WO2024077489A1 - 改进热淋清颗粒生物学活性的方法 - Google Patents

改进热淋清颗粒生物学活性的方法 Download PDF

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WO2024077489A1
WO2024077489A1 PCT/CN2022/124673 CN2022124673W WO2024077489A1 WO 2024077489 A1 WO2024077489 A1 WO 2024077489A1 CN 2022124673 W CN2022124673 W CN 2022124673W WO 2024077489 A1 WO2024077489 A1 WO 2024077489A1
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extract
polygonum capitatum
methanol
chromatographic peaks
retention time
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PCT/CN2022/124673
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English (en)
French (fr)
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唐靖雯
卢礼平
潘梅
梁斌
张丽艳
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贵州威门药业股份有限公司
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Priority to PCT/CN2022/124673 priority Critical patent/WO2024077489A1/zh
Publication of WO2024077489A1 publication Critical patent/WO2024077489A1/zh

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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)
    • A61K36/70—Polygonaceae (Buckwheat family), e.g. spineflower or dock
    • A61K36/704—Polygonum, e.g. knotweed
    • 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
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • 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
    • A61P13/04—Drugs for disorders of the urinary system for urolithiasis
    • 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
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • 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
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86

Definitions

  • the present invention belongs to the field of traditional Chinese medicine, and relates to a method for preparing an extract of a medicinal material Polygonum capitatum for preparing a traditional Chinese medicine Relinqing granule.
  • the present invention relates to an effective part obtained from an alcohol extract of a medicinal material Polygonum capitatum, which is a raw material of Relinqing, and a method for preparing the effective part, and also relates to the use of the effective part.
  • the method for preparing Relinqing granules of the present invention can significantly improve the biological activity of the prepared Relinqing granules.
  • Relinqing Granules As the raw material of Relinqing Granules, Polygonum capitatum Buch-Ham ex D. Don is a perennial herbaceous plant of the Polygonaceae family. It has the effects of clearing away heat and dampness, promoting diuresis and relieving stranguria, and has a significant effect in the clinical treatment of urinary tract infections.
  • the Chinese patent medicine preparation "Relinqing Granules" made with it as the raw material was included in the national basic medical insurance catalog in 2004 and in the Guizhouzhouzhouzhou medicine catalog in 2012.
  • Polygonum capitatum is a commonly used medicine in ethnic minority areas, mainly used for pyelonephritis, urinary tract infection, diuresis and stranguria. Related pharmacological studies are rare, and there are only a few reports by Ren Guangyou et al. Ren Guangyou used a rat bacterial pyelonephritis model to conduct experiments. The results showed that the WBC and BLD in the urine of rats in the Polygonum capitatum water extract group were significantly reduced compared with the control group, indicating that the Polygonum capitatum water extract has a certain anti-inflammatory effect on pyelonephritis. Ren Guangyou et al.
  • the Chinese patent application publication specification CN1054899A (Chinese patent application number 90107810.7, published on October 2, 1991) discloses a production process of Miganling granules and syrups.
  • the production process is to use four seasons grass as raw material, boil it with water for 30-60 minutes, filter and concentrate the extract, decompress and concentrate the supernatant into a paste, extract it with 60-70% ethanol, and vacuum dry the ethanol extract to obtain four seasons red extract, which is further prepared into granules or syrups, which are believed to have the effects of detoxification, blood stasis, diuresis, and stranguria.
  • CN 1481832A (Chinese patent application No. 02129686.3, published on March 17, 2004) and CN 1483466A (Chinese patent application No. 03146381.9, published on March 24, 2004) disclose extracts of Polygonum capitatum, which are basically prepared by the following steps: a. the fresh or dried product of the whole plant of Polygonum capitatum is decocted twice with water or refluxed extracted with an alcohol-water mixture for two to three times, each time for 1-2 hours, the decoctions are combined, filtered and concentrated to a relative density of 1.2 at 20°C, and spray-dried or dried under reduced pressure; or b.
  • the extract is obtained by supercritical carbon dioxide extraction of the whole plant of Polygonum capitatum and its water-extracted residue. It is believed that the extract can be used for antibacterial, anti-inflammatory, analgesic, diuretic, treatment of urinary system stones, treatment of pyelonephritis and prostatitis.
  • the present invention aims to obtain an effective product from Polygonum capitatum.
  • the present inventors have found that the extract obtained by extracting Polygonum capitatum with alcohol and eluting it with a specific macroporous resin has an expected effect in terms of biological activity, especially the biological activity is significantly improved.
  • the present invention is completed based on this discovery.
  • the first aspect of the present invention provides a Polygonum capitatum extract, which is basically prepared by a method comprising the following steps:
  • the methanol eluate obtained in step (3) is a 50% methanol eluate (referred to as: fraction E in the present invention).
  • the methanol eluate obtained in step (3) is a 50%-60% methanol eluate (abbreviated as: fraction F in the present invention).
  • the methanol eluate obtained in step (3) is a 60%-80% methanol eluate (referred to as: fraction G in the present invention).
  • the extract of Polygonum capitatum obtained in step (3) is a mixture of 50%-80% methanol eluates (hereinafter referred to as "fraction Y").
  • the fraction Y may be a mixture of dried products of fractions E to G, or a dried product of a mixture obtained by eluting with 50%-80% methanol and then drying to remove the solvent.
  • the Polygonum capitatum extract wherein part E contains lignans and/or flavonoid glycosides, for example, contains a mixture of lignans and flavonoid glycosides.
  • the Polygonum capitatum extract contains flavonoid glycosides in the fraction F.
  • the Polygonum capitatum extract contains flavonoid aglycones in the fraction G.
  • the Polygonum capitatum extract contains lignans and/or flavonoid glycosides and/or flavonoid aglycones in part Y, such as quercetin-3-O- ⁇ -D-pyranoglucoside, quercetin-pyranoglucoside, punicalagin B/carpinusin, quercetin, and kaempferol 3-(2-galloyl glucoside).
  • lignans and/or flavonoid glycosides and/or flavonoid aglycones in part Y, such as quercetin-3-O- ⁇ -D-pyranoglucoside, quercetin-pyranoglucoside, punicalagin B/carpinusin, quercetin, and kaempferol 3-(2-galloyl glucoside).
  • the Polygonum capitatum extract according to the first aspect of the present invention is measured by ultra-high performance liquid chromatography-time of flight-mass spectrometry (hereinafter referred to as UPLC-TOF-MS), and the results are as follows:
  • chromatographic peaks of flavonoid glycosides are displayed between retention times 12.8-13.8 min (e.g., about 13.3 min), between retention times 13.8-14.8 min (e.g., about 14.3 min), and between retention times 14.8-15.8 min (e.g., about 15.3 min);
  • test solution Preparation of test solution: Weigh an appropriate amount of Polygonum capitatum extract powder, add 70% methanol to make a suspension with a concentration of about 5 mg/ml, sonicate to dissolve as much as possible, dilute the suspension 10 times, filter, and inject 2 ⁇ l for mass spectrometry detection;
  • Chromatographic column Acquity BEH C18 column (2.1 ⁇ 100 mm, 1.7 ⁇ m), column temperature: 40°C; flow rate: 0.35ml/min; injection volume: 2 ⁇ l; mass spectrometry conditions: ion source: ESI source; drying gas temperature: 180°C; capillary voltage: 4500eV; detection mode: negative ion mode; spray pressure: 2.5bar; drying gas (N2) flow rate: 8L/min; scanning range: 100-2000amu; collision energy: 10ev; 0.1% formic acid aqueous solution as mobile phase A, 0.1% formic acid acetonitrile solution as mobile phase B, gradient elution according to the following program: 95%A at 0 min, 95%A at 0.5 min, 81.5%A at 20 min, 0%A at 28 min, 0%A at 30 min, 95%A at 30.1 min, 95%A at 32 min.
  • the Polygonum capitatum extract according to the first aspect of the present invention is measured according to the ultra performance liquid chromatography-time of flight-mass spectrometry method, and the results are: (e) chromatographic peaks of flavonoid glycosides (such as quercetin-3-O- ⁇ -D-pyranoglucoside, punicalagin B/carpinusin) are displayed between retention times of 12.8-13.8 min (for example, at about 13.3 min) and between retention times of 14.8-15.8 min (for example, at about 15.3 min), and/or chromatographic peaks of lignans (such as nudiposide) are displayed between retention times of 14.4-15.4 min (for example, at about 14.9 min).
  • flavonoid glycosides such as quercetin-3-O- ⁇ -D-pyranoglucoside, punicalagin B/carpinusin
  • the Polygonum capitatum extract according to the first aspect of the present invention is measured according to the ultra high performance liquid chromatography-time of flight-mass spectrometry method, and the results are: (f) chromatographic peaks of flavonoid glycosides (such as quercetin-3-O- ⁇ -D-pyranoglucoside, quercetin-pyranoglucoside, and punicalagin B/carpinusin) are displayed between retention times of 12.8-13.8 min (for example, at about 13.3 min), between retention times of 13.8-14.8 min (for example, at about 14.3 min), and between retention times of 14.8-15.8 min (for example, at about 15.3 min).
  • flavonoid glycosides such as quercetin-3-O- ⁇ -D-pyranoglucoside, quercetin-pyranoglucoside, and punicalagin B/carpinusin
  • the Polygonum capitatum extract according to the first aspect of the present invention is measured according to the ultra-high performance liquid chromatography-time-of-flight-mass spectrometry method, and the results are: (g) chromatographic peaks of flavonoid aglycones (such as quercetin and kaempferol 3-(2-galloyl glucoside)) are displayed between retention times of 16.6-17.6 min (for example, at about 17.1 min) and between retention times of 21.4-22.4 min (for example, at about 21.9 min).
  • flavonoid aglycones such as quercetin and kaempferol 3-(2-galloyl glucoside
  • the Polygonum capitatum extract according to the first aspect of the present invention is measured by ultra-high performance liquid chromatography-time of flight-mass spectrometry, and the results are:
  • the Polygonum capitatum extract according to the first aspect of the present invention has the characteristics described in any embodiment of the second aspect of the present invention.
  • the second aspect of the present invention provides a Polygonum capitatum extract, which is measured by ultra-high performance liquid chromatography-time of flight-mass spectrometry (abbreviated as UPLC-TOF-MS in the present invention), and the results are:
  • chromatographic peaks of flavonoid glycosides are displayed between retention times 12.8-13.8 min (e.g., about 13.3 min), between retention times 13.8-14.8 min (e.g., about 14.3 min), and between retention times 14.8-15.8 min (e.g., about 15.3 min);
  • test solution Preparation of test solution: Weigh an appropriate amount of Polygonum capitatum extract powder, add 70% methanol to make a suspension with a concentration of about 5 mg/ml, sonicate to dissolve as much as possible, dilute the suspension 10 times, filter, and inject 2 ⁇ l for mass spectrometry detection;
  • Chromatographic column Acquity BEH C18 column (2.1 ⁇ 100 mm, 1.7 ⁇ m), column temperature: 40°C; flow rate: 0.35ml/min; injection volume: 2 ⁇ l; mass spectrometry conditions: ion source: ESI source; drying gas temperature: 180°C; capillary voltage: 4500eV; detection mode: negative ion mode; spray pressure: 2.5bar; drying gas (N2) flow rate: 8L/min; scanning range: 100-2000amu; collision energy: 10ev; 0.1% formic acid aqueous solution as mobile phase A, 0.1% formic acid acetonitrile solution as mobile phase B, gradient elution according to the following program: 95%A at 0 min, 95%A at 0.5 min, 81.5%A at 20 min, 0%A at 28 min, 0%A at 30 min, 95%A at 30.1 min, 95%A at 32 min.
