WO2020244493A1 - 蓟属植物的有机提取物及其应用与组合物 - Google Patents

蓟属植物的有机提取物及其应用与组合物 Download PDF

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WO2020244493A1
WO2020244493A1 PCT/CN2020/093875 CN2020093875W WO2020244493A1 WO 2020244493 A1 WO2020244493 A1 WO 2020244493A1 CN 2020093875 W CN2020093875 W CN 2020093875W WO 2020244493 A1 WO2020244493 A1 WO 2020244493A1
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minutes
extract
tuberculosis
thistle
cirsium
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PCT/CN2020/093875
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English (en)
French (fr)
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何建明
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浙江我武生物科技股份有限公司
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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)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • 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
    • 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
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8624—Detection of slopes or peaks; baseline correction
    • G01N30/8631—Peaks
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
    • G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
    • 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
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • 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

Definitions

  • the invention relates to the field of pharmacy, in particular to an organic extract of thistle plant and its application and composition.
  • Mycobacterium tuberculosis can infect the lungs and cause tuberculosis. It can also infect extrapulmonary organs such as the intestine, peritoneum, kidney, bladder, ureter, pleura, bone, joints, brain, meninges, reproductive system, etc. Extrapulmonary tuberculosis is more common in people with weakened immunity. Tuberculosis includes primary tuberculosis, hematogenous tuberculosis and secondary tuberculosis. Primary pulmonary tuberculosis, also known as primary tuberculosis, is more common in children. The classic lesions include tuberculosis inflammation of the primary lung, draining lymphatic vessels, and hilar or mediastinal lymph nodes.
  • the three are collectively called primary syndrome.
  • Low-grade fever, night sweats, fatigue, and weight loss are common in patients with tuberculosis.
  • High fever may occur when the lesion progresses rapidly.
  • the other symptoms vary depending on the site of infection: patients with tuberculosis often cough, expectoration, hemoptysis, chest pain, shortness of breath, and may also have allergic reactions.
  • Intestinal tuberculosis patients are mostly oral infections, that is, patients with open tuberculosis or throat tuberculosis swallowing sputum containing Mycobacterium tuberculosis.
  • the first-line treatment drugs for tuberculosis include isoniazid, rifampicin, pyrazineamide, streptomycin and ethambutol.
  • these drugs generally have drug resistance and severe liver damage. Therefore, there is an urgent need in the art to find a natural medicine that can resist Mycobacterium tuberculosis to overcome the limitations of existing medicines.
  • one aspect of the present invention provides an extract of thistle plant, which is an organic phase extract; preferably, the organic extract uses ethanol, acetone, acetic acid
  • the thistle plant is selected from thistle (Cirsium japonicum), wild thistle (Cirsium maackii), green thistle (Cirsium chinense), Cirsium (Cirsium setosum), line leaf thistle (Cirsium lineare) ) And Hangzhou thistle (Cirsium tianmushanicum).
  • Another aspect of the present invention also provides an extract of thistle genus plant, the extract is characterized by having a characteristic ultraviolet absorption peak with a peak time between 35-40 minutes, wherein the peak time is performed at a wavelength of 254 nm Detected by high performance liquid chromatography.
  • the extract has characteristic ultraviolet absorption peaks with peak times of 35.8 ⁇ 0.3 minutes, 36.6 ⁇ 0.3 minutes, and 37.7 ⁇ 0.3 minutes; more preferably, the extract has peak times respectively Characteristic ultraviolet absorption peaks of 35.8 ⁇ 0.2 minutes, 36.6 ⁇ 0.2 minutes, and 37.7 ⁇ 0.2 minutes; more preferably, the extract has characteristic peak times of 35.8 ⁇ 0.1 minutes, 36.6 ⁇ 0.1 minutes, and 37.7 ⁇ 0.1 minutes, respectively UV absorption peak.
  • the extract further has a characteristic ultraviolet absorption peak with a peak time of 37.0 ⁇ 0.3 minutes, preferably 37.0 ⁇ 0.2 minutes, more preferably 37.0 ⁇ 0.1 minutes, and a peak time of 38.9 ⁇ 0.3
  • a characteristic ultraviolet absorption peak with a peak time of 37.0 ⁇ 0.3 minutes, preferably 37.0 ⁇ 0.2 minutes, more preferably 37.0 ⁇ 0.1 minutes, and a peak time of 38.9 ⁇ 0.3
  • One or more characteristic ultraviolet absorption peaks among the characteristic ultraviolet absorption peaks of minutes preferably 38.9 ⁇ 0.2 minutes, more preferably 38.9 ⁇ 0.1 minutes;
  • the extract also has a characteristic UV absorption peak with a peak time between 30-35 minutes, and/or a characteristic UV absorption peak with a peak time between 40-45 minutes; preferably Ground, the extract has a characteristic ultraviolet absorption peak with a peak time of 34.2 ⁇ 0.3 minutes, preferably 34.2 ⁇ 0.2 minutes, more preferably 34.2 ⁇ 0.1 minutes, and a peak time of 32.5 ⁇ 0.3 minutes, preferably 32.5 ⁇ 0.2 minutes , More preferably, the characteristic ultraviolet absorption peak of 32.5 ⁇ 0.1 minutes, the peak time is 31.5 ⁇ 0.3 minutes, preferably 31.5 ⁇ 0.2 minutes, more preferably the characteristic ultraviolet absorption peak of 31.5 ⁇ 0.1 minutes, the peak time is 42.0 ⁇ 0.3 Minutes, preferably 42.0 ⁇ 0.2 minutes, more preferably 42.0 ⁇ 0.1 minutes characteristic ultraviolet absorption peak, and peak time of 43.8 ⁇ 0.3 minutes, preferably 43.8 ⁇ 0.2 minutes, more preferably 43.8 ⁇ 0.1 minutes characteristic ultraviolet absorption One or more characteristic ultraviolet absorption peaks in the peak;
  • the detection conditions of the high performance liquid chromatography method are:
  • the extract has characteristic ultraviolet absorption peaks with peak times as follows: 35.8 ⁇ 0.1 minutes, 36.6 ⁇ 0.1 minutes, and 37.7 ⁇ 0.1 minutes, preferably 34.2 ⁇ 0.1 minutes, 35.8 ⁇ 0.1 Minutes, 36.6 ⁇ 0.1 minutes, 37.0 ⁇ 0.1 minutes, 37.7 ⁇ 0.1 minutes and 42.0 ⁇ 0.1 minutes, more preferably 31.5 ⁇ 0.1 minutes, 32.5 ⁇ 0.1 minutes, 34.2 ⁇ 0.1 minutes, 35.8 ⁇ 0.1 minutes, 36.6 ⁇ 0.1 minutes, 37.0 ⁇ 0.1 minutes, 37.7 ⁇ 0.1 minutes, 38.9 ⁇ 0.1 minutes, 42.0 ⁇ 0.1 minutes and 43.8 ⁇ 0.1 minutes; or there are characteristic UV absorption peaks with peak times as shown below: 35.8 ⁇ 0.2 minutes, 36.6 ⁇ 0.2 minutes and 37.7 ⁇ 0.2 minutes, preferably 34.2 ⁇ 0.2 minutes, 35.8 ⁇ 0.2 minutes, 36.6 ⁇ 0.2 minutes, 37.0 ⁇ 0.2 minutes, 37.7 ⁇ 0.2 minutes and 42.0 ⁇ 0.2 minutes, more preferably 31.5 ⁇ 0.2 minutes, 32.5 ⁇ 0.2 minutes, 34.2 ⁇ 0.2 minutes , 35.8 ⁇ 0.2 minutes, 35.8 ⁇
  • the Thistle plant extract has characteristic ultraviolet absorption peaks with peak times as follows: 35.6 ⁇ 0.1 minutes, 36.6 ⁇ 0.1 minutes, 37.8 ⁇ 0.1 minutes, 38.9 ⁇ 0.1 minutes, 42.0 ⁇ 0.1 minutes and 43.7 ⁇ 0.1 minutes, optionally at 38.2 ⁇ 0.1 minutes and 46.8 ⁇ 0.1 minutes, there are also characteristic ultraviolet absorption peaks.
  • the Thistle plant extract has characteristic ultraviolet absorption peaks with peak times as follows: 34.2 ⁇ 0.1 minutes, 35.9 ⁇ 0.1 minutes, 36.6 ⁇ 0.1 minutes, 36.9 ⁇ 0.1 minutes, and 37.6 ⁇ 0.1 minutes, optionally at 32.9 ⁇ 0.1 minutes, 33.2 ⁇ 0.1 minutes, 34.5 ⁇ 0.1 minutes and 38.1 ⁇ 0.1 minutes, there are also characteristic absorption peaks.
  • the Thistle plant extract has ultraviolet characteristic ultraviolet absorption peaks with peak times as follows: 31.5 ⁇ 0.1 minutes, 32.5 ⁇ 0.1 minutes, 34.3 ⁇ 0.1 minutes, 35.8 ⁇ 0.1 minutes, 36.6 ⁇ 0.1 minutes, 37.0 ⁇ 0.1 minutes, and 37.6 ⁇ 0.1 minutes, optionally 29.9 ⁇ 0.1 minutes, 30.3 ⁇ 0.1 minutes, 31.0 ⁇ 0.1 minutes, 32.0 ⁇ 0.1 minutes, 33.2 ⁇ 0.1 minutes, 33.9 ⁇ 0.1 minutes, 35.2 ⁇ 0.1 minutes, 35.7 ⁇ 0.1 minutes, 36.1 ⁇ 0.1 minutes, 40.0 ⁇ 0.1 minutes, 40.7 ⁇ 0.1 minutes and 41.2 ⁇ 0.1 minutes also have ultraviolet characteristic absorption peaks.
  • the thistle plant is selected from thistle (Cirsium japonicum), wild thistle (Cirsium maackii), green thistle (Cirsium chinense), Cirsium (Cirsium setosum), line leaf thistle (Cirsium lineare) ) And Hangzhou thistle (Cirsium tianmushanicum).
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the extract described in any of the embodiments herein and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition is composed of a therapeutically effective amount of any of the The extract described in the embodiment and a pharmaceutically acceptable carrier are composed.
  • the present invention also provides the application of the extract according to any embodiment of the present invention in the preparation of anti-Mycobacterium tuberculosis drugs or treatment drugs for tuberculosis.
  • the Mycobacterium tuberculosis is selected from the group consisting of Mycobacterium tuberculosis human and Mycobacterium bovis; more preferably, the Mycobacterium tuberculosis is selected from the drug-resistant Mycobacterium tuberculosis.
  • the tuberculosis is caused by Mycobacterium tuberculosis; more preferably, the tuberculosis is selected from pulmonary tuberculosis or extrapulmonary tuberculosis; still preferably, the tuberculosis is pulmonary tuberculosis, liver tuberculosis, gastric tuberculosis Or intestinal tuberculosis.
  • the present invention also provides a method for treating tuberculosis, the method comprising administering to a patient a therapeutically effective amount of the extract according to any embodiment of the present invention, or administering to the patient the pharmaceutical composition according to any embodiment of the present invention .
  • the tuberculosis is caused by Mycobacterium tuberculosis; more preferably, the Mycobacterium tuberculosis is selected from the group consisting of Mycobacterium tuberculosis human and Mycobacterium bovis; still more preferably, the tuberculosis Mycobacteria are selected from drug-resistant human Mycobacterium tuberculosis.
