0.4 g/kg), medium dose group (0.8 g/kg), high dose group (1.6 g/kg). Except for the blank control group, 40% carbon tetrachloride vegetable oil solution was subcutaneously injected at 1 ml/kg twice a week for 3 consecutive months, and high fat diet and 5% ethanol aqueous solution were simultaneously administered. The test substance was administered by gavage once a day for 3 consecutive months.
  • ALT alanine aminotransferase
  • PC-III type III procollagen
  • HA hyaluronic acid
  • LH laminin
  • the mazin extract had protective effects on liver injury induced by carbon tetrachloride in rats with experimental liver fibrosis at 0.8g/kg and 1.6g/kg, especially in At high doses, each indicator improved significantly.
  • sham control group (0.5% scutellaria
  • model control group (0.5% scutellaria
  • positive control group (dexamethasone).
  • mazin extract prepared according to the method of Example 1
  • low dose group 0.4 g/kg
  • medium dose group 0.8 g/kg
  • high dose group 1.6 g/kg
  • All animals were isolated by intraperitoneal injection of 10% chloral hydrate (350 mg/kg).
  • 0.4 mL of bleomycin physiological saline solution was injected at 5 mg/kg.
  • the sham-operated control group was only injected with normal saline. .
  • test substance was administered the next day after the operation, once a day for 30 consecutive days.
  • the animals were sacrificed by cervical dislocation and the lungs were taken.
  • the hydroxyproline content was determined after partial organ homogenization, and some pathological observations were performed.
  • the results of measurement of hydroxyproline content in lung tissue are shown in Table 10.
  • 60 SD rats (200-240 g, male) were randomly divided into blank control group, model control group, positive control group (dexamethasone, 1 mg/kg) and mazin extract group according to body weight (according to the examples).
  • the method of 1 was prepared, 0.8 g/kg), the polyene phosphatidylcholine group (1 ml/kg), and the combination group (Machia extract 0.8 g/kg + polyene phosphatidylcholine 1 ml/kg). Except for the blank control group, 40% carbon tetrachloride vegetable oil solution was subcutaneously injected at 1 ml/kg twice a week for 3 consecutive months, and high fat diet and 5% ethanol aqueous solution were simultaneously administered.
  • test substance is administered by gavage or injection once a day for 3 consecutive months. Blood was taken from the abdominal aorta on the next day after the end of administration, and plasma was separated to determine the levels of alanine aminotransferase (ALT), type III procollagen (PC-III), hyaluronic acid (HA) and laminin (LH); The animals were then sacrificed and the liver was taken for pathological examination. The results of serum biochemical tests are shown in Table 11.
  • SD rats (200-240 g, male) 60 were randomly divided into sham control group, model control group, positive control group (dexamethasone, 1 mg/kg) and mazin extract according to body weight stratification (according to the examples).
  • sham-operated control group In addition to the sham-operated control group, 0.4 mL of bleomycin physiological saline solution was injected at 5 mg/kg. The sham-operated control group was only injected with normal saline. . The test substance was administered by intragastric administration or injection the next day after the operation, once a day for 30 consecutive days. The animals were sacrificed by cervical dislocation and the lungs were taken. The hydroxyproline content was determined after partial organ homogenization, and some pathological observations were performed. The results of measurement of hydroxyproline content in lung tissue are shown in Table 12.
  • 40g of polyvinyl alcohol 124 is swollen in 400ml of purified water; another 100g of melamine ethanol extract is dissolved in 400ml of ethanol, and then 100ml of glycerin is added, stirred evenly, slowly added to the polyvinyl alcohol solution, stirred and filtered, and then Add ethanol to the filter to 1000ml, that is.
  • Example 1 1 g of the ethanol extract of the horse mace prepared in Example 1 (1) was weighed, dissolved in a 5% isopropanol solution to 10 ml, and filtered by a 0.22 ⁇ m filter. 1 ml of the above solution was sterilized, added to 9 ml of PDA medium which was cooled and cooled to about 50 ° C, shaken well, and quickly poured into a Petri dish having a diameter of 6 cm, and allowed to stand to prepare a 10 mg/ml mazax extract. The PDA culture plate of the object. A blank control PDA culture plate was prepared using the same volume of 5% isopropanol solution.
  • the cut inoculum was transferred into the above-mentioned drug-containing PDA culture plate and cultured at 25 ° C.
  • the diameter of the colonies on all the culture plates was determined by the cross method, and the inhibition rate was calculated after the calibration. .
  • the results are shown in Table 13.
  • the ethanol extract of the horses has obvious inhibitory effects on various fungi tested. Since the tested fungi are common pathogenic fungi and have obvious representativeness, the results suggest that the alcohol extract of the horseshoe has strong antifungal activity and is expected to be applied to the preparation of antifungal drugs.
  • the scale was shaken, and the absorbance was measured at a wavelength of 540 nm, and the total triterpene content in the test solution (including triterpenoids in terms of citric acid) was calculated. It was calculated that the triterpenoid component in 1 g of the mazin extract contained 23 mg.
  • ethanol extract of the whole plant of vestibule Take 300 g of the ethanol extract of the whole plant of vestibule, and add suitable excipients, such as: 100 g of microcrystalline cellulose, 57.5 g of lactose, 20 g of croscarmellose sodium, etc., and tablet into tablets.
  • suitable excipients such as: 100 g of microcrystalline cellulose, 57.5 g of lactose, 20 g of croscarmellose sodium, etc.
  • the ethyl acetate extract of the stem and leaf of the vestibule is taken, and a suitable auxiliary material such as lactose, compressible starch, carboxymethyl starch, microcrystalline cellulose, etc. is added to prepare a capsule.
  • a suitable auxiliary material such as lactose, compressible starch, carboxymethyl starch, microcrystalline cellulose, etc.
  • auxiliary materials such as: octadecyl alcohol, glyceryl monostearate, glycerin, stearic acid, etc. to make an ointment.
  • auxiliary materials such as: mixed fatty acid glyceride, PEG, beeswax, etc. to make a suppository.
  • the spleen of Kunming mice was aseptically placed in a dish containing appropriate amount of RPMI1640 medium, the connective tissue was removed, ground with a syringe needle, filtered through a 200-mesh sieve, transferred to a centrifuge tube, and washed with RPMI1640 to collect cells. Add 1 ml of red blood cell lysate. After 5-10 min at 4 ° C, the cells were centrifuged at 1500 r/min for 5 min, the supernatant was discarded, washed twice with RPMI 1640 medium, and finally the cells were suspended in 1 ml of RPMI1640 complete medium.
  • the survival rate was determined by trypan blue staining, and the results showed that the survival rate of splenocytes was more than 95%.
  • the above spleen cell suspension was appropriately diluted, and the cell density was adjusted to 2 ⁇ 10 6 /ml for use.
  • a suspension of 2 x 10 6 /ml spleen cells was added to a 96-well plate at 100 ⁇ l per well.
  • a blank group, a control group, and a mazin extract group were set.
  • 100 ⁇ l of RPMI1640 complete medium was added to each well.
  • 100 ⁇ l of RPMI1640 complete medium solution containing different concentrations of mazin extract was added to each well; a blank group was added, and only the medium was contained.
  • the above 96-well plates were placed in a 5% CO 2 incubator at 37 ° C for 60 h. The cell plate was taken out, the liquid in the well was aspirated, and washed with PBS three times.
  • a suspension of 2 x 10 6 /ml spleen cells was added to a 96-well plate at 100 ⁇ l per well.
  • a blank group, a control group, a ConA group, and a mazin extract group were set.
  • 100 ⁇ l of RPMI1640 complete medium was added to each well.
  • 100 ⁇ l of RPMI1640 complete medium solution containing 25 ⁇ g/ml ConA was added to each well.
  • 100 ⁇ l of each well was extracted with different concentrations of mazax.
  • RPMI1640 complete medium solution 25 ⁇ g/ml ConA in solution
  • a blank group containing only medium was used in the control group.
  • the above 96-well plates were placed in a 5% CO 2 incubator at 37 ° C for 60 h.
  • the cell plate was taken out, the liquid in the well was aspirated, and washed with PBS three times.
  • 100 ⁇ l of the medium and 20 ⁇ l of MTT solution (5 mg ⁇ ml -1 ) were added, and the cells were again cultured in a humidity environment of 37 ° C and a volume fraction of 5% CO 2 .
  • the cell culture plate was taken out, the supernatant was carefully aspirated, 150 ⁇ l of dimethyl sulfoxide was added, and the plate was shaken on a microplate shaker for 10 min to completely dissolve the crystal formamidine, and then ELISA.
  • the 490 nm colorimetric assay was performed on the detector. The results were calculated according to formula (1).
  • the average survival rate of mouse spleen cells in the ConA group and the scorpion extract group was calculated according to formula (1).
  • the galanga extract was induced to ConA. Proliferation rate of mouse spleen cells in vitro.
  • a suspension of 2 x 10 6 /ml spleen cells was added to a 96-well plate at 100 ⁇ l per well.
  • a blank group, a control group, an LPS group, and a mazin extract group were set.
  • 100 ⁇ l of RPMI1640 complete medium was added to each well.
  • 100 ⁇ l of RPMI1640 complete medium solution containing 20 ⁇ g/ml LPS was added to each well.
  • 100 ⁇ l of each well was extracted with different concentrations of mazax.
  • the above 96-well plates were placed in a 5% CO 2 incubator at 37 ° C for 60 h.
  • the cell plate was taken out, the liquid in the well was aspirated, and washed with PBS three times.
  • 100 ⁇ l of the medium and 20 ⁇ l of MTT solution (5 mg ⁇ ml -1 ) were added, and the cells were again cultured in a humidity environment of 37 ° C and a volume fraction of 5% CO 2 .
  • the cell culture plate was taken out, the supernatant was carefully aspirated, 150 ⁇ l of dimethyl sulfoxide was added, and the plate was shaken on a microplate shaker for 10 min to completely dissolve the crystal formamidine, and then ELISA.
  • the 490 nm colorimetric assay was performed on the detector. The results were calculated. According to formula (1), the average survival rate of mouse spleen cells in the LPS group and the scorpion extract group was calculated. Then, according to formula (2), the Lappa extract was small for LPS induction. Proliferation rate of mouse spleen cells in vitro.
  • mice Eighty KM mice were randomly divided into 8 groups according to body weight, which were suspension control group (0.5% tragacanth), model control group (0.5% tragacanth), and positive control group (Yunzhi polysaccharide). ), ethyl acetate extract low-dose group, ethyl acetate extract high-dose group, ethanol extract group, methanol extract group, petroleum ether extract group.
  • the test drug or suspension is administered intragastrically once a day for 14 days. Except for the suspension control group, the cyclophosphamide physiological saline solution was administered intraperitoneally at 25 mg/kg on the 8th, 10th, and 12th day after administration, resulting in low immunity.
  • Ethyl acetate extract of E. striata was administered intragastrically at a dose of 0.1 g/kg and above for 14 days, which significantly increased the phagocytic ability of peritoneal macrophages in immunosuppressed mice, and the intensity of 0.4 g/kg dose group and Yunzhi polysaccharide group There was no significant difference; ethanol, methanol and petroleum ether extract 1.2g/kg could also effectively enhance the phagocytic ability of peritoneal macrophages in immunocompromised mice, suggesting that these four extracts have better immune enhancement in immunocompromised animals. effect.
  • mice Eighty KM mice were randomly divided into 8 groups according to body weight, which were suspension control group (0.5% tragacanth), model control group (0.5% tragacanth), and positive control group (Yunzhi polysaccharide). ), ethyl acetate extract low-dose group, ethyl acetate extract high-dose group, ethanol extract group, methanol extract group, petroleum ether extract group.
  • the test drug or suspension is administered intragastrically once a day for 14 days. Except for the suspension control group, the cyclophosphamide physiological saline solution was administered intraperitoneally at 25 mg/kg on the 8th, 10th, and 12th day after administration, resulting in low immunity.
  • DNFB 2,4-dinitrofluorobenzene
  • mice Eighty KM mice were randomly divided into 8 groups according to body weight stratification, which were suspension control group (0.5% scutellaria gum), enhancement positive control group (Yunzhi polysaccharide), and inhibition positive control group (ring Phosphoramide), ethyl acetate extract low-dose group, ethyl acetate extract high-dose group, ethanol extract group, methanol extract group, petroleum ether extract group. Except the inhibitory positive control group was given a single subcutaneous injection on the 13th day, the other groups were given the test drug or suspension once a day for 14 days.
  • suspension control group (0.5% scutellaria gum
  • enhancement positive control group Yunzhi polysaccharide
  • inhibition positive control group ring Phosphoramide
  • ethyl acetate extract low-dose group ethyl acetate extract high-dose group
  • ethanol extract group ethyl acetate extract high-dose group
  • ethanol extract group ethyl acetate extract high-dose
  • E. coli extract of 0.4g/kg was administered intragastrically for 14 days, which could inhibit the phagocytic ability of normal mouse peritoneal macrophages to a certain extent, but no obvious effect was observed in 0.1g/kg; ethanol, methanol, The petroleum ether extract 1.2g/kg also showed different degrees of inhibitory effects, suggesting that the four types of extracts have mild immunosuppressive effects on normal animals.
  • mice Eighty KM mice were randomly divided into 8 groups according to body weight stratification, which were suspension control group (0.5% scutellaria gum), enhancement positive control group (Yunzhi polysaccharide), and inhibition positive control group (ring Phosphoramide), ethyl acetate extract low-dose group, ethyl acetate extract high-dose group, ethanol extract group, methanol extract group, petroleum ether extract group. Except the inhibitory positive control group was given a single subcutaneous injection on the 13th day, the other groups were given the test drug or suspended by intragastric administration. Agent, once a day for 14 days.
  • suspension control group (0.5% scutellaria gum
  • enhancement positive control group Yunzhi polysaccharide
  • inhibition positive control group ring Phosphoramide
  • ethyl acetate extract low-dose group ethyl acetate extract high-dose group
  • ethanol extract group ethyl acetate extract high-dose group
  • ethanol extract group ethy
  • DNFB 2,4-dinitrofluorobenzene
  • mice Eighty KM mice were randomly divided into 8 groups according to body weight stratification, which were suspension control group (0.5% scutellaria), model control group (0.5% scutellaria), and positive control group (Telecoma sinensis). Glycoside), ethyl acetate extract low-dose group, ethyl acetate extract high-dose group, ethanol extract group, methanol extract group, petroleum ether extract group. Except for the suspension control group, 0.5 ml/intraperitoneal injection of pristane was administered intraperitoneally with drug or suspension once daily for 30 days. Blood was collected from the eyelids 24 hours after the last drug, and the serum was separated by centrifugation at 4 ° C. The anti-dsDNA antibody levels in the serum were determined by ELISA.
  • Another group of animals were also grouped and replicated. On the 24th day after pristane injection, the animals in each group were intraperitoneally injected with 5% chicken red blood cell suspension (0.2 ml/mouse), and continued to continue until the suspension control group. On the 30th day after the injection of pristane. 20 ⁇ l of blood was taken from the eyelids 24 h after the last administration, and 1 ml of physiological saline was added, followed by adding 0.5 ml of 4% chicken red blood cell suspension and 0.5 ml of 10% guinea pig serum, mixing, incubating at 37 ° C for 0.5 h, centrifugation at 3000 rpm. 10 min, 1 ml of the supernatant was taken, 3 ml of Duchen solution was added, and the color was 540 nm.
  • the ethyl acetate extract of 0.1g/kg can effectively reduce the abnormally elevated hemolysin level.
  • the methanol, petroleum ether extract 1.2g/kg was similar to the ethyl acetate extract, suggesting that the four types of extracts have significant inhibitory effects on abnormal immune function.
  • the scale was shaken, and the absorbance was measured at a wavelength of 540 nm, and the total triterpene content in the test solution (including triterpenoids in terms of citric acid) was calculated. It was calculated that the triterpenoid component in 1 g of the mazin extract contained 23 mg.
  • ethanol extract of the whole plant of vestibule Take 300 g of the ethanol extract of the whole plant of vestibule, and add suitable excipients, such as: 100 g of microcrystalline cellulose, 57.5 g of lactose, 20 g of croscarmellose sodium, etc., and tablet into tablets.
  • suitable excipients such as: 100 g of microcrystalline cellulose, 57.5 g of lactose, 20 g of croscarmellose sodium, etc.
  • the ethyl acetate extract of the stem and leaf of the vestibule is taken, and a suitable auxiliary material such as lactose, compressible starch, carboxymethyl starch, microcrystalline cellulose, etc. is added to prepare a capsule.
  • a suitable auxiliary material such as lactose, compressible starch, carboxymethyl starch, microcrystalline cellulose, etc.
  • auxiliary materials such as: octadecyl alcohol, glyceryl monostearate, glycerin, stearic acid, etc. to make an ointment.
  • auxiliary materials such as: mixed fatty acid glyceride, PEG, beeswax, etc. to make a suppository.
  • Eighty SD rats were randomly divided into 8 groups according to body weight, which were model control group (0.5% tragacanth), positive control group (ranitidine), ethyl acetate extract low dose group, acetic acid. Ethyl extract extract group, ethyl acetate extract high dose group, ethanol extract group, methanol extract group, petroleum ether extract group.
  • the test substance was administered by gavage once a day for 3 consecutive days.
  • the pylorus ligation was performed 1 h after the last administration.
  • the animals were sacrificed by cervical vertebrae 15 h after operation.
  • the stomach was removed and fixed with 1% formaldehyde for 20 min. The degree of mucosal damage was observed by stereomicroscope and the ulcer index and ulcer inhibition rate were calculated.