  • the Polygonum capitatum extract according to the second aspect of the present invention is measured according to the ultra performance liquid chromatography-time of flight-mass spectrometry method, and the results are: (e) chromatographic peaks of flavonoid glycosides (such as quercetin-3-O- ⁇ -D-pyranoglucoside, punicalagin B/carpinusin) are displayed between retention times of 12.8-13.8 min (for example, at about 13.3 min) and between retention times of 14.8-15.8 min (for example, at about 15.3 min), and/or chromatographic peaks of lignans (such as nudiposide) are displayed between retention times of 14.4-15.4 min (for example, at about 14.9 min).
  • flavonoid glycosides such as quercetin-3-O- ⁇ -D-pyranoglucoside, punicalagin B/carpinusin
  • the Polygonum capitatum extract according to the second aspect of the present invention is measured according to the ultra high performance liquid chromatography-time of flight-mass spectrometry method, and the results are: (f) chromatographic peaks of flavonoid glycosides (such as quercetin-3-O- ⁇ -D-pyranoglucoside, quercetin-pyranoglucoside, punicalagin B/carpinusin) are displayed between retention times of 12.8-13.8 min (for example, at about 13.3 min), between retention times of 13.8-14.8 min (for example, at about 14.3 min), and between retention times of 14.8-15.8 min (for example, at about 15.3 min).
  • flavonoid glycosides such as quercetin-3-O- ⁇ -D-pyranoglucoside, quercetin-pyranoglucoside, punicalagin B/carpinusin
  • the Polygonum capitatum extract according to the second aspect of the present invention is measured according to the ultra-high performance liquid chromatography-time-of-flight-mass spectrometry method, and the results are: (g) chromatographic peaks of flavonoid aglycones (such as quercetin and kaempferol 3-(2-galloyl glucoside)) are shown between retention times 16.6-17.6 min (for example, at about 17.1 min) and between retention times 21.4-22.4 min (for example, at about 21.9 min).
  • flavonoid aglycones such as quercetin and kaempferol 3-(2-galloyl glucoside
  • the Polygonum capitatum extract according to the second aspect of the present invention is measured by ultra-high performance liquid chromatography-time of flight-mass spectrometry, and the results are:
  • the Polygonum capitatum extract according to the second aspect of the present invention is basically prepared by a method comprising the following steps:
  • the methanol eluate obtained in step (3) is a 50% methanol eluate (referred to as: fraction E in the present invention).
  • the methanol eluate obtained in step (3) is a 50%-60% methanol eluate (referred to as: fraction F in the present invention).
  • the methanol eluate obtained in step (3) is a 60%-80% methanol eluate (referred to as: fraction G in the present invention).
  • the extract of Polygonum capitatum obtained in step (3) is a mixture of 50%-80% methanol eluates (hereinafter referred to as "fraction Y").
  • the fraction Y may be a mixture of dried products of fractions E to G, or a dried product of a mixture obtained by eluting with 50%-80% methanol and then drying to remove the solvent.
  • the Polygonum capitatum extract wherein part E contains lignans and/or flavonoid glycosides, for example, contains a mixture of lignans and flavonoid glycosides.
  • the Polygonum capitatum extract contains flavonoid glycosides in the fraction F.
  • the Polygonum capitatum extract contains flavonoid aglycones in the fraction G.
  • the Polygonum capitatum extract contains lignans and/or flavonoid glycosides and/or flavonoid aglycones in part Y, such as quercetin-3-O- ⁇ -D-pyranoglucoside, quercetin-pyranoglucoside, punicalagin B/carpinusin, quercetin, and kaempferol 3-(2-galloyl glucoside).
  • lignans and/or flavonoid glycosides and/or flavonoid aglycones in part Y, such as quercetin-3-O- ⁇ -D-pyranoglucoside, quercetin-pyranoglucoside, punicalagin B/carpinusin, quercetin, and kaempferol 3-(2-galloyl glucoside).
  • the Polygonum capitatum extract according to the second aspect of the present invention has the characteristics described in any embodiment of the first aspect of the present invention.
  • the third aspect of the present invention provides a method for improving the biological activities of Relinqing granules in clearing away heat and dampness, promoting diuresis and relieving stranguria, treating urinary tract infection or stones, pyelonephritis, detoxifying, dispersing blood stasis, antibacterial, anti-inflammatory, analgesic, and anti-prostatitis.
  • the method comprises administering a therapeutically effective amount of Polygonum capitatum extract to a subject in need thereof, wherein the Polygonum capitatum extract is prepared according to the following steps:
  • the methanol eluate obtained in step (3) is a 50% methanol eluate (referred to as: part E in the present invention).
  • the methanol eluate obtained in step (3) is a 50%-60% methanol eluate (referred to as: part F in the present invention).
  • the methanol eluate obtained in step (3) is a 60%-80% methanol eluate (referred to as: part G in the present invention).
  • the methanol eluate obtained in step (3) is a mixture of 50%-80% methanol eluates (hereinafter referred to as "fraction Y").
  • fraction Y may be a mixture of dried products of fractions E to G, or may be a dried product of a mixture obtained by eluting with 50%-80% methanol and then drying to remove the solvent.
  • part E contains lignans and/or flavonoid glycosides, for example, contains a mixture of lignans and flavonoid glycosides.
  • part F contains flavonoid glycosides.
  • part G contains flavonoid aglycones.
  • part Y contains lignans and/or flavonoid glycosides and/or flavonoid aglycones, such as quercetin-3-O- ⁇ -D-pyranoglucoside, quercetin-pyranoglucoside, punicalagin B/carpinusin, quercetin, and kaempferol 3-(2-galloyl glucoside).
  • lignans and/or flavonoid glycosides and/or flavonoid aglycones such as quercetin-3-O- ⁇ -D-pyranoglucoside, quercetin-pyranoglucoside, punicalagin B/carpinusin, quercetin, and kaempferol 3-(2-galloyl glucoside).
  • the obtained Polygonum capitatum extract has the characteristics described in any embodiment of the first aspect or the second aspect of the present invention.
  • the Polygonum capitatum extract is measured by ultra-high performance liquid chromatography-time of flight-mass spectrometry (hereinafter referred to as UPLC-TOF-MS), and the result is:
  • chromatographic peaks of flavonoid glycosides are displayed between retention times 12.8-13.8 min (e.g., about 13.3 min), between retention times 13.8-14.8 min (e.g., about 14.3 min), and between retention times 14.8-15.8 min (e.g., about 15.3 min);
  • test solution Preparation of test solution: Weigh an appropriate amount of Polygonum capitatum extract powder, add 70% methanol to make a suspension with a concentration of about 5 mg/ml, sonicate to dissolve as much as possible, dilute the suspension 10 times, filter, and inject 2 ⁇ l for mass spectrometry detection;
  • Chromatographic column Acquity BEH C18 column (2.1 ⁇ 100 mm, 1.7 ⁇ m), column temperature: 40 °C; flow rate: 0.35 ml/min; injection volume: 2 ⁇ l; mass spectrometry conditions: ion source: ESI source; drying gas temperature: 180 °C; capillary voltage: 4500 eV; detection mode: negative ion mode; spray pressure: 2.5 bar; drying gas (N2) flow rate: 8 L/min; scanning range: 100-2000 amu; collision energy: 10 ev; 0.1% formic acid aqueous solution as mobile phase A, 0.1% formic acid acetonitrile solution as mobile phase B, gradient elution according to the following program: 95% A at 0 min, 95% A at 0.5 min, 81.5% A at 20 min, 0% A at 28 min, 0% A at 30 min, 0% A at 30.1 min, 95% A at 32 min.
  • the Polygonum capitatum extract is measured according to the ultra performance liquid chromatography-time of flight-mass spectrometry method, and the results are: (e) chromatographic peaks of flavonoid glycosides (such as quercetin-3-O- ⁇ -D-pyranoglucoside, punicalagin B/carpinusin) are displayed between retention times of 12.8-13.8 min (for example, at about 13.3 min) and between retention times of 14.8-15.8 min (for example, at about 15.3 min), and/or chromatographic peaks of lignans (such as nudiposide) are displayed between retention times of 14.4-15.4 min (for example, at about 14.9 min).
  • flavonoid glycosides such as quercetin-3-O- ⁇ -D-pyranoglucoside, punicalagin B/carpinusin
  • the Polygonum capitatum extract is measured according to the ultra performance liquid chromatography-time of flight-mass spectrometry method, and the results are: (f) chromatographic peaks of flavonoid glycosides (such as quercetin-3-O- ⁇ -D-pyranoglucoside, quercetin-pyranoglucoside, punicalagin B/carpinusin) are displayed between retention times of 12.8-13.8 min (for example, at about 13.3 min), between retention times of 13.8-14.8 min (for example, at about 14.3 min), and between retention times of 14.8-15.8 min (for example, at about 15.3 min).
  • flavonoid glycosides such as quercetin-3-O- ⁇ -D-pyranoglucoside, quercetin-pyranoglucoside, punicalagin B/carpinusin
  • the Polygonum capitatum extract is measured according to the ultra performance liquid chromatography-time of flight-mass spectrometry method, and the results are: (g) chromatographic peaks of flavonoid aglycones (such as quercetin and kaempferol 3-(2-galloyl glucoside)) are displayed between retention times of 16.6-17.6 min (for example, at about 17.1 min) and between retention times of 21.4-22.4 min (for example, at about 21.9 min).
  • flavonoid aglycones such as quercetin and kaempferol 3-(2-galloyl glucoside
  • the Polygonum capitatum extract is measured according to the ultra performance liquid chromatography-time of flight-mass spectrometry method, and the results are: (e) chromatographic peaks of flavonoid glycosides (such as quercetin-3-O- ⁇ -D-pyranoglucoside, punicalagin B/carpinusin) are displayed between retention times of 12.8-13.8 min (for example, at about 13.3 min) and 14.8-15.8 min (for example, at about 15.3 min), and/or chromatographic peaks of lignans (such as nudiposide) are displayed between retention times of 14.4-15.4 min (for example, at about 14.9 min); (f) chromatographic peaks of flavonoid glycosides (such as quercetin-3-O- ⁇ -D-pyranoglucoside, punicalagin B/carpinusin) are displayed between retention times of 12.8-13.8 min (for example, at about 13.3 min).
  • flavonoid glycosides such as quercetin-3-O
  • chromatographic peaks of flavonoid glycosides are displayed between retention times 13.8-14.8 min (e.g., about 14.3 min), and between retention times 14.8-15.8 min (e.g., about 15.3 min); and/or, (g) chromatographic peaks of flavonoid aglycones (e.g., quercetin, kaempferol 3-(2-galloyl glucoside)) are displayed between retention times 16.6-17.6 min (e.g., about 17.1 min) and between retention times 21.4-22.4 min (e.g., about 21.9 min).
  • flavonoid glycosides e.g., quercetin-3-O- ⁇ -D-pyranoglucopyranoside, quercetin-pyranoglucopyranoside, punicalagin B/carpinusin
  • retention times 13.8-14.8 min e.g., about 14.3 min
  • retention times 14.8-15.8 min e.g., about 15.3 min
  • the fourth aspect of the present invention provides the use of the Polygonum capitatum extract described in the first aspect or the second aspect of the present invention in the preparation of medicines for clearing away heat and dampness, diuretics and stranguria, medicines for urinary tract infection or stones, medicines for pyelonephritis, medicines for detoxification and dispersing blood stasis, antibacterial, anti-inflammatory, analgesic, and prostatitis.
  • the fifth aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the Polygonum capitatum extract according to any embodiment of the first aspect or the second aspect of the present invention and an optional pharmaceutically acceptable carrier.
  • the pharmaceutical composition according to the fifth aspect of the present invention is in the form of a preparation for oral or injection administration.
  • the pharmaceutical composition is in the form of tablets, capsules, granules, pills, oral liquids, injections (water injections and/or powder injections), etc.
