CN111349140A - Preparation method and quality control method of high-purity rhizoma paridis saponin VII - Google Patents
Preparation method and quality control method of high-purity rhizoma paridis saponin VII Download PDFInfo
- Publication number
- CN111349140A CN111349140A CN202010345991.2A CN202010345991A CN111349140A CN 111349140 A CN111349140 A CN 111349140A CN 202010345991 A CN202010345991 A CN 202010345991A CN 111349140 A CN111349140 A CN 111349140A
- Authority
- CN
- China
- Prior art keywords
- purity
- methanol
- paris
- saponin vii
- vii
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000002360 preparation method Methods 0.000 title claims abstract description 27
- 150000007949 saponins Chemical class 0.000 title claims description 122
- 229930182490 saponin Natural products 0.000 title claims description 121
- 239000001397 quillaja saponaria molina bark Substances 0.000 title claims description 114
- 238000003908 quality control method Methods 0.000 title abstract description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 122
- 241000244987 Daiswa polyphylla Species 0.000 claims abstract description 116
- FBFJAXUYHGSVFN-IYUYFXHASA-N 68124-04-9 Chemical compound C([C@@]12[C@H]([C@@]3([C@@]4(C)CC[C@@H]5[C@@]6(C)CC[C@@H](CC6=CC[C@H]5[C@@H]4C[C@@H]3O2)O[C@H]2[C@@H]([C@@H](O)[C@H](O[C@H]3[C@@H]([C@H](O)[C@@H](O[C@H]4[C@@H]([C@H](O)[C@@H](O)[C@H](C)O4)O)[C@H](C)O3)O)[C@@H](CO)O2)O[C@H]2[C@@H]([C@H](O)[C@@H](O)[C@H](C)O2)O)O)C)C[C@@H](C)CO1 FBFJAXUYHGSVFN-IYUYFXHASA-N 0.000 claims abstract description 71
- FBFJAXUYHGSVFN-UHFFFAOYSA-N 25(R)-pennogenin-3-O-alpha-L-rhamnopyranosyl-(14)-alpha-L-rhamnopyranosyl-(14)-[alpha-L-rhamnopyranosyl-(12)]-beta-D-glucopyranoside Natural products O1C2CC3C4CC=C5CC(OC6C(C(O)C(OC7C(C(O)C(OC8C(C(O)C(O)C(C)O8)O)C(C)O7)O)C(CO)O6)OC6C(C(O)C(O)C(C)O6)O)CCC5(C)C4CCC3(C)C2(O)C(C)C21CCC(C)CO2 FBFJAXUYHGSVFN-UHFFFAOYSA-N 0.000 claims abstract description 70
- 238000000034 method Methods 0.000 claims abstract description 49
- 239000012535 impurity Substances 0.000 claims abstract description 32
- 238000000605 extraction Methods 0.000 claims abstract description 28
- 238000010992 reflux Methods 0.000 claims abstract description 21
- 239000000463 material Substances 0.000 claims abstract description 17
- 230000008569 process Effects 0.000 claims abstract description 14
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 153
- 239000000243 solution Substances 0.000 claims description 71
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 55
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 48
- 238000010828 elution Methods 0.000 claims description 45
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 38
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 38
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 claims description 36
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 33
- 239000000284 extract Substances 0.000 claims description 32
- 238000004809 thin layer chromatography Methods 0.000 claims description 32
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 30
- 238000001514 detection method Methods 0.000 claims description 29
- 238000002156 mixing Methods 0.000 claims description 29
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 24
- 239000002904 solvent Substances 0.000 claims description 24
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 23
- 239000002021 butanolic extract Substances 0.000 claims description 23
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 22
- 230000014759 maintenance of location Effects 0.000 claims description 21
- 239000013078 crystal Substances 0.000 claims description 19
- 239000003208 petroleum Substances 0.000 claims description 18
- 238000010606 normalization Methods 0.000 claims description 17
- 238000010898 silica gel chromatography Methods 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 15
- 239000003480 eluent Substances 0.000 claims description 13
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 12
- 238000010438 heat treatment Methods 0.000 claims description 12
- 238000010298 pulverizing process Methods 0.000 claims description 11
- MHLMRBVCMNDOCW-UHFFFAOYSA-N acetic acid;butan-1-ol;hydrate Chemical compound O.CC(O)=O.CCCCO MHLMRBVCMNDOCW-UHFFFAOYSA-N 0.000 claims description 10
- LIOIDYIXMHPGGB-UHFFFAOYSA-N chloroform;formic acid;methanol Chemical compound OC.OC=O.ClC(Cl)Cl LIOIDYIXMHPGGB-UHFFFAOYSA-N 0.000 claims description 10
- SIHHLZPXQLFPMC-UHFFFAOYSA-N chloroform;methanol;hydrate Chemical compound O.OC.ClC(Cl)Cl SIHHLZPXQLFPMC-UHFFFAOYSA-N 0.000 claims description 10
- 239000000706 filtrate Substances 0.000 claims description 10
- 238000004262 preparative liquid chromatography Methods 0.000 claims description 10
- KIWBPDUYBMNFTB-UHFFFAOYSA-N Ethyl hydrogen sulfate Chemical compound CCOS(O)(=O)=O KIWBPDUYBMNFTB-UHFFFAOYSA-N 0.000 claims description 9
- 238000007605 air drying Methods 0.000 claims description 9
- 239000012153 distilled water Substances 0.000 claims description 9
- 235000011869 dried fruits Nutrition 0.000 claims description 9
- 238000001914 filtration Methods 0.000 claims description 9
- 238000004237 preparative chromatography Methods 0.000 claims description 9
- 238000005507 spraying Methods 0.000 claims description 9
- 239000007864 aqueous solution Substances 0.000 claims description 8
- 238000002347 injection Methods 0.000 claims description 5
- 239000007924 injection Substances 0.000 claims description 5
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical group O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 5
- 239000012846 chemical reference substance Substances 0.000 abstract description 34
- 239000003814 drug Substances 0.000 abstract description 24
- 238000011160 research Methods 0.000 abstract description 19
- 238000000926 separation method Methods 0.000 abstract description 16
- 238000004519 manufacturing process Methods 0.000 abstract description 8
- 230000008901 benefit Effects 0.000 abstract description 5
- 238000007689 inspection Methods 0.000 abstract description 5
- 241000234280 Liliaceae Species 0.000 abstract description 3
- 238000013461 design Methods 0.000 abstract description 3
- 238000009776 industrial production Methods 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract 1
- 235000017709 saponins Nutrition 0.000 description 113
- 239000000523 sample Substances 0.000 description 33
- 239000000126 substance Substances 0.000 description 28
- 239000012071 phase Substances 0.000 description 25
- 239000000047 product Substances 0.000 description 17
- 238000004458 analytical method Methods 0.000 description 10
- 238000005259 measurement Methods 0.000 description 9
- 229940079593 drug Drugs 0.000 description 8
- 150000001875 compounds Chemical class 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 6
- 241000245032 Trillium Species 0.000 description 6
- 150000002500 ions Chemical class 0.000 description 6
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 description 6
- 239000011347 resin Substances 0.000 description 6
- 229920005989 resin Polymers 0.000 description 6
- 150000005856 steroid saponins Chemical class 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000011161 development Methods 0.000 description 5
- 241000411851 herbal medicine Species 0.000 description 5
- 238000001819 mass spectrum Methods 0.000 description 5
- 239000013558 reference substance Substances 0.000 description 5
- 229940126680 traditional chinese medicines Drugs 0.000 description 5
- 241000509517 Ypsilandra Species 0.000 description 4
- 238000000862 absorption spectrum Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 4
- 239000000741 silica gel Substances 0.000 description 4
- 229910002027 silica gel Inorganic materials 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- 238000005481 NMR spectroscopy Methods 0.000 description 3
- 244000184734 Pyrus japonica Species 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 239000000287 crude extract Substances 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000004445 quantitative analysis Methods 0.000 description 3
- 230000003595 spectral effect Effects 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 239000012085 test solution Substances 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 239000012259 ether extract Substances 0.000 description 2
- 239000002024 ethyl acetate extract Substances 0.000 description 2
- 238000003810 ethyl acetate extraction Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000002329 infrared spectrum Methods 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- VYQNWZOUAUKGHI-UHFFFAOYSA-N monobenzone Chemical compound C1=CC(O)=CC=C1OCC1=CC=CC=C1 VYQNWZOUAUKGHI-UHFFFAOYSA-N 0.000 description 2
- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical compound [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 description 2
- 238000002953 preparative HPLC Methods 0.000 description 2
- JUJWROOIHBZHMG-RALIUCGRSA-N pyridine-d5 Chemical compound [2H]C1=NC([2H])=C([2H])C([2H])=C1[2H] JUJWROOIHBZHMG-RALIUCGRSA-N 0.000 description 2
- 238000004451 qualitative analysis Methods 0.000 description 2
- 238000013441 quality evaluation Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 2
- 238000002211 ultraviolet spectrum Methods 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 240000005528 Arctium lappa Species 0.000 description 1
- 235000001553 Betula platyphylla Nutrition 0.000 description 1
- 241001313086 Betula platyphylla Species 0.000 description 1
- 241000001609 Conradina brevifolia Species 0.000 description 1
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- 238000004566 IR spectroscopy Methods 0.000 description 1
- 241000604742 Machilus thunbergii Species 0.000 description 1
- 235000004267 Persea thunbergii Nutrition 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 241000438048 Stenophylla Species 0.000 description 1
- 238000004847 absorption spectroscopy Methods 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 239000002034 butanolic fraction Substances 0.000 description 1
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 150000001793 charged compounds Chemical class 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000005238 degreasing Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000010829 isocratic elution Methods 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- QGBPKJFJAVDUNC-UHFFFAOYSA-N methyl 4-methoxy-3-oxobutanoate Chemical compound COCC(=O)CC(=O)OC QGBPKJFJAVDUNC-UHFFFAOYSA-N 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 229930188006 polyphyllin Natural products 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 238000005464 sample preparation method Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 229930002600 steroidal saponin Natural products 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 238000001195 ultra high performance liquid chromatography Methods 0.000 description 1
- 238000002137 ultrasound extraction Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
- C07J71/0005—Oxygen-containing hetero ring
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Organic Chemistry (AREA)
- Steroid Compounds (AREA)
Abstract
The invention relates to a preparation method and a quality control method of high-purity paris saponin VII, which takes dry shells of Yunnan paris polyphylla, Paris polyphylla or Paris polyphylla of Liliaceae as raw materials, uses ethanol solution for reflux extraction, and establishes an analytical determination method for batch extraction process, purity and content and impurity inspection of a chemical reference substance of the paris saponin VII, thereby establishing the technical standard of the chemical reference substance of the paris saponin VII and providing scientific basis and guarantee for the quality standard research of the chemical reference substance as a traditional Chinese medicine and medicinal materials and preparations. The method has the advantages of reasonable process design, simple process, high separation speed, short production period, high purity of the obtained product, content of more than 98 percent, controllable quality, suitability for industrial production and good application prospect.
Description
Technical Field
The invention relates to the technical field of compound preparation, in particular to a preparation method and a quality control method of high-purity paris saponin VII.
