WO2024077490A1 - Method for improving the quality of polygonum capitatum extract and quality of relinqing granules - Google Patents
Method for improving the quality of polygonum capitatum extract and quality of relinqing granules Download PDFInfo
- Publication number
- WO2024077490A1 WO2024077490A1 PCT/CN2022/124674 CN2022124674W WO2024077490A1 WO 2024077490 A1 WO2024077490 A1 WO 2024077490A1 CN 2022124674 W CN2022124674 W CN 2022124674W WO 2024077490 A1 WO2024077490 A1 WO 2024077490A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- extract
- polygonum capitatum
- thin layer
- water
- preparation
- Prior art date
Links
- 241000764065 Persicaria capitata Species 0.000 title claims abstract description 237
- 239000000284 extract Substances 0.000 title claims abstract description 137
- 238000000034 method Methods 0.000 title claims abstract description 113
- 239000008187 granular material Substances 0.000 title claims abstract description 91
- 239000000463 material Substances 0.000 claims abstract description 148
- 238000002360 preparation method Methods 0.000 claims abstract description 103
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 77
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 71
- 238000004809 thin layer chromatography Methods 0.000 claims abstract description 53
- 239000000706 filtrate Substances 0.000 claims abstract description 37
- 238000001035 drying Methods 0.000 claims abstract description 36
- 238000001914 filtration Methods 0.000 claims abstract description 25
- 238000012360 testing method Methods 0.000 claims abstract description 25
- 238000000605 extraction Methods 0.000 claims abstract description 24
- 238000002156 mixing Methods 0.000 claims abstract description 22
- 239000006286 aqueous extract Substances 0.000 claims abstract description 20
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- 239000008107 starch Substances 0.000 claims abstract description 17
- 235000019698 starch Nutrition 0.000 claims abstract description 17
- 238000010992 reflux Methods 0.000 claims abstract description 13
- 238000001694 spray drying Methods 0.000 claims abstract description 12
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 123
- 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 54
- 239000012085 test solution Substances 0.000 claims description 46
- 238000011161 development Methods 0.000 claims description 44
- 239000002904 solvent Substances 0.000 claims description 30
- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 claims description 28
- 239000000243 solution Substances 0.000 claims description 28
- 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 27
- 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 27
- 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 27
- 235000005875 quercetin Nutrition 0.000 claims description 27
- 229960001285 quercetin Drugs 0.000 claims description 27
- JMGZEFIQIZZSBH-UHFFFAOYSA-N Bioquercetin Natural products CC1OC(OCC(O)C2OC(OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5)C(O)C2O)C(O)C(O)C1O JMGZEFIQIZZSBH-UHFFFAOYSA-N 0.000 claims description 26
- IVTMALDHFAHOGL-UHFFFAOYSA-N eriodictyol 7-O-rutinoside Natural products OC1C(O)C(O)C(C)OC1OCC1C(O)C(O)C(O)C(OC=2C=C3C(C(C(O)=C(O3)C=3C=C(O)C(O)=CC=3)=O)=C(O)C=2)O1 IVTMALDHFAHOGL-UHFFFAOYSA-N 0.000 claims description 26
- FDRQPMVGJOQVTL-UHFFFAOYSA-N quercetin rutinoside Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 FDRQPMVGJOQVTL-UHFFFAOYSA-N 0.000 claims description 26
- 235000005493 rutin Nutrition 0.000 claims description 26
- IKGXIBQEEMLURG-BKUODXTLSA-N rutin Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@@H]1OC[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 IKGXIBQEEMLURG-BKUODXTLSA-N 0.000 claims description 26
- ALABRVAAKCSLSC-UHFFFAOYSA-N rutin Natural products CC1OC(OCC2OC(O)C(O)C(O)C2O)C(O)C(O)C1OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5 ALABRVAAKCSLSC-UHFFFAOYSA-N 0.000 claims description 26
- 229960004555 rutoside Drugs 0.000 claims description 26
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 21
- 239000012088 reference solution Substances 0.000 claims description 18
- 239000000843 powder Substances 0.000 claims description 17
- XQWWRAXBCYLTTI-UHFFFAOYSA-N butan-2-one ethyl acetate formic acid hexane Chemical compound C(=O)O.CC(CC)=O.C(C)(=O)OCC.CCCCCC XQWWRAXBCYLTTI-UHFFFAOYSA-N 0.000 claims description 15
- 239000000203 mixture Substances 0.000 claims description 15
- 229940074391 gallic acid Drugs 0.000 claims description 14
- 235000004515 gallic acid Nutrition 0.000 claims description 14
- 239000004952 Polyamide Substances 0.000 claims description 8
- 229920002647 polyamide Polymers 0.000 claims description 8
- 239000007921 spray Substances 0.000 claims description 8
- 238000009210 therapy by ultrasound Methods 0.000 claims description 7
- 239000002024 ethyl acetate extract Substances 0.000 claims description 6
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- ZWEHNKRNPOVVGH-UHFFFAOYSA-N 2-Butanone Chemical compound CCC(C)=O ZWEHNKRNPOVVGH-UHFFFAOYSA-N 0.000 description 4
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- 239000009429 Ginkgo biloba extract Substances 0.000 description 2
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- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
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- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
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- 235000006533 astragalus Nutrition 0.000 description 2
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- PFKIBRPYVNVMRU-UHFFFAOYSA-N cyclosieversioside F Natural products CC(C)(O)C1COC(C)(C1)C2C(O)CC3(C)C4CC(OC5OC(CO)C(O)C(O)C5O)C6C(C)(C)C(CCC67CC47CCC23C)OC8OCC(O)C(O)C8O PFKIBRPYVNVMRU-UHFFFAOYSA-N 0.000 description 2
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- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 2
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- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
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- RDEIXVOBVLKYNT-VQBXQJRRSA-N (2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2r,3r,6s)-3-amino-6-(1-aminoethyl)oxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-5-methyl-4-(methylamino)oxane-3,5-diol;(2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2r,3r,6s)-3-amino-6-(aminomethyl)oxan-2-yl]o Chemical compound OS(O)(=O)=O.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H](CC[C@@H](CN)O2)N)[C@@H](N)C[C@H]1N.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H](CC[C@H](O2)C(C)N)N)[C@@H](N)C[C@H]1N.O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N RDEIXVOBVLKYNT-VQBXQJRRSA-N 0.000 description 1
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- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- GSEMDSHDLFBDML-UHFFFAOYSA-N O.OC=O.CCOC(C)=O Chemical compound O.OC=O.CCOC(C)=O GSEMDSHDLFBDML-UHFFFAOYSA-N 0.000 description 1
- 241000978468 Persicaria runcinata Species 0.000 description 1
- 241001079007 Phellodendron chinense Species 0.000 description 1
- 244000292697 Polygonum aviculare Species 0.000 description 1
- 235000006386 Polygonum aviculare Nutrition 0.000 description 1
- 101150107345 Rhag gene Proteins 0.000 description 1
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- 239000003463 adsorbent Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000002686 anti-diuretic effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- YBHILYKTIRIUTE-UHFFFAOYSA-N berberine Chemical compound C1=C2CC[N+]3=CC4=C(OC)C(OC)=CC=C4C=C3C2=CC2=C1OCO2 YBHILYKTIRIUTE-UHFFFAOYSA-N 0.000 description 1
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- LIOIDYIXMHPGGB-UHFFFAOYSA-N chloroform;formic acid;methanol Chemical compound OC.OC=O.ClC(Cl)Cl LIOIDYIXMHPGGB-UHFFFAOYSA-N 0.000 description 1
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- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 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
-
- 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
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
-
- 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
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
- G01N30/95—Detectors specially adapted therefor; Signal analysis
Definitions
- the present invention belongs to the field of traditional Chinese medicine, relates to the field of quality evaluation of traditional Chinese medicine, and specifically relates to a quality evaluation method of Relinqing granules and its raw material Polygonum capitatum, especially relates to a method for determining the fingerprint of Relinqing granules and its raw material Polygonum capitatum, and more specifically relates to a method for evaluating the quality of Relinqing granules and Polygonum capitatum from different origins through thin layer chromatography fingerprint.
- the present invention essentially provides a method for improving the quality of Polygonum capitatum extract and Relinqing granules.
- Polygonum capitatum is a commonly used Miao medicine in Guizhou, and is one of the "six major Miao medicines” and “seven major Chinese medicine industry chains” that are key to the modernization of traditional Chinese medicine in Guizhouzhou.
- the applicant of the present invention has established standardized planting bases for Polygonum capitatum in Shibing, Guizhou and Wudang District, Guiyang, and has passed the national GAP certification.
- CN101091749A discloses a Polygonum capitatum medicinal material, an extract and a quality control method thereof, wherein the Polygonum capitatum medicinal material has an HPLC fingerprint spectrum, contains 13 characteristic peaks, and the chromatographic conditions are: chromatographic column: HYPERSIL ODS chromatographic column (4.6mm ⁇ 150mm, 5 ⁇ m); column temperature: 25°C; mobile phase: acetonitrile (A)-0.4% phosphoric acid (B) solution binary gradient elution, weight percentage: 0-30%: 100-70%; flow rate: 0.8mL ⁇ min -1 ; detection wavelength: 310nm; injection volume 20 ⁇ L.
- the quality control method of the invention investigates and compares Polygonum capitatum from different origins, and constructs the HPLC fingerprint spectrum of Polygonum capitatum.
- CN102296118A discloses a method for identifying Polygonum capitatum and Polygonum nepalensis using DNA molecular marker identification technology.
- Polygonum capitatum a representative ethnic medicine in Guizhou
- RAPD analysis method to analyze the genetic diversity of Polygonum capitatum and Polygonum nepalensis; and on the basis of RAPD experiments, the RAPD method is converted into SCAR markers, providing an accurate, reliable, fast and stable method for the identification of Polygonum capitatum and Polygonum nepalensis.
- DNA molecular marker identification technology is also not suitable for conventional quality evaluation and control of medicinal materials.
- the fingerprint of traditional Chinese medicine refers to the chromatogram or spectrum of a certain type or several types of components that are common and characteristic in a certain type of Chinese herbal medicine or Chinese patent medicine.
- the fingerprint of traditional Chinese medicine is of great significance for effectively controlling the quality of Chinese herbal medicine or Chinese patent medicine.
- the main manufacturers of Japanese Kampo medicine had already adopted high-performance liquid fingerprint to control quality within the company.
- the medical effect of ginkgo leaf extract is the result of the overall effect of the substance group obtained from the extract, and the high-performance liquid fingerprint method is also used for such a whole quality control.
- the current standard for evaluating the quality of traditional Chinese medicine is to use spectral or chromatographic methods to identify and determine one or several effective ingredients, active ingredients or index ingredients, as well as routine inspection items specified in the pharmacopoeia.
- the setting of these quality standards is imitating the model of chemical drugs.
- Other countries such as the United Kingdom, India, the American Herbal Codex, the Chinese medicine in the Japanese Pharmacopoeia, and the German Herbal Monographs edited by the German Commission E also adopt basically the same content.
- their active ingredients are single compounds with clear structures and clear structure-activity relationships. Their content and purity directly express their effectiveness and safety.
- the characteristic of traditional Chinese medicine is compound compatibility, and the high or low content of any single effective or active ingredient cannot express its overall efficacy.
- astragaloside IV contained in Astragalus is currently the most common target selected as the quality standard for identification and content determination, but there is no evidence to prove the clear connection between astragaloside IV and the functions and indications of Astragalus.
- Coptis chinensis, Phellodendron chinense, and Tripterygium wilfordii all contain berberine, which is generally used as the target of detection, but the functions and indications of the three are completely different. The situation of compound preparations is even more complicated.
- the non-one-to-one nonlinear theory and practice of traditional Chinese medicine indicate that the quality of traditional Chinese medicine should adopt some kind of macro-comprehensive quality evaluation method.
- One object of the present invention is to provide a method for improving the quality of Polygonum capitatum extract and Relinqing granules.
- Another object of the present invention is to provide a method suitable for routine use for evaluating the quality of Relinqing granules and their raw material Polygonum capitatum, in particular, a method that can be used for authenticity identification and/or preliminary quality determination of Relinqing granules and their Polygonum capitatum medicinal materials.
- TLC thin layer chromatography
- This method can effectively and objectively and accurately evaluate the quality of Relinqing granules and their Polygonum capitatum medicinal materials, and provide a reference for improving the quality standards of Relinqing granules and Polygonum capitatum medicinal materials, as well as the selection and breeding of excellent seed sources and standardized production of Polygonum capitatum medicinal materials.
- the present invention is completed based on this discovery.
- the first aspect of the present invention provides a method for improving the quality of Relinqing granules, which is implemented by preparing and testing a Polygonum capitatum extract to obtain a high-quality Relinqing granule preparation, comprising the following steps:
- step (2) adding 6-8 times the amount of water to the residue obtained in step (1) and boiling and extracting twice, each time for 1 hour, filtering, concentrating the filtrate, and drying to obtain an aqueous extract;
- the method according to the first aspect of the present invention further comprises determining the quality of the obtained Polygonum capitatum extract and/or Relinqing granules by the following thin layer chromatography method:
- the general operating specification of the "thin layer chromatography" may not be limited.
- the thin layer chromatography may be a method recorded in, but not limited to, the following documents: Appendix VI B of Part I of the Pharmacopoeia of the People's Republic of China 2010 edition, Appendix VB of Part II of the Pharmacopoeia of the People's Republic of China 2010 edition, Appendix VI B of Part I of the Pharmacopoeia of the People's Republic of China 2005 edition, Appendix VB of Part II of the Pharmacopoeia of the People's Republic of China 2005 edition, etc.
- the Polygonum capitatum test solution is prepared by the following method: take the Polygonum capitatum medicinal material powder, put it into a volumetric flask, add an appropriate amount of water, ultrasonically treat it, cool it, dilute it to the scale with water, filter it, extract the filtrate with ethyl acetate, evaporate the ethyl acetate solution, add ethyl acetate to dissolve the residue, and obtain the test solution.
- the ultrasonic treatment in step (a) is carried out at a power of 250 W and a frequency of 33 kHz for 15 to 60 min, preferably 20 to 40 min, for example about 30 min.
- the Polygonum capitatum test solution is prepared by the following method: take 2 g of Polygonum capitatum medicinal material powder, put it into a 25 mL volumetric flask, add 25 mL of water, and ultrasonically treat it at a power of 250 W and a frequency of 33 kHz for 30 min, cool it, dilute it to the scale with water, filter it, extract the filtrate with ethyl acetate for 3 times, 25 mL each time, combine the ethyl acetate solutions, evaporate them, add 5 mL of ethyl acetate to dissolve the residue, and obtain the test solution.
- step (a) further includes the operation of preparing reference solutions of rutin, quercetin and gallic acid, respectively, the steps of which are: accurately weighing appropriate amounts of rutin, quercetin and gallic acid reference substances dried to constant weight, respectively, and using methanol to prepare reference solutions with concentrations of 0.5 ⁇ 0.05 mg/mL, 0.1 ⁇ 0.02 mg/mL, and 0.1 ⁇ 0.02 mg/mL, respectively.
- the volume of the spotting solution is 2-10 ⁇ L, preferably 2-6 ⁇ L, such as 4 ⁇ L, such as 5 ⁇ L.
- step (b) the spotting solution is spotted at a position 10 mm away from the bottom edge of the thin layer plate.
- the expansion distance is 7 to 15 cm, such as 7 to 12 cm, such as 7 to 10 cm, preferably 8 cm.
- step (b) a mixed solution of n-hexane-ethyl acetate-butanone-formic acid in a volume ratio of 7:6.5:2.5:3 is used for secondary development.
- step (b) the development is carried out by adding the above-mentioned developing solvent to one side of a double-trough developing box for pre-balancing, at a temperature of 20°C and a relative humidity of less than 88% (preferably less than 72%, more preferably less than 50%).
- step (b) is performed in the following manner: using a polyamide thin layer plate, taking 4 ⁇ L of the test solution and the reference solution, and spotting them at a distance of 10 mm from the bottom edge of the thin layer plate using a semi-automatic spotter; using a mixed solution of n-hexane-ethyl acetate-butanone-formic acid in a volume ratio of 7:6.5:2.5:3 as a developing agent; adding the above-mentioned developing solvent to one side of a double-slot developing box at a temperature of 20°C and a relative humidity of less than 88% for pre-equilibration for 15 minutes, developing upward with a development distance of 8 cm; and using a mixed solution of n-hexane-ethyl acetate-butanone-formic acid in a volume ratio of 7:6.5:2.5:3 as a developing agent for secondary development.
- the inspection in step (c), can be visual observation, photographic processing and/or scanning by a scanner.
- Photographic processing and/or scanning by a scanner can easily obtain spots and chromatographic peaks on the unfolded thin layer plate, which is useful for further evaluating the results.
- the method of the first aspect of the present invention also includes step (d) of analyzing the thin layer chromatogram: the medicinal material corresponding to the thin layer chromatographic fingerprint with 5 common peaks/spots can be preliminarily determined to be the authentic Polygonum capitatum medicinal material, while the medicinal material without 5 common peaks/spots can be determined to be a counterfeit.
- step (d) if the 3rd and 4th peaks/spots of the medicinal material correspond to the peaks/spots shown by rutin, it can be preliminarily determined that the medicinal material is the authentic Polygonum capitatum medicinal material, otherwise it can be determined as a counterfeit.
- step (d) if the peak/spot No. 4 of the medicinal material corresponds to the peak/spot displayed by quercetin, it can be preliminarily determined that the medicinal material is the authentic Polygonum capitatum medicinal material, otherwise it can be determined as a counterfeit.
- step (d) if the 3rd and 4th peaks/spots of the medicinal material correspond to the peaks/spots displayed by rutin, and the 4th peak/spot corresponds to the peak/spot displayed by quercetin, then it can be preliminarily determined that the medicinal material is the authentic Polygonum capitatum medicinal material, otherwise it can be determined as a counterfeit.
- step (d) under the conditions of the same sampling amount and the same spotting amount, if the intensities of the five common peaks/spots of a batch of medicinal materials are stronger than those of other batches of medicinal materials developed at the same time, it indicates that the quality of this batch of medicinal materials is better than that of other batches of medicinal materials.
- the method of the first aspect of the present invention can be applied to the existing Polygonum capitatum preparations and the Polygonum capitatum extracts used to prepare the preparations by making slight changes in the preparation process of the test solution.
- the preparation method of the Relinqing Granules included in the 2010 edition of the Chinese Pharmacopoeia records the extraction method of Polygonum capitatum.
- the extract i.e., the Polygonum capitatum extract
- the sugar-free or sugar-containing Relinqing Granules can be further prepared by adding an appropriate amount of starch and/or sucrose.
- commercially available Polygonum capitatum preparations such as Relinqing tablets and capsules are all prepared by obtaining extracts using or similarly using the Polygonum capitatum extraction method recorded in the Relinqing Granules included in the 2010 edition of the Chinese Pharmacopoeia, and then adding conventional pharmaceutical excipients to prepare tablets and capsules. These conventional pharmaceutical excipients usually do not show chromatographic spots under the TLC method of the present invention.
- the term "Polygonum capitatum preparation” has the same meaning as the terms such as Polygonum capitatum extract, Relinqing granules, Relinqing tablets, and Relinqing capsules in terms of achieving the purpose of the present invention (i.e., these samples are suitable for determining thin layer chromatography fingerprints using the method of the present invention).
- the second aspect of the present invention provides a method for determining the fingerprint of a Polygonum capitatum preparation (e.g., Relinqing granules) and a Polygonum capitatum extract used to prepare the preparation, which is determined by thin layer chromatography and comprises the following steps:
- the hot leaching granules are prepared according to the following steps:
- step (2) adding 6-8 times the amount of water to the residue obtained in step (1) and boiling and extracting twice, each time for 1 hour, filtering, concentrating the filtrate, and drying to obtain an aqueous extract;
- the step (a) further optionally comprises a step of dissolving the Polygonum capitatum preparation or the Polygonum capitatum extract of the preparation with water before extraction. After the dissolving step, extraction is performed with ethyl acetate as described in step (a).
- the test solution is prepared by the following method: take the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation, put it into a volumetric flask, add water to dissolve it, dilute to the scale with water, filter it, extract the filtrate with ethyl acetate, evaporate the ethyl acetate solution, add ethyl acetate to dissolve the residue, and obtain the test solution.
- step (a) further includes the operation of preparing reference solutions of rutin, quercetin and gallic acid, respectively, the steps of which are: accurately weighing appropriate amounts of rutin, quercetin and gallic acid reference substances dried to constant weight, respectively, and using methanol to prepare reference solutions with concentrations of 0.5 ⁇ 0.05 mg/mL, 0.1 ⁇ 0.02 mg/mL and 0.1 ⁇ 0.02 mg/mL, respectively.
