WO2024077489A1 - Method for improving biological activity of relinqing granules - Google Patents
Method for improving biological activity of relinqing granules Download PDFInfo
- Publication number
- 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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- WO
- WIPO (PCT)
- Prior art keywords
- extract
- polygonum capitatum
- methanol
- chromatographic peaks
- retention time
- Prior art date
Links
- 239000008187 granular material Substances 0.000 title claims abstract description 13
- 238000000034 method Methods 0.000 title claims description 58
- 230000004071 biological effect Effects 0.000 title claims description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 327
- 239000000284 extract Substances 0.000 claims abstract description 126
- 241000764065 Persicaria capitata Species 0.000 claims abstract description 122
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 87
- 238000002360 preparation method Methods 0.000 claims abstract description 40
- 238000010828 elution Methods 0.000 claims abstract description 27
- 238000001035 drying Methods 0.000 claims abstract description 26
- 239000000203 mixture Substances 0.000 claims abstract description 23
- 239000000706 filtrate Substances 0.000 claims abstract description 15
- 238000001914 filtration Methods 0.000 claims abstract description 8
- 206010037596 Pyelonephritis Diseases 0.000 claims abstract description 7
- 238000010992 reflux Methods 0.000 claims abstract description 7
- 208000004880 Polyuria Diseases 0.000 claims abstract description 6
- 230000035619 diuresis Effects 0.000 claims abstract description 6
- 239000008280 blood Substances 0.000 claims abstract description 5
- 210000004369 blood Anatomy 0.000 claims abstract description 5
- 201000007094 prostatitis Diseases 0.000 claims abstract description 5
- 230000014759 maintenance of location Effects 0.000 claims description 97
- 229930182486 flavonoid glycoside Natural products 0.000 claims description 48
- 150000007955 flavonoid glycosides Chemical class 0.000 claims description 48
- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 claims description 34
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 claims description 34
- IYRMWMYZSQPJKC-UHFFFAOYSA-N kaempferol Chemical compound C1=CC(O)=CC=C1C1=C(O)C(=O)C2=C(O)C=C(O)C=C2O1 IYRMWMYZSQPJKC-UHFFFAOYSA-N 0.000 claims description 34
- 229930013686 lignan Natural products 0.000 claims description 31
- 235000009408 lignans Nutrition 0.000 claims description 31
- 150000005692 lignans Chemical class 0.000 claims description 31
- QCCCZTBAYDERBM-XSZDUELUSA-N carpinusin Natural products O[C@@H]1OC[C@@H]2O[C@@H](OC(=O)c3cc(O)c(O)c(O)c3c4c(O)c(O)c(O)cc14)[C@H]5OC(=O)c6cc(O)c(O)c7O[C@@]8(O)C(=O)C=C([C@@H](c67)C8(O)O)C(=O)O[C@H]2[C@H]5OC(=O)c9cc(O)c(O)c(O)c9 QCCCZTBAYDERBM-XSZDUELUSA-N 0.000 claims description 28
- QCCCZTBAYDERBM-NLPUGBIRSA-N carpinusin Chemical compound O([C@@H]1[C@@H]2OC(=O)C3=CC(=O)C4(O)OC=5C(O)=C(O)C=C(C=5C3C4(O)O)C(=O)O[C@@H]1[C@H]1O[C@@H]2COC(C2=CC(O)=C(O)C(O)=C2C2=C(O)C(O)=C(O)C=C2C(=O)O1)O)C(=O)C1=CC(O)=C(O)C(O)=C1 QCCCZTBAYDERBM-NLPUGBIRSA-N 0.000 claims description 28
- 229920000241 Punicalagin Polymers 0.000 claims description 27
- ZJVUMAFASBFUBG-OGJBWQGYSA-N punicalagin Chemical compound C([C@H]1O[C@@H]([C@@H]2OC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)O[C@H]2[C@@H]1OC(=O)C1=CC(O)=C(O)C(O)=C11)O)OC(=O)C2=CC(O)=C(O)C(O)=C2C2=C(O)C(O)=C(OC3=O)C4=C2C(=O)OC2=C4C3=C1C(O)=C2O ZJVUMAFASBFUBG-OGJBWQGYSA-N 0.000 claims description 27
- LMIBIMUSUFYFJN-RSVYENFWSA-N punicalagin Natural products O[C@@H]1O[C@@H]2COC(=O)c3cc(O)c(O)c(O)c3c4c(O)cc5OC(=O)c6c(c(O)c(O)c7OC(=O)c4c5c67)c8c(O)c(O)c(O)cc8C(=O)O[C@H]2[C@@H]9OC(=O)c%10cc(O)c(O)c(O)c%10c%11c(O)c(O)c(O)cc%11C(=O)O[C@@H]19 LMIBIMUSUFYFJN-RSVYENFWSA-N 0.000 claims description 27
- ZRKSVMFLACVUIU-UHFFFAOYSA-N punicalagin isomer Natural products OC1=C(O)C(=C2C3=4)OC(=O)C=4C4=C(O)C(O)=C3OC(=O)C2=C1C1=C(O)C(O)=C(O)C=C1C(=O)OC1C2OC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC2C(O)OC1COC(=O)C1=CC4=C(O)C(O)=C1O ZRKSVMFLACVUIU-UHFFFAOYSA-N 0.000 claims description 27
- 229930003935 flavonoid Natural products 0.000 claims description 23
- 150000002215 flavonoids Chemical class 0.000 claims description 23
- 235000017173 flavonoids Nutrition 0.000 claims description 23
- 239000007788 liquid Substances 0.000 claims description 21
- 238000001269 time-of-flight mass spectrometry Methods 0.000 claims description 20
- UBSCDKPKWHYZNX-UHFFFAOYSA-N Demethoxycapillarisin Natural products C1=CC(O)=CC=C1OC1=CC(=O)C2=C(O)C=C(O)C=C2O1 UBSCDKPKWHYZNX-UHFFFAOYSA-N 0.000 claims description 17
- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 claims description 17
- HWTZYBCRDDUBJY-UHFFFAOYSA-N Rhynchosin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=CC(O)=C(O)C=C2O1 HWTZYBCRDDUBJY-UHFFFAOYSA-N 0.000 claims description 17
- 229930182478 glucoside Natural products 0.000 claims description 17
- 150000008131 glucosides Chemical class 0.000 claims description 17
- 235000008777 kaempferol Nutrition 0.000 claims description 17
- UXOUKMQIEVGVLY-UHFFFAOYSA-N morin Natural products OC1=CC(O)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 UXOUKMQIEVGVLY-UHFFFAOYSA-N 0.000 claims description 17
- 235000005875 quercetin Nutrition 0.000 claims description 17
- 229960001285 quercetin Drugs 0.000 claims description 17
- 238000004949 mass spectrometry Methods 0.000 claims description 15
- 230000000844 anti-bacterial effect Effects 0.000 claims description 12
- 238000001514 detection method Methods 0.000 claims description 12
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 12
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- 230000003110 anti-inflammatory effect Effects 0.000 claims description 11
- 238000002347 injection Methods 0.000 claims description 10
- 239000007924 injection Substances 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 8
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 6
- XBJFCYDKBDVADW-UHFFFAOYSA-N acetonitrile;formic acid Chemical compound CC#N.OC=O XBJFCYDKBDVADW-UHFFFAOYSA-N 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 235000019253 formic acid Nutrition 0.000 claims description 6
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- 239000012085 test solution Substances 0.000 claims description 6