Background
The chemical reference substance is also called as a standard substance and is a real object reference for the quality standard research, quality detection and quality control of traditional Chinese medicines, the research of the chemical reference substance of the traditional Chinese medicines is a very important part of the standardization research of the traditional Chinese medicines, and the chemical reference substance of the traditional Chinese medicines plays an extremely important role in the quality evaluation of products, particularly in the quality control of medicine production and is the basis and the core of the quality control of the traditional Chinese medicines.
Paris saponin VII is a saponin chemical component, is one of active components of plants such as Yunnan Paris polyphylla, Paris polyphylla Smith, etc., and is also an index component for standard quality control of many plants and medicines. Although corresponding national drug standard substances exist at present, systematic research on the paris saponin VII traditional Chinese medicine chemical reference substance is not reported at home and abroad, the paris saponin VII chemical reference substance is researched according to the technical requirements of the traditional Chinese medicine chemical reference substance (for content measurement), and an analytical measurement method for checking the batch extraction process, the purity, the content and the impurities of the paris saponin VII chemical reference substance is established, so that the technical standard of the paris saponin VII chemical reference substance is established, and scientific basis and guarantee are provided for the quality standard research of the paris saponin VII as the traditional Chinese medicine chemical reference substance and medicinal materials and preparations.
Rhizoma paridis saponin VII is prepared from rhizoma paridis Yunnanensis of LiliaceaeParis polyphyllaSmith var. yunnanensis (Franch.) handParis polyphyllaSmith var. chinensis (Franch.) Hara, Paris polyphyllaParis polyphyllaThe active substance obtained by separation in Smith et al is known from the open literature, and the extraction separation process and content determination method of the paris saponin VII mainly comprise the following methods:
1. [ PROBLEM ] study of chemical composition of rhizoma paridis (Huangxian, Gaoyuan, Manshuli, Yanyanjin, Wang Yanli & the chemical composition of rhizoma paridis [ J ]. study of Chinese journal of Chinese medicine, 2009,34(14): 1812-1815): heating and reflux-extracting 2.1 kg of whole plant of rhizoma paridis respectively with 95% and 60% ethanol for 4 times, each for 3 hr, mixing filtrates, recovering ethanol under reduced pressure to obtain extract, adding appropriate amount of water, suspending, and extracting with petroleum ether, ethyl acetate and water saturated n-butanol respectively. Subjecting n-butanol fraction to silica gel column crude separation, gradient eluting with chloroform-methanol system (100: 0, 95: 5, 9: 1, 8: 2: 0.2, 7: 3: 0.3, 6: 4: 0.4, methanol) to obtain 10 components, and subjecting the 7 th component to PTLC to obtain rhizoma paridis saponin VII.
2. [ PROBLEMS ] separation and structural identification of steroid saponin component in Paris polyphylla Smith (Liu shou Bo, Zhanghao, Yongzheng, Xue Dan.) separation and structural identification of steroid saponin component in Paris polyphylla Smith & Waixi J. Pharmacology, 2010,25(05):508 one 511): weighing 5.3 kg of dry rhizome coarse powder of long-drug paris polyphylla, repeatedly percolating and extracting at room temperature by using 70% ethanol (20 times of the amount of medicinal materials in total), merging extracting solutions, concentrating under reduced pressure until no alcohol smell exists, adding a proper amount of water for suspension and dispersion, sequentially extracting by using petroleum ether and ethyl acetate, adding a large amount of water into the residual water layer solution for dilution, passing through macroporous resin D101, sequentially eluting by using 30%, 50%, 70% and 95% ethanol, and respectively recovering the eluent to obtain dry extract. 8 g of 70% elution part was subjected to silica gel column chromatography, and gradient elution was carried out using chloroform-methanol system (6: 1, 5:1, 4:1, 3: 1) to obtain 5 fractions (Fr.7-11). Fr.9 is subjected to chromatography treatment by pre-packed silica gel medium pressure column, gradient elution by chloroform-methanol system (5: 1, 4:1, 3: 1), and purification by Sephadex LH-20 to obtain 73mg of paris saponin VII.
3. [ PROBLEMS ] chemical components of Paris polyphylla (rhizome of Manysantin Seisabellina, Wangying, Li Xue jiao, Li Xia, Huang Lu Qi, Xiaopegen, Gao Wen Yuan.) and their anti-tumor activities [ J ] Chinese herbal medicine, 2017,48(06): 1093-: 3.2 kg of rhizome of paris polyphylla, crushing, and sequentially heating and refluxing with 90% ethanol and 60% ethanol for 3 times and 2 hours each time. Mixing extractive solutions, and recovering ethanol under reduced pressure to obtain extract. And suspending the extract in water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, and recovering solvent to obtain 6.8 g of petroleum ether extract, 38.6 g of ethyl acetate extract and 74.1 g of n-butanol extract. Separating the ethyl acetate extraction part with silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate (10: 0 → 0: 10) to obtain 6 components (Fr. A-F), sequentially passing Fr.C through silica gel column chromatography (dichloromethane-methanol 10: 0 → 5: 5) and gel Sephadex LH-20 column chromatography (dichloromethane-methanol 1: 1), and purifying with preparative liquid phase (70% methanol) to obtain paridis saponin VII (9 mg).
4. [ PROBLEM ] chemical composition of rhizoma paridis Anoectochili Roxburghii (Liuhai, Huangyun, Zhang Ting, Wangqiang, Xiao Qing, chemical composition of rhizoma paridis Anoectochili Roxburghii [ J ] academic newspaper of Chinese pharmacy, 2006(05): 409-: drying rhizome of rhizoma paridis Roxburghii 10 kg, pulverizing, extracting with 5 times of industrial ethanol under reflux for 3 times (each for 3 hr), mixing extractive solutions, concentrating under reduced pressure until no ethanol smell exists, and dissolving in water. And (3) putting the water solution on a macroporous resin D101 column, collecting 40-80% ethanol eluent, and recovering to obtain about 50 g of extract. Subjecting the obtained extract to repeated silica gel column chromatography (chloroform-methanol gradient elution) to obtain parts A and B, and repeatedly separating and purifying parts A and B with Sephadex LH-20 (chloroform-methanol) and ODS (methanol-water) to obtain rhizoma paridis saponin VII (203 mg).
5. [ PROBLEMS ] separation and identification of chemical components of steroid saponins of Paris polyphylla (YIHONGXIANG, XUEDAN, Machilus thunbergii, Chengning, Chenyang, Zhang Hao.) separation and identification of chemical components of steroid saponins of Paris polyphylla [ J ]. university of Sichuan (medical edition), 2008(03): 485-: drying rhizome of Paris polyphylla (dried rhizome of Paris polyphylla Smith) 15 kg, pulverizing, repeatedly percolating with 70% ethanol at room temperature (total 20 times of the amount of the medicinal materials), mixing extractive solutions, concentrating under reduced pressure until no alcohol smell is produced, and adding water for dispersion. Degreasing the water solution with petroleum ether, and sequentially extracting with ethyl acetate and n-butanol. And (3) putting the n-butanol extraction part on a macroporous resin HPD100 column, collecting 40-80% ethanol eluent, and recovering to dry to obtain about 80 g of extract. And purifying the obtained extract by repeated silica gel column chromatography (chloroform-methanol gradient elution) and Sephadex LH-20 (chloroform: methanol (V/V) 1: 1 isocratic elution) to obtain the paris saponin VII (435 mg).
6. [ PROBLEMS ] chemical components of Yunnan rhizoma paridis (Zhangyubo, Wuxia, Li Yalan, Queen could, chemical components of Yunnan rhizoma paridis [ J ]. proceedings of Chinese university (Nature science and medical edition), 2014,35(01): 66-72): dried rhizome of Yunnan rhizoma paridis 10.0 kg, pulverizing, percolating with 40L 70% ethanol, mixing extractive solutions, and concentrating under reduced pressure to obtain extract 1.5 kg. Dispersing the extract with water, passing the supernatant through HP-20 type macroporous adsorbent resin, and sequentially eluting with water and ethanol with volume fractions of 30%, 60% and 90%. Concentrating the elution part of ethanol with the volume fraction of 30% under reduced pressure to obtain 141 g of extract, performing gradient elution on the obtained extract through a silica gel column by using V chloroform to V methanol = 97: 3-1: 1, analyzing and merging the obtained flows through TLC to obtain 7 fractions (Fr.1-7) Fr.4, sequentially passing through a reversed-phase ODS column, Sephadex LH-20 and preparative HPLC, and separating and purifying to obtain the paris saponin VII (18.1 mg).
7. Chinese patent: preparation methods of rhizoma paridis saponin II and rhizoma paridis saponin VII are disclosed in application numbers: 201710587402.X, applicants: kunming plant institute of Chinese academy of sciences, abstract: crushing the Yunnan rhizoma paridis peel, carrying out ultrasonic extraction by using water or 1-99% ethanol, and concentrating an extracting solution to obtain a crude extract. Adding water into the crude extract to prepare suspension, sequentially extracting with ethyl acetate and n-butanol respectively, concentrating the extractive solution to obtain ethyl acetate extraction part and n-butanol extraction part, subjecting the n-butanol extraction part to Sephadex LH-20 gel column chromatography, and eluting with methanol to obtain part rich in paridis saponin II and VII; or subjecting the crude extract to D101 macroporous resin chromatography, eluting with water, 80% ethanol, 95% ethanol and acetone respectively, wherein the 80% ethanol eluate fraction is concentrated to obtain eluate, collecting the 80% ethanol eluate fraction, adding 100mL water to obtain suspension, sequentially extracting with petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether extract fraction, ethyl acetate extract fraction and n-butanol extract fraction, wherein the n-butanol extract fraction is rich in parin saponins II and VII. The part rich in rhizoma paridis saponin II and VII is prepared by preparative HPLC to obtain compound rhizoma paridis saponin VII and rhizoma paridis saponin II.
8. Chinese patent: a preparation process of rhizoma paridis saponin series, application number: 201710982092.1, Applicant: shanghai-sourced leaf Biotechnology, Inc., abstract: pulverizing rhizoma paridis, soaking in ethanol solution, and heating to obtain extractive solution; concentrating the extract to small volume, performing static adsorption by using macroporous resin, then filling the column, eluting by using ethanol solution to remove impurities, and collecting the saponin part of the total weight; dissolving obtained paridis saponin with methanol, preparing with reverse phase C18 medium pressure column, eluting mobile phase with acetonitrile solution, collecting mixed part of paridis saponin VI and VII, separating the mixed part of paridis saponin VI and VII with normal phase silica gel, and eluting with mixed solution of dichloromethane and methanol to obtain purified paridis saponin VI and VII.
9. [ PROBLEMS ] research on measuring the content of 4 types of paridis saponins in Paris polyphylla root tubers by using an HPLC method (Korean Flat, Raney river, Wanghaiming, Duwei, Dingjian, Zhang guan morning. research on measuring the content of 4 types of paridis saponins in Paris polyphylla root tubers by using an HPLC method [ J ]. university of Dalian nationality, 2018,20(01): 17-20): establishing a method for measuring the content of 4 types of rhizoma paridis saponins such as rhizoma paridis saponin VII in rhizoma paridis root tuber by HPLC.