  • the method of the present invention obtains the "Polygonum capitatum extract", which term can also be referred to as the "Polygonum capitatum effective component" of the present invention or the “Polygonum capitatum effective part” of the present invention.
  • the MCI macroporous resin can be the MCI GEL series produced by Mitsubishi Corporation of Japan.
  • the MCI GEL series reverse phase separation filler is designed based on Mitsubishi Chemical Diaion and Sepabeads macroporous adsorption resins. Because based on modern HPLC high pressure liquid chromatography separation technology, smaller particles have higher chromatographic separation performance and are widely used for the separation of natural products and fermentation products.
  • MCI macroporous resin has two types of fillers: polystyrene and acrylic type. The particle size range of polystyrene type resin is 4 ⁇ m-100 ⁇ m, and the particle size range of methacrylate type resin is 4 ⁇ m-31 ⁇ m.
  • MCI GEL reverse phase fine separation filler is a resin filler that can be used to separate saponin components, generally eluted with methanol water or ethanol water. Furthermore, MCI is generally used to remove chlorophyll from components with low polarity, and has a good effect on removing chlorophyll from petroleum ether and chloroform.
  • the MCI macroporous resin preferably used is a polystyrene type, including but not limited to MCI GEL CHP 10M, MCI GEL CHP 5C, MCI GEL CHP 55A, MCI GEL CHP 55Y, MCI GEL CHP 20Y, MCI GEL CHP 20P, MCI GEL CHP 20SS.
  • the macroporous resin of model MCI GEL CHP 20P (75 ⁇ 150 ⁇ m) is preferably used, which is the most widely used model. It is a porous polystyrene polymer, which shows a wide range of reverse phase adsorption.
  • the purpose of the present invention is to overcome the deficiencies of the prior art and provide a highly effective and low-toxic anti-inflammatory active ingredient (effective part) of Polygonum capitatum and its application.
  • the present invention focuses on the pharmacological effects of Polygonum capitatum, clarifies its action characteristics and screens the pharmacological effects of different extraction parts of Polygonum capitatum.
  • Samples of different extraction parts of Polygonum capitatum are used to screen the effective components (effective parts) and pharmacological action characteristics of Polygonum capitatum through anti-inflammatory, analgesic and antibacterial pharmacological experiments.
  • the invention achieves the above object through the following technical solutions:
  • the invention provides an effective component (effective part) of Polygonum capitatum, which is obtained by the following steps: (1) adding 65-75% ethanol in an amount of 6-8 times the amount of the fresh or dry aerial part of Polygonum capitatum to reflux extract twice, each time for about 1.5 hours), filtering, concentrating and drying the filtrate to obtain an alcohol extract; (2) suspending the alcohol extract in methanol, ultrasonically treating for about 1.5 hours, centrifuging, and loading the supernatant onto an MCI macroporous resin (MCI GEL CHP 20P) column (the amount of resin used for every 100g of extract is about 2 liters); (3) gradient eluting with water, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, and 100% methanol in sequence (for example, the amount of each solvent used is about 1 column volume), collecting the eluates of each part, recovering the solvent, and drying the parts obtained by eluting with different solvents to obtain methanol eluates at any concentration interval or concentration point
  • the methanol eluate obtained in the above step (3) may be a 50% methanol eluate (part E); the methanol eluate obtained in the above step (3) may be a 50%-60% methanol eluate (part F); the methanol eluate obtained in the above step (3) may be a 60%-80% methanol eluate (part G); the methanol eluate obtained in the above step (3) may be a mixture of 50%-80% methanol eluates (part Y).
  • the part Y may be a mixture of dried products from parts E to G, or may be a dried product of a mixed solution obtained by eluting with 50%-80% methanol after solvent removal and drying.
  • the fraction E obtained in the above step (3) contains lignans and/or flavonoid glycosides, for example, a mixture of lignans and flavonoid glycosides; the fraction F obtained in the above step (3) contains flavonoid glycosides; the fraction G obtained in the above step (3) contains flavonoid aglycones; the fraction Y obtained in the above step (3) contains lignans and/or flavonoid glycosides and/or flavonoid aglycones, for example, quercetin-3-O- ⁇ -D-pyranoglucoside, quercetin-pyranoglucoside, punicalagin B/carpinusin, quercetin, and kaempferol 3-(2-galloyl glucoside).
  • the effective component (effective part) of Polygonum capitatum provided by the present invention was used in an experiment on the effect of xylene on mouse ear swelling.
  • the extract obtained from 5g of Polygonum capitatum dry product/kg body weight was administered by gavage for 5 consecutive days, and the experiment was conducted 60 minutes after the last administration.
  • the results showed that the effective component had a significant anti-inflammatory effect, and the highest inflammation inhibition rate could reach 56% (effective part Y); in the mouse hot plate pain experiment, the effective component could prolong the latency time of the mouse hot plate licking reaction (compared with the control group, P ⁇ 0.05), and the highest delay rate could reach 16.6% (effective part Y).
  • the effective part obtained by the method of the present invention can significantly enhance the anti-inflammatory effect compared with the water extract.
  • the preparation method of "Polygonum capitatum water extract” is: decoct the dried Polygonum capitatum with 10 times water (W/W) twice, each time for 1.5 hours, filter, combine the filtrate, concentrate the filtrate to a relative density of 1.1 at 20°C, and spray-dry the concentrate to obtain fine powder of more than 100 mesh.
  • the Polygonum capitatum water extract is also used as a control test drug in the following test examples.
  • each effective part is as follows: (1) adding 7 times the amount of 70% ethanol to the fresh or dried aerial part of Polygonum capitatum for reflux extraction twice, each time for about 1.5 hours, filtering, concentrating the filtrate, and drying to obtain an alcohol extract; (2) suspending the alcohol extract in methanol, ultrasonically treating for about 1.5 hours, centrifuging, and loading the supernatant onto an MCI macroporous resin (MCI GEL CHP 20P) column (the amount of resin used for every 100g of extract is about 2 liters); (3) gradient elution is performed in sequence with water, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, and 100% methanol (each solvent is used in an amount of about 1 column volume), collecting the eluates of each part, recovering the solvent, and drying the parts obtained by elution with different solvents to obtain a 50% methanol eluate as part E, a 50%-60% methanol eluate as part F, The 60%-80% m
  • the dosage of the test when expressed in g/kg, unless otherwise specified, it refers to the weight of the corresponding test drug (such as the extract of the present invention) converted into the dry product of Polygonum capitatum administered per kg of animal body weight.
  • the effective components of the Polygonum capitatum of the present invention can be combined with auxiliary materials acceptable in drug manufacturing to prepare various common preparations and sustained-release preparations, controlled-release preparations, targeted preparations and the like.
  • the yield of each part is basically the same as that in Preparation Example 1.
  • the inventors found that the use of methacrylate type MCI resin in the above Preparation Examples 1-3 could not obtain a good separation effect, and the yield of part Y was less than 5%.
  • mice SPF Kunming mice, weighing 18-22 g, both male and female.
  • Test drugs, dosage and administration method The various effective parts obtained from the above "Preparation Example 1: Effective components of Polygonum capitatum” and the control sample Polygonum capitatum water extract (see above) were administered at a dosage of 5 g of the corresponding test drug per kg of animal body weight, equivalent to the weight of Polygonum capitatum dry product. Each test drug was suspended in physiological saline before use to a concentration equivalent to 0.4 ml per oral administration to each animal, and a control group was set up to be administered with the same volume of physiological saline.
  • mice Both male and female mice were used and randomly divided into groups, with 10 animals in each test drug group.
  • the drugs were administered for 5 consecutive days. 60 minutes after the last administration, 0.1 ml of xylene was applied to the right ear of each mouse to induce inflammation.
  • the mice were killed 30 minutes later, and the left and right ear pieces were taken with a 6 mm diameter puncher. They were immediately weighed with an electronic balance, and the weight difference between the two ear pieces was used as the swelling degree (mg).
  • the inhibition rate (%) of each test drug group was calculated by the following formula:
  • Inhibition rate (%) [(swelling degree of control group - swelling degree of drug-treated group) ⁇ swelling degree of control group] ⁇ 100%
  • the inhibition rate is graded: an inhibition rate below 10% is grade A, an inhibition rate of 10-20% is grade AA, an inhibition rate of 20-30% is grade AAA, an inhibition rate of 30-40% is grade AAAA, an inhibition rate of 40-50% is grade AAAAA, and an inhibition rate greater than 50% is grade AAAAAA.
  • the results of the effects of different Polygonum capitatum samples on xylene-induced ear swelling in mice are as follows: the inhibition rate of site E is graded AAAA*, the inhibition rate of site F is graded AAAA**, the inhibition rate of site G is graded AAAAA**, the inhibition rate of site Y is graded AAAAA**, and the inhibition rate of Polygonum capitatum water extract is graded AAAA**.
  • Swelling degree (mg) compared with the animals in the solvent control group: * P ⁇ 0.05, ** P ⁇ 0.01.
  • the various parts of Preparation Example 2 and Preparation Example 3 were used as test drugs.
  • the UPLC-TOF-MS determination method is as follows:
  • test solution Preparation of test solution: Weigh an appropriate amount of Polygonum capitatum extract powder, add 70% methanol to make a suspension with a concentration of about 5 mg/ml, sonicate to dissolve as much as possible, dilute the suspension 10 times, filter, and inject 2 ⁇ l for mass spectrometry detection;
  • Chromatographic column Acquity BEH C18 column (2.1 ⁇ 100 mm, 1.7 ⁇ m), column temperature: 40°C; flow rate: 0.35ml/min; injection volume: 2 ⁇ l; mass spectrometry conditions: ion source: ESI source; drying gas temperature: 180°C; capillary voltage: 4500eV; detection mode: negative ion mode; spray pressure: 2.5bar; drying gas (N2) flow rate: 8L/min; scanning range: 100-2000amu; collision energy: 10ev; 0.1% formic acid aqueous solution as mobile phase A, 0.1% formic acid acetonitrile solution as mobile phase B, gradient elution according to the following program: 95%A at 0 min, 95%A at 0.5 min, 81.5%A at 20 min, 0%A at 28 min, 0%A at 30 min, 95%A at 30.1 min, 95%A at 32 min.
  • the effective parts of Polygonum capitatum provided by the present invention include the following part substances and characterization data: peak 16 has a t R of 13.3 min, a molecular formula of C 21 H 20 O 12 , a measured mass ( m / z , [MH] - )463.0858, a theoretical mass ( m / z , [MH] - )463.0882, and the compound is quercetin-3-O- ⁇ -D-pyranoglucoside; peak 18 has a t R of 14.3 min, a molecular formula of C 21 H 20 O 12 , a measured mass ( m / z , [MH] - )463.0852, a theoretical mass ( m / z , [MH] - )463.0882, and the compound is quercetin-pyranoglucoside (isomer of 16); peak 19 has a t R of 14.9 min, a molecular formula of C 27 H 36 O 12 ,
  • the 50% methanol eluate is fraction E (yield 2.84%)
  • the 50%-60% methanol eluate is fraction F (yield 2.26%)
  • the 60%-80% methanol eluate is fraction G (yield 6.24%)
  • the 50%-80% methanol eluate is fraction Y (yield 10.42%).
  • the 50% methanol eluate is fraction E (yield 2.88%), the 50%-60% methanol eluate is fraction F (yield 2.04%), the 60%-80% methanol eluate is fraction G (yield 6.32%), and the 50%-80% methanol eluate is fraction Y (yield 10.98%).