- 208000019206 urinary tract infection Diseases 0.000 claims description 5
- 230000001760 anti-analgesic effect Effects 0.000 claims description 4
- 208000009911 Urinary Calculi Diseases 0.000 claims description 3
- 230000001737 promoting effect Effects 0.000 claims description 3
- 239000008346 aqueous phase Substances 0.000 claims 4
- 239000012071 phase Substances 0.000 claims 2
- 239000002904 solvent Substances 0.000 abstract description 40
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- 229920005989 resin Polymers 0.000 abstract description 35
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 31
- 239000006228 supernatant Substances 0.000 abstract description 13
- 238000000605 extraction Methods 0.000 abstract description 9
- 238000011068 loading method Methods 0.000 abstract description 6
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- 206010061218 Inflammation Diseases 0.000 abstract description 3
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- 230000005764 inhibitory process Effects 0.000 description 20
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 19
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- QOKISIDHCPDXMC-UHFFFAOYSA-N (+)-5'-methoxyisolacriciresinol-9'-beta-D-xylopyranoside Natural products C=1C(OC)=C(O)C(OC)=CC=1C1C=2C=C(O)C(OC)=CC=2CC(CO)C1COC1OCC(O)C(O)C1O QOKISIDHCPDXMC-UHFFFAOYSA-N 0.000 description 13
- GWDZRGQRNHELQM-NHJKQUFYSA-N (2r,3r,4s,5r)-2-[[(1r,2s,3s)-7-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-3-(hydroxymethyl)-6,8-dimethoxy-1,2,3,4-tetrahydronaphthalen-2-yl]methoxy]oxane-3,4,5-triol Chemical compound C([C@@H]1[C@@H](CO)CC=2C=C(C(=C(OC)C=2[C@H]1C=1C=C(OC)C(O)=C(OC)C=1)O)OC)O[C@@H]1OC[C@@H](O)[C@H](O)[C@H]1O GWDZRGQRNHELQM-NHJKQUFYSA-N 0.000 description 13
- GWDZRGQRNHELQM-VXZQQKRVSA-N Lyoniside Natural products O(C[C@@H]1[C@@H](c2cc(OC)c(O)c(OC)c2)c2c(OC)c(O)c(OC)cc2C[C@H]1CO)[C@H]1[C@@H](O)[C@@H](O)[C@@H](O)CO1 GWDZRGQRNHELQM-VXZQQKRVSA-N 0.000 description 13
- GWDZRGQRNHELQM-QWMLTHFVSA-N Nudiposide Natural products O(C[C@H]1[C@H](c2cc(OC)c(O)c(OC)c2)c2c(OC)c(O)c(OC)cc2C[C@@H]1CO)[C@@H]1[C@H](O)[C@H](O)[C@@H](O)CO1 GWDZRGQRNHELQM-QWMLTHFVSA-N 0.000 description 13
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- 241000700159 Rattus Species 0.000 description 2
- 241000607764 Shigella dysenteriae Species 0.000 description 2
- 241000191967 Staphylococcus aureus Species 0.000 description 2
- 241000194017 Streptococcus Species 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 229930002875 chlorophyll Natural products 0.000 description 2
- 235000019804 chlorophyll Nutrition 0.000 description 2
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 2
- 239000003405 delayed action preparation Substances 0.000 description 2
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- 230000002949 hemolytic effect Effects 0.000 description 2
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- 150000008163 sugars Chemical class 0.000 description 2
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- 206010059517 Bacterial pyelonephritis Diseases 0.000 description 1
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- 244000025254 Cannabis sativa Species 0.000 description 1
- 238000012449 Kunming mouse Methods 0.000 description 1
- 240000001307 Myosotis scorpioides Species 0.000 description 1
- 241000588652 Neisseria gonorrhoeae Species 0.000 description 1
- 241000219050 Polygonaceae Species 0.000 description 1
- 244000294611 Punica granatum Species 0.000 description 1
- 235000014360 Punica granatum Nutrition 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 238000002814 agar dilution Methods 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000001882 diuretic effect Effects 0.000 description 1
- 229940030606 diuretics Drugs 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000007905 drug manufacturing Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- 239000000469 ethanolic extract Substances 0.000 description 1
- 230000003631 expected effect Effects 0.000 description 1
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- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
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- 239000006187 pill Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 238000003815 supercritical carbon dioxide extraction Methods 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
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- 238000003809 water extraction Methods 0.000 description 1
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Classifications
-
- 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
The use of a Polygonum capitatum Buch-Ham ex D. Don extract in the preparation of Relinqing granules for clearing heat and removing dampness, inducing diuresis for treating strangurtia, treating urinary system infections or stones, treating pyelonephritis, achieving detoxication, dissipating blood stasis, resisting bacteria, resisting inflammation, easing pain and resisting prostatitis. The Polygonum capitatum Buch-Ham ex D. Don extract is prepared according to the following steps: adding ethanol to fresh or dried overground parts of Polygonum capitatum Buch-Ham ex D. Don, performing reflux extraction and then filtration, and concentrating and drying a filtrate to obtain an alcoholic extract; suspending the alcoholic extract in methanol, performing ultrasonic treatment and centrifugation, and loading a macroporous resin column with a supernatant; and performing gradient elution with water and methanol in sequence, collecting eluents of various parts, recycling a solvent, and drying the parts, which are eluted with different solvents, to obtain methanol eluates and a mixture thereof.
Description
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。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. Specifically, 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. In particular, the method for preparing Relinqing granules of the present invention can significantly improve the biological activity of the prepared Relinqing granules.
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，在临床上治疗泌尿系统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。
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 Guizhou Province basic medicine catalog in 2012.