- the volume of the spotting solution is 2-10 ⁇ L, preferably 2-6 ⁇ L, such as 4 ⁇ L, such as 5 ⁇ L.
- step (b) the spotting solution is spotted at a position 10 mm away from the bottom edge of the thin layer plate.
- the expansion distance is 7 to 15 cm, such as 7 to 12 cm, such as 7 to 10 cm, preferably 8 cm.
- step (b) a mixed solution of n-hexane-ethyl acetate-butanone-formic acid in a volume ratio of 7:6.5:2.5:3 is used for secondary development.
- step (b) the development is carried out by adding the above-mentioned developing solvent to one side of a double-trough developing box for pre-balancing, at a temperature of 20°C and a relative humidity of less than 88% (preferably less than 72%, more preferably less than 50%).
- step (b) is performed as follows: using a polyamide thin layer plate, taking 4 ⁇ L of the test solution and the reference solution, and spotting them at a distance of 10 mm from the bottom edge of the thin layer plate using a semi-automatic spotter; using a mixed solution of n-hexane-ethyl acetate-butanone-formic acid in a volume ratio of 7:6.5:2.5:3 as a developing agent; adding the above-mentioned developing solvent to one side of a double-slot developing box at a temperature of 20°C and a relative humidity of less than 88% for pre-equilibration for 15 minutes, developing upward with a development distance of 8 cm; and using a mixed solution of n-hexane-ethyl acetate-butanone-formic acid in a volume ratio of 7:6.5:2.5:3 as a developing agent for secondary development.
- the inspection in step (c), can be visual observation, photographic processing and/or scanning by a scanner.
- Photographic processing and/or scanning by a scanner can easily obtain spots and chromatographic peaks on the unfolded thin layer plate, which is useful for further evaluating the results.
- the method of the second aspect of the present invention also includes step (d) of analyzing the thin layer chromatogram: the medicinal material corresponding to the thin layer chromatographic fingerprint with 5 common peaks/spots can be preliminarily judged as the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation is normal; if there are no 5 common peaks/spots, it can be judged as the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation is abnormal.
- step (d) if the 3rd and 4th peaks/spots of the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation correspond to the peaks/spots displayed by rutin, it can be preliminarily determined that the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation is normal, otherwise it can be determined to be abnormal. Based on the results of this method, other methods can be used to further verify the intrinsic quality of the product.
- step (d) if the peak/spot No. 4 of the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation corresponds to the peak/spot shown by quercetin, it can be preliminarily determined that the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation is normal, otherwise it can be determined to be abnormal.
- step (d) if the 3rd and 4th peaks/spots of the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation correspond to the peaks/spots shown by rutin, and the 4th peak/spot corresponds to the peak/spot shown by quercetin, then it can be preliminarily determined that the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation is normal, otherwise it can be determined to be abnormal.
- step (d) if the 5 common peaks/spot intensities of a batch of Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation are stronger than those of other batches of Polygonum capitatum preparations or the Polygonum capitatum extracts used to prepare the preparation under the conditions of the same sampling amount and the same spotting amount, then it indicates that the quality of the batch of Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation is better than that of other batches of Polygonum capitatum preparations or the Polygonum capitatum extracts used to prepare the preparation.
- the third aspect of the present invention provides a method for preparing Relinqing granules, comprising the following steps: (1) extracting the dry aerial part of the medicinal material Polygonum capitatum with ethanol by reflux, filtering, concentrating and drying the filtrate to obtain an alcohol extract; (2) decocting and extracting the filter residue obtained in step (1) by adding water, filtering, concentrating and drying the filtrate to obtain an aqueous extract; (3) mixing the alcohol extract and the aqueous extract, spray drying to remove moisture, and obtaining a Polygonum capitatum extract; (4) mixing the Polygonum capitatum extract with an appropriate amount of starch, granulating with water, and drying to obtain a preparation of Relinqing granules.
- the method according to the third aspect of the present invention comprises the following steps: (1) adding 7-10 times the amount of ethanol containing 0.75% acetic acid to the dry aerial part of the medicinal material of Polygonum capitatum, reflux extraction twice, each time for 1 hour, filtering, concentrating the filtrate, and drying to obtain an alcohol extract; (2) adding 6-8 times the amount of water to the filter residue obtained in step (1) to boil and extract twice, each time for 1 hour, filtering, concentrating the filtrate, and drying to obtain an aqueous extract; (3) mixing the alcohol extract and the aqueous extract, spray drying to remove moisture (moisture content is reduced to less than 3%), and obtaining a Polygonum capitatum extract; (4) mixing the Polygonum capitatum extract obtained from 1000 grams of the medicinal material of Polygonum capitatum with an appropriate amount of starch to a total amount of 400 grams, granulating with water, drying, and obtaining a preparation of hot linqing granules, and packaging, with a packing amount of 4 grams per
- the fourth aspect of the present invention provides a hot linqing granule, which is prepared by a method comprising the following steps: (1) extracting the dry aerial part of the medicinal material Polygonum capitatum with ethanol by reflux, filtering, concentrating and drying the filtrate to obtain an alcohol extract; (2) decocting and extracting the filter residue obtained in step (1) by adding water, filtering, concentrating and drying the filtrate to obtain an aqueous extract; (3) mixing the alcohol extract and the aqueous extract, spray drying to remove moisture, and obtaining a Polygonum capitatum extract; (4) mixing the Polygonum capitatum extract with an appropriate amount of starch, granulating with water, and drying to obtain a preparation of hot linqing granules.
- the Relinqing granules are prepared by a method comprising the following steps: (1) adding 7-10 times the amount of ethanol containing 0.75% acetic acid to the dry aerial part of the medicinal material of Polygonum capitatum, reflux extraction twice, each time for 1 hour, filtering, concentrating the filtrate, and drying to obtain an alcohol extract; (2) adding 6-8 times the amount of water to the filter residue obtained in step (1) to boil and extract twice, each time for 1 hour, filtering, concentrating the filtrate, and drying to obtain an aqueous extract; (3) mixing the alcohol extract and the aqueous extract, spray drying to remove moisture (moisture content is reduced to less than 3%), and obtaining a Polygonum capitatum extract; (4) mixing the Polygonum capitatum extract obtained from 1000 grams of the medicinal material of Polygonum capitatum with an appropriate amount of starch to a total amount of 400 grams, granulating with water, and drying to obtain a preparation of Relinqing granule
- any embodiment of any aspect of the present invention are also applicable to any other embodiment of the aspect and any embodiment of other aspects, as long as they do not contradict each other; of course, when applicable to each other, the corresponding features may be appropriately modified if necessary.
- 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.
- 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 herein. If the terms and phrases mentioned are inconsistent with the known meanings, the meanings expressed in the present invention shall prevail.
- the method of the present invention shows that the thin layer chromatography of the medicinal material of Polygonum capitatum is composed of 5 common peaks, which constitute the basic framework of the thin layer chromatography fingerprint.
- peaks 3 and 4 are rutin, and peak 4 is quercetin; since the above common peaks are contained in each batch of medicinal materials, the above common peaks should be used as characteristic components for the identification of Polygonum capitatum medicinal materials.
- the difference in the quality of medicinal materials of different batches of Polygonum capitatum samples from the same origin and different origins can be reflected from the spot color depth of these peaks.
- the five peaks of Polygonum capitatum and Relinqing granules can be used as characteristic peaks for authenticity identification of Polygonum capitatum and quality control of preparations.
- the peak heights of the samples differ, reflecting the quantitative differences in certain chemical components in the samples; but the peak shapes are consistent, reflecting that there is no major difference in quality.
- the thin layer chromatography fingerprint determination conditions of Polygonum capitatum medicinal materials are determined as follows: water is used as the extraction solvent, ultrasonic treatment, and ethyl acetate is used as the extraction solvent after extraction; n-hexane-ethyl acetate-butanone-formic acid (7:6.5:2.5:3) is used as the developing agent, secondary development, pre-equilibration for 15 minutes, and upward development; development distance: 8 cm; temperature 20°C, relative humidity controlled to 32% ⁇ 72%. Color development is performed with aluminum chloride test solution. After evaporating the solvent, the fluorescent thin layer chromatography is inspected under ultraviolet light (320nm).
- the results of the present invention show that the thin layer chromatograms and contour diagrams of 12 batches of Polygonum capitatum medicinal materials produced in Shibing, Guizhou are very similar, all with 5 spots and corresponding 5 peaks; for medicinal materials from different origins, the peaks have differences in peak intensity, reflecting the differences in the amount of chemical components contained in Polygonum capitatum from different origins.
- the counterfeit product of Polygonum capitatum, Chijingsan does not have the above characteristics, indicating that the method of the present invention can well distinguish counterfeit Polygonum capitatum products and can be used for authenticity identification of Polygonum capitatum medicinal materials.
- the thin layer chromatography fingerprint method of the present invention can not only reflect the characteristic components of the Polygonum capitatum medicinal material, but also reflect the differences between medicinal materials from different sources, and can also be used to identify the authenticity of medicinal materials; at the same time, the method is simple and easy to operate, and is convenient for promotion and application. It can be used for rapid inspection in procurement and production.
- the present invention can be further described by the following examples, however, the scope of the present invention is not limited to the following examples. Professionals in the field can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.
- the present invention generally and/or specifically describes the materials and test methods used in the test. Although many materials and operating methods used to achieve the purpose of the present invention are well known in the art, the present invention is still described as detailed as possible.
- Reagents and reagents Methanol, ethanol, chloroform, petroleum ether, cyclohexane, ethyl acetate, acetone, formic acid, n-hexane, butanone, etc. are all analytically pure and can be used as developing reagents and extracting solvents.
- Rutin batch number: 110725-200513, for content determination
- quercetin batch number: 111538-200403, for content determination
- gallic acid reference substance Batch number: 110831-200302, for content determination
- Methanol, tetrahydrofuran, and acetonitrile were chromatographically pure; water was purified water; other reagents were all analytically pure.
- Medicinal materials 12 batches of Polygonum capitatum medicinal materials were collected from the Polygonum capitatum standardized planting research and GAP trial demonstration base in Shibing, Guizhou
- the medicinal material samples No. 1 to 12 were from Shibing, Guizhou, and the medicinal material samples No. 13 to 23 were from Xingyi, Guizhou, Qinglong, Guizhou, Panxian, Guizhou, Bijie, Guizhou, Shuicheng, Guizhou, Luodian, Hunan, Shizong, Yunnan, Tianlin, Guangxi, Nanchuan, Chongqing, Nanchuan, Sichuan, and Chijinsan (counterfeit).
- the Guiyang University of Traditional Chinese Medicine identified them as the dried whole herb or aerial part of Polygonum capitatum Buch.-Ham.ex D.Don, a Polygonaceae plant; the counterfeit Chijinsan was the dried whole herb or aerial part of Polygonum runcinatum Buch.-Ham.var.sinense Hemsl., a Polygonaceae plant.
- Relinqing granules are sugar-containing Relinqing granules prepared according to the method under the item "Relinqing granules" in the 2010 edition of the Chinese Pharmacopoeia, using Polygonum capitatum produced in Shibing as the medicinal material.
- Test Example 1 The most preferred thin layer chromatography analysis conditions determined by the present invention
- the most preferred thin layer chromatography analysis conditions and operating methods can be determined as follows.
- Polygonum capitatum take 2g of the medicinal material powder of Polygonum capitatum, put it into a 25mL volumetric flask, add 25mL of water, ultrasonically treat (power 250W, frequency 33kHz) for 30min, cool, dilute to the scale with water, filter, extract the filtrate with ethyl acetate for 3 times, 25mL each time, combine the ethyl acetate solutions, evaporate to dryness, add 5mL of ethyl acetate to dissolve the residue, and use it as the test solution.
- ultrasonically treat power 250W, frequency 33kHz
- the Polygonum capitatum extract or Relinqing preparation such as Relinqing granules: take the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation (equivalent to 2 grams of the extract or preparation of the medicinal material), put it into a volumetric flask, add 25mL of water to dissolve or suspend it, make up to the scale with water, filter, extract the filtrate with ethyl acetate for 3 times, 25mL each time, combine the ethyl acetate solutions, evaporate to dryness, add 5mL of ethyl acetate to dissolve the residue, and use it as the test solution.
- Thin layer plate polyamide thin layer plate (Qingdao Ocean Chemical Plant). Take 4 ⁇ L of the above test solution and reference solution, and use a semi-automatic spotter to spot 10 mm from the bottom of the thin layer plate.
- Development system n-hexane-ethyl acetate-butanone-formic acid (7:6.5:2.5:3).
- Development method add the above development solvent to one side of the double-slot development box for pre-equilibration for 15 minutes, and develop upward; development distance 8 cm; temperature 20 ° C, control relative humidity to less than 72%.
- Detection conditions Spray with AlCl 3 test solution for color development, and after waving off the residual solvent on the thin layer plate, examine the fluorescent thin layer chromatogram under ultraviolet light (320 nm).
- test example 1 The following test further verifies the method of the present invention, and in the following test examples, unless otherwise specified, the method of this test example 1 can be used to conduct the test.
- the Polygonum capitatum medicinal material No. 1 was used for testing.
- ultrasonic treatment power 250W, frequency 33kHz
- For the water-treated sample it was extracted with ethyl acetate three times after ultrasonic treatment. After treatment (if necessary), it was concentrated to a concentration of 0.4g medicinal material per ml of the test solution to obtain 6 test sample solutions obtained by different extraction methods.
- the reference solutions of rutin and quercetin were prepared according to the method described in Test Example 1.
- S1 n-hexane-ethyl acetate-butanone-formic acid (7:6.5:2.5:3), secondary development
- S2 toluene-ethyl acetate-formic acid (10:11:3.1)
- S3 cyclohexane-ethyl acetate-formic acid (8:4:0.4)
- S4 water-ethanol-butanone-acetylacetone (65:15:15:5)
- S5 chloroform-methanol-formic acid (15:4:0.5)
- S6 cyclohexane-ethyl acetate-formic acid (5:5:1)
- S7 ethyl acetate-formic acid-water (8:1:1), secondary development.
- the thin layer chromatograms (320nm) of different extraction solvents and different development systems of Polygonum capitatum medicinal materials are shown in Figure 1 of the patent document. Taking the intuitive clarity and effective information content of the thin layer chromatogram as the investigation index, 6 different extraction solvents and 7 different development systems were compared. The results showed that different extraction solvents had a greater impact on the thin layer chromatography of Polygonum capitatum medicinal materials, among which water extraction and ethyl acetate extraction had the best effect and large spot capacity (see S1 in Figure 1 of the patent document); S2 ⁇ S7 development solvent systems had poor separation effect, serious tailing, and small spot capacity.
- the thin layer chromatogram of Polygonum capitatum medicinal materials obtained by S1 had better separation and large spot capacity, so n-hexane-ethyl acetate-butanone-formic acid (7:6.5:2.5:3) was selected for secondary development as the development solvent system, which was preferred.
- the inventor also found that for the development conditions of S1, if a single development was used or the developing agent did not contain butanone, the development effect was far worse than that of S1, and 5 obvious spots could not be separated.
- the test solution is prepared by first extracting with water and then extracting with ethyl acetate
- the existing Polygonum capitatum preparations or the Polygonum capitatum extracts used to prepare the preparations are mostly obtained by extracting the Polygonum capitatum medicinal materials with water or aqueous ethanol and removing the solvent. Therefore, for the existing Polygonum capitatum preparations or the Polygonum capitatum extracts used to prepare the preparations, the fingerprint spectrum can also be determined using the method of the first aspect of the present invention.
- step (a) of the first aspect of the method of the present invention when preparing the thin layer spotting solution, the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation is directly extracted with ethyl acetate (or if necessary, the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation can be dissolved in water before extraction), the ethyl acetate extract is evaporated to dryness, and the residue is dissolved with ethyl acetate, that is, as the test solution of the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation, the other steps are the same as the other steps of the first aspect of the method of the present invention.
- the second aspect of the present invention provides a method for determining the fingerprint of a Polygonum capitatum preparation and a Polygonum capitatum extract used to prepare the preparation, which is determined according to thin layer chromatography and includes the following steps: (a) Preparation of thin layer spotting solution: weigh the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation, extract with ethyl acetate, evaporate the ethyl acetate extract to dryness, and dissolve the residue with ethyl acetate as the Polygonum capitatum test solution; and optionally prepare reference solutions of rutin, quercetin and gallic acid respectively; and steps (b), (c), and (d) as described in any embodiment of the first aspect of the present invention.
- step (a) also optionally includes the step of dissolving the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation with water before extraction, and then extracting with ethyl acetate.
- the figure depicts the effect of relative humidity (RH, 32% and 88%) on the TLC behavior of Polygonum capitatum medicinal material (320nm).
- the four developed bands in the figure are from left to right: 1. Polygonum capitatum sample; 2. Relinqing granule sample; 3. Rutin; 4. Quercetin.
- two spotting conditions of RH72% and RH50% were also used. The results showed that the TLC behavior of Polygonum capitatum medicinal material under these two conditions was similar to that of RH32%. It can be seen that when the relative humidity (RH) is less than 88%, the TLC behavior of Polygonum capitatum medicinal material is not significantly affected.
- the relative humidity (RH) is 88% or higher, the TLC behavior of Polygonum capitatum medicinal material is significantly affected, the separation is reduced, the spots are reduced, and the chromatogram cannot be identified. Therefore, the relative humidity (RH) is less than 88%, especially when it is 32% ⁇ 72%. Relinqing granules also show similar results to medicinal materials.
- the balance between the thin layer plate and the developing agent vapor is mainly manifested in the pre-adsorption of the developing agent vapor by the thin layer from the beginning to the end of the development, and the degree of adsorption of each part of the thin layer is different.
- a multi-component developing agent selective adsorption will occur, which will affect the chromatographic separation. Therefore, when using a multi-component developing agent, the thin layer and the developing chamber are often pre-saturated to obtain a chromatogram with better reproducibility.
- S1 was used as the solvent system, and the solution obtained from the sample with the medicinal material number 1 was used as the test solution.
- the test was conducted using the medicinal material No. 1. After waving off the residual solvent on the thin layer plate, the fluorescent thin layer chromatogram was examined under a UV lamp at 320nm. The fluorescent thin layer chromatogram was imported into the Chromafinger Chromatographic Fingerprint Solution Software, and a grayscale scan was generated and integrated to obtain the scan profile of rutin, quercetin and Polygonum capitatum medicinal material samples, as shown in Figure 6 of the patent document. From the results in the figure, it can be seen that among the five main peaks of the medicinal material No. 1, the 3rd and 4th main peaks correspond to the two main peaks of rutin, and the 4th main peak also corresponds to the main peak of quercetin.
- Figure 8 depicts the thin layer chromatograms and profile scanning overlays (320 nm) of 12 batches of Polygonum capitatum medicinal materials from different origins.
- the 14 chromatographic bands in the thin layer chromatogram are from left to right: 1. Polygonum capitatum samples from Shibing, Guizhou; 2. Xingyi, Guizhou; 3. Qinglong, Guizhou; 4. Panxian, Guizhou; 5. Bijie, Guizhou; 6. Shuicheng, Guizhou; 7. Luodian, Guizhou; 8.
- Test Example 10 Thin layer chromatogram and profile scan of Polygonum capitatum and its counterfeit Chijingsan
- FIG. 9 depicts the thin layer chromatogram and profile scan (320nm) of the Polygonum capitatum medicinal material and the counterfeit Polygonum capitatum medicinal material from Shibing.
- the four chromatographic bands in the thin layer chromatogram (a) represent the test samples from left to right: 1. Polygonum capitatum sample from Shibing; 2. Chijinsan; 3. Rutin; 4. Quercetin.
- the profile scan of the Polygonum capitatum medicinal material from Shibing (b) shows 5 typical characteristic peaks, while the counterfeit Chijinsan does not show characteristic peaks at the corresponding positions.
- Extract powder A obtained from 1000 grams of medicinal materials was added with an appropriate amount of starch to a total amount of 400 grams, mixed, granulated, dried, and made into Relinqing granules, which were packaged in 4 grams per bag.
- Extract powder A obtained from another 1000 grams of medicinal materials was added with an appropriate amount of sucrose powder to a total amount of 800 grams, mixed, granulated, dried, and made into sugar-containing Relinqing granules, which were packaged in 8 grams per bag.