10. [ PROBLEMS ] qualitative and quantitative analysis of main steroid saponin components in Long-column Paris polyphylla (Huangyuan, Kangliping, Penhuasheng, Liu Da, Hao Qingxiu, Zhao Jia Liu, Chenmin, Huang Qi, qualitative and quantitative analysis of main steroid saponin components in Long-column Paris polyphylla [ J ] Chinese traditional medicine journal, 2017,42(18): 3452-: HPLC-UV is established to simultaneously determine the content of 8 types of paris polyphylla saponins such as paris polyphylla saponin VII in 77 long-column paris polyphylla with different sources.
11. [ PROBLEM ] UPLC method simultaneously measures the content of 11 saponins in Yunnan rhizoma paridis cultivated product ( Keke, Gaoygming, Chi ceramic, Liujie, Chuangnong, Zhengjian, Mashuangcheng. UPLC method simultaneously measures the content of 11 saponins in Yunnan rhizoma paridis cultivated product [ J ]. J. Med. 2017,37(09): 1572-1577): and (3) establishing an ultra-high performance liquid chromatography and simultaneously determining the contents of 11 steroidal saponins such as paris polyphylla saponin VII in the paris polyphylla cultivation product.
12. [ PROBLEMS ] measurement and differential analysis of the content of 3 saponin components in different parts of Ypsilandra japonica at different production places (Chengshai, Zhang Mei, Yuan, Rosen, Quga. measurement and differential analysis of the content of 3 saponin components in different parts of Ypsilandra japonica at different production places [ J ] pharmacy China, 2020,31(03):325 + 329): establishing high performance liquid chromatography for measuring and determining contents of rhizoma paridis saponin II, VI and VII in Ypsilandra, and comparing content difference of the 3 kinds of saponin components in different parts of Ypsilandra.
13. [ PROBLEMS ] orthogonal test preferably comprises extraction process research of Paris polyphylla saponin VII in Trillium alba (Liapulodes, Zhang Yao Yu, Zhao Li Chun, Zhang Chun Ge, Liu Yao Yong. orthogonal test preferably comprises extraction process research of Paris polyphylla saponin VII in Trillium alba [ J ] specialty product research, 2019,41(02): 1-5): an orthogonal test method is adopted to investigate the influence of 3 factors of material-liquid ratio, extraction time and extraction temperature on the content of the paris polyphylla saponin VII in the rhizomes of the Trillium albopictus. And (3) determining the content of the paris saponin VII in the trillium baihua by an HPLC method. The optimal extraction process conditions of the paris polyphylla saponin VII in the rhizomes of the white flower trillium are that the material-liquid ratio is 1: 9, the extraction time is 4 h and the extraction temperature is 90 ℃. Under the condition, the content of the paris saponin VII in the rhizomes of the trillium baihuaxianro is measured to be 0.931 mg/g.
The method discusses the extraction and separation method of the paris saponin VII from different angles. The separation purity is higher, but most of the purity does not meet the requirements of the chemical reference substances of the traditional Chinese medicine, namely the purity is more than 98 percent, and the requirements of the high-purity paris saponin VII chemical reference substances cannot be met; the determination of the paris polyphylla saponin VII and the research on the quality control and evaluation of the paris polyphylla saponin VII chemical reference substance have no relevant reports.
In the future, various products, whether in domestic or international markets, need to be developed and improved by high-level quality standards and high-level analysis testing technologies, otherwise the market is lost, the quality standards and detection methods of the products become more and more important, and the 'safe, effective and quality-controllable' medication standards become an international consensus, the drug production should be developed around the center, the core of the drug production is the quality standard control level, and the chemical reference substances play a key role. However, most of the traditional Chinese medicine materials and preparations thereof cannot clarify the chemical substance basis of the action and control the quality because the chemical components are unknown or have no chemical reference substances, and also cannot be accepted by the modern civilization society, and become the key of restriction that Chinese herbal medicines and natural medicines are difficult to enter the international medicine market, and the technical barriers bring difficulties to the development of the traditional Chinese medicine industry, so that the research on the chemical components of the Chinese herbal medicines and the standardized research on the quality standards are necessary ways for the modernization development of the traditional Chinese medicine, and have important significance for clarifying the material basis of the action of the Chinese herbal medicines, the establishment of the production and processing technology of the Chinese herbal medicine preparation, the identification of counterfeit products and the like. The quality standard research of the paris polyphylla saponin VII is carried out, a standardized analysis test method is established, and a detection index and an analysis method for controlling the quality of the paris polyphylla saponin VII with high technical level are formulated, so that the paris polyphylla saponin VII is scientific and standardized, the international competitiveness is enhanced, conditions are created for the traditional Chinese medicine to enter the international market, and the method has great practical significance and academic value.
Disclosure of Invention
The invention aims to provide a preparation method and a quality control method of high-purity paris saponin VII, which solve the problem of a high-purity paris saponin VII chemical reference substance.
The paris polyphylla saponin VII is used as a chemical reference substance of plants, medicinal materials and products thereof, is a technical key for quality control, and a high-purity reference substance is needed by numerous enterprises, scientific research and inspection departments, so that the market demand is high. The invention carries out the preparation of the paris saponin VII traditional Chinese medicine chemical standard substance and the quality control technical research thereof, solves the problem of the high-purity paris saponin VII chemical reference substance, and has great practical significance and academic value.
The invention is realized by the following technical scheme:
a preparation method of high-purity rhizoma paridis saponin VII is characterized in that: the method comprises the following steps:
1) taking dried fruit shells of Yunnan rhizoma paridis, rhizoma paridis or rhizoma paridis, pulverizing, extracting with ethanol under reflux at 75-85 deg.C, filtering the extractive solution, mixing filtrates, and recovering ethanol to obtain extract;
2) suspending the extract with distilled water with the volume of 1-1.5 times, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, standing, combining n-butanol layers, and concentrating to obtain an n-butanol extract;
3) subjecting the n-butanol extract to silica gel column chromatography, gradient eluting with chloroform-methanol system, and collecting fraction containing parinunin VII;
4) detecting the fractions by using a thin-layer chromatography, mixing, and concentrating to obtain crude crystals of the paris saponin VII with the purity of 80-90 percent by weight;
5) separating and purifying the crude crystals of the paris polyphylla saponin VII by high performance preparative liquid chromatography, and collecting the paris polyphylla saponin VII components;
6) and detecting each part of collected eluent by using a high performance liquid chromatography, combining the paris polyphylla saponin VII with the same retention time and the purity of 90-98%, combining the paris polyphylla saponin VII with the same retention time and the purity of more than 98%, and respectively carrying out reduced pressure concentration to obtain the paris polyphylla saponin VII with the purity of 90-98% and the purity of more than 98%.
The process flow of the present invention is shown in FIG. 1.
Specifically, in the step 1), the adding amount of the ethanol is 5-20 times of the weight of the medicinal materials, the concentration of the ethanol is 70-90%, and the reflux extraction times are 3-8 times and 1-3 hours each time.
The gradient elution conditions in the step 3) are as follows: for 0-A minutes, the elution system is chloroform-methanol, and the ratio of the chloroform to the methanol is 100: 1-20: 1; A-B minutes, wherein an elution system is chloroform-methanol, and the ratio of the chloroform-methanol to the methanol is 3: 1-5: 1; the number of the A is 90-160, and the number of the B is 120-200.
The parameters of the thin layer chromatography in the step 4) are as follows:
thin-layer plate: silica gel G;
three developer systems: the system is uniform, the volume ratio of chloroform-methanol-formic acid is 3:1:0.1, the system is two, the volume ratio of n-butyl alcohol-acetic acid-water is 4:1:5 (upper layer solution), the system is three, the volume ratio of chloroform-methanol-water is 65:35:10 (lower layer solution);
sample application: preparing 1 mg/mL solution with methanol, and performing gradient spotting on the same silica gel G plate according to different spotting amounts of 20 μ G, 40 μ G, 60 μ G, 80 μ G and 100 μ G; placing the unfolding cylinders to unfold respectively, wherein the unfolding distance is 15 cm;
positioning: spraying 10% ethanol sulfate solution, air drying, heating at 105 deg.C until the spots are clearly developed, and inspecting in sunlight; as a result, in thin layer chromatography, a single orange-red spot can be seen, three developing solvent systems and 5 gradient sample application with different concentrations are all single spots, and no impurity spot is seen.
The parameters of the high performance liquid preparative chromatography of the step 5) are as follows: the chromatographic column is a C-18 column, acetonitrile-water is used as a mobile phase for elution, and the volume ratio of the chromatographic column to the acetonitrile-water is 55-75: 25-45, the flow rate is 5-10 mL/min, the detection wavelength is 203-210 nm, and the column temperature is 25-35 ℃.
The high performance liquid chromatography of the step 6) is specifically as follows:
the chromatographic column is C-18 with the diameter of 4.6 × 250 mm and the diameter of 5 mu m, the flow rate is 0.8-1.2 ml/min, the sample injection amount is 10-20 mu l, the quantitative determination is carried out by an area normalization method, and the system condition is one of the following three conditions:
the first condition is as follows: the mobile phase is acetonitrile-water, the volume ratio of the acetonitrile-water to the water is 35-40: 60-65, and the detection wavelength is 203 nm;
and a second condition: the mobile phase is methanol-water, the volume ratio of the methanol to the water is 75-80: 20-25, and the detection wavelength is 210 nm;
and (3) carrying out a third condition: the flow is methanol-0.1% phosphoric acid water solution, the volume ratio of the methanol to the phosphoric acid water solution is 60-80: 20-40, and the detection wavelength is 203-205 nm.
Preferably, in the mobile phase of the first system condition, the volume ratio of acetonitrile to water is 38: 62; in the mobile phase under the second system condition, the volume ratio of methanol to water is 75: 25; in the mobile phase of the third system condition, the volume ratio of the methanol to the 0.1% phosphoric acid aqueous solution is 75: 25.
The high-purity rhizoma paridis saponin VII prepared by the method has the purity higher than 98 percent detected by high performance liquid chromatography.
The preparation method of the high-purity paris polyphylla saponin VII is also suitable for extracting the high-purity paris polyphylla saponin VII from other varieties of paris polyphylla. Paris polyphylla, the academic name:Paris polyphyllais a plant of genus Paris of family Liliaceae. The main variants also include: rhizome of Paris polyphylla (C. brevifolia (C.))Paris polyphyllaSm. var. apendicula Hara), rhizoma paridis (rhizoma paridis) with missing petalsParis polyphyllaSm. var. apetala hand. -Mzt.), and Paris polyphylla (C. var. japonica, Paris polyphylla (C. var. lappa hand.), (C. M. Zt.)Paris polyphyllaSm. var. latifolia Wang et Chang, var. nov), Paris polyphylla (Paris polyphylla)Paris polyphyllaSm. stenophylla Franch, paris polyphylla (rhizoma paridis)Paris polyphyllaSm. var, thibetica (Franch.) Hara), rhizoma paridis (rhizoma paridis with wide petals)Paris polyphyllaSm. var. yunnanensis (Franch.) Hand.) and the like.