  • Ethyl acetate extract of E. striata was administered intragastrically twice at a dose of 0.2 g/kg and above, which significantly inhibited the degree of gastric ulcer induced by pylorus ligation, and the intensity of the 0.4 g/kg dose group was similar to that of ranitidine. There was no significant difference in the 60mg/kg group; ethanol, methanol and petroleum ether extract 1.2g/kg could also effectively inhibit the degree of gastric ulcer, suggesting that the four types of extracts have better anti-gastric ulcer effects.
  • TNBS-induced experimental colitis in rats may result in increased inflammatory cells, elevated levels of inflammatory cytokines, and ulceration of the colon surface.
  • Ethyl acetate extract of O. chinensis at 0.2g/kg and above can inhibit the increase of leukocyte and important inflammatory factor TNF- ⁇ , and reduce the formation of ulcer surface; ethanol, methanol, petroleum ether extract 1.2g/kg and ethyl acetate The effects of the extracts were similar, suggesting that the four types of extracts have better anti-colitis effects.
  • 120 SD rats were randomly divided into 10 groups according to body weight, which were normal control group (0.5% western tragacanth), model control group (0.5% western tragacanth), and positive control group (Sanjiu Weitai Granules). ), ethyl acetate extract low-dose group, ethyl acetate extract dose group, ethyl acetate extract high-dose group, ethanol extract group, methanol extract group, petroleum ether extract group. All animals were given 0.02% ammonia water by gavage once a day for 90 consecutive days. At the same time, the test substance was administered by gavage once a day for 90 consecutive days. The next day after the last administration, the cervical vertebrae were sacrificed.
  • the small stomach wall was taken, fixed in 10% formalin, embedded in paraffin, sectioned, and stained with HE and PAS.
  • the HE stained tablets were observed for inflammatory reaction and scored, and the thickness of the gastric mucosa was measured; the PAS stained sheet was used to measure the thickness of the positive layer to characterize the thickness of the mucus layer.
  • the results are shown in Table 21.
  • the ethyl acetate extract of the horseshoe has the effect of inhibiting the degree of inflammation and increasing the thickness of the gastric mucosa and mucus layer by the experimental chronic gastritis caused by ammonia water, especially in inhibiting inflammation and increasing the thickness of the mucus layer; ethanol,
  • the action of methanol and petroleum ether extract 1.2g/kg was similar to that of ethyl acetate extract, suggesting that the four types of extracts have better anti-chronic gastritis effects.
  • mice 50 SD rats were randomly divided into 5 groups according to body weight, which were model control group (0.5% tragacanth), positive control group (ranitidine), ethyl acetate extract group, Sanjiu stomach. Thai group, joint application group.
  • the test substance was administered by gavage once a day for 3 consecutive days.
  • the pylorus ligation was performed 1 h after the last administration.
  • the animals were sacrificed by cervical vertebrae 15 h after operation.
  • the stomach was removed and fixed with 1% formaldehyde for 20 min.
  • the degree of mucosal damage was observed by stereomicroscope and the ulcer index and ulcer inhibition rate were calculated.
  • the use of the mazin extract of the present invention or its protomedical mazin has significant antitumor activity, antifungal activity, anti-fibrosis, and two-way immunomodulation, as well as treatment of oral and digestive tract inflammation or ulceration. efficacy.

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Abstract

马甲子以及马甲子提取物的新用途，包括在制备具有抗纤维化作用，抗真菌作用，抗肿瘤作用，双向免疫调节作用以及治疗口腔及消化道炎症或（和）溃疡的药物中的用途。

Description

马甲子提取物及其制备方法和用途 技术领域
本发明属于医药领域，具体涉及马甲子提取物及其制备方法和用途。
背景技术
马甲子(Paliurus ramosissimus(Lour.)Poir)为落叶灌木，是一种常见药用植物，据报道枝叶根花果均可药用。性味苦、平，无毒。能除寒活血，发表解热，消肿，治跌打损伤及心腹疼痛。收载于《中药大辞典》及一些地方药物志。
本发明的发明人在研究过程中发现马甲子及其提取物具有良好的抗纤维化，抗真菌活性、抗肿瘤活性，治疗口腔及消化道炎症或(和)溃疡相关疾病，并具有双向免疫调节作用。
发明内容
本发明所解决的第一个技术问题是提供马甲子的新用途。
应用时，马甲子采用马甲子全株植物或其中任一部位；其中，药用部位可为根、茎、叶、花、果任一部位或其混合。
具体的，马甲子新的药用用途是在制备具有抗纤维化，抗真菌活性、抗肿瘤活性，治疗口腔及消化道炎症或(和)溃疡相关疾病，以及具有双向免疫调节作用的药物中的用途。
本发明所解决的第二个技术问题是提供一种新的马甲子提取物。鉴于药材的采集期和保存便利性，为了便于临床用药，可以马甲子制成提取物后应用。
具体的，本发明马甲子提取物是以马甲子全株植物或其中任一部位为原料药，采用常规提取方法制备而得。本发明所得马甲子提取物主要成分包括黄酮类、萜类、生物碱类、香豆素类；进一步还包括上述黄酮、萜、生物碱、香豆素的苷及其单体成分；还包括多糖类和纤维素。
本发明所解决的第三个技术问题是提供本发明马甲子提取物的制备方法如下：
方法一：
A、以马甲子全株植物或其中任一部位为原料药；
B、以溶剂提取、干燥、即得。
上述技术方案中，步骤A所述以马甲子作为原料药是采用其鲜品、冷冻干燥品、有机溶媒预处理品。
上述技术方案中，步骤B所述溶剂为甲醇、乙醇、异丙醇、乙酸乙酯或石油醚；优选甲醇或乙醇。
上述技术方案中，步骤B所述提取采用浸渍、回流或渗漉提取。
上述技术方案中，步骤B所述干燥为减压干燥、冷冻干燥、喷雾干燥或微波干燥。
方法二：
A、以马甲子全株植物或其中任一部位为原料药；
B、以溶剂a提取，滤液浓缩，得浓缩液；
C、以溶剂b萃取步骤B所得滤液，得液相，干燥，即得；
或，浓缩或干燥步骤B所得滤液得提取物1，以溶剂b提取，干燥提取液，即得。
上述技术方案中，步骤A所述以马甲子作为原料药是采用其鲜品、冷冻干燥品、有机溶媒预处理品。
上述技术方案中，步骤B所述溶剂a为甲醇或乙醇、异丙醇；优选甲醇或乙醇。
上述技术方案中，步骤C所述溶剂b为乙酸乙酯或石油醚。
上述技术方案中，步骤B及步骤C所述提取采用浸渍法、回流法、渗漉法或萃取法提取。
上述技术方案中，步骤B及步骤C所述干燥为减压干燥、冷冻干燥、喷雾干燥或微波干燥。
采用上述方法制备的提取物，如按方法一制备所得，则以步骤B所用溶剂命名为对应的提取物，如马甲子乙醇提取物，马甲子甲醇提取物、马甲子异丙醇提取物、马甲子乙酸乙酯提取物、马甲子石油醚提取物。如按方法二制备所得，则以步骤C所用溶剂b命名为对应的提取物，如马甲子石油醚提取物，马甲子乙酸乙酯提取物；而方法二步骤C所得提取物1则以方法二步骤B所用溶剂a命名对应的提取物，如马甲子乙醇提取物，马甲子甲醇提取物、马甲子异丙醇提取物。
上述技术方案中，方法一的步骤B所使用的溶剂与马甲子的用量关系为，溶剂添加量为马甲子质量的1-20倍；优选的，溶剂添加量为马甲子质量的5-15倍；更进一步的，溶剂添加量为马甲子质量的8-10倍。
上述技术方案中，方法二的步骤B所使用的溶剂a与马甲子的用量关系为，溶剂a添加量为马甲子质量的1-20倍；优选的，溶剂a添加量为马甲子质量的5-15倍；更进一步的，溶剂a添加量为马甲子质量的8-10倍。
优选的，溶剂或溶剂a采用甲醇或乙醇时，甲醇或乙醇添加量为马甲子质量的1-20倍；优选的，甲醇或乙醇添加量为马甲子质量的5-15倍；更进一步的，优选甲醇或乙醇添加量为马甲子质量的8-10倍。
其中，所述乙醇的浓度为10-95％。优选的，采用乙醇浓度为50-95％。最优选的，采用乙醇浓度为95％。
上述技术方案中，原料马甲子在用有机溶媒处理前必须为鲜品或冷冻干燥品。原因是，发明人在研究过程中发现马甲子晾晒或烘干的干品没有相应的活性，故选择马甲子鲜品为原料。但是鲜品不利于保存、运输，故发明人研究后发现，采用冷冻干燥和有机溶媒预处理马甲子鲜品，有利于保存药用活性成分，效果与鲜品相当，可以满足本发明马甲子提取物的新用途，利于保存与运输。
具体的，采用有机溶媒处理的方式是：取马甲子用有机溶媒浸泡，干燥即可。
其中，所述有机溶媒为乙醇、甲醇、乙酸乙酯、石油醚、异丙醇等；优选甲醇或乙醇。
本发明提取物可采用口服(包括颊内或舌下)、经鼻、局部(包括颊内、舌下或经皮)、不经肠道(包括皮下、皮内、肌肉内、关节内、滑膜内、胸骨内、鞘内、病灶内、静脉内或皮内注射或输注)途径给药。这些制剂可通过药剂学技术中已知的任何方法，例如通过使活性 成分与载体或赋形剂结合来制备。
本发明所解决的第四个技术问题是提供由本发明马甲子提取物制备而成的药物制剂，即本发明马甲子提取物加入药物学上可接受的辅料可制备成多种途径给药的药物制剂。为了便于应用，本发明提取物可以采用常规方法制成常规口服制剂、注射制剂及外用制剂。例如通过口服(包括颊内或舌下)、经鼻、局部(包括颊内、舌下或经皮)、不经肠道(包括皮下、皮内、肌肉内、关节内、滑膜内、胸骨内、鞘内、病灶内、静脉内或皮内注射或输注)途径给药。这些制剂可通过药剂学技术中已知的任何方法，例如通过使活性成分与载体或赋形剂结合来制备。例如片剂、胶囊剂、颗粒剂、微丸、微球、滴丸、控释制剂、缓释制剂或注射剂。
适于口服给药的药物制剂可呈现为独立单位，诸如胶囊、片剂、粉末或颗粒；水性或非水性液体中的溶液、悬浮液、水包油型液体乳液或油包水型乳液。可以以单位剂量形式制备口服液体，如溶液、糖浆和酏剂。糖浆可通过使化合物溶解于经适当调味的水溶液中来制备，而酏剂是经由使用无毒载体(vehicle)来制备。还可添加增溶剂和乳化剂(诸如乙氧基化异十八醇和聚氧化乙烯山梨糖醇醚)、防腐剂、调味添加剂(诸如薄荷油或天然甜味剂，或糖精或其它人工甜味剂)及类似物。适当时，用于口服给药的剂量单位制剂可以是微胶囊化的也可通过涂布或包埋颗粒物质于聚合物、蜡或类似物中来制备制剂以延长或持续释放。还可以以脂质体给药系统(诸如小单层脂质体、大单层脂质体和多层脂质体)的形式给药，脂质体可由各种各样的磷脂，如胆固醇、硬脂胺或磷脂酰胆碱形成。
适于经皮给药的药物制剂可以以意欲保持与接受者表皮紧密接触较长时段的独立贴片形式呈现。适于局部给药的药物制剂可配制为软膏剂、乳膏、悬浮液、洗剂、粉末、溶液、糊剂、凝胶、喷雾剂、气雾剂、搽剂或油剂。
本发明所解决的第五个技术问题是提供本发明马甲子提取物的药用新用途。