  • the UPLC-TOF-MS determination method is as follows:
  • test solution Preparation of test solution: Weigh an appropriate amount of Polygonum capitatum extract powder, add 70% methanol to make a suspension with a concentration of about 5 mg/ml, sonicate to dissolve as much as possible, dilute the suspension 10 times, filter, and inject 2 ⁇ l for mass spectrometry detection;
  • Chromatographic column Acquity BEH C18 column (2.1 ⁇ 100 mm, 1.7 ⁇ m), column temperature: 40 °C; flow rate: 0.35 ml/min; injection volume: 2 ⁇ l; mass spectrometry conditions: ion source: ESI source; drying gas temperature: 180 °C; capillary voltage: 4500 eV; detection mode: negative ion mode; spray pressure: 2.5 bar; drying gas (N2) flow rate: 8 L/min; scanning range: 100-2000 amu; collision energy: 10 ev; 0.1% formic acid aqueous solution as mobile phase A, 0.1% formic acid acetonitrile solution as mobile phase B, gradient elution according to the following program: 95% A at 0 min, 95% A at 0.5 min, 81.5% A at 20 min, 0% A at 28 min, 0% A at 30 min, 0% A at 30.1 min, 95% A at 32 min.
  • Test Example 4 Antibacterial test of the Polygonum capitatum extract obtained in Preparation Examples 11 to 13

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Abstract

一种头花蓼提取物在制备清热利湿、利尿通淋、治疗泌尿***感染或结石、治疗肾盂肾炎、解毒、散瘀、抗菌、抗炎、镇痛、抗***炎的热淋清颗粒中的用途，所述头花蓼提取物是按照以下步骤制备得到的：将头花蓼地上部分鲜品或干品加乙醇回流提取，过滤，使滤液浓缩、干燥、得醇浸膏；使醇浸膏混悬于甲醇中，超声处理，离心，将上清液加载到打孔树脂柱上；用水、甲醇依次进行梯度洗脱，收集各部分洗脱液，回收溶剂，由不同溶剂洗脱获得的部位干燥，得到甲醇洗脱物及其混合物。

Description

改进热淋清颗粒生物学活性的方法 技术领域
本发明属于中药领域，涉及一种用于制备中药热淋清颗粒的药材头花蓼的提取物的方法。具体地，本发明涉及由热淋清的原料即药材头花蓼的醇提取物所获得的有效部位，以及该有效部位的制备方法，还涉及所述有效部位的用途。尤其是，本发明制备热淋清颗粒的方法能够使得制备的热淋清颗粒生物学活性得以显著提高。
 背景技术
作为热淋清颗粒的原料药材，头花蓼( Polygonum capitatum Buch-Ham ex D. Don)为蓼科蓼属多年生草本植物。具有清热利湿、利尿通淋之功效，在临床上治疗泌尿***感染有着显著的效果。以其为原料制成的中成药制剂“热淋清颗粒”2004年进入国家基本医疗保险目录，2012年进入贵州省基本药物目录。
头花蓼是少数民族地区的常用药，主要用于肾盂肾炎、尿道感染、利尿通淋等症。相关的药理学研究少见，目前仅有任光友等几篇报道。任光友采用大鼠细菌性肾盂肾炎模型进行实验，结果头花蓼水提取物组大鼠尿液中的WBC和BLD与对照组比较明显减少，显示头花蓼水提物对肾盂肾炎具有一定的抗炎作用。任光友等以小鼠腹腔注射大肠杆菌菌液观察5天内小鼠死亡情况，结果对照组死亡率为100%，头花蓼组死亡率分别为20%和50%，显示头花蓼水提物能够对抗大肠杆菌引起的感染。任光友等以头花蓼水提物灌胃给药予家兔，结果，头花蓼水提物组与对照组比较， 体温无显著性差异 ，但能降低静脉注射伤寒副伤寒菌苗引起的家兔的发热体温。任光友等，以头花蓼水提物分别灌胃给药家兔、大鼠，与空白组和速尿对照组比较尿量。结果显示头花蓼水提物对家兔和大鼠无明显利尿作用。徐英春等人采用琼脂稀释法检测了头花蓼对10株淋病奈瑟球菌(***)的体外抑菌活性，结果头花蓼对***有抑菌活性。其对10株***的最小抑菌浓度范围为8~32g/L，平均值为11.2g/L。
中国专利申请公开说明书CN1054899A(中国专利申请号90107810.7，公开日1991年10月2日)中公开了一种泌感灵冲剂、糖浆生产工艺。该生产工艺是以四季草为原料用水煎煮30-60分钟，提取液过滤浓缩，将其上清液减压浓缩成膏，再用60-70%乙醇提取，将乙醇提取液真空干燥，得四季红浸膏，进一步配制成冲剂或糖浆剂，据信其具有解毒、散瘀、利尿、通淋的功效。
中华人民共和国***药典委员会1998年编的中华人民共和国***《药品标准—中药成方制剂》(第十七册)中收载了名为“热淋清颗粒”的中成药制剂，其是通过将头花蓼加水煎煮两次后过滤浓缩制成的。
CN 1481832A (中国专利申请号02129686.3，公开日2004年3月17日)和CN 1483466A (中国专利申请号03146381.9，公开日2004年3月24日)公开了头花蓼提取物，其基本上是由以下步骤制备的：a.由头花蓼全草的鲜品或干品用水分两次煎煮或者用醇水混合物分二至三次回流提取，每次1-2小时，合并煎煮液，过滤浓缩至20°C时的相对密度为1.2，喷雾干燥或减压干燥得到；或者，b.由头花蓼全草及其水提药渣经二氧化碳超临界萃取得到。据信此提取物可用于抗菌、抗炎、镇痛、利尿、治疗泌尿***结石、治疗肾盂肾炎以及***炎。
 技术问题
尽管目前有诸多关于制备头花蓼提取物的报道，然而本领域技术人员仍然期待从头花蓼获得有效的产品。尤其是赋予头花蓼提取物显著更优的生物学活性。
 技术解决方案
本发明目的是期待从头花蓼获得一种有效的产品。本发明人发现，使用醇提取头花蓼，得到的提取物经特定的大孔树脂洗脱所获得的洗脱物，其在生物活性方面具有令人期待的效果，尤其是生物学活性得以显著改善。本发明基于此发现而得以完成。
为此，本发明第一方面提供了一种头花蓼提取物，其基本上由包括以下步骤的方法制备得到：
(1)将头花蓼地上部分鲜品或干品加5-15倍量(例如5-12倍量，例如6-10倍量)的50~90%(例如60~80%，例如65~75%，例如约70%)乙醇回流提取1-3次(例如2次)，每次1-3小时(例如约1.5小时)，过滤，使滤液浓缩、干燥，得醇浸膏；所述乙醇中还包含0.75%乙酸；
(2)使醇浸膏混悬于甲醇中，超声处理0.5~5小时(例如0.5~3小时，例如1~2小时，例如约1.5小时)，离心，将上清液加载到MCI大孔树脂柱(例如，每100g浸膏使用树脂的量为0.5-4升，例如1-3升，例如1.5-2.5升，例如2升)上；
(3)用水，10%、20%、30%、35%、40%、45%、50%、60%、70%、80%、100%甲醇依次进行梯度洗脱(例如每种溶剂用量为0.5~5倍柱体积，例如0.5~2.5倍柱体积，例如0.5~2倍柱体积，例如1倍柱体积)，收集各部分洗脱液，回收溶剂，由不同溶剂洗脱获得的部位干燥，得到50%-80%之间任意浓度间隔或浓度点的甲醇洗脱物，或者它们的混合物，即得。
根据本发明第一方面的头花蓼提取物，其中步骤(3)所得甲醇洗脱物是50%甲醇洗脱物(在本发明中可简称为：部位E)。
根据本发明第一方面的头花蓼提取物，其中步骤(3)所得甲醇洗脱物是50%-60%甲醇洗脱物(在本发明中可简称为：部位F)。
根据本发明第一方面的头花蓼提取物，其中步骤(3)所得甲醇洗脱物是60%-80%甲醇洗脱物(在本发明中可简称为：部位G)。
根据本发明第一方面的头花蓼提取物，其中步骤(3)所得甲醇洗脱物是50%-80%之间甲醇洗脱物的混合物(在本发明中可简称为：部位Y)。在本发明中，该部位Y可以是部位E至部位G干燥品的混合物，亦可以是50%-80%甲醇洗脱所得混合液经除溶剂干燥后的干燥品。
根据本发明第一方面的头花蓼提取物，其中部位E中含有木脂素和/或黄酮苷类，例如含有木脂素和黄酮苷混合物。
根据本发明第一方面的头花蓼提取物，其中部位F中含有黄酮苷类。
根据本发明第一方面的头花蓼提取物，其中部位G中含有黄酮苷元类。
根据本发明第一方面的头花蓼提取物，其中部位Y中含有木脂素和/或黄酮苷类和/或黄酮苷元类，例如槲皮素-3-O-β-D-吡喃葡萄糖苷、槲皮素-吡喃葡萄糖苷、石榴皮素B/carpinusin、槲皮苷、山奈酚3-(2-没食子酰基葡萄糖苷)。
根据本发明第一方面的头花蓼提取物，其照超高效液相色谱-飞行时间-质谱联用法(在本发明中可简称为UPLC-TOF-MS)测定，结果：
(e)在保留时间12.8-13.8min之间(例如约13.3min处)和保留时间14.8-15.8min之间(例如约15.3min处)显示黄酮苷类(例如槲皮素-3-O-β-D-吡喃葡萄糖苷、石榴皮素B/carpinusin)的色谱峰，和/或在保留时间14.4-15.4min之间(例如约14.9min处)显示木脂素(例如nudiposide)的色谱峰；
(f)在保留时间12.8-13.8min之间(例如约13.3min处)、保留时间13.8-14.8min之间(例如约14.3min处)和保留时间14.8-15.8min之间(例如约15.3min处)显示黄酮苷类(例如槲皮素-3-O-β-D-吡喃葡萄糖苷、槲皮素-吡喃葡萄糖苷、石榴皮素B/carpinusin)的色谱峰；
(g)在保留时间16.6-17.6min之间(例如约17.1min处)和保留时间21.4-22.4min之间(例如约21.9min处)显示黄酮苷元(例如槲皮苷、山奈酚3-(2-没食子酰基葡萄糖苷))的色谱峰；和/或
(y)显示以上(e)至(g)中所示任一或全部色谱峰；
其中，UPLC-TOF-MS的测定方法如下：
(i)供试液配制：称取适量头花蓼提取物粉末, 加入70%甲醇制成浓度约5mg/ml的混悬液，超声使尽量溶解，混悬液稀释10倍，过滤，进样2μl作质谱检测；
(ii)色谱及质谱分析条件：