The invention relates to a Chinese medicinal preparation prepared from Yunnan rhizoma paridis of LiliaceaeParis polyphyllaSmith var. yunnanensis (Franch.) handParis polyphyllaSmith var. chinensis (Franch.) Hara or Paris polyphyllaParis polyphyllaThe paris saponin VII is prepared by extracting, separating, refining and purifying Smith fruit shells, and has the following chemical name, molecular formula and structural formula:
the name of Chinese: rhizoma paridis saponin VII;
the chemical name is pennogenin-3-O- α -L-rhamnopyranosyl (1 → 4) - [ α -L-rhamnopyranosyl (1 → 2) ] - α 0-D-glucoside and pennogenin-3-O- α -L-rhamnopyranosyl (1 → 4) - α -L-rhamnopyranosyl (1 → 4) - [ α -L-rhamnopyranosyl (1 → 2) ] - β -D-glucoside (pennogenin-3-O- α -L-rhamnopyranosyl- (1 → 4) -O- α -L-rhamnopyranosyl- (1 → 4) - [ O- α -L-rhamnopyranosyl- (1 → 2) ] -O- β -D-glucopyranoside)
The name of English: chonglou Saponin VII;
the molecular formula is as follows: c51H82O21；
The structural formula is as follows:
the quality control method of the product of the paris saponin VII comprises the following steps:
firstly, content and purity determination:
precisely weighing a proper amount of reference substance dried at 105 ℃ to constant weight, adding methanol solution to prepare a solution containing 1 mg per 1 ml, injecting 20 mul (about equivalent to 20 mug) of sample under the determination condition, injecting into a liquid chromatograph, respectively recording chromatogram with preferred 3 mobile phase solvent systems until the peak-out retention time of the main component is more than 2.5 times, calculating the content by an area normalization method and the self-contrast of the main component, and determining the content of the reference substance by a result system to be more than 98%. And (4) impurity inspection, wherein in chromatograms recorded in different systems, except for a solvent peak, the sum of the areas of impurity peaks is less than 2.0%. The results are shown in Table 1. The chromatographic purity of the candidate chemical reference substance of the paris saponin VII is 98.69 (n = 3) measured by adopting a self-contrast method, and the total content of impurities is below 2.0 percent.
TABLE 1 quantitative analysis of HPLC (System ①, System ②, System ③) normalized method
Secondly, detecting the peak purity:
taking a proper amount of reference substances, and performing peak purity inspection on a high performance liquid chromatograph by using a diode array DAD detector according to a mobile phase system, wherein the HPLC chromatographic peak of the paris polyphylla saponin VII is more than 98%, the chromatogram is a single peak, and the three-dimensional chromatogram and the 5-point spectrogram are completely overlapped to show that the chromatogram is a single pure substance peak. The results are shown in FIGS. 3, 4 and 5.
The present invention examines the HPLC chromatographic analysis methodology as follows:
chromatographic conditions are as follows: an Agilent ZORBAX SB-C18 chromatographic column is adopted, methanol-0.1% phosphoric acid aqueous solution (75: 25) is taken as a mobile phase, the detection wavelength is 203nm, and the sample injection amount is 20 mu l; the flow rate was 1 mL/min.
First, linear relationship investigation
Taking the candidate chemical reference substance of the paris polyphylla saponin VII, drying at 105 ℃ to constant weight, precisely weighing about 10 mg, placing in a 10mL volumetric flask, adding methanol to dilute to scale, shaking up, and preparing into a reference substance stock solution with the concentration of 998.40 mug/mL. Respectively sucking 1 ml, 1.2 ml, 1.4 ml, 1.6 ml, 1.8 ml and 2.0 ml of reference stock solutions, placing in a 10ml volumetric flask, adding methanol to dilute, shaking, injecting into a liquid chromatograph under the above chromatographic conditions, measuring peak area, drawing a standard curve with the peak area as ordinate and the sample concentration as abscissa, and calculating to obtain a regression equation ofY=3198.0X+64.34，R= 0.9999. The paris saponin VII candidate chemical reference substance has a good linear relation within the sampling amount of 9.98-19.97 mu g.
Second, investigation of reproducibility, stability and precision
Taking 6 parts of the same test sample, respectively measuring according to the method and the chromatographic system, recording the chromatogram and the peak area integral value of the paris polyphylla saponin VII, calculating the content, and indicating that the method has good reproducibility, wherein the average content of the result is 99.83 percent, and the RSD is 0.21 percent.
Taking the same test solution, standing at room temperature for 2, 4, 6, 8, 10, 12 and 24 hours, carrying out sample injection determination according to a method, recording a chromatogram and a peak area integral value of the paris polyphylla saponin VII, wherein the result shows that RSD is 0.082%, and the test solution has good stability within 24 hours.
And continuously feeding the same sample solution for 6 times, recording the chromatogram and the peak area of the paris saponin VII, and indicating that the RSD is 0.54 percent and the precision of the instrument is good.
Third, durability examination of the method
The method adopts 3 chromatographic columns of different manufacturers and brands to respectively measure retention time, theoretical plate number, separation degree and impurity separation effect, under the analysis conditions, the paris polyphylla saponin VII peak and other impurity peaks reach baseline separation, the separation degree is more than 1.5, and when the theoretical plate number is not less than 4000 according to the paris polyphylla saponin VII peak, the measurement requirement can be met, see table 2.
TABLE 2 Retention time, number of theoretical plates and degree of separation
The invention has the beneficial effects that:
1. the compound prepared by the invention is confirmed to be the paris saponin VII by spectrum and spectrum analysis. The invention firstly crushes dried shells of Yunnan rhizoma paridis, rhizoma paridis or rhizoma paridis foliatae of Liliaceae plants, uses ethanol solution for reflux extraction, combines extracting solutions, concentrates, uses petroleum ether, ethyl acetate and n-butanol to extract after an extract is suspended by water, and then stands, combines n-butanol layers and concentrates to obtain an n-butanol extract. Subjecting the n-butanol extract to silica gel column chromatography, gradient eluting with chloroform-methanol, detecting with Thin Layer Chromatography (TLC), collecting eluate containing paridis saponin VII, mixing, and concentrating under reduced pressure to obtain coarse crystals of paridis saponin VII; separating and purifying by using a high performance liquid chromatography (PHPLC) method, and detecting each collected eluent by using High Performance Liquid Chromatography (HPLC) to respectively obtain the paris polyphylla saponin VII with the purity of between 90 and 98 percent and the paris polyphylla saponin VII with the purity of more than 98 percent.
2. The invention has reasonable design and simple process, the paris polyphylla saponin VII with higher purity can be obtained by extracting with an alcohol-water solvent and performing silica gel column chromatography once, and finally the paris polyphylla saponin VII chemical reference substance with the purity of more than 98 percent is prepared by high performance preparative liquid chromatography, and the method is simple and easy to implement.
3. According to the invention, the paris saponin VII chemical reference substance is researched, and the analytical determination method for batch extraction process, purity and content and impurity inspection of the paris saponin VII chemical reference substance is established, so that the technical standard of the paris saponin VII chemical reference substance is established, and scientific basis and guarantee are provided for the quality standard research of the paris saponin VII chemical reference substance as a traditional Chinese medicine chemical reference substance and medicinal materials and preparations. The research result of the invention can provide more complete basic chemical basis for the paris polyphylla saponin VII chemical reference substance, grasp the chemical information and the analysis and test technology thereof, is beneficial to further development and utilization of related products, develops products with high technology and high added value for developing special products in China, improves the market competitiveness and can generate potential and immeasurable social benefit and economic benefit.
4. The invention adopts thin-layer chromatography and high performance liquid chromatography to carry out purity check, content measurement and quality control, thereby ensuring the quality of the product. The method has the advantages of reasonable process design, simple process, high separation speed, short production period, high purity of the obtained product, controllable quality, suitability for industrial production and good application prospect.
5. The invention prepares the paris saponin VII with purity meeting the requirement of a chemical reference substance and content of more than 98 percent from the shells of paris polyphylla, paris polyphylla or paris polyphylla, solves the problem of supplying the paris saponin VII chemical reference substance, and provides scientific basis and guarantee for the quality control of paris polyphylla and other medicines containing paris polyphylla saponin VII components.
Drawings
FIG. 1 is a flow chart of the preparation process of high-purity paris saponin VII;
FIGS. 2-1, 2-2, and 2-3 are thin layer chromatograms of three development systems of Paris saponin VII, respectively; wherein, the developing agent in the figure 2-1 is chloroform-methanol-formic acid with a volume ratio of 3:1:0.1, the developing agent in the figure 2-2 is n-butanol-acetic acid-water with a volume ratio of 4:1:5, and the developing agent in the figure 2-3 is chloroform-methanol-water with a volume ratio of 65:35: 10;
FIG. 3 is a high performance liquid chromatogram of Paris saponin VII;
FIG. 4 is a 3-dimensional spectrogram of high performance liquid chromatography of rhizoma paridis saponin VII;
FIG. 5 is a spectrum of paris saponin VII 5 point;
FIG. 6 is an infrared spectrogram of polyphyllin VII;
FIG. 7 is an ultraviolet absorption spectrum of the paris saponin VII;
FIG. 8 shows Paris saponin VII1H-NMR spectrum;
FIG. 9 shows Paris saponin VII13C-NMR spectrum;
FIGS. 10-1 and 10-2 are mass spectrograms of paris saponin VII.
Detailed Description
In order to make the technical solutions and advantages of the present invention clearer, the following describes the technical solutions of the present invention clearly and completely in combination with the embodiments of the present invention. It should be understood that the examples of the present invention are for illustrative purposes and not intended to limit the present invention. Simple modifications of the invention in accordance with its spirit fall within the scope of the claimed invention. Unless otherwise stated, the percentage of the amount of methanol in the present invention is a volume percentage, and v/v represents a volume ratio of the solution.
Example 1
A preparation method of high-purity rhizoma paridis saponin VII is characterized in that: the method comprises the following steps:
1) taking 5 kg of dried fruit shell of Yunnan rhizoma paridis, pulverizing, extracting with ethanol under reflux at 75 deg.C, filtering the extractive solution, mixing filtrates, and recovering ethanol to obtain extract;
2) suspending the extract with 1.2 times of distilled water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, standing, mixing n-butanol layers, and concentrating to obtain n-butanol extract;
3) subjecting the n-butanol extract to silica gel column chromatography, gradient eluting with chloroform-methanol system, and collecting fraction containing parinunin VII;
4) detecting the fractions by using a thin-layer chromatography, mixing, and concentrating to obtain crude crystals of the paris saponin VII with the purity of 80-90 percent by weight;
5) separating and purifying the crude crystals of the paris polyphylla saponin VII by high performance preparative liquid chromatography, and collecting the paris polyphylla saponin VII components;
6) and detecting each part of collected eluent by using a high performance liquid chromatography, combining the paris polyphylla saponin VII with the same retention time and the purity of 90-98%, combining the paris polyphylla saponin VII with the same retention time and the purity of more than 98%, and respectively carrying out reduced pressure concentration to obtain the paris polyphylla saponin VII with the purity of 95% and the purity of 99.6%.