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(淋球菌)的体外抑菌活性，结果头花蓼对淋球菌有抑菌活性。其对10株淋球菌的最小抑菌浓度范围为8~32g/L，平均值为11.2g/L。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. injected Escherichia coli liquid into mice intraperitoneally and observed the death of mice within 5 days. The results showed that the mortality rate of the control group was 100%, and the mortality rate of the Polygonum capitatum group was 20% and 50%, respectively, indicating that the Polygonum capitatum water extract can fight against the infection caused by Escherichia coli. Ren Guangyou et al. administered the Polygonum capitatum water extract to rabbits by gavage. The results showed that there was no significant difference in body temperature between the Polygonum capitatum water extract group and the control group, but it could reduce the fever body temperature of rabbits caused by intravenous injection of typhoid and paratyphoid vaccines. Ren Guangyou et al. administered water extract of Polygonum capitatum to rabbits and rats by gavage, and compared urine volume with that of blank group and furosemide control group. The results showed that water extract of Polygonum capitatum had no obvious diuretic effect on rabbits and rats. Xu Yingchun et al. used agar dilution method to detect the in vitro antibacterial activity of Polygonum capitatum against 10 strains of Neisseria gonorrhoeae (gonococci), and the results showed that Polygonum capitatum had antibacterial activity against gonococci. The minimum inhibitory concentration ranged from 8 to 32 g/L for 10 strains of gonococci, with an average of 11.2 g/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。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.
中华人民共和国卫生部药典委员会1998年编的中华人民共和国卫生部《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。The "Drug Standards - Chinese Medicine Formula Preparations" (Volume 17) compiled by the Pharmacopoeia Committee of the Ministry of Health of the People's Republic of China in 1998 includes a Chinese medicine preparation called "Relinqing Granules", which is made by decocting Polygonum capitatum with water twice and then filtering and concentrating it.
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、治疗泌尿系统结石、治疗肾盂肾炎以及前列腺炎。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.
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。Although there are many reports on the preparation of Polygonum capitatum extracts, those skilled in the art still expect to obtain effective products from Polygonum capitatum, especially to endow Polygonum capitatum extracts with significantly better biological activities.
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。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.
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：To this end, the first aspect of the present invention provides a Polygonum capitatum extract, which is basically prepared by a method comprising the following steps:
(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；(1) adding 5-15 times (e.g., 5-12 times, e.g., 6-10 times) 50-90% (e.g., 60-80%, e.g., 65-75%, e.g., about 70%) ethanol to the fresh or dried aerial parts of Polygonum capitatum, reflux extracting 1-3 times (e.g., 2 times), each time for 1-3 hours (e.g., about 1.5 hours), filtering, concentrating and drying the filtrate to obtain an alcohol extract; the ethanol further comprising 0.75% acetic acid;
(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；(2) suspending the alcohol extract in methanol, ultrasonically treating for 0.5 to 5 hours (e.g., 0.5 to 3 hours, e.g., 1 to 2 hours, e.g., about 1.5 hours), centrifuging, and loading the supernatant onto an MCI macroporous resin column (e.g., the amount of resin used per 100 g of extract is 0.5 to 4 liters, e.g., 1 to 3 liters, e.g., 1.5 to 2.5 liters, e.g., 2 liters);
(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。(3) Gradient elution is performed in sequence using water, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, and 100% methanol (for example, the amount of each solvent used is 0.5 to 5 column volumes, for example, 0.5 to 2.5 column volumes, for example, 0.5 to 2 column volumes, for example, 1 column volume), the eluate from each portion is collected, the solvent is recovered, and the portions obtained by elution with different solvents are dried to obtain methanol eluates at any concentration interval or concentration point between 50% and 80%, or a mixture thereof.
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)。According to the Polygonum capitatum extract of the first aspect of the present invention, the methanol eluate obtained in step (3) is a 50% methanol eluate (referred to as: fraction E in the present invention).
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)。According to the Polygonum capitatum extract of the first aspect of the present invention, the methanol eluate obtained in step (3) is a 50%-60% methanol eluate (abbreviated as: fraction F in the present invention).
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)。According to the Polygonum capitatum extract of the first aspect of 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).
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。According to the first aspect of 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"). In the present invention, 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.
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。According to the first aspect of the present invention, the Polygonum capitatum extract, wherein part E contains lignans and/or flavonoid glycosides, for example, contains a mixture of lignans and flavonoid glycosides.
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。According to the first aspect of the present invention, the Polygonum capitatum extract contains flavonoid glycosides in the fraction F.
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。According to the first aspect of the present invention, the Polygonum capitatum extract contains flavonoid aglycones in the fraction G.
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)。According to the first aspect of the present invention, the Polygonum capitatum extract contains lignans and/or flavonoid glycosides and/or flavonoid aglycones in part Y, such as quercetin-3-O-β-D-pyranoglucoside, quercetin-pyranoglucoside, punicalagin B/carpinusin, quercetin, and kaempferol 3-(2-galloyl glucoside).
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：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:
(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；(e) showing chromatographic peaks of flavonoid glycosides (e.g., quercetin-3-O-β-D-pyranoglucopyranoside, punicalagin B/carpinusin) at retention times between 12.8-13.8 min (e.g., about 13.3 min) and between 14.8-15.8 min (e.g., about 15.3 min), and/or showing chromatographic peaks of lignans (e.g., nudiposide) at retention times between 14.4-15.4 min (e.g., about 14.9 min);
(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；(f) chromatographic peaks of flavonoid glycosides (e.g., quercetin-3-O-β-D-pyranoglucopyranoside, quercetin-pyranoglucopyranoside, punicalagin B/carpinusin) 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);
(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(g) showing chromatographic peaks of flavonoid aglycones (e.g., quercetin, kaempferol 3-(2-galloyl glucoside)) 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); and/or
(y)显示以上(e)至(g)中所示任一或全部色谱峰；(y) showing any or all of the chromatographic peaks shown in (e) to (g) above;
其中，UPLC-TOF-MS的测定方法如下：Among them, the determination method of UPLC-TOF-MS is as follows:
(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；(i) 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 μl for mass spectrometry detection;
(ii)色谱及质谱分析条件：(ii) Chromatographic and mass spectrometric analysis conditions:
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。Chromatographic column: Acquity BEH C18 column (2.1
× 100 mm, 1.7μm), column temperature: 40℃; flow rate: 0.35ml/min; injection volume: 2μl; mass spectrometry conditions: ion source: ESI source; drying gas temperature: 180℃; 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.
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。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-β-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).
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。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-β-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).