色谱柱：Acquity BEH C18 柱 (2.1 × 100 mm, 1.7μm)， 柱温：40℃；流速：0.35ml/min；进样量：2μl；质谱条件：离子源：ESI源；干燥气体温度：180℃；毛细管电压：4500eV；检测模式：负离子模式；喷雾压力：2.5bar；干燥气(N2)流速：8L/min；扫描范围：100-2000amu；碰撞能量: 10ev；以0.1%甲酸水溶液为流动相A，0.1%甲酸乙腈溶液为流动相B，按以下规定的程序进行梯度洗脱：0 min时95%A、0.5 min时95%A、20 min时81.5%A、28 min时0%A、30 min时0%A、30.1 min时95%A、32 min时95%A。
根据本发明第一方面的头花蓼提取物，其照所述超高效液相色谱-飞行时间-质谱联用法测定，结果：(e)在保留时间12.8-13.8min之间(例如约13.3min处)和保留时间14.8-15.8min之间(例如约15.3min处)显示黄酮苷类(例如槲皮素-3-O-β-D-吡喃葡萄糖苷、石榴皮素B/carpinusin)的色谱峰，和/或在保留时间14.4-15.4min之间(例如约14.9min处)显示木脂素(例如nudiposide)的色谱峰。
根据本发明第一方面的头花蓼提取物，其照所述超高效液相色谱-飞行时间-质谱联用法测定，结果：(f)在保留时间12.8-13.8min之间(例如约13.3min处)、保留时间13.8-14.8min之间(例如约14.3min处)和保留时间14.8-15.8min之间(例如约15.3min处)显示黄酮苷类(例如槲皮素-3-O-β-D-吡喃葡萄糖苷、槲皮素-吡喃葡萄糖苷、石榴皮素B/carpinusin)的色谱峰。
根据本发明第一方面的头花蓼提取物，其照所述超高效液相色谱-飞行时间-质谱联用法测定，结果：(g)在保留时间16.6-17.6min之间(例如约17.1min处)和保留时间21.4-22.4min之间(例如约21.9min处)显示黄酮苷元(例如槲皮苷、山奈酚3-(2-没食子酰基葡萄糖苷))的色谱峰。
根据本发明第一方面的头花蓼提取物，其照所述超高效液相色谱-飞行时间-质谱联用法测定，结果：
(e)在保留时间12.8-13.8min之间(例如约13.3min处)和保留时间14.8-15.8min之间(例如约15.3min处)显示黄酮苷类(例如槲皮素-3-O-β-D-吡喃葡萄糖苷、石榴皮素B/carpinusin)的色谱峰，和/或在保留时间14.4-15.4min之间(例如约14.9min处)显示木脂素(例如nudiposide)的色谱峰；
(f)在保留时间12.8-13.8min之间(例如约13.3min处)、保留时间13.8-14.8min之间(例如约14.3min处)和保留时间14.8-15.8min之间(例如约15.3min处)显示黄酮苷类(例如槲皮素-3-O-β-D-吡喃葡萄糖苷、槲皮素-吡喃葡萄糖苷、石榴皮素B/carpinusin)的色谱峰；和/或
(g)在保留时间16.6-17.6min之间(例如约17.1min处)和保留时间21.4-22.4min之间(例如约21.9min处)显示黄酮苷元(例如槲皮苷、山奈酚3-(2-没食子酰基葡萄糖苷))的色谱峰。
根据本发明第一方面的头花蓼提取物，其具有本发明第二方面任一实施方案所述特征。
本发明第二方面提供了一种头花蓼提取物，其照超高效液相色谱-飞行时间-质谱联用法(在本发明中可简称为UPLC-TOF-MS)测定，结果：
(e)在保留时间12.8-13.8min之间(例如约13.3min处)和保留时间14.8-15.8min之间(例如约15.3min处)显示黄酮苷类(例如槲皮素-3-O-β-D-吡喃葡萄糖苷、石榴皮素B/carpinusin)的色谱峰，和/或在保留时间14.4-15.4min之间(例如约14.9min处)显示木脂素(例如nudiposide)的色谱峰；
(f)在保留时间12.8-13.8min之间(例如约13.3min处)、保留时间13.8-14.8min之间(例如约14.3min处)和保留时间14.8-15.8min之间(例如约15.3min处)显示黄酮苷类(例如槲皮素-3-O-β-D-吡喃葡萄糖苷、槲皮素-吡喃葡萄糖苷、石榴皮素B/carpinusin)的色谱峰；
(g)在保留时间16.6-17.6min之间(例如约17.1min处)和保留时间21.4-22.4min之间(例如约21.9min处)显示黄酮苷元(例如槲皮苷、山奈酚3-(2-没食子酰基葡萄糖苷))的色谱峰；和/或
(y)显示以上(e)至(g)中所示任一或全部色谱峰；
其中，UPLC-TOF-MS的测定方法如下：
(i)供试液配制：称取适量头花蓼提取物粉末, 加入70%甲醇制成浓度约5mg/ml的混悬液，超声使尽量溶解，混悬液稀释10倍，过滤，进样2μl作质谱检测；
(ii)色谱及质谱分析条件：
色谱柱：Acquity BEH C18 柱 (2.1 × 100 mm, 1.7μm)， 柱温：40℃；流速：0.35ml/min；进样量：2μl；质谱条件：离子源：ESI源；干燥气体温度：180℃；毛细管电压：4500eV；检测模式：负离子模式；喷雾压力：2.5bar；干燥气(N2)流速：8L/min；扫描范围：100-2000amu；碰撞能量: 10ev；以0.1%甲酸水溶液为流动相A，0.1%甲酸乙腈溶液为流动相B，按以下规定的程序进行梯度洗脱：0 min时95%A、0.5 min时95%A、20 min时81.5%A、28 min时0%A、30 min时0%A、30.1 min时95%A、32 min时95%A。
根据本发明第二方面的头花蓼提取物，其照所述超高效液相色谱-飞行时间-质谱联用法测定，结果：(e)在保留时间12.8-13.8min之间(例如约13.3min处)和保留时间14.8-15.8min之间(例如约15.3min处)显示黄酮苷类(例如槲皮素-3-O-β-D-吡喃葡萄糖苷、石榴皮素B/carpinusin)的色谱峰，和/或在保留时间14.4-15.4min之间(例如约14.9min处)显示木脂素(例如nudiposide)的色谱峰。
根据本发明第二方面的头花蓼提取物，其照所述超高效液相色谱-飞行时间-质谱联用法测定，结果：(f)在保留时间12.8-13.8min之间(例如约13.3min处)、保留时间13.8-14.8min之间(例如约14.3min处)和保留时间14.8-15.8min之间(例如约15.3min处)显示黄酮苷类(例如槲皮素-3-O-β-D-吡喃葡萄糖苷、槲皮素-吡喃葡萄糖苷、石榴皮素B/carpinusin)的色谱峰。
根据本发明第二方面的头花蓼提取物，其照所述超高效液相色谱-飞行时间-质谱联用法测定，结果：(g)在保留时间16.6-17.6min之间(例如约17.1min处)和保留时间21.4-22.4min之间(例如约21.9min处)显示黄酮苷元(例如槲皮苷、山奈酚3-(2-没食子酰基葡萄糖苷))的色谱峰。
根据本发明第二方面的头花蓼提取物，其照所述超高效液相色谱-飞行时间-质谱联用法测定，结果：
(e)在保留时间12.8-13.8min之间(例如约13.3min处)和保留时间14.8-15.8min之间(例如约15.3min处)显示黄酮苷类(例如槲皮素-3-O-β-D-吡喃葡萄糖苷、石榴皮素B/carpinusin)的色谱峰，和/或在保留时间14.4-15.4min之间(例如约14.9min处)显示木脂素(例如nudiposide)的色谱峰；
(f)在保留时间12.8-13.8min之间(例如约13.3min处)、保留时间13.8-14.8min之间(例如约14.3min处)和保留时间14.8-15.8min之间(例如约15.3min处)显示黄酮苷类(例如槲皮素-3-O-β-D-吡喃葡萄糖苷、槲皮素-吡喃葡萄糖苷、石榴皮素B/carpinusin)的色谱峰；和/或
(g)在保留时间16.6-17.6min之间(例如约17.1min处)和保留时间21.4-22.4min之间(例如约21.9min处)显示黄酮苷元(例如槲皮苷、山奈酚3-(2-没食子酰基葡萄糖苷))的色谱峰。
根据本发明第二方面的头花蓼提取物，其基本上由包括以下步骤的方法制备得到：
(1)将头花蓼地上部分鲜品或干品加5-15倍量(例如5-12倍量，例如6-10倍量)的50~90%(例如60~80%，例如65~75%，例如约70%)乙醇回流提取1-3次(例如2次)，每次1-3小时(例如约1.5小时)，过滤，使滤液浓缩、干燥，得醇浸膏；所述乙醇中还包含0.75%乙酸；
(2)使醇浸膏混悬于甲醇中，超声处理0.5~5小时(例如0.5~3小时，例如1~2小时，例如约1.5小时)，离心，将上清液加载到MCI大孔树脂柱(例如，每100g浸膏使用树脂的量为0.5-4升，例如1-3升，例如1.5-2.5升，例如2升)上；
(3)用水，10%、20%、30%、35%、40%、45%、50%、60%、70%、80%、100%甲醇依次进行梯度洗脱(例如每种溶剂用量为0.5~5倍柱体积，例如0.5~2.5倍柱体积，例如0.5~2倍柱体积，例如1倍柱体积)，收集各部分洗脱液，回收溶剂，由不同溶剂洗脱获得的部位干燥，得到50%-80%之间任意浓度间隔或浓度点的甲醇洗脱物，或者它们的混合物，即得。
根据本发明第二方面的头花蓼提取物，其中步骤(3)所得甲醇洗脱物是50%甲醇洗脱物(在本发明中可简称为：部位E)。
根据本发明第二方面的头花蓼提取物，其中步骤(3)所得甲醇洗脱物是50%-60%甲醇洗脱物(在本发明中可简称为：部位F)。
根据本发明第二方面的头花蓼提取物，其中步骤(3)所得甲醇洗脱物是60%-80%甲醇洗脱物(在本发明中可简称为：部位G)。
根据本发明第二方面的头花蓼提取物，其中步骤(3)所得甲醇洗脱物是50%-80%之间甲醇洗脱物的混合物(在本发明中可简称为：部位Y)。在本发明中，该部位Y可以是部位E至部位G干燥品的混合物，亦可以是50%-80%甲醇洗脱所得混合液经除溶剂干燥后的干燥品。
根据本发明第二方面的头花蓼提取物，其中部位E中含有木脂素和/或黄酮苷类，例如含有木脂素和黄酮苷混合物。
根据本发明第二方面的头花蓼提取物，其中部位F中含有黄酮苷类。
根据本发明第二方面的头花蓼提取物，其中部位G中含有黄酮苷元类。
根据本发明第二方面的头花蓼提取物，其中部位Y中含有木脂素和/或黄酮苷类和/或黄酮苷元类，例如槲皮素-3-O-β-D-吡喃葡萄糖苷、槲皮素-吡喃葡萄糖苷、石榴皮素B/carpinusin、槲皮苷、山奈酚3-(2-没食子酰基葡萄糖苷)。
根据本发明第二方面的头花蓼提取物，其具有本发明第一方面任一实施方案所述特征。
本发明第三方面提供了改进热淋清颗粒的清热利湿、利尿通淋、泌尿***感染或结石、肾盂肾炎、解毒、散瘀、抗菌、抗炎、镇痛、抗***炎的生物学活性的方法，该方法包括给有需要的受试者施用治疗有效量的头花蓼提取物，所述头花蓼提取物是按照以下步骤制备得到的：
(1)将头花蓼地上部分鲜品或干品加5-15倍量(例如5-12倍量，例如6-10倍量)的50~90%(例如60~80%，例如65~75%，例如约70%)乙醇回流提取1-3次(例如2次)，每次1-3小时(例如约1.5小时)，过滤，使滤液浓缩、干燥，得醇浸膏；所述乙醇中还包含0.75%乙酸；