- Extract powder A equivalent to 1000g of medicinal materials add a proper amount of a mixture of starch, lactose and microcrystalline cellulose in a weight ratio of 1:1:1, until the total amount is 200g, mix well, granulate, dry, add a proper amount of magnesium stearate, press into tablets, and make hot leaching tablets, each weighing 0.5g.
- the thin layer plate was placed on a CS-9301PC dual-wavelength flying spot thin layer chromatography scanner and scanned in a zigzag manner at a wavelength of 320 nm to obtain the scanning profile overlay of rutin, quercetin, gallic acid and Polygonum capitatum preparation samples, and photographed.
- Figure 10 of the patent document shows the thin layer chromatograms (a) and profile scan overlays (b) of 12 batches of sugar-containing Relinqing granules.
- 1 to 12 are 12 batches of Relinqing granule samples
- 13 is rutin
- 14 is quercetin
- 15 is gallic acid.
- Figure 11 of the patent document shows the thin layer chromatograms (a) and profile scan overlays (b) of 12 batches of sugar-free Relinqing granules.
- Relinqing Granules1 Referring to the method under "Relinqing Granules" in Part I of the 2010 edition of the Chinese Pharmacopoeia, take an appropriate amount of Polygonum capitatum medicinal material, add water and boil twice, each time for 1.5 hours, filter the decoction, combine the filtrates, concentrate to an appropriate amount, filter, spray dry, and obtain extract powder A; take the extract powder A obtained from 1000 grams of medicinal material, add an appropriate amount of starch to a total amount of 400 grams, mix well, granulate, dry, and make Relinqing granules, and package, with 4 grams per bag (equivalent to 1 gram of medicinal material per bag).
- Relinqing Granules2 (1) Add 7-10 times the amount of ethanol containing 0.75% acetic acid to the dry aerial part of the medicinal material of Polygonum capitatum, reflux and extract twice, each time for 1 hour, filter, concentrate the filtrate, and dry to obtain an alcohol extract; (2) Add 6-8 times the amount of water to the filter residue obtained in step (1) and boil and extract twice, each time for 1 hour, filter, concentrate the filtrate, and dry to obtain an aqueous extract; (3) Mix the alcohol extract and the aqueous extract, spray dry to remove moisture (moisture content is reduced to less than 3%), and obtain a Polygonum capitatum extract; (4) Mix the Polygonum capitatum extract obtained from 1000 grams of the medicinal material of Polygonum capitatum with an appropriate amount of starch to a total amount of 400 grams, granulate with water, dry, and obtain a preparation of Relinqing Granules, and package them, with a packing amount of 4 grams per bag (equivalent to 1 gram of medicinal material per
- the experimental strains were first inoculated on the plate and cultured in a constant temperature and humidity incubator at 37 °C for 24 h.
- the characteristic strains were selected and inoculated into the liquid culture medium and shaken at 37 °C for 24 h.
- the suspension was diluted to 0.5 McFarland concentration, i.e. 10 ⁇ 6-10 ⁇ 8 CFU/mL, by McFarland turbidimetry for standby use; 20g of purified agar powder was added to 1L nutrient broth medium, and 1L of double distilled water was added and the pH was adjusted to 7.4. Sterilize at 121 °C for 30 min and then take out.
- the culture medium is cooled to about 45 °C, pour the culture medium into a sterile culture dish on the clean bench for condensation to obtain the experimental agar plate culture medium.
- the concentration was 12.5 ⁇ 0.012 mg/mL; gentamicin sulfate was used as the positive control and DMSO was used as the negative control; the mixture was cultured at 37°C for 24 h, and then transferred to agar plate medium.
- the micropores without bacterial growth after 24 h were the minimum inhibitory concentration (MIC) of the drug (the experimental method can be referred to: Hu Lu, Zhang Jin, Lin Liangcai, et al. Study on the material basis of the antibacterial effect of Polygonum capitatum based on spectrum-effect relationship [J]. Chinese Medicinal Materials, 2016, 39(9): 2037-2040).
- the results are as follows: the MIC values of Relinqing Granules 1 against Escherichia coli and Pseudomonas aeruginosa were 2476 mg/L and 9314 mg/L, respectively, and the MIC values of Relinqing Granules 2 against Escherichia coli and Pseudomonas aeruginosa were 153 mg/L and 1127 mg/L, respectively. The results show that the Relinqing Granules prepared by the method of the present invention exhibit significantly better biological activity.
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Abstract
A method for improving the quality of a Polygonum capitatum extract and quality of Relinqing granules, said method comprising the following steps: (1) adding ethanol to a dry product of the overground part of a Polygonum capitatum medicinal material to perform reflux extraction, performing filtration, concentrating a filtrate, and drying same to obtain an alcohol extract; (2) adding water to filter residues obtained in step (1), decocting and extracting same, performing filtration, concentrating a filtrate, and drying same to obtain an aqueous extract; (3) mixing the alcohol extract and the aqueous extract, and carrying out spray drying to remove water to obtain a Polygonum capitatum extract; and (4) mixing the Polygonum capitatum extract with a proper amount of starch, performing granulation with water, drying same to obtain a Relinqing granular preparation, and carrying out thin-layer chromatography and fingerprint tests on the Polygonum capitatum extract and/or the Relinqing granules.
Description
本发明属于中药领域，涉及中药质量评价领域，具体涉及一种热淋清颗粒及其原料药材头花蓼的质量评价方法，特别涉及热淋清颗粒及其原料药材头花蓼指纹图谱的测定方法，更特别地具体涉及一种通过薄层色谱指纹图谱对热淋清颗粒及不同产地头花蓼进行质量评价的方法。基于本发明的方法，本发明本质上提供了一种提高头花蓼提取物和热淋清颗粒质量的方法。The present invention belongs to the field of traditional Chinese medicine, relates to the field of quality evaluation of traditional Chinese medicine, and specifically relates to a quality evaluation method of Relinqing granules and its raw material Polygonum capitatum, especially relates to a method for determining the fingerprint of Relinqing granules and its raw material Polygonum capitatum, and more specifically relates to a method for evaluating the quality of Relinqing granules and Polygonum capitatum from different origins through thin layer chromatography fingerprint. Based on the method of the present invention, the present invention essentially provides a method for improving the quality of Polygonum capitatum extract and Relinqing granules.
作为热淋清颗粒的药材，已知头花蓼为蓼科植物头花蓼
Polygonum
capitatum Buch.-Ham. ex D.Don的干燥全草或地上部分，收载于《贵州省中药材民族药材质量标准》2003年版，具清热利湿、抗菌消炎、利尿通淋等功效(贵州省食品药品监督管理局. 贵州省中药材民族药材质量标准[M].贵阳：贵州科技出版社，2003:147)。
As the medicinal material of Relinqing Granules, it is known that Polygonum capitatum Buch.-Ham. ex D.Don is the dried whole herb or aerial part of the Polygonum capitatum Buch.-Ham. ex D.Don plant of the Polygonaceae family, which is included in the 2003 edition of "Quality Standards for Traditional Chinese Medicines and Ethnic Medicinal Materials in Guizhou Province". It has the effects of clearing away heat and dampness, antibacterial and anti-inflammatory, diuretic and relieving stranguria (Guizhou Provincial Food and Drug Administration. Quality Standards for Traditional Chinese Medicines and Ethnic Medicinal Materials in Guizhou Province [M]. Guiyang: Guizhou Science and Technology Press, 2003:147).
头花蓼是贵州常用苗药，是贵州省中药现代化重点发展的“六大苗药”和重点培育发展的“七大中药产业链”的品种之一。本发明的申请人已在贵州施秉及贵阳乌当区建立了头花蓼规范化种植基地，并通过国家GAP认证。Polygonum capitatum is a commonly used Miao medicine in Guizhou, and is one of the "six major Miao medicines" and "seven major Chinese medicine industry chains" that are key to the modernization of traditional Chinese medicine in Guizhou Province. The applicant of the present invention has established standardized planting bases for Polygonum capitatum in Shibing, Guizhou and Wudang District, Guiyang, and has passed the national GAP certification.
现在有关头花蓼GAP种植及化学成分等方面的研究已取得一定成果(孙长生，韩见宇，杨锦纲，等. 头花蓼GAP种植基地的环境质量评价[J]. 中药研究与信息，2005, 7(2):
27~30)，但头花蓼药材现行质量标准较为简单，仅以单一成分槲皮素为指标进行评价，无法达到真实、全面反应药材内在品质的目的。At present, research on GAP planting and chemical composition of Polygonum capitatum has achieved certain results (Sun Changsheng, Han Jianyu, Yang Jingang, et al. Environmental quality evaluation of GAP planting base of Polygonum capitatum [J]. Chinese Medicine Research and Information, 2005, 7(2):
27~30). However, the current quality standard of Polygonum capitatum medicinal material is relatively simple, and only a single component quercetin is used as an indicator for evaluation, which cannot achieve the purpose of truly and comprehensively reflecting the intrinsic quality of the medicinal material.
CN101091749A公开了一种头花蓼药材、提取物及其质量控制方法，其中的头花蓼药材具有如HPLC指纹图谱，含有13个特征峰，其中色谱条件为:色谱柱:依利特HYPERSIL ODS色谱柱(4.6mm×150mm，5μm)；柱温：25℃；流动相：乙腈(A)-0.4%磷酸(B)溶液二元梯度洗脱，重量百分比为：0-30%:100-70%；流速:0.8mL·min
-1；检测波长：310nm；进样量20μL。该发明质控制方法对不同产地头花蓼进行了考察比较，构建了头花蓼HPLC的指纹图谱。
CN101091749A discloses a Polygonum capitatum medicinal material, an extract and a quality control method thereof, wherein the Polygonum capitatum medicinal material has an HPLC fingerprint spectrum, contains 13 characteristic peaks, and the chromatographic conditions are: chromatographic column: HYPERSIL ODS chromatographic column (4.6mm×150mm, 5μm); column temperature: 25℃; mobile phase: acetonitrile (A)-0.4% phosphoric acid (B) solution binary gradient elution, weight percentage: 0-30%: 100-70%; flow rate: 0.8mL·min -1 ; detection wavelength: 310nm; injection volume 20μL. The quality control method of the invention investigates and compares Polygonum capitatum from different origins, and constructs the HPLC fingerprint spectrum of Polygonum capitatum.
CN102296118A公开了一种DNA分子标记鉴别技术鉴别头花蓼与尼泊尔蓼的方法，在该发明中，以贵州具有代表性的民族药头花蓼为研究对象，探索使用RAPD分析方法对头花蓼与尼泊尔蓼进行遗传多样性分析；并在RAPD的实验基础上，将RAPD方法转化为SCAR标记，为头花蓼与尼泊尔蓼的药材鉴定提供了准确可靠、快速稳定的方法。DNA分子标记鉴别技术同样不适合药材的常规质量评价和控制。CN102296118A discloses a method for identifying Polygonum capitatum and Polygonum nepalensis using DNA molecular marker identification technology. In this invention, Polygonum capitatum, a representative ethnic medicine in Guizhou, is used as the research object to explore the use of RAPD analysis method to analyze the genetic diversity of Polygonum capitatum and Polygonum nepalensis; and on the basis of RAPD experiments, the RAPD method is converted into SCAR markers, providing an accurate, reliable, fast and stable method for the identification of Polygonum capitatum and Polygonum nepalensis. DNA molecular marker identification technology is also not suitable for conventional quality evaluation and control of medicinal materials.
中药指纹图谱是指某种中药材或中成药中所共有的、具有特征性的某类或数类成分的色谱或光谱的图谱。在现阶段中药的有效成分绝大多数没有明确的情况下，中药指纹图谱对于有效控制中药材或中成药的质量具有重要的意义。日本汉方药主要生产企业在20世纪80年代就已经在企业内部采用高效液相指纹图谱控制质量。德国、法国在对银杏叶提取物联合开发的过程中，发现银杏叶提取物的医疗作用是提取物所得物质群的整体作用结果，而对这样一个整体的质量控制，亦采用高效液相指纹图谱方法。美国FDA最近几年制定的植物草药指南中已经明确把指纹图谱作为混合物质群的质量控制方法(FDA．Guidance of Industy：Botanical Drug(Draft)．2000 August)。随着研究的深入，人们发现，作为中医理论的实践产物，中药，尤其是复方中药，其中所含的任一成分都不能代表其整体疗效。人们逐渐认识到，现行的参照西药(合成药)质量控制模式的质量标准不能恰当地反映中药内在的质量。从发展趋势看，从现行的质量控制模式向一种综合的、宏观的、可量化的鉴别与主要有效成分含量测定结合已是发展的趋势。The fingerprint of traditional Chinese medicine refers to the chromatogram or spectrum of a certain type or several types of components that are common and characteristic in a certain type of Chinese herbal medicine or Chinese patent medicine. At present, when most of the effective ingredients of Chinese medicine are not clear, the fingerprint of traditional Chinese medicine is of great significance for effectively controlling the quality of Chinese herbal medicine or Chinese patent medicine. In the 1980s, the main manufacturers of Japanese Kampo medicine had already adopted high-performance liquid fingerprint to control quality within the company. In the process of joint development of ginkgo leaf extract, Germany and France found that the medical effect of ginkgo leaf extract is the result of the overall effect of the substance group obtained from the extract, and the high-performance liquid fingerprint method is also used for such a whole quality control. In the botanical herbal medicine guidelines formulated by the US FDA in recent years, fingerprints have been clearly used as a quality control method for mixed substance groups (FDA. Guidance of Industry: Botanical Drug (Draft). August 2000). With the deepening of research, people have found that as a practical product of traditional Chinese medicine theory, Chinese medicine, especially compound Chinese medicine, does not contain any component that represents its overall efficacy. People gradually realize that the current quality standards based on the quality control model of Western medicine (synthetic medicine) cannot properly reflect the intrinsic quality of Chinese medicine. From the development trend, it is a trend to move from the current quality control model to a comprehensive, macroscopic, quantifiable identification combined with the determination of the content of the main active ingredients.
中药质量评价的现行标准是利用光谱或色谱手段鉴别和测定某一种或几种有效成分、活性成分或指标成分，以及药典规定的常规检查项目。显然，这些质量标准的设置是模仿了化学药品的模式。其它国家如英国、印度、美国草药典、日本药局方中的汉药及德国Commission
E编辑的德国草药专论等也采用了基本相同的内容。对于化学药品而言，其药效成分为结构清晰的单一化合物，构效关系明确，其含量和纯度直接表达其有效及安全性。然而，中医用药的特点是复方配伍，任何单一的有效或活性成分的含量高低均不能表达其整体的疗效。例如，黄芪所含的黄芪甲苷(aastraga loside IV)是当前被选择为质量标准的鉴别和含量测定的最为常见的目标，但并没有依据证明黄芪甲苷与黄芪的功能主治的明确联系。同样，黄连、黄柏、三棵针均含小檗碱，一般都以它作为检测的目标，但是三者的功能主治却截然不同。复方制剂的情况就更加复杂。中医这种不是一对一的非线性的理论和实践说明中药质量应该采用某种宏观的综合的质量评价手段。The current standard for evaluating the quality of traditional Chinese medicine is to use spectral or chromatographic methods to identify and determine one or several effective ingredients, active ingredients or index ingredients, as well as routine inspection items specified in the pharmacopoeia. Obviously, the setting of these quality standards is imitating the model of chemical drugs. Other countries such as the United Kingdom, India, the American Herbal Codex, the Chinese medicine in the Japanese Pharmacopoeia, and the German Herbal Monographs edited by the German Commission
E also adopt basically the same content. For chemical drugs, their active ingredients are single compounds with clear structures and clear structure-activity relationships. Their content and purity directly express their effectiveness and safety. However, the characteristic of traditional Chinese medicine is compound compatibility, and the high or low content of any single effective or active ingredient cannot express its overall efficacy. For example, astragaloside IV contained in Astragalus is currently the most common target selected as the quality standard for identification and content determination, but there is no evidence to prove the clear connection between astragaloside IV and the functions and indications of Astragalus. Similarly, Coptis chinensis, Phellodendron chinense, and Tripterygium wilfordii all contain berberine, which is generally used as the target of detection, but the functions and indications of the three are completely different. The situation of compound preparations is even more complicated. The non-one-to-one nonlinear theory and practice of traditional Chinese medicine indicate that the quality of traditional Chinese medicine should adopt some kind of macro-comprehensive quality evaluation method.
然而就头花蓼药材的质量评价，例如就产地差异对头花蓼药材进行质量评价，或者对药材的真伪进行鉴别，以及就由头花蓼制备得到的提取物或者制剂，例如热淋清颗粒，迄今为止尚无一从整体上控制样品质量的适用的常规检验方法。因此，本领域仍然需要有一从整体上控制头花蓼药材提取物或者制剂例如热淋清颗粒质量评价的方法，特别是可以用于头花蓼药材真伪鉴别以及药材、提取物或制剂简便、宏观、可行的质量判定方法。However, there is no conventional test method for overall quality control of samples for quality evaluation of Polygonum capitatum medicinal materials, such as quality evaluation of Polygonum capitatum medicinal materials based on differences in origin, or identification of the authenticity of medicinal materials, as well as extracts or preparations prepared from Polygonum capitatum, such as Relinqing Granules. Therefore, there is still a need in the art for a method for overall quality evaluation of extracts or preparations of Polygonum capitatum medicinal materials, such as Relinqing Granules, especially a simple, macroscopic, and feasible quality determination method that can be used for authenticity identification of Polygonum capitatum medicinal materials and medicinal materials, extracts or preparations.
基于上述现有待解决的技术问题，本领域技术人员迫切期待提供了一种提高头花蓼提取物和热淋清颗粒质量的方法。Based on the above-mentioned existing technical problems to be solved, those skilled in the art are eager to provide a method for improving the quality of Polygonum capitatum extract and Relinqing granules.
本发明的一个目的在于提供一种提高头花蓼提取物和热淋清颗粒质量的方法。本发明的另一个目的在于提供一种适合常规使用的用于热淋清颗粒及其原料药材头花蓼质量评价的方法，特别是可以用于热淋清颗粒及其头花蓼药材真伪鉴别和/或初步质量判定的方法。本发明人发现采用一种独特的薄层色谱法(TLC)方法可以有效地实现至少一个上述的目的，该方法可以有效地为客观、准确地评价热淋清颗粒及其头花蓼药材的品质，为热淋清颗粒及头花蓼药材质量标准提升以及头花蓼药材优良种源选育、规范化生产提供参考依据。本发明基于此发现而得以完成。One object of the present invention is to provide a method for improving the quality of Polygonum capitatum extract and Relinqing granules. Another object of the present invention is to provide a method suitable for routine use for evaluating the quality of Relinqing granules and their raw material Polygonum capitatum, in particular, a method that can be used for authenticity identification and/or preliminary quality determination of Relinqing granules and their Polygonum capitatum medicinal materials. The inventors have discovered that a unique thin layer chromatography (TLC) method can effectively achieve at least one of the above-mentioned purposes. This method can effectively and objectively and accurately evaluate the quality of Relinqing granules and their Polygonum capitatum medicinal materials, and provide a reference for improving the quality standards of Relinqing granules and Polygonum capitatum medicinal materials, as well as the selection and breeding of excellent seed sources and standardized production of Polygonum capitatum medicinal materials. The present invention is completed based on this discovery.
为此，本发明第一方面提供了提高热淋清颗粒质量的方法，该方法通过制备并检测头花蓼提取物以获取高品质的热淋清颗粒制剂来实施，包括如下步骤：To this end, the first aspect of the present invention provides a method for improving the quality of Relinqing granules, which is implemented by preparing and testing a Polygonum capitatum extract to obtain a high-quality Relinqing granule preparation, comprising the following steps:
(1)将头花蓼药材的地上部分干品加7-10倍量的包含0.75%乙酸的乙醇回流提取2次，每次1小时，过滤，使滤液浓缩、干燥，得醇浸膏；(1) adding 7-10 times the amount of ethanol containing 0.75% acetic acid to the dry aerial part of the medicinal material of Polygonum capitatum, reflux extraction twice, each time for 1 hour, filtering, concentrating the filtrate, and drying to obtain an alcohol extract;
(2)使步骤(1)所得滤渣加6-8倍量水煎煮提取2次，每次1小时，过滤，使滤液浓缩、干燥，得水浸膏；(2) adding 6-8 times the amount of water to the residue obtained in step (1) and boiling and extracting twice, each time for 1 hour, filtering, concentrating the filtrate, and drying to obtain an aqueous extract;
(3)使所述醇浸膏和水浸膏混合，喷雾干燥除去水分(水分降至3%以下)，得到头花蓼提取物，(3) mixing the alcohol extract and the water extract, and spray drying to remove water (water content is reduced to less than 3%) to obtain a Polygonum capitatum extract,
(4)使相当于由1000克头花蓼药材制得的头花蓼提取物与适量淀粉混合至总量为400克，用水制粒，干燥，得到热淋清颗粒的制剂，包装，每袋装量4克(相当于每袋包含药材1克)。(4) The extract of Polygonum capitatum obtained from 1000 g of the medicinal material of Polygonum capitatum is mixed with an appropriate amount of starch to a total amount of 400 g, granulated with water, dried to obtain a preparation of Relinqing granules, and packaged, with a filling amount of 4 g per bag (equivalent to 1 g of the medicinal material per bag).