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。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).
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：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:
(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；(e) showing chromatographic peaks of flavonoid glycosides (e.g., quercetin-3-O-β-D-pyranoglucopyranoside, punicalagin B/carpinusin) at retention times between 12.8-13.8 min (e.g., about 13.3 min) and between 14.8-15.8 min (e.g., about 15.3 min), and/or showing chromatographic peaks of lignans (e.g., nudiposide) at retention times between 14.4-15.4 min (e.g., about 14.9 min);
(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(f) showing chromatographic peaks of flavonoid glycosides (e.g., quercetin-3-O-β-D-pyranoglucopyranoside, quercetin-pyranoglucopyranoside, punicalagin B/carpinusin) at retention times between 12.8-13.8 min (e.g., about 13.3 min), between 13.8-14.8 min (e.g., about 14.3 min), and between 14.8-15.8 min (e.g., about 15.3 min); and/or
(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。(g) Chromatographic peaks of flavonoid aglycones (eg, quercetin, kaempferol 3-(2-galloyl glucoside)) are shown between retention times 16.6-17.6 min (eg, about 17.1 min) and between retention times 21.4-22.4 min (eg, about 21.9 min).
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。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.
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：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:
(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；(e) showing chromatographic peaks of flavonoid glycosides (e.g., quercetin-3-O-β-D-pyranoglucopyranoside, punicalagin B/carpinusin) at retention times between 12.8-13.8 min (e.g., about 13.3 min) and between 14.8-15.8 min (e.g., about 15.3 min), and/or showing chromatographic peaks of lignans (e.g., nudiposide) at retention times between 14.4-15.4 min (e.g., about 14.9 min);
(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；(f) chromatographic peaks of flavonoid glycosides (e.g., quercetin-3-O-β-D-pyranoglucopyranoside, quercetin-pyranoglucopyranoside, punicalagin B/carpinusin) 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);
(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(g) showing chromatographic peaks of flavonoid aglycones (e.g., quercetin, kaempferol 3-(2-galloyl glucoside)) 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); and/or
(y)显示以上(e)至(g)中所示任一或全部色谱峰；(y) showing any or all of the chromatographic peaks shown in (e) to (g) above;
其中，UPLC-TOF-MS的测定方法如下：Among them, the determination method of UPLC-TOF-MS is as follows:
(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；(i) 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 μl for mass spectrometry detection;
(ii)色谱及质谱分析条件：(ii) Chromatographic and mass spectrometric analysis conditions:
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。Chromatographic column: Acquity BEH C18 column (2.1
× 100 mm, 1.7μm), column temperature: 40℃; flow rate: 0.35ml/min; injection volume: 2μl; mass spectrometry conditions: ion source: ESI source; drying gas temperature: 180℃; 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.
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。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-β-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).
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。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-β-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).
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。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).
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：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:
(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；(e) showing chromatographic peaks of flavonoid glycosides (e.g., quercetin-3-O-β-D-pyranoglucopyranoside, punicalagin B/carpinusin) at retention times between 12.8-13.8 min (e.g., about 13.3 min) and between 14.8-15.8 min (e.g., about 15.3 min), and/or showing chromatographic peaks of lignans (e.g., nudiposide) at retention times between 14.4-15.4 min (e.g., about 14.9 min);
(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(f) showing chromatographic peaks of flavonoid glycosides (e.g., quercetin-3-O-β-D-pyranoglucopyranoside, quercetin-pyranoglucopyranoside, punicalagin B/carpinusin) at retention times between 12.8-13.8 min (e.g., about 13.3 min), between 13.8-14.8 min (e.g., about 14.3 min), and between 14.8-15.8 min (e.g., about 15.3 min); and/or
(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。(g) Chromatographic peaks of flavonoid aglycones (eg, quercetin, kaempferol 3-(2-galloyl glucoside)) are shown between retention times 16.6-17.6 min (eg, about 17.1 min) and between retention times 21.4-22.4 min (eg, about 21.9 min).
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：The Polygonum capitatum extract according to the second aspect of the present invention is basically prepared by a method comprising the following steps:
(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；(1) adding 5-15 times (e.g., 5-12 times, e.g., 6-10 times) 50-90% (e.g., 60-80%, e.g., 65-75%, e.g., about 70%) ethanol to the fresh or dried aerial parts of Polygonum capitatum, reflux extracting 1-3 times (e.g., 2 times), each time for 1-3 hours (e.g., about 1.5 hours), filtering, concentrating and drying the filtrate to obtain an alcohol extract; the ethanol further comprising 0.75% acetic acid;
(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；(2) suspending the alcohol extract in methanol, ultrasonically treating for 0.5 to 5 hours (e.g., 0.5 to 3 hours, e.g., 1 to 2 hours, e.g., about 1.5 hours), centrifuging, and loading the supernatant onto an MCI macroporous resin column (e.g., the amount of resin used per 100 g of extract is 0.5 to 4 liters, e.g., 1 to 3 liters, e.g., 1.5 to 2.5 liters, e.g., 2 liters);
(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。(3) Gradient elution is performed in sequence using water, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, and 100% methanol (for example, the amount of each solvent used is 0.5 to 5 column volumes, for example, 0.5 to 2.5 column volumes, for example, 0.5 to 2 column volumes, for example, 1 column volume), the eluate from each portion is collected, the solvent is recovered, and the portions obtained by elution with different solvents are dried to obtain methanol eluates at any concentration interval or concentration point between 50% and 80%, or a mixture thereof.
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)。According to the Polygonum capitatum extract of the second aspect of the present invention, the methanol eluate obtained in step (3) is a 50% methanol eluate (referred to as: fraction E in the present invention).
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)。According to the Polygonum capitatum extract of the second aspect of 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).
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)。According to the Polygonum capitatum extract of the second aspect of 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).
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。According to the second aspect of 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"). In the present invention, 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.
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。According to the second aspect of the present invention, the Polygonum capitatum extract, wherein part E contains lignans and/or flavonoid glycosides, for example, contains a mixture of lignans and flavonoid glycosides.
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。According to the second aspect of the present invention, the Polygonum capitatum extract contains flavonoid glycosides in the fraction F.
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。According to the second aspect of the present invention, the Polygonum capitatum extract contains flavonoid aglycones in the fraction G.
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)。According to the second aspect of the present invention, the Polygonum capitatum extract contains lignans and/or flavonoid glycosides and/or flavonoid aglycones in part Y, such as quercetin-3-O-β-D-pyranoglucoside, quercetin-pyranoglucoside, punicalagin B/carpinusin, quercetin, and kaempferol 3-(2-galloyl glucoside).