(2)使醇浸膏混悬于甲醇中，超声处理0.5~5小时(例如0.5~3小时，例如1~2小时，例如约1.5小时)，离心，将上清液加载到MCI大孔树脂柱(例如，每100g浸膏使用树脂的量为0.5-4升，例如1-3升，例如1.5-2.5升，例如2升)上；
(3)用水，10%、20%、30%、35%、40%、45%、50%、60%、70%、80%、100%甲醇依次进行梯度洗脱(例如每种溶剂用量为0.5~5倍柱体积，例如0.5~2.5倍柱体积，例如0.5~2倍柱体积，例如1倍柱体积)，收集各部分洗脱液，回收溶剂，由不同溶剂洗脱获得的部位干燥，得到50%-80%之间任意浓度间隔或浓度点的甲醇洗脱物，或者它们的混合物，即得。
根据本发明第三方面的方法，其中步骤(3)所得甲醇洗脱物是50%甲醇洗脱物(在本发明中可简称为：部位E)。
根据本发明第三方面的方法，其中步骤(3)所得甲醇洗脱物是50%-60%甲醇洗脱物(在本发明中可简称为：部位F)。
根据本发明第三方面的方法，其中步骤(3)所得甲醇洗脱物是60%-80%甲醇洗脱物(在本发明中可简称为：部位G)。
根据本发明第三方面的方法，其中步骤(3)所得甲醇洗脱物是50%-80%之间甲醇洗脱物的混合物(在本发明中可简称为：部位Y)。在本发明中，该部位Y可以是部位E至部位G干燥品的混合物，亦可以是50%-80%甲醇洗脱所得混合液经除溶剂干燥后的干燥品。
根据本发明第三方面的方法，其中部位E中含有木脂素和/或黄酮苷类，例如含有木脂素和黄酮苷混合物。
根据本发明第三方面的方法，其中部位F中含有黄酮苷类。
根据本发明第三方面的方法，其中部位G中含有黄酮苷元类。
根据本发明第三方面的方法，其中部位Y中含有木脂素和/或黄酮苷类和/或黄酮苷元类，例如槲皮素-3-O-β-D-吡喃葡萄糖苷、槲皮素-吡喃葡萄糖苷、石榴皮素B/carpinusin、槲皮苷、山奈酚3-(2-没食子酰基葡萄糖苷)。
根据本发明第三方面的方法，其中所得头花蓼提取物具有本发明第一方面或第二方面任一实施方案所述特征。
根据本发明第三方面的方法，其中所述头花蓼提取物照超高效液相色谱-飞行时间-质谱联用法(在本发明中可简称为UPLC-TOF-MS)测定，结果：
(e)在保留时间12.8-13.8min之间(例如约13.3min处)和保留时间14.8-15.8min之间(例如约15.3min处)显示黄酮苷类(例如槲皮素-3-O-β-D-吡喃葡萄糖苷、石榴皮素B/carpinusin)的色谱峰，和/或在保留时间14.4-15.4min之间(例如约14.9min处)显示木脂素(例如nudiposide)的色谱峰；
(f)在保留时间12.8-13.8min之间(例如约13.3min处)、保留时间13.8-14.8min之间(例如约14.3min处)和保留时间14.8-15.8min之间(例如约15.3min处)显示黄酮苷类(例如槲皮素-3-O-β-D-吡喃葡萄糖苷、槲皮素-吡喃葡萄糖苷、石榴皮素B/carpinusin)的色谱峰；
(g)在保留时间16.6-17.6min之间(例如约17.1min处)和保留时间21.4-22.4min之间(例如约21.9min处)显示黄酮苷元(例如槲皮苷、山奈酚3-(2-没食子酰基葡萄糖苷))的色谱峰；和/或
(y)显示以上(e)至(g)中所示任一或全部色谱峰；
其中，UPLC-TOF-MS的测定方法如下：
(i)供试液配制：称取适量头花蓼提取物粉末, 加入70%甲醇制成浓度约5mg/ml的混悬液，超声使尽量溶解，混悬液稀释10倍，过滤，进样2μl作质谱检测；
(ii)色谱柱：Acquity BEH C18 柱 (2.1 × 100 mm, 1.7μm)， 柱温：40℃；流速：0.35ml/min；进样量：2μl；质谱条件：离子源：ESI源；干燥气体温度：180℃；毛细管电压：4500eV；检测模式：负离子模式；喷雾压力：2.5bar；干燥气(N2)流速：8L/min；扫描范围：100-2000amu；碰撞能量: 10ev；以0.1%甲酸水溶液为流动相A，0.1%甲酸乙腈溶液为流动相B，按以下规定的程序进行梯度洗脱：0 min时95%A、0.5 min时95%A、20 min时81.5%A、28 min时0%A、30 min时0%A、30.1 min时95%A、32 min时95%A。
根据本发明第三方面的方法，其中所述头花蓼提取物照所述超高效液相色谱-飞行时间-质谱联用法测定，结果：(e)在保留时间12.8-13.8min之间(例如约13.3min处)和保留时间14.8-15.8min之间(例如约15.3min处)显示黄酮苷类(例如槲皮素-3-O-β-D-吡喃葡萄糖苷、石榴皮素B/carpinusin)的色谱峰，和/或在保留时间14.4-15.4min之间(例如约14.9min处)显示木脂素(例如nudiposide)的色谱峰。
根据本发明第三方面的方法，其中所述头花蓼提取物照所述超高效液相色谱-飞行时间-质谱联用法测定，结果：(f)在保留时间12.8-13.8min之间(例如约13.3min处)、保留时间13.8-14.8min之间(例如约14.3min处)和保留时间14.8-15.8min之间(例如约15.3min处)显示黄酮苷类(例如槲皮素-3-O-β-D-吡喃葡萄糖苷、槲皮素-吡喃葡萄糖苷、石榴皮素B/carpinusin)的色谱峰。
根据本发明第三方面的方法，其中所述头花蓼提取物照所述超高效液相色谱-飞行时间-质谱联用法测定，结果：(g)在保留时间16.6-17.6min之间(例如约17.1min处)和保留时间21.4-22.4min之间(例如约21.9min处)显示黄酮苷元(例如槲皮苷、山奈酚3-(2-没食子酰基葡萄糖苷))的色谱峰。
根据本发明第三方面的方法，其中所述头花蓼提取物照所述超高效液相色谱-飞行时间-质谱联用法测定，结果：(e)在保留时间12.8-13.8min之间(例如约13.3min处)和保留时间14.8-15.8min之间(例如约15.3min处)显示黄酮苷类(例如槲皮素-3-O-β-D-吡喃葡萄糖苷、石榴皮素B/carpinusin)的色谱峰，和/或在保留时间14.4-15.4min之间(例如约14.9min处)显示木脂素(例如nudiposide)的色谱峰；(f)在保留时间12.8-13.8min之间(例如约13.3min处)、保留时间13.8-14.8min之间(例如约14.3min处)和保留时间14.8-15.8min之间(例如约15.3min处)显示黄酮苷类(例如槲皮素-3-O-β-D-吡喃葡萄糖苷、槲皮素-吡喃葡萄糖苷、石榴皮素B/carpinusin)的色谱峰；和/或，(g)在保留时间16.6-17.6min之间(例如约17.1min处)和保留时间21.4-22.4min之间(例如约21.9min处)显示黄酮苷元(例如槲皮苷、山奈酚3-(2-没食子酰基葡萄糖苷))的色谱峰。
本发明第四方面提供本发明第一方面或第二方面所述头花蓼提取物在制备清热利湿用药，利尿通淋用药，泌尿***感染或结石用药，肾盂肾炎用药，解毒、散瘀用药，抗菌、抗炎、镇痛用药，***炎用药的药物中的用途。
本发明第五方面提供了一种药物组合物，其中包含本发明第一方面或第二方面任一实施方案所述的头花蓼提取物以及任选的药学可接受的载体。
根据本发明第五方面的药物组合物，其呈口服或注射给药的制剂形式。在一个实施方案中，所述药物组合物呈片剂、胶囊剂、颗粒剂、丸剂、口服液剂、注射剂(水针和/或粉针)等的形式。
本发明任一方面或该任一方面的任一实施方案所具有的任一技术特征同样适用其它任一实施方案或其它任一方面的任一实施方案，只要它们不会相互矛盾，当然在相互之间适用时，必要的话可对相应特征作适当修饰。下面对本发明的各个方面和特点作进一步的描述。
本发明所引述的所有文献，它们的全部内容通过引用并入本文，并且如果这些文献所表达的含义与本发明不一致时，以本发明的表述为准。此外，本发明使用的各种术语和短语具有本领域技术人员公知的一般含义，即便如此，本发明仍然希望在此对这些术语和短语作更详尽的说明和解释，提及的术语和短语如有与公知含义不一致的，以本发明所表述的含义为准。
在本发明中，本发明方法得到“头花蓼提取物”，该术语亦可称为本发明的“头花蓼有效组分”或者本发明的“头花蓼有效部位”。
在本发明中，MCI大孔树脂可以是是由日本三菱公司生产的MCI GEL系列，MCI GEL系列反相分离填料是在三菱化学Diaion和Sepabeads大孔吸附树脂基础上设计的，因为基于现代的HPLC高压液相色谱分离技术，较小的颗粒有更高的色谱分离性能，广泛地用于天然产物和发酵产物的分离。MCI大孔树脂有两种填料：聚苯乙烯和丙烯酸型，聚苯乙烯型的树脂粒经范围为4μm-100μm，甲基丙烯酸酯型树脂的粒经范围为4μm－31μm。此外MCI系列不仅有大孔型的树脂，还有阴离子型及阳离子型等型号树脂。MCI GEL 反相精细分离填料是一种树脂类填料，可用于分离皂苷类成分，一般用甲醇水或乙醇水洗脱。再者MCI一般用来去处极性小的成分中的叶绿素，对石油醚部分和氯仿部分去处叶绿素的效果很好。在本发明中，优选使用的MCI大孔树脂是聚苯乙烯型，其包括但不限于MCI GEL CHP 10M、MCI GEL CHP 5C、MCI GEL CHP 55A、MCI GEL CHP 55Y、MCI GEL CHP 20Y、MCI GEL CHP 20P、MCI GEL CHP 20SS。此外，在本发明中，如未另外特别说明，优选地使用到型号为MCI GEL CHP 20P（75~150μm）的大孔树脂，其是最为广泛使用的型号。它是一种多孔性的聚苯乙烯高聚物，它所显示的反相吸附作用广泛，除了对高极性的糖、氨基酸基本没有吸附作用外，对大多数次生代谢产物有不同程度吸附，这使得它一方面能有效地除去对水溶性干扰极大的糖，氨基酸，另一方面又能分离不同类型的化合物。
本发明目的在于克服现有技术的不足，提供一种高效低毒的头花蓼抗炎有效成分(有效部位)与应用。
本发明对头花蓼药效作用进行聚焦，弄清其作用特点同时对头花蓼不同提取部位的药效作用进行筛选，采用头花蓼不同提取部位的样品通过抗炎、镇痛、抗菌药理实验进行了头花蓼有效组分(有效部位)与药效作用特点的筛选。
在一个实施方案中，发明通过以下技术方案实现上述目的：
本发明提供了一种头花蓼有效组分(有效部位)，是由以下步骤获得：(1)将头花蓼地上部分鲜品或干品加6-8倍量的65~75%乙醇回流提取2次，每次约1.5小)，过滤，使滤液浓缩、干燥，得醇浸膏；(2)使醇浸膏混悬于甲醇中，超声处理约1.5小时)，离心，将上清液加载到MCI大孔树脂(MCI GEL CHP 20P)柱(每100g浸膏使用树脂的量为约2升)上；(3)用水，10%、20%、30%、35%、40%、45%、50%、60%、70%、80%、100%甲醇依次进行梯度洗脱(例如每种溶剂用量为约1倍柱体积)，收集各部分洗脱液，回收溶剂，由不同溶剂洗脱获得的部位干燥，得到50%-80%之间任意浓度间隔或浓度点的甲醇洗脱物，或者它们的混合物，即得。