本发明马甲子提取物，包括马甲子甲醇提取物、马甲子乙醇提取物、马甲子乙酸乙酯提取物、马甲子石油醚提取物，均可应用于制备具有抗纤维化，抗真菌活性、抗肿瘤活性，治疗口腔及消化道炎症或(和)溃疡相关疾病，以及具有双向免疫调节作用的药物中的制药新用途。包括单独使用马甲子提取物或以马甲子提取物为主要活性成分的药物组合物在上述制药新用途。
其中：
本发明马甲子提取物在制备抗肿瘤活性的药物中的应用时，还可以加入其它具有治疗肿瘤作用的中药、西药联合使用，包括：放疗、免疫疗法、DNA损伤的化疗剂、干扰细胞复制的化疗剂和免疫调节药物，具体为拓扑异构酶I抑制剂、拓扑异构酶II抑制剂、烷化剂、DNA嵌合剂、DNA嵌入剂和自由基生成剂、干扰细胞复制的化疗剂、蛋白酪氨酸激酶抑制剂、蛋白酶抑制剂、与癌症中过表达的蛋白结合从而下调细胞复制的抗体、蛋白或酶抑制剂。其它具有治疗肿瘤的中药、西药可以选自伊立替康、拓扑替康、喜树碱及其类似物或代谢物、阿霉素、依托泊苷、替尼泊苷、道诺霉素、美法仑、苯丁酸氮芥、白消安、塞替哌、异环磷酰胺、亚硝基脲氮芥、洛莫司汀、司莫司汀、链脲菌素、氨烯咪胺、甲氨蝶呤、丝裂霉素C、环磷酰胺、顺铂、奥沙利铂、卡铂、博来霉素、5-氟尿嘧啶、卡培他滨、吉西他滨、氟达拉 滨、阿糖胞苷、巯嘌呤、硫鸟嘌呤、喷司他丁、羟基脲、紫杉醇、紫杉萜及其相关类似物、长春新碱、长春碱及有关类似物、沙立度胺及有关类似物、甲磺酸伊马替尼、吉非替尼、硼替佐米、曲妥单抗、利妥昔单抗、西妥昔单抗、贝伐单抗、华蟾素、姬松茸、珍珠梅乙酸乙酯提取物、荔枝核水提液、当雷公藤红素、猪苓多糖、羧甲基茯苓多糖、泽泻醇提浓缩液、甘草多糖、当归总多糖、补骨脂素、五味子多糖。
本发明马甲子提取物在制备抗纤维化的药物中的应用时，还可以加入其它具有治疗纤维化的中药、西药联合使用。其它具有治疗纤维化的中药、西药选自：三七总甙、川芎嗪、丹参、刺五加注射液、川芎嗪注射液、红花注射液、银杏黄酮苷、生脉、丹参注射液、双黄连、香丹注射液、益气活血颗粒、抗纤颗粒、肺康颗粒、百合固金丸、虫草蛤蚧散合洋参丸、黄芪、西红花、生地黄、三七、绞股蓝、姜黄、黄葵、苦杏仁甙、汉防己甲素、大黄素、恩替卡韦、拉米夫定、β-胡萝卜素、维生素E、磷脂酰胆碱、S-腺苷蛋氨酸、前列地尔、地诺前列酮、秋水仙碱、雌激素、血管紧张素II受体阻断药、交感神经系统抑制剂、干扰素、脯氨酰-4-羟化酶抑制剂、肝素、水飞蓟素、熊去氧胆酸。所述纤维化包括肺纤维化、肾纤维化、肝纤维化、心肌纤维化。
本发明马甲子提取物在制备具有双向免疫调节作用的药物中的应用时，具体是指在制备免疫功能低下或(和)自身免疫性疾病药物的应用，尤其是免疫功能异常引起的免疫功能低下或(和)自身免疫性疾病。其中，所述免疫功能异常的免疫功能低下，包括感冒易感、体虚、肿瘤、艾滋病等。所述免疫功能异常的自身免疫性疾病，包括类风湿性关节炎、红斑狼疮、硬皮病、甲状腺机能亢进、青少年糖尿病、原发性血小板紫癜、自身免疫性溶血性贫血、溃疡性结肠炎、慢性肝病等。
上述技术方案中，当马甲子提取物应用于制备抗肿瘤活性的药物时，可以采用马甲子全株植物或其中任一部位为原料药，优选叶作为原料药。
以马甲子全株，包括根、茎、叶全部或其中任一部位为原料时，马甲子提取物可以采用如下方法制备得到：取马甲子全株，包括根、茎、叶全部或其中任一部位的鲜品或者冷冻干燥品，用有机溶媒预处理马甲子鲜品或者冷冻干燥品后备用，在制备时再将上述预处理的马甲子鲜品或者冷冻干燥品用有机溶媒提取。或将马甲子鲜品或冻干品直接用有机溶媒提取，得到马甲子提取物，用有机溶媒提取的方法包括但不限于浸渍法、回流法、渗漉法等本领域常规的提取方法。
所述马甲子在用有机溶媒处理前优选鲜品或冷冻干燥品。
所述有机溶媒选自甲醇、乙醇、异丙醇、乙酸乙酯和石油醚等，优选50-95％的乙醇，再优选95％的乙醇。
马甲子提取物优选采用如下方法制备：取新鲜马甲子全株，加入8-10倍重量份的95％乙醇浸渍1天，然后将马甲子粉碎，再加入6-10倍重量份的95％乙醇浸渍提取2-3天，收集提取液，减压回收溶剂，得到的提取物浓缩液，干燥即得马甲子乙醇提取物，其中干燥方法包括但不限于减压干燥、冷冻干燥、喷雾干燥、微波干燥。
更优选新鲜马甲子茎叶，加入8倍重量份的95％乙醇浸泡1天，然后将马甲子粉碎，再加入10倍重量份的95％乙醇提取，回流提取，收集提取液，60℃减压回收乙醇至无醇味， 得到的提取物浓缩液、干燥后得马甲子乙醇提取物。
更优选新鲜马甲子茎叶，加入8倍重量份的甲醇浸泡1天，然后将马甲子粉碎，再加入10倍重量份的甲醇提取，回流提取，收集提取液，60℃减压回收甲醇，得到的提取物浓缩液、干燥后得马甲子甲醇提取物。
进一步讲，还可以将马甲子乙醇提取物用水分散后，依次用石油醚、乙酸乙酯萃取，浓缩后干燥得到石油醚提取物或者乙酸乙酯提取物。
进一步地，所述醇提的具体步骤为：
(1)取马甲子叶片，加入5-15倍量的50％～95％乙醇浸泡；
(2)将叶片进行粉碎，再加入5-15倍量的50％～95％乙醇浸渍，提取醇溶性成分；
(3)收集提取液，30～70℃下减压回收乙醇至无醇味，得到的提取物；
(4)将提取物浓缩液进一步冷冻干燥即得马甲子叶片醇提物。
进一步地，所述中药提取物通过石油醚或乙酸乙酯进行萃取处理。将该中药提取物依次用石油醚、乙酸乙酯、正丁醇进行萃取，回收溶剂并冻干，得到3个极性部位的萃取物，均用异丙醇溶解，然后测试其对肿瘤细胞的抑制效果，结果该中药提取物的石油醚部位和乙酸乙酯部位具有很好的抗肿瘤活性，正丁醇提取部位显示无抗肿瘤活性，乙酸乙酯部位表现更好的抗肿瘤活性。
进一步地，所述马甲子提取物主要成分包括三萜类，黄酮类，生物碱类以及香豆素类。
上述技术方案中，当马甲子提取物应用于制备抗纤维化的药物时，采用马甲子全株植物或其中任一部位为原料药，优选叶作为原料药。
以马甲子全株，包括根、茎、叶全部或其中任一部位为原料时，马甲子提取物可以采用如下方法制备得到：取马甲子全株，包括根、茎、叶全部或其中任一部位的鲜品或者冷冻干燥品，用有机溶媒预处理马甲子鲜品或者冷冻干燥品后备用，在制备时再将上述预处理的马甲子鲜品或者冷冻干燥品用有机溶媒提取。或将马甲子鲜品或冻干品直接用有机溶媒提取，得到马甲子提取物，用有机溶媒提取的方法包括但不限于浸渍法、回流法、渗漉法等本领域常规的提取方法。
所述马甲子在用有机溶媒处理前优选鲜品或冷冻干燥品。
所述有机溶媒选自甲醇、10-100％乙醇，优选95％的乙醇。
马甲子提取物优选采用如下方法制备：取新鲜马甲子全株，加入8-10倍重量份的95％乙醇浸渍1天，然后将马甲子粉碎，再加入6-10倍重量份的95％乙醇浸渍提取2-3天，收集提取液，减压回收溶剂，得到的提取物浓缩液，干燥即得马甲子乙醇提取物，其中干燥方法包括但不限于减压干燥、冷冻干燥、喷雾干燥、微波干燥。
更优选新鲜马甲子茎叶，加入8倍重量份的95％乙醇浸泡1天，然后将马甲子粉碎，再加入10倍重量份的95％乙醇提取，回流提取，收集提取液，60℃减压回收乙醇至无醇味，得到的提取物浓缩液、干燥后得马甲子乙醇提取物。
更优选新鲜马甲子茎叶，加入8倍重量份的甲醇浸泡1天，然后将马甲子粉碎，再加入10倍重量份的甲醇提取，回流提取，收集提取液，60℃减压回收甲醇，得到的提取物浓缩液、干燥后得马甲子甲醇提取物。
进一步讲，还可以将马甲子乙醇提取物用水分散后，依次用石油醚、乙酸乙酯萃取，浓缩后干燥得到石油醚提取物或者乙酸乙酯提取物。
上述技术方案中，当马甲子提取物应用于制备抗真菌的药物时，采用马甲子全株植物或其中任一部位为原料药，优选叶作为原料药。
马甲子提取物的制备方法为：取马甲子全株植物或其中任一部位，加入马甲子体积的1-20倍的甲醇或乙醇提取，或者将甲醇或乙醇提取物进一步用乙酸乙酯或石油醚提取；提取液浓缩后干燥，制得马甲子提取物。
所述的马甲子优选鲜品、冷冻干燥品或甲醇或乙醇处理的样品；所述的提取方法为浸渍、回流或渗漉；所述的干燥方法为减压干燥、冷冻干燥、喷雾干燥或微波干燥。
所述制得的马甲子提取物包括萜类、黄酮类、生物碱类、香豆素类及萜类、黄酮类、生物碱类、香豆素类的苷类和单体。
所述的马甲子提取物的各类制剂包括马甲子提取物片剂、颗粒剂、软膏剂、凝胶剂、涂膜剂、搽剂、洗剂和喷雾剂。
本发明提供的具有抗真菌活性的马甲子提取物和各类制剂在抗真菌活性的应用。所述马甲子提取物为马甲子甲醇、乙醇、乙酸乙酯、石油醚或类似溶媒提取物。以马甲子鲜品、冷冻干燥品、甲醇(乙醇)或其他有机溶媒预处理样品为原料，以确保有效成分的稳定。
上述技术方案中，当马甲子提取物应用于制备治疗免疫功能低下或(和)自身免疫性疾病的药物时，采用马甲子全株植物或其中任一部位为原料药，优选叶作为原料药。
马甲子提取物的制备方法如下：取马甲子全株植物或其中任一部位，加入马甲子体积的1-20倍的甲醇或乙醇提取，或者将甲醇或乙醇提取物进一步用乙酸乙酯或石油醚提取；提取液浓缩后干燥，制得马甲子提取物。
所述的马甲子提取物，马甲子优选鲜品、冷冻干燥品或甲醇、乙醇、异丙醇等预处理处理的样品；提取方法包括浸渍、回流或渗漉；干燥方法包括减压干燥、冷冻干燥、喷雾干燥或微波干燥。
所述的马甲子提取物，成分包括萜类、黄酮类、生物碱类、香豆素类、多糖类、纤维素类以及萜类、黄酮类、生物碱类、香豆素类的苷类和单体。
所述的马甲子提取物的制剂，包括马甲子提取物的片剂、胶囊剂、贴膏剂、颗粒剂、注射剂、栓剂、软膏剂、凝胶剂、涂膜剂、搽剂、洗剂和喷雾剂。
所述的马甲子提取物的应用，应用于制备治疗免疫功能低下相关疾病或(和)自身免疫性疾病的药物，包括单独使用马甲子提取物或以马甲子提取物为主要活性成分的药物组合物在制备治疗免疫功能低下相关疾病或(和)自身免疫性疾病药物中的用途。
上述技术方案中，当马甲子提取物应用于制备治疗口腔及消化道炎症或和溃疡的药物时，采用马甲子全株植物或其中任一部位为原料药，优选叶作为原料药。
马甲子提取物的制备方法如下：取马甲子叶、马甲子茎叶或马甲子全株植物的鲜品，加入马甲子质量1-20倍的甲醇或乙醇提取；或者进一步将甲醇或乙醇提取物用乙酸乙酯或石油醚提取；提取液浓缩后干燥，制得马甲子提取物；成分包括萜类、黄酮类、生物碱类、香豆素类、多糖类和纤维素类以及萜、黄酮、生物碱、香豆素的苷类和单体。
所述的马甲子提取物，将马甲子鲜品或冷冻干燥品直接用有机溶媒提取，或将马甲子鲜品用有机溶媒预处理后再提取；有机溶媒包括甲醇、乙醇、异丙醇、乙酸乙酯、石油醚；所述提取方法包括浸渍、回流或渗漉；所述干燥方法包括减压干燥、冷冻干燥、喷雾干燥或微波干燥。
所述的马甲子提取物的制剂，包括片剂、胶囊剂、贴膏剂、颗粒剂、注射剂、栓剂、软膏剂、凝胶剂、涂膜剂、搽剂、洗剂和喷雾剂。
马甲子提取物的应用包括单独使用马甲子提取物或以马甲子提取物为主要活性成分的药物组合物在制备治疗口腔及消化道炎症或/和溃疡药物中的用途。
具体实施方式
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明，说明但不限制本发明。
以下为本发明马甲子提取物的成分研究
样品供试液的配制：取提取物样品1g，溶于25ml无水乙醇中，离心。取上清液2ml，无水乙醇稀释5倍，至终浓度为0.008g/ml。
(1)三萜类成分的检识
A.L-B反应
将样品溶于乙酸酐中，加浓硫酸－乙酸酐(1：20)数滴，可产生黄→红→紫→蓝等颜色变化，最后褪色，表明含有三萜类化合物。
B.Kahlenberg反应
将样品的氯仿或醇溶液点于滤纸上，喷20％五氯化锑的氯仿溶液(或三氯化锑饱和的氯仿溶液)，干燥后60－70℃加热，显蓝色，表明含有三萜类化合物。
C.R-H反应
将样品供试液滴在滤纸上，喷出25％三氯乙酸乙醇溶液，加热至100℃，显红色，逐渐变为紫色，表明含有三萜类化合物。
D.Salkowki反应
将样品溶于氯仿，加入浓硫酸后，在硫酸层呈现红色或蓝色，氯仿层有绿色荧光出现，表明含有三萜类化合物。
E.Tschugaeff反应
将样品溶于冰乙酸中，加乙酰氯数滴及氯化锌结晶数粒，稍加热，则呈现淡红色或紫红色，表明含有三萜类化合物。
(2)黄酮类成分的检识
A.盐酸－镁粉还原反应(还原反应显色)
取少量样品溶于1mL乙醇中，加入少许镁粉及浓盐酸，振荡片刻，观察反应颜色呈紫红色，表明含有黄酮类化合物。
B.三氯化铝反应(金属离子的络合反应显色)
用玻璃棒蘸取样品供试液涂于滤纸上，吹干，喷1％的三氯化铝乙醇溶液，吹干，观察现象。置紫外灯下显鲜黄色，表明含有黄酮类化合物。
C.三氯化铁反应(金属离子的络合反应显色)
用玻璃棒蘸取供试液涂于滤纸上，吹干，在紫外灯下观察荧光后，喷3％的三氯化铁乙醇溶液，吹干，出现暗蓝色荧光斑点，再经氨熏后转变为棕色荧光斑点，表明含有黄酮类化合物。
D.碱性试剂显色
用玻璃棒蘸取供试液涂于滤纸上，干燥后，喷以氢氧化钠水溶液或暴露于氨蒸汽中，在日光灯下观察时，氨蒸汽能使样品色点转变为亮黄色，表明含有黄酮类化合物。
(3)生物碱类成分的检识
A.改良碘化铋钾(Dragendorff)法
①次硝酸铋0.85g溶于10ml冰醋酸与40ml水中；②碘化钾8g溶于20ml水中。试液①与试液②等量混合置棕色瓶内保存作贮备液。用前将1ml贮备液、4ml冰醋酸与12ml水混合。将样品供试液加入上述试剂中，呈红棕色溶液，加蒸馏水一摇即有沉淀产生，表明含有生物碱类化合物。
B.碘-碘化钾(Wagner)法
碘1g与碘化钾10g溶于50ml水，加2ml乙酸，加水至100ml。取上述试剂适量，加入1ml样品供试液即显棕褐色，表明含有生物碱类化合物。
C.硅钨酸(Bertrand)法
5g硅钨酸溶于100ml水中，加浓盐酸少量调至pH＝2左右。取上述试剂适量，加入1ml样品供试液即显棕褐色，表明含有生物碱类化合物。
(4)香豆素类成分的检识
A.异羟肟酸铁反应
①a.盐酸羟胺20g溶于50ml水，用乙醇稀释至200ml，冷处保存；b.氢氧化钾50g溶于少许水中，加500ml乙醇。②氯化铁(FeCl3·6H2O)10g溶于20ml36％盐酸溶液中，加乙醚200ml摇匀，置密闭容器内保存。使用时将试液①a.与①b.按1：2混合，滤去沉淀，滤液放冰箱保存。用玻璃棒蘸取样品供试液涂于滤纸上，先喷ab混合试液，稍干，再喷试液②，即显红色，表明含有香豆素类化合物。
B.重氮化试剂反应
①对硝基苯胺0.35g，溶于浓盐酸5ml中，加水至50ml；②亚硝酸钠5g，加水50ml。取①、②液等量在冰水浴中混合后使用。取少量样品供试液滴加重氮化试剂，即显橙红色，表明含有香豆素类化合物。
经过上述研究，确定本发明马甲子提取物主要成分包括黄酮类、萜类、生物碱类、香豆素类，以及上述黄酮、萜、生物碱、香豆素的苷及其单体成分，还含有多糖类和纤维素。
一、马甲子叶提取物的抗肿瘤活性
1、马甲子叶片提取
(1)取马甲子鲜叶1kg，加入8-10倍量的95％乙醇浸泡1-2天后(作用：1、使鲜叶中的活性酶系失活，避免对有效成分的破坏；2、使鲜叶硬度增加，更利于下一步的粉碎流程)，将鲜叶进行粉碎，再加入10倍量的95％乙醇(60～100％乙醇提取)浸渍3天提取醇 溶性成分。收集提取液，30～40℃下加压回收乙醇至无醇味，得到的提取物浓缩液进一步冷冻干燥即得马甲子鲜叶醇提物。
(2)将提取残渣晾干，加入8～10倍量的水浸渍3天提取水溶性成分，收集提取液，冷冻干燥即得马甲子鲜叶水提物。
(3)马甲子叶干品(室温自然干燥)按照上述方法进行提取，获得马甲子叶干品醇提物及水提物。
(4)马甲子鲜叶片经过乙醇浸泡，之后置于避光处晾干，获得干叶片，所得马甲子经过乙醇浸泡并晾干的叶片按照上述方法进行提取，获得醇提物及水提物。
2、抗肿瘤活性