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。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.
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、泌尿系统感染或结石、肾盂肾炎、解毒、散瘀、抗菌、抗炎、镇痛、抗前列腺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：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:
(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；(1) adding 5-15 times (e.g., 5-12 times, e.g., 6-10 times) 50-90% (e.g., 60-80%, e.g., 65-75%, e.g., about 70%) ethanol to the fresh or dried aerial parts of Polygonum capitatum, reflux extracting 1-3 times (e.g., 2 times), each time for 1-3 hours (e.g., about 1.5 hours), filtering, concentrating and drying the filtrate to obtain an alcohol extract; the ethanol further comprising 0.75% acetic acid;
(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；(2) suspending the alcohol extract in methanol, ultrasonically treating for 0.5 to 5 hours (e.g., 0.5 to 3 hours, e.g., 1 to 2 hours, e.g., about 1.5 hours), centrifuging, and loading the supernatant onto an MCI macroporous resin column (e.g., the amount of resin used per 100 g of extract is 0.5 to 4 liters, e.g., 1 to 3 liters, e.g., 1.5 to 2.5 liters, e.g., 2 liters);
(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。(3) Gradient elution is performed in sequence using water, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, and 100% methanol (for example, the amount of each solvent used is 0.5 to 5 column volumes, for example, 0.5 to 2.5 column volumes, for example, 0.5 to 2 column volumes, for example, 1 column volume), the eluate from each portion is collected, the solvent is recovered, and the portions obtained by elution with different solvents are dried to obtain methanol eluates at any concentration interval or concentration point between 50% and 80%, or a mixture thereof.
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)。According to the method of the third aspect of the present invention, the methanol eluate obtained in step (3) is a 50% methanol eluate (referred to as: part E in the present invention).
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)。According to the method of the third aspect of 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).
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)。According to the method of the third aspect of 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).
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。According to the method of the third aspect of the present invention, the methanol eluate obtained in step (3) is a mixture of 50%-80% methanol eluates (hereinafter referred to as "fraction Y"). In the present invention, 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.
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。According to the method of the third aspect of the present invention, part E contains lignans and/or flavonoid glycosides, for example, contains a mixture of lignans and flavonoid glycosides.
根据本发明第三方面的方法，其中部位F中含有黄酮苷类。According to the method of the third aspect of the present invention, part F contains flavonoid glycosides.
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。According to the method of the third aspect of the present invention, part G contains flavonoid aglycones.
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)。According to the method of the third aspect of the present invention, part Y contains lignans and/or flavonoid glycosides and/or flavonoid aglycones, such as quercetin-3-O-β-D-pyranoglucoside, quercetin-pyranoglucoside, punicalagin B/carpinusin, quercetin, and kaempferol 3-(2-galloyl glucoside).
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。According to the method of the third aspect of the present invention, the obtained Polygonum capitatum extract has the characteristics described in any embodiment of the first aspect or the second aspect of the present invention.
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：According to the method of the third 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:
(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；(e) showing chromatographic peaks of flavonoid glycosides (e.g., quercetin-3-O-β-D-pyranoglucopyranoside, punicalagin B/carpinusin) at retention times between 12.8-13.8 min (e.g., about 13.3 min) and between 14.8-15.8 min (e.g., about 15.3 min), and/or showing chromatographic peaks of lignans (e.g., nudiposide) at retention times between 14.4-15.4 min (e.g., about 14.9 min);
(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；(f) chromatographic peaks of flavonoid glycosides (e.g., quercetin-3-O-β-D-pyranoglucopyranoside, quercetin-pyranoglucopyranoside, punicalagin B/carpinusin) 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);
(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(g) showing chromatographic peaks of flavonoid aglycones (e.g., quercetin, kaempferol 3-(2-galloyl glucoside)) 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); and/or
(y)显示以上(e)至(g)中所示任一或全部色谱峰；(y) showing any or all of the chromatographic peaks shown in (e) to (g) above;
其中，UPLC-TOF-MS的测定方法如下：Among them, the determination method of UPLC-TOF-MS is as follows:
(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；(i) 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 μl for mass spectrometry detection;
(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。(ii) Chromatographic column: Acquity BEH C18 column (2.1 × 100 mm, 1.7 μm), column temperature: 40 °C; flow rate: 0.35 ml/min; injection volume: 2 μl; 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.
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。According to the method of the third aspect of the present invention, 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-β-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).
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。According to the method of the third aspect of the present invention, 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-β-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).
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。According to the method of the third aspect of the present invention, 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).
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。According to the method of the third aspect of the present invention, 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-β-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-β-D-pyranoglucoside, punicalagin B/carpinusin) are displayed between retention times of 12.8-13.8 min (for example, at about 13.3 min). (g) chromatographic peaks of flavonoid glycosides (e.g., quercetin-3-O-β-D-pyranoglucopyranoside, quercetin-pyranoglucopyranoside, punicalagin B/carpinusin) 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).
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，泌尿系统感染或结石用药，肾盂肾炎用药，解毒、散瘀用药，抗菌、抗炎、镇痛用药，前列腺炎用药的药物中的用途。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.
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。The fifth aspect of the present invention provides 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.
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。The pharmaceutical composition according to the fifth aspect of the present invention is in the form of a preparation for oral or injection administration. In one embodiment, the pharmaceutical composition is in the form of tablets, capsules, granules, pills, oral liquids, injections (water injections and/or powder injections), etc.
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。Any technical feature of any aspect of the present invention or any embodiment of the aspect is also applicable to any other embodiment or any other embodiment of any other aspect, as long as they do not contradict each other. Of course, when they are applicable to each other, the corresponding features can be appropriately modified if necessary. The various aspects and features of the present invention are further described below.
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。All documents cited in the present invention are incorporated herein by reference in their entirety, and if the meanings expressed in these documents are inconsistent with the present invention, the description of the present invention shall prevail. In addition, the various terms and phrases used in the present invention have the general meanings known to those skilled in the art. Even so, the present invention still hopes to provide a more detailed description and explanation of these terms and phrases. If the terms and phrases mentioned are inconsistent with the known meanings, the meanings expressed in the present invention shall prevail.
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”。In the present invention, 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.
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。 In 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 4μm-100μm, and the particle size range of methacrylate type resin is 4μm-31μm. In addition, the MCI series has not only macroporous resins, but also anionic and cationic resins. 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. In the present invention, 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. In addition, in the present invention, unless otherwise specified, the macroporous resin of model MCI GEL CHP 20P (75~150μm) 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. In addition to having basically no adsorption effect on highly polar sugars and amino acids, it has different degrees of adsorption on most secondary metabolites, which enables it to effectively remove sugars and amino acids that have great interference with water solubility on the one hand, and on the other hand, it can separate different types of compounds.