以上步骤(3)所得甲醇洗脱物可以是50%甲醇洗脱物(部位E)；以上步骤(3)所得甲醇洗脱物可以是50%-60%甲醇洗脱物(部位F)；以上步骤(3)所得甲醇洗脱物可以是60%-80%甲醇洗脱物(部位G)；以上步骤(3)所得甲醇洗脱物可以是50%-80%之间甲醇洗脱物的混合物(部位Y)。在本发明中，该部位Y可以是部位E至部位G干燥品的混合物，亦可以是50%-80%甲醇洗脱所得混合液经除溶剂干燥后的干燥品。
以上步骤(3)所得部位E中含有木脂素和/或黄酮苷类，例如含有木脂素和黄酮苷混合物；以上步骤(3)所得部位F中含有黄酮苷类；以上步骤(3)所得部位G中含有黄酮苷元类；以上步骤(3)所得部位Y中含有木脂素和/或黄酮苷类和/或黄酮苷元类，例如槲皮素-3-O-β-D-吡喃葡萄糖苷、槲皮素-吡喃葡萄糖苷、石榴皮素B/carpinusin、槲皮苷、山奈酚3-(2-没食子酰基葡萄糖苷)。
本发明所提供的头花蓼有效组分(有效部位)，对二甲苯致小鼠耳肿胀的影响实验，按5g头花蓼干品所得提取物/kg体重灌胃给药，连续5天，末次给药60min后实验，有显著的抗炎作用，最高炎症抑制率可达56%(有效部位Y)；对于小鼠热板致痛实验，能延长小鼠热板添足反应潜伏时间(与对照组比较P<0.05)，最高延时率可达16.6%(有效部位Y)。
本发明方法获得的有效部位可以使抗炎作用比水提物明显增强。本发明提取物与水提工艺的提取物比较，能非常显著地提高其抗炎效果，其实验结果如下(n=10)：头花蓼水提物(剂量5 g/kg)肿胀抑制率29.2％、有效部位E(剂量2.5 g/kg)肿胀抑制率43.3％、有效部位F(剂量2.5 g/kg)肿胀抑制率37.2％、有效部位G(剂量2.5 g/kg)肿胀抑制率36.4％、有效部位Y(剂量2.5 g/kg)肿胀抑制率39.58％。
其中，“头花蓼水提物”制备方法是：将头花蓼干品用10倍水(W/W)分成二次煎煮，每次1.5小时，过滤，合并滤液，将滤液浓缩至20℃时相对密度为1.1，浓缩液喷雾干燥得100目以上细粉。该头花蓼水提物还用于下文试验例作为对照试药。
其中，各有效部位制备方法是：(1)将头花蓼地上部分鲜品或干品加7倍量的70%乙醇回流提取2次，每次约1.5小时，过滤，使滤液浓缩、干燥，得醇浸膏；(2)使醇浸膏混悬于甲醇中，超声处理约1.5小时，离心，将上清液加载到MCI大孔树脂(MCI GEL CHP 20P)柱(每100g浸膏使用树脂的量为约2升)上；(3)用水，10%、20%、30%、35%、40%、45%、50%、60%、70%、80%、100%甲醇依次进行梯度洗脱(每种溶剂用量为约1倍柱体积)，收集各部分洗脱液，回收溶剂，由不同溶剂洗脱获得的部位干燥，得到50%甲醇洗脱物为部位E，50%-60%甲醇洗脱物是部位F， 60%-80%甲醇洗脱物是部位G，50%-80%甲醇洗脱物是部位Y。
在本发明中，以g/kg表示试验的给药剂量时，如无另外说明，是指每kg动物体重给予相应试药(例如本发明提取物)折合成头花蓼干品的重量。
本发明头花蓼有效组分可以与药物制造上可以接受的辅料组合制备成各种常用制剂和缓释剂、控释剂、靶向制剂等。
 具体实施方式
为了能够更清楚地理解本发明的技术内容，特举以下实施例详细说明，但本发明的实施方式不限于此。
A、提取物制备例部分
制备例1：头花蓼有效组分
(1)将头花蓼地上部分干品10Kg，加7倍量的70%乙醇回流提取2次，每次约1.5小时，过滤，使滤液浓缩、干燥，得醇浸膏(885g)；(2)使醇浸膏(45g)混悬于甲醇中，超声处理约1.5小时，离心，将上清液加载到MCI大孔树脂(MCI GEL CHP 20P)柱(每100g浸膏使用树脂的量为约2升)上；(3)用水，10%、20%、30%、35%、40%、45%、50%、60%、70%、80%、100%甲醇依次进行梯度洗脱(每种溶剂用量为约1倍柱体积)，收集各部分洗脱液，回收溶剂，由不同溶剂洗脱获得的部位干燥，得到50%甲醇洗脱物为部位E(得率2.8%)，50%-60%甲醇洗脱物是部位F(得率2.0%)，60%-80%甲醇洗脱物是部位G(得率6.1%)，50%-80%甲醇洗脱物是部位Y(得率10.9%)。
在下文中，如无另外说明，所用部位E、F、G、Y试样是用本制备例1的产物。
制备例2：头花蓼有效组分
(1)将头花蓼地上部分干品10Kg，加6倍量的60%乙醇回流提取3次，每次约1小时，过滤，使滤液浓缩、干燥，得醇浸膏(935g)；(2)使醇浸膏(45g)混悬于甲醇中，超声处理约2小时，离心，将上清液加载到MCI大孔树脂(MCI GEL CHP 20P)柱(每100g浸膏使用树脂的量为约3升)上；(3)用水，10%、20%、30%、35%、40%、45%、50%、60%、70%、80%、100%甲醇依次进行梯度洗脱(每种溶剂用量为约2倍柱体积)，收集各部分洗脱液，回收溶剂，由不同溶剂洗脱获得的部位干燥，得到50%甲醇洗脱物为部位E，50%-60%甲醇洗脱物是部位F，60%-80%甲醇洗脱物是部位G，50%-80%甲醇洗脱物是部位Y。各部位的得率基本与制备例1相同。
制备例3：头花蓼有效组分
(1)将头花蓼地上部分干品10Kg，加10倍量的80%乙醇回流提取1次3小时，过滤，使滤液浓缩、干燥，得醇浸膏(818g)；(2)使醇浸膏(45g)混悬于甲醇中，超声处理约1小时，离心，将上清液加载到MCI大孔树脂(MCI GEL CHP 55A)柱(每100g浸膏使用树脂的量为约1升)上；(3)用水，10%、20%、30%、35%、40%、45%、50%、60%、70%、80%、100%甲醇依次进行梯度洗脱(每种溶剂用量为约0.5倍柱体积)，收集各部分洗脱液，回收溶剂，由不同溶剂洗脱获得的部位干燥，得到50%甲醇洗脱物为部位E，50%-60%甲醇洗脱物是部位F，60%-80%甲醇洗脱物是部位G，50%-80%甲醇洗脱物是部位Y。各部位的得率基本与制备例1相同。另外，发明人发现，在以上制备例1-3中使用甲基丙烯酸酯型MCI树脂无法获得良好的分离效果，部位Y得率低于5%。
B、药效学试验例部分
试验例1：二甲苯致小鼠耳肿胀实验，考察头花蓼提取物的抗炎效果
动物：SPF级昆明种小鼠，体重18～22g，雌雄兼用。
试药、剂量和给药方法：以上文“制备例1：头花蓼有效组分”所得的各种有效部位和对照试样头花蓼水提物(见上文)，剂量为每kg动物体重给予相应试药折合成头花蓼干品的重量为5g。各试药临用前用生理盐水混悬至相当于每只动物每次灌胃给药0.4ml的浓度，同时设置灌胃同体积生理盐水的对照组。
试验方法：小鼠雌雄兼用，随机分组，每一试药组10只动物；连续给药5天，末次给药60min后，用二甲苯0.1 ml/只涂右耳致炎，30min后处死小鼠，用直径6 mm的打孔器取左右两耳片。立即用电子天平称重，以两耳片重量差作为肿胀度(mg)，以下式计算各试药组的抑制率(%)：
抑制率(%)=[(对照组肿胀度-给药组肿胀度)÷对照组肿胀度]×100%
其中肿胀度(mg)=右耳片重-左耳片重
结果进行统计分析。
对抑制率进行分级，抑制率低于10%为A级，抑制率10~20%为AA级，抑制率20~30%为AAA级，抑制率30~40%为AAAA级，抑制率40~50%为AAAAA级，抑制率大于50%为AAAAAA级。
不同的头花蓼试样对二甲苯致小鼠耳肿胀的影响的结果为：部位E抑制率分级AAAA*，部位F抑制率分级AAAA**，部位G抑制率分级AAAAA**，部位Y抑制率分级AAAAA**，头花蓼水提物抑制率分级AAAA**。注：肿胀度(mg)，与溶媒对照组动物比较：* P<0.05，** P<0.01。平行试验中使用制备例2和制备例3的各部位作为试药。
试验例2：不同头花蓼提取物的抗菌实验
采用常规抗菌试验方法，使用MH肉汤培养基对绿脓杆菌、大肠杆菌M421C1ST、痢疾杆菌、金黄色葡萄球菌、溶血性链球菌进行试验，以上文“制备例1”所得的各样品和头花蓼水提物作为试药，测试它们对这些细胞的敏感性。
结果显示，部位E、部位F、部位G、部位Y各样品对五种细菌的MIC在1-4mg/ml之间，头花蓼水提物的MIC在10-25 mg/ml之间。
C、化学分析例部分
使用UPLC-TOF-MS法测试制备例1所得醇提取物和各部位。
UPLC-TOF-MS的测定方法如下：
(i)供试液配制：称取适量头花蓼提取物粉末, 加入70%甲醇制成浓度约5mg/ml的混悬液，超声使尽量溶解，混悬液稀释10倍，过滤，进样2μl作质谱检测；
(ii)色谱及质谱分析条件：
色谱柱：Acquity BEH C18 柱 (2.1 × 100 mm, 1.7μm)， 柱温：40℃；流速：0.35ml/min；进样量：2μl；质谱条件：离子源：ESI源；干燥气体温度：180℃；毛细管电压：4500eV；检测模式：负离子模式；喷雾压力：2.5bar；干燥气(N2)流速：8L/min；扫描范围：100-2000amu；碰撞能量: 10ev；以0.1%甲酸水溶液为流动相A，0.1%甲酸乙腈溶液为流动相B，按以下规定的程序进行梯度洗脱：0 min时95%A、0.5 min时95%A、20 min时81.5%A、28 min时0%A、30 min时0%A、30.1 min时95%A、32 min时95%A。
结果：(e)部位E样品显示，在保留时间12.8-13.8min之间(例如约13.3min处)和保留时间14.8-15.8min之间(例如约15.3min处)显示黄酮苷类(例如槲皮素-3-O-β-D-吡喃葡萄糖苷、石榴皮素B/carpinusin)的色谱峰，和/或在保留时间14.4-15.4min之间(例如约14.9min处)显示木脂素(例如nudiposide)的色谱峰，参见图1；(f)部位F样品显示，在保留时间12.8-13.8min之间(例如约13.3min处)、保留时间13.8-14.8min之间(例如约14.3min处)和保留时间14.8-15.8min之间(例如约15.3min处)显示黄酮苷类(例如槲皮素-3-O-β-D-吡喃葡萄糖苷、槲皮素-吡喃葡萄糖苷、石榴皮素B/carpinusin)的色谱峰，参见图2；(g)部位G样品显示，(g)在保留时间16.6-17.6min之间(例如约17.1min处)和保留时间21.4-22.4min之间(例如约21.9min处)显示黄酮苷元(例如槲皮苷、山奈酚3-(2-没食子酰基葡萄糖苷))的色谱峰，参见图3。
本发明提供的头花蓼各有效部位，其中所包含的部位物质及其表征数据如下：峰16的 t R为13.3min、分子式C 21H 20O 12、实测质量( m/z, [M-H] -)463.0858、理论质量( m/z, [M-H] -)463.0882、化合物为槲皮素-3-O-β-D-吡喃葡萄糖苷，峰18的 t R为14.3 min、分子式C 21H 20O 12、实测质量( m/z, [M-H] -)463.0852、理论质量( m/z, [M-H] -)463.0882、化合物为槲皮素-吡喃葡萄糖苷 (16的异构体)，峰19的 t R为14.9 min、分子式C 27H 36O 12、实测质量( m/z, [M-H] -)551.2131、理论质量( m/z, [M-H] -)551.2134、化合物为nudiposide，峰20的 t R为15.3 min、分子式C 41H 28O 27、实测质量( m/z, [M-H] -)951.0745、理论质量( m/z, [M-H] -)951.0745、化合物为石榴皮素B/carpinusin，峰21的 t R为17.1 min、分子式C 21H 20O 11、实测质量( m/z, [M-H] -)447.0911、理论质量( m/z, [M-H] -)447.0933、化合物为槲皮苷，峰23的 t R为21.9 min、分子式C 28H 24O 15、实测质量( m/z, [M-H] -)599.1036、理论质量( m/z, [M-H] -)599.1042、化合物为山奈酚3-(2-没食子酰基葡萄糖苷)。