In the step 1), the adding amount of the ethanol is 12 times of the weight of the medicinal materials, the concentration of the ethanol is 85%, and the reflux extraction times are 5 times, and each time lasts for 1.5 hours.
The gradient elution conditions in the step 3) are as follows: 0-90 minutes, wherein an elution system is chloroform-methanol, and the ratio of the chloroform to the methanol is 100: 1-20: 1; and (3) performing elution for 90-120 minutes, wherein the elution system is chloroform-methanol, and the ratio of the chloroform to the methanol is 5: 1.
The parameters of the thin layer chromatography in the step 4) are as follows:
thin-layer plate: silica gel G;
three developer systems: the system is uniform, the volume ratio of chloroform-methanol-formic acid is 3:1:0.1, the system is two, the volume ratio of n-butyl alcohol-acetic acid-water is 4:1:5 (upper layer solution), the system is three, the volume ratio of chloroform-methanol-water is 65:35:10 (lower layer solution);
sample application: preparing 1 mg/mL solution with methanol, and performing gradient spotting on the same silica gel G plate according to different spotting amounts of 20 μ G, 40 μ G, 60 μ G, 80 μ G and 100 μ G; placing the unfolding cylinders to unfold respectively, wherein the unfolding distance is 15 cm;
positioning: spraying 10% ethanol sulfate solution, air drying, heating at 105 deg.C until the spots are clearly developed, and inspecting in sunlight; as a result, in thin layer chromatography, a single orange-red spot can be seen, three developing solvent systems and 5 gradient sample application with different concentrations are all single spots, and no impurity spot is seen.
The parameters of the high performance liquid preparative chromatography of the step 5) are as follows: the chromatographic column is a C-18 column, acetonitrile-water is used as a mobile phase for elution, the volume ratio of the chromatographic column to the acetonitrile-water is 75:25, the flow rate is 5mL/min, the detection wavelength is 203nm, and the column temperature is 25 ℃.
The high performance liquid chromatography of the step 6) is specifically as follows:
chromatographic column C-18 of 4.6 × 250 mm and 5 μm, flow rate of 1.0 ml/min, sample amount of 20 μ l, quantitative determination by area normalization method, system conditions of methanol-0.1% phosphoric acid aqueous solution flowing, volume ratio of the two at 80:20, and detection wavelength of 203 nm;
quantitative determination by area normalization method can ensure that the peak of the main component (the paris saponin VII) is not less than 98.0 percent, and if an impurity peak exists, the sum of the peak areas of all impurities except the solvent peak is not more than 2.0 percent.
Example 2
A preparation method of high-purity rhizoma paridis saponin VII is characterized in that: the method comprises the following steps:
1) taking 3 kg of dried shells of the Paris polyphylla, crushing, extracting with ethanol under reflux at the extraction temperature of 78 ℃, filtering the extracting solution, combining the filtrates, and recovering the ethanol to obtain an extract;
2) suspending the extract with 1.3 times of distilled water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, standing, mixing n-butanol layers, and concentrating to obtain n-butanol extract;
3) subjecting the n-butanol extract to silica gel column chromatography, gradient eluting with chloroform-methanol system, and collecting fraction containing parinunin VII;
4) detecting the fractions by using a thin-layer chromatography, mixing, and concentrating to obtain crude crystals of the paris saponin VII with the purity of 80-90 percent by weight;
5) separating and purifying the crude crystals of the paris polyphylla saponin VII by high performance preparative liquid chromatography, and collecting the paris polyphylla saponin VII components;
6) and detecting each part of collected eluent by using a high performance liquid chromatography, combining the paris polyphylla saponin VII with the same retention time and the purity of 90-98%, combining the paris polyphylla saponin VII with the same retention time and the purity of more than 98%, and respectively carrying out reduced pressure concentration to obtain the paris polyphylla saponin VII with the purity of 96% and the purity of 98.5%.
In the step 1), the adding amount of the ethanol is 20 times of the weight of the medicinal materials, the concentration of the ethanol is 70%, and the reflux extraction times are 8 times, and each time lasts for 2 hours.
The gradient elution conditions in the step 3) are as follows: 0-120 minutes, wherein an elution system is chloroform-methanol, and the ratio of the chloroform to the methanol is 100: 1-20: 1; and (3) 120-160 minutes, wherein an elution system is chloroform-methanol, and the ratio of the chloroform to the methanol is 4: 1.
The parameters of the thin layer chromatography in the step 4) are as follows:
thin-layer plate: silica gel G;
three developer systems: the system is uniform, the volume ratio of chloroform-methanol-formic acid is 3:1:0.1, the system is two, the volume ratio of n-butyl alcohol-acetic acid-water is 4:1:5 (upper layer solution), the system is three, the volume ratio of chloroform-methanol-water is 65:35:10 (lower layer solution);
sample application: preparing 1 mg/mL solution with methanol, and performing gradient spotting on the same silica gel G plate according to different spotting amounts of 20 μ G, 40 μ G, 60 μ G, 80 μ G and 100 μ G; placing the unfolding cylinders to unfold respectively, wherein the unfolding distance is 15 cm;
positioning: spraying 10% ethanol sulfate solution, air drying, heating at 105 deg.C until the spots are clearly developed, and inspecting in sunlight; as a result, in thin layer chromatography, a single orange-red spot can be seen, three developing solvent systems and 5 gradient sample application with different concentrations are all single spots, and no impurity spot is seen.
The parameters of the high performance liquid preparative chromatography of the step 5) are as follows: the chromatographic column is a C-18 column, acetonitrile-water is used as a mobile phase for elution, the volume ratio of the chromatographic column to the acetonitrile-water is 70:30, the flow rate is 7 mL/min, the detection wavelength is 205nm, and the column temperature is 30 ℃.
The high performance liquid chromatography of the step 6) is specifically as follows:
chromatographic column C-18 of 4.6 × 250 mm and 5 μm, flow rate of 1.1 ml/min, sample amount of 15 μ l, quantitative determination by area normalization method, system conditions that the mobile phase is acetonitrile-water, the volume ratio of the two is 38:62, and the detection wavelength is 203 nm;
quantitative determination by area normalization method can ensure that the peak of the main component (the paris saponin VII) is not less than 98.0 percent, and if an impurity peak exists, the sum of the peak areas of all impurities except the solvent peak is not more than 2.0 percent.
Example 3
A preparation method of high-purity rhizoma paridis saponin VII is characterized in that: the method comprises the following steps:
1) taking 5 kg of dried fruit shell of Yunnan rhizoma paridis, pulverizing, extracting with ethanol under reflux at 80 deg.C, filtering the extractive solution, mixing filtrates, and recovering ethanol to obtain extract;
2) suspending the extract with distilled water of 1 time volume, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, standing, mixing n-butanol layers, and concentrating to obtain n-butanol extract;
3) subjecting the n-butanol extract to silica gel column chromatography, gradient eluting with chloroform-methanol system, and collecting fraction containing parinunin VII;
4) detecting the fractions by using a thin-layer chromatography, mixing, and concentrating to obtain crude crystals of the paris saponin VII with the purity of 80-90 percent by weight;
5) separating and purifying the crude crystals of the paris polyphylla saponin VII by high performance preparative liquid chromatography, and collecting the paris polyphylla saponin VII components;
6) and detecting each part of collected eluent by using a high performance liquid chromatography, combining the paris polyphylla saponin VII with the same retention time and the purity of 90-98%, combining the paris polyphylla saponin VII with the same retention time and the purity of more than 98%, and respectively carrying out reduced pressure concentration to obtain the paris polyphylla saponin VII with the purity of 97% and the purity of 99.8%.
In the step 1), the adding amount of the ethanol is 10 times of the weight of the medicinal materials, the concentration of the ethanol is 75%, and the reflux extraction times are 3 times, and each time lasts for 3 hours.
The gradient elution conditions in the step 3) are as follows: 0-100 minutes, wherein an elution system is chloroform-methanol, and the ratio of the chloroform to the methanol is 100: 1-20: 1; and (3) 100-140 minutes, wherein the elution system is chloroform-methanol, and the ratio of the chloroform to the methanol is 5: 1.
The parameters of the thin layer chromatography in the step 4) are as follows:
thin-layer plate: silica gel G;
three developer systems: the system is uniform, the volume ratio of chloroform-methanol-formic acid is 3:1:0.1, the system is two, the volume ratio of n-butyl alcohol-acetic acid-water is 4:1:5 (upper layer solution), the system is three, the volume ratio of chloroform-methanol-water is 65:35:10 (lower layer solution);
sample application: preparing 1 mg/mL solution with methanol, and performing gradient spotting on the same silica gel G plate according to different spotting amounts of 20 μ G, 40 μ G, 60 μ G, 80 μ G and 100 μ G; placing the unfolding cylinders to unfold respectively, wherein the unfolding distance is 15 cm;
positioning: spraying 10% ethanol sulfate solution, air drying, heating at 105 deg.C until the spots are clearly developed, and inspecting in sunlight; as a result, in thin layer chromatography, a single orange-red spot can be seen, three developing solvent systems and 5 gradient sample application with different concentrations are all single spots, and no impurity spot is seen.
The parameters of the high performance liquid preparative chromatography of the step 5) are as follows: the chromatographic column is a C-18 column, acetonitrile-water is used as a mobile phase for elution, the volume ratio of the chromatographic column to the water is 60:40, the flow rate is 8mL/min, the detection wavelength is 203nm, and the column temperature is 25 ℃.
The high performance liquid chromatography of the step 6) is specifically as follows:
chromatographic column C-18 of 4.6 × 250 mm and 5 μm, flow rate of 1.2 ml/min, sample amount of 15 μ l, and quantitative determination by area normalization method, wherein the system conditions comprise that the mobile phase is methanol-water, the volume ratio of the two is 75:25, and the detection wavelength is 210 nm;
quantitative determination by area normalization method can ensure that the peak of the main component (the paris saponin VII) is not less than 98.0 percent, and if an impurity peak exists, the sum of the peak areas of all impurities except the solvent peak is not more than 2.0 percent.
Example 4
A preparation method of high-purity rhizoma paridis saponin VII is characterized in that: the method comprises the following steps:
1) taking 6 kg of dried fruit shell of Paris polyphylla, pulverizing, extracting with ethanol under reflux at 78 deg.C, filtering the extractive solution, mixing filtrates, and recovering ethanol to obtain extract;
2) suspending the extract with 1.1 times of distilled water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, standing, mixing n-butanol layers, and concentrating to obtain n-butanol extract;
3) subjecting the n-butanol extract to silica gel column chromatography, gradient eluting with chloroform-methanol system, and collecting fraction containing parinunin VII;
4) detecting the fractions by using a thin-layer chromatography, mixing, and concentrating to obtain crude crystals of the paris saponin VII with the purity of 80-90 percent by weight;
5) separating and purifying the crude crystals of the paris polyphylla saponin VII by high performance preparative liquid chromatography, and collecting the paris polyphylla saponin VII components;
6) and detecting each part of collected eluent by using a high performance liquid chromatography, combining the paris polyphylla saponin VII with the same retention time and the purity of 90-98%, combining the paris polyphylla saponin VII with the same retention time and the purity of more than 98%, and respectively concentrating under reduced pressure to obtain the paris polyphylla saponin VII with the purity of 94% and the purity of 98.2%.