根据本发明第一方面的方法，其中还包括对制得的头花蓼提取物和/或热淋清颗粒照以下薄层色谱法测定其质量：The method according to the first aspect of the present invention further comprises determining the quality of the obtained Polygonum capitatum extract and/or Relinqing granules by the following thin layer chromatography method:
(a)薄层点样液制备：称取头花蓼药材粉末或头花蓼提取物或热淋清颗粒，加水超声处理，过滤，滤液用乙酸乙酯萃取，将乙酸乙酸萃取液蒸干，残渣用乙酸乙酯使溶解，作为供试品溶液；以及任选地分别制备芦丁、槲皮苷和没食子酸的对照品溶液；(a) Preparation of thin layer spotting solution: weigh the powder of Polygonum capitatum medicinal material or Polygonum capitatum extract or Relinqing granules, add water for ultrasonic treatment, filter, extract the filtrate with ethyl acetate, evaporate the ethyl acetate extract to dryness, dissolve the residue with ethyl acetate, and prepare the test solution; and optionally prepare the reference solution of rutin, quercetin and gallic acid respectively;
(b)薄层层析：采用聚酰胺薄层板，取上述供试品溶液和任选的对照品溶液点样于薄层板上，用体积比为7:6.5:2.5:3的正己烷-乙酸乙酯-丁酮-甲酸混合液作为展开剂进行展开；(b) Thin layer chromatography: using a polyamide thin layer plate, spot the test solution and the optional reference solution on the plate, and develop with a mixture of n-hexane-ethyl acetate-butanone-formic acid in a volume ratio of 7:6.5:2.5:3 as a developing solvent;
(c)显色：向挥干的薄层板上喷以显色液AlCl
3试液，挥尽薄层板上的残余溶剂，置320nm的紫外光灯下检视荧光薄层色谱，得到头花蓼药材的指纹图谱。
(c) Color development: Spray the dried thin layer plate with AlCl 3 test solution to evaporate the residual solvent on the thin layer plate, and examine the fluorescent thin layer chromatogram under a 320nm ultraviolet lamp to obtain the fingerprint of Polygonum capitatum.
在本发明中，所述“薄层色谱法”的一般操作规范可以不作限定。在第一方面方法的一个实施方案中，所述的薄层色谱法可以是记载于包括但不限于下列文件中的方法：中华人民共和国药典2010年版一部附录VI B、中华人民共和国药典2010年版二部附录VB、中华人民共和国药典2005年版一部附录VI B、中华人民共和国药典2005年版二部附录VB等。In the present invention, the general operating specification of the "thin layer chromatography" may not be limited. In one embodiment of the first aspect method, the thin layer chromatography may be a method recorded in, but not limited to, the following documents: Appendix VI B of Part I of the Pharmacopoeia of the People's Republic of China 2010 edition, Appendix VB of Part II of the Pharmacopoeia of the People's Republic of China 2010 edition, Appendix VI B of Part I of the Pharmacopoeia of the People's Republic of China 2005 edition, Appendix VB of Part II of the Pharmacopoeia of the People's Republic of China 2005 edition, etc.
根据本发明第一方面的方法，其中步骤(a)中，所述头花蓼供试品溶液是用如下方法配制的：取头花蓼药材粉末，至容量瓶中，加水适量，超声处理，放冷，用水定容至刻度，滤过，滤液用乙酸乙酯萃取，使乙酸乙酯液蒸干，残渣加乙酸乙酯使溶解，即得供试品溶液。According to the method of the first aspect of the present invention, in step (a), the Polygonum capitatum test solution is prepared by the following method: take the Polygonum capitatum medicinal material powder, put it into a volumetric flask, add an appropriate amount of water, ultrasonically treat it, cool it, dilute it to the scale with water, filter it, extract the filtrate with ethyl acetate, evaporate the ethyl acetate solution, add ethyl acetate to dissolve the residue, and obtain the test solution.
根据本发明第一方面的方法，其中步骤(a)中超声处理是以功率250W、频率33kHz的条件超声处理15~60min，优选20~40min，例如约30min。According to the method of the first aspect of the present invention, the ultrasonic treatment in step (a) is carried out at a power of 250 W and a frequency of 33 kHz for 15 to 60 min, preferably 20 to 40 min, for example about 30 min.
根据本发明第一方面的方法，其中步骤(a)中，所述头花蓼供试品溶液是用如下方法配制的：取头花蓼药材粉末2g，至25mL容量瓶中，加水25mL，以功率250W、频率33kHz超声处理30min，放冷，用水定容至刻度，滤过，滤液用乙酸乙酯萃取3次，每次25mL，合并乙酸乙酯液，蒸干，残渣加5mL乙酸乙酯使溶解，即得供试品溶液。According to the method of the first aspect of the present invention, in step (a), the Polygonum capitatum test solution is prepared by the following method: take 2 g of Polygonum capitatum medicinal material powder, put it into a 25 mL volumetric flask, add 25 mL of water, and ultrasonically treat it at a power of 250 W and a frequency of 33 kHz for 30 min, cool it, dilute it to the scale with water, filter it, extract the filtrate with ethyl acetate for 3 times, 25 mL each time, combine the ethyl acetate solutions, evaporate them, add 5 mL of ethyl acetate to dissolve the residue, and obtain the test solution.
根据本发明第一方面的方法，其中步骤(a)中，还包括分别制备芦丁、槲皮苷和没食子酸的对照品溶液的操作，其步骤是：分别精密称取干燥至恒重的芦丁、槲皮苷、没食子酸对照品适量，用甲醇制成浓度分别为0.5±0.05
mg/mL、0.1±0.02
mg/mL、0.1±0.02
mg/mL的对照品溶液。According to the method of the first aspect of the present invention, step (a) further includes the operation of preparing reference solutions of rutin, quercetin and gallic acid, respectively, the steps of which are: accurately weighing appropriate amounts of rutin, quercetin and gallic acid reference substances dried to constant weight, respectively, and using methanol to prepare reference solutions with concentrations of 0.5±0.05
mg/mL, 0.1±0.02
mg/mL, and 0.1±0.02
mg/mL, respectively.
根据本发明第一方面的方法，其中步骤(b)中，点样液的体积为2~10µL，优选2~6µL，例如4µL，例如5µL。According to the method of the first aspect of the present invention, in step (b), the volume of the spotting solution is 2-10 µL, preferably 2-6 µL, such as 4 µL, such as 5 µL.
根据本发明第一方面的方法，其中步骤(b)中，点样液点样于距薄层板底边10mm处。According to the method of the first aspect of the present invention, in step (b), the spotting solution is spotted at a position 10 mm away from the bottom edge of the thin layer plate.
根据本发明第一方面的方法，其中步骤(b)中，展开距离为7~15cm，例如7~12cm，例如7~10cm，优选8cm。According to the method of the first aspect of the present invention, in step (b), the expansion distance is 7 to 15 cm, such as 7 to 12 cm, such as 7 to 10 cm, preferably 8 cm.
根据本发明第一方面的方法，其中步骤(b)中，使用体积比为7:6.5:2.5:3的正己烷-乙酸乙酯-丁酮-甲酸混合液进行二次展开。According to the method of the first aspect of the present invention, in step (b), a mixed solution of n-hexane-ethyl acetate-butanone-formic acid in a volume ratio of 7:6.5:2.5:3 is used for secondary development.
根据本发明第一方面的方法，其中步骤(b)中，所述展开是在双槽展开箱的一侧加入上述展开溶剂进行预平衡，温度20℃，相对湿度至小于88%(优选小于72%，更优选小于50%)的条件下进行的。According to the method of the first aspect of the present invention, in step (b), the development is carried out by adding the above-mentioned developing solvent to one side of a double-trough developing box for pre-balancing, at a temperature of 20°C and a relative humidity of less than 88% (preferably less than 72%, more preferably less than 50%).
根据本发明第一方面的方法，其中步骤(b)是以如下方式操作的：采用聚酰胺薄层板，取供试品溶液、对照品溶液各4
µL，用半自动点样仪点于距薄层板底边10mm处；用体积比为7:6.5:2.5:3的正己烷-乙酸乙酯-丁酮-甲酸混合液作为展开剂；在温度20℃、相对湿度至小于88%的条件下于双槽展开箱的一侧加入上述展开溶剂预平衡15min，上行展开，展距8cm；用体积比为7:6.5:2.5:3的正己烷-乙酸乙酯-丁酮-甲酸混合液作为展开剂进行二次展开，即可。According to the method of the first aspect of the present invention, step (b) is performed in the following manner: using a polyamide thin layer plate, taking 4 µL of the test solution and the reference solution, and spotting them at a distance of 10 mm from the bottom edge of the thin layer plate using a semi-automatic spotter; using a mixed solution of n-hexane-ethyl acetate-butanone-formic acid in a volume ratio of 7:6.5:2.5:3 as a developing agent; adding the above-mentioned developing solvent to one side of a double-slot developing box at a temperature of 20°C and a relative humidity of less than 88% for pre-equilibration for 15 minutes, developing upward with a development distance of 8 cm; and using a mixed solution of n-hexane-ethyl acetate-butanone-formic acid in a volume ratio of 7:6.5:2.5:3 as a developing agent for secondary development.
根据本发明第一方面的方法，其中步骤(c)中，所述检视可以是肉眼观察、照相处理和/或扫描仪扫描。照相处理和/或扫描仪扫描可以容易获得展开的薄层板上的斑点和色谱峰，对于进一步对结果进行评价是有益的。According to the method of the first aspect of the present invention, in step (c), the inspection can be visual observation, photographic processing and/or scanning by a scanner. Photographic processing and/or scanning by a scanner can easily obtain spots and chromatographic peaks on the unfolded thin layer plate, which is useful for further evaluating the results.
根据本发明第一方面的方法，其中还包括步骤(d)对薄层色谱图进行分析：包括5个共有峰/斑点的薄层色谱指纹图谱所对应的药材可初步判定为头花蓼药材正品，无5个共有峰/斑点则可判定为伪品。According to the method of the first aspect of the present invention, it also includes step (d) of analyzing the thin layer chromatogram: the medicinal material corresponding to the thin layer chromatographic fingerprint with 5 common peaks/spots can be preliminarily determined to be the authentic Polygonum capitatum medicinal material, while the medicinal material without 5 common peaks/spots can be determined to be a counterfeit.
根据本发明第一方面的方法，其中步骤(d)中，如果药材的第3、4号峰/斑点与芦丁显示的峰/斑点对应，则可初步判定该药材为头花蓼药材正品，否则可判定为伪品。According to the method of the first aspect of the present invention, in step (d), if the 3rd and 4th peaks/spots of the medicinal material correspond to the peaks/spots shown by rutin, it can be preliminarily determined that the medicinal material is the authentic Polygonum capitatum medicinal material, otherwise it can be determined as a counterfeit.
根据本发明第一方面的方法，其中步骤(d)中，如果药材的第4号峰/斑点与槲皮苷显示的峰/斑点对应，则可初步判定该药材为头花蓼药材正品，否则可判定为伪品。According to the method of the first aspect of the present invention, in step (d), if the peak/spot No. 4 of the medicinal material corresponds to the peak/spot displayed by quercetin, it can be preliminarily determined that the medicinal material is the authentic Polygonum capitatum medicinal material, otherwise it can be determined as a counterfeit.
根据本发明第一方面的方法，其中步骤(d)中，如果药材的第3、4号峰/斑点与芦丁显示的峰/斑点对应，并且第4号峰/斑点与槲皮苷显示的峰/斑点对应，则可初步判定该药材为头花蓼药材正品，否则可判定为伪品。According to the method of the first aspect of the present invention, in step (d), if the 3rd and 4th peaks/spots of the medicinal material correspond to the peaks/spots displayed by rutin, and the 4th peak/spot corresponds to the peak/spot displayed by quercetin, then it can be preliminarily determined that the medicinal material is the authentic Polygonum capitatum medicinal material, otherwise it can be determined as a counterfeit.
根据本发明第一方面的方法，其中步骤(d)中，相同取样量、相同点样量的条件下，如果某批次药材的5个共有峰/斑点强度与同时展开的其它批次药材相比更强，则表明该批次药材质量比其它批次药材更好。According to the method of the first aspect of the present invention, in step (d), under the conditions of the same sampling amount and the same spotting amount, if the intensities of the five common peaks/spots of a batch of medicinal materials are stronger than those of other batches of medicinal materials developed at the same time, it indicates that the quality of this batch of medicinal materials is better than that of other batches of medicinal materials.
由于本发明第一方面的方法中制备供试液时是使用水为溶剂作为提取液，而现有的头花蓼制剂以及制备该制剂的头花蓼提取物多是使用水或者含水乙醇作为溶剂提取得到，因此本发明第一方面的方法在供试品溶液的制备过程中作微小的变化即可适用于现有的头花蓼制剂以及制备该制剂的头花蓼提取。例如，中国药典2010年版收载的热淋清颗粒，其制备方法中记载了头花蓼的提取方法，使用该方法，由药材提取至获得浸膏(即头花蓼提取物)，加上适量淀粉和/或蔗糖可以进一步制成无糖型或者含糖型的热淋清颗粒。此外，市售的热淋清片、胶囊剂等头花蓼制剂，它们均是用或者类似地用中国药典2010年版收载的热淋清颗粒所记载的头花蓼提取方法获得浸膏，再加入常规药用辅料制备成片剂和胶囊剂，这些常规药用辅料通常在本发明TLC方法下不会显示色谱斑点，因此，在本发明的一个实施方案中，术语“头花蓼制剂”，其在为实现本发明目的的意义方面，其含义与头花蓼提取物、热淋清颗粒、热淋清片、热淋清胶囊等术语等同(即这些样品均适合使用本发明方法来测定薄层色谱指纹图谱)。Since water is used as a solvent as an extracting solution when preparing the test solution in the method of the first aspect of the present invention, and the existing Polygonum capitatum preparations and the Polygonum capitatum extracts used to prepare the preparations are mostly extracted using water or aqueous ethanol as solvents, the method of the first aspect of the present invention can be applied to the existing Polygonum capitatum preparations and the Polygonum capitatum extracts used to prepare the preparations by making slight changes in the preparation process of the test solution. For example, the preparation method of the Relinqing Granules included in the 2010 edition of the Chinese Pharmacopoeia records the extraction method of Polygonum capitatum. Using this method, the extract (i.e., the Polygonum capitatum extract) is obtained by extracting the medicinal material, and the sugar-free or sugar-containing Relinqing Granules can be further prepared by adding an appropriate amount of starch and/or sucrose. In addition, commercially available Polygonum capitatum preparations such as Relinqing tablets and capsules are all prepared by obtaining extracts using or similarly using the Polygonum capitatum extraction method recorded in the Relinqing Granules included in the 2010 edition of the Chinese Pharmacopoeia, and then adding conventional pharmaceutical excipients to prepare tablets and capsules. These conventional pharmaceutical excipients usually do not show chromatographic spots under the TLC method of the present invention. Therefore, in one embodiment of the present invention, the term "Polygonum capitatum preparation" has the same meaning as the terms such as Polygonum capitatum extract, Relinqing granules, Relinqing tablets, and Relinqing capsules in terms of achieving the purpose of the present invention (i.e., these samples are suitable for determining thin layer chromatography fingerprints using the method of the present invention).
因此，本发明第二方面提供了一种头花蓼制剂(例如热淋清颗粒)以及制备该制剂的头花蓼提取物的指纹图谱的测定方法，其是照薄层色谱法测定，包括以下步骤：Therefore, the second aspect of the present invention provides a method for determining the fingerprint of a Polygonum capitatum preparation (e.g., Relinqing granules) and a Polygonum capitatum extract used to prepare the preparation, which is determined by thin layer chromatography and comprises the following steps:
(a)薄层点样液制备：称取头花蓼制剂或者制备该制剂的头花蓼提取物，用乙酸乙酯萃取，将乙酸乙酸萃取液蒸干，残渣用乙酸乙酯使溶解，作为供试品溶液；以及任选地分别制备芦丁、槲皮苷和没食子酸的对照品溶液；(a) Preparation of thin layer spotting solution: Weigh the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation, extract with ethyl acetate, evaporate the ethyl acetate extract to dryness, and dissolve the residue with ethyl acetate as the test solution; and optionally prepare reference solutions of rutin, quercetin and gallic acid respectively;
(b)薄层层析：采用聚酰胺薄层板，取上述供试品溶液和任选的对照品溶液点样于薄层板上，用体积比为7:6.5:2.5:3的正己烷-乙酸乙酯-丁酮-甲酸混合液作为展开剂进行展开；(b) Thin layer chromatography: using a polyamide thin layer plate, spot the test solution and the optional reference solution on the plate, and develop with a mixture of n-hexane-ethyl acetate-butanone-formic acid in a volume ratio of 7:6.5:2.5:3 as a developing solvent;
(c)显色：向挥干的薄层板上喷以显色液AlCl
3试液，挥尽薄层板上的残余溶剂，置320nm的紫外光灯下检视荧光薄层色谱，得到头花蓼制剂或者制备该制剂的头花蓼提取物的指纹图谱。
(c) Color development: Spray the evaporated thin layer plate with a color developing solution of AlCl 3 test solution to evaporate the residual solvent on the thin layer plate, and examine the fluorescent thin layer chromatogram under a 320 nm ultraviolet lamp to obtain the fingerprint of the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation.
根据本发明第二方面的方法，其中所述热淋清颗粒是按照如下步骤的方法制备得到的：According to the method of the second aspect of the present invention, the hot leaching granules are prepared according to the following steps:
(1)将头花蓼药材的地上部分干品加7-10倍量的包含0.75%乙酸的乙醇回流提取2次，每次1小时，过滤，使滤液浓缩、干燥，得醇浸膏；(1) adding 7-10 times the amount of ethanol containing 0.75% acetic acid to the dry aerial part of the medicinal material of Polygonum capitatum, reflux extraction twice, each time for 1 hour, filtering, concentrating the filtrate, and drying to obtain an alcohol extract;
(2)使步骤(1)所得滤渣加6-8倍量水煎煮提取2次，每次1小时，过滤，使滤液浓缩、干燥，得水浸膏；(2) adding 6-8 times the amount of water to the residue obtained in step (1) and boiling and extracting twice, each time for 1 hour, filtering, concentrating the filtrate, and drying to obtain an aqueous extract;
(3)使所述醇浸膏和水浸膏混合，喷雾干燥除去水分(水分降至3%以下)，得到头花蓼提取物，(3) mixing the alcohol extract and the water extract, and spray drying to remove water (water content is reduced to less than 3%) to obtain a Polygonum capitatum extract,
(4)使相当于由1000克头花蓼药材制得的头花蓼提取物与适量淀粉混合至总量为400克，用水制粒，干燥，得到热淋清颗粒的制剂，包装，每袋装量4克(相当于每袋包含药材1克)。(4) The extract of Polygonum capitatum obtained from 1000 g of the medicinal material of Polygonum capitatum is mixed with an appropriate amount of starch to a total amount of 400 g, granulated with water, dried to obtain a preparation of Relinqing granules, and packaged, with a filling amount of 4 g per bag (equivalent to 1 g of the medicinal material per bag).
根据本发明第二方面的方法，其中所述步骤(a)中还任选地包括在萃取之前用水溶解所述头花蓼制剂或者制备该制剂的头花蓼提取物的步骤。在此溶解步骤之后再如步骤(a)所述用乙酸乙酯进行萃取。According to the method of the second aspect of the present invention, the step (a) further optionally comprises a step of dissolving the Polygonum capitatum preparation or the Polygonum capitatum extract of the preparation with water before extraction. After the dissolving step, extraction is performed with ethyl acetate as described in step (a).