  • the tuberculosis is selected from pulmonary tuberculosis or extrapulmonary tuberculosis; more preferably, the tuberculosis is pulmonary tuberculosis, liver tuberculosis, gastric tuberculosis or intestinal tuberculosis.
  • the present invention also provides a method for preparing thistle plant extract, the method comprising the step of using an organic solution to extract thistle plant; preferably, the organic solution is selected from ethanol, acetone, ethyl acetate, dichloromethane, One or more of chloroform, petroleum ether, n-hexane and cyclohexane.
  • the root part of thistle plant is used for extraction.
  • the thistle plant is selected from thistle (Cirsium japonicum), wild thistle (Cirsium maackii), green thistle (Cirsium chinense), Cirsium (Cirsium setosum), line leaf thistle (Cirsium lineare) ) And Hangzhou thistle (Cirsium tianmushanicum).
  • Figure 1 shows the HPLC profile of the ethanol extract of root in Example 4.
  • Figure 2 shows the HPLC profile of the radix petroleum ether extract in Example 4.
  • FIG. 3 shows the HPLC profile of the C01-34 component in Example 4.
  • Figure 4 shows the HPLC profile of the C01-84 component in Example 4.
  • Figure 5 shows the HPLC profile of the C01-159 component in Example 4.
  • the sum of the parts of each component in the composition may be 100 parts by weight.
  • the numerical range “a-b” represents an abbreviated representation of any combination of real numbers between a and b, where both a and b are real numbers.
  • the numerical range “0-5" means that all real numbers between "0-5" have been listed in this article, and "0-5" is only an abbreviation of these numerical combinations.
  • the integer value range "a-b” represents an abbreviated representation of any combination of integers between a and b, where both a and b are integers.
  • the integer value range "1-N" means 1, 2...N, where N is an integer.
  • “combination thereof” means a multi-component mixture of the respective elements, such as two, three, four, and up to the largest possible multi-component mixture.
  • the “range” disclosed herein is in the form of lower and upper limits. There can be one or more lower limits, and one or more upper limits, respectively. The given range is limited by selecting a lower limit and an upper limit. The selected lower and upper limits define the boundaries of the particular range. All ranges that can be defined in this way are inclusive and combinable, that is, any lower limit can be combined with any upper limit to form a range. For example, the ranges of 60-120 and 80-110 are listed for specific parameters, and the ranges of 60-110 and 80-120 are also expected. In addition, if the minimum range values 1 and 2 are listed, and if the maximum range values 3, 4, and 5 are listed, the following ranges can all be expected: 1-3, 1-4, 1-5, 2- 3, 2-4, and 2-5.
  • the present invention provides an organic extract of thistle plant.
  • the thistle is a plant of the Compositae family, the thistle described herein includes the thistle plants known in the art, including but not limited to thistle (Cirsium japonicum), wild thistle (Cirsium maackii), green thistle (Cirsium chinense), Cirsium setosum, Cirsium lineare and Cirsium tianmushanicum.
  • thistle plants are plants of the genus Thistle grown in Shanghai, Zhejiang City, Jiangsu province and Anhui province, and more preferably are plants of the genus Thistle grown in Yiwu, Zhejiang City.
  • the part of thistle plant used for extraction may preferably be a root.
  • the organic extract refers to the extract obtained by extraction with an organic solvent, usually in the organic phase.
  • Organic solvents suitable for the present invention include, but are not limited to, any one or more of ethanol, acetone, ethyl acetate, dichloromethane, chloroform, petroleum ether, n-hexane, and cyclohexane.
  • the preferred organic solvent is one or more of ethanol, petroleum ether, n-hexane and cyclohexane.
  • the mass percentage concentration of the organic solvent may be 20-100%, preferably 40-100%, more preferably 60-100%, most preferably 80-100%.
  • ethanol or an aqueous ethanol solution having a concentration of 20-100% by mass, preferably 40-100%, more preferably 60-100%, and most preferably 80-100% can be used for extraction.
  • the Thistle plant extract is an ethanol extract, petroleum ether extract, n-hexane extract or cyclohexane extract of the root of thistle plant.
  • the present invention provides a type of thistle plant extract, which is characterized by having a characteristic ultraviolet absorption peak with a peak time between 35-40 minutes, wherein the peak time is Detected by high performance liquid chromatography (HPLC) at a wavelength of 254nm.
  • the extract has characteristic ultraviolet absorption peaks with peak times of 35.8 ⁇ 0.3 minutes, 36.6 ⁇ 0.3 minutes, and 37.7 ⁇ 0.3 minutes; more preferably, the extract has peak times of 35.8 ⁇ 0.2 minutes, 36.6 ⁇ 0.3 minutes, respectively.
  • the extract also has a characteristic ultraviolet absorption peak with a peak time of 37.0 ⁇ 0.3 minutes, preferably 37.0 ⁇ 0.2 minutes, more preferably 37.0 ⁇ 0.1 minutes, and a peak time of 38.9 ⁇ 0.3 minutes, preferably 38.9
  • the extract also has a characteristic ultraviolet absorption peak with a peak time between 30-35 minutes, and/or a characteristic ultraviolet absorption peak with a peak time between 40-45 minutes.
  • the extract has a characteristic ultraviolet absorption peak with a peak time of 34.2 ⁇ 0.3 minutes, preferably 34.2 ⁇ 0.2 minutes, more preferably 34.2 ⁇ 0.1 minutes, and a peak time of 32.5 ⁇ 0.3 minutes, preferably 32.5 ⁇ 0.2
  • the characteristic ultraviolet absorption peak of 32.5 ⁇ 0.1 minutes, the peak time is 31.5 ⁇ 0.3 minutes, preferably 31.5 ⁇ 0.2 minutes, more preferably the characteristic UV absorption peak of 31.5 ⁇ 0.1 minutes
  • the peak time is 42.0 ⁇
  • the Thistle plant extract of the present invention has characteristic ultraviolet absorption peaks with peak times as follows: 35.8 ⁇ 0.1 minutes, 36.6 ⁇ 0.1 minutes and 37.7 ⁇ 0.1 minutes, preferably 34.2 ⁇ 0.1 minutes, 35.8 ⁇ 0.1 minutes, 36.6 ⁇ 0.1 minutes, 37.0 ⁇ 0.1 minutes, 37.7 ⁇ 0.1 minutes and 42.0 ⁇ 0.1 minutes, more preferably 31.5 ⁇ 0.1 minutes, 32.5 ⁇ 0.1 minutes, 34.2 ⁇ 0.1 minutes, 35.8 ⁇ 0.1 minutes, 36.6 ⁇ 0.1 Minutes, 37.0 ⁇ 0.1 minutes, 37.7 ⁇ 0.1 minutes, 38.9 ⁇ 0.1 minutes, 42.0 ⁇ 0.1 minutes and 43.8 ⁇ 0.1 minutes; or, with the characteristic UV absorption peaks with peak time as shown below: 35.8 ⁇ 0.2 minutes, 36.6 ⁇ 0.2 Minutes and 37.7 ⁇ 0.2 minutes, preferably 34.2 ⁇ 0.2 minutes, 35.8 ⁇ 0.2 minutes, 36.6 ⁇ 0.2 minutes, 37.0 ⁇ 0.2 minutes, 37.7 ⁇ 0.2 minutes and 42.0 ⁇ 0.2 minutes, more preferably 31.5 ⁇ 0.2 minutes, 32.5 ⁇ 0.2 minutes, 34.2 ⁇ 0.2 minutes,
  • Thistle plant extract of the present invention may also be a purified extract, such as an extract purified by preparative high performance liquid chromatography (HPLC), or an appropriate method (such as using a filler for Separation and purification by silica gel column chromatography) collected specific fractions.
  • HPLC high performance liquid chromatography
  • an appropriate method such as using a filler for Separation and purification by silica gel column chromatography
  • the Thistle extract of the present invention can be a mixture of different organic solvent extracts in any ratio, or a mixture of different fractions obtained by purification of different organic solvent extracts, or a mixture of the same organic solvent extract.
  • a mixture of different fractions obtained by purification in any ratio may also be a mixture of fractions obtained by purification and an unpurified organic solvent extract in any ratio.
  • the Thistle plant extract of the present invention has characteristic ultraviolet absorption peaks with peak times as follows: 35.6 ⁇ 0.1 minutes, 36.6 ⁇ 0.1 minutes, 37.8 ⁇ 0.1 minutes, 38.9 ⁇ 0.1 minutes, 42.0 ⁇ 0.1 Minutes and 43.7 ⁇ 0.1 minutes, optionally 38.2 ⁇ 0.1 minutes and 46.8 ⁇ 0.1 minutes, also have characteristic ultraviolet absorption peaks.
  • the Thistle plant extract of the present invention has characteristic ultraviolet absorption peaks with peak times as follows: 34.2 ⁇ 0.1 minutes, 35.9 ⁇ 0.1 minutes, 36.6 ⁇ 0.1 minutes, 36.9 ⁇ 0.1 minutes, and 37.6 ⁇ 0.1 Minutes, optionally at 32.9 ⁇ 0.1 minutes, 33.2 ⁇ 0.1 minutes, 34.5 ⁇ 0.1 minutes and 38.1 ⁇ 0.1 minutes, also have characteristic absorption peaks.
  • the Thistle extract of the present invention has ultraviolet characteristic ultraviolet absorption peaks with peak times as follows: 31.5 ⁇ 0.1 minutes, 32.5 ⁇ 0.1 minutes, 34.3 ⁇ 0.1 minutes, 35.8 ⁇ 0.1 minutes, 36.6 ⁇ 0.1 minutes, 37.0 ⁇ 0.1 minutes and 37.6 ⁇ 0.1 minutes, optional among 29.9 ⁇ 0.1 minutes, 30.3 ⁇ 0.1 minutes, 31.0 ⁇ 0.1 minutes, 32.0 ⁇ 0.1 minutes, 33.2 ⁇ 0.1 minutes, 33.9 ⁇ 0.1 minutes, 35.2 ⁇ 0.1 minutes , 35.7 ⁇ 0.1 minutes, 36.1 ⁇ 0.1 minutes, 40.0 ⁇ 0.1 minutes, 40.7 ⁇ 0.1 minutes and 41.2 ⁇ 0.1 minutes also have ultraviolet characteristic absorption peaks.
  • such extracts are extracts purified by preparative high performance liquid chromatography (HPLC).
  • the extract of the present invention can be used to fight against Mycobacterium tuberculosis and to treat tuberculosis caused by Mycobacterium tuberculosis.
  • Mycobacterium tuberculosis is preferably selected from the group consisting of Mycobacterium tuberculosis human and Mycobacterium bovis, and more preferably drug-resistant Mycobacterium tuberculosis.
  • tuberculosis is selected from pulmonary tuberculosis or extrapulmonary tuberculosis.
  • the tuberculosis is pulmonary tuberculosis, liver tuberculosis, gastric tuberculosis or intestinal tuberculosis.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the Thistle plant extract described herein and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition consists of a therapeutically effective amount of the Thistle It consists of plant extracts and pharmaceutically acceptable carriers.
  • the term "comprising" means that the pharmaceutical composition may also contain any other components, and these components may be present in any content, as long as the group is present at that content
  • the ingredients are acceptable to the human body and have no negative influence on the activity of the active ingredient (the extract of thistle plant) in the pharmaceutical composition of the present invention.