In the step 1), the adding amount of the ethanol is 15 times of the weight of the medicinal materials, the concentration of the ethanol is 80%, and the reflux extraction times are 6 times and 2 hours each time.
The gradient elution conditions in the step 3) are as follows: 0-160 minutes, wherein an elution system is chloroform-methanol, and the ratio of the chloroform to the methanol is 100: 1-20: 1; 160-200 minutes, and the elution system is chloroform-methanol, and the ratio of the chloroform to the methanol is 3: 1.
The parameters of the thin layer chromatography in the step 4) are as follows:
thin-layer plate: silica gel G;
three developer systems: the system is uniform, the volume ratio of chloroform-methanol-formic acid is 3:1:0.1, the system is two, the volume ratio of n-butyl alcohol-acetic acid-water is 4:1:5 (upper layer solution), the system is three, the volume ratio of chloroform-methanol-water is 65:35:10 (lower layer solution);
sample application: preparing 1 mg/mL solution with methanol, and performing gradient spotting on the same silica gel G plate according to different spotting amounts of 20 μ G, 40 μ G, 60 μ G, 80 μ G and 100 μ G; placing the unfolding cylinders to unfold respectively, wherein the unfolding distance is 15 cm;
positioning: spraying 10% ethanol sulfate solution, air drying, heating at 105 deg.C until the spots are clearly developed, and inspecting in sunlight; as a result, in thin layer chromatography, a single orange-red spot can be seen, three developing solvent systems and 5 gradient sample application with different concentrations are all single spots, and no impurity spot is seen.
The parameters of the high performance liquid preparative chromatography of the step 5) are as follows: the chromatographic column is a C-18 column, acetonitrile-water is used as a mobile phase for elution, the volume ratio of the chromatographic column to the acetonitrile-water is 55:45, the flow rate is 10 mL/min, the detection wavelength is 210 nm, and the column temperature is 30 ℃.
The high performance liquid chromatography of the step 6) is specifically as follows:
chromatographic column C-18 of 4.6 × 250 mm and 5 μm, flow rate of 0.8 ml/min, sample amount of 20 μ l, and quantitative determination by area normalization method, wherein the system conditions comprise that the mobile phase is methanol-water with volume ratio of 80:20, and the detection wavelength is 210 nm;
quantitative determination by area normalization method can ensure that the peak of the main component (the paris saponin VII) is not less than 98.0 percent, and if an impurity peak exists, the sum of the peak areas of all impurities except the solvent peak is not more than 2.0 percent.
Example 5
A preparation method of high-purity rhizoma paridis saponin VII is characterized in that: the method comprises the following steps:
1) pulverizing dried fruit shell of Paris polyphylla (dried fruit shell of Paris polyphylla Smith) Smith, extracting with ethanol under reflux at 77 deg.C, filtering the extractive solution, mixing filtrates, and recovering ethanol to obtain extract;
2) suspending the extract with 1.2 times of distilled water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, standing, mixing n-butanol layers, and concentrating to obtain n-butanol extract;
3) subjecting the n-butanol extract to silica gel column chromatography, gradient eluting with chloroform-methanol system, and collecting fraction containing parinunin VII;
4) detecting the fractions by using a thin-layer chromatography, mixing, and concentrating to obtain crude crystals of the paris saponin VII with the purity of 80-90 percent by weight;
5) separating and purifying the crude crystals of the paris polyphylla saponin VII by high performance preparative liquid chromatography, and collecting the paris polyphylla saponin VII components;
6) and detecting each part of collected eluent by using a high performance liquid chromatography, combining the paris polyphylla saponin VII with the same retention time and the purity of 90-98%, combining the paris polyphylla saponin VII with the same retention time and the purity of more than 98%, and respectively carrying out reduced pressure concentration to obtain the paris polyphylla saponin VII with the purity of 95% and the purity of 98.3%.
In the step 1), the adding amount of the ethanol is 8 times of the weight of the medicinal materials, the concentration of the ethanol is 80%, and the reflux extraction times are 4 times, and each time lasts for 2.5 hours.
The gradient elution conditions in the step 3) are as follows: 0-110 minutes, wherein an elution system is chloroform-methanol, and the ratio of the chloroform to the methanol is 100: 1-20: 1; and (3) performing elution for 110-140 minutes, wherein the elution system is chloroform-methanol, and the ratio of the chloroform to the methanol is 5: 1.
The parameters of the thin layer chromatography in the step 4) are as follows:
thin-layer plate: silica gel G;
three developer systems: the system is uniform, the volume ratio of chloroform-methanol-formic acid is 3:1:0.1, the system is two, the volume ratio of n-butyl alcohol-acetic acid-water is 4:1:5 (upper layer solution), the system is three, the volume ratio of chloroform-methanol-water is 65:35:10 (lower layer solution);
sample application: preparing 1 mg/mL solution with methanol, and performing gradient spotting on the same silica gel G plate according to different spotting amounts of 20 μ G, 40 μ G, 60 μ G, 80 μ G and 100 μ G; placing the unfolding cylinders to unfold respectively, wherein the unfolding distance is 15 cm;
positioning: spraying 10% ethanol sulfate solution, air drying, heating at 105 deg.C until the spots are clearly developed, and inspecting in sunlight; as a result, in thin layer chromatography, a single orange-red spot can be seen, three developing solvent systems and 5 gradient sample application with different concentrations are all single spots, and no impurity spot is seen.
The parameters of the high performance liquid preparative chromatography of the step 5) are as follows: the chromatographic column is a C-18 column, acetonitrile-water is used as a mobile phase for elution, the volume ratio of the chromatographic column to the acetonitrile-water is 75:25, the flow rate is 5mL/min, the detection wavelength is 205nm, and the column temperature is 35 ℃.
The high performance liquid chromatography of the step 6) is specifically as follows:
chromatographic column C-18 of 4.6 × 250 mm and 5 μm, flow rate of 1.1 ml/min, sample amount of 10 μ l, and quantitative determination by area normalization method, wherein the system conditions comprise flowing methanol-0.1% phosphoric acid aqueous solution, volume ratio of the two is 75:25, and detection wavelength is 203 nm;
quantitative determination by area normalization method can ensure that the peak of the main component (the paris saponin VII) is not less than 98.0 percent, and if an impurity peak exists, the sum of the peak areas of all impurities except the solvent peak is not more than 2.0 percent.
Example 6
A preparation method of high-purity rhizoma paridis saponin VII is characterized in that: the method comprises the following steps:
1) taking 2 kg of dried fruit shell of Paris polyphylla, pulverizing, extracting with ethanol under reflux at 76 deg.C, filtering the extractive solution, mixing filtrates, and recovering ethanol to obtain extract;
2) suspending the extract with 1.5 times of distilled water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, standing, mixing n-butanol layers, and concentrating to obtain n-butanol extract;
3) subjecting the n-butanol extract to silica gel column chromatography, gradient eluting with chloroform-methanol system, and collecting fraction containing parinunin VII;
4) detecting the fractions by using a thin-layer chromatography, mixing, and concentrating to obtain crude crystals of the paris saponin VII with the purity of 80-90 percent by weight;
5) separating and purifying the crude crystals of the paris polyphylla saponin VII by high performance preparative liquid chromatography, and collecting the paris polyphylla saponin VII components;
6) and detecting each part of collected eluent by using a high performance liquid chromatography, combining the paris polyphylla saponin VII with the same retention time and the purity of 90-98%, combining the paris polyphylla saponin VII with the same retention time and the purity of more than 98%, and respectively carrying out reduced pressure concentration to obtain the paris polyphylla saponin VII with the purity of 96% and the purity of 99.1%.
In the step 1), the adding amount of the ethanol is 18 times of the weight of the medicinal materials, the concentration of the ethanol is 75%, and the reflux extraction times are 7 times and 2 hours each time.
The gradient elution conditions in the step 3) are as follows: 0-150 minutes, wherein an elution system is chloroform-methanol, and the ratio of the chloroform to the methanol is 100: 1-20: 1; 150-180 minutes, and the elution system is chloroform-methanol, and the ratio of the chloroform to the methanol is 4: 1.
The parameters of the thin layer chromatography in the step 4) are as follows:
thin-layer plate: silica gel G;
three developer systems: the system is uniform, the volume ratio of chloroform-methanol-formic acid is 3:1:0.1, the system is two, the volume ratio of n-butyl alcohol-acetic acid-water is 4:1:5 (upper layer solution), the system is three, the volume ratio of chloroform-methanol-water is 65:35:10 (lower layer solution);
sample application: preparing 1 mg/mL solution with methanol, and performing gradient spotting on the same silica gel G plate according to different spotting amounts of 20 μ G, 40 μ G, 60 μ G, 80 μ G and 100 μ G; placing the unfolding cylinders to unfold respectively, wherein the unfolding distance is 15 cm;
positioning: spraying 10% ethanol sulfate solution, air drying, heating at 105 deg.C until the spots are clearly developed, and inspecting in sunlight; as a result, in thin layer chromatography, a single orange-red spot can be seen, three developing solvent systems and 5 gradient sample application with different concentrations are all single spots, and no impurity spot is seen.
The parameters of the high performance liquid preparative chromatography of the step 5) are as follows: the chromatographic column is a C-18 column, acetonitrile-water is used as a mobile phase for elution, the volume ratio of the chromatographic column to the acetonitrile-water is 70:30, the flow rate is 8mL/min, the detection wavelength is 210 nm, and the column temperature is 30 ℃.
The high performance liquid chromatography of the step 6) is specifically as follows:
a chromatographic column with the diameter of 5 mu m and the diameter of C-18 of 4.6 × 250 mm, the flow rate of 1.2 ml/min, the sample injection quantity of 20 mu l, the quantitative determination by an area normalization method, and the system conditions that the flowing is methanol-0.1 percent phosphoric acid aqueous solution, the volume ratio of the methanol to the phosphoric acid aqueous solution is 60:40, and the detection wavelength is 205 nm;
quantitative determination by area normalization method can ensure that the peak of the main component (the paris saponin VII) is not less than 98.0 percent, and if an impurity peak exists, the sum of the peak areas of all impurities except the solvent peak is not more than 2.0 percent.
Example 7
A preparation method of high-purity rhizoma paridis saponin VII is characterized in that: the method comprises the following steps:
1) taking 5 kg of dried fruit shell of paris polyphylla, crushing, extracting with ethanol under reflux at the extraction temperature of 78 ℃, filtering an extracting solution, combining filtrates, and recovering ethanol to obtain an extract;
2) suspending the extract with 1.5 times of distilled water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, standing, mixing n-butanol layers, and concentrating to obtain n-butanol extract;
3) subjecting the n-butanol extract to silica gel column chromatography, gradient eluting with chloroform-methanol system, and collecting fraction containing parinunin VII;
4) detecting the fractions by using a thin-layer chromatography, mixing, and concentrating to obtain crude crystals of the paris saponin VII with the purity of 80-90 percent by weight;
5) separating and purifying the crude crystals of the paris polyphylla saponin VII by high performance preparative liquid chromatography, and collecting the paris polyphylla saponin VII components;
6) and detecting each part of collected eluent by using a high performance liquid chromatography, combining the paris polyphylla saponin VII with the same retention time and the purity of 90-98%, combining the paris polyphylla saponin VII with the same retention time and the purity of more than 98%, and respectively carrying out reduced pressure concentration to obtain the paris polyphylla saponin VII with the purity of 92% and the purity of 98.1%.