制备例11：头花蓼有效组分
(1)将头花蓼地上部分干品10Kg，加7倍量的70%乙醇回流提取2次，每次约1.5小时，过滤，使滤液浓缩、干燥，得醇浸膏(885g)；所述乙醇中还包含0.75%乙酸；(2)使醇浸膏(45g)混悬于甲醇中，超声处理约1.5小时，离心，将上清液加载到MCI大孔树脂(MCI GEL CHP 20P)柱(每100g浸膏使用树脂的量为约2升)上；(3)用水，10%、20%、30%、35%、40%、45%、50%、60%、70%、80%、100%甲醇依次进行梯度洗脱(每种溶剂用量为约1倍柱体积)，收集各部分洗脱液，回收溶剂，由不同溶剂洗脱获得的部位干燥，得到50%甲醇洗脱物为部位E(得率2.91%)，50%-60%甲醇洗脱物是部位F(得率1.97%)，60%-80%甲醇洗脱物是部位G(得率6.14%)，50%-80%甲醇洗脱物是部位Y(得率10.77%)。
制备例12：头花蓼有效组分
(1)将头花蓼地上部分干品10Kg，加6倍量的60%乙醇回流提取3次，每次约1小时，过滤，使滤液浓缩、干燥，得醇浸膏(935g)；所述乙醇中还包含0.75%乙酸；(2)使醇浸膏(45g)混悬于甲醇中，超声处理约2小时，离心，将上清液加载到MCI大孔树脂(MCI GEL CHP 20P)柱(每100g浸膏使用树脂的量为约3升)上；(3)用水，10%、20%、30%、35%、40%、45%、50%、60%、70%、80%、100%甲醇依次进行梯度洗脱(每种溶剂用量为约2倍柱体积)，收集各部分洗脱液，回收溶剂，由不同溶剂洗脱获得的部位干燥，得到50%甲醇洗脱物为部位E，50%-60%甲醇洗脱物是部位F，60%-80%甲醇洗脱物是部位G，50%-80%甲醇洗脱物是部位Y。
得到50%甲醇洗脱物为部位E(得率2.84%)，50%-60%甲醇洗脱物是部位F(得率2.26%)，60%-80%甲醇洗脱物是部位G(得率6.24%)，50%-80%甲醇洗脱物是部位Y(得率10.42%)。
制备例13：头花蓼有效组分
(1)将头花蓼地上部分干品10Kg，加10倍量的80%乙醇回流提取1次3小时，过滤，使滤液浓缩、干燥，得醇浸膏(818g)；所述乙醇中还包含0.75%乙酸；(2)使醇浸膏(45g)混悬于甲醇中，超声处理约1小时，离心，将上清液加载到MCI大孔树脂(MCI GEL CHP 55A)柱(每100g浸膏使用树脂的量为约1升)上；(3)用水，10%、20%、30%、35%、40%、45%、50%、60%、70%、80%、100%甲醇依次进行梯度洗脱(每种溶剂用量为约0.5倍柱体积)，收集各部分洗脱液，回收溶剂，由不同溶剂洗脱获得的部位干燥，得到50%甲醇洗脱物为部位E，50%-60%甲醇洗脱物是部位F，60%-80%甲醇洗脱物是部位G，50%-80%甲醇洗脱物是部位Y。
得到50%甲醇洗脱物为部位E(得率2.88%)，50%-60%甲醇洗脱物是部位F(得率2.04%)，60%-80%甲醇洗脱物是部位G(得率6.32%)，50%-80%甲醇洗脱物是部位Y(得率10.98%)。
试验例3：使用UPLC-TOF-MS法测试制备例11~13所得醇提取物和各部位。
UPLC-TOF-MS的测定方法如下：
(i)供试液配制：称取适量头花蓼提取物粉末, 加入70%甲醇制成浓度约5mg/ml的混悬液，超声使尽量溶解，混悬液稀释10倍，过滤，进样2μl作质谱检测；
(ii)色谱柱：Acquity BEH C18 柱 (2.1 × 100 mm, 1.7μm)， 柱温：40℃；流速：0.35ml/min；进样量：2μl；质谱条件：离子源：ESI源；干燥气体温度：180℃；毛细管电压：4500eV；检测模式：负离子模式；喷雾压力：2.5bar；干燥气(N2)流速：8L/min；扫描范围：100-2000amu；碰撞能量: 10ev；以0.1%甲酸水溶液为流动相A，0.1%甲酸乙腈溶液为流动相B，按以下规定的程序进行梯度洗脱：0 min时95%A、0.5 min时95%A、20 min时81.5%A、28 min时0%A、30 min时0%A、30.1 min时95%A、32 min时95%A。
结果：(e)部位E样品显示，在保留时间12.5-13.9min之间和保留时间14.6-15.8min之间显示黄酮苷类的色谱峰，在保留时间14.5-15.7min之间显示木脂素的色谱峰；(f)部位F样品显示，在保留时间12.2-13.5min之间、保留时间13.9-14.7min之间、保留时间15.0-15.9min之间显示槲皮素-3-O-β-D-吡喃葡萄糖苷、槲皮素-吡喃葡萄糖苷、石榴皮素B/carpinusin的色谱峰；(g)部位G样品显示，在保留时间16.3-17.4min之间和保留时间21.6-22.5min之间显示槲皮苷、山奈酚3-(2-没食子酰基葡萄糖苷)的色谱峰。
试验例4：制备例11~13所得头花蓼提取物的抗菌实验
采用常规抗菌试验方法，使用MH肉汤培养基对绿脓杆菌、大肠杆菌M421C1ST、痢疾杆菌、金黄色葡萄球菌、溶血性链球菌进行试验，以上文制备例11~13所得的各样品和头花蓼水提物作为试药，测试它们对这些细胞的敏感性。结果显示，制备例11~13部位E对五种细菌的MIC在0.6-0.9mg/ml之间，制备例11~13部位F对五种细菌的MIC在0.2-0.6mg/ml之间，制备例11~13部位G对五种细菌的MIC在0.07-0.13mg/ml之间，而头花蓼水提物的MIC在10-25 mg/ml之间，这些结果表明制备例11~13所得头花蓼提取物具有显著更优异的抗菌活性。

Claims (1)

  1. 改进热淋清颗粒的清热利湿、利尿通淋、泌尿***感染或结石、肾盂肾炎、解毒、散瘀、抗菌、抗炎、镇痛、抗***炎的生物学活性的方法，该方法包括给有需要的受试者施用治疗有效量的头花蓼提取物，所述头花蓼提取物是按照以下步骤制备得到的：(1)将头花蓼地上部分鲜品或干品加5-15倍量的50~90%乙醇回流提取1-3次，每次1-3小时，过滤，使滤液浓缩、干燥，得醇浸膏；所述乙醇中还包含0.75%乙酸；(2)使醇浸膏混悬于甲醇中，超声处理0.5~5小时，离心，将上清液加载到MCI大孔树脂柱上；(3)用水，10%、20%、30%、35%、40%、45%、50%、60%、70%、80%、100%甲醇依次进行梯度洗脱，收集各部分洗脱液，回收溶剂，由不同溶剂洗脱获得的部位干燥，得到50%-80%之间任意浓度间隔或浓度点的甲醇洗脱物，或者它们的混合物，即得。
    2．根据权利要求1的方法，其中步骤(3)所得甲醇洗脱物是50%、50%-60%、或60%-80%甲醇洗脱物。
    3．根据权利要求1的方法，其中步骤(3)所得甲醇洗脱物是50%-80%之间甲醇洗脱物的混合物。
    4．根据权利要求1的方法，其中部位E中含有木脂素和/或黄酮苷类，例如含有木脂素和黄酮苷混合物。
    5．根据权利要求1的方法，其中部位F中含有黄酮苷类、部位G中含有黄酮苷元类、部位Y中含有木脂素和/或黄酮苷类和/或黄酮苷元类，例如槲皮素-3-O-β-D-吡喃葡萄糖苷、槲皮素-吡喃葡萄糖苷、石榴皮素B/carpinusin、槲皮苷、山奈酚3-(2-没食子酰基葡萄糖苷)。
    6．根据权利要求1的方法，所述头花蓼提取物照超高效液相色谱-飞行时间-质谱联用法测定，结果：(e)在保留时间12.8-13.8min之间和保留时间14.8-15.8min之间显示黄酮苷类的色谱峰，和/或在保留时间14.4-15.4min之间显示木脂素的色谱峰；(f)在保留时间12.8-13.8min之间、保留时间13.8-14.8min之间和保留时间14.8-15.8min之间显示黄酮苷类的色谱峰；(g)在保留时间16.6-17.6min之间和保留时间21.4-22.4min之间显示黄酮苷元的色谱峰；和/或(y)显示以上(e)至(g)中所示任一或全部色谱峰；其中，UPLC-TOF-MS的测定方法如下：
    (i)供试液配制：称取适量头花蓼提取物粉末, 加入70%甲醇制成浓度约5mg/ml的混悬液，超声使尽量溶解，混悬液稀释10倍，过滤，进样2μl作质谱检测；
    (ii)色谱柱：Acquity BEH C18 柱 (2.1 × 100 mm, 1.7μm)， 柱温：40℃；流速：0.35ml/min；进样量：2μl；质谱条件：离子源：ESI源；干燥气体温度：180℃；毛细管电压：4500eV；检测模式：负离子模式；喷雾压力：2.5bar；干燥气(N2)流速：8L/min；扫描范围：100-2000amu；碰撞能量: 10ev；以0.1%甲酸水溶液为流动相A，0.1%甲酸乙腈溶液为流动相B，按以下规定的程序进行梯度洗脱：0 min时95%A、0.5 min时95%A、20 min时81.5%A、28 min时0%A、30 min时0%A、30.1 min时95%A、32 min时95%A。
    7．根据权利要求1的方法，其中所述头花蓼提取物照所述超高效液相色谱-飞行时间-质谱联用法测定，结果：(e)在保留时间12.8-13.8min之间和保留时间14.8-15.8min之间显示黄酮苷类的色谱峰，和/或在保留时间14.4-15.4min之间显示木脂素的色谱峰。
    8．根据权利要求1的方法，其中所述头花蓼提取物照所述超高效液相色谱-飞行时间-质谱联用法测定，在保留时间12.8-13.8min之间、保留时间13.8-14.8min之间和保留时间14.8-15.8min之间显示黄酮苷类的色谱峰。
    9．根据权利要求1的方法，其中所述头花蓼提取物照所述超高效液相色谱-飞行时间-质谱联用法测定，在保留时间16.6-17.6min之间和保留时间21.4-22.4min之间显示黄酮苷元的色谱峰。
    10．根据权利要求1的方法，其中所述头花蓼提取物照所述超高效液相色谱-飞行时间-质谱联用法测定，结果：(e)在保留时间12.8-13.8min之间和保留时间14.8-15.8min之间显示黄酮苷类的色谱峰，和/或在保留时间14.4-15.4min之间显示木脂素的色谱峰；(f)在保留时间12.8-13.8min之间、保留时间13.8-14.8min之间和保留时间14.8-15.8min之间显示黄酮苷类的色谱峰；和/或，(g)在保留时间16.6-17.6min之间和保留时间21.4-22.4min之间显示黄酮苷元的色谱峰。
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