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。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.
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。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.
在一个实施方案中，发明通过以下技术方案实现上述目的：In one embodiment, the invention achieves the above object through the following technical solutions:
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。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 between 50% and 80%, or a mixture thereof.
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。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). In the present invention, 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.
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)。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-β-D-pyranoglucoside, quercetin-pyranoglucoside, punicalagin B/carpinusin, quercetin, and kaempferol 3-(2-galloyl glucoside).
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)。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).
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％。The effective part obtained by the method of the present invention can significantly enhance the anti-inflammatory effect compared with the water extract. Compared with the extract of the water extraction process, the extract of the present invention can significantly improve its anti-inflammatory effect, and the experimental results are as follows (n=10): the swelling inhibition rate of the water extract of Polygonum capitatum (dosage 5 g/kg) is 29.2%, the swelling inhibition rate of the effective part E (dosage 2.5 g/kg) is 43.3%, the swelling inhibition rate of the effective part F (dosage 2.5 g/kg) is 37.2%, the swelling inhibition rate of the effective part G (dosage 2.5 g/kg) is 36.4%, and the swelling inhibition rate of the effective part Y (dosage 2.5 g/kg) is 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。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.
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。The preparation method of 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% methanol eluate is fraction G, and the 50%-80% methanol eluate is fraction 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。In the present invention, when the dosage of the test is 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.
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。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.
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。In order to more clearly understand the technical content of the present invention, the following embodiments are given to explain in detail, but the embodiments of the present invention are not limited thereto.
A、提取物制备例部分A. Extract Preparation Examples
制备例1：头花蓼有效组分Preparation Example 1: Effective components of Polygonum capitatum
(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%)。(1) 10 kg of the dry aerial part of Polygonum capitatum was added with 7 times the amount of 70% ethanol and refluxed for extraction twice, each time for about 1.5 hours, filtered, concentrated and dried the filtrate to obtain an alcohol extract (885 g); (2) the alcohol extract (45 g) was suspended in methanol, ultrasonically treated for about 1.5 hours, centrifuged, and the supernatant was loaded onto 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 carried out in sequence with water, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, and 100% methanol (the amount of each solvent used is about 1 column volume), the eluate of each part is collected, the solvent is recovered, and the fractions obtained by elution with different solvents are dried to obtain 50% methanol eluate as fraction E (yield 2.8%), 50%-60% methanol eluate as fraction F (yield 2.0%), 60%-80% methanol eluate as fraction G (yield 6.1%), and 50%-80% methanol eluate as fraction Y (yield 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。In the following, unless otherwise specified, the samples of sites E, F, G, and Y used are the products of Preparation Example 1.
制备例2：头花蓼有效组分Preparation Example 2: Effective components of Polygonum capitatum
(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。(1) 10 kg of the dry aerial part of Polygonum capitatum was added with 6 times the amount of 60% ethanol and refluxed for extraction for 3 times, each time for about 1 hour, filtered, concentrated and dried the filtrate to obtain an alcohol extract (935 g); (2) the alcohol extract (45 g) was suspended in methanol, ultrasonically treated for about 2 hours, centrifuged, and the supernatant was loaded onto MCI macroporous resin (MCI GEL CHP 20P) column (the amount of resin used for every 100g of extract is about 3 liters); (3) gradient elution is carried out in sequence with water, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, and 100% methanol (the amount of each solvent used is about 2 times the column volume), the eluate of each part is collected, the solvent is recovered, and the parts obtained by elution with different solvents are dried to obtain 50% methanol eluate as part E, 50%-60% methanol eluate as part F, 60%-80% methanol eluate as part G, and 50%-80% methanol eluate as part Y. The yield of each part is basically the same as that in Preparation Example 1.
制备例3：头花蓼有效组分Preparation Example 3: Effective components of Polygonum capitatum
(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%。(1) 10 kg of the dry aerial part of Polygonum capitatum was added with 10 times the amount of 80% ethanol and refluxed for 3 hours, filtered, concentrated and dried the filtrate to obtain an alcohol extract (818 g); (2) the alcohol extract (45 g) was suspended in methanol, ultrasonically treated for about 1 hour, centrifuged, and the supernatant was loaded onto MCI macroporous resin (MCI GEL CHP 55A) column (the amount of resin used for every 100g of extract is about 1 liter); (3) gradient elution is performed in sequence with water, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, and 100% methanol (the amount of each solvent used is about 0.5 column volume), the eluate of each part is collected, the solvent is recovered, and the parts obtained by elution with different solvents are dried to obtain 50% methanol eluate as part E, 50%-60% methanol eluate as part F, 60%-80% methanol eluate as part G, and 50%-80% methanol eluate as part Y. The yield of each part is basically the same as that in Preparation Example 1. In addition, 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%.
B、药效学试验例部分B. Pharmacodynamics Test Examples
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Experimental Example 1: Xylene-induced ear swelling experiment in mice to investigate the anti-inflammatory effect of Polygonum capitatum extract
动物：SPF级昆明种小鼠，体重18～22g，雌雄兼用。Animals: SPF Kunming mice, weighing 18-22 g, both male and female.
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。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.
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(%)：Test method: 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:
抑制率(%)=[(对照组肿胀度-给药组肿胀度)÷对照组肿胀度]×100%Inhibition rate (%) = [(swelling degree of control group - swelling degree of drug-treated group) ÷ swelling degree of control group] × 100%
其中肿胀度(mg)=右耳片重-左耳片重Swelling degree (mg) = right ear weight - left ear weight
结果进行统计分析。The results were statistically analyzed.
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。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.
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。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**. Note: Swelling degree (mg), compared with the animals in the solvent control group: * P<0.05, **
P<0.01. In the parallel test, the various parts of Preparation Example 2 and Preparation Example 3 were used as test drugs.
试验例2：不同头花蓼提取物的抗菌实验Test Example 2: Antibacterial Experiment of Different Polygonum capitatum Extracts
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。Conventional antibacterial test methods were used to test Pseudomonas aeruginosa, Escherichia coli M421C1ST, Shigella dysenteriae, Staphylococcus aureus, and hemolytic Streptococcus using MH broth culture medium. The samples obtained in the above "Preparation Example 1" and the water extract of Polygonum capitatum were used as test drugs to test their sensitivity to these cells.