比较马甲子叶片提取物的抗肿瘤活性研究。取处于对数生长期的多种肿瘤细胞(采用的肿瘤细胞株有如下几种：宫颈癌细胞株Hela；人肝癌细胞株SMMC-7721；人肺癌细胞株A549；人结肠癌细胞株Caco-2；白血病细胞株K562；胃癌细胞株MGC-803；)，2000rpm离心5min，用含有含10％胎牛血清的相应培养液将沉淀细胞浓度调为1×105个/ml细胞悬液后，将细胞接种于96孔培养板。每孔加细胞悬液200μl，然后分别加入一定浓度无菌的提取物溶液，使每孔内的提取物终浓度为0.02、0.1、0.2、0.4、0.5、0.8、1.0、2.0mg/ml，混匀后置37℃、5％CO2孵箱中培养24h后，析出培养液并用PBS洗涤两次，再向每孔加入5mg/ml的MTT磷酸缓冲液20μl以及150μl培养基，同样条件下继续培养4h后终止培养。2000rpm离心5min，然后弃去培养板孔内的培养液，每孔加入150μl DMSO，震荡10min，使形成的甲臜颗粒充分溶解后，酶标仪检测吸光值。选择测定波长为570nm。计算提取物对肿瘤细胞的IC50。结果见表1。
表1马甲子提取物对不同肿瘤细胞的抑制作用：
Figure PCTCN2014089895-appb-000001
由结果可知，马甲子鲜叶醇提物及马甲子经过乙醇浸泡并晾干的叶片醇提取物具有良好的肿瘤抑制效果，其他几种提取物并不具有抗肿瘤活性，分析原因可能与活性提取物的溶解性以及稳定性有关。
3、马甲子叶片醇提物的成分含量测定
(1)三萜类成分含量测定
取本品粗粉约0.1g，各三份，加入10ml容量瓶内，加乙酸乙酯至刻度，再从中精密吸取4ml溶液加入10ml容量瓶内，挥干溶剂后，加入5％香草醛-冰醋酸0.4ml、高氯酸1.6ml， 混匀，并由乙酸乙酯稀释至刻度，置7O℃恒温水浴中加热15min，冷却至室温，并转移至10ml容量瓶中加乙酸乙酯稀释至刻度，摇匀，在540nm波长处测定吸收度，计算供试品溶液中总三萜含量(含三萜以美洲茶酸计)。经计算1g醇提物中三萜类含0.052±0.010g。
(2)黄酮类成分含量测定
取本品粗粉约0.1g，各三份(相当于2g生药)，精密称定，置50ml量瓶中，加乙醇适量，超声溶解，放冷，加乙醇至刻度，摇匀。精密量取1ml，置10ml量瓶中，加水至刻度，摇匀。精密量取3ml，置25ml量瓶中，测定吸光度，计算供试品溶液中总黄酮含量(含黄酮量以芦丁计)。经计算1g醇提物中总黄酮含0.325±0.043g。
(3)生物碱类成分含量测定
取样品粉末约1g，各三份，精密称定，置具塞锥形瓶中，以18％氨水2ml润湿1h，加乙醚2氯仿2乙醇(25∶8∶2.5)混合溶剂30ml，超声提取20min，倾出上清液至小锥形瓶中，再加入上述混合溶剂30ml，冷浸放置半小时,再超声震荡提取20min，滤过，以相同溶剂15ml分3次洗涤残渣和滤纸,合并滤液于锥形瓶中，于60℃水浴上蒸干，准确加入10ml氯仿使其全部溶解,再准确吸取5mL转移至小分液漏斗中，加6mL氯仿，2ml缓冲液(pH＝5.0，0.2M邻苯二甲酸氢钾缓冲液)。用1mmol·L-1溴麝香草酚蓝溶液滴定，并不断震摇；接近终点时，分出氯仿层，再加入新鲜氯仿5ml，继续滴定并不断震摇，静置分层，至水层略显黄色即为终点。计算总生物碱(含生物碱以马甲子碱B计)。经计算1g醇提物中生物碱类含量为0.028±0.007g。
(4)香豆素类成分含量测定
经测定，香豆素类成分含量为：1％～10％。
4、马甲子叶片醇提物3种极性部位抗肿瘤活性研究
将马甲子叶片醇提物依次用石油醚、乙酸乙酯、正丁醇进行萃取，回收溶剂并冻干，得到3个极性部位的萃取物，均用异丙醇溶解。取处于对数生长期的各种肿瘤细胞(采用的肿瘤细胞株有如下几种：宫颈癌细胞株Hela；人肝癌细胞株SMMC-7721；人肺癌细胞株A549；人结肠癌细胞株Caco-2；白血病细胞株K562；胃癌细胞株MGC-803；)，2000rpm离心5min，用含有含10％胎牛血清的相应培养液将沉淀细胞浓度调为1×105个/ml细胞悬液后，将细胞接种于96孔培养板。每孔加细胞悬液200μl，然后分别加入一定浓度无菌的提取物溶液，使每孔内的提取物终浓度为0.002、0.01、0.02、0.04、0.05、0.08、0.1、0.2mg/ml，混匀后置37℃、5％CO2孵箱中培养24h后，析出培养液并用PBS洗涤两次，再向每孔加入5mg/ml的MTT磷酸缓冲液20μl以及150μl培养基，同样条件下继续培养4h后终止培养。2000rpm离心5min，然后弃去培养板孔内的培养液，每孔加入150μl DMSO，震荡10min，使形成的甲臜颗粒充分溶解后，酶标仪检测吸光值。选择测定波长为570nm。计算提取物对肿瘤细胞的IC50。结果见表2。由结果可知，马甲子叶片醇提物的石油醚部位和乙酸乙酯部位具有很好的抗肿瘤活性，特别是乙酸乙酯部位表现出更好的活性，正丁醇提取部位在试验浓度范围内无法计算IC50。我们将进一步对乙酸乙酯部位进行研究。
表2马甲子叶片醇提物不同极性部位对肿瘤细胞的抑制作用：
细胞株 石油醚部位 乙酸乙酯部位 正丁醇提取部位
Hela 0.0492mg/ml 0.0139mg/ml ——
SMMC-7721 0.0993mg/ml 0.0365mg/ml ——
A549 0.0142mg/ml 0.0075mg/ml ——
Caco-2 0.0545mg/ml 0.0778mg/ml ——
MGC-803 0.0702mg/ml 0.0305mg/ml ——
5、乙酸乙酯极性部位对荷艾氏腹水瘤小鼠的作用效果研究
对小鼠移植瘤S180的抑制作用：
取昆明种小鼠，于右前腋皮下常规接种S180悬液0.2ml(约1×106个瘤细胞)，接种24h后，将小鼠随机分组编号，对照组鼠数为10只，实验组共三组，分为高剂量组、中剂量组、低剂量组，每组10只，每组灌胃给药，每天1次，共14次。阳性对照组每天灌胃环磷酰胺(85mg/kg)，给药量、次数、时间同实验组。末次给药后24h，处死动物，称体重后完整剥离瘤块并称重，计算抑瘤率，结果见表3。
表3乙酸乙酯极性部位对荷艾氏腹水瘤小鼠的作用：
Figure PCTCN2014089895-appb-000002
*：与对照组相比，P<0.05，差异具有统计学意义。
二、马甲子全株提取物的抗肿瘤活性
实施例1
取马甲子全株1kg，加入8倍量的95％乙醇浸渍1天后，然后进行粉碎，再加入10倍量的95％乙醇浸渍2天提取，收集提取液，50℃减压回收乙醇至无醇味，干燥即得马甲子乙醇提取物。
实施例2
取马甲子茎叶1kg，加入10倍量的95％乙醇浸渍1天后，然后进行粉碎，再加入10倍量的95％乙醇回流提取，收集提取液，60℃减压回收乙醇至无醇味，干燥即得马甲子乙醇提取物。将马甲子乙醇提取物加水分散后，依次用石油醚、乙酸乙酯萃取，得到马甲子石油醚提取物和乙酸乙酯提取物。
实施例3
取马甲子全株1kg，加入10倍量的甲醇浸渍1天后，然后进行粉碎，再加入8倍量的甲醇浸渍2天，收集提取液，40℃下减压回收甲醇，干燥即得马甲子甲醇提取物，将马甲子甲醇提取物加水分散后，依次用石油醚、乙酸乙酯萃取，得到马甲子石油醚提取物和乙酸乙酯提取物。
实施例4
取实施例2的马甲子石油醚提取物0.1g，三份，加入10ml容量瓶内，加乙酸乙酯至刻 度，再从中精密吸取4ml溶液加入10ml容量瓶内，挥干溶剂后，加入5％香草醛-冰醋酸0.4ml、高氯酸1.6ml，混匀，并由乙酸乙酯稀释至刻度，置70℃恒温水浴中加热15min，冷却至室温，并转移至10ml容量瓶中加乙酸乙酯稀释至刻度，摇匀，在540nm波长处测定吸收度，计算供试品溶液中总三萜含量(含三萜以美洲茶酸计)。经计算1g马甲子提取物中三萜类成分含23mg。
取实施例2的马甲子石油醚提取物0.1g，三份，精密称定，置50ml量瓶中，加乙醇适量，超声溶解，放冷，加乙醇至刻度，摇匀。精密量取1ml，置10ml量瓶中，加水至刻度，摇匀。精密量取3ml，置25ml量瓶中，在510nm波长处测定吸光度，计算供试品溶液中总黄酮含量(含黄酮量以芦丁计)。经计算1g马甲子提取物中总黄酮含103mg。
取实施例2的马甲子石油醚提取物0.1g，三份，精密称定，置具塞锥形瓶中,以18％氨水2ml润湿1h，加乙醚2氯仿2乙醇(25∶8∶2.5)混合溶剂30ml，超声提取20min，倾出上清液至小锥形瓶中，再加入上述混合溶剂30ml，冷浸放置半小时,再超声震荡提取20min，滤过,以相同溶剂15ml分3次洗涤残渣和滤纸,合并滤液于锥形瓶中，于60℃水浴上蒸干，准确加入10ml氯仿使其全部溶解,再准确吸取5mL转移至小分液漏斗中，加6mL氯仿，2ml缓冲液(pH＝5.0，0.2M邻苯二甲酸氢钾缓冲液)。用1mmol·L-1溴麝香草酚蓝溶液滴定,并不断震摇；接近终点时，分出氯仿层，再加入新鲜氯仿5ml，继续滴定并不断震摇,静置分层，至水层略显黄色即为终点。计算总生物碱(含生物碱以马甲子碱B计)。经计算1g马甲子提取物中生物碱含21mg。
取实施例2的马甲子石油醚提取物0.1g，三份，精密称定，置50ml量瓶中，加乙醇适量，超声溶解，放冷，加乙醇至刻度，摇匀。精密量取1ml，置10ml量瓶中，加70％乙醇至刻度，摇匀。精密量取3ml，置25ml量瓶中，在340nm波长处测定测定吸光度，计算供试品溶液中总香豆素含量(含香豆素量以伞形花内酯计)。经计算1g马甲子提取物中总香豆素含10.2mg。
实施例5
取实施例2的马甲子乙酸乙酯提取物0.1g，三份，加入10ml容量瓶内，加乙酸乙酯至刻度，再从中精密吸取4ml溶液加入10ml容量瓶内，挥干溶剂后，加入5％香草醛-冰醋酸0.4ml、高氯酸1.6ml，混匀，并由乙酸乙酯稀释至刻度，置70℃恒温水浴中加热15min，冷却至室温，并转移至10ml容量瓶中加乙酸乙酯稀释至刻度，摇匀，在540nm波长处测定吸收度，计算供试品溶液中总三萜含量(含三萜以美洲茶酸计)。经计算1g马甲子提取物中三萜类成分含108mg。
取实施例2的马甲子乙酸乙酯提取物0.1g，三份，精密称定，置50ml量瓶中，加乙醇适量，超声溶解，放冷，加乙醇至刻度，摇匀。精密量取1ml，置10ml量瓶中，加水至刻度，摇匀。精密量取3ml，置25ml量瓶中，在510nm波长处测定吸光度，计算供试品溶液中总黄酮含量(含黄酮量以芦丁计)。经计算1g马甲子提取物中总黄酮含497mg。
取实施例2的马甲子乙酸乙酯提取物0.1g，三份，精密称定，置具塞锥形瓶中,以18％氨水2ml润湿1h，加乙醚2氯仿2乙醇(25∶8∶2.5)混合溶剂30ml，超声提取20min，倾出上清液至小锥形瓶中，再加入上述混合溶剂30ml，冷浸放置半小时,再超声震荡提取 20min，滤过,以相同溶剂15ml分3次洗涤残渣和滤纸,合并滤液于锥形瓶中，于60℃水浴上蒸干，准确加入10ml氯仿使其全部溶解,再准确吸取5mL转移至小分液漏斗中，加6mL氯仿，2ml缓冲液(pH＝5.0，0.2M邻苯二甲酸氢钾缓冲液)。用1mmol·L-1溴麝香草酚蓝溶液滴定,并不断震摇；接近终点时，分出氯仿层，再加入新鲜氯仿5ml，继续滴定并不断震摇,静置分层，至水层略显黄色即为终点。计算总生物碱(含生物碱以马甲子碱B计)。经计算1g马甲子提取物中生物碱含107mg。
取实施例2的马甲子乙酸乙酯提取物0.1g，三份，精密称定，置50ml量瓶中，加乙醇适量，超声溶解，放冷，加乙醇至刻度，摇匀。精密量取1ml，置10ml量瓶中，加70％乙醇至刻度，摇匀。精密量取3ml，置25ml量瓶中，在340nm波长处测定测定吸光度，计算供试品溶液中总香豆素含量(含香豆素量以伞形花内酯计)。经计算1g马甲子提取物中总香豆素含186mg。
1、马甲子乙醇提取物体外抗肿瘤活性研究
取处于对数生长期的各种肿瘤细胞(采用的肿瘤细胞株有：宫颈癌细胞株Hela；人肝癌细胞株SMMC-7721；人肺癌细胞株A549；人结肠癌细胞株Caco-2；白血病细胞株K562；胃癌细胞株MGC-803；)，2000rpm离心5min，用含有含10％胎牛血清的相应培养液将沉淀细胞浓度调为1×105个/ml细胞悬液后，将细胞接种于96孔培养板。每孔加细胞悬液200μl，然后分别加入一定浓度无菌的提取物溶液，使每孔内的提取物终浓度为0.02、0.1、0.2、0.4、0.5、0.8、1.0、2.0mg/ml，混匀后置37℃、5％CO2孵箱中培养24h后，吸出培养液并用PBS洗涤两次，再向每孔加入5mg/ml的MTT磷酸缓冲液20μl以及150μl培养基，同样条件下继续培养4h后终止培养。2000rpm离心5min，然后弃去培养板孔内的培养液，每孔加入150μl DMSO，震荡10min，使形成的甲臜颗粒充分溶解后，选择测定波长为570nm，酶标仪检测吸光值。计算实施例1的马甲子乙醇提取物对肿瘤细胞的IC50，结果见表4。由结果可知，马甲子全株提取物具有良好的肿瘤抑制效果。
表4马甲子乙醇提取物对不同肿瘤细胞的抑制作用
细胞株 马甲子乙醇提取物
Hela 0.0216mg/ml
SMMC-7721 0.1452mg/ml
A549 0.0101mg/ml
Caco-2 0.0875mg/ml
MGC-803 0.1862mg/ml
2、取实施例2马甲子3种提取物的体外抗肿瘤活性研究
将实施例1的马甲子乙醇提取物依次用石油醚、乙酸乙酯、正丁醇进行萃取，回收溶剂并干燥，分别得到马甲子石油醚、乙酸乙酯、正丁醇提取物，均用异丙醇溶解。取处于对数生长期的各种肿瘤细胞(采用的肿瘤细胞株有：宫颈癌细胞株Hela；人肝癌细胞株SMMC-7721；人肺癌细胞株A549；人结肠癌细胞株Caco-2；白血病细胞株K562；胃癌细胞株MGC-803；)，2000rpm离心5min，用含有含10％胎牛血清的相应培养液将沉淀细胞浓度调为1×105个/ml细胞悬液后，将细胞接种于96孔培养板。每孔加细胞悬液200μl，然 后分别加入一定浓度无菌的提取物溶液，使每孔内的提取物终浓度为0.002、0.01、0.02、0.04、0.05、0.08、0.1、0.2mg/ml，混匀后置37℃、5％CO2孵箱中培养24h后，吸出培养液并用PBS洗涤两次，再向每孔加入5mg/ml的MTT磷酸缓冲液20μl以及150μl培养基，同样条件下继续培养4h后终止培养。2000rpm离心5min，然后弃去培养板孔内的培养液，每孔加入150μl DMSO，震荡10min，使形成的甲臜颗粒充分溶解后，选择测定波长为570nm，酶标仪检测吸光值。计算提取物对肿瘤细胞的IC50，结果见表5。由结果可知，马甲子石油醚提取物和乙酸乙酯提取物具有很好的抗肿瘤活性，特别是乙酸乙酯部位表现出更好的活性，正丁醇提取物在试验浓度范围内无法计算IC50。
表5马甲子不同提取物对肿瘤细胞的抑制作用
细胞株 石油醚提取物 乙酸乙酯提取物 正丁醇提取物
Hela 0.0357mg/ml 0.0102mg/ml -
SMMC-7721 0.1242mg/ml 0.0357mg/ml -
A549 0.0156mg/ml 0.0064mg/ml -
Caco-2 0.0915mg/ml 0.0623mg/ml -
MGC-803 0.0812mg/ml 0.0487mg/ml -
“-”：试验浓度范围内无法计算IC50
3、马甲子乙醇、甲醇、石油醚及乙酸乙酯提取物对荷S180小鼠的影响
选取接种8d健康状况良好的S180瘤源小鼠，腹部皮肤消毒后抽取腹水，以无菌生理盐水按1:4(腹水体积：生理盐水体积)混悬备用。雄性昆明种小鼠82只，18～20g，按体重分层随机均分为7组，分别为模型对照组(0.5％西黄芪胶)、阳性对照组(环磷酰胺，CTX)、乙酸乙酯提取物小剂量组、乙酸乙酯提取物大剂量组、乙醇提取物组、甲醇提取物组、石油醚提取物组，均在其右侧腋部皮下接种0.2ml前述混悬液。2h后模型对照组和药物组分别灌胃给予受试物或混悬剂，每日一次，连续14日；阳性对照组腹腔注射给予CTX，隔日一次，共7次。末次给药后24h颈椎脱臼处死小鼠，剥离瘤块称重，并计算抑瘤率((1—实验组平均瘤重/模型对照组平均瘤重)*100％)，结果见表6。
表6马甲子全株提取物对荷S180小鼠的影响
Figure PCTCN2014089895-appb-000003
Figure PCTCN2014089895-appb-000004
与模型对照组比较，*P＜0.05，**P＜0.01
实验结果表明，马甲子全株提取物乙酸乙酯提取物1.6g/kg灌胃给予，可显著抑制S180 在小鼠体内的生长，且1.6g/kg剂量作用强度与环磷酰胺40mg/kg隔日腹腔给予类似；乙醇、甲醇、石油醚提取物4.8g/kg也可有效降低瘤重、抑瘤率均超过50％，提示该四类提取物均具有较好的抗肿瘤活性。
4、马甲子乙醇、甲醇、石油醚及乙酸乙酯提取物对荷艾氏腹水瘤小鼠的影响
选取接种8d健康状况良好的艾氏腹水瘤瘤源小鼠，腹部皮肤消毒后抽取腹水，以无菌生理盐水混悬为4×106/ml备用。雄性昆明种小鼠82只，18～20g，按体重分层随机均分为7组，分别为模型对照组(0.5％西黄芪胶)、阳性对照组(环磷酰胺，CTX)、乙酸乙酯提取物小剂量组、乙酸乙酯提取物大剂量组、乙醇提取物组、甲醇提取物组、石油醚提取物组，均在其腹腔内接种0.2ml前述混悬液。2h后模型对照组和药物组分别灌胃给予受试物或混悬剂，每日一次，持续给药直至动物垂死；阳性对照组腹腔注射给予CTX，隔日一次，共7次。动物垂死时即脱颈椎处死，垂死时间计为生存天数；处死后称量体重，随后放尽腹水，再次称重，其差值计为腹水重量，结果见表7。
表7马甲子全株提取物对荷艾氏腹水瘤小鼠的影响
Figure PCTCN2014089895-appb-000005
Figure PCTCN2014089895-appb-000006
与模型对照组比较，*P＜0.05，**P＜0.01
实验结果表明，马甲子全株提取物乙酸乙酯提取物在0.4g/kg及以上剂量灌胃给予，可显著抑制艾氏腹水瘤在小鼠体内的生长，延长动物生存时间，且1.6g/kg剂量抑制腹水量作用强度与环磷酰胺类似，而后者不能延长动物生存时间，显示一定的独特优势；乙醇、甲醇、石油醚提取物4.8g/kg也可有效延长生存时间，抑制腹水生成，提示该四类提取物均具有较好的抗肿瘤活性。
5、马甲子乙酸乙酯提取物与紫杉醇及华蟾素联合应用对荷S180小鼠的影响
选取接种8d健康状况良好的S180瘤源小鼠，腹部皮肤消毒后抽取腹水，以无菌生理盐水按1:4(腹水体积：生理盐水体积)混悬备用。雄性昆明种小鼠84只，18～20g，按体重分层随机均分为7组，分别为模型对照组(0.5％西黄芪胶)、阳性对照组(环磷酰胺，CTX)、乙酸乙酯提取物组、紫杉醇组、华蟾素组、与紫杉醇联合用药组、与华蟾素联合用药组，均在其右侧腋部皮下接种0.2ml前述混悬液。2h后模型对照组和药物组分别灌胃或静脉注射给予受试物或混悬剂，每日一次，连续14日；阳性对照组腹腔注射给予CTX，隔日一次，共7次。末次给药后24h颈椎脱臼处死小鼠，剥离瘤块称重，并计算抑瘤率((1—实验组平均瘤重/模型对照组平均瘤重)*100％)，结果见表8。
表8马甲子全株乙酸乙酯提取物与紫杉醇及华蟾素联合应用对荷S180小鼠的影响
Figure PCTCN2014089895-appb-000007
Figure PCTCN2014089895-appb-000008
与模型对照组比较，*P＜0.05，**P＜0.01；与紫杉醇组比较，@P＜0.05；与华蟾素组比较，#P＜0.05；
实验结果表明，马甲子全株乙酸乙酯提取物0.4g/kg灌胃给予，对S180在小鼠体内的生长无明显抑制作用，紫杉醇5mg/kg、华蟾素1ml/kg则显示一定效果。但同样剂量的马甲子全株乙酸乙酯提取物分别与紫杉醇5mg/kg、华蟾素1ml/kg合用，则均可显著提高紫杉醇的抑瘤率，提示马甲子全株提取物与其他抗肿瘤药物联合应用可提高这些药物的抗肿瘤药效。
三、马甲子提取物的抗纤维化活性
实施例1
取马甲子全株1kg，加入8倍量的95％乙醇浸渍1天后，然后进行粉碎，再加入10倍量的95％乙醇浸渍2天提取，收集提取液，50℃减压回收乙醇至无醇味，干燥即得马甲子乙醇提取物。
实施例2
取马甲子茎叶1kg，加入10倍量的95％乙醇浸渍1天后，然后进行粉碎，再加入10倍量的95％乙醇回流提取，收集提取液，60℃减压回收乙醇至无醇味，干燥即得马甲子乙醇提取物。将马甲子乙醇提取物加水分散后，依次用石油醚、乙酸乙酯萃取，得到马甲子石油醚提取物和乙酸乙酯提取物。
实施例3
取马甲子全株1kg，加入10倍量的甲醇浸渍1天后，然后进行粉碎，再加入8倍量的甲醇浸渍2天，收集提取液，40℃下减压回收甲醇，干燥即得马甲子甲醇提取物，将马甲子甲醇提取物加水分散后，依次用石油醚、乙酸乙酯萃取，得到马甲子石油醚提取物和乙酸乙酯提取物。
实施例4
取实施例2的马甲子石油醚提取物0.1g，三份，加入10ml容量瓶内，加乙酸乙酯至刻度，再从中精密吸取4ml溶液加入10ml容量瓶内，挥干溶剂后，加入5％香草醛-冰醋酸0.4ml、高氯酸1.6ml，混匀，并由乙酸乙酯稀释至刻度，置70℃恒温水浴中加热15min，冷却至室 温，并转移至10ml容量瓶中加乙酸乙酯稀释至刻度，摇匀，在540nm波长处测定吸收度，计算供试品溶液中总三萜含量(含三萜以美洲茶酸计)。经计算1g马甲子提取物中三萜类成分含23mg。
取实施例2的马甲子石油醚提取物0.1g，三份，精密称定，置50ml量瓶中，加乙醇适量，超声溶解，放冷，加乙醇至刻度，摇匀。精密量取1ml，置10ml量瓶中，加水至刻度，摇匀。精密量取3ml，置25ml量瓶中，在510nm波长处测定吸光度，计算供试品溶液中总黄酮含量(含黄酮量以芦丁计)。经计算1g马甲子提取物中总黄酮含103mg。
取实施例2的马甲子石油醚提取物0.1g，三份，精密称定，置具塞锥形瓶中,以18％氨水2ml润湿1h，加乙醚2氯仿2乙醇(25∶8∶2.5)混合溶剂30ml，超声提取20min，倾出上清液至小锥形瓶中，再加入上述混合溶剂30ml，冷浸放置半小时,再超声震荡提取20min，滤过,以相同溶剂15ml分3次洗涤残渣和滤纸,合并滤液于锥形瓶中，于60℃水浴上蒸干，准确加入10ml氯仿使其全部溶解,再准确吸取5mL转移至小分液漏斗中，加6mL氯仿，2ml缓冲液(pH＝5.0，0.2M邻苯二甲酸氢钾缓冲液)。用1mmol·L-1溴麝香草酚蓝溶液滴定,并不断震摇；接近终点时，分出氯仿层，再加入新鲜氯仿5ml，继续滴定并不断震摇,静置分层，至水层略显黄色即为终点。计算总生物碱(含生物碱以马甲子碱B计)。经计算1g马甲子提取物中生物碱含21mg。
取实施例2的马甲子石油醚提取物0.1g，三份，精密称定，置50ml量瓶中，加乙醇适量，超声溶解，放冷，加乙醇至刻度，摇匀。精密量取1ml，置10ml量瓶中，加70％乙醇至刻度，摇匀。精密量取3ml，置25ml量瓶中，在340nm波长处测定测定吸光度，计算供试品溶液中总香豆素含量(含香豆素量以伞形花内酯计)。经计算1g马甲子提取物中总香豆素含10.2mg。
实施例5
取实施例2的马甲子乙酸乙酯提取物0.1g，三份，加入10ml容量瓶内，加乙酸乙酯至刻度，再从中精密吸取4ml溶液加入10ml容量瓶内，挥干溶剂后，加入5％香草醛-冰醋酸0.4ml、高氯酸1.6ml，混匀，并由乙酸乙酯稀释至刻度，置70℃恒温水浴中加热15min，冷却至室温，并转移至10ml容量瓶中加乙酸乙酯稀释至刻度，摇匀，在540nm波长处测定吸收度，计算供试品溶液中总三萜含量(含三萜以美洲茶酸计)。经计算1g马甲子提取物中三萜类成分含108mg。
取实施例2的马甲子乙酸乙酯提取物0.1g，三份，精密称定，置50ml量瓶中，加乙醇适量，超声溶解，放冷，加乙醇至刻度，摇匀。精密量取1ml，置10ml量瓶中，加水至刻度，摇匀。精密量取3ml，置25ml量瓶中，在510nm波长处测定吸光度，计算供试品溶液中总黄酮含量(含黄酮量以芦丁计)。经计算1g马甲子提取物中总黄酮含497mg。
取实施例2的马甲子乙酸乙酯提取物0.1g，三份，精密称定，置具塞锥形瓶中,以18％氨水2ml润湿1h，加乙醚2氯仿2乙醇(25∶8∶2.5)混合溶剂30ml，超声提取20min，倾出上清液至小锥形瓶中，再加入上述混合溶剂30ml，冷浸放置半小时,再超声震荡提取20min，滤过,以相同溶剂15ml分3次洗涤残渣和滤纸,合并滤液于锥形瓶中，于60℃水浴上蒸干，准确加入10ml氯仿使其全部溶解,再准确吸取5mL转移至小分液漏斗中，加6mL 氯仿，2ml缓冲液(pH＝5.0，0.2M邻苯二甲酸氢钾缓冲液)。用1mmol·L-1溴麝香草酚蓝溶液滴定,并不断震摇；接近终点时，分出氯仿层，再加入新鲜氯仿5ml，继续滴定并不断震摇,静置分层，至水层略显黄色即为终点。计算总生物碱(含生物碱以马甲子碱B计)。经计算1g马甲子提取物中生物碱含107mg。
取实施例2的马甲子乙酸乙酯提取物0.1g，三份，精密称定，置50ml量瓶中，加乙醇适量，超声溶解，放冷，加乙醇至刻度，摇匀。精密量取1ml，置10ml量瓶中，加70％乙醇至刻度，摇匀。精密量取3ml，置25ml量瓶中，在340nm波长处测定测定吸光度，计算供试品溶液中总香豆素含量(含香豆素量以伞形花内酯计)。经计算1g马甲子提取物中总香豆素含186mg。
1、对大鼠肝纤维化的影响
SD大鼠60只(200-240g，雄性)，按体重分层随机均分成空白对照组(0.5％西黄芪胶)、模型对照组(0.5％西黄芪胶)、阳性对照组(地塞米松，1mg/kg)、马甲子提取物(按照实施例1的方法制备得到)低剂量组(0.4g/kg)、中剂量组(0.8g/kg)、高剂量组(1.6g/kg)。除空白对照组外，均按1ml/kg皮下注射40％四氯化碳植物油溶液，每周2次，连续3月，同时给予高脂饲料以及5％的乙醇水溶液。灌胃给予受试物，每日1次，连续3月。给药结束后次日腹主动脉取血，分离血浆，测定丙氨酸转氨酶(ALT)、Ⅲ型前胶原(PC-Ⅲ)、透明质酸(HA)和层黏连蛋白(LH)水平；随后处死动物，取肝脏进行病理学检查。血清生化检测结果如表9。
表9马甲子提取物对实验性肝纤维化大鼠肝脏及血液生化指标的影响(n＝10，
Figure PCTCN2014089895-appb-000009
)
组别 ALT(U/L) PC-Ⅲ(μg/L) HA(μg/L) LN(μg/L)
正常对照 43±4** 9.5±1.7** 131±29** 12±2**
模型对照 678±123 32.2±6.9 319±71 86±19
阳性对照 456±98** 20.8±3.1** 280±54 62±20*
马甲子提取物(0.4g/kg) 617±107 30.5±6.2 289±62 80±23
马甲子提取物(0.8g/kg) 412±126** 25.1±4.2* 263±69 59±17*
马甲子提取物(1.6g/kg) 335±138** 18.5±4.8** 217±58** 41±9**
与模型对照组比较，*P<0.05，**P<0.01
结果显示，与模型组相比，马甲子提取物在0.8g/kg及1.6g/kg时，对四氯化碳所致实验性肝纤维化大鼠肝损伤有一定的保护作用，特别是在大剂量时，各指标明显改善。
病理学检查显示，模型组大鼠肝细胞水样变性明显，有明显的肝细胞坏死和脂肪变性，表现为明显肝纤维化；马甲子提取物高剂量和中剂量组肝细胞水样变性和脂肪变性程度较模型组明显降低，提示其对肝纤维化有良好抑制作用。
2、对大鼠肺纤维化的影响
SD大鼠(200-240g，雄性)60只，按体重分层随机均分成假手术对照组(0.5％西黄芪胶)、模型对照组(0.5％西黄芪胶)、阳性对照组(地塞米松，1mg/kg)、马甲子提取物(按照实施例1的方法制备得到)低剂量组(0.4g/kg)、中剂量组(0.8g/kg)、高剂量组(1.6g/kg)。 所有动物以10％水合氯醛(350mg/kg)腹腔注射麻醉分离气管，除假手术对照组外，均按5mg/kg注入博莱霉素生理盐水溶液0.4mL，假手术对照组仅注入生理盐水。手术后次日开始给予受试物，每日1次，连续30日。颈椎脱臼处死动物，取肺脏，部分脏器匀浆后测定羟脯氨酸含量，部分进行病理学观察。肺组织中羟脯氨酸含量测定结果如表10所示。
表10各组大鼠肺脏羟脯氨酸含量变化(n＝10，
Figure PCTCN2014089895-appb-000010
)
组别 羟脯氨酸含量(mg/gpro)
假手术对照 10.3±3.8**
模型对照 18.4±5.0
阳性对照 13.6±3.3*
马甲子提取物(0.4g/kg) 16.5±4.7
马甲子提取物(0.8g/kg) 14.1±6.2*
马甲子提取物(1.6g/kg) 12.3±3.7*
与模型对照组比较，*P<0.05，**P<0.01
结果显示，与模型组相比，马甲子提取物在0.8g/kg及1.6g/kg时，能有效降低模型大鼠肺组织中羟脯氨酸含量，提示其对肺纤维化有良好的抑制作用。
病理学检查结果显示，模型组大鼠出现明显肺纤维化病变，表现为肺间质大量成纤维细胞，大片纤维结缔组织沉积，肺泡结构破坏，部分肺泡腔消失；与模型组相比，马甲子提取物各剂量组大鼠纤维结缔组织沉积较少，肺间质成纤维细胞亦较少，且大剂量组更为显著，提示该提取物是一种潜在的肺纤维化治疗药物。
3、马甲子提取物联合多烯磷脂酰胆碱对大鼠肝纤维化的影响
SD大鼠60只(200-240g，雄性)，按体重分层随机均分成空白对照组、模型对照组、阳性对照组(地塞米松，1mg/kg)、马甲子提取物组(按照实施例1的方法制备得到，0.8g/kg)、多烯磷脂酰胆碱组(1ml/kg)、联合用药组(马甲子提取物0.8g/kg+多烯磷脂酰胆碱1ml/kg)。除空白对照组外，均按1ml/kg皮下注射40％四氯化碳植物油溶液，每周2次，连续3月，同时给予高脂饲料以及5％的乙醇水溶液。灌胃或注射给予受试物，每日1次，连续3月。给药结束后次日腹主动脉取血，分离血浆，测定丙氨酸转氨酶(ALT)、Ⅲ型前胶原(PC-Ⅲ)、透明质酸(HA)和层黏连蛋白(LH)水平；随后处死动物，取肝脏进行病理学检查。血清生化检测结果如表11。
表11联合用药对实验性肝纤维化大鼠肝脏及血液生化指标的影响(n＝10，
Figure PCTCN2014089895-appb-000011
)
组别 ALT(U/L) PC-Ⅲ(μg/L) HA(μg/L) LN(μg/L)
正常对照 42±3** 9.7±1.9** 129±32** 11±4**
模型对照 684±114 31.7±8.0 310±63 87±21
阳性对照 478±90** 20.6±3.8** 285±57 65±23*
马甲子提取物 423±98** 24.5±3.9* 271±60 60±18**
多烯磷脂酰胆碱 443±130** 27.0±4.8 284±78 63±18*
联合用药 315±102**# 18.6±5.1**## 186±67**## 47±11**#
与模型对照组比较，*P<0.05，**P<0.01；与多烯磷脂酰胆碱组比较，#P<0.05，##P<0.01
4、马甲子提取物联合川芎嗪对大鼠肺纤维化的影响
SD大鼠(200-240g，雄性)60只，按体重分层随机均分成假手术对照组、模型对照组、阳性对照组(地塞米松，1mg/kg)、马甲子提取物(按照实施例1的方法制备得到，0.8g/kg，op)、川芎嗪组(40mg/kg，ip)、联合用药组(马甲子提取物0.8g/kg，op；川芎嗪注射液40mg/kg，ip)。所有动物以10％水合氯醛(350mg/kg)腹腔注射麻醉分离气管，除假手术对照组外，均按5mg/kg注入博莱霉素生理盐水溶液0.4mL，假手术对照组仅注入生理盐水。手术后次日开始灌胃或注射给予受试物，每日1次，连续30日。颈椎脱臼处死动物，取肺脏，部分脏器匀浆后测定羟脯氨酸含量，部分进行病理学观察。肺组织中羟脯氨酸含量测定结果如表12所示。