根据本发明第二方面的方法，其中步骤(a)中，所述供试品溶液是用如下方法配制的：取头花蓼制剂或者制备该制剂的头花蓼提取物，至容量瓶中，加水溶解，用水定容至刻度，滤过，滤液用乙酸乙酯萃取，使乙酸乙酯液蒸干，残渣加乙酸乙酯使溶解，即得供试品溶液。According to the method of the second aspect of the present invention, in step (a), the test solution is prepared by the following method: take the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation, put it into a volumetric flask, add water to dissolve it, dilute to the scale with water, filter it, extract the filtrate with ethyl acetate, evaporate the ethyl acetate solution, add ethyl acetate to dissolve the residue, and obtain the test solution.
根据本发明第二方面的方法，其中步骤(a)中，还包括分别制备芦丁、槲皮苷和没食子酸的对照品溶液的操作，其步骤是：分别精密称取干燥至恒重的芦丁、槲皮苷、没食子酸对照品适量，用甲醇制成浓度分别为0.5±0.05
mg/mL、0.1±0.02
mg/mL、0.1±0.02
mg/mL的对照品溶液。According to the method of the second aspect of the present invention, step (a) further includes the operation of preparing reference solutions of rutin, quercetin and gallic acid, respectively, the steps of which are: accurately weighing appropriate amounts of rutin, quercetin and gallic acid reference substances dried to constant weight, respectively, and using methanol to prepare reference solutions with concentrations of 0.5±0.05 mg/mL, 0.1±0.02 mg/mL and 0.1±0.02 mg/mL, respectively.
根据本发明第二方面的方法，其中步骤(b)中，点样液的体积为2~10µL，优选2~6µL，例如4µL，例如5µL。According to the method of the second aspect of the present invention, in step (b), the volume of the spotting solution is 2-10 µL, preferably 2-6 µL, such as 4 µL, such as 5 µL.
根据本发明第二方面的方法，其中步骤(b)中，点样液点样于距薄层板底边10mm处。According to the method of the second aspect of the present invention, in step (b), the spotting solution is spotted at a position 10 mm away from the bottom edge of the thin layer plate.
根据本发明第二方面的方法，其中步骤(b)中，展开距离为7~15cm，例如7~12cm，例如7~10cm，优选8cm。According to the method of the second aspect of the present invention, in step (b), the expansion distance is 7 to 15 cm, such as 7 to 12 cm, such as 7 to 10 cm, preferably 8 cm.
根据本发明第二方面的方法，其中步骤(b)中，使用体积比为7:6.5:2.5:3的正己烷-乙酸乙酯-丁酮-甲酸混合液进行二次展开。According to the method of the second aspect of the present invention, in step (b), a mixed solution of n-hexane-ethyl acetate-butanone-formic acid in a volume ratio of 7:6.5:2.5:3 is used for secondary development.
根据本发明第二方面的方法，其中步骤(b)中，所述展开是在双槽展开箱的一侧加入上述展开溶剂进行预平衡，温度20℃，相对湿度至小于88%(优选小于72%，更优选小于50%)的条件下进行的。According to the method of the second aspect of the present invention, in step (b), the development is carried out by adding the above-mentioned developing solvent to one side of a double-trough developing box for pre-balancing, at a temperature of 20°C and a relative humidity of less than 88% (preferably less than 72%, more preferably less than 50%).
根据本发明第二方面的方法，其中步骤(b)是以如下方式操作的：采用聚酰胺薄层板，取供试品溶液、对照品溶液各4
µL，用半自动点样仪点于距薄层板底边10mm处；用体积比为7:6.5:2.5:3的正己烷-乙酸乙酯-丁酮-甲酸混合液作为展开剂；在温度20℃、相对湿度至小于88%的条件下于双槽展开箱的一侧加入上述展开溶剂预平衡15min，上行展开，展距8cm；用体积比为7:6.5:2.5:3的正己烷-乙酸乙酯-丁酮-甲酸混合液作为展开剂进行二次展开，即可。According to the method of the second aspect of the present invention, step (b) is performed as follows: using a polyamide thin layer plate, taking 4 µL of the test solution and the reference solution, and spotting them at a distance of 10 mm from the bottom edge of the thin layer plate using a semi-automatic spotter; using a mixed solution of n-hexane-ethyl acetate-butanone-formic acid in a volume ratio of 7:6.5:2.5:3 as a developing agent; adding the above-mentioned developing solvent to one side of a double-slot developing box at a temperature of 20°C and a relative humidity of less than 88% for pre-equilibration for 15 minutes, developing upward with a development distance of 8 cm; and using a mixed solution of n-hexane-ethyl acetate-butanone-formic acid in a volume ratio of 7:6.5:2.5:3 as a developing agent for secondary development.
根据本发明第二方面的方法，其中步骤(c)中，所述检视可以是肉眼观察、照相处理和/或扫描仪扫描。照相处理和/或扫描仪扫描可以容易获得展开的薄层板上的斑点和色谱峰，对于进一步对结果进行评价是有益的。According to the method of the second aspect of the present invention, in step (c), the inspection can be visual observation, photographic processing and/or scanning by a scanner. Photographic processing and/or scanning by a scanner can easily obtain spots and chromatographic peaks on the unfolded thin layer plate, which is useful for further evaluating the results.
根据本发明第二方面的方法，其中还包括步骤(d)对薄层色谱图进行分析：包括5个共有峰/斑点的薄层色谱指纹图谱所对应的药材可初步判定为该头花蓼制剂或者制备该制剂的头花蓼提取物正常，无5个共有峰/斑点则可判定为该头花蓼制剂或者制备该制剂的头花蓼提取物异常。According to the method of the second aspect of the present invention, it also includes step (d) of analyzing the thin layer chromatogram: the medicinal material corresponding to the thin layer chromatographic fingerprint with 5 common peaks/spots can be preliminarily judged as the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation is normal; if there are no 5 common peaks/spots, it can be judged as the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation is abnormal.
根据本发明第二方面的方法，其中步骤(d)中，如果该头花蓼制剂或者制备该制剂的头花蓼提取物的第3、4号峰/斑点与芦丁显示的峰/斑点对应，则可初步判定该头花蓼制剂或者制备该制剂的头花蓼提取物正常，否则可判定异常。基于此方法的结果可采用其它方法作进一步验证产品内在质量。According to the method of the second aspect of the present invention, in step (d), if the 3rd and 4th peaks/spots of the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation correspond to the peaks/spots displayed by rutin, it can be preliminarily determined that the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation is normal, otherwise it can be determined to be abnormal. Based on the results of this method, other methods can be used to further verify the intrinsic quality of the product.
根据本发明第二方面的方法，其中步骤(d)中，如果该头花蓼制剂或者制备该制剂的头花蓼提取物的第4号峰/斑点与槲皮苷显示的峰/斑点对应，则可初步判定该头花蓼制剂或者制备该制剂的头花蓼提取物正常，否则可判定异常。According to the method of the second aspect of the present invention, in step (d), if the peak/spot No. 4 of the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation corresponds to the peak/spot shown by quercetin, it can be preliminarily determined that the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation is normal, otherwise it can be determined to be abnormal.
根据本发明第二方面的方法，其中步骤(d)中，如果该头花蓼制剂或者制备该制剂的头花蓼提取物的第3、4号峰/斑点与芦丁显示的峰/斑点对应，并且第4号峰/斑点与槲皮苷显示的峰/斑点对应，则可初步判定该头花蓼制剂或者制备该制剂的头花蓼提取物正常，否则可判定异常。According to the method of the second aspect of the present invention, in step (d), if the 3rd and 4th peaks/spots of the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation correspond to the peaks/spots shown by rutin, and the 4th peak/spot corresponds to the peak/spot shown by quercetin, then it can be preliminarily determined that the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation is normal, otherwise it can be determined to be abnormal.
根据本发明第二方面的方法，其中步骤(d)中，如果某批次头花蓼制剂或者制备该制剂的头花蓼提取物的在相同取样量、相同点样量的条件下，5个共有峰/斑点强度与同时展开的其它批次头花蓼制剂或者制备该制剂的头花蓼提取物相比更强，则表明该批次头花蓼制剂或者制备该制剂的头花蓼提取物质量比其它批次头花蓼制剂或者制备该制剂的头花蓼提取物更好。According to the method of the second aspect of the present invention, in step (d), if the 5 common peaks/spot intensities of a batch of Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation are stronger than those of other batches of Polygonum capitatum preparations or the Polygonum capitatum extracts used to prepare the preparation under the conditions of the same sampling amount and the same spotting amount, then it indicates that the quality of the batch of Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation is better than that of other batches of Polygonum capitatum preparations or the Polygonum capitatum extracts used to prepare the preparation.
进一步的，本发明第三方面提供了一种制备热淋清颗粒的方法，包括如下步骤：(1)将头花蓼药材的地上部分干品加乙醇回流提取，过滤，使滤液浓缩、干燥，得醇浸膏；(2)使步骤(1)所得滤渣加水煎煮提取，过滤，使滤液浓缩、干燥，得水浸膏；(3)使所述醇浸膏和水浸膏混合，喷雾干燥除去水分，得到头花蓼提取物，(4)使头花蓼提取物与适量淀粉混合，用水制粒，干燥，得到热淋清颗粒的制剂。Furthermore, the third aspect of the present invention provides a method for preparing Relinqing granules, comprising the following steps: (1) extracting the dry aerial part of the medicinal material Polygonum capitatum with ethanol by reflux, filtering, concentrating and drying the filtrate to obtain an alcohol extract; (2) decocting and extracting the filter residue obtained in step (1) by adding water, filtering, concentrating and drying the filtrate to obtain an aqueous extract; (3) mixing the alcohol extract and the aqueous extract, spray drying to remove moisture, and obtaining a Polygonum capitatum extract; (4) mixing the Polygonum capitatum extract with an appropriate amount of starch, granulating with water, and drying to obtain a preparation of Relinqing granules.
根据本发明第三方面的方法，包括如下步骤：(1)将头花蓼药材的地上部分干品加7-10倍量的包含0.75%乙酸的乙醇回流提取2次，每次1小时，过滤，使滤液浓缩、干燥，得醇浸膏；(2)使步骤(1)所得滤渣加6-8倍量水煎煮提取2次，每次1小时，过滤，使滤液浓缩、干燥，得水浸膏；(3)使所述醇浸膏和水浸膏混合，喷雾干燥除去水分(水分降至3%以下)，得到头花蓼提取物，(4)使相当于由1000克头花蓼药材制得的头花蓼提取物与适量淀粉混合至总量为400克，用水制粒，干燥，得到热淋清颗粒的制剂，包装，每袋装量4克(相当于每袋包含药材1克)。The method according to the third aspect of the present invention comprises the following steps: (1) adding 7-10 times the amount of ethanol containing 0.75% acetic acid to the dry aerial part of the medicinal material of Polygonum capitatum, reflux extraction twice, each time for 1 hour, filtering, concentrating the filtrate, and drying to obtain an alcohol extract; (2) adding 6-8 times the amount of water to the filter residue obtained in step (1) to boil and extract twice, each time for 1 hour, filtering, concentrating the filtrate, and drying to obtain an aqueous extract; (3) mixing the alcohol extract and the aqueous extract, spray drying to remove moisture (moisture content is reduced to less than 3%), and obtaining a Polygonum capitatum extract; (4) mixing the Polygonum capitatum extract obtained from 1000 grams of the medicinal material of Polygonum capitatum with an appropriate amount of starch to a total amount of 400 grams, granulating with water, drying, and obtaining a preparation of hot linqing granules, and packaging, with a packing amount of 4 grams per bag (equivalent to 1 gram of the medicinal material per bag).
进一步的，本发明第四方面提供了一种热淋清颗粒，其是由包括如下步骤的方法制备得到的：(1)将头花蓼药材的地上部分干品加乙醇回流提取，过滤，使滤液浓缩、干燥，得醇浸膏；(2)使步骤(1)所得滤渣加水煎煮提取，过滤，使滤液浓缩、干燥，得水浸膏；(3)使所述醇浸膏和水浸膏混合，喷雾干燥除去水分，得到头花蓼提取物，(4)使头花蓼提取物与适量淀粉混合，用水制粒，干燥，得到热淋清颗粒的制剂。Furthermore, the fourth aspect of the present invention provides a hot linqing granule, which is prepared by a method comprising the following steps: (1) extracting the dry aerial part of the medicinal material Polygonum capitatum with ethanol by reflux, filtering, concentrating and drying the filtrate to obtain an alcohol extract; (2) decocting and extracting the filter residue obtained in step (1) by adding water, filtering, concentrating and drying the filtrate to obtain an aqueous extract; (3) mixing the alcohol extract and the aqueous extract, spray drying to remove moisture, and obtaining a Polygonum capitatum extract; (4) mixing the Polygonum capitatum extract with an appropriate amount of starch, granulating with water, and drying to obtain a preparation of hot linqing granules.
根据本发明第四方面的热淋清颗粒，其是由包括如下步骤的方法制备得到的：(1)将头花蓼药材的地上部分干品加7-10倍量的包含0.75%乙酸的乙醇回流提取2次，每次1小时，过滤，使滤液浓缩、干燥，得醇浸膏；(2)使步骤(1)所得滤渣加6-8倍量水煎煮提取2次，每次1小时，过滤，使滤液浓缩、干燥，得水浸膏；(3)使所述醇浸膏和水浸膏混合，喷雾干燥除去水分(水分降至3%以下)，得到头花蓼提取物，(4)使相当于由1000克头花蓼药材制得的头花蓼提取物与适量淀粉混合至总量为400克，用水制粒，干燥，得到热淋清颗粒的制剂，包装，每袋装量4克(相当于每袋包含药材1克)。According to the fourth aspect of the present invention, the Relinqing granules are prepared by a method comprising the following steps: (1) adding 7-10 times the amount of ethanol containing 0.75% acetic acid to the dry aerial part of the medicinal material of Polygonum capitatum, reflux extraction twice, each time for 1 hour, filtering, concentrating the filtrate, and drying to obtain an alcohol extract; (2) adding 6-8 times the amount of water to the filter residue obtained in step (1) to boil and extract twice, each time for 1 hour, filtering, concentrating the filtrate, and drying to obtain an aqueous extract; (3) mixing the alcohol extract and the aqueous extract, spray drying to remove moisture (moisture content is reduced to less than 3%), and obtaining a Polygonum capitatum extract; (4) mixing the Polygonum capitatum extract obtained from 1000 grams of the medicinal material of Polygonum capitatum with an appropriate amount of starch to a total amount of 400 grams, granulating with water, and drying to obtain a preparation of Relinqing granules, and packaging, with a packing amount of 4 grams per bag (equivalent to 1 gram of the medicinal material per bag).
本发明任一方面的任一实施方案所具有的特征同样适用于该方面的其它任一实施方案以及其它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。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。The features of any embodiment of any aspect of the present invention are also applicable to any other embodiment of the aspect and any embodiment of other aspects, as long as they do not contradict each other; of course, when applicable to each other, the corresponding features may be appropriately modified if necessary. 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 herein. 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，提及%时，如未另外说明，是指重量/重量百分数。In the present invention, when % is mentioned, unless otherwise specified, it means weight/weight percentage.
在本发明中，提及展开剂混合液时，它们的比率是以体积比表示的。In the present invention, when referring to the developer mixture, their ratio is expressed as a volume ratio.
使用本发明的方法，表明头花蓼药材薄层色谱由5个共有峰构成了薄层色谱指纹图谱的基本骨架，通过对照品实验，确定3、4号峰为芦丁，4号峰为槲皮苷；由于以上共有峰各批药材均含有，因此上述共有峰应作为头花蓼药材鉴定的特征性成分。同一产地不同批次和不同产地头花蓼样品的药材质量的差异，可以从这几个峰的斑点颜色深度得到反映。贵州施秉产12批头花蓼药材样品中这几个斑点深度相似及峰面积大小相当，说明这12批样品相似性较大，品质差异小；不同产地样品相似性较大，但斑点深度略浅及峰面积差异较大，表明品质差异较大；头花蓼的伪品赤惊散没有上述5个共有峰，表明本发明的色谱条件和方法可以将伪品区别。The method of the present invention shows that the thin layer chromatography of the medicinal material of Polygonum capitatum is composed of 5 common peaks, which constitute the basic framework of the thin layer chromatography fingerprint. Through the control product experiment, it is determined that peaks 3 and 4 are rutin, and peak 4 is quercetin; since the above common peaks are contained in each batch of medicinal materials, the above common peaks should be used as characteristic components for the identification of Polygonum capitatum medicinal materials. The difference in the quality of medicinal materials of different batches of Polygonum capitatum samples from the same origin and different origins can be reflected from the spot color depth of these peaks. The depth of these spots in 12 batches of Polygonum capitatum medicinal material samples produced in Shibing, Guizhou is similar and the peak area size is comparable, indicating that the similarity of these 12 batches of samples is large and the quality difference is small; the similarity of samples from different origins is large, but the spot depth is slightly shallow and the peak area difference is large, indicating that the quality difference is large; the counterfeit product of Polygonum capitatum, Chijingsan, does not have the above 5 common peaks, indicating that the chromatographic conditions and methods of the present invention can distinguish counterfeits.
因此头花蓼药材和热淋清颗粒的五个峰可以作为特征峰用于头花蓼药材的真伪鉴别以及制剂质量控制。各样品在峰高上存在差异，反映了样品中某些化学成分上有量的差异；但峰形一致，体现了它们在质上没有大的差别。Therefore, the five peaks of Polygonum capitatum and Relinqing granules can be used as characteristic peaks for authenticity identification of Polygonum capitatum and quality control of preparations. The peak heights of the samples differ, reflecting the quantitative differences in certain chemical components in the samples; but the peak shapes are consistent, reflecting that there is no major difference in quality.
在本发明的一个实施方案中，头花蓼药材薄层色谱指纹图谱测定条件确定为：以水为提取溶剂，超声处理，乙酸乙酯萃取后为提取溶剂；以正己烷-乙酸乙酯-丁酮-甲酸(7:6.5:2.5:3)作为展开剂，二次展开，预平衡15min，上行展开；展距：8cm；温度20℃，控制相对湿度至32%~72%。用三氯化铝试液显色。挥尽溶剂后置紫外光灯(320nm)下检视荧光薄层色谱。本发明的结果显示，贵州施秉产的12批头花蓼药材的薄层色谱图和轮廓图十分相似，均有5个斑点和对应的5个峰；对于不同产地的药材而言，各峰在峰强度上有差异，体现了不同产地头花蓼所含化学成分在量上有差别。此外，头花蓼的伪品赤惊散没有上述特征，说明本发明方法可以很好的区分头花蓼伪品，可以用于头花蓼药材的真伪鉴别。In one embodiment of the present invention, the thin layer chromatography fingerprint determination conditions of Polygonum capitatum medicinal materials are determined as follows: water is used as the extraction solvent, ultrasonic treatment, and ethyl acetate is used as the extraction solvent after extraction; n-hexane-ethyl acetate-butanone-formic acid (7:6.5:2.5:3) is used as the developing agent, secondary development, pre-equilibration for 15 minutes, and upward development; development distance: 8 cm; temperature 20°C, relative humidity controlled to 32%~72%. Color development is performed with aluminum chloride test solution. After evaporating the solvent, the fluorescent thin layer chromatography is inspected under ultraviolet light (320nm). The results of the present invention show that the thin layer chromatograms and contour diagrams of 12 batches of Polygonum capitatum medicinal materials produced in Shibing, Guizhou are very similar, all with 5 spots and corresponding 5 peaks; for medicinal materials from different origins, the peaks have differences in peak intensity, reflecting the differences in the amount of chemical components contained in Polygonum capitatum from different origins. In addition, the counterfeit product of Polygonum capitatum, Chijingsan, does not have the above characteristics, indicating that the method of the present invention can well distinguish counterfeit Polygonum capitatum products and can be used for authenticity identification of Polygonum capitatum medicinal materials.
本发明的薄层色谱指纹图谱法不仅能反映头花蓼药材的特征性成分，也能反映不同来源药材之间的差异，还可以用于鉴别药材的真伪；同时该方法简单易于操作，便于推广应用。可用于采购及生产中的快速检验。The thin layer chromatography fingerprint method of the present invention can not only reflect the characteristic components of the Polygonum capitatum medicinal material, but also reflect the differences between medicinal materials from different sources, and can also be used to identify the authenticity of medicinal materials; at the same time, the method is simple and easy to operate, and is convenient for promotion and application. It can be used for rapid inspection in procurement and production.
通过下面的实施例可以对本发明进行进一步的描述, 然而，本发明的范围并不限于下述实施例。本领域的专业人员能够理解,
在不背离本发明的精神和范围的前提下,
可以对本发明进行各种变化和修饰。本发明对试验中所使用到的材料以及试验方法进行一般性和/或具体的描述。虽然为实现本发明目的所使用的许多材料和操作方法是本领域公知的，但是本发明仍然在此作尽可能详细描述。The present invention can be further described by the following examples, however, the scope of the present invention is not limited to the following examples. Professionals in the field can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention. The present invention generally and/or specifically describes the materials and test methods used in the test. Although many materials and operating methods used to achieve the purpose of the present invention are well known in the art, the present invention is still described as detailed as possible.