  • the term "pharmaceutically acceptable carrier” should be compatible with the active ingredient (the extract of the Thistle plant) in the pharmaceutical composition of the present invention, that is, it can be blended with it without Under normal circumstances, the efficacy of the drug will be greatly reduced.
  • terapéuticaally effective amount means that the active ingredient (the extract of the Thistle plant) in the pharmaceutical composition can play a role in treating diseases such as tuberculosis.
  • the administration mode of the pharmaceutical composition is via the digestive tract, via the respiratory tract, transdermal, transmucosal, or injection.
  • the subject of administration of the pharmaceutical composition is a primate, more preferably a human.
  • the dosage form of the pharmaceutical composition is a dosage form for administration through the digestive tract, a dosage form for administration through the respiratory tract, a dosage form for transdermal administration, a dosage form for transmucosal administration, and an injection dosage form; more preferably, the dosage form of the pharmaceutical composition is selected From oral liquids, tablets, capsules, powders, granules, pills, eye drops, nasal drops, suppositories, pills, liniments, ointments, patches, pastes, sprays, aerosols, powders Spray, inhalation solution (suspension), injection, powder injection, water injection, large infusion dosage form, etc.
  • the application also provides the application of the Thistle plant extract described herein in the preparation of anti-Mycobacterium tuberculosis drugs or drugs for the treatment of tuberculosis, or the application of the Thistle plant extract in the treatment of tuberculosis, or the use of An extract of thistle plant for treating tuberculosis, or a method for treating tuberculosis with an extract of the thistle plant is provided.
  • the tuberculosis is caused by Mycobacterium tuberculosis.
  • the Mycobacterium tuberculosis is selected from the group consisting of Mycobacterium tuberculosis human and Mycobacterium bovis; and more preferably, the Mycobacterium tuberculosis is selected from the drug-resistant Mycobacterium tuberculosis.
  • the tuberculosis is selected from pulmonary tuberculosis or extrapulmonary tuberculosis; more preferably, the tuberculosis is pulmonary tuberculosis, liver tuberculosis, gastric tuberculosis or intestinal tuberculosis.
  • the subject of the application or method is a primate (such as a monkey or a human), preferably a human.
  • the present invention also provides a method for preparing thistle plant extract, which includes the step of extracting thistle plant by using an organic solution.
  • the organic solvents suitable for the present invention include but are not limited to any one or more of ethanol, acetone, ethyl acetate, methylene chloride, chloroform, petroleum ether, n-hexane and cyclohexane.
  • the preferred organic solvent is one or more of ethanol, petroleum ether, n-hexane and cyclohexane.
  • the mass percentage concentration of the organic solvent may be 20-100%, preferably 40-100%, more preferably 60-100%, most preferably 80-100%.
  • ethanol or an aqueous ethanol solution having a mass percentage concentration of 20-100%, preferably 40-100%, more preferably 60-100%, and most preferably 80-100% can be used.
  • the Thistle plants suitable for the method of the present invention may be various plants of the Thistle family known in the art, including but not limited to thistle (Cirsium japonicum), Cirsium maackii, Green thistle (Cirsium chinense), Cirsium setosum), Cirsium lineare and Cirsium tianmushanicum.
  • thistle Cirsium japonicum
  • Cirsium maackii Green thistle (Cirsium chinense)
  • Cirsium setosum Cirsium lineare
  • Cirsium tianmushanicum Cirsium tianmushanicum.
  • the present invention uses thistle plants grown in Shanghai, Zhejiang City, Jiangsuzhou and Anhui province, more preferably in Yiwu, Zhejiang Province.
  • the present invention uses the roots of thistle plants for extraction.
  • the dried thistle plants can be crushed by conventional methods and then soaked in organic solvents.
  • the volume-to-mass ratio of the organic solvent to thistle plant may be in the range of 5:1-40:1, preferably in the range of 10:1-30:1.
  • the amount of organic solvents can be adjusted appropriately.
  • the soaking time can be in the range of 4-48 hours, preferably in the range of 8-36 hours, more preferably in the range of 12-24.
  • the soaking time can be adjusted appropriately according to the type and amount of organic solvent and the amount of thistle.
  • the mixture can be appropriately stirred as needed.
  • an extract is obtained, that is, the Thistle plant extract described herein is obtained.
  • the extract is obtained by using methods such as centrifugation and/or filtration.
  • a percolation method can also be used to gradually obtain an extract while soaking and extracting.
  • the method of the present invention further includes a step of purifying the extract.
  • a step of purifying the extract for example, preparative high performance liquid phase (HPLC) can be used for purification.
  • HPLC preparative high performance liquid phase
  • column chromatography can be used for purification, such as separation and purification using column chromatography with silica gel as the filler, and the fractions with the peak time described above are collected to form the extract of the present invention alone, or combined After these fractions, the extract of the present invention is formed.
  • the present invention uses petroleum ether as a solvent to extract the roots of thistle plants.
  • the volume-to-mass ratio of petroleum ether to thistle plant is in the range of 10:1 to 30:1.
  • column chromatography separation (such as using silica gel packing) is eluted with a gradient of petroleum ether and petroleum ether/ethyl acetate.
  • the usage amount of petroleum ether can be 1-2BV; the petroleum ether/ethyl acetate gradient is 20:1, 15:1, 10:1, 5:1, and 1:1, and the amount can be respectively It is 1.5-2.5BV, 1.5-2.5BV, 1.5-2.5BV, 2.5-3.5BV and 1.5-2.5BV.
  • 0.02-0.1 BV is collected for each fraction, and these fractions are collected separately or combined as the extract of thistle plant of the present invention.
  • the present invention also includes the Thistle plant extract prepared by the method of the present invention and its medical uses, such as the various uses described above.
  • Thistle (Cirsium japonicum) purchased from Yiwu, Zhejiang province, take its roots, wash, dry naturally, and crush. Put the crushed roots into a glass column with a sieve, add 5 times the amount (L/kg) of ethanol, soak for 12 hours, maintain a uniform speed for extraction at room temperature, and constantly add fresh ethanol. The ratio of solvent volume (L) to mass (kg) of medicinal materials is 20:1. The percolation extracts were combined, concentrated under reduced pressure to a solvent-free extract and weighed to obtain root ethanol extract.
  • Thistle (Cirsium japonicum) purchased from Yiwu, Zhejiang province, take its roots, wash, dry naturally, and crush. Put the crushed roots into a round-bottom flask, add 10 times the amount (L/kg) of water, heat at 100°C to reflux for extraction, and filter to obtain an extract. It was concentrated under reduced pressure to a solvent-free extract and weighed to obtain a root water extract.
  • Thistle (Cirsium japonicum) purchased from Yiwu, Zhejiang province, take its roots, wash, dry naturally, and crush. Put the crushed roots into a glass column with a sieve, add 5 times the amount (L/kg) of petroleum ether, soak for 12 hours, maintain a uniform speed at room temperature for percolation extraction, and constantly add fresh petroleum ether. The ratio of solvent volume (L) to mass (kg) of medicinal materials is 20:1. The percolation extracts are combined, concentrated under reduced pressure to a solvent-free extract and weighed to obtain a root petroleum ether extract.
  • Thistle (Cirsium japonicum) purchased from Yiwu, Zhejiang province, take its roots, wash, dry naturally, and crush. Put the crushed roots into a round bottom flask, add 10 times the amount (L/kg) of n-hexane, stir at room temperature and low speed for 12 hours, and filter to obtain the extract; continue to add 10 times the amount (L/kg) of n-hexane, and stir at room temperature and low speed for 12 Hours, filtered to obtain the extract. The extracts were combined, concentrated under reduced pressure into a solvent-free extract and weighed to obtain root n-hexane extract.
  • Thistle (Cirsium japonicum) purchased from Yiwu, Zhejiang province, take its roots, wash, dry naturally, and crush. Put the crushed roots into a glass column with a sieve, add 5 times the amount (L/kg) of cyclohexane, soak for 12 hours, maintain a uniform speed at room temperature for percolation extraction, and constantly add fresh cyclohexane. The ratio of solvent volume (L) to mass (kg) of medicinal materials is 20:1. The percolation extracts are combined, concentrated under reduced pressure into a solvent-free extract and weighed to obtain the root cyclohexane extract.
  • Thistle (Cirsium japonicum) purchased from Yiwu, Zhejiang province, took its stems, washed, naturally dried, and crushed. Put the crushed stems into a glass column with a sieve, add 5 times the amount (L/kg) of ethanol, soak for 12 hours, maintain a uniform speed for extraction at room temperature, and constantly add fresh ethanol. The ratio of solvent volume (L) to mass (kg) of medicinal materials is 20:1. The percolation extracts were combined, concentrated under reduced pressure into a solvent-free extract and weighed to obtain the stem ethanol extract.
  • Thistle (Cirsium japonicum) purchased from Yiwu, Zhejiang province, took its stems, washed, naturally dried, and crushed. Put the crushed stems into a round bottom flask, add 10 times the amount (L/kg) of water, heat at 100°C to reflux for extraction, and filter to obtain the extract. It was concentrated under reduced pressure to a solvent-free extract and weighed to obtain a stem water extract.
  • Thistle (Cirsium japonicum) purchased from Yiwu, Zhejiang province, the leaves are taken, washed, air dried, and crushed. Put the crushed leaves into a glass column with a sieve, add 5 times the amount (L/kg) of petroleum ether, soak for 12 hours, maintain a uniform speed at room temperature for percolation extraction, and constantly add fresh petroleum ether. The ratio of solvent volume (L) to mass (kg) of medicinal materials is 20:1. The percolation extracts were combined, concentrated under reduced pressure into a solvent-free extract and weighed to obtain a leaf petroleum ether extract.
  • Thistle (Cirsium japonicum) purchased from Yiwu, Zhejiang province, the leaves are taken, washed, air dried, and crushed. Put the crushed leaves into a round bottom flask, add 10 times the amount (L/kg) of water, heat at 100°C to reflux for extraction, and filter to obtain the extract. The extract was concentrated under reduced pressure into a solvent-free extract and weighed to obtain leaf water extract.
  • Combination and concentration Combine fractions 1-22, 23-27, 28-33, 34-83, 84-158, 159-177, 178-248 and 249, respectively, and concentrate under reduced pressure to obtain components C01-1, C01 -23, C01-28, C01-34, C01-84, C01-159, C01-178 and C01-249.
  • Solution preparation The extracts or subcomponents obtained in Example 1 and Example 2 were all dissolved in DMSO to prepare a mother liquor of 25.6 mg/mL. On the day of the experiment, the mother liquor was diluted 50 times with 7H9+10% ADC liquid medium (7H9 medium was purchased from: Beijing Solebold Technology Co., Ltd.; ADC enrichment liquid was purchased from: Shanghai Guandao Biological Engineering Co., Ltd.) liquid.
  • Inoculation plate preparation Add 100 ⁇ L of 7H9+10% ADC liquid medium to each well of a 96-well plate, add 100 ⁇ L of compound working solution in column 1, and pipette 100 ⁇ L of liquid from column 1 to column 2. After pipetting evenly, Pipette 100 ⁇ L to the third column, and perform 2-fold serial dilutions sequentially, 10 times in total, and remove 100 ⁇ L from each well in the 11th column to obtain an inoculation plate.
  • Mycobacterium tuberculosis was obtained from Beijing Tuberculosis and Thoracic Tumor Research Institute. Use a disposable sterile inoculating loop to scrape the colonies grown on the neutral Roche medium for 3 to 4 weeks, and transfer them to a sterile glass bead and 100 ⁇ L 5% Tween-80. Use a vortex shaker to fully shake and homogenize the bacteria for 30 seconds, and then let stand for 15 minutes. Sterile normal saline was added until the turbidity of the obtained bacterial suspension reached 1 McFarland (McFarland turbidity), and it was diluted 20 times with 7H9+10% ADC liquid medium to obtain a bacterial inoculum.
  • Bacterial inoculation and culture Add the bacterial inoculum solution to each well of the inoculation plate containing 100 ⁇ L of compounds of different concentrations. After culturing at 37°C for 9 days, add 20 ⁇ L of Alamar Blue to each well and incubate for another 24 hours.
  • the minimum inhibitory concentration (MIC) is the minimum drug concentration that can completely inhibit the discoloration of Alamar Blue by naked eyes, or by fluorescence detection (Ex/Em, 530nm/590nm), the inhibition rate is >90 The minimum drug concentration produced by% reduced Alamar Blue.
  • Inhibition rate % 100%-(detection hole fluorescence value-background fluorescence value)/(growth control well fluorescence value-background fluorescence value) ⁇ 100%.
  • the results of the anti-tuberculosis drug susceptibility test are shown in Table 1.
  • the activity of the root extract of thistle plant is much greater than that of the stem and leaf extract, and the stem and leaf extract has almost no anti-tuberculosis activity (MIC>128 ⁇ g/mL).
  • the activity of the root organic phase extract of thistle plant is much greater than that of the root water phase extract.
  • some among the sub-components obtained by the extraction of root petroleum ether by silica gel column chromatography, some have strong anti-tuberculosis activity, and the C01-34, C01-84 and C01-159 components have better anti-tuberculosis activity.
  • the MICs of the listed positive drugs streptomycin and ethambutol are >32 ⁇ g/mL and >160 ⁇ g/mL respectively, that is, the extract of thistle plant of the present invention has significant advantages.
  • Root ethanol extract 64 32 32 Root water extract >128 >128 >128 Root Petroleum Ether Extract 64 16 16 Root hexane extract 64 16 16 Root cyclohexane extract 64 16 16 16 Stem ethanol extract >128 >128 >128 Stem water extract >128 >128 >128 Leaf petroleum ether extract >128 >128 >128 Leaf water extract >128 >128 C01-1 64 64 32 C01-23 128 128 >128
  • Example 4 High performance liquid chromatography (HPLC) pattern determination
  • root ethanol extract root petroleum ether extract, C01-34, C01-84 and C01-159 components (obtained in Example 1) with better anti-tuberculosis effect, HPLC profile determination was performed.
  • the inventor believes that the peak time of the characteristic ultraviolet absorption peak of the extract with anti-tuberculosis active ingredients at a wavelength of 254nm is concentrated in 30-50 minutes, preferably in 30-45 Minutes, more preferably concentrated in 35-40 minutes.
  • the peak time of the characteristic ultraviolet absorption peak of the extract with anti-tuberculosis active ingredients at a wavelength of 254nm is 35.8 ⁇ 0.3 minutes, 36.6 ⁇ 0.3 minutes, 37.7 ⁇ 0.3 minutes, preferably 34.2 ⁇ 0.3 minutes, 35.8 ⁇ 0.3 minutes, 36.6 ⁇ 0.3 minutes, 37.0 ⁇ 0.3 minutes, 37.7 ⁇ 0.3 minutes, 42.0 ⁇ 0.3 minutes, more preferably 31.5 ⁇ 0.3 minutes, 32.5 ⁇ 0.3 minutes, 34.2 ⁇ 0.3 minutes, 35.8 ⁇ 0.3 minutes, 36.6 ⁇ 0.3 minutes, 37.0 ⁇ 0.3 minutes, 37.7 ⁇ 0.3 minutes, 38.9 ⁇ 0.3 minutes, 42.0 ⁇ 0.3 minutes, 43.8 ⁇ 0.3 minutes.

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Abstract

公开了蓟属植物的有机提取物，使用乙醇、丙酮、乙酸乙酯、二氯甲烷、三氯甲烷、石油醚、正己烷、环己烷中的一种或多种提取获得，为蓟属植物根部位的提取物，该提取物具有出峰时间在35-40分钟之间的特征紫外吸收峰，其中该出峰时间以254nm波长进行高效液相色谱法检测得到。还涉及所述提取物在制备抗结核分枝杆菌或结核病治疗药物中的应用。

Description

蓟属植物的有机提取物及其应用与组合物 技术领域
本发明涉及制药领域，尤其涉及一种蓟属植物的有机提取物及其应用与组合物。
背景技术
结核分枝杆菌可感染肺部导致肺结核，亦可感染肺外器官如肠、腹膜、肾、膀胱、输尿管、胸膜、骨、关节、脑、脑膜、生殖系统等。肺外结核多见于免疫力低下者。肺结核包括原发型肺结核、血行播散型肺结核和继发型肺结核。原发型肺结核又称初染结核，多见于儿童，经典病变包括肺部原发灶、引流淋巴管和肺门或纵膈淋巴结的结核性炎症，三者联合称为原发综合征。结核病患者常见低热、盗汗、乏力、体重减轻，病灶急剧进展时可出现高热，其余症状因感染部位不同而各异：肺结核患者常见咳嗽、咳痰、咯血、胸痛、气急，亦可出现过敏反应和无反应性结核。肠结核患者多为经口感染，即开放性肺结核或喉结核患者吞下含有结核分枝杆菌的痰液引起。多发生于回盲部，导致腹痛、腹泻、便秘、腹部肿块，可并发肠梗阻、瘘管、脓肿、急性肠穿孔等。结核性腹膜炎常见腹痛、腹水、腹壁柔韧感或腹部肿块以及腹泻。
目前，结核病的一线治疗药物包括异烟肼、利福平、比嗪酰胺、链霉素和乙胺丁醇等。但这些药物普遍存在耐药性以及较为严重的肝损。因此，本领域迫切需要寻找到一种能够抗结核分枝杆菌的天然药物，以克服现有药物的局限性。
发明内容
为了解决上述技术问题，本发明一方面提供了一种蓟属植物的提取物，所述蓟属植物的提取物为有机相提取物；优选地，所述有机提取物是使用乙醇、丙酮、乙酸乙酯、二氯甲烷、三氯甲烷、石油醚、正己烷和环己烷中的一种或多种进行提取获得的有机提取物；优选地，所述蓟属植物的提取物为蓟属植物根部位的提取物。
在一个或多个实施方案中，所述蓟属植物选自蓟(Cirsium japonicum)、野蓟(Cirsium maackii)、绿蓟(Cirsium chinense)、刺儿菜(Cirsium setosum)、线叶蓟(Cirsium lineare)和杭蓟(Cirsium tianmushanicum)。
本发明另一方面还提供一种蓟属植物提取物，该提取物的特征是，具有出峰时间在35-40分钟之间的特征紫外吸收峰，其中，该出峰时间是以254nm波长进行高效液相色谱法检测得到。
在一个或多个实施方案中，该提取物具有出峰时间分别为35.8±0.3分钟、36.6±0.3分钟和37.7±0.3分钟的特征紫外吸收峰；更优选地，该提取物具有出峰时间分别为35.8±0.2分钟、36.6±0.2分钟和37.7±0.2分钟的特征紫外吸收峰；更优选地，该提取物具有出峰时间分别为35.8±0.1分钟、36.6±0.1分钟和37.7±0.1分钟的特征紫外吸收峰。