表12联合用药对大鼠肺脏羟脯氨酸的影响(n＝10，
Figure PCTCN2014089895-appb-000012
)
组别 羟脯氨酸含量(mg/gpro)
假手术对照 10.1±3.4**
模型对照 17.9±4.3
阳性对照 13.4±3.7*
马甲子提取物 12.9±4.9*
川芎嗪 14.6±5.7
联合用药 9.8±2.9**#
与模型对照组比较，*P<0.05，**P<0.01；与川芎嗪组比较，#P<0.05
结果显示，单独应用川芎嗪40mg/kg对实验性肺纤维化无明显药效，而联合用药则能有效降低模型大鼠肺组织中羟脯氨酸含量，其与川芎嗪40mg/kg相比有显著性差异，提示马甲子提取物可有效提高其他肺纤维治疗药物的疗效。
四、马甲子提取物的抗真菌活性
(一)实施例马甲子提取物的制备
1、马甲子乙醇提取物的制备
(1)取马甲子全株植物鲜品1kg，加入马甲子体积的8倍的95％(体积浓度)乙醇浸泡1-2天后，将叶、根和茎粉碎，再用马甲子体积的12倍95％乙醇提取浸渍3天，收集提取液，减压回收乙醇，浓缩液冷冻干燥即得马甲子乙醇提取物。
(2)取马甲子全株植物鲜品(或其中任一部位)1kg，粉碎，加入马甲子体积的8倍的95％乙醇回流提取3次，收集提取液，减压回收乙醇，浓缩液冷冻干燥即得马甲子乙醇提取物。
(3)取马甲子全株植物鲜品(根、茎、叶等)1kg，冷冻干燥后粉碎(也可粉碎后冷冻干燥)，加入马甲子体积的10倍的95％乙醇回流提取3次，收集提取液，减压回收乙醇，浓缩液冷冻干燥即得马甲子乙醇提取物。
2、马甲子甲醇提取物的制备
取马甲子全株植物鲜品1kg，粉碎，加入马甲子体积的6倍的甲醇回流提取3次，收 集提取液，减压回收甲醇，浓缩液冷冻干燥即得马甲子甲醇提取物。
3、马甲子石油醚提取物的制备
(1)马甲子乙醇提取物的浓缩溶液，用石油醚萃取，回收石油醚干燥。
(2)马甲子甲醇提物干燥样品，加10倍水混悬，用石油醚萃取，回收石油醚后干燥。
(3)马甲子乙醇提取物干燥样品，用10倍石油醚回流提取，回收石油醚干燥。
4、马甲子乙酸乙酯提取物的制备
(1)马甲子乙醇提取物的浓缩溶液，用10倍石油醚萃取后再用10倍乙酸乙酯萃取，回收乙酸乙酯后干燥。
(2)马甲子甲醇提物干燥样品，加10倍倍水混悬，用10倍石油醚萃取后再用10倍乙酸乙酯萃取。回收乙酸乙酯后干燥。
(3)马甲子乙醇提取物干燥样品，用6倍乙酸乙酯回流提取2次，回收乙酸乙酯后干燥。
5、马甲子乙酸乙酯提取物片剂的制备
取300g马甲子乙酸乙酯提取物，粉碎后，过40目筛，加入100g微晶纤维素，57.5g乳糖，20g交联羧甲基纤维素钠，混匀后，均匀喷洒体积浓度为95％乙醇溶液适量，湿法挤压制粒，过24目筛，50℃烘干后，再加入20g交联羧甲基纤维素钠和2.5g的硬脂酸镁，充分混匀后，压片，得到马甲子乙酸乙酯提取物片剂；
6、马甲子乙醇提取物颗粒剂的制备
取1000g马甲子乙醇提取物，粉碎后，过40目筛，加入4000g糖粉，混匀后，均匀喷洒体积浓度为95％乙醇溶液适量，湿法挤压制粒，过24目筛，50℃烘干后，整粒，得到马甲子乙醇提取物颗粒剂；
7、马甲子甲醇提取物软膏剂的制备
称取115g十八醇、115g白凡士林、70g单硬脂酸甘油酯，加热融化，得到油相，加入40g马甲子甲醇提取物；另取100g甘油、15g十二烷基硫酸钠、0.01g盐酸半胱氨酸，加入650ml水溶解，得到水相；分别加热至75℃～80℃，搅拌下将水相缓缓加入油相，然后继续搅拌15分钟，得到马甲子甲醇提取物软膏剂。
8、马甲子乙醇提取物凝胶剂的制备
取10g卡波姆撒入420ml纯化水中，搅拌使溶胀，加入100ml丙二醇搅拌溶解，搅拌下滴加18g三乙醇胺，制成凝胶基质；另取100g马甲子乙醇提取物与2g羟苯乙酯溶解于350ml乙醇中，搅拌下加入凝胶基质中，搅匀即得。
9、马甲子乙醇提取物涂膜剂的制备
取40g聚乙烯醇124溶胀于400ml纯化水中；另取100g马甲子乙醇提取物溶于400ml乙醇中，再加入100ml甘油，搅拌均匀后缓缓加入聚乙烯醇溶液中，搅匀后过滤，再自滤器上加乙醇至1000ml，即得。
10、马甲子石油醚提取物搽剂的制备
取100g马甲子石油醚提取物细粉置于研钵中，加入500ml花生油研匀，再缓缓加入饱和氢氧化钙水溶液至1000ml，研磨至均匀白色乳状物即得。
11、马甲子乙醇提取物洗剂的制备
取100g马甲子乙醇提取物细粉置于研钵中，加入50ml甘油和适量纯化水，研磨成糊状，逐渐加入纯化水至全量混匀即得。
(二)马甲子提取物的抗真菌活性研究
称取实例1(1)中制备的马甲子乙醇提取物1g，用5％异丙醇溶液溶解配制至10ml，0.22μm滤膜过滤除菌。取1ml除菌上述溶液，加入到9ml熔融后冷却至约50℃的PDA培养基中，充分摇匀，迅速倒入直径为6cm的培养皿中，静置，制成含有10mg/ml马甲子提取物的PDA培养平板。用相同体积的5％异丙醇溶液制成空白对照PDA培养平板。
将切取好的菌块接种体移入上述含药PDA培养平板，25℃恒温培养，待对照组菌落接近培养皿边缘时，用十字交叉法测定所有培养平板上的菌落直径，校正后计算抑菌率。
结果如表13所示。马甲子醇提物对受试的各类真菌具有明显的抑制作用。由于受试真菌均为常见致病真菌，具有明显的代表性，该结果提示马甲子醇提物具有较强抗真菌活性，可望应用于抗真菌类药物的制备。
表13马甲子醇提物对各种真菌的抑制效果(n＝3，
Figure PCTCN2014089895-appb-000013
)
菌种 抑菌率(％)
白色念球菌 87.53±8.36
红色毛癣菌 80.46±10.24
须癣毛癣菌 72.69±11.53
紫色毛癣菌 90.16±14.36
断发毛癣菌 54.39±8.67
疣状毛癣菌 42.63±12.81
猪小孢子菌 60.54±10.33
犬小孢子菌 61.32±7.65
申克孢子丝菌 76.87±15.69
紧密着色霉菌 66.63±17.65
皮炎外瓶霉 62.65±8.87
裴氏着色霉菌 53.13±10.08
五、马甲子叶提取物治疗免疫功能低下或(和)自身免疫性疾病
(一)马甲子提取物的制备
1、全株乙醇提取物的制备
取马甲子全株1kg，加入8倍量的95％乙醇浸渍1天后，然后进行粉碎，再加入10倍量的95％乙醇浸渍2天提取，收集提取液，50℃减压回收乙醇至无醇味，干燥即得马甲子乙醇提取物。
2、茎叶乙醇、石油醚和乙酸乙酯提取物的制备
取马甲子茎叶1kg，加入10倍量的95％乙醇浸渍1天后，然后进行粉碎，再加入10 倍量的95％乙醇回流提取，收集提取液，60℃减压回收乙醇至无醇味，干燥即得马甲子乙醇提取物。将马甲子乙醇提取物加水分散后，依次用石油醚、乙酸乙酯萃取，得到马甲子石油醚提取物和乙酸乙酯提取物。
3、全株石油醚和乙酸乙酯提取物的制备
取马甲子全株1kg，加入10倍量的甲醇浸渍1天后，然后进行粉碎，再加入8倍量的甲醇浸渍2天，收集提取液，40℃下减压回收甲醇，干燥即得马甲子甲醇提取物，将马甲子甲醇提取物加水分散后，依次用石油醚、乙酸乙酯萃取，得到马甲子石油醚提取物和乙酸乙酯提取物。
4、成分定量分析
取马甲子茎叶石油醚提取物0.1g，三份，加入10ml容量瓶内，加乙酸乙酯至刻度，再从中精密吸取4ml溶液加入10ml容量瓶内，挥干溶剂后，加入5％香草醛-冰醋酸0.4ml、高氯酸1.6ml，混匀，并由乙酸乙酯稀释至刻度，置70℃恒温水浴中加热15min，冷却至室温，并转移至10ml容量瓶中加乙酸乙酯稀释至刻度，摇匀，在540nm波长处测定吸收度，计算供试品溶液中总三萜含量(含三萜以美洲茶酸计)。经计算1g马甲子提取物中三萜类成分含23mg。
取马甲子茎叶石油醚提取物0.1g，三份，精密称定，置50ml量瓶中，加乙醇适量，超声溶解，放冷，加乙醇至刻度，摇匀。精密量取1ml，置10ml量瓶中，加水至刻度，摇匀。精密量取3ml，置25ml量瓶中，在510nm波长处测定吸光度，计算供试品溶液中总黄酮含量(含黄酮量以芦丁计)。经计算1g马甲子提取物中总黄酮含103mg。
取马甲子茎叶石油醚提取物0.1g，三份，精密称定，置具塞锥形瓶中,以18％氨水2ml润湿1h，加乙醚2氯仿2乙醇(25∶8∶2.5)混合溶剂30ml，超声提取20min，倾出上清液至小锥形瓶中，再加入上述混合溶剂30ml，冷浸放置半小时,再超声震荡提取20min，滤过,以相同溶剂15ml分3次洗涤残渣和滤纸,合并滤液于锥形瓶中，于60℃水浴上蒸干，准确加入10ml氯仿使其全部溶解,再准确吸取5mL转移至小分液漏斗中，加6mL氯仿，2ml缓冲液(pH＝5.0，0.2M邻苯二甲酸氢钾缓冲液)。用1mmol·L-1溴麝香草酚蓝溶液滴定,并不断震摇；接近终点时，分出氯仿层，再加入新鲜氯仿5ml，继续滴定并不断震摇,静置分层，至水层略显黄色即为终点。计算总生物碱(含生物碱以马甲子碱B计)。经计算1g马甲子提取物中生物碱含21mg。
取马甲子茎叶石油醚提取物0.1g，三份，精密称定，置50ml量瓶中，加乙醇适量，超声溶解，放冷，加乙醇至刻度，摇匀。精密量取1ml，置10ml量瓶中，加70％乙醇至刻度，摇匀。精密量取3ml，置25ml量瓶中，在340nm波长处测定测定吸光度，计算供试品溶液中总香豆素含量(含香豆素量以伞形花内酯计)。经计算1g马甲子提取物中总香豆素含10.2mg。
取马甲子茎叶乙酸乙酯提取物0.1g，三份，加入10ml容量瓶内，加乙酸乙酯至刻度，再从中精密吸取4ml溶液加入10ml容量瓶内，挥干溶剂后，加入5％香草醛-冰醋酸0.4ml、高氯酸1.6ml，混匀，并由乙酸乙酯稀释至刻度，置70℃恒温水浴中加热15min，冷却至室温，并转移至10ml容量瓶中加乙酸乙酯稀释至刻度，摇匀，在540nm波长处测定吸收度， 计算供试品溶液中总三萜含量(含三萜以美洲茶酸计)。经计算1g马甲子提取物中三萜类成分含108mg。
取马甲子茎叶乙酸乙酯提取物0.1g，三份，精密称定，置50ml量瓶中，加乙醇适量，超声溶解，放冷，加乙醇至刻度，摇匀。精密量取1ml，置10ml量瓶中，加水至刻度，摇匀。精密量取3ml，置25ml量瓶中，在510nm波长处测定吸光度，计算供试品溶液中总黄酮含量(含黄酮量以芦丁计)。经计算1g马甲子提取物中总黄酮含497mg。
取马甲子全株乙酸乙酯提取物0.1g，三份，精密称定，置具塞锥形瓶中,以18％氨水2ml润湿1h，加乙醚2氯仿2乙醇(25∶8∶2.5)混合溶剂30ml，超声提取20min，倾出上清液至小锥形瓶中，再加入上述混合溶剂30ml，冷浸放置半小时,再超声震荡提取20min，滤过,以相同溶剂15ml分3次洗涤残渣和滤纸,合并滤液于锥形瓶中，于60℃水浴上蒸干，准确加入10ml氯仿使其全部溶解,再准确吸取5mL转移至小分液漏斗中，加6mL氯仿，2ml缓冲液(pH＝5.0，0.2M邻苯二甲酸氢钾缓冲液)。用1mmol·L-1溴麝香草酚蓝溶液滴定,并不断震摇；接近终点时，分出氯仿层，再加入新鲜氯仿5ml，继续滴定并不断震摇,静置分层，至水层略显黄色即为终点。计算总生物碱(含生物碱以马甲子碱B计)。经计算1g马甲子提取物中生物碱含107mg。
取马甲子全株乙酸乙酯提取物0.1g，三份，精密称定，置50ml量瓶中，加乙醇适量，超声溶解，放冷，加乙醇至刻度，摇匀。精密量取1ml，置10ml量瓶中，加70％乙醇至刻度，摇匀。精密量取3ml，置25ml量瓶中，在340nm波长处测定测定吸光度，计算供试品溶液中总香豆素含量(含香豆素量以伞形花内酯计)。经计算1g马甲子提取物中总香豆素含186mg。
(二)马甲子制剂的制备
1、片剂的制备
取马甲子全株乙醇提取物300g，加入适宜辅料，如：100g微晶纤维素，57.5g乳糖，20g交联羧甲基纤维素钠等，压片制成片剂。
2、胶囊剂的制备
取马甲子茎叶乙酸乙酯提取物，加入适宜辅料，如：乳糖、可压性淀粉、羧甲基淀粉、微晶纤维素、等制成胶囊剂。
3、颗粒剂的制备
取马甲子全株甲醇提取物，加入适宜辅料，如：乳糖、淀粉、甲基纤维素、羟甲基纤维素、羟丙基甲基纤维素、羟丙基纤维素、聚乙烯吡咯烷酮、微粉硅胶等制成颗粒剂。
4、软膏剂的制备
取马甲子茎叶石油醚提取物，加入适宜辅料，如：十八醇、单硬脂酸甘油脂、甘油、硬脂酸等制成软膏剂。
5、栓剂的制备
取马甲子全株乙醇提物物，加入适宜辅料，如：混合脂肪酸甘油酯、PEG、蜂蜡等制成栓剂。
(三)体外细胞学研究
1.马甲子提取物对小鼠脾淋巴细胞体外增殖的影响
1.1.小鼠脾淋巴细胞的制备
无菌取昆明小鼠脾脏，置于盛有适量RPMI1640培养基的平皿中，除去结缔组织，用注射器针芯研磨，经200目筛网过滤，转移至离心管中，用RPMI1640洗涤后，收集细胞，加入1ml红细胞裂解液。4℃下放置5-10min后，1500r/min离心5min，弃取上清，用RPMI1640培养基洗涤2次，最后将细胞悬浮于1ml RPMI1640完全培养基中。经台盼蓝染色测定存活率，结果显示脾细胞存活率大于95％。将上述脾细胞悬液适当稀释，调整细胞密度为2×106个/ml备用。
1.2.马甲子提取物对小鼠脾淋巴细胞体外增殖的影响
将2×106个/ml的脾细胞悬液，按照每孔100μl加入96孔板中。设置空白组、对照组以及马甲子提取物组。对照组每孔再加入100μl的RPMI1640完全培养基；马甲子提取物组中，每孔再加入100μl含有不同浓度马甲子提取物的RPMI1640完全培养基溶液；另设空白组，只含有培养基。将上述96孔板置于5％CO2、37℃孵箱中培养60h。取出细胞板，将孔内液体吸出，用PBS洗涤3次后，加入100μl培养基和20μlMTT液(5mg·ml－1)，再次置于37℃、体积分数为5％CO2的湿度环境下培养4h后，取出细胞培养板，小心吸出上清液，加入150μl二甲基亚砜，将培养板置于微孔板振荡器上振摇10min，使结晶物甲瓒充分溶解后，于酶联免疫检测仪上490nm比色，记录结果，按照公式(1)计算马甲子提取物对小鼠脾细胞体外平均存活率。
Figure PCTCN2014089895-appb-000014
结果显示，马甲子提取物浓度为0.004mg/ml、0.02mg/ml、0.2mg/ml时，小鼠脾细胞的平均存活率为117.85％、107.21％以及77.46％。结果表明低浓度马甲子提取物能促进小鼠脾细胞体外增殖，而高浓度马甲子提取物则对脾细胞的增殖具有抑制作用。
2.马甲子提取物对ConA诱导的脾细胞体外增殖的影响
将2×106个/ml的脾细胞悬液，按照每孔100μl加入96孔板中。设置空白组、对照组、ConA组以及马甲子提取物组。对照组每孔再加入100μl的RPMI1640完全培养基；ConA组每孔再加入100μl含有25μg/ml ConA的RPMI1640完全培养基溶液；马甲子提取物组中，每孔再加入100μl含有不同浓度马甲子提取物的RPMI1640完全培养基溶液(溶液中同时含有25μg/ml ConA)；另设空白组，只含有培养基。将上述96孔板置于5％CO2、37℃孵箱中培养60h。取出细胞板，将孔内液体吸出，用PBS洗涤3次后，加入100μl培养基和20μlMTT液(5mg·ml－1)，再次置于37℃、体积分数为5％CO2的湿度环境下培养4h后，取出细胞培养板，小心吸出上清液，加入150μl二甲基亚砜，将培养板置于微孔板振荡器上振摇10min，使结晶物甲瓒充分溶解后，于酶联免疫检测仪上490nm比色，记录结果，按照公式(1)计算ConA组和马甲子提取物组对小鼠脾细胞体外平均存活率；再按照公式(2)计算马甲子提取物对ConA诱导的小鼠脾细胞体外增殖的增殖率。
Figure PCTCN2014089895-appb-000015