In the step 1), the adding amount of the ethanol is 12 times of the weight of the medicinal materials, the concentration of the ethanol is 85%, and the reflux extraction times are 5 times, and each time lasts for 1.5 hours.
The gradient elution conditions in the step 3) are as follows: 0-160 minutes, wherein an elution system is chloroform-methanol, and the ratio of the chloroform to the methanol is 100: 1-20: 1; 160-200 minutes, and the elution system is chloroform-methanol, and the ratio of the chloroform to the methanol is 3: 1.
The parameters of the thin layer chromatography in the step 4) are as follows:
thin-layer plate: silica gel G;
three developer systems: the system is uniform, the volume ratio of chloroform-methanol-formic acid is 3:1:0.1, the system is two, the volume ratio of n-butyl alcohol-acetic acid-water is 4:1:5 (upper layer solution), the system is three, the volume ratio of chloroform-methanol-water is 65:35:10 (lower layer solution);
sample application: preparing 1 mg/mL solution with methanol, and performing gradient spotting on the same silica gel G plate according to different spotting amounts of 20 μ G, 40 μ G, 60 μ G, 80 μ G and 100 μ G; placing the unfolding cylinders to unfold respectively, wherein the unfolding distance is 15 cm;
positioning: spraying 10% ethanol sulfate solution, air drying, heating at 105 deg.C until the spots are clearly developed, and inspecting in sunlight; as a result, in thin layer chromatography, a single orange-red spot can be seen, three developing solvent systems and 5 gradient sample application with different concentrations are all single spots, and no impurity spot is seen.
The parameters of the high performance liquid preparative chromatography of the step 5) are as follows: the chromatographic column is a C-18 column, acetonitrile-water is used as a mobile phase for elution, and the volume ratio of the chromatographic column to the acetonitrile-water is 65:35, the flow rate is 8mL/min, the detection wavelength is 205nm, and the column temperature is 30 ℃.
The high performance liquid chromatography of the step 6) is specifically as follows:
chromatographic column C-18 of 4.6 × 250 mm and 5 μm, flow rate of 1.0 ml/min, sample amount of 20 μ l, and quantitative determination by area normalization method, wherein the system conditions comprise flowing methanol-0.1% phosphoric acid aqueous solution, volume ratio of the two is 75:25, and detection wavelength is 203 nm;
quantitative determination by area normalization method can ensure that the peak of the main component (the paris saponin VII) is not less than 98.0 percent, and if an impurity peak exists, the sum of the peak areas of all impurities except the solvent peak is not more than 2.0 percent.
A sample of the products prepared in examples 1-7 was subjected to the following structural confirmation:
physical and chemical constants: the paris polyphylla saponin VII is powdered toner, has a melting point of 260-262 ℃, and is easily soluble in methanol and chloroform.
The spectral data were identified as follows:
1. infrared absorption Spectrum (IR)
The instrument comprises the following steps: bruker tesser 27 FTIR; the IR spectrum of the polystyrene film is corrected and detected by an instrument, and accords with the regulation of the 2015 edition of Chinese pharmacopoeia;
the sample preparation method comprises the following steps: taking a proper amount of samples, and tabletting potassium bromide;
the measurement data are shown in Table 3, and the measured infrared absorption spectrum is shown in FIG. 6.
TABLE 3 Paris saponin VII Infrared Spectroscopy data
And (3) analysis:
3423 cm-1: -stretching vibrations of OH; 1130. 1053, 979 and 892 cm-1: side chains of 25D-spirostanes.
2. Ultraviolet absorption Spectroscopy (UV)
The instrument comprises the following steps: japanese Shimadzu UV-2550 ultraviolet spectrometer;
the instrument is corrected and calibrated to meet the regulation of 2015 edition of Chinese pharmacopoeia;
solvent: analytically pure methanol;
test solution preparation: taking a proper amount of samples, and adding methanol to prepare a solution containing 50 mu g of methanol per 1 mL;
the measurement data are shown in Table 4, and the measured ultraviolet absorption spectrum is shown in FIG. 7.
TABLE 4 Paris saponin VII UV data
| Solvent(s) | Maximum absorption |
| Methanol | End absorption |
And (3) analysis:
the ultraviolet spectrum is end absorption, and the structure does not contain an unsaturated conjugated system from an ultraviolet spectrogram.
3. Nuclear magnetic resonance spectroscopy
（1）1H-NMR nuclear magnetic resonance spectrum
The instrument comprises the following steps: BRUKER Dre-500, Germany;
solvent: pyridine-d 5TMS is internal standard;
measurement data shown in Table 51The H-NMR spectrum is shown in FIG. 8.
TABLE 51H-NMR nuclear magnetic resonance spectrum data table
（2）13C-NMR nuclear magnetic resonance spectrum
The instrument comprises the following steps: BRUKER 125 MHz, Germany;
solvent: pyridine-d 5TMS is internal standard;
measured data are shown in Table 613The C-NMR spectrum is shown in FIG. 9.
TABLE 613C-NMR nuclear magnetic resonance spectrum data
| Position of | Chemical shift (ppm) | |
| 1 | 37.9 | 37.9 |
| 2 | 30.5 | 30.5 |
| 3 | 78.1 | 78.1 |
| 4 | 39.3 | 39.3 |
| 5 | 141.1 | 141.1 |
| 6 | 122.2 | 122.2 |
| 7 | 32.8 | 32.8 |
| 8 | 30.8 | 30.8 |
| 9 | 50.6 | 50.6 |
| 10 | 37.5 | 37.5 |
| 11 | 21.3 | 21.3 |
| 12 | 32.7 | 32.4 |
| 13 | 45.5 | 45.5 |
| 14 | 53.4 | 53.4 |
| 15 | 32.5 | 32.7 |
| 16 | 90.4 | 90.3 |
| 17 | 90.5 | 90.5 |
| 18 | 17.5 | 17.5 |
| 19 | 19.8 | 19.8 |
| 20 | 45.1 | 45.1 |
| 21 | 10.2 | 10.2 |
| 22 | 110.2 | 110.2 |
| 23 | 32.2 | 32.2 |
| 24 | 29.2 | 29.1 |
| 25 | 30.5 | 30.5 |
| 26 | 67.1 | 67.0 |
| 27 | 17.7 | 17.7 |
| 3- |
||
| 1 | 100.7 | 100.6 |
| 2 | 78.4 | 78.3 |
| 3 | 78.0 | 77.9 |
| 4 | 78.3 | 78.3 |
| 5 | 77.4 | 77.3 |
| 6 | 61.5 | 61.5 |
| 4-Rha′′ | ||
| 1 | 102.5 | 102.5 |
| 2 | 72.9 | 72.9 |
| 3 | 73.3 | 73.4 |
| 4 | 74.5 | 74.5 |
| 5 | 69.9 | 69.9 |
| 6 | 19.3 | 19.0 |
| 4′-Rha′′′ | ||
| 1 | 103.7 | 103.7 |
| 2 | 73.7 | 73.7 |
| 3 | 73.2 | 73.3 |
| 4 | 80.8 | 80.8 |
| 5 | 68.7 | 68.6 |
| 6 | 19.0 | 19.2 |
| 2-Glc′′′′ | ||
| 1 | 102.6 | 102.5 |
| 2 | 73.0 | 73.0 |
| 3 | 73.2 | 73.2 |
| 4 | 74.4 | 74.4 |
| 5 | 70.8 | 70.8 |
| 6 | 18.8 | 18.8 |
4. Mass Spectrum (MS)
The instrument comprises the following steps: thermo QE FOCUS liquid chromatography-mass spectrometry
And (3) testing conditions are as follows: mobile phase methanol-0.1% aqueous formic acid, 75: 25; an ionization mode HESI source; a negative ion mode; ionization energy (N) CE 30 eV; Full-MS resolution 70000, dd-MS2Resolution 17500. The measurement data are shown in Table 7, and the mass spectrum is shown in FIG. 10.
TABLE 7 ESI/MS Mass Spectrometry data
And (3) measuring results: sample excimer ion peakm/z1075.53442[M+CHOOH-H]-The MS mass number of the compound is 1030, and the molecular formula of the compound with the paris saponin VII is C51H82O21And (6) matching.
The main cleavage pathways: ion M/z 1029.52734[ M-H ] of sample]-The fragment ion has M/z 883.47095 of [ M-H-146]-737.41284 is [ M-H-146 × 2 ]]-Are respectively lost1 molecule and 2 molecules of rhamnosyl C6H10O4 -Fragment ion of (2), i.e. C45H72O17、C39H62O13The structure of the compound accords with that of the paris polyphylla saponin VII.
The molecular weight of the sample is 1030 and the molecular formula C is synthesized by analyzing each MS data51H82O21The calculated unsaturation degree is omega = (2 × 51+ 2-82)/2 =11, which accords with the structure of the product.
And (4) conclusion: the peak of HR-ESI/MS molecular ion is 1029.52734[ M-H ] measured by mass spectrum]-The molecular weight is 1030, and the structural unit contains C = C, OH and CH3-, C-O, benzene ring. This is demonstrated by IR, UV, NMR, MS, and the mass spectrum characteristic fragment ions also demonstrate their chemical structure.
And (3) combining all the spectral data, wherein all the physical and chemical constants and the spectral data are consistent with the structure of the paris polyphylla saponin VII compound and basically consistent with literature values, and determining the paris polyphylla saponin VII.
As a result: the chemical reference substance of the paris polyphylla saponin VII which is separated and purified by the invention is subjected to infrared spectrum, ultraviolet spectrum, nuclear magnetic resonance, mass spectrum and physical and chemical detection to confirm the chemical structure. Detection by TLC of 5 different concentrations in 3 development systems; 3 mobile phase systems and 3 HPLC detections with different wavelengths, the result meets the requirements of chemical reference substances for traditional Chinese medicine content determination, and the content is more than 98%.
Claims (9)
1. A preparation method of high-purity rhizoma paridis saponin VII is characterized in that: the method comprises the following steps:
1) pulverizing dried fruit shell of rhizoma paridis Yunnanensis, rhizoma paridis, or rhizoma paridis, reflux-extracting with ethanol, filtering extractive solution, mixing filtrates, and recovering ethanol to obtain extract;
2) suspending the extract with distilled water with the volume of 1-1.5 times, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, standing, combining n-butanol layers, and concentrating to obtain an n-butanol extract;
3) subjecting the n-butanol extract to silica gel column chromatography, gradient eluting with chloroform-methanol system, and collecting fraction containing parinunin VII;
4) detecting the fractions by using a thin-layer chromatography, mixing, and concentrating to obtain crude crystals of the paris saponin VII with the purity of 80-90 percent by weight;
5) separating and purifying the crude crystals of the paris polyphylla saponin VII by high performance preparative liquid chromatography, and collecting the paris polyphylla saponin VII components;
6) and detecting each part of collected eluent by using a high performance liquid chromatography, combining the paris polyphylla saponin VII with the same retention time and the purity of 90-98%, combining the paris polyphylla saponin VII with the same retention time and the purity of more than 98%, and respectively carrying out reduced pressure concentration to obtain the paris polyphylla saponin VII with the purity of 90-98% and the purity of more than 98%.