在一个或多个实施方案中，该提取物还具有出峰时间为37.0±0.3分钟、优选为37.0±0.2分钟、更优选为37.0±0.1分钟的特征紫外吸收峰，出峰时间为38.9±0.3分钟、优选为38.9±0.2分钟、更优选为38.9±0.1分钟的特征紫外吸收峰中的一个或多个特征紫外吸收峰；
在一个或多个实施方案中，该提取物还具有出峰时间在30-35分钟之间的特征紫外吸收峰，和/或出峰时间在40-45分钟之间的特征紫外吸收峰；优选地，该提取物具有出峰时间为34.2±0.3分钟、优选为34.2±0.2分钟、更优选为34.2±0.1分钟的特征紫外吸收峰，出峰时间为32.5±0.3分钟、优选为32.5±0.2分钟、更优选为32.5±0.1分钟的特征紫外吸收峰，出峰时间为31.5±0.3分钟、优选为31.5±0.2分钟、更优选为31.5±0.1分钟的特征紫外吸收峰，出峰时间为42.0±0.3分钟、优选为42.0±0.2分钟、更优选为42.0±0.1分钟的特征紫外吸收峰，以及出峰时间为43.8±0.3分钟、优选为43.8±0.2分钟、更优选为43.8±0.1分钟的特征紫外吸收峰中的一个或多个特征紫外吸收峰；
其中，所述高效液相色谱法的检测条件为：
柱温：35℃，
检测器：紫外检测器，
检测波长：254nm，
色谱柱：phenomenex luna 4.6×250mm，5μm，
进样量：1-20μL，
流动相：0.1％冰醋酸水溶液、乙腈，
流动相流速：1mL/分钟，
梯度洗脱条件：
时间/分钟 乙腈/体积百分比 0.1％冰醋酸水溶液/体积百分比
0 12 88
8 40 60
18 70 30
55 100 0
。
在一个或多个实施方案中，所述提取物具有出峰时间如下所示的特征紫外吸收峰：35.8±0.1分钟、36.6±0.1分钟和37.7±0.1分钟，优选34.2±0.1分钟、35.8±0.1分钟、36.6±0.1分钟、37.0±0.1分钟、37.7±0.1分钟和42.0±0.1分钟，更优选31.5±0.1分钟、32.5±0.1分钟、34.2±0.1分钟、35.8±0.1分钟、36.6±0.1分钟、37.0±0.1分钟、37.7±0.1分钟、38.9±0.1分钟、42.0±0.1分钟和43.8±0.1分钟；或者有出峰时间如下所示的特征紫外吸收峰：35.8±0.2分钟、36.6±0.2分钟和37.7±0.2分钟，优选34.2±0.2分钟、35.8±0.2分钟、36.6±0.2分钟、37.0±0.2分钟、37.7±0.2分钟和42.0±0.2分钟，更优选31.5±0.2分钟、32.5±0.2分钟、34.2±0.2分钟、35.8±0.2分钟、36.6±0.2分钟、37.0±0.2分钟、37.7±0.2分钟、38.9±0.2分钟、42.0±0.2分钟和43.8±0.2分钟；或者具有出峰时间如下所示的特征紫外吸收峰：35.8±0.3分钟、36.6±0.3分钟和37.7±0.3分钟，优选34.2±0.3分钟、35.8±0.3分钟、36.6±0.3分钟、37.0±0.3分钟、37.7±0.3分钟和42.0±0.3分钟，更优选31.5±0.3分钟、32.5±0.3分钟、34.2±0.3分钟、35.8±0.3分钟、36.6±0.3分钟、37.0±0.3分钟、37.7±0.3分钟、38.9±0.3分钟、42.0±0.3分钟和43.8±0.3分钟。
在一个或多个实施方案中，所述蓟属植物提取物具有出峰时间如下所示的特征紫外吸收峰：35.6±0.1分钟、36.6±0.1分钟、37.8±0.1分钟、38.9±0.1分钟、42.0±0.1分钟和43.7±0.1分钟，任选在38.2±0.1分钟和46.8±0.1分钟还具有特征紫外吸收峰。
在一个或多个实施方案中，所述蓟属植物提取物具有出峰时间如下所示的特征紫外吸收峰：34.2±0.1分钟、35.9±0.1分钟、36.6±0.1分钟、36.9±0.1分钟和37.6±0.1分钟，任选在32.9±0.1分钟、33.2±0.1分钟、34.5±0.1分钟和38.1±0.1分 钟还具有特征吸收峰。
在一个或多个实施方案中，所述蓟属植物提取物具有出峰时间如下所示的紫外特征紫外吸收峰：31.5±0.1分钟、32.5±0.1分钟、34.3±0.1分钟、35.8±0.1分钟、36.6±0.1分钟、37.0±0.1分钟和37.6±0.1分钟，任选在29.9±0.1分钟、30.3±0.1分钟、31.0±0.1分钟、32.0±0.1分钟、33.2±0.1分钟、33.9±0.1分钟、35.2±0.1分钟、35.7±0.1分钟、36.1±0.1分钟、40.0±0.1分钟、40.7±0.1分钟以及41.2±0.1分钟还具有紫外特征吸收峰。
在一个或多个实施方案中，所述蓟属植物选自蓟(Cirsium japonicum)、野蓟(Cirsium maackii)、绿蓟(Cirsium chinense)、刺儿菜(Cirsium setosum)、线叶蓟(Cirsium lineare)和杭蓟(Cirsium tianmushanicum)。
本发明还提供一种药物组合物，它包含治疗有效量的本文任一实施方案所述的提取物和药学上可接受的载体；优选地，所述药物组合物由治疗有效量的本文任一实施方案所述的提取物和药学上可接受的载体组成。
本发明还提供本发明任一实施方案所述的提取物在制备抗结核分枝杆菌药物或结核病治疗药物中的应用。
在一个或多个实施方案中，所述结核分枝杆菌选自人结核分枝杆菌和牛型分枝杆菌；更优选，所述结核分枝杆菌选自耐药型人结核分枝杆菌。
在一个或多个实施方案中，所述结核病由结核分枝杆菌引起；更优选地，所述结核病选自肺结核或者肺外结核；还要优选地，所述结核病为肺结核、肝结核、胃结核或肠结核。
本发明还提供一种治疗结核病的方法，所述方法包括对患者施用治疗有效量的本发明任一实施方案所述的提取物，或者对患者施用本发明任一实施方案所述的药物组合物。
在一个或多个实施方案中，所述结核病是由结核分枝杆菌引起的；更优选，所述结核分枝杆菌选自人结核分枝杆菌和牛型分枝杆菌；还要优选，所述结核分枝杆菌选自耐药型人结核分枝杆菌。
在一个或多个实施方案中，所述结核病选自肺结核或者肺外结核；更优选地，所述结核病为肺结核、肝结核、胃结核或肠结核。
本发明还提供一种制备蓟属植物提取物的方法，所述方法包括采用有机溶液提 取蓟属植物的步骤；优选地，所述有机溶液选自乙醇、丙酮、乙酸乙酯、二氯甲烷、三氯甲烷、石油醚、正己烷和环己烷中的一种或多种。
在一个或多个实施方案中，使用蓟属植物根部位进行提取。
在一个或多个实施方案中，所述蓟属植物选自蓟(Cirsium japonicum)、野蓟(Cirsium maackii)、绿蓟(Cirsium chinense)、刺儿菜(Cirsium setosum)、线叶蓟(Cirsium lineare)和杭蓟(Cirsium tianmushanicum)。
附图说明
图1显示实施例4中根乙醇提取物的HPLC图谱。
图2显示实施例4中根石油醚提取物的HPLC图谱。
图3显示实施例4中C01-34组分的HPLC图谱。
图4显示实施例4中C01-84组分的HPLC图谱。
图5显示实施例4中C01-159组分的HPLC图谱。
具体实施方案
在本说明书中，如果没有特别的说明，所涉及的各组分或其优选组分可以相互组合形成新的技术方案。
在本说明书中，如果没有特别的说明，本文所提到的所有实施方案以及优选实施方案可以相互组合形成新的技术方案。
在本说明书中，如果没有特别的说明，本文所提到的所有技术特征以及优选特征可以相互组合形成新的技术方案。
在本说明书中，如果没有相反的说明，组合物中各组分的含量之和为100％。
在本说明书中，如果没有相反的说明，组合物中各组分的份数之和可以为100重量份。
在本说明书中，除非有其他说明，数值范围“a-b”表示a到b之间的任意实数组合的缩略表示，其中a和b都是实数。例如数值范围“0-5”表示本文中已经全部列出了“0-5”之间的全部实数，“0-5”只是这些数值组合的缩略表示。
在本说明书中，除非有其他说明，整数数值范围“a-b”表示a到b之间的任意整数组合的缩略表示，其中a和b都是整数。例如整数数值范围“1-N”表示1、 2……N，其中N是整数。
在本说明书中，除非有其他说明，“其组合”表示所述各元件的多组分混合物，例如两种、三种、四种以及直到最大可能的多组分混合物。
如果没有特别指出，本说明书所用的术语“一种”指“至少一种”。
如果没有特别指出，本说明书所述的百分数(包括重量百分数)的基准都是所述组合物的总重量。
本文所公开的“范围”以下限和上限的形式。可以分别为一个或多个下限，和一个或多个上限。给定范围是通过选定一个下限和一个上限进行限定的。选定的下限和上限限定了特别范围的边界。所有可以这种方式进行限定的范围是包含和可组合的，即任何下限可以与任何上限组合形成一个范围。例如，针对特定参数列出了60－120和80－110的范围，理解为60－110和80－120的范围也是预料到的。此外，如果列出的最小范围值1和2，和如果列出了最大范围值3，4和5，则下面的范围可全部预料到：1－3、1－4、1－5、2－3、2－4、和2－5。
具体而言，本发明提供了一种蓟属植物的有机提取物。蓟属植物为菊目菊科植物，本文所述的蓟属植物包括本领域周知的蓟属植物，包括但不限于蓟(Cirsium japonicum)、野蓟(Cirsium maackii)、绿蓟(Cirsium chinense)、刺儿菜(Cirsium setosum)、线叶蓟(Cirsium lineare)和杭蓟(Cirsium tianmushanicum)。在本发明优选的实施方案中，蓟属植物为生长于上海市、浙江省、江苏省和安徽省、更优选为生长于浙江省义乌的蓟属植物。用于提取的蓟属植物部位优选可以是根部。
本文中，有机提取物指采用有机溶剂进行提取获得的提取物，通常位于有机相内。适用于本发明的有机溶剂包括但不限于乙醇、丙酮、乙酸乙酯、二氯甲烷、三氯甲烷、石油醚、正己烷和环己烷中的任意一种或多种。优选的有机溶剂为乙醇、石油醚、正己烷和环己烷中的一种或多种。当使用水溶性有机溶剂时，该有机溶剂的质量百分比浓度可以是20-100％，优选40-100％，更优选60-100％，最优选80-100％。例如，可使用质量百分比浓度为20-100％、优选40-100％、更优选60-100％、最优选80-100％的乙醇或乙醇水溶液进行提取。
在本发明优选的实施方案中，所述蓟属植物提取物是蓟属植物根部的乙醇提取物、石油醚提取物、正己烷提取物或环己烷提取物。
在本发明一些实施方案中，本发明提供一类蓟属植物提取物，该提取物的特征是，具有出峰时间在35-40分钟之间的特征紫外吸收峰，其中，该出峰时间是以254nm波长进行高效液相色谱法(HPLC)检测得到。优选地，该提取物具有出峰时间35.8±0.3分钟、36.6±0.3分钟和37.7±0.3分钟的特征紫外吸收峰；更优选地，该提取物具有出峰时间分别为35.8±0.2分钟、36.6±0.2分钟和37.7±0.2分钟的特征紫外吸收峰；更优选地，该提取物具有出峰时间分别为35.8±0.1分钟、36.6±0.1分钟和37.7±0.1分钟的特征紫外吸收峰。任选地，该提取物还具有出峰时间为37.0±0.3分钟、优选为37.0±0.2分钟、更优选为37.0±0.1分钟的特征紫外吸收峰，出峰时间为38.9±0.3分钟、优选为38.9±0.2分钟、更优选为38.9±0.1分钟的特征紫外吸收峰中的一个或多个特征紫外吸收峰。任选地，该提取物还具有出峰时间在30-35分钟之间的特征紫外吸收峰，和/或出峰时间在40-45分钟之间的特征紫外吸收峰。优选地，该提取物具有出峰时间为34.2±0.3分钟、优选为34.2±0.2分钟、更优选为34.2±0.1分钟的特征紫外吸收峰，出峰时间为32.5±0.3分钟、优选为32.5±0.2分钟、更优选为32.5±0.1分钟的特征紫外吸收峰，出峰时间为31.5±0.3分钟、优选为31.5±0.2分钟、更优选为31.5±0.1分钟的特征紫外吸收峰，出峰时间为42.0±0.3分钟、优选为42.0±0.2分钟、更优选为42.0±0.1分钟的特征紫外吸收峰，以及出峰时间为43.8±0.3分钟、优选为43.8±0.2分钟、更优选为43.8±0.1分钟的特征紫外吸收峰中的一个或多个特征紫外吸收峰。