结果显示，马甲子提取物浓度为0.004mg/ml、0.02mg/ml、0.2mg/ml时，相对于ConA组的相对增值率分别为15.11％、5.69％和-14.35％，说明马甲子提取物低浓度时对ConA诱导的小鼠脾细胞体外增殖有促进作用，而高浓度时则表现出抑制作用。
3.马甲子提取物对LPS诱导的脾细胞体外增殖的影响
将2×106个/ml的脾细胞悬液，按照每孔100μl加入96孔板中。设置空白组、对照组、LPS组以及马甲子提取物组。对照组每孔再加入100μl的RPMI1640完全培养基；LPS组每孔再加入100μl含有20μg/ml LPS的RPMI1640完全培养基溶液；马甲子提取物组中，每孔再加入100μl含有不同浓度马甲子提取物的RPMI1640完全培养基溶液(溶液中同时含有20μg/ml LPS)；另设空白组，只含有培养基。将上述96孔板置于5％CO2、37℃孵箱中培养60h。取出细胞板，将孔内液体吸出，用PBS洗涤3次后，加入100μl培养基和20μlMTT液(5mg·ml－1)，再次置于37℃、体积分数为5％CO2的湿度环境下培养4h后，取出细胞培养板，小心吸出上清液，加入150μl二甲基亚砜，将培养板置于微孔板振荡器上振摇10min，使结晶物甲瓒充分溶解后，于酶联免疫检测仪上490nm比色，记录结果，按照公式(1)计算LPS组和马甲子提取物组对小鼠脾细胞体外平均存活率；再按照公式(2)计算马甲子提取物对LPS诱导的小鼠脾细胞体外增殖的增殖率。
结果显示，马甲子提取物浓度为0.004mg/ml、0.02mg/ml、0.2mg/ml时，相对于LPS组的相对增值率分别为34.37％、20.48％和-15.60％，说明马甲子提取物低浓度时对LPS诱导的小鼠脾细胞体外增殖有促进作用，而高浓度时则表现出抑制作用，这与上述马甲子提取物对ConA诱导的脾细胞体外增殖具双向免疫调节的结论是一致的。
(四)药理学验证
1、乙醇、甲醇、石油醚提取物及乙酸乙酯提取物对免疫低下小鼠非特异性免疫功能的影响(腹腔巨噬细胞吞噬法)
KM小鼠80只，按体重分层随机均分为8组，分别为混悬剂对照组(0.5％西黄芪胶)、模型对照组(0.5％西黄芪胶)、阳性对照组(云芝多糖)、乙酸乙酯提取物小剂量组、乙酸乙酯提取物大剂量组、乙醇提取物组、甲醇提取物组、石油醚提取物组。灌胃给予试验药物或混悬剂，每日1次，连续14d。除混悬剂对照组外，给药第8、10、12d按25mg/kg腹腔注射给予环磷酰胺生理盐水溶液，造成免疫低下。第13d、14d分别腹腔注射6％淀粉溶液；至第14d末次给药后1h，腹腔注射5％鸡红细胞生理盐水悬液1ml，30min后，颈椎脱臼处死动物，经腹腔注入生理盐水2ml，并轻揉腹部。1min后剪开腹部，吸取腹腔洗液1ml，平均分滴于2片载玻片上，放入湿盒内，37℃温育30min。后于生理盐水中漂洗，晾干，并以1:1丙酮-甲醇溶液固定，4％(v/v)Giemsa-PBS染色3min，蒸馏水漂洗晾干，镜检，计算吞噬百分率。
表14马甲子提取物对免疫低下小鼠腹腔巨噬细胞吞噬功能的影响(n＝10,
Figure PCTCN2014089895-appb-000016
)
Figure PCTCN2014089895-appb-000017
与模型对照组比较，*P＜0.05，**P＜0.01
结果如表14所示。马甲子乙酸乙酯提取物在0.1g/kg及以上剂量灌胃给予14日，可显著提高免疫抑制小鼠腹腔巨噬细胞的吞噬能力，且0.4g/kg剂量组作用强度与云芝多糖组无显著性差异；乙醇、甲醇、石油醚提取物1.2g/kg也可有效增强免疫低下小鼠腹腔巨噬细胞的吞噬能力，提示该四类提取物对免疫低下动物均具有较好的免疫增强作用。
2、乙醇、甲醇、石油醚提取物及乙酸乙酯提取物对免疫低下小鼠特异性免疫功能的影响(2，4-二硝氟苯所致耳肿胀法)
KM小鼠80只，按体重分层随机均分为8组，分别为混悬剂对照组(0.5％西黄芪胶)、模型对照组(0.5％西黄芪胶)、阳性对照组(云芝多糖)、乙酸乙酯提取物小剂量组、乙酸乙酯提取物大剂量组、乙醇提取物组、甲醇提取物组、石油醚提取物组。灌胃给予试验药物或混悬剂，每日1次，连续14d。除混悬剂对照组外，给药第8、10、12d按25mg/kg腹腔注射给予环磷酰胺生理盐水溶液，造成免疫低下。给药第9d以1％2，4-二硝氟苯(DNFB)溶液(取100mgDNFB加入到1：1的丙酮-植物油混合物中混匀，定容至10ml)25μl涂抹小鼠腹部。至第13d给药后1h，取10μl1％DNFB溶液涂抹于小鼠左耳；涂抹后24h，即末次给药后1h，颈椎脱臼处死动物，耳片称重，计算耳肿胀度。
表15马甲子提取物对免疫低下小鼠DNFB所致耳肿胀的影响(n＝10,
Figure PCTCN2014089895-appb-000018
)
Figure PCTCN2014089895-appb-000019
与模型对照组比较，*P＜0.05，**P＜0.01
结果如表15所示。马甲子乙酸乙酯提取物可提高免疫低下小鼠的DNFB所致耳肿胀程度，提示其对免疫低下动物的细胞免疫功能具有增强作用；乙醇、甲醇、石油醚提取物1.2g/kg与乙酸乙酯提取物作用类似，提示该四类提取物均具有较好的增强免疫低下动物特异性免疫的作用。
3、乙醇、甲醇、石油醚提取物及乙酸乙酯提取物对正常小鼠非特异性免疫功能的影响(腹腔巨噬细胞吞噬法)
KM小鼠80只，按体重分层随机均分为8组，分别为混悬剂对照组(0.5％西黄芪胶)、增强作用阳性对照组(云芝多糖)、抑制作用阳性对照组(环磷酰胺)、乙酸乙酯提取物小剂量组、乙酸乙酯提取物大剂量组、乙醇提取物组、甲醇提取物组、石油醚提取物组。除抑制作用阳性对照组于第13d一次性皮下注射给予外，其余各组灌胃给予试验药物或混悬剂，每日1次，连续14d。第13d、14d分别腹腔注射6％淀粉溶液；至第14d末次给药后1h，腹腔注射5％鸡红细胞生理盐水悬液1ml，30min后，颈椎脱臼处死动物，经腹腔注入生理盐水2ml，并轻揉腹部。1min后剪开腹部，吸取腹腔洗液1ml，平均分滴于2片载玻片上，放入湿盒内，37℃温育30min。后于生理盐水中漂洗，晾干，并以1:1丙酮-甲醇溶液固定，4％(v/v)Giemsa-PBS染色3min，蒸馏水漂洗晾干，镜检，计算吞噬百分率。
表16马甲子提取物对正常小鼠腹腔巨噬细胞吞噬功能的影响(n＝10,
Figure PCTCN2014089895-appb-000020
)
Figure PCTCN2014089895-appb-000021
与混悬剂对照组比较，*P＜0.05，**P＜0.01
结果如表16所示。马甲子乙酸乙酯提取物0.4g/kg剂量灌胃给予14日，可在一定程度抑制正常小鼠腹腔巨噬细胞的吞噬能力，而0.1g/kg则未观察到明显作用；乙醇、甲醇、石油醚提取物1.2g/kg也表现不同程度的抑制效应，提示该四类提取物对正常动物有轻度的免疫抑制作用。
4、乙醇、甲醇、石油醚提取物及乙酸乙酯提取物对正常小鼠特异性免疫功能的影响(2，4-二硝氟苯所致耳肿胀法)
KM小鼠80只，按体重分层随机均分为8组，分别为混悬剂对照组(0.5％西黄芪胶)、增强作用阳性对照组(云芝多糖)、抑制作用阳性对照组(环磷酰胺)、乙酸乙酯提取物小剂量组、乙酸乙酯提取物大剂量组、乙醇提取物组、甲醇提取物组、石油醚提取物组。除抑制作用阳性对照组于第13d一次性皮下注射给予外，其余各组灌胃给予试验药物或混悬 剂，每日1次，连续14d。给药第9d以1％2，4-二硝氟苯(DNFB)溶液(取100mgDNFB加入到1：1的丙酮-植物油混合物中混匀，定容至10ml)25μl涂抹小鼠腹部。至第13d给药后1h，取10μl1％DNFB溶液涂抹于小鼠左耳；涂抹后24h，即末次给药后1h，颈椎脱臼处死动物，耳片称重，计算耳肿胀度。
表17马甲子提取物对正常小鼠DNFB所致耳肿胀的影响(n＝10,
Figure PCTCN2014089895-appb-000022
)
Figure PCTCN2014089895-appb-000023
与混悬剂对照组比较，*P＜0.05，**P＜0.01
结果如表17所示。马甲子乙酸乙酯提取物0.1g/kg即可抑制正常小鼠DNFB所致耳肿胀程度，提示其对正常动物具有一定的细胞免疫功能抑制作用；乙醇、甲醇、石油醚提取物1.2g/kg与乙酸乙酯提取物作用类似，提示该四类提取物均具有一定的特异性免疫抑制作用。
5、乙醇、甲醇、石油醚提取物及乙酸乙酯提取物对小鼠实验性红斑狼疮的治疗作用及对细胞免疫的影响
KM小鼠80只，按体重分层随机均分为8组，分别为混悬剂对照组(0.5％西黄芪胶)、模型对照组(0.5％西黄芪胶)、阳性对照组(雷公藤多苷)、乙酸乙酯提取物小剂量组、乙酸乙酯提取物大剂量组、乙醇提取物组、甲醇提取物组、石油醚提取物组。除混悬剂对照组外均按0.5ml/只腹腔注射降植烷(pristane)，灌胃给予药物或混悬剂，每日1次，连续30d。末次药后24h眼眶采血，4℃冷冻离心分离血清，ELISA测定血清中抗dsDNA抗体水平。
另取一批动物同样分组及复制模型，注射pristane后第24日，除混悬剂对照组外，各组动物腹腔注射5％鸡红细胞生理盐水悬液0.2ml/只，并继续给药持续至注射pristane后第30日。末次给药后24h于眼眶取血20μl，加入1ml生理盐水中，随后分别加入4％鸡红细胞生理盐水悬液0.5ml和10％豚鼠血清0.5ml,混匀后于37℃孵育0.5h，3000rpm离心10min，取上清液1ml，加入3ml都氏液，540nm比色。
表18马甲子提取物对实验性红斑狼疮小鼠的影响(n＝10,
Figure PCTCN2014089895-appb-000024
)
Figure PCTCN2014089895-appb-000025
与模型对照组比较，*P＜0.05，**P＜0.01
结果如表18所示。马甲子乙酸乙酯提取物0.4g/kg可有效降低实验性红斑狼疮小鼠血清中dsDNA抗体水平，提示其可用于红斑狼疮治疗；乙醇、甲醇、石油醚提取物1.2g/kg与乙酸乙酯提取物作用类似，提示该四类提取物均具有一定的对红斑狼疮的作用。红斑狼疮可表现为体液免疫异常升高，本实验显示模型动物溶血素水平显著高于正常动物，马甲子乙酸乙酯提取物0.1g/kg即可有效降低此异常升高的溶血素水平，乙醇、甲醇、石油醚提取物1.2g/kg与乙酸乙酯提取物作用类似，提示该四类提取物均对异常免疫功能亢进有显著抑制作用。
六、马甲子提取物治疗口腔及消化道炎症或和溃疡
(一)马甲子提取物的制备
1、全株乙醇提取物的制备
取马甲子全株1kg，加入8倍量的95％乙醇浸渍1天后，然后进行粉碎，再加入10倍量的95％乙醇浸渍2天提取，收集提取液，50℃减压回收乙醇至无醇味，干燥即得马甲子乙醇提取物。
2、茎叶乙醇、石油醚和乙酸乙酯提取物的制备
取马甲子茎叶1kg，加入10倍量的95％乙醇浸渍1天后，然后进行粉碎，再加入10倍量的95％乙醇回流提取，收集提取液，60℃减压回收乙醇至无醇味，干燥即得马甲子乙醇提取物。将马甲子乙醇提取物加水分散后，依次用石油醚、乙酸乙酯萃取，得到马甲子石油醚提取物和乙酸乙酯提取物。
3、全株石油醚和乙酸乙酯提取物的制备
取马甲子全株1kg，加入10倍量的甲醇浸渍1天后，然后进行粉碎，再加入8倍量的甲醇浸渍2天，收集提取液，40℃下减压回收甲醇，干燥即得马甲子甲醇提取物，将马甲子甲醇提取物加水分散后，依次用石油醚、乙酸乙酯萃取，得到马甲子石油醚提取物和乙酸乙酯提取物。
4、成分定量分析
取马甲子茎叶石油醚提取物0.1g，三份，加入10ml容量瓶内，加乙酸乙酯至刻度，再从中精密吸取4ml溶液加入10ml容量瓶内，挥干溶剂后，加入5％香草醛-冰醋酸0.4ml、高氯酸1.6ml，混匀，并由乙酸乙酯稀释至刻度，置70℃恒温水浴中加热15min，冷却至室温，并转移至10ml容量瓶中加乙酸乙酯稀释至刻度，摇匀，在540nm波长处测定吸收度，计算供试品溶液中总三萜含量(含三萜以美洲茶酸计)。经计算1g马甲子提取物中三萜类成分含23mg。
取马甲子茎叶石油醚提取物0.1g，三份，精密称定，置50ml量瓶中，加乙醇适量，超声溶解，放冷，加乙醇至刻度，摇匀。精密量取1ml，置10ml量瓶中，加水至刻度，摇匀。精密量取3ml，置25ml量瓶中，在510nm波长处测定吸光度，计算供试品溶液中总黄酮含量(含黄酮量以芦丁计)。经计算1g马甲子提取物中总黄酮含103mg。
取马甲子茎叶石油醚提取物0.1g，三份，精密称定，置具塞锥形瓶中,以18％氨水2ml润湿1h，加乙醚2氯仿2乙醇(25∶8∶2.5)混合溶剂30ml，超声提取20min，倾出上清液至小锥形瓶中，再加入上述混合溶剂30ml，冷浸放置半小时,再超声震荡提取20min，滤过,以相同溶剂15ml分3次洗涤残渣和滤纸,合并滤液于锥形瓶中，于60℃水浴上蒸干，准确加入10ml氯仿使其全部溶解,再准确吸取5mL转移至小分液漏斗中，加6mL氯仿，2ml缓冲液(pH＝5.0，0.2M邻苯二甲酸氢钾缓冲液)。用1mmol·L-1溴麝香草酚蓝溶液滴定,并不断震摇；接近终点时，分出氯仿层，再加入新鲜氯仿5ml，继续滴定并不断震摇,静置分层，至水层略显黄色即为终点。计算总生物碱(含生物碱以马甲子碱B计)。经计算1g马甲子提取物中生物碱含21mg。
取马甲子茎叶石油醚提取物0.1g，三份，精密称定，置50ml量瓶中，加乙醇适量，超声溶解，放冷，加乙醇至刻度，摇匀。精密量取1ml，置10ml量瓶中，加70％乙醇至刻度，摇匀。精密量取3ml，置25ml量瓶中，在340nm波长处测定测定吸光度，计算供试品溶液中总香豆素含量(含香豆素量以伞形花内酯计)。经计算1g马甲子提取物中总香豆素含10.2mg。
取马甲子茎叶乙酸乙酯提取物0.1g，三份，加入10ml容量瓶内，加乙酸乙酯至刻度，再从中精密吸取4ml溶液加入10ml容量瓶内，挥干溶剂后，加入5％香草醛-冰醋酸0.4ml、高氯酸1.6ml，混匀，并由乙酸乙酯稀释至刻度，置70℃恒温水浴中加热15min，冷却至室温，并转移至10ml容量瓶中加乙酸乙酯稀释至刻度，摇匀，在540nm波长处测定吸收度，计算供试品溶液中总三萜含量(含三萜以美洲茶酸计)。经计算1g马甲子提取物中三萜类成分含108mg。
取马甲子茎叶乙酸乙酯提取物0.1g，三份，精密称定，置50ml量瓶中，加乙醇适量，超声溶解，放冷，加乙醇至刻度，摇匀。精密量取1ml，置10ml量瓶中，加水至刻度，摇匀。精密量取3ml，置25ml量瓶中，在510nm波长处测定吸光度，计算供试品溶液中总黄酮含量(含黄酮量以芦丁计)。经计算1g马甲子提取物中总黄酮含497mg。
取马甲子全株乙酸乙酯提取物0.1g，三份，精密称定，置具塞锥形瓶中,以18％氨水2ml润湿1h，加乙醚2氯仿2乙醇(25∶8∶2.5)混合溶剂30ml，超声提取20min，倾出上清液至小锥形瓶中，再加入上述混合溶剂30ml，冷浸放置半小时,再超声震荡提取20min，滤过, 以相同溶剂15ml分3次洗涤残渣和滤纸,合并滤液于锥形瓶中，于60℃水浴上蒸干，准确加入10ml氯仿使其全部溶解,再准确吸取5mL转移至小分液漏斗中，加6mL氯仿，2ml缓冲液(pH＝5.0，0.2M邻苯二甲酸氢钾缓冲液)。用1mmol·L-1溴麝香草酚蓝溶液滴定,并不断震摇；接近终点时，分出氯仿层，再加入新鲜氯仿5ml，继续滴定并不断震摇,静置分层，至水层略显黄色即为终点。计算总生物碱(含生物碱以马甲子碱B计)。经计算1g马甲子提取物中生物碱含107mg。
取马甲子全株乙酸乙酯提取物0.1g，三份，精密称定，置50ml量瓶中，加乙醇适量，超声溶解，放冷，加乙醇至刻度，摇匀。精密量取1ml，置10ml量瓶中，加70％乙醇至刻度，摇匀。精密量取3ml，置25ml量瓶中，在340nm波长处测定测定吸光度，计算供试品溶液中总香豆素含量(含香豆素量以伞形花内酯计)。经计算1g马甲子提取物中总香豆素含186mg。