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。The results showed that the MICs of the samples from parts E, F, G and Y against the five bacteria were between 1-4 mg/ml, and the MIC of the water extract of Polygonum capitatum was between 10-25 mg/ml.
C、化学分析例部分C. Chemical Analysis Examples
使用UPLC-TOF-MS法测试制备例1所得醇提取物和各部位。The alcohol extract and various parts obtained in Preparation Example 1 were tested using the UPLC-TOF-MS method.
UPLC-TOF-MS的测定方法如下：The UPLC-TOF-MS determination method is as follows:
(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；(i) 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 μl for mass spectrometry detection;
(ii)色谱及质谱分析条件：(ii) Chromatographic and mass spectrometric analysis conditions:
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。Chromatographic column: Acquity BEH C18 column (2.1
× 100 mm, 1.7μm), column temperature: 40℃; flow rate: 0.35ml/min; injection volume: 2μl; mass spectrometry conditions: ion source: ESI source; drying gas temperature: 180℃; 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.
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。Results: (e) The sample from site E showed chromatographic peaks of flavonoid glycosides (e.g., quercetin-3-O-β-D-pyranoglucoside, punicalagin B/carpinusin) at retention times of 12.8-13.8 min (e.g., about 13.3 min) and 14.8-15.8 min (e.g., about 15.3 min), and/or chromatographic peaks of lignans (e.g., nudiposide) at retention times of 14.4-15.4 min (e.g., about 14.9 min), see Figure 1; (f) The sample from site F showed chromatographic peaks of lignans (e.g., nudiposide) at retention times of 12.8-13.8 min (e.g., about 13.3 min), 13.8-15.8 min (e.g., about 15.3 min), and/or chromatographic peaks of lignans (e.g., nudiposide) at retention times of 14.4-15.4 min (e.g., about 14.9 min), see Figure 1; .8-14.8min (e.g., at about 14.3min) and at retention times of 14.8-15.8min (e.g., at about 15.3min) show chromatographic peaks of flavonoid glycosides (e.g., quercetin-3-O-β-D-pyranoglucoside, quercetin-pyranoglucoside, punicalagin B/carpinusin), see Figure 2; (g) the sample at position G shows that (g) chromatographic peaks of flavonoid aglycones (e.g., quercetin, kaempferol 3-(2-galloyl glucoside)) are displayed between retention times of 16.6-17.6min (e.g., at about 17.1min) and between retention times of 21.4-22.4min (e.g., at about 21.9min), see Figure 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)。
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-β-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 , a measured mass ( m/z , [MH] - )551.2131, theoretical mass ( m/z , [MH] - )551.2134, the compound is nudiposide, the t R of peak 20 is 15.3 min, the molecular formula is C 41 H 28 O 27 , the measured mass ( m/z , [MH] - )951.0745, the theoretical mass ( m/z , [MH] - )951.0745, the compound is punica granatum B/carpinusin, the t R of peak 21 is 17.1 min, the molecular formula is C 21 H 20 O 11 , the measured mass ( m/z , [MH] - )447.0911, the theoretical mass ( m/z , [MH] - )447.0933, the compound is quercetin, the t R of peak 23 is 21.9 min, the molecular formula is C 28 H 24 O 15 , measured mass ( m / z , [MH] - )599.1036, theoretical mass ( m / z , [MH] - )599.1042, the compound is kaempferol 3-(2-galloyl glucoside).
制备例11：头花蓼有效组分Preparation Example 11: Effective components of Polygonum capitatum
(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%)。(1) 10 kg of the dry aerial part of Polygonum capitatum was added with 7 times the amount of 70% ethanol and refluxed for extraction twice, each time for about 1.5 hours, filtered, concentrated and dried the filtrate to obtain an alcohol extract (885 g); the ethanol also contained 0.75% acetic acid; (2) the alcohol extract (45 g) was suspended in methanol, ultrasonically treated for about 1.5 hours, centrifuged, and the supernatant was loaded onto 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 (the amount of each solvent used is about 1 column volume), the eluate of each part is collected, the solvent is recovered, and the parts obtained by elution with different solvents are dried to obtain 50% methanol eluate as part E (yield 2.91%), 50%-60% methanol eluate as part F (yield 1.97%), 60%-80% methanol eluate as part G (yield 6.14%), and 50%-80% methanol eluate as part Y (yield 10.77%).
制备例12：头花蓼有效组分Preparation Example 12: Effective components of Polygonum capitatum
(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。(1) 10 kg of the dry aerial part of Polygonum capitatum was added with 6 times the amount of 60% ethanol and refluxed for extraction for 3 times, each time for about 1 hour, filtered, concentrated and dried the filtrate to obtain an alcohol extract (935 g); the ethanol also contained 0.75% acetic acid; (2) the alcohol extract (45 g) was suspended in methanol, ultrasonically treated for about 2 hours, centrifuged, and the supernatant was loaded onto MCI macroporous resin (MCI GEL CHP
20P) column (the amount of resin used for every 100g of extract is about 3 liters); (3) gradient elution is performed in sequence with water, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, and 100% methanol (the amount of each solvent used is about 2 times the column volume), the eluate of each part is collected, the solvent is recovered, and the parts obtained by elution with different solvents are dried to obtain 50% methanol eluate as part E, 50%-60% methanol eluate as part F, 60%-80% methanol eluate as part G, and 50%-80% methanol eluate as part 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%)。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%), and the 50%-80% methanol eluate is fraction Y (yield 10.42%).
制备例13：头花蓼有效组分Preparation Example 13: Effective components of Polygonum capitatum
(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。(1) 10 kg of the dry aerial part of Polygonum capitatum was added with 10 times the amount of 80% ethanol and refluxed for extraction for 3 hours, filtered, concentrated and dried the filtrate to obtain an alcohol extract (818 g); the ethanol also contained 0.75% acetic acid; (2) the alcohol extract (45 g) was suspended in methanol, ultrasonically treated for about 1 hour, centrifuged, and the supernatant was loaded onto MCI macroporous resin (MCI GEL CHP
55A) column (the amount of resin used for every 100g of extract is about 1 liter); (3) gradient elution is performed in sequence with water, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, and 100% methanol (the amount of each solvent used is about 0.5 times the column volume), the eluate of each part is collected, the solvent is recovered, and the parts obtained by elution with different solvents are dried to obtain 50% methanol eluate as part E, 50%-60% methanol eluate as part F, 60%-80% methanol eluate as part G, and 50%-80% methanol eluate as part 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%)。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%).
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。Experimental Example 3: The alcohol extracts and various parts obtained in Preparation Examples 11 to 13 were tested using the UPLC-TOF-MS method.