在一些优选的实施方案中，本发明的蓟属植物提取物具有出峰时间如下所示的特征紫外吸收峰：35.8±0.1分钟、36.6±0.1分钟和37.7±0.1分钟，优选34.2±0.1分钟、35.8±0.1分钟、36.6±0.1分钟、37.0±0.1分钟、37.7±0.1分钟和42.0±0.1分钟，更优选31.5±0.1分钟、32.5±0.1分钟、34.2±0.1分钟、35.8±0.1分钟、36.6±0.1分钟、37.0±0.1分钟、37.7±0.1分钟、38.9±0.1分钟、42.0±0.1分钟和43.8±0.1分钟；或者，具有出峰时间如下所示的特征紫外吸收峰：35.8±0.2分钟、36.6±0.2分钟和37.7±0.2分钟，优选34.2±0.2分钟、35.8±0.2分钟、36.6±0.2分钟、37.0±0.2分钟、37.7±0.2分钟和42.0±0.2分钟，更优选31.5±0.2分钟、32.5±0.2分钟、34.2±0.2分钟、35.8±0.2分钟、36.6±0.2分钟、37.0±0.2分钟、37.7±0.2分钟、38.9±0.2分钟、42.0±0.2分钟和43.8±0.2分钟；或者，具有出峰时间如下所示的特征紫外吸收峰：35.8±0.3分钟、36.6±0.3分钟和37.7±0.3分钟，优选34.2±0.3分钟、35.8±0.3分钟、 36.6±0.3分钟、37.0±0.3分钟、37.7±0.3分钟和42.0±0.3分钟，更优选31.5±0.3分钟、32.5±0.3分钟、34.2±0.3分钟、35.8±0.3分钟、36.6±0.3分钟、37.0±0.3分钟、37.7±0.3分钟、38.9±0.3分钟、42.0±0.3分钟和43.8±0.3分钟。
应理解的是，本发明的蓟属植物提取物还可以是经纯化的提取物，如可以是经制备型高效液相色谱法(HPLC)纯化的提取物，或者采用适宜方法(如使用填料为硅胶的柱色谱法进行分离纯化)收集的具体流份。
如本文所示，经纯化的提取物，其不同流份都有可能具有本文所述的生物学活性(抗结核分枝杆菌的活性)。因此，本发明的蓟属植物提取物可以是不同有机溶剂提取物的任意比例的混合物，或者是不同有机溶剂提取物经纯化获得的不同流份的任意比例的混合物，或者是同一有机溶剂提取物经纯化获得的不同流份的任意比例的混合物，也可以是经纯化获得的流份与未经纯化的有机溶剂提取物的任意比例的混合物。
在一些实施方案中，本发明的蓟属植物提取物具有出峰时间如下所示的特征紫外吸收峰：35.6±0.1分钟、36.6±0.1分钟、37.8±0.1分钟、38.9±0.1分钟、42.0±0.1分钟和43.7±0.1分钟，任选在38.2±0.1分钟和46.8±0.1分钟还具有特征紫外吸收峰。
在一些实施方案中，本发明的蓟属植物提取物具有出峰时间如下所示的特征紫外吸收峰：34.2±0.1分钟、35.9±0.1分钟、36.6±0.1分钟、36.9±0.1分钟和37.6±0.1分钟，任选在32.9±0.1分钟、33.2±0.1分钟、34.5±0.1分钟和38.1±0.1分钟还具有特征吸收峰。
在一些实施方案中，本发明的蓟属植物提取物具有出峰时间如下所示的紫外特征紫外吸收峰：31.5±0.1分钟、32.5±0.1分钟、34.3±0.1分钟、35.8±0.1分钟、36.6±0.1分钟、37.0±0.1分钟和37.6±0.1分钟，任选在29.9±0.1分钟、30.3±0.1分钟、31.0±0.1分钟、32.0±0.1分钟、33.2±0.1分钟、33.9±0.1分钟、35.2±0.1分钟、35.7±0.1分钟、36.1±0.1分钟、40.0±0.1分钟、40.7±0.1分钟以及41.2±0.1分钟还具有紫外特征吸收峰。
在优选的实施方案中，这类提取物是经制备型高效液相色谱法(HPLC)纯化得到的提取物。
本文中，除非另有说明，否则本文所述的特征紫外吸收峰是采用以下所述的高 效液相色谱法(HPLC)检测条件检测得到：
柱温：35℃，
检测器：紫外检测器，
检测波长：254nm，
色谱柱：phenomenex luna 4.6×250mm，5μm，
进样量：1-20μL，
流动相：0.1％冰醋酸水溶液、乙腈，
流动相流速：1mL/分钟，
梯度洗脱条件：
时间/分钟 乙腈/体积百分比 0.1％冰醋酸水溶液/体积百分比
0 12 88
8 40 60
18 70 30
55 100 0
。
应理解的是，虽然本申请实施例具体披露了纯化蓟属植物提取物的方法，并给出了一些提取物相应的出峰时间，但采用其它纯化技术纯化蓟属植物有机溶剂提取物、且以本文所述检测方法检测所获得的出峰时间在本文所述范围内的提取物也应落入本文所述的范围。
本发明的提取物可用于抗结核分枝杆菌，用以治疗由结核分枝杆菌引起的结核病。本文中，结核分枝杆菌优选选自人结核分枝杆菌和牛型分枝杆菌，更优选为耐药型人结核分枝杆菌。本文中，结核病选自肺结核或者肺外结核。优选地，结核病为肺结核、肝结核、胃结核或肠结核。
本发明提供了一种药物组合物，它包含治疗有效量的本文所述的蓟属植物提取物和药学上可接受的载体；优选地，所述药物组合物由治疗有效量的所述蓟属植物的提取物和药学上可接受的载体组成。
在本说明书中，除非另有说明，术语“包含……”指的是所述药物组合物中还可以含有任何其它组分，这些组分可以以任何含量存在，只要以该含量存在的该组分是人体可接受的，并对于本发明药物组合物中活性成分(所述蓟属植物的提取物)的活性没有负面影响即可。
在本说明书中，除非另有说明，术语“药学上可接受的载体”应当与本发明药 物组合物中的活性成分(所述蓟属植物的提取物)相容，即能与其共混而不会在通常情况下大幅度降低药物的疗效。可作为“药学上可接受的载体”包括但不限于：糖类，如乳糖、葡萄糖、蔗糖、海藻糖；淀粉，如玉米淀粉、土豆淀粉；纤维素或其衍生物，如羧甲基纤维素钠、乙基纤维素、甲基纤维素；西黄蓍胶粉末；明胶；滑石粉；凡士林；固体润滑剂，如硬脂酸、硬脂酸镁；硫酸钙；植物油，如花生油、棉籽油、芝麻油、橄榄油、玉米油、可可油；醇类，如乙醇、丙二醇、甘油、山梨糖醇、甘露糖醇、聚乙二醇；氨基己酸，海藻酸；乳化剂，如吐温；润湿剂，如月桂基硫酸钠；着色剂；调味剂；压片剂；抗氧化剂；药用防腐剂；无热原水；等渗盐溶液；缓冲液等，或其组合。
术语“治疗有效量”表示所述药物组合物中的活性成分(所述蓟属植物的提取物)可以起到治疗疾病(例如结核病)的作用。
优选地，所述药物组合物的给药方式为经消化道给药、经呼吸道给药、经皮给药、经粘膜给药、注射给药。所述药物组合物的给药对象为灵长类动物，更优选为人类。所述药物组合物的剂型为经消化道给药剂型、经呼吸道给药剂型、经皮给药剂型、经粘膜给药剂型、注射给药剂型；更优选地，所述药物组合物的剂型选自口服液、片剂、胶囊剂、散剂、颗粒剂、丸剂、滴眼剂、滴鼻剂、栓剂、滴丸剂、搽剂、软膏剂、贴剂、糊剂、喷雾剂、气雾剂、粉雾剂、吸入用溶液(混悬液)、注射剂、粉针剂、水针剂、大输液剂型等。
本申请还提供本文所述蓟属植物提取物在制备抗结核分枝杆菌药物或治疗结核病的药物中的应用，或者提供所述蓟属植物的提取物在治疗结核病中的应用，或者提供用于治疗结核病的蓟属植物的提取物，或者提供所述蓟属植物的提取物治疗结核病的方法。优选地，所述结核病由结核分枝杆菌引起。更优选，所述结核分枝杆菌选自人结核分枝杆菌和牛型分枝杆菌；还要优选，所述结核分枝杆菌选自耐药型人结核分枝杆菌。优选地，所述结核病选自肺结核或者肺外结核；更优选地，所述结核病为肺结核、肝结核、胃结核或肠结核。
在本申请的一个实施方案中，所述应用或方法的对象是灵长类动物(例如猴或人)，优选是人。
本发明还提供了一种制备蓟属植物提取物的方法，包括采用有机溶液提取蓟属植物的步骤。如前文所述，适用于本发明的有机溶剂包括但不限于乙醇、丙酮、乙 酸乙酯、二氯甲烷、三氯甲烷、石油醚、正己烷和环己烷中的任意一种或多种。优选的有机溶剂为乙醇、石油醚、正己烷和环己烷中的一种或多种。当使用水溶性有机溶剂时，该有机溶剂的质量百分比浓度可以是20-100％，优选40-100％，更优选60-100％，最优选80-100％。例如，可使用质量百分比浓度为20-100％、优选40-100％、更优选60-100％、最优选80-100％的乙醇或乙醇水溶液。
适用于本发明方法的蓟属植物可以是本领域周知的各种蓟属植物，包括但不限于蓟(Cirsium japonicum)、野蓟(Cirsium maackii)、绿蓟(Cirsium chinense)、刺儿菜(Cirsium setosum)、线叶蓟(Cirsium lineare)和杭蓟(Cirsium tianmushanicum)。在本发明优选的实施方案中，本发明使用生长于上海市、浙江省、江苏省和安徽省、更优选为生长于浙江省义乌的蓟属植物。在优选的实施方案中，本发明使用蓟属植物的根部进行提取。
提取之前，可采用常规的方法将干的蓟属植物粉碎，然后用有机溶剂浸泡。通常，有机溶剂与蓟属植物的体积质量比可在5:1-40:1的范围内，优选在10:1-30:1的范围内。根据有机溶剂种类的不同，可适当调整有机溶剂的用量。浸泡时间可在4-48小时的范围内，优选在8-36小时的范围内、更优选在12-24的范围内。可根据有机溶剂的种类和用量以及蓟属植物的量适当调整浸泡时间。浸泡时，根据需要可适当搅拌该混合物。浸泡结束后，获得浸提液，即获得本文所述的蓟属植物提取物。通过采用例如离心和/或过滤等方式获得浸提液。当然，本领域技术人员熟知的，也可以采用渗漉的方法，在浸泡提取的同时即逐步获得浸提液。
在一些实施方案中，本发明的方法还包括对浸提液进行纯化的步骤。例如，可采用制备型高效液相(HPLC)进行纯化。
在一些实施方案中，可采用柱层析进行纯化，如使用填料为硅胶的柱色谱法进行分离纯化，收集具有前文所述出峰时间的流份，单独形成本发明的提取物，或合并多个流份后形成本发明的提取物。
在优选的实施方案中，本发明使用石油醚作为溶剂提取蓟属植物的根。优选地，石油醚与蓟属植物的体积质量比在10：1到30：1的范围内。获得蓟属植物的石油醚提取物后，对其进行柱层析分离。在特别优选的实施方案中，柱层析分离(如采用硅胶填料)时，以石油醚和石油醚/乙酸乙酯梯度洗脱。其中，洗脱时，石油醚的使用量可以为1-2BV；石油醚/乙酸乙酯梯度依次为20：1、15：1、10：1、5：1 和1：1，其用量可分别为1.5-2.5BV、1.5-2.5BV、1.5-2.5BV、2.5-3.5BV和1.5-2.5BV。每个流份收集0.02-0.1BV，分别或合并这些流份，作为本发明蓟属植物的提取物。
本发明还包括采用本发明的方法制备得到的蓟属植物提取物及其医药用途，如前文所述的各种用途。
下面将结合实施例进一步详细地描述本发明。然而应当理解，列举这些实施例只是为了起说明作用，而并不是用来限制本发明的范围。
实施例1：蓟属植物提取物的制备
1、根乙醇提取物
购自浙江义乌的蓟(Cirsium japonicum)，取其根部，洗净、自然晾干、粉碎。将粉碎的根装入带筛玻璃柱，加入5倍量(L/kg)乙醇，浸泡12小时后，保持均匀的速度室温渗漉提取，并不断补充新鲜乙醇。总共使用的溶剂体积(L)与药材的质量(kg)比为20：1。合并渗漉提取液，减压浓缩成无溶剂的浸膏并称重，得到根乙醇提取物。
2、根水提取物
购自浙江义乌的蓟(Cirsium japonicum)，取其根部，洗净、自然晾干、粉碎。将粉碎的根装入圆底烧瓶，加入10倍量(L/kg)水，100℃加热回流提取，过滤得到提取液。减压浓缩成无溶剂的浸膏并称重，得到根水提取物。
3、根石油醚提取物
购自浙江义乌的蓟(Cirsium japonicum)，取其根部，洗净、自然晾干、粉碎。将粉碎的根装入带筛玻璃柱，加入5倍量(L/kg)石油醚，浸泡12小时后，保持均匀的速度室温渗漉提取，并不断补充新鲜石油醚。总共使用的溶剂体积(L)与药材的质量(kg)比为20：1。合并渗漉提取液，减压浓缩成无溶剂的浸膏并称重，得到根石油醚提取物。
4、根正己烷提取物
购自浙江义乌的蓟(Cirsium japonicum)，取其根部，洗净、自然晾干、粉碎。将粉碎的根装入圆底烧瓶，加入10倍量(L/kg)正己烷，常温低速搅拌12小时，过滤得到提取液；继续加入10倍量(L/kg)正己烷，常温低速搅拌12小时，过滤得到提取液。合并提取液，减压浓缩成无溶剂的浸膏并称重，得到根正己烷提取物。