(二)马甲子制剂的制备
1、片剂的制备
取马甲子全株乙醇提取物300g，加入适宜辅料，如：100g微晶纤维素，57.5g乳糖，20g交联羧甲基纤维素钠等，压片制成片剂。
2、胶囊剂的制备
取马甲子茎叶乙酸乙酯提取物，加入适宜辅料，如：乳糖、可压性淀粉、羧甲基淀粉、微晶纤维素、等制成胶囊剂。
3、颗粒剂的制备
取马甲子全株甲醇提取物，加入适宜辅料，如：乳糖、淀粉、甲基纤维素、羟甲基纤维素、羟丙基甲基纤维素、羟丙基纤维素、聚乙烯吡咯烷酮、微粉硅胶等制成颗粒剂。
4、软膏剂的制备
取马甲子茎叶石油醚提取物，加入适宜辅料，如：十八醇、单硬脂酸甘油脂、甘油、硬脂酸等制成软膏剂。
5、栓剂的制备
取马甲子全株乙醇提物物，加入适宜辅料，如：混合脂肪酸甘油酯、PEG、蜂蜡等制成栓剂。
(三)马甲子提取物用途的药理学验证
1、乙醇、甲醇、石油醚提取物及乙酸乙酯提取物对幽门结扎所致大鼠实验性胃溃疡的影响
SD大鼠80只，按体重分层随机均分为8组，分别为模型对照组(0.5％西黄芪胶)、阳性对照组(雷尼替丁)、乙酸乙酯提取物小剂量组、乙酸乙酯提取物中剂量组、乙酸乙酯提取物大剂量组、乙醇提取物组、甲醇提取物组、石油醚提取物组。灌胃给予受试物，每日1次，连续3日。末次给药后1h行幽门结扎术，术后15h脱颈椎处死动物，取胃，以1％甲醛固定20min后剖开，以体视显微镜观察粘膜损伤程度，计算溃疡指数和溃疡抑制率。
表19马甲子提取物对幽门结扎所致大鼠胃溃疡的影响(n＝10,
Figure PCTCN2014089895-appb-000026
)
Figure PCTCN2014089895-appb-000027
与模型对照组比较，*P＜0.05，**P＜0.01
结果如表19所示。马甲子乙酸乙酯提取物在0.2g/kg及以上剂量灌胃给予3次，即可显著抑制幽门结扎所致的大鼠胃溃疡程度，且0.4g/kg剂量组作用强度与雷尼替丁60mg/kg组无显著性差异；乙醇、甲醇、石油醚提取物1.2g/kg也可有效抑制胃溃疡程度，提示该四类提取物均具有较好的抗胃溃疡作用。
2、乙醇、甲醇、石油醚提取物及乙酸乙酯提取物对2,4,6-三硝基甲苯磺酸(TNBS)所致大鼠实验性结肠炎的影响
SD大鼠90只，按体重分层随机均分为9组，分别为假手术对照组(0.5％西黄芪胶)、模型对照组(0.5％西黄芪胶)、阳性对照组(地塞米松)、乙酸乙酯提取物小剂量组、乙酸乙酯提取物中剂量组、乙酸乙酯提取物大剂量组、乙醇提取物组、甲醇提取物组、石油醚提取物组。动物禁食24h后以戊巴比妥钠麻醉，除假手术对照组外，用TNBS和40％乙醇灌肠，复制实验性结肠炎模型；造模后6h，给予受试物。给药第5d，尾静脉取血行白细胞计数。第6d以乌拉坦麻醉，腹主动脉取血后脱颈椎处死动物，从肛门向上截取9cm结肠，在冰浴中沿肠系膜缘剪开肠腔，漂洗内容物，测量溃疡面积，计算溃疡面积百分比；结肠称重后刮取结肠黏膜，ELISA测定肿瘤坏死因子(TNF-α)浓度。
表20马甲子提取物对TNBS所致大鼠实验性结肠炎的影响(n＝10,
Figure PCTCN2014089895-appb-000028
)
Figure PCTCN2014089895-appb-000029
与模型对照组比较，*P＜0.05，**P＜0.01
结果如表21所示。TNBS所致大鼠实验性结肠炎可出现炎性细胞增加、炎性细胞因子水平上升及结肠表面溃疡。马甲子乙酸乙酯提取物0.2g/kg及以上剂量可抑制白细胞及重要致炎因子TNF-α的升高，减少溃疡面形成；乙醇、甲醇、石油醚提取物1.2g/kg与乙酸乙酯提取物作用类似，提示该四类提取物均具有较好的抗结肠炎作用。
3、乙醇、甲醇、石油醚提取物及乙酸乙酯提取物对氨水所致大鼠实验性慢性胃炎的影响
SD大鼠120只，按体重分层随机均分为10组，分别为正常对照组(0.5％西黄芪胶)、模型对照组(0.5％西黄芪胶)、阳性对照组(三九胃泰颗粒)、乙酸乙酯提取物小剂量组、乙酸乙酯提取物中剂量组、乙酸乙酯提取物大剂量组、乙醇提取物组、甲醇提取物组、石油醚提取物组。所有动物均灌胃给予0.02％氨水，每日1次，连续90日；同时灌胃给予受试物，每日1次，连续90日。末次给药后次日脱颈椎处死，取胃小弯胃壁，10％福尔马林固定，石蜡包埋、切片，HE及PAS染色。HE染色片观察炎症反应情况并计分，测量胃体部粘膜厚度；PAS染色片测量阳性层厚度以表征粘液层厚度。
表21马甲子提取物对氨水所致大鼠实验性慢性胃炎的影响(n＝12,
Figure PCTCN2014089895-appb-000030
)
Figure PCTCN2014089895-appb-000031
与模型对照组比较，*P＜0.05，**P＜0.01；；与阳性对照组(三九胃泰颗粒组)比较，#P＜0.05
结果如表21所示。马甲子乙酸乙酯提取物对氨水所致的实验性慢性胃炎具有抑制炎症程度、增加胃粘膜及粘液层厚度的作用，尤其是在抑制炎症及增加粘液层厚度方面药效更为显著；乙醇、甲醇、石油醚提取物1.2g/kg与乙酸乙酯提取物作用类似，提示该四类提取物均具有较好的抗慢性胃炎作用。
4、乙酸乙酯提取物与三九胃泰联合应用对幽门结扎所致大鼠实验性胃溃疡的影响
SD大鼠50只，按体重分层随机均分为5组，分别为模型对照组(0.5％西黄芪胶)、阳性对照组(雷尼替丁)、乙酸乙酯提取物组、三九胃泰组、联合应用组。灌胃给予受试物，每日1次，连续3日。末次给药后1h行幽门结扎术，术后15h脱颈椎处死动物，取胃，以1％甲醛固定20min后剖开，以体视显微镜观察粘膜损伤程度，计算溃疡指数和溃疡抑制率。
表22马甲子乙酸乙酯提取物与三九胃泰联合应用对幽门结扎所致大鼠胃溃疡的影响(n＝10,
Figure PCTCN2014089895-appb-000032
)
Figure PCTCN2014089895-appb-000033
与模型对照组比较，*P＜0.05，**P＜0.01；与三九胃泰组比较，#P＜0.05，##P＜0.01
结果如表22所示。马甲子乙酸乙酯提取物在0.2g/kg灌胃给予3次，即可显著抑制幽门结扎所致的大鼠胃溃疡程度；单独应用三九胃泰对该模型无明显影响，但与本发明提取物合用则可显著增强抗胃溃疡药效，提示其与抗胃溃疡药物或组方联合应用可有协同作用。·
5、乙酸乙酯提取物与三九胃泰联合应用对氨水所致大鼠实验性慢性胃炎的影响
本试验与药效学试验2同时进行，研究结果见表21，共用正常对照组及模型对照组。结果显示，三九胃泰可显著增加粘液层厚度，减少炎症，但对粘膜层厚度无明显影响。与本发明提取物联合应用后，除可在一定程度上增强三九胃泰已有作用外，还可有效增加粘膜层厚度，提示与其他治疗慢性胃炎的药物或组方有协同作用。
综上，采用本发明马甲子提取物或其原型药材马甲子是有显著的抗肿瘤活性、抗真菌活性、抗纤维化、并具有双向免疫调节作用，以及治疗口腔及消化道炎症或和溃疡的功效。

Claims (37)

  1. 马甲子在制备具有抗纤维化的药物中的用途。
  2. 马甲子在制备具有抗真菌活性的药物中的用途。
  3. 马甲子在制备具有抗肿瘤活性的药物中的用途。
  4. 马甲子在制备治疗口腔及消化道炎症或(和)溃疡相关疾病的药物中的用途。
  5. 马甲子在制备具有双向免疫调节作用的药物中的用途。
  6. 根据权利要求1-5任一项所述的用途，其特征在于：马甲子采用马甲子全株植物或其中任一部位；
    优选的，所述部位为根、茎、叶、花、果任一部位或其混合；
    最优选的，所述部位为叶。
  7. 马甲子提取物的制备方法，其特征在于：以马甲子全株植物或其中任一部位为原料药，采用常规提取方法制备而得。
  8. 根据权利要求7所述的马甲子提取物的制备方法，其特征在于：
    制备方法一
    A、以马甲子全株植物或其中任一部位为原料药；
    B、以溶剂提取、干燥、即得；
  9. 根据权利要求7所述的马甲子提取物的制备方法，其特征在于：
    制备方法二
    A、以马甲子全株植物或其中任一部位为原料药；
    B、以溶剂a提取，滤液浓缩，得浓缩液；
    C、以溶剂b萃取步骤B所得浓缩液，得液相，干燥，即得；
    或，干燥步骤B所得浓缩液得提取物1，以溶剂b提取，干燥，即得。
  10. 根据权利要求8或9所述的马甲子提取物的制备方法，其特征在于：步骤A所述以马甲子作为原料药是采用其鲜品、冷冻干燥品、有机溶媒预处理品；
    优选的，有机溶媒预处理品的制备方法是：取马甲子用有机溶媒浸泡；
    进一步优选的，所述有机溶媒包括乙醇、甲醇、乙酸乙酯和石油醚、异丙醇等；优选甲醇或乙醇。
  11. 根据权利要求9所述的马甲子提取物的制备方法，其特征在于：所述有机溶媒预处理品的有机溶媒包括甲醇、乙醇、异丙醇、乙酸乙酯或石油醚；优选甲醇或乙醇。
  12. 根据权利要求8所述的马甲子提取物的制备方法，其特征在于：所述制备方法一中的步骤B所述溶剂包括甲醇、乙醇、异丙醇、乙酸乙酯或石油醚；优选甲醇或乙醇。
  13. 根据权利要求8所述的马甲子提取物的制备方法，其特征在于：所述制备方法一中的步骤B所述提取方法采用浸渍、回流或渗漉提取。
  14. 根据权利要求8所述的马甲子提取物的制备方法，其特征在于：所述制备方法一中的步骤B所述干燥方法采用减压干燥、冷冻干燥、喷雾干燥或微波干燥。
  15. 根据权利要求8所述的马甲子提取物的制备方法，其特征在于：所述制备方法二 中的步骤B所述溶剂a包括甲醇或乙醇、异丙醇；优选甲醇或乙醇。
  16. 根据权利要求8所述的马甲子提取物的制备方法，其特征在于：所述制备方法二中的步骤C所述溶剂b包括乙酸乙酯或石油醚。
  17. 根据权利要求8所述的马甲子提取物的制备方法，其特征在于：所述制备方法二中的步骤B及步骤C所述提取采用浸渍法、回流法、渗漉法或萃取法提取。
  18. 根据权利要求8所述的马甲子提取物的制备方法，其特征在于：所述制备方法二中的步骤B及步骤C所述干燥采用减压干燥、冷冻干燥、喷雾干燥或微波干燥。
  19. 根据权利要求8所述的马甲子提取物的制备方法，其特征在于：
    所述制备方法一的步骤B所使用的溶剂与马甲子的用量关系为，溶剂添加量为马甲子质量的1-20倍；
    优选的，溶剂添加量为马甲子质量的5-15倍；
    进一步优选的，溶剂添加量为马甲子质量的8-10倍；
    优选的，溶剂采用甲醇或乙醇时，甲醇或乙醇添加量为马甲子质量的1-20倍；
    所述方法二的步骤B所使用的溶剂a与马甲子的用量关系为，溶剂a添加量为马甲子质量的1-20倍；
    优选的，溶剂a添加量为马甲子质量的5-15倍；
    进一步优选的，溶剂a添加量为马甲子质量的8-10倍。
  20. 根据权利要求10、11、14或18任一项所述的马甲子提取物的制备方法，其特征在于：所述乙醇的浓度为10-95％；
    优选的，采用乙醇浓度为50-95％；
    最优选的，采用乙醇浓度为95％。
  21. 权利要求7-19任一项所述的制备方法所得的马甲子提取物。
  22. 根据权利要求20所述的马甲子提取物，其特征在于：主要成分包括黄酮类、萜类、生物碱类、香豆素类；
    进一步的优选包括上述黄酮类、萜类、生物碱类、香豆素类的苷及其单体成分以及多糖类和纤维素。
  23. 权利要求20或21所述的马甲子提取物的药物组合物，其特征在于：以马甲子提取物为活性成分，加入药学上可接受的辅料制成的制剂。
  24. 根据权利要求22所述的马甲子提取物的药物组合物，其特征在于；所述制剂为口服制剂、注射制剂或外用制剂；或，所述制剂为控释制剂或缓释制剂；
    优选的，所述口服制剂片剂、胶囊剂、颗粒剂、微丸、微球、滴丸；
    优选的，所述外用制剂为贴膏剂、软膏剂、凝胶剂、涂膜剂、搽剂、栓剂、洗剂或喷雾剂。
  25. 权利要求20或21所述的马甲子提取物在制备具有抗纤维化的药物中的用途。
  26. 根据权利要求24所述的用途，其特征在于：所述纤维化包括肺纤维化、肾纤维化、肝纤维化、心肌纤维化。
  27. 根据权利要求24所述的用途，其特征在于：加入其它具有治疗纤维化的中药、西 药联合使用。
  28. 根据权利要求26所述的用途，其特征在于：其它具有治疗纤维化的中药、西药选自：三七总甙、川芎嗪、丹参、刺五加注射液、川芎嗪注射液、红花注射液、银杏黄酮苷、生脉、丹参注射液、双黄连、香丹注射液、益气活血颗粒、抗纤颗粒、肺康颗粒、百合固金丸、虫草蛤蚧散合洋参丸、黄芪、西红花、生地黄、三七、绞股蓝、姜黄、黄葵、苦杏仁甙、汉防己甲素、大黄素、恩替卡韦、拉米夫定、β-胡萝卜素、维生素E、磷脂酰胆碱、S-腺苷蛋氨酸、前列地尔、地诺前列酮、秋水仙碱、雌激素、血管紧张素II受体阻断药、交感神经系统抑制剂、干扰素、脯氨酰-4-羟化酶抑制剂、肝素、水飞蓟素、熊去氧胆酸。
  29. 权利要求20或21所述的马甲子提取物在制备具有抗肿瘤活性的药物中的用途。
  30. 根据权利要求28所述的用途，其特征在于：加入其它具有治疗肿瘤的中药、西药联合使用。
  31. 根据权利要求29所述的用途，其特征在于：其它具有治疗肿瘤的中药、西药选自：放疗、免疫疗法、DNA损伤的化疗剂、干扰细胞复制的化疗剂和免疫调节药物；
    优选的，其它具有治疗肿瘤的中药、西药选自拓扑异构酶I抑制剂、拓扑异构酶II抑制剂、烷化剂、DNA嵌合剂、DNA嵌入剂和自由基生成剂、干扰细胞复制的化疗剂、蛋白酪氨酸激酶抑制剂、蛋白酶抑制剂、与癌症中过表达的蛋白结合从而下调细胞复制的抗体、蛋白或酶抑制剂；其它具有治疗肿瘤的中药、西药可以选自伊立替康、拓扑替康、喜树碱及其类似物或代谢物、阿霉素、依托泊苷、替尼泊苷、道诺霉素、美法仑、苯丁酸氮芥、白消安、塞替哌、异环磷酰胺、亚硝基脲氮芥、洛莫司汀、司莫司汀、链脲菌素、氨烯咪胺、甲氨蝶呤、丝裂霉素C、环磷酰胺、顺铂、奥沙利铂、卡铂、博来霉素、5-氟尿嘧啶、卡培他滨、吉西他滨、氟达拉滨、阿糖胞苷、巯嘌呤、硫鸟嘌呤、喷司他丁、羟基脲、紫杉醇、紫杉萜及其相关类似物、长春新碱、长春碱及有关类似物、沙立度胺及有关类似物、甲磺酸伊马替尼、吉非替尼、硼替佐米、曲妥单抗、利妥昔单抗、西妥昔单抗、贝伐单抗、华蟾素、姬松茸、珍珠梅乙酸乙酯提取物、荔枝核水提液、当雷公藤红素、猪苓多糖、羧甲基茯苓多糖、泽泻醇提浓缩液、甘草多糖、当归总多糖、补骨脂素、五味子多糖。
  32. 权利要求20或21所述的马甲子提取物在制备具有抗真菌活性的药物中的用途。
  33. 权利要求20或21所述的马甲子提取物在制备治疗口腔及消化道炎症或(和)溃疡相关疾病的药物中的用途。
  34. 权利要求20或21所述的马甲子提取物在制备具有双向免疫调节作用的药物中的用途。
  35. 根据权利要求33所述的用途，其特征在于：双向免疫调节作用是针对免疫功能异常引起的免疫功能低下或(和)自身免疫性疾病。
  36. 根据权利要求34所述的用途，其特征在于：所述免疫功能异常的免疫功能低下，包括感冒易感、体虚、肿瘤或艾滋病。
  37. 根据权利要求34所述的用途，其特征在于：所述免疫功能异常的自身免疫性疾病，包括类风湿性关节炎、红斑狼疮、硬皮病、甲状腺机能亢进、青少年糖尿病、原发性血小板紫癜、自身免疫性溶血性贫血、溃疡性结肠炎或慢性肝病。
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CN109198182A (zh) * 2018-10-25 2019-01-15 杨凌金石牧业有限责任公司 一种用于畜禽饲料的中药复方添加剂
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