本文涉及的一些图谱参见201210044662X(其在下文中称为专利文献)中所涉及的图谱。For some of the graphs involved in this article, please refer to the graphs involved in 201210044662X (hereinafter referred to as patent document).
A、测试仪器与试药A. Test instruments and test drugs
仪器：LINOMAT V半自动点样仪(瑞士卡玛公司)、CAMAG Reprostar3薄层色谱成像系统(瑞士卡玛公司)、CS-9301PC双波长飞点薄层色谱扫描仪(日本岛津公司)、AB204-S 型电子天平(梅特勒-托利多公司)、CH-250 型超声波清洗机(天津恒奥科技公司，功率250W，频率33kHz)、CZX-GF101-4-S-Ⅱ型电热恒温鼓风干燥箱(上海贺德实验设备有限公司)，双槽展开箱，Chromafinger色谱指纹图谱解决方案软件(珠海科曼中药研究有限公司)。Instruments: LINOMAT V semi-automatic spotter (Kama, Switzerland), CAMAG Reprostar3 thin-layer chromatography imaging system (Kama, Switzerland), CS-9301PC dual-wavelength flying-spot thin-layer chromatography scanner (Shimadzu, Japan), AB204-S electronic balance (Mettler-Toledo), CH-250 ultrasonic cleaning machine (Tianjin Hengao Technology Co., Ltd., power 250W, frequency 33kHz), CZX-GF101-4-S-Ⅱ electric constant temperature blast drying oven (Shanghai Hede Experimental Equipment Co., Ltd.), double-slot development box, Chromafinger chromatographic fingerprint solution software (Zhuhai Keman Chinese Medicine Research Co., Ltd.).
试剂、试药：甲醇、乙醇、氯仿、石油醚、环己烷、乙酸乙酯、丙酮、甲酸、正己烷、丁酮等均为分析纯，可作为展开试剂可提取溶剂使用。芦丁(批号：110725-200513，含量测定用)、槲皮苷(批号：111538-200403，含量测定用)、没食子酸对照品(批号：110831-200302，含量测定用)，均购于中国药品生物制品检定所。甲醇、四氢呋喃、乙腈为色谱纯；水为纯净水；其它试剂均为分析纯。Reagents and reagents: Methanol, ethanol, chloroform, petroleum ether, cyclohexane, ethyl acetate, acetone, formic acid, n-hexane, butanone, etc. are all analytically pure and can be used as developing reagents and extracting solvents. Rutin (batch number: 110725-200513, for content determination), quercetin (batch number: 111538-200403, for content determination), and gallic acid reference substance (batch number: 110831-200302, for content determination) were all purchased from the National Institute for the Control of Pharmaceutical and Biological Products. Methanol, tetrahydrofuran, and acetonitrile were chromatographically pure; water was purified water; other reagents were all analytically pure.
药材：12批头花蓼药材采自贵州施秉头花蓼规范化种植研究与GAP试验示范基地Medicinal materials: 12 batches of Polygonum capitatum medicinal materials were collected from the Polygonum capitatum standardized planting research and GAP trial demonstration base in Shibing, Guizhou
编号1~12的药材样品来源于贵州施秉，编号13~23的药材样品分别来源于贵州兴义、贵州晴隆、贵州盘县、贵州毕节、贵州水城、贵州罗甸、湖南古丈、云南师宗、广西田林、重庆南川、四川南川、赤胫散(伪品)。经贵阳中医学院鉴定为蓼科植物头花蓼
Polygonum capitatum
Buch.-Ham.ex D.Don的干燥全草或地上部分；伪品赤胫散为蓼科植物赤胫散
Polygonum runcinatum Buch.-Ham.var.sinense
Hemsl.的干燥全草或地上部分。
The medicinal material samples No. 1 to 12 were from Shibing, Guizhou, and the medicinal material samples No. 13 to 23 were from Xingyi, Guizhou, Qinglong, Guizhou, Panxian, Guizhou, Bijie, Guizhou, Shuicheng, Guizhou, Luodian, Hunan, Shizong, Yunnan, Tianlin, Guangxi, Nanchuan, Chongqing, Nanchuan, Sichuan, and Chijinsan (counterfeit). The Guiyang University of Traditional Chinese Medicine identified them as the dried whole herb or aerial part of Polygonum capitatum Buch.-Ham.ex D.Don, a Polygonaceae plant; the counterfeit Chijinsan was the dried whole herb or aerial part of Polygonum runcinatum Buch.-Ham.var.sinense Hemsl., a Polygonaceae plant.
在下文中，使用到以上编号1~24的各药材，如果未特别提及其药材编号，是指使用编号为1号的药材进行处理。In the following text, when the medicinal materials numbered 1 to 24 are used, if their medicinal material numbers are not specifically mentioned, it means that the medicinal material numbered 1 is used for treatment.
B、药材试验例部分B. Medicinal Materials Test Examples
在本药材试验例部分，必要时使用到热淋清颗粒样品作为比较用的考察对象，如未另外说明，所用热淋清颗粒是照中国药典2010年版一部“热淋清颗粒”项下的方法以施秉出产头花蓼为药材的制备得到的含糖型热淋清颗粒。In the medicinal material test examples, when necessary, samples of Relinqing granules are used as comparative investigation objects. Unless otherwise specified, the Relinqing granules used are sugar-containing Relinqing granules prepared according to the method under the item "Relinqing granules" in the 2010 edition of the Chinese Pharmacopoeia, using Polygonum capitatum produced in Shibing as the medicinal material.
试验例1、本发明确定的最优选的薄层色谱分析条件Test Example 1: The most preferred thin layer chromatography analysis conditions determined by the present invention
根据本发明上下文，可以确定最优选的薄层色谱分析条件和操作方法，具体如下。According to the context of the present invention, the most preferred thin layer chromatography analysis conditions and operating methods can be determined as follows.
(1)对照品溶液的制备：(1) Preparation of reference solution:
分别精密称取干燥至恒重的芦丁、槲皮苷、没食子酸对照品适量，用甲醇制成浓度分别为0.5200
mg/mL、0.1005 mg/mL、0.1042 mg/mL的对照品溶液。Accurately weigh appropriate amounts of rutin, quercetin, and gallic acid reference substances dried to constant weight, and use methanol to prepare reference substance solutions with concentrations of 0.5200 mg/mL, 0.1005 mg/mL, and 0.1042 mg/mL, respectively.
(2)供试品溶液的制备：(2) Preparation of test solution:
对于头花蓼药材：取头花蓼药材粉末2g，至25mL容量瓶中，加水25mL，超声处理(功率250W，频率33kHz)30min，放冷，用水定容至刻度，滤过，滤液用乙酸乙酯萃取3次，每次25mL，合并乙酸乙酯液，蒸干，残渣加5mL乙酸乙酯使溶解，作为供试品溶液。For the medicinal material Polygonum capitatum: take 2g of the medicinal material powder of Polygonum capitatum, put it into a 25mL volumetric flask, add 25mL of water, ultrasonically treat (power 250W, frequency 33kHz) for 30min, cool, dilute to the scale with water, filter, extract the filtrate with ethyl acetate for 3 times, 25mL each time, combine the ethyl acetate solutions, evaporate to dryness, add 5mL of ethyl acetate to dissolve the residue, and use it as the test solution.
对于头花蓼提取物或者热淋清制剂如热淋清颗粒：取头花蓼制剂或者制备该制剂的头花蓼提取物(相当于2克药材量的提取物或制剂)，至容量瓶中，加水25mL溶解或混悬，用水定容至刻度，滤过，滤液用乙酸乙酯萃取3次，每次25mL，合并乙酸乙酯液，蒸干，残渣加5mL乙酸乙酯使溶解，作为供试品溶液。For the Polygonum capitatum extract or Relinqing preparation such as Relinqing granules: take the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation (equivalent to 2 grams of the extract or preparation of the medicinal material), put it into a volumetric flask, add 25mL of water to dissolve or suspend it, make up to the scale with water, filter, extract the filtrate with ethyl acetate for 3 times, 25mL each time, combine the ethyl acetate solutions, evaporate to dryness, add 5mL of ethyl acetate to dissolve the residue, and use it as the test solution.
(3)薄层色谱条件：(3) Thin layer chromatography conditions:
薄层板：聚酰胺薄层板(青岛海洋化工厂)。点样取上述供试品溶液、对照品溶液各4
µL，用半自动点样仪点于距薄层板底边10mm处。展开系统：正己烷-乙酸乙酯-丁酮-甲酸(7:6.5:2.5:3)。展开方式：于双槽展开箱的一侧加入上述展开溶剂预平衡15min，上行展开；展距8cm；温度20℃，控制相对湿度至小于72%。用展开剂正己烷-乙酸乙酯-丁酮-甲酸(7:6.5:2.5:3)二次展开。检测条件：喷以AlCl
3试液显色，挥尽薄层板上的残留溶剂后置紫外光灯(320nm)下检视荧光薄层色谱。
Thin layer plate: polyamide thin layer plate (Qingdao Ocean Chemical Plant). Take 4 µL of the above test solution and reference solution, and use a semi-automatic spotter to spot 10 mm from the bottom of the thin layer plate. Development system: n-hexane-ethyl acetate-butanone-formic acid (7:6.5:2.5:3). Development method: add the above development solvent to one side of the double-slot development box for pre-equilibration for 15 minutes, and develop upward; development distance 8 cm; temperature 20 ° C, control relative humidity to less than 72%. Use the developing agent n-hexane-ethyl acetate-butanone-formic acid (7:6.5:2.5:3) for secondary development. Detection conditions: Spray with AlCl 3 test solution for color development, and after waving off the residual solvent on the thin layer plate, examine the fluorescent thin layer chromatogram under ultraviolet light (320 nm).
下面的试验对本发明方法作进一步验证，并且在下面的试验例中，如未另外说明，可以采用本试验例1的方法进行试验。The following test further verifies the method of the present invention, and in the following test examples, unless otherwise specified, the method of this test example 1 can be used to conduct the test.
试验例2：不同提取溶剂、不同展开系统对头花蓼药材薄层色谱的影响Experimental Example 2: Effects of different extraction solvents and different development systems on thin-layer chromatography of Polygonum capitatum
用编号为1号的头花蓼药材进行测试。分别以乙酸乙酯、乙醇、甲醇、丙酮、正丁醇、水6种溶剂为提取溶剂，采用超声(功率250W，频率33kHz)30min的方式处理，对于水处理的试样，超声处理后用乙酸乙酯萃取3次，处理之后(必要时)浓缩到每ml供试液中含有0.4g药材的浓度，得到不同提取方法获得的6个供试品溶液。另照试验例1所记载的方法分别制备芦丁、槲皮苷的对照品溶液。The Polygonum capitatum medicinal material No. 1 was used for testing. Six solvents, including ethyl acetate, ethanol, methanol, acetone, n-butanol, and water, were used as extraction solvents, and ultrasonic treatment (power 250W, frequency 33kHz) was used for 30min. For the water-treated sample, it was extracted with ethyl acetate three times after ultrasonic treatment. After treatment (if necessary), it was concentrated to a concentration of 0.4g medicinal material per ml of the test solution to obtain 6 test sample solutions obtained by different extraction methods. In addition, the reference solutions of rutin and quercetin were prepared according to the method described in Test Example 1.
将以上6个供试品溶液和2个对照品溶液点于同一聚酰胺薄层板上，共制备7个点样板，分别用以下7种溶剂系统(S1至S7)作为展开剂进行展开：S1：正己烷-乙酸乙酯-丁酮-甲酸(7:6.5:2.5:3)，二次展开；S2：甲苯-乙酸乙酯-甲酸(10:11:3.1)；S3：环己烷-醋酸乙酯-甲酸(8:4:0.4)；S4：水-乙醇-丁酮-乙酰丙酮(65:15:15:5)；S5：氯仿-甲醇-甲酸(15:4:0.5)；S6：环己烷-乙酸乙酯-甲酸(5:5:1)；S7：乙酸乙酯-甲酸-水(8:1:1)，二次展开。The above 6 test sample solutions and 2 reference sample solutions were spotted on the same polyamide thin layer plate to prepare 7 spot plates in total, and the following 7 solvent systems (S1 to S7) were used as developing agents for development: S1: n-hexane-ethyl acetate-butanone-formic acid (7:6.5:2.5:3), secondary development; S2: toluene-ethyl acetate-formic acid (10:11:3.1); S3: cyclohexane-ethyl acetate-formic acid (8:4:0.4); S4: water-ethanol-butanone-acetylacetone (65:15:15:5); S5: chloroform-methanol-formic acid (15:4:0.5); S6: cyclohexane-ethyl acetate-formic acid (5:5:1); S7: ethyl acetate-formic acid-water (8:1:1), secondary development.
头花蓼药材不同提取溶剂、不同展开系统薄层色谱图(320nm)见专利文献之图1。以薄层色谱图直观的清晰度和有效信息量为考察指标，对6种不同提取溶剂、7种不同展开系统进行比较，结果表明：不同提取溶剂对头花蓼药材薄层色谱影响较大，其中以水提取，乙酸乙酯萃取效果最好，斑点容量大(见专利文献之图1中的S1)；S2~S7展开溶剂系统分离效果较差，拖尾较严重，斑点容量较小，S1得到的头花蓼药材薄层色谱分离度较好、斑点容量大，故选择正己烷-乙酸乙酯-丁酮-甲酸(7:6.5:2.5:3)二次展开，作为展开溶剂系统是优选的。发明人还发现，对于S1展开条件，改用一次展开或者展开剂中不含有丁酮，则展开效果远比S1的差，无法分出5个明显的斑点。The thin layer chromatograms (320nm) of different extraction solvents and different development systems of Polygonum capitatum medicinal materials are shown in Figure 1 of the patent document. Taking the intuitive clarity and effective information content of the thin layer chromatogram as the investigation index, 6 different extraction solvents and 7 different development systems were compared. The results showed that different extraction solvents had a greater impact on the thin layer chromatography of Polygonum capitatum medicinal materials, among which water extraction and ethyl acetate extraction had the best effect and large spot capacity (see S1 in Figure 1 of the patent document); S2~S7 development solvent systems had poor separation effect, serious tailing, and small spot capacity. The thin layer chromatogram of Polygonum capitatum medicinal materials obtained by S1 had better separation and large spot capacity, so n-hexane-ethyl acetate-butanone-formic acid (7:6.5:2.5:3) was selected for secondary development as the development solvent system, which was preferred. The inventor also found that for the development conditions of S1, if a single development was used or the developing agent did not contain butanone, the development effect was far worse than that of S1, and 5 obvious spots could not be separated.
另外，尽管乙酸乙酯、丙酮、正丁醇三种溶剂提取所得的提取试样亦能显示出5个明显的斑点，但重复性差，不具有可操作性，作为常规检测是不恰当的。In addition, although the extraction samples obtained by extraction with three solvents, ethyl acetate, acetone and n-butanol, also showed 5 obvious spots, their repeatability was poor and they were not operable, so they were not suitable for routine detection.
此外，对于先用水提取，然后用乙酸乙酯萃取制备供试液的情况，本领域技术人员清楚，现有的头花蓼制剂或者制备该制剂的头花蓼提取物，多是使用水提取或者含水乙醇提取头花蓼药材，除溶剂后得到的。因此，对于现有的头花蓼制剂或者制备该制剂的头花蓼提取物，亦可以使用本发明第一方面的方法进行指纹图谱的测定。即，只要在本发明第一方面方法步骤(a)中，进行薄层点样液制备时，直接用乙酸乙酯萃取头花蓼制剂或者制备该制剂的头花蓼提取物(或者必要时，可以在萃取之前用水溶解所述头花蓼制剂或者制备该制剂的头花蓼提取物)，将乙酸乙酸萃取液蒸干，残渣用乙酸乙酯溶解，即作为头花蓼制剂或者制备该制剂的头花蓼提取物的供试品溶液，即可，其它步骤与本发明第一方面方法的其它步骤相同。为此，本发明第二方面提供了一种头花蓼制剂以及制备该制剂的头花蓼提取的指纹图谱的测定方法，其是照薄层色谱法测定，包括以下步骤：(a)薄层点样液制备：称取头花蓼制剂或者制备该制剂的头花蓼提取物，用乙酸乙酯萃取，将乙酸乙酸萃取液蒸干，残渣用乙酸乙酯使溶解，作为头花蓼供试品溶液；以及任选地分别制备芦丁、槲皮苷和没食子酸的对照品溶液；以及如本发明第一方面任一实施方案所述的步骤(b)、步骤(c)、和步骤(d)。在本发明第二方面的方法中，其中所述步骤(a)中还任选地包括在萃取之前用水溶解所述头花蓼制剂或者制备该制剂的头花蓼提取物，然后用乙酸乙酯进行萃取的步骤。In addition, for the case where the test solution is prepared by first extracting with water and then extracting with ethyl acetate, it is clear to those skilled in the art that the existing Polygonum capitatum preparations or the Polygonum capitatum extracts used to prepare the preparations are mostly obtained by extracting the Polygonum capitatum medicinal materials with water or aqueous ethanol and removing the solvent. Therefore, for the existing Polygonum capitatum preparations or the Polygonum capitatum extracts used to prepare the preparations, the fingerprint spectrum can also be determined using the method of the first aspect of the present invention. That is, as long as in step (a) of the first aspect of the method of the present invention, when preparing the thin layer spotting solution, the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation is directly extracted with ethyl acetate (or if necessary, the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation can be dissolved in water before extraction), the ethyl acetate extract is evaporated to dryness, and the residue is dissolved with ethyl acetate, that is, as the test solution of the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation, the other steps are the same as the other steps of the first aspect of the method of the present invention. To this end, the second aspect of the present invention provides a method for determining the fingerprint of a Polygonum capitatum preparation and a Polygonum capitatum extract used to prepare the preparation, which is determined according to thin layer chromatography and includes the following steps: (a) Preparation of thin layer spotting solution: weigh the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation, extract with ethyl acetate, evaporate the ethyl acetate extract to dryness, and dissolve the residue with ethyl acetate as the Polygonum capitatum test solution; and optionally prepare reference solutions of rutin, quercetin and gallic acid respectively; and steps (b), (c), and (d) as described in any embodiment of the first aspect of the present invention. In the method of the second aspect of the present invention, step (a) also optionally includes the step of dissolving the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation with water before extraction, and then extracting with ethyl acetate.
试验例3：相对湿度(RH)对头花蓼药材薄层色谱行为的影响Experimental Example 3: Effect of relative humidity (RH) on the thin layer chromatography behavior of Polygonum capitatum
在点样过程中，吸附剂表面能可逆地吸收水分，如空气湿度过大，薄层活度会降低，影响分离效果，因此，本实验以S1为溶剂系统展开，以药材编号为1号的样品所得溶液为供试品溶液，考察了相对湿度对头花蓼药材薄层色谱行为的影响，同时与热淋清颗粒的薄层色谱行为作比较，结果见专利文献之图2。不同相对湿度可按如下配置设置：相对湿度32%(硫酸68 mL、100 mL)，相对湿度88%(硫酸10.8 mL、100 mL)。During the spotting process, the adsorbent surface can reversibly absorb water. If the air humidity is too high, the thin layer activity will decrease, affecting the separation effect. Therefore, this experiment was carried out with S1 as the solvent system, and the solution obtained from the sample with the medicinal material number 1 was used as the test solution. The effect of relative humidity on the thin layer chromatography behavior of Polygonum capitatum medicinal material was investigated, and the thin layer chromatography behavior of Relinqing granules was compared. The results are shown in Figure 2 of the patent document. Different relative humidity can be set as follows: relative humidity 32% (sulfuric acid 68 mL, 100 mL), relative humidity 88% (sulfuric acid 10.8 mL, 100 mL).