5、根环己烷提取物
购自浙江义乌的蓟(Cirsium japonicum)，取其根部，洗净、自然晾干、粉碎。将粉碎的根装入带筛玻璃柱，加入5倍量(L/kg)环己烷，浸泡12小时后，保持均匀的速度室温渗漉提取，并不断补充新鲜环己烷。总共使用的溶剂体积(L)与药材的质量(kg)比为20：1。合并渗漉提取液，减压浓缩成无溶剂的浸膏并称重，得到根环己烷提取物。
6、茎乙醇提取物
购自浙江义乌的蓟(Cirsium japonicum)，取其茎部，洗净、自然晾干、粉碎。将粉碎的茎装入带筛玻璃柱，加入5倍量(L/kg)乙醇，浸泡12小时后，保持均匀的速度室温渗漉提取，并不断补充新鲜乙醇。总共使用的溶剂体积(L)与药材的质量(kg)比为20：1。合并渗漉提取液，减压浓缩成无溶剂的浸膏并称重，得到茎乙醇提取物。
7、茎水提取物
购自浙江义乌的蓟(Cirsium japonicum)，取其茎部，洗净、自然晾干、粉碎。将粉碎的茎装入圆底烧瓶，加入10倍量(L/kg)水，100℃加热回流提取，过滤得到提取液。减压浓缩成无溶剂的浸膏并称重，得到茎水提取物。
8、叶石油醚提取物
购自浙江义乌的蓟(Cirsium japonicum)，取其叶子，洗净、自然晾干、粉碎。将粉碎的叶子装入带筛玻璃柱，加入5倍量(L/kg)石油醚，浸泡12小时后，保持均匀的速度室温渗漉提取，并不断补充新鲜石油醚。总共使用的溶剂体积(L)与药材的质量(kg)比为20：1。合并渗漉提取液，减压浓缩成无溶剂的浸膏并称 重，得到叶石油醚提取物。
9、叶水提取物
购自浙江义乌的蓟(Cirsium japonicum)，取其叶子，洗净、自然晾干、粉碎。将粉碎的叶子装入圆底烧瓶，加入10倍量(L/kg)水，100℃加热回流提取，过滤得到提取液。减压浓缩成无溶剂的浸膏并称重，得到叶水提取物。
实施例2：根石油醚提取物子组分的制备
色谱分离：实施例1获得的根石油醚提取物用硅胶(200-300目)柱色谱进行分离，以石油醚(1.2BV)和石油醚/乙酸乙酯梯度(20:1(2BV)、15:1(2BV)、10：1(2BV)、5:1(3BV)、1:1(2BV))洗脱，每个流份收集0.049BV，共得到249个流份。
合并、浓缩：分别合并1～22、23～27、28～33、34～83、84～158、159～177、178～248和249流份，减压浓缩，得到组分C01-1、C01-23、C01-28、C01-34、C01-84、C01-159、C01-178和C01-249。
实施例3：抗结核活性测试
溶液的配制：实施例1与实施例2中获得的提取物或子组分均溶于DMSO配制成25.6mg/mL母液。实验当天，母液用7H9+10％ADC液体培养基(7H9培养基购自：北京索莱宝科技有限公司；ADC增菌液购自：上海冠导生物工程有限公司)稀释50倍，配成工作液。
接种板准备：96孔板中每孔加入100μL 7H9+10％ADC液体培养基，第1列加入100μL化合物工作液，吹打均匀后，从第1列吸取100μL液体加入第2列，吹打均匀后，吸取100μL到第3列，依次进行2倍梯度稀释，共10次，第11列每孔移除100μL，得到接种板。
细菌接种液准备：结核分枝杆菌来自北京市结核病胸部肿瘤研究所。用一次性无菌接种环刮取中性罗氏培养基上生长3～4周的菌落，转移至带有消毒玻璃珠及100μL 5％吐温-80的磨菌瓶中。使用涡旋振荡器充分震荡、摇均菌体30秒后静置15分钟。加入无菌生理盐水，直至获得的菌悬液浊度达到1McFarland(麦式浊度)，用7H9+10％ADC液体培养基稀释20倍，得到细菌接种液。
细菌接种和培养：在含有100μL不同浓度化合物的接种板的每孔中加入细菌接种液，37℃培养9天后，每孔加入20μL Alamar Blue(阿尔玛蓝)，再培养24小时。
MIC值读取：最小抑菌浓度(MIC)即为通过肉眼观察能够完全抑制Alamar Blue(阿尔玛蓝)变色的最小药物浓度，或通过荧光检测(Ex/Em，530nm/590nm)抑制率>90％还原型Alamar Blue生成的最小药物浓度。
抑制率计算：抑制率％＝100％-(检测孔荧光值-背景荧光值)/(生长对照孔荧光值-背景荧光值)×100％。
抗结核药敏试验结果如表1所示。蓟属植物的根提取物的活性远大于茎与叶提取物的活性，茎与叶提取物几乎无抗结核活性(MIC＞128μg/mL)。蓟属植物的根有机相提取物的活性远大于根水相提取物的活性。其中，根石油醚提取物用硅胶柱色谱分离得到的各子组分中，部分具有较强的抗结核活性，其中C01-34，C01-84和C01-159组分的抗结核活性较好。尤其对于耐药株(M3)，已上市阳性药品链霉素、乙氨丁醇MIC分别＞32μg/mL、＞160μg/mL，即本发明所述蓟属植物的提取物具有显著的优势。
表1：石油醚部位抗结核活性(MIC，μg/mL)
样品 减毒株 敏感株(S17) 耐药株(M3)
根乙醇提取物 64 32 32
根水提取物 ＞128 ＞128 ＞128
根石油醚提取物 64 16 16
根正己烷提取物 64 16 16
根环己烷提取物 64 16 16
茎乙醇提取物 ＞128 ＞128 ＞128
茎水提取物 ＞128 ＞128 ＞128
叶石油醚提取物 ＞128 ＞128 ＞128
叶水提取物 ＞128 ＞128 ＞128
C01-1 64 64 32
C01-23 128 128 ＞128
C01-28 64 128 64
C01-34 8 4 2
C01-84 32 32 16
C01-159 8 16 32
C01-178 128 128 ＞128
C01-249 ＞128 ＞128 ＞128
实施例4：高效液相色谱法(HPLC)图谱测定
对于抗结核效果较好的根乙醇提取物、根石油醚提取物、C01-34、C01-84和C01-159组分(实施例1获得)进行HPLC图谱测定。
1、色谱条件：
设备：岛津LC-20A高效液相色谱仪
柱温：35℃
检测器：SPD-20AV紫外检测器
检测波长：254nm
色谱柱：phenomenex luna 4.6×250mm，5μm
进样量：20μL
流动相：A(0.1％冰醋酸水溶液)，B(乙腈)
流动相流速：1mL/min
梯度洗脱条件：
时间(分钟) B(体积％) A(体积％)
0 12 88
8 40 60
18 70 30
55 100 0
2、检测结果：
HPLC检测结果见图1-图5与表2-表6。
表2：根乙醇提取物HPLC出峰结果
峰# 保留时间(分钟) 面积 高度 面积(％) 高度(％)
1 7.306 39160 2990 0.643 0.660
2 8.597 84905 2234 1.394 0.493
3 9.821 95301 6737 1.565 1.488
4 12.305 1178163 112002 19.347 24.737
5 13.573 112561 8427 1.848 1.861
6 13.893 167701 10854 2.754 2.397
7 14.149 59557 5556 0.978 1.227
8 14.971 862704 38231 14.167 8.444
9 16.684 308999 13697 5.074 3.025
10 17.110 75556 5193 1.241 1.147
11 17.520 217011 8781 3.564 1.939
12 19.462 355425 29682 5.836 6.556
13 21.532 49331 7288 0.810 1.610
14 23.090 92827 4112 1.524 0.908
15 29.923 20217 1582 0.332 0.349
16 30.290 19787 1897 0.325 0.419
17 31.501 33093 2765 0.543 0.611
18 32.568 57992 3536 0.952 0.781
19 32.853 50378 3785 0.827 0.836
20 33.186 23266 1785 0.382 0.394
21 34.104 161640 15034 2.654 3.320
22 34.418 21637 2167 0.355 0.479
23 35.534 69939 6406 1.148 1.415
24 35.794 127545 10361 2.094 2.288
25 36.012 99087 8430 1.627 1.862
26 36.577 240184 17950 3.944 3.964
27 36.896 341809 30400 5.613 6.714
28 37.650 251254 21119 4.126 4.664
29 38.056 30770 2410 0.505 0.532
30 38.799 146459 11677 2.405 2.579
31 41.962 273143 25974 4.485 5.736
32 43.689 114462 10561 1.880 2.333
33 45.747 107780 8971 1.770 1.981
34 46.699 149021 7212 2.447 1.593
35 49.209 51026 2972 0.838 0.656
总计   6089688 452778 100.000 100.000
表3：根石油醚提取物HPLC出峰结果
峰# 保留时间(分钟) 面积 高度 面积(％) 高度(％)
1 30.358 133052 7441 2.107 1.890
2 31.571 165286 10346 2.618 2.628
3 32.643 204998 11756 3.247 2.986
4 32.912 127179 9111 2.014 2.314
5 34.117 343455 27898 5.440 7.087
6 34.458 116132 8737 1.839 2.219
7 35.876 942789 28900 14.932 7.342
8 36.556 611473 41510 9.684 10.545
9 36.863 810487 63680 12.836 16.177
10 37.619 735141 45212 11.643 11.485
11 38.040 144054 8425 2.281 2.140
12 38.751 294405 20166 4.663 5.123
13 41.987 559095 43693 8.855 11.100
14 43.714 226992 17119 3.595 4.349
15 45.780 400110 29521 6.337 7.499
16 46.723 363097 13877 5.751 3.525
17 49.222 136285 6256 2.158 1.589
总计   6314029 393648 100.000 100.000
表4：C01-34组分HPLC出峰结果
峰# 保留时间(分钟) 面积 高度 面积(％) 高度(％)
1 35.681 1367752 117857 3.35 3.897
2 36.646 4923542 365106 12.059 12.074
3 37.878 326112 29027 0.799 0.96
4 38.274 434852 38610 1.065 1.277
5 38.926 4940657 269386 12.101 8.908
6 42.054 20792974 1553895 50.929 51.385
7 43.788 6862902 571415 16.81 18.896
8 46.808 1178599 78730 2.887 2.603
总计   40827389 3024026 100 100
表5：C01-84组分HPLC出峰结果
峰# 保留时间(分钟) 面积 高度 面积(％) 高度(％)