UPLC-TOF-MS的测定方法如下：The UPLC-TOF-MS determination method is as follows:
(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；(i) 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 μl for mass spectrometry detection;
(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。(ii) Chromatographic column: Acquity BEH C18 column (2.1 × 100 mm, 1.7 μm), column temperature: 40 °C; flow rate: 0.35 ml/min; injection volume: 2 μl; 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.
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。Results: (e) The samples from site E showed chromatographic peaks of flavonoid glycosides between retention times of 12.5-13.9 min and 14.6-15.8 min, and chromatographic peaks of lignans between retention times of 14.5-15.7 min; (f) The samples from site F showed chromatographic peaks of quercetin-3-O-β-D-pyranoglucoside, quercetin-pyranoglucoside, and punicalagin B/carpinusin between retention times of 12.2-13.5 min, 13.9-14.7 min, and 15.0-15.9 min; (g) The samples from site G showed chromatographic peaks of quercetin and kaempferol 3-(2-galloyl glucoside) between retention times of 16.3-17.4 min and 21.6-22.5 min.
试验例4：制备例11~13所得头花蓼提取物的抗菌实验Test Example 4: Antibacterial test of the Polygonum capitatum extract obtained in Preparation Examples 11 to 13
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。Conventional antibacterial test methods were used to test Pseudomonas aeruginosa, Escherichia coli M421C1ST, Shigella dysenteriae, Staphylococcus aureus, and hemolytic Streptococcus using MH broth medium. The samples obtained in Preparation Examples 11 to 13 and the water extract of Polygonum capitatum were used as test drugs to test their sensitivity to these cells. The results showed that the MIC of Part E of Preparation Examples 11 to 13 against five bacteria was between 0.6-0.9 mg/ml, the MIC of Part F of Preparation Examples 11 to 13 against five bacteria was between 0.2-0.6 mg/ml, the MIC of Part G of Preparation Examples 11 to 13 against five bacteria was between 0.07-0.13 mg/ml, and the MIC of the water extract of Polygonum capitatum was between 10-25 mg/ml. These results showed that the extract of Polygonum capitatum obtained in Preparation Examples 11 to 13 had significantly better antibacterial activity.
Claims (1)
- 改进热淋清颗粒的清热利湿、利尿通淋、泌尿系统感染或结石、肾盂肾炎、解毒、散瘀、抗菌、抗炎、镇痛、抗前列腺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。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: (1) adding 50-90% ethanol in an amount of 5-15 times the amount of fresh or dried aerial parts of Polygonum capitatum to reflux and extract 1-3 times, each time for 1-3 hours, filtering, concentrating the filtrate, and drying to obtain an alcohol extract; the ethanol further comprises The invention relates to a method for preparing the aqueous phase of the present invention, wherein the aqueous phase of the aqueous phase of the present invention comprises the steps of: (1) suspending the aqueous phase of ...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。2. The method according to claim 1, wherein the methanol eluate obtained in step (3) is 50%, 50%-60%, or 60%-80% methanol eluate.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。3. The method according to claim 1, wherein the methanol eluate obtained in step (3) is a mixture of methanol eluates with a content of between 50% and 80%.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。4. The method according to claim 1, wherein the fraction E contains lignans and/or flavonoid glycosides, for example, a mixture of lignans and flavonoid glycosides.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)。5. The method according to claim 1, wherein the part F contains flavonoid glycosides, the part G contains flavonoid aglycones, and the part Y contains lignans and/or flavonoid glycosides and/or flavonoid aglycones, such as quercetin-3-O-β-D-pyranoglucopyranoside, quercetin-pyranoglucopyranoside, punicalagin B/carpinusin, quercetin, kaempferol 3-(2-galloyl glucoside).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：6. According to the method of claim 1, the Polygonum capitatum extract is measured by ultra-high performance liquid chromatography-time of flight-mass spectrometry, and the results are as follows: (e) chromatographic peaks of flavonoid glycosides are shown between retention time 12.8-13.8 min and between retention time 14.8-15.8 min, and/or chromatographic peaks of lignans are shown between retention time 14.4-15.4 min; (f) chromatographic peaks of flavonoid glycosides are shown between retention time 12.8-13.8 min, between retention time 13.8-14.8 min, and between retention time 14.8-15.8 min; (g) chromatographic peaks of flavonoid aglycones are shown between retention time 16.6-17.6 min and between retention time 21.4-22.4 min; and/or (y) any or all of the chromatographic peaks shown in (e) to (g) above; wherein the determination method of UPLC-TOF-MS is as follows:(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；(i) 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 μl for mass spectrometry detection;(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。(ii) Chromatographic column: Acquity BEH C18 column (2.1 × 100 mm, 1.7μm), column temperature: 40℃; flow rate: 0.35ml/min; injection volume: 2μl; mass spectrometry conditions: ion source: ESI source; drying gas temperature: 180℃; 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.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。7. The method according to claim 1, wherein the Polygonum capitatum extract is measured by ultra-high performance liquid chromatography-time of flight-mass spectrometry, and the result is: (e) chromatographic peaks of flavonoid glycosides are shown between retention times of 12.8-13.8 min and between retention times of 14.8-15.8 min, and/or chromatographic peaks of lignans are shown between retention times of 14.4-15.4 min.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。8. The method according to claim 1, wherein the Polygonum capitatum extract shows chromatographic peaks of flavonoid glycosides between retention times 12.8-13.8 min, between retention times 13.8-14.8 min, and between retention times 14.8-15.8 min when measured by ultra performance liquid chromatography-time of flight-mass spectrometry.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。9. The method according to claim 1, wherein the Polygonum capitatum extract shows chromatographic peaks of flavonoid aglycones between retention times 16.6-17.6 min and between retention times 21.4-22.4 min as measured by ultra-high performance liquid chromatography-time of flight-mass spectrometry.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。10. The method according to claim 1, wherein the Polygonum capitatum extract is measured by ultra-high performance liquid chromatography-time-of-flight-mass spectrometry, and the results show that: (e) the chromatographic peaks of flavonoid glycosides are shown between retention time 12.8-13.8 min and between retention time 14.8-15.8 min, and/or the chromatographic peaks of lignans are shown between retention time 14.4-15.4 min; (f) the chromatographic peaks of flavonoid glycosides are shown between retention time 12.8-13.8 min, between retention time 13.8-14.8 min and between retention time 14.8-15.8 min; and/or, (g) the chromatographic peaks of flavonoid aglycones are shown between retention time 16.6-17.6 min and between retention time 21.4-22.4 min.
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