图中描绘了点样时的相对湿度(RH，32%和88%)对头花蓼药材薄层层析行为的影响(320nm)，图中四条展开带中从左至右依次是：1.头花蓼样品；2.热淋清颗粒样品；3.芦丁；4.槲皮苷。在另外的试验中，还使用了RH72%、RH50%两种点样条件，结果显示这两种条件下对头花蓼药材薄层层析行为与RH32%相似。可见：相对湿度(RH)小于88%时，对头花蓼药材薄层色谱行为影响不明显，当相对湿度(RH)为88%或更高时，对头花蓼药材薄层色谱行为有显著影响，分离度降低，斑点减少，色谱无法辨认，故相对湿度(RH)小于88%为宜，特别是在32%~72%条件下是优选的。热淋清颗粒亦显示出与药材类似的结果。The figure depicts the effect of relative humidity (RH, 32% and 88%) on the TLC behavior of Polygonum capitatum medicinal material (320nm). The four developed bands in the figure are from left to right: 1. Polygonum capitatum sample; 2. Relinqing granule sample; 3. Rutin; 4. Quercetin. In other experiments, two spotting conditions of RH72% and RH50% were also used. The results showed that the TLC behavior of Polygonum capitatum medicinal material under these two conditions was similar to that of RH32%. It can be seen that when the relative humidity (RH) is less than 88%, the TLC behavior of Polygonum capitatum medicinal material is not significantly affected. When the relative humidity (RH) is 88% or higher, the TLC behavior of Polygonum capitatum medicinal material is significantly affected, the separation is reduced, the spots are reduced, and the chromatogram cannot be identified. Therefore, the relative humidity (RH) is less than 88%, especially when it is 32%~72%. Relinqing granules also show similar results to medicinal materials.
试验例4：温度对头花蓼药材薄层色谱行为的影响Experimental Example 4: Effect of Temperature on Thin-Layer Chromatographic Behavior of Polygonum capitatum
为考察温度对头花蓼薄层色谱行为的影响，本实验以S1为溶剂系统展开，以药材编号为1号的样品所得溶液为供试品溶液，同时与热淋清颗粒的薄层色谱行为作比较，将展开槽放在恒温干燥箱中，设置温度为10℃、20℃、30℃，结果见专利文献之图3。In order to investigate the effect of temperature on the thin layer chromatographic behavior of Polygonum capitatum, this experiment was carried out with S1 as the solvent system, and the solution obtained from the sample with medicinal material number 1 was used as the test solution. At the same time, the thin layer chromatographic behavior was compared with that of Relinqing Granules. The developing tank was placed in a constant temperature drying oven, and the temperature was set at 10℃, 20℃, and 30℃. The results are shown in Figure 3 of the patent document.
结果表明：温度对头花蓼药材薄层色谱行为影响很明显，当温度为20℃时，头花蓼药材薄层色谱分离度较好、斑点清晰可见，故环境温度应控制在20℃左右。本领域技术人员理解，在本发明的一个实施方案中，一般而言在20±2℃是有利的。热淋清颗粒亦显示出与药材类似的结果。The results show that temperature has a significant effect on the TLC behavior of Polygonum capitatum medicinal materials. When the temperature is 20°C, the TLC separation of Polygonum capitatum medicinal materials is good and the spots are clearly visible, so the ambient temperature should be controlled at around 20°C. Those skilled in the art understand that in one embodiment of the present invention, generally speaking, 20±2°C is beneficial. Relinqing granules also showed similar results to medicinal materials.
试验例5：预饱和时间对头花蓼药材薄层色谱行为的影响Experimental Example 5: Effect of presaturation time on thin layer chromatography behavior of Polygonum capitatum
薄层板和展开剂蒸汽间的平衡主要表现在从展开开始至结束过程中薄层对展开剂蒸汽的预吸附，而薄层各个部分吸附的程度是不同的。当用多元展开剂时，又将发生选择性吸附现象，这些都会影响色谱分离。因此在用多元展开剂时，经常将薄层及展开室进行预饱和，以便得到重现性较好的色谱图。本实验以S1为溶剂系统，以药材编号为1号的样品所得溶液为供试品溶液，同时与热淋清颗粒的薄层色谱行为作比较，分别对饱和15min、30min、45min进行了对比，结果见专利文献之图4。结果表明：饱和15min、30min、45min薄层色谱效果基本相同，饱和时间增加，分离度并未明显提高，因此饱和时间确定为15min即可。热淋清颗粒亦显示出与药材类似的结果。The balance between the thin layer plate and the developing agent vapor is mainly manifested in the pre-adsorption of the developing agent vapor by the thin layer from the beginning to the end of the development, and the degree of adsorption of each part of the thin layer is different. When using a multi-component developing agent, selective adsorption will occur, which will affect the chromatographic separation. Therefore, when using a multi-component developing agent, the thin layer and the developing chamber are often pre-saturated to obtain a chromatogram with better reproducibility. In this experiment, S1 was used as the solvent system, and the solution obtained from the sample with the medicinal material number 1 was used as the test solution. At the same time, it was compared with the thin layer chromatography behavior of the hot linqing granules, and the saturation 15min, 30min, and 45min were compared respectively. The results are shown in Figure 4 of the patent document. The results show that the saturated 15min, 30min, and 45min thin layer chromatography effects are basically the same. The separation degree is not significantly improved when the saturation time increases, so the saturation time can be determined to be 15min. The hot linqing granules also showed similar results to the medicinal materials.
试验例6：显色对头花蓼药材薄层色谱的影响Experimental Example 6: Effect of color development on thin layer chromatography of Polygonum capitatum medicinal material
为比较显色剂对头花蓼药材薄层色谱的影响，本实验以S1为溶剂系统，以药材编号为1号的样品所得溶液为供试品溶液，同时与热淋清颗粒的薄层色谱行为作比较，对分别使用AlCl
3试液、1%三氯化铁试液、AlCl
3乙醇试液作为显色剂显色进行了考察。结果见专利文献之图5。结果表明：用1%三氯化铁显色后无荧光，AlCl
3试液喷雾显色后，斑点荧光颜色明显，斑点数清晰；而用AlCl
3乙醇试液的显色效果并未明显改善。因此，可以选择用AlCl
3试液喷雾显色。热淋清颗粒亦显示出与药材类似的结果。
In order to compare the effect of color developer on TLC of Polygonum capitatum medicinal material, this experiment used S1 as solvent system, and the solution obtained from the sample with medicinal material number 1 as the test solution. At the same time, the TLC behavior of Relinqing granules was compared, and the color development using AlCl 3 test solution, 1% ferric chloride test solution, and AlCl 3 ethanol test solution as color developers was investigated. The results are shown in Figure 5 of the patent document. The results show that there is no fluorescence after color development with 1% ferric chloride, and the color of the spots is obvious and the number of spots is clear after spraying with AlCl 3 test solution; while the color development effect of AlCl 3 ethanol test solution is not significantly improved. Therefore, it is possible to choose to spray with AlCl 3 test solution for color development. Relinqing granules also show similar results to the medicinal materials.
试验例7：头花蓼药材薄层色谱扫描轮廓图分析Experimental Example 7: Thin layer chromatography scanning profile analysis of Polygonum capitatum medicinal material
使用编号为1的药材进行测试，在挥尽薄层板上的残留溶剂后，置紫外光灯320nm下检视荧光薄层色谱。将荧光薄层色谱图导入Chromafinger
色谱指纹图谱解决方案软件，生成灰度扫描图并积分，得芦丁、槲皮苷和头花蓼药材样品扫描轮廓图，见专利文献之图6。从图中结果可见，编号为1的药材的5个主峰中，第3、4号主峰与芦丁的两个主峰对应，第4号主峰还与槲皮苷的主峰对应。对于其它批次的药材，以及市售热淋清颗粒(无糖型)，其薄层扫描图与1号药材和两个对照品在峰位上对应，仅在峰强上有差别。结果表明，头花蓼药材及其制剂的薄层色谱图像，以320nm下的荧光色谱信息最丰富，头花蓼药材可以明显观察到5个荧光斑点，头花蓼制剂可以明显观察到5个荧光斑点。扫描轮廓图中的特征峰与斑点相对应。The test was conducted using the medicinal material No. 1. After waving off the residual solvent on the thin layer plate, the fluorescent thin layer chromatogram was examined under a UV lamp at 320nm. The fluorescent thin layer chromatogram was imported into the Chromafinger
Chromatographic Fingerprint Solution Software, and a grayscale scan was generated and integrated to obtain the scan profile of rutin, quercetin and Polygonum capitatum medicinal material samples, as shown in Figure 6 of the patent document. From the results in the figure, it can be seen that among the five main peaks of the medicinal material No. 1, the 3rd and 4th main peaks correspond to the two main peaks of rutin, and the 4th main peak also corresponds to the main peak of quercetin. For other batches of medicinal materials, as well as the commercially available Relinqing Granules (sugar-free), their thin layer scans correspond to the peak positions of medicinal material No. 1 and the two reference substances, with only a difference in peak intensity. The results show that the thin layer chromatographic images of Polygonum capitatum medicinal materials and their preparations have the richest fluorescence chromatographic information at 320nm. Five fluorescent spots can be clearly observed in the Polygonum capitatum medicinal materials, and five fluorescent spots can be clearly observed in the Polygonum capitatum preparations. The characteristic peaks in the scan profile correspond to the spots.
试验例8：12批贵州施秉头花蓼药材薄层色谱图及轮廓扫描叠加图Experimental Example 8: TLC and profile scanning overlay of 12 batches of Polygonum aviculare from Shibing, Guizhou
采用表1中记载的编号为1至12的12批贵州施秉出产的头花蓼药材，参考试验例6的方法进行薄层色谱分析，结果见专利文献之图7。描绘了12批贵州施秉出产的头花蓼药材薄层色谱图及轮廓扫描叠加图(320nm)，薄层色谱图(图中a)中14个色谱带从左至右依次是：1~12.编号为1~12的12批施秉头花蓼样品，13.芦丁；14. 槲皮苷。其中显示12个批次的同一地出产的药材，5个特征峰的位置和强度基本上相同，显示在轮廓扫描叠加图(图中b)中亦相当吻合。Twelve batches of Polygonum capitatum medicinal materials produced in Shibing, Guizhou, numbered 1 to 12 and recorded in Table 1, were used for thin layer chromatography analysis according to the method of Experimental Example 6, and the results are shown in Figure 7 of the patent document. TLCs and profile scan overlays (320 nm) of 12 batches of Polygonum capitatum medicinal materials produced in Shibing, Guizhou are depicted. The 14 chromatographic bands in the thin layer chromatogram (a in the figure) are from left to right: 1~12. 12 batches of Polygonum capitatum samples numbered 1~12 in Shibing, 13. Rutin; 14. Quercetin. It shows that the positions and intensities of the five characteristic peaks of the 12 batches of medicinal materials produced in the same place are basically the same, which is also quite consistent in the profile scan overlay (b in the figure).
试验例9：12批不同产地头花蓼药材薄层色谱图及轮廓扫描叠加图Experimental Example 9: TLC and profile scanning overlays of 12 batches of Polygonum capitatum medicinal materials from different origins
采用表1中记载的编号为12至23的12批不同产地出产的头花蓼药材，参考试验例6的方法进行薄层色谱分析，结果见专利文献之图8。图8描绘了12批不同产地头花蓼药材薄层色谱图及轮廓扫描叠加图(320nm)，薄层色谱图(图中a)中14个色谱带从左至右依次是：1.贵州施秉头花蓼样品；2.贵州兴义；3.贵州晴隆；4.贵州盘县；5.贵州毕节；6.贵州水城；7.贵州罗甸；8.湖南古丈；9.云南师宗；10.广西田林;11.重庆南川；12.四川南川;13.芦丁；14.槲皮苷。其中显示12个批次的不同产地出产的药材，5个特征峰的位置基本上相同，但强度差异大，峰位置显示在轮廓扫描叠加图(图中b)中比较吻合，但强度变化较明显。Twelve batches of Polygonum capitatum medicinal materials from different origins, numbered 12 to 23 and recorded in Table 1, were used for thin layer chromatography analysis according to the method of Experimental Example 6, and the results are shown in Figure 8 of the patent document. Figure 8 depicts the thin layer chromatograms and profile scanning overlays (320 nm) of 12 batches of Polygonum capitatum medicinal materials from different origins. The 14 chromatographic bands in the thin layer chromatogram (a in the figure) are from left to right: 1. Polygonum capitatum samples from Shibing, Guizhou; 2. Xingyi, Guizhou; 3. Qinglong, Guizhou; 4. Panxian, Guizhou; 5. Bijie, Guizhou; 6. Shuicheng, Guizhou; 7. Luodian, Guizhou; 8. Guzhang, Hunan; 9. Shizong, Yunnan; 10. Tianlin, Guangxi; 11. Nanchuan, Chongqing; 12. Nanchuan, Sichuan; 13. Rutin; 14. Quercetin. It shows that there are 12 batches of medicinal materials produced in different places. The positions of the five characteristic peaks are basically the same, but the intensities vary greatly. The peak positions are relatively consistent in the contour scan overlay diagram (b in the figure), but the intensity changes are more obvious.
试验例10：头花蓼药材及其伪品赤胫散薄层色谱图及轮廓扫描图Test Example 10: Thin layer chromatogram and profile scan of Polygonum capitatum and its counterfeit Chijingsan
采用表1中记载的编号为1的头花蓼药材和编号为24的伪品赤胫散，参考试验例6的方法进行薄层色谱分析，结果见专利文献之图9。图9描绘了施秉头花蓼药材及头花蓼伪品薄层色谱图及轮廓扫描图(320nm)，薄层色谱图(a)中四个色谱带从左至右依次表示的检品为：1.施秉头花蓼样品；2.
赤胫散；3.芦丁；4.槲皮苷。施秉头花蓼药材轮廓扫描图(b)显示出5个典型的特征峰，而伪品赤胫散在相应位置未显示特征峰。The Polygonum capitatum medicinal material numbered 1 and the counterfeit Chijinsan numbered 24 recorded in Table 1 were used for thin layer chromatography analysis according to the method of Experimental Example 6, and the results are shown in Figure 9 of the patent document. Figure 9 depicts the thin layer chromatogram and profile scan (320nm) of the Polygonum capitatum medicinal material and the counterfeit Polygonum capitatum medicinal material from Shibing. The four chromatographic bands in the thin layer chromatogram (a) represent the test samples from left to right: 1. Polygonum capitatum sample from Shibing; 2. Chijinsan; 3. Rutin; 4. Quercetin. The profile scan of the Polygonum capitatum medicinal material from Shibing (b) shows 5 typical characteristic peaks, while the counterfeit Chijinsan does not show characteristic peaks at the corresponding positions.
在以上试验例1-10中，使用热淋清颗粒的薄层色谱行为进行对照时，该热淋清颗粒在各种试验条件下与制备它的头花蓼药材显示出相同的试验结果。In the above Test Examples 1-10, when the thin layer chromatography behavior of Relinqing Granules was used for comparison, the Relinqing Granules showed the same test results as the Polygonum capitatum medicinal material used to prepare it under various test conditions.
C、提取物或制剂实施例部分C. Extract or Preparation Examples
参照中国药典2010年版一部“热淋清颗粒”项下的方法，取头花蓼12.5kg，加水煎煮二次，每次1.5小时，煎液滤过，滤液合并，浓缩至适量，滤过，喷雾干燥，得浸膏粉A。取相当于1000克药材得到的浸膏粉A，加适量淀粉至总量成400克，混匀，制粒，干燥，制成热淋清颗粒，包装，每袋4克。另取相当于1000克药材得到的浸膏粉A，加适量蔗糖粉至总量成800克，混匀，制粒，干燥，制成含糖型热淋清颗粒，包装，每袋8克。另取相当于1000克药材得到的浸膏粉A，加淀粉、乳糖、微晶纤维素三者以重量比1:1:1配得的混合物适量，至总量成200克，混匀，制粒，干燥，加入适量硬脂酸镁，压片，制成热淋清片，每片重0.5g。以上方法各制备12批的制剂样品。According to the method under "Relinqing Granules" in the 2010 edition of the Chinese Pharmacopoeia, 12.5 kg of Polygonum capitatum was taken, decocted twice with water, each time for 1.5 hours, the decoction was filtered, the filtrates were combined, concentrated to an appropriate amount, filtered, and spray-dried to obtain extract powder A. Extract powder A obtained from 1000 grams of medicinal materials was added with an appropriate amount of starch to a total amount of 400 grams, mixed, granulated, dried, and made into Relinqing granules, which were packaged in 4 grams per bag. Extract powder A obtained from another 1000 grams of medicinal materials was added with an appropriate amount of sucrose powder to a total amount of 800 grams, mixed, granulated, dried, and made into sugar-containing Relinqing granules, which were packaged in 8 grams per bag. Take extract powder A equivalent to 1000g of medicinal materials, add a proper amount of a mixture of starch, lactose and microcrystalline cellulose in a weight ratio of 1:1:1, until the total amount is 200g, mix well, granulate, dry, add a proper amount of magnesium stearate, press into tablets, and make hot leaching tablets, each weighing 0.5g. Prepare 12 batches of preparation samples by the above method.
照上文“B、药材试验例部分”试验例1所述的方法，对以上分别由浸膏粉A制备得到的无糖型热淋清颗粒、含糖型热淋清颗粒、和热淋清片，对其进行薄层色谱分析。According to the method described in Experimental Example 1 of "B. Medicinal Materials Experimental Examples" above, the sugar-free Relinqing granules, sugar-containing Relinqing granules, and Relinqing tablets prepared from the extract powder A were subjected to thin layer chromatography analysis.
将薄层板置于CS-9301PC双波长飞点薄层色谱扫描仪上在320nm波长，以锯齿方式扫描得芦丁、槲皮苷、没食子酸和头花蓼制剂样品扫描轮廓叠加图，并照相。The thin layer plate was placed on a CS-9301PC dual-wavelength flying spot thin layer chromatography scanner and scanned in a zigzag manner at a wavelength of 320 nm to obtain the scanning profile overlay of rutin, quercetin, gallic acid and Polygonum capitatum preparation samples, and photographed.
专利文献之图10显示了12批含糖热淋清颗粒薄层色谱图(a)及轮廓扫描叠加图(b)，薄层照片显示的层析带中，从左至右1～12分别是12批热淋清颗粒样品，13为芦丁，14为槲皮苷，15为没食子酸。专利文献之图11显示了12批无糖热淋清颗粒薄层色谱图(a)及轮廓扫描叠加图(b)，薄层照片显示的层析带中，从左至右1～12分别是12批热淋清颗粒样品，13为芦丁，14为槲皮苷，15为没食子酸。专利文献之图12显示了12批热淋清片薄层色谱图(a)及轮廓扫描叠加图(b)，薄层照片显示的层析带中，从左至右1～12分别是12批热淋清颗粒样品。另外，用浸膏粉A为试药进行测试，结果显示与图10基本相同的薄层结果。结果表明，不同配方、不同批次制剂的薄层扫描结果一致。Figure 10 of the patent document shows the thin layer chromatograms (a) and profile scan overlays (b) of 12 batches of sugar-containing Relinqing granules. In the chromatographic bands shown in the thin layer photos, from left to right, 1 to 12 are 12 batches of Relinqing granule samples, 13 is rutin, 14 is quercetin, and 15 is gallic acid. Figure 11 of the patent document shows the thin layer chromatograms (a) and profile scan overlays (b) of 12 batches of sugar-free Relinqing granules. In the chromatographic bands shown in the thin layer photos, from left to right, 1 to 12 are 12 batches of Relinqing granule samples, 13 is rutin, 14 is quercetin, and 15 is gallic acid. Figure 12 of the patent document shows the thin layer chromatograms (a) and profile scan overlays (b) of 12 batches of Relinqing tablets. In the chromatographic bands shown in the thin layer photos, from left to right, 1 to 12 are 12 batches of Relinqing granule samples. In addition, the extract powder A was used as the test drug, and the results showed the same thin layer results as Figure 10. The results showed that the thin layer scanning results of preparations with different formulas and batches were consistent.
D、制备热淋清颗粒D. Preparation of hot leaching granules
使用同一批采自贵州毕节的头花蓼制备如下2批热淋清颗粒：The same batch of Polygonum capitatum collected from Bijie, Guizhou was used to prepare the following two batches of Relinqing granules:
热淋清颗粒①：参照中国药典2010年版一部“热淋清颗粒”项下的方法，取头花蓼药材适量，加水煎煮二次，每次1.5小时，煎液滤过，滤液合并，浓缩至适量，滤过，喷雾干燥，得浸膏粉A；取相当于1000克药材得到的浸膏粉A，加适量淀粉至总量成400克，混匀，制粒，干燥，制成热淋清颗粒，包装，每袋装量4克(相当于每袋包含药材1克)。Relinqing Granules①: Referring to the method under "Relinqing Granules" in Part I of the 2010 edition of the Chinese Pharmacopoeia, take an appropriate amount of Polygonum capitatum medicinal material, add water and boil twice, each time for 1.5 hours, filter the decoction, combine the filtrates, concentrate to an appropriate amount, filter, spray dry, and obtain extract powder A; take the extract powder A obtained from 1000 grams of medicinal material, add an appropriate amount of starch to a total amount of 400 grams, mix well, granulate, dry, and make Relinqing granules, and package, with 4 grams per bag (equivalent to 1 gram of medicinal material per bag).