2. The process for producing a high-purity Paris saponin VII according to claim 1, wherein: in the step 1), the adding amount of the ethanol is 5-20 times of the weight of the medicinal materials, the concentration of the ethanol is 70-90%, and the reflux extraction times are 3-8 times and 1-3 hours each time.
3. The process for producing a high-purity Paris saponin VII according to claim 1, wherein: the gradient elution conditions in the step 3) are as follows: for 0-A minutes, the elution system is chloroform-methanol, and the ratio of the chloroform to the methanol is 100: 1-20: 1; A-B minutes, wherein an elution system is chloroform-methanol, and the ratio of the chloroform-methanol to the methanol is 5: 1-3: 1; the number of the A is 90-160, and the number of the B is 120-200.
4. The process for producing a high-purity Paris saponin VII according to claim 1, wherein: the parameters of the thin layer chromatography in the step 4) are as follows:
thin-layer plate: silica gel G;
three developer systems: the system is uniform, the volume ratio of chloroform-methanol-formic acid is 3:1:0.1, the system is two, the volume ratio of n-butyl alcohol-acetic acid-water is 4:1:5 (upper layer solution), the system is three, the volume ratio of chloroform-methanol-water is 65:35:10 (lower layer solution);
sample application: preparing 1 mg/mL solution with methanol, and performing gradient spotting on the same silica gel G plate according to different spotting amounts of 20 μ G, 40 μ G, 60 μ G, 80 μ G and 100 μ G; placing the unfolding cylinders to unfold respectively, wherein the unfolding distance is 15 cm;
positioning: spraying 10% ethanol sulfate solution, air drying, heating at 105 deg.C until the spots are clearly developed, and inspecting in sunlight; as a result, in thin layer chromatography, a single orange-red spot can be seen, three developing solvent systems and 5 gradient sample application with different concentrations are all single spots, and no impurity spot is seen.
5. The process for producing a high-purity Paris saponin VII according to claim 1, wherein: the parameters of the high performance liquid preparative chromatography of the step 5) are as follows: the chromatographic column is a C-18 column, acetonitrile-water is used as a mobile phase for elution, and the volume ratio of the chromatographic column to the acetonitrile-water is 55-75: 25-45, the flow rate is 5-10 mL/min, the detection wavelength is 203-210 nm, and the column temperature is 25-35 ℃.
6. The process for producing a high-purity Paris saponin VII according to claim 1, wherein: the high performance liquid chromatography of the step 6) is specifically as follows:
the chromatographic column is C-18 with the diameter of 4.6 × 250 mm and the diameter of 5 mu m, the flow rate is 0.8-1.2 ml/min, the sample injection amount is 10-20 mu l, the quantitative determination is carried out by an area normalization method, and the system condition is one of the following three conditions:
the first condition is as follows: the mobile phase is acetonitrile-water, the volume ratio of the acetonitrile-water to the water is 35-40: 60-65, and the detection wavelength is 203 nm;
and a second condition: the mobile phase is methanol-water, the volume ratio of the methanol to the water is 75-80: 20-25, and the detection wavelength is 210 nm;
and (3) carrying out a third condition: the flow is methanol-0.1% phosphoric acid water solution, the volume ratio of the methanol to the phosphoric acid water solution is 60-80: 20-40, and the detection wavelength is 203-205 nm.
7. The process for producing a high-purity Paris saponin VII according to claim 6, wherein: in the mobile phase under the third system condition, the volume ratio of the methanol to the 0.1% phosphoric acid aqueous solution is 60-80: 20-40.
8. The process for producing a high-purity Paris saponin VII according to claim 6, wherein: the obtained high-purity rhizoma paridis saponin VII has a purity of more than 98% detected by high performance liquid chromatography.
9. The process for producing high-purity Paris saponin VII according to claim 1, which is used for extracting high-purity Paris saponin VII from other varieties of Paris polyphylla.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010345991.2A CN111349140B (en) | 2020-04-27 | 2020-04-27 | Preparation method and quality control method of high-purity rhizoma paridis saponin VII |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010345991.2A CN111349140B (en) | 2020-04-27 | 2020-04-27 | Preparation method and quality control method of high-purity rhizoma paridis saponin VII |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111349140A true CN111349140A (en) | 2020-06-30 |
| CN111349140B CN111349140B (en) | 2022-12-27 |
Family
ID=71191588
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010345991.2A Active CN111349140B (en) | 2020-04-27 | 2020-04-27 | Preparation method and quality control method of high-purity rhizoma paridis saponin VII |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111349140B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114668773A (en) * | 2022-04-08 | 2022-06-28 | 山东中医药大学 | Application of rhizoma paridis extract in resisting Climiya Congo hemorrhagic fever virus |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106353417A (en) * | 2016-08-14 | 2017-01-25 | 西南民族大学 | UPLC (ultra-performance liquid chromatography) detection method of multiple steroid saponins in Yunnan rhizoma paridis or its polygerm strain |
| CN107312061A (en) * | 2017-07-18 | 2017-11-03 | 中国科学院昆明植物研究所 | Chonglou saponin II and chonglou saponin VII preparation method |
| CN107686502A (en) * | 2017-10-20 | 2018-02-13 | 上海源叶生物科技有限公司 | A kind of preparation technology of chonglou saponin series |
| CN110464789A (en) * | 2018-05-09 | 2019-11-19 | 成都众宜坊农业开发有限公司 | A kind of preparation method of chonglou saponin |
| CN110540558A (en) * | 2019-07-17 | 2019-12-06 | 广西壮族自治区中医药研究院 | Preparation and quality detection method of high-purity phlorizin |
-
2020
- 2020-04-27 CN CN202010345991.2A patent/CN111349140B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106353417A (en) * | 2016-08-14 | 2017-01-25 | 西南民族大学 | UPLC (ultra-performance liquid chromatography) detection method of multiple steroid saponins in Yunnan rhizoma paridis or its polygerm strain |
| CN107312061A (en) * | 2017-07-18 | 2017-11-03 | 中国科学院昆明植物研究所 | Chonglou saponin II and chonglou saponin VII preparation method |
| CN107686502A (en) * | 2017-10-20 | 2018-02-13 | 上海源叶生物科技有限公司 | A kind of preparation technology of chonglou saponin series |
| CN110464789A (en) * | 2018-05-09 | 2019-11-19 | 成都众宜坊农业开发有限公司 | A kind of preparation method of chonglou saponin |
| CN110540558A (en) * | 2019-07-17 | 2019-12-06 | 广西壮族自治区中医药研究院 | Preparation and quality detection method of high-purity phlorizin |
Non-Patent Citations (3)
| Title |
|---|
| YA-SHU CHEN 等: "Growth inhibition by pennogenyl saponins from Rhizoma paridis on hepatoma xenografts in nude mice", 《STEROIDS》 * |
| 尹鸿翔等: "狭叶重楼的主要甾体皂苷类化学成分的分离及鉴定", 《四川大学学报(医学版)》 * |
| 罗廷顺等: "重楼HPLC指纹图谱及4种重楼皂苷含量测定", 《时珍国医国药》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114668773A (en) * | 2022-04-08 | 2022-06-28 | 山东中医药大学 | Application of rhizoma paridis extract in resisting Climiya Congo hemorrhagic fever virus |
| CN114668773B (en) * | 2022-04-08 | 2023-10-03 | 山东中医药大学 | Application of paris polyphylla extract in resisting Crimedes congo hemorrhagic fever virus |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111349140B (en) | 2022-12-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Cheng et al. | Recent advances in chemical analysis of licorice (Gan-Cao) | |
| CN102796066B (en) | Flavone compound and preparation method and application thereof | |
| Wen et al. | Chemical characteristics of saponins from Paris fargesii var. brevipetala and cytotoxic activity of its main ingredient, paris saponin H | |
| Chen et al. | Separation of six compounds including two n‐butyrophenone isomers and two stibene isomers from Rheum tanguticum Maxim by recycling high speed counter‐current chromatography and preparative high‐performance liquid chromatography | |
| CN109912680B (en) | Oleane-type triterpenoid saponin and extraction separation method and application thereof | |
| CN108003214B (en) | Saponin compound extracted from rhizoma bolbostemmae, and its preparation method and application | |
| CN107271577A (en) | A kind of analysis of effective component method of stilbene Siberian cocklebur warm kidney medicine for eliminating bursa | |
| Zhang et al. | Hierarchical extraction and simultaneous determination of flavones and triterpenes in different parts of Trichosanthes kirilowii Maxim. by ultra-high-performance liquid chromatography coupled with tandem mass spectrometry | |
| CN105566414B (en) | The method that four kinds of flavone glycosides are isolated and purified from waxberry flesh | |
| CN110526952B (en) | Preparation method for extracting flavonoid glycoside from pteris crassipes | |
| CN111349140B (en) | Preparation method and quality control method of high-purity rhizoma paridis saponin VII | |
| CN113759034B (en) | Method for establishing ophiopogon decoction substance reference | |
| CN101791366A (en) | Method for testing quality of Discorea nipponica Makino in different places and medicinal materials of same genera | |
| CN110540558B (en) | Preparation of high-purity phlorizin and quality detection method thereof | |
| CN103145775A (en) | Preparation and quality control method for high purity cleidion brevipetiolatum glycoside A | |
| CN101703669B (en) | Smilax china effective fractions and extraction as well as purification process thereof | |
| CN114573456B (en) | Kaurane diterpenoid compound and preparation method and application thereof | |
| CN104892620B (en) | A kind of preparation method of high-purity karanjin | |
| CN111233807B (en) | Preparation method and quality control method of high-purity kaempferol-7, 4' -dimethyl ether | |
| CN111116700B (en) | Method for extracting, separating and purifying dioscin from chrysanthemum leaves | |
| Banzouzi et al. | Cogniauxia podolaena: bioassay-guided fractionation of defoliated stems, isolation of active compounds, antiplasmodial activity and cytotoxicity | |
| CN106317157A (en) | Polyhydroxy diketone cucuribitane triterpene as well as preparation method and application thereof | |
| Wang et al. | Optimization of the extraction conditions and simultaneous quantification of six flavonoid glycosides in Flos Chrysanthemi by RP-LC | |
| CN102628839B (en) | Preparation of high-purity camellianin and its quality control method | |
| CN102526561B (en) | Detection method for Sanjin preparation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20200630 Assignee: Guangxi Houde Pharmaceutical Co.,Ltd. Assignor: GUANGXI INSTITUTE OF CHINESE MEDICINE & PHARMACEUTICAL SCIENCE Contract record no.: X2023980045210 Denomination of invention: Preparation method and quality control method of a high-purity Paris saponin VII Granted publication date: 20221227 License type: Common License Record date: 20231102 |
|
| EE01 | Entry into force of recordation of patent licensing contract |