1 32.953 378876 32121 4.536 4.662
2 33.283 118617 11293 1.42 1.639
3 34.217 1862267 166814 22.296 24.213
4 34.507 183183 18915 2.193 2.745
5 35.917 762467 64538 9.129 9.368
6 36.6 884790 78284 10.593 11.363
7 36.986 1996637 144028 23.905 20.905
8 37.694 1939589 154345 23.222 22.403
9 38.15 225910 18612 2.705 2.701
总计   8352337 688951 100 100
表6：C01-159组分HPLC出峰结果
峰# 保留时间(分钟) 面积 高度 面积(％) 高度(％)
1 29.993 788938 52159 1.786 1.727
2 30.376 1391002 91550 3.150 3.031
3 31.013 603079 36863 1.366 1.220
4 31.502 3406533 233149 7.713 7.719
5 32.032 546416 29342 1.237 0.971
6 32.545 6200899 388165 14.040 12.851
7 33.249 1134082 55405 2.568 1.834
8 33.923 1345130 91446 3.046 3.028
9 34.354 1460842 71987 3.308 2.383
10 35.205 1875686 92416 4.247 3.060
11 35.750 2591330 176131 5.867 5.831
12 35.860 2947609 298668 6.674 9.888
13 36.146 5590577 439389 12.658 14.547
14 36.685 1490338 86874 3.374 2.876
15 37.041 8280789 649573 18.750 21.505
16 37.649 1656504 71904 3.751 2.381
17 40.063 1404068 62822 3.179 2.080
18 40.767 980713 55184 2.221 1.827
19 41.277 470779 37495 1.066 1.241
总计   44165315 3020521 100.000 100.000
综合共同出峰时间、出峰面积等因素，发明人认为具有抗结核活性成分的提取物在254nm波长下具有的特征紫外吸收峰的出峰时间集中在30-50分钟，优选集中在30-45分钟，更优选集中在35-40分钟。更进一步，具有抗结核活性成分的提取物在254nm波长下具有的特征紫外吸收峰的出峰时间分别为：35.8±0.3分钟、36.6±0.3分钟、37.7±0.3分钟，优选为34.2±0.3分钟、35.8±0.3分钟、36.6±0.3分 钟、37.0±0.3分钟、37.7±0.3分钟、42.0±0.3分钟，更优选为31.5±0.3分钟、32.5±0.3分钟、34.2±0.3分钟、35.8±0.3分钟、36.6±0.3分钟、37.0±0.3分钟、37.7±0.3分钟、38.9±0.3分钟、42.0±0.3分钟、43.8±0.3分钟。
尽管本发明描述了具体的例子，但是有一点对于本领域技术人员来说是明显的，即在不脱离本发明的精神和范围的前提下可对本发明作各种变化和改动。因此，所附权利要求覆盖了所有这些在本发明范围内的变动。

Claims (10)

  1. 蓟属植物的有机提取物；优选地，所述有机提取物是使用乙醇、丙酮、乙酸乙酯、二氯甲烷、三氯甲烷、石油醚、正己烷和环己烷中的一种或多种进行提取获得的有机提取物；优选地，所述蓟属植物的提取物为蓟属植物根部位的提取物。
  2. 如权利要求1所述的有机提取物，其特征在于，所述蓟属植物选自蓟(Cirsium japonicum)、野蓟(Cirsium maackii)、绿蓟(Cirsium chinense)、刺儿菜(Cirsium setosum)、线叶蓟(Cirsium lineare)和杭蓟(Cirsium tianmushanicum)。
  3. 一种蓟属植物提取物，该提取物的特征是，具有出峰时间在35-40分钟之间的特征紫外吸收峰，其中，该出峰时间是以254nm波长进行高效液相色谱法检测得到；
    优选地，该提取物具有出峰时间分别为35.8±0.3分钟、36.6±0.3分钟和37.7±0.3分钟的特征紫外吸收峰；更优选地，该提取物具有出峰时间分别为35.8±0.2分钟、36.6±0.2分钟和37.7±0.2分钟的特征紫外吸收峰；更优选地，该提取物具有出峰时间分别为35.8±0.1分钟、36.6±0.1分钟和37.7±0.1分钟的特征紫外吸收峰；
    任选地，该提取物还具有出峰时间为37.0±0.3分钟、优选为37.0±0.2分钟、更优选为37.0±0.1分钟的特征紫外吸收峰，出峰时间为38.9±0.3分钟、优选为38.9±0.2分钟、更优选为38.9±0.1分钟的特征紫外吸收峰中的一个或多个特征紫外吸收峰；
    任选地，该提取物还具有出峰时间在30-35分钟之间的特征紫外吸收峰，和/或出峰时间在40-45分钟之间的特征紫外吸收峰；优选地，该提取物具有出峰时间为34.2±0.3分钟、优选为34.2±0.2分钟、更优选为34.2±0.1分钟的特征紫外吸收峰，出峰时间为32.5±0.3分钟、优选为32.5±0.2分钟、更优选为32.5±0.1分钟的特征紫外吸收峰，出峰时间为31.5±0.3分钟、优选为31.5±0.2分钟、更优选为31.5±0.1分钟的特征紫外吸收峰，出峰时间为42.0±0.3分钟、优选为42.0±0.2分钟、更优选为42.0±0.1分钟的特征紫外吸收峰，以及出峰时间为43.8±0.3分钟、优选为43.8±0.2分钟、更优选为43.8±0.1分钟的特征紫外吸收峰中的一个或多个特征 紫外吸收峰；
    其中，所述高效液相色谱法的检测条件为：
    柱温：35℃，
    检测器：紫外检测器，
    检测波长：254nm，
    色谱柱：phenomenex luna 4.6×250mm，5μm，
    进样量：1-20μL，
    流动相：0.1％冰醋酸水溶液、乙腈，
    流动相流速：1mL/分钟，
    梯度洗脱条件：
    时间/分钟 乙睛/体积百分比 0.1％冰醋酸水溶液/体积百分比 0 12 88 8 40 60 18 70 30 55 100 0
    。
  4. 如权利要求3所述的蓟属植物提取物，其特征在于，所述提取物具有出峰时间如下所示的特征紫外吸收峰：35.8±0.1分钟、36.6±0.1分钟和37.7±0.1分钟，优选34.2±0.1分钟、35.8±0.1分钟、36.6±0.1分钟、37.0±0.1分钟、37.7±0.1分钟和42.0±0.1分钟，更优选31.5±0.1分钟、32.5±0.1分钟、34.2±0.1分钟、35.8±0.1分钟、36.6±0.1分钟、37.0±0.1分钟、37.7±0.1分钟、38.9±0.1分钟、42.0±0.1分钟和43.8±0.1分钟；或者具有出峰时间如下所示的特征紫外吸收峰：35.8±0.2分钟、36.6±0.2分钟和37.7±0.2分钟，优选34.2±0.2分钟、35.8±0.2分钟、36.6±0.2分钟、37.0±0.2分钟、37.7±0.2分钟和42.0±0.2分钟，更优选31.5±0.2分钟、32.5±0.2分钟、34.2±0.2分钟、35.8±0.2分钟、36.6±0.2分钟、37.0±0.2分钟、37.7±0.2分钟、38.9±0.2分钟、42.0±0.2分钟和43.8±0.2分钟；或者具有出峰时间如下所示的特征紫外吸收峰：35.8±0.3分钟、36.6±0.3分钟和37.7±0.3分钟，优选34.2±0.3分钟、35.8±0.3分钟、36.6±0.3分钟、37.0±0.3分钟、37.7±0.3分钟和42.0±0.3分钟，更优选31.5±0.3分钟、32.5±0.3分钟、34.2±0.3分钟、35.8±0.3分钟、36.6±0.3分钟、37.0±0.3分钟、37.7±0.3分钟、38.9±0.3分钟、42.0±0.3分钟和43.8±0.3分钟。
  5. 如权利要求3所述的蓟属植物提取物，其特征在于，
    所述蓟属植物提取物具有出峰时间如下所示的特征紫外吸收峰：35.6±0.1分钟、36.6±0.1分钟、37.8±0.1分钟、38.9±0.1分钟、42.0±0.1分钟和43.7±0.1分钟，任选在38.2±0.1分钟和46.8±0.1分钟还具有特征紫外吸收峰；或
    所述蓟属植物提取物具有出峰时间如下所示的特征紫外吸收峰：34.2±0.1分钟、35.9±0.1分钟、36.6±0.1分钟、36.9±0.1分钟和37.6±0.1分钟，任选在32.9±0.1分钟、33.2±0.1分钟、34.5±0.1分钟和38.1±0.1分钟还具有特征吸收峰；或
    所述蓟属植物提取物具有出峰时间如下所示的紫外特征紫外吸收峰：31.5±0.1分钟、32.5±0.1分钟、34.3±0.1分钟、35.8±0.1分钟、36.6±0.1分钟、37.0±0.1分钟和37.6±0.1分钟，任选在29.9±0.1分钟、30.3±0.1分钟、31.0±0.1分钟、32.0±0.1分钟、33.2±0.1分钟、33.9±0.1分钟、35.2±0.1分钟、35.7±0.1分钟、36.1±0.1分钟、40.0±0.1分钟、40.7±0.1分钟以及41.2±0.1分钟还具有紫外特征吸收峰。
  6. 如权利要求3-5中任一项所述的蓟属植物提取物，其特征在于，所述蓟属植物选自蓟(Cirsium japonicum)、野蓟(Cirsium maackii)、绿蓟(Cirsium chinense)、刺儿菜(Cirsium setosum)、线叶蓟(Cirsium lineare)和杭蓟(Cirsium tianmushanicum)。
  7. 一种药物组合物，它包含治疗有效量的权利要求1-6中任一项所述的提取物和药学上可接受的载体；优选地，所述药物组合物由治疗有效量的权利要求1-6中任一项所述的提取物和药学上可接受的载体组成。
  8. 权利要求1-6中任一项所述的提取物在制备抗结核分枝杆菌药物或结核病治疗药物中的应用；
    优选地，所述结核分枝杆菌选自人结核分枝杆菌和牛型分枝杆菌；更优选，所述结核分枝杆菌选自耐药型人结核分枝杆菌；
    优选地，所述结核病由结核分枝杆菌引起；更优选地，所述结核病选自肺结核或者肺外结核；还要优选地，所述结核病为肺结核、肝结核、胃结核或肠结核。
  9. 一种治疗结核病的方法，其特征在于，所述方法包括对患者施用治疗有效量的权利要求1-6中任一项所述的提取物，或者对患者施用权利要求7所述的药物组合物；
    优选地，所述结核病是由结核分枝杆菌引起的；更优选，所述结核分枝杆菌选自人结核分枝杆菌和牛型分枝杆菌；还要优选，所述结核分枝杆菌选自耐药型人结 核分枝杆菌；
    优选地，所述结核病选自肺结核或者肺外结核；更优选地，所述结核病为肺结核、肝结核、胃结核或肠结核。
  10. 一种制备蓟属植物提取物的方法，其特征在于，所述方法包括采用有机溶液提取蓟属植物的步骤；优选地，所述有机溶液选自乙醇、丙酮、乙酸乙酯、二氯甲烷、三氯甲烷、石油醚、正己烷和环己烷中的一种或多种；
    优选地，使用蓟属植物根部位进行提取；
    优选地，所述蓟属植物选自蓟(Cirsium japonicum)、野蓟(Cirsium maackii)、绿蓟(Cirsium chinense)、刺儿菜(Cirsium setosum)、线叶蓟(Cirsium lineare)和杭蓟(Cirsium tianmushanicum)。
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