热淋清颗粒②：(1)将头花蓼药材的地上部分干品加7-10倍量的包含0.75%乙酸的乙醇回流提取2次，每次1小时，过滤，使滤液浓缩、干燥，得醇浸膏；(2)使步骤(1)所得滤渣加6-8倍量水煎煮提取2次，每次1小时，过滤，使滤液浓缩、干燥，得水浸膏；(3)使所述醇浸膏和水浸膏混合，喷雾干燥除去水分(水分降至3%以下)，得到头花蓼提取物，(4)使相当于由1000克头花蓼药材制得的头花蓼提取物与适量淀粉混合至总量为400克，用水制粒，干燥，得到热淋清颗粒的制剂，包装，每袋装量4克(相当于每袋包含药材1克)。Relinqing Granules②: (1) Add 7-10 times the amount of ethanol containing 0.75% acetic acid to the dry aerial part of the medicinal material of Polygonum capitatum, reflux and extract twice, each time for 1 hour, filter, concentrate the filtrate, and dry to obtain an alcohol extract; (2) Add 6-8 times the amount of water to the filter residue obtained in step (1) and boil and extract twice, each time for 1 hour, filter, concentrate the filtrate, and dry to obtain an aqueous extract; (3) Mix the alcohol extract and the aqueous extract, spray dry to remove moisture (moisture content is reduced to less than 3%), and obtain a Polygonum capitatum extract; (4) Mix the Polygonum capitatum extract obtained from 1000 grams of the medicinal material of Polygonum capitatum with an appropriate amount of starch to a total amount of 400 grams, granulate with water, dry, and obtain a preparation of Relinqing Granules, and package them, with a packing amount of 4 grams per bag (equivalent to 1 gram of medicinal material per bag).
E、生物学性能考察E. Biological performance investigation
本试验的目的是测定热淋清颗粒①和热淋清颗粒②的MIC值The purpose of this test is to determine the MIC value of Relinqing Granules ① and Relinqing Granules ②
实验用菌种首先接种于平板上，在37 ℃恒温恒湿培养箱中培养24 h。挑选特征菌种接于液体培养基中，于37 ℃条件下，振摇24 h。采用麦氏比浊法将其稀释至0.5麦氏浓度，即10^6-10^8 CFU/mL菌悬液备用；向1L营养肉汤培养基中加入20g纯化琼脂粉，加入1L双蒸水并调节PH=7.4。于121℃条件下，灭菌30 min后取出。待培养基冷却至45 ℃左右时，在超净工作台将培养基倒入无菌培养皿中冷凝，即得实验用琼脂平板培养基。The experimental strains were first inoculated on the plate and cultured in a constant temperature and humidity incubator at 37 °C for 24 h. The characteristic strains were selected and inoculated into the liquid culture medium and shaken at 37 °C for 24 h. The suspension was diluted to 0.5 McFarland concentration, i.e. 10^6-10^8 CFU/mL, by McFarland turbidimetry for standby use; 20g of purified agar powder was added to 1L nutrient broth medium, and 1L of double distilled water was added and the pH was adjusted to 7.4. Sterilize at 121 °C for 30 min and then take out. When the culture medium is cooled to about 45 °C, pour the culture medium into a sterile culture dish on the clean bench for condensation to obtain the experimental agar plate culture medium.
在超净工作台先向无菌的96孔板中每孔中加入50 μL液体培养基和50μL的菌悬液，将待测药物(热淋清颗粒①和热淋清颗粒②)用移液枪精确量取100 μL加入到96孔板中的第一孔混匀，并吸取100 μL移至第二孔混匀，以此类推至最后一孔混匀吸取100 μL弃掉，使每孔保持100 μL。即得浓度为12.5~0.012
mg/mL；以硫酸庆大霉素为阳性对照，DMSO为阴性对照；37℃培养24 h，分别转种至琼脂平板培养基，24 h后无细菌生长的微孔则为药物最低抑菌浓度(MIC)(试验方法可参考：胡露，张锦，蔺良才，等.基于谱效关系的头花蓼抑菌作用物质基础研究[J].中药材，2016，39(9)：2037-2040)，结果如下：热淋清颗粒①对大肠埃希菌和铜绿假单胞菌的MIC值分别为2476mg/L和9314mg/L，热淋清颗粒②对大肠埃希菌和铜绿假单胞菌的MIC值分别为153mg/L和1127mg/L。结果显示本发明方法制备得到的热淋清颗粒呈现显著更优良的生物学活性。On a clean bench, add 50 μL of liquid culture medium and 50 μL of bacterial suspension to each well of a sterile 96-well plate. Use a pipette to accurately measure 100 μL of the drug to be tested (Relinqing Granules ① and Relinqing Granules ②) and add it to the first well of the 96-well plate to mix. Then, pipette 100 μL to the second well to mix, and so on to the last well. Mix and pipette 100 μL and discard it, so that each well retains 100 μL. The concentration was 12.5~0.012
mg/mL; gentamicin sulfate was used as the positive control and DMSO was used as the negative control; the mixture was cultured at 37℃ for 24 h, and then transferred to agar plate medium. The micropores without bacterial growth after 24 h were the minimum inhibitory concentration (MIC) of the drug (the experimental method can be referred to: Hu Lu, Zhang Jin, Lin Liangcai, et al. Study on the material basis of the antibacterial effect of Polygonum capitatum based on spectrum-effect relationship [J]. Chinese Medicinal Materials, 2016, 39(9): 2037-2040). The results are as follows: the MIC values of Relinqing Granules ① against Escherichia coli and Pseudomonas aeruginosa were 2476 mg/L and 9314 mg/L, respectively, and the MIC values of Relinqing Granules ② against Escherichia coli and Pseudomonas aeruginosa were 153 mg/L and 1127 mg/L, respectively. The results show that the Relinqing Granules prepared by the method of the present invention exhibit significantly better biological activity.
对于本领域技术人员而言，显然本发明不限于上述示范性实施例的细节，而且在不背离本发明的精神或基本特征的情况下，能够以其他的具体形式实现本发明。因此，无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的，本发明的范围由所附权利要求而不是上述说明限定，因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.
Claims (1)
- 提高热淋清颗粒质量的方法，该方法通过制备并检测头花蓼提取物以获取高品质的热淋清颗粒制剂来实施，包括如下步骤：(1)将头花蓼药材的地上部分干品加乙醇回流提取，过滤，使滤液浓缩、干燥，得醇浸膏；(2)使步骤(1)所得滤渣加水煎煮提取，过滤，使滤液浓缩、干燥，得水浸膏；(3)使所述醇浸膏和水浸膏混合，喷雾干燥除去水分，得到头花蓼提取物；(4)使头花蓼提取物与适量淀粉混合，用水制粒，干燥，得到热淋清颗粒的制剂。A method for improving the quality of Relinqing granules, the method is implemented by preparing and testing a Polygonum capitatum extract to obtain a high-quality Relinqing granule preparation, comprising the following steps: (1) extracting the dry aerial part of the Polygonum capitatum medicinal material with ethanol by reflux, filtering, concentrating and drying the filtrate to obtain an alcohol extract; (2) decocting and extracting the filter residue obtained in step (1) by adding water, filtering, concentrating and drying the filtrate to obtain an aqueous extract; (3) mixing the alcohol extract and the aqueous extract, spray drying to remove moisture, and obtaining a Polygonum capitatum extract; (4) mixing the Polygonum capitatum extract with an appropriate amount of starch, granulating with water, and drying to obtain a preparation of Relinqing granules.2．根据权利要求1的方法，其中还包括对制得的头花蓼提取物和/或热淋清颗粒照以下薄层色谱法测定其质量：2. The method according to claim 1, further comprising determining the quality of the obtained Polygonum capitatum extract and/or Relinqing granules by the following thin layer chromatography method:(a)薄层点样液制备：称取头花蓼药材粉末或头花蓼提取物或热淋清颗粒，加水超声处理，过滤，滤液用乙酸乙酯萃取，将乙酸乙酸萃取液蒸干，残渣用乙酸乙酯使溶解，作为供试品溶液；以及任选地分别制备芦丁、槲皮苷和没食子酸的对照品溶液；(a) Preparation of thin layer spotting solution: weigh the powder of Polygonum capitatum medicinal material or Polygonum capitatum extract or Relinqing granules, add water for ultrasonic treatment, filter, extract the filtrate with ethyl acetate, evaporate the ethyl acetate extract to dryness, dissolve the residue with ethyl acetate, and prepare the test solution; and optionally prepare the reference solution of rutin, quercetin and gallic acid respectively;(b)薄层层析：采用聚酰胺薄层板，取上述供试品溶液和任选的对照品溶液点样于薄层板上，用体积比为7:6.5:2.5:3的正己烷-乙酸乙酯-丁酮-甲酸混合液作为展开剂进行展开；(b) Thin layer chromatography: using a polyamide thin layer plate, spot the test solution and the optional reference solution on the plate, and develop with a mixture of n-hexane-ethyl acetate-butanone-formic acid in a volume ratio of 7:6.5:2.5:3 as a developing solvent;(c)显色：向挥干的薄层板上喷以显色液AlCl 3试液，挥尽薄层板上的残余溶剂，置320nm的紫外光灯下检视荧光薄层色谱，得到头花蓼药材的指纹图谱。 (c) Color development: Spray the dried thin layer plate with AlCl 3 test solution to evaporate the residual solvent on the thin layer plate, and examine the fluorescent thin layer chromatogram under a 320nm ultraviolet lamp to obtain the fingerprint of Polygonum capitatum.3．根据权利要求1的方法，步骤(a)中，所述头花蓼供试品溶液是用如下方法配制的：取头花蓼药材粉末，至容量瓶中，加水适量，超声处理，放冷，用水定容至刻度，滤过，滤液用乙酸乙酯萃取，使乙酸乙酯液蒸干，残渣加乙酸乙酯使溶解，即得供试品溶液。3. According to the method of claim 1, in step (a), the Polygonum capitatum test solution is prepared by the following method: taking the Polygonum capitatum medicinal material powder, putting it into a volumetric flask, adding an appropriate amount of water, ultrasonically treating, cooling, diluting to scale with water, filtering, extracting the filtrate with ethyl acetate, evaporating the ethyl acetate solution, and dissolving the residue with ethyl acetate to obtain the test solution.4．根据权利要求1的方法，其中步骤(a)中，所述头花蓼供试品溶液是用如下方法配制的：取头花蓼药材粉末2g，至25mL容量瓶中，加水25mL，以功率250W、频率33kHz超声处理30min，放冷，用水定容至刻度，滤过，滤液用乙酸乙酯萃取3次，每次25mL，合并乙酸乙酯液，蒸干，残渣加5mL乙酸乙酯使溶解，即得供试品溶液。4. The method according to claim 1, wherein in step (a), the Polygonum capitatum test solution is prepared by the following method: 2 g of Polygonum capitatum powder is taken into a 25 mL volumetric flask, 25 mL of water is added, and ultrasonic treatment is performed at a power of 250 W and a frequency of 33 kHz for 30 min, the mixture is cooled, the volume is adjusted to the mark with water, and the mixture is filtered. The filtrate is extracted with ethyl acetate for 3 times, 25 mL each time, the ethyl acetate solutions are combined, evaporated to dryness, and 5 mL of ethyl acetate is added to the residue to dissolve it, thereby obtaining the test solution.5．根据权利要求1的方法，其中还包括步骤(d)对薄层色谱图进行分析：包括5个共有峰/斑点的薄层色谱指纹图谱所对应的药材可初步判定为头花蓼药材正品，无5个共有峰/斑点则可判定为伪品。5. The method according to claim 1, further comprising step (d) analyzing the thin layer chromatogram: the medicinal material corresponding to the thin layer chromatographic fingerprint with 5 common peaks/spots can be preliminarily determined to be the authentic Polygonum capitatum medicinal material, and the medicinal material without 5 common peaks/spots can be determined to be a counterfeit.6．根据权利要求1的方法，制备所述热淋清颗粒包括如下步骤：(1)将头花蓼药材的地上部分干品加7-10倍量的包含0.75%乙酸的乙醇回流提取2次，每次1小时，过滤，使滤液浓缩、干燥，得醇浸膏；(2)使步骤(1)所得滤渣加6-8倍量水煎煮提取2次，每次1小时，过滤，使滤液浓缩、干燥，得水浸膏；(3)使所述醇浸膏和水浸膏混合，喷雾干燥除去水分，得到头花蓼提取物；(4)使相当于由1000克头花蓼药材制得的头花蓼提取物与适量淀粉混合至总量为400克，用水制粒，干燥，得到热淋清颗粒的制剂。6. According to the method of claim 1, the preparation of the Relinqing granules comprises the following steps: (1) adding 7-10 times the amount of ethanol containing 0.75% acetic acid to the dry aerial part of the medicinal material of Polygonum capitatum, reflux extraction twice, each time for 1 hour, filtering, concentrating the filtrate, and drying to obtain an alcohol extract; (2) adding 6-8 times the amount of water to the filter residue obtained in step (1) to decoct and extract twice, each time for 1 hour, filtering, concentrating the filtrate, and drying to obtain a water extract; (3) mixing the alcohol extract and the water extract, spray drying to remove water, and obtaining a Polygonum capitatum extract; (4) mixing the Polygonum capitatum extract obtained from 1000 grams of the medicinal material of Polygonum capitatum with an appropriate amount of starch to a total amount of 400 grams, granulating with water, and drying to obtain a preparation of Relinqing granules.7．测定权利要求1~6任一项所述头花蓼制剂热淋清颗粒或制备该制剂的头花蓼提取物的指纹图谱的测定方法，包括以下步骤：7. A method for determining the fingerprint of the Polygonum capitatum preparation Relinqing granules or the Polygonum capitatum extract used to prepare the preparation according to any one of claims 1 to 6, comprising the following steps:(a)薄层点样液制备：称取头花蓼制剂或者制备该制剂的头花蓼提取物，用乙酸乙酯萃取，将乙酸乙酸萃取液蒸干，残渣用乙酸乙酯使溶解，作为供试品溶液；以及任选地分别制备芦丁、槲皮苷和没食子酸的对照品溶液；(a) Preparation of thin layer spotting solution: Weigh the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation, extract with ethyl acetate, evaporate the ethyl acetate extract to dryness, and dissolve the residue with ethyl acetate as the test solution; and optionally prepare reference solutions of rutin, quercetin and gallic acid respectively;(b)薄层层析：采用聚酰胺薄层板，取上述供试品溶液和任选的对照品溶液点样于薄层板上，用体积比为7:6.5:2.5:3的正己烷-乙酸乙酯-丁酮-甲酸混合液作为展开剂进行展开；(b) Thin layer chromatography: using a polyamide thin layer plate, spot the test solution and the optional reference solution on the plate, and develop with a mixture of n-hexane-ethyl acetate-butanone-formic acid in a volume ratio of 7:6.5:2.5:3 as a developing solvent;(c)显色：向挥干的薄层板上喷以显色液AlCl 3试液，挥尽薄层板上的残余溶剂，置320nm的紫外光灯下检视荧光薄层色谱，得到头花蓼制剂或者制备该制剂的头花蓼提取物的指纹图谱。 (c) Color development: Spray the evaporated thin layer plate with a color developing solution of AlCl 3 test solution to evaporate the residual solvent on the thin layer plate, and examine the fluorescent thin layer chromatogram under a 320 nm ultraviolet lamp to obtain the fingerprint of the Polygonum capitatum preparation or the Polygonum capitatum extract used to prepare the preparation.8．制备热淋清颗粒的方法，包括如下步骤：(1)将头花蓼药材的地上部分干品加乙醇回流提取，过滤，使滤液浓缩、干燥，得醇浸膏；(2)使步骤(1)所得滤渣加水煎煮提取，过滤，使滤液浓缩、干燥，得水浸膏；(3)使所述醇浸膏和水浸膏混合，喷雾干燥除去水分，得到头花蓼提取物，(4)使头花蓼提取物与适量淀粉混合，用水制粒，干燥，得到热淋清颗粒的制剂。8. A method for preparing Relinqing granules, comprising the following steps: (1) extracting the dry aerial part of the medicinal material Polygonum capitatum by reflux with ethanol, filtering, concentrating and drying the filtrate to obtain an alcohol extract; (2) boiling and extracting the filter residue obtained in step (1), filtering, concentrating and drying the filtrate to obtain an aqueous extract; (3) mixing the alcohol extract and the aqueous extract, spray drying to remove water, and obtaining a Polygonum capitatum extract; (4) mixing the Polygonum capitatum extract with an appropriate amount of starch, granulating with water, and drying to obtain a preparation of Relinqing granules.9．根据权利要求8的方法，包括如下步骤：(1)将头花蓼药材的地上部分干品加7-10倍量的包含0.75%乙酸的乙醇回流提取2次，每次1小时，过滤，使滤液浓缩、干燥，得醇浸膏；(2)使步骤(1)所得滤渣加6-8倍量水煎煮提取2次，每次1小时，过滤，使滤液浓缩、干燥，得水浸膏；(3)使所述醇浸膏和水浸膏混合，喷雾干燥除去水分，得到头花蓼提取物；(4)使相当于由1000克头花蓼药材制得的头花蓼提取物与适量淀粉混合至总量为400克，用水制粒，干燥，得到热淋清颗粒的制剂。9. The method according to claim 8, comprising the following steps: (1) adding 7-10 times the amount of ethanol containing 0.75% acetic acid to the dry aerial part of the medicinal material of Polygonum capitatum, reflux extraction twice, each time for 1 hour, filtering, concentrating the filtrate, and drying to obtain an alcohol extract; (2) adding 6-8 times the amount of water to the filter residue obtained in step (1) to decoct and extract twice, each time for 1 hour, filtering, concentrating the filtrate, and drying to obtain a water extract; (3) mixing the alcohol extract and the water extract, spray drying to remove water, and obtaining a Polygonum capitatum extract; (4) mixing the Polygonum capitatum extract obtained from 1000 grams of the medicinal material of Polygonum capitatum with an appropriate amount of starch to a total amount of 400 grams, granulating with water, and drying to obtain a preparation of hot linqing granules.10．一种热淋清颗粒，其是由包括如下步骤的方法制备得到的：(1)将头花蓼药材的地上部分干品加乙醇回流提取，过滤，使滤液浓缩、干燥，得醇浸膏；(2)使步骤(1)所得滤渣加水煎煮提取，过滤，使滤液浓缩、干燥，得水浸膏；(3)使所述醇浸膏和水浸膏混合，喷雾干燥除去水分，得到头花蓼提取物；(4)使头花蓼提取物与适量淀粉混合，用水制粒，干燥，得到热淋清颗粒的制剂。10. A Relinqing granule, which is prepared by a method comprising the following steps: (1) extracting the dry aerial part of the medicinal material Polygonum capitatum by reflux with ethanol, filtering, concentrating the filtrate, and drying to obtain an alcohol extract; (2) boiling the filter residue obtained in step (1) with water, filtering, concentrating the filtrate, and drying to obtain an aqueous extract; (3) mixing the alcohol extract and the aqueous extract, spray drying to remove water, and obtaining a Polygonum capitatum extract; (4) mixing the Polygonum capitatum extract with an appropriate amount of starch, granulating with water, and drying to obtain a preparation of Relinqing granules.
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| CN102526219A (en) * | 2012-01-05 | 2012-07-04 | 贵州威门药业股份有限公司 | Extract for Relinqing Granule, as well as preparation method and application of same |
| CN102608248A (en) * | 2012-02-27 | 2012-07-25 | 贵州威门药业股份有限公司 | Relinqing granules and polygonum capitatum thin-layer fingerprint chromatogram determination method |
| CN103550311A (en) * | 2013-10-30 | 2014-02-05 | 浙江百草中药饮片有限公司 | Polygonum capitatum extract as well as preparation method and application of polygonum capitatum extract |
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| CN102526219A (en) * | 2012-01-05 | 2012-07-04 | 贵州威门药业股份有限公司 | Extract for Relinqing Granule, as well as preparation method and application of same |
| CN102608248A (en) * | 2012-02-27 | 2012-07-25 | 贵州威门药业股份有限公司 | Relinqing granules and polygonum capitatum thin-layer fingerprint chromatogram determination method |
| CN103550311A (en) * | 2013-10-30 | 2014-02-05 | 浙江百草中药饮片有限公司 | Polygonum capitatum extract as well as preparation method and application of polygonum capitatum extract |
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