CN105891403A - Anemone flaccida medicinal material HPLC-UV characteristic spectrum construction method - Google Patents

Anemone flaccida medicinal material HPLC-UV characteristic spectrum construction method Download PDF

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CN105891403A
CN105891403A CN201610488312.0A CN201610488312A CN105891403A CN 105891403 A CN105891403 A CN 105891403A CN 201610488312 A CN201610488312 A CN 201610488312A CN 105891403 A CN105891403 A CN 105891403A
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peak
characteristic spectrum
rhizoma anemones
medical material
anemones flaccidae
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CN105891403B (en
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范春林
王英
杨伟群
叶文才
裴红
鲁展雨
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GUANGZHOU KANGHE PHARMACEUTICAL CO Ltd
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GUANGZHOU KANGHE PHARMACEUTICAL CO Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample

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Abstract

The invention belongs to the field of analysis and detection of traditional Chinese medicines and discloses an anemone flaccida medicinal material high performance liquid chromatography (HPLC) characteristic spectrum construction method for solving quality control problem of an anemone flaccida medicinal material. The method comprises the following steps: (1) preparing a reference solution; (2) preparing a test solution; (3) establishing chromatographic conditions; and (4) constructing characteristic spectrum. The method disclosed by the invention has the advantages of simple operation, accuracy, reliability and good repeatability, and multiple main effective ingredients in the anemone flaccida medicinal material can be more comprehensively detected, so that the anemone flaccida medicinal material HPLC-UV characteristic spectrum is constructed.

Description

Rhizoma Anemones flaccidae medical material HPLC-UV characteristic spectrum construction method
Technical field
The present invention relates to Chinese medicine quality control field, particularly relate to the construction method of Chinese medicine Rhizoma Anemones flaccidae characteristic spectrum.
Background technology
Chinese medicine quality controls to refer to use necessary analysis method and the quality of detection means monitoring Chinese medicine, to ensure The safety of Chinese medicine medication, effectiveness, stability and controllability.Owing to Chinese crude drug is of a great variety, chemical composition Complexity, the level of development causing Chinese medicine quality to control cannot meet the development of modern TCM industry always.Existing Chinese medicine quality control model many foreigns chemical drugs quality control model set up, just for some or several Composition carries out assay, and this can not reflect complexity and the globality of Chinese medicine well.Therefore, Chinese medicine is promoted Quality standard, strengthen quality control, guarantee Chinese medicine drug safety, it is achieved Chinese medicine " safely, effectively, Stable, controlled " (Xu Xiang etc., journal of shanghai Chinese medicine, 2006,40 (3): 50-51).
Chinese medicine fingerprint mean Chinese crude drug or Chinese medicine preparation appropriately processed after, use certain analysis method to obtain To can embody Chinese crude drug or the collection of illustrative plates of Chinese medicine preparation overall permanence, and combine mathematical statistics method, by phase Evaluate Chinese crude drug and the quality of the quality of the pharmaceutical preparations thereof like degree, the kind of contained chemical composition in Chinese medicine can be reflected more comprehensively And quantity, thus reflect Chinese medicine inherent quality.Chinese medicine fingerprint is more and more applied to the mirror of Chinese herbal medicine Not and quality control, World Health Organization (WHO) (WHO), FDA (Food and Drug Adminstration) (FDA), British Herbal Pharmacopoeia, Medicinal plants association of Germany etc. all accepts the method for quality control of finger printing, shows that finger printing has become international Generally acknowledge control Chinese medicine and natural drug quality effective ways (Yi Lunchao etc., chromatograph, 2008,26 (2): 166-171)。
2010 editions " Chinese Pharmacopoeia " establishes HPLC " fingerprint/characteristic spectrum " method first, to control portion Dividing the quality of herbal species, 2010 editions " Chinese Pharmacopoeia " records HPLC finger printing totally 13, such as Radix Notoginseng Total saponins, Oleum Curcumae and centella general glycoside etc., record HPLC characteristic spectrum totally 7, including Radix Ginseng total saponins, Herba Artemisiae Scopariae extract and Oleum Rhododendri Daurici etc..In 2015 editions " Chinese Pharmacopoeias ", record HPLC finger printing totally 22 , on the basis of 2010 editions " Chinese Pharmacopoeia ", add Xuezhikang sheet, KANGGONGYAN PIAN, QINKAILING ZHUSHEYE etc., Record HPLC characteristic spectrum totally 38, on the basis of 2010 editions " Chinese Pharmacopoeia " add Rhizoma Et Radix Notopterygii, Lignum Aquilariae Resinatum, Antivirus oral liquid, YINHUANG PIAN etc..The application of finger printing, can effectively control Chinese medicine quality, promotes that Chinese medicine is existing Dai Hua.But different sources, different collecting season, different pharmacy corporation processing of crude drugs techniques etc. cause in same Medicine uses same " finger printing " to control, and difficulty is bigger.Therefore, " characteristic spectrum " method is used to control Chinese medicine quality, only makes regulation to the relative retention time at main common characteristic peak, ignores the peak that peak area is less, More meet the feature of Chinese medicine, be also the Main way of Chinese medicine quality control development.
Rhizoma Anemones flaccidae is being dried of Ranunculaceae thimbleweed woods shade Anemone cathayensis Kitag. Anemone flaccida Fr.Schmidt Rhizome, has another name called unregistered land thunder, gold beading, Scolopendra Radix Notoginseng etc., and it is warm in nature, acrid in the mouth, micro-hardship, main product in Hubei and Guizhou, additionally, all there are distribution (Nanjing University of Traditional Chinese Medicine, Chinese medicine voluminous dictionary (in the ground such as Sichuan, Zhejiang, Hunan Two editions), 2005:1115), tool removing toxic substances, effect of wind-damp dispelling, it is clinically used for treating rheumatic arthralgia, traumatic injury Deng disease, in the treatment being widely used in rheumatic and rheumatoid arthritis among the people.
Phytochemistry and pharmaceutical research show, oleanane-type triterpene saponin is the main active of Rhizoma Anemones flaccidae (Han Lintao etc., Chinese crude drug, 2009,32 (7): 1059-1062), there is antiinflammatory, antiallergic, antihistaminic and exempt from The effect such as epidemic disease regulation (Bing Feihong etc., China Medicine University's journal, 2008,39 (6): 496-499).With the total soap of Rhizoma Anemones flaccidae Glycosides be raw material make Chinese medicine five kind new medicine " Rhizoma Anemones flaccidae treating rheumatism capsule " (Bing Feihong etc., Hubei Journal of Traditional Chinese Medicine, 2005,27 (1): 48-49), there is good resisting rheumatoid arthritis effect, currently carried out for III phase clinical Research, has wide development prospect.
At present, the quality standard research to Rhizoma Anemones flaccidae is concentrated mainly on application spectrophotometry (UV-Vis) Method mensuration total saponin content (Wang Aimin, China Dispensary, 2007,18 (21): 1654-1655) and apply efficient liquid phase Chromatograph (HPLC) method measure single compounds content in Rhizoma Anemones flaccidae (Zhao Huinan etc., Chinese crude drug, 2013,36 (10): 1632-1634), and the characteristic constituents in Rhizoma Anemones flaccidae is not studied, lack the entirety of Radix Linderae material over the ground Quality evaluation.
Summary of the invention
Based on this, a kind of method that it is an object of the invention to build Rhizoma Anemones flaccidae medical material HPLC-UV characteristic spectrum, Total quality evaluation for Rhizoma Anemones flaccidae medical material provides effective ways, is the quality assurance of Rhizoma Anemones flaccidae treating rheumatism capsule further Provide material base.
The construction method of the Rhizoma Anemones flaccidae medical material HPLC-UV characteristic spectrum that the present invention relates to, the concrete technical side of employing Case is as follows:
(1) preparation of reference substance solution
Precision weighs drying under reduced pressure to the anhuienoside E of constant weight, glycoside St-14a, hemsgiganoside B, flaccidoside II, hederasaponin B reference substance are appropriate, and add 30% acetonitrile making concentration is 0.2～0.5 mg·mL-1Mixing reference substance solution, filter, to obtain final product.
(2) preparation of need testing solution
Take Rhizoma Anemones flaccidae medical material sample powder about 0.5g, accurately weighed, put in tool plug conical flask, accurate addition 50～100% methanol, weigh, supersound extraction, let cool, supply the weight of less loss, shake up, with 0.22 μm filter Membrane filtration mistake, takes subsequent filtrate, to obtain final product.
(3) foundation of HPLC-UV chromatographic condition
Chromatographic column: octadecylsilane chemically bonded silica post；Flowing phase: acetonitrile-0.01% trifluoroacetic acid solution (30:70)；Detection wavelength: 200～220nm；Flow velocity: 0.8～1.2mL min-1；Column temperature: 20～50 DEG C；Enter Sample amount: 2～20 μ L.
(4) structure of characteristic spectrum
Precision draws need testing solution and reference substance solution respectively, injects high performance liquid chromatograph and is measured, note Record chromatogram, uses " similarity evaluation (2004A version) " software to institute's Dry Sack Spectrogram processes, and obtains the comparison characteristic spectrum of Rhizoma Anemones flaccidae medical material, uses the method calculating relative retention time true Surely the identification at peak is had.
High performance liquid chromatograph applied in step (3) is Agilent 1260 or Ultimate 3000, chromatographic column For Phenomenex Luna C18Post, specification is 4.6mm × 250mm, 5 μm, or Cosmosil 5C18-MS-II post, specification is 4.6mm × 250mm, 5 μm, or Ultimate XB-C18Post, specification is 4.6mm×250mm,5μm。
Characteristic spectrum obtained by above-mentioned Rhizoma Anemones flaccidae medical material HPLC-UV characteristic spectrum construction method there are 5 features Total peak, wherein with No. 4 peak flaccidoside II for object of reference peak, peak 1 is anhuienoside E, relatively Retention time 0.46；Peak 2 is glycoside St-14a, relative retention time 0.53；Peak 3 is hemsgiganoside B, relative retention time 0.66；Peak 4 is flaccidoside II, relative retention time 1.00；Peak 5 is Hederasaponin B: relative retention time 1.07.The relative retention time of each chromatographic peak should be at setting Within ± 10%.
The present invention is on the basis of Rhizoma Anemones flaccidae is carried out cycle chemistry composition Study, is provided by HPLC-UV method A kind of construction method of the HPLC-UV characteristic spectrum of Rhizoma Anemones flaccidae medical material multi-target ingredient.The present invention has operation Simply, the advantage such as the most reliable, reproducible, can compared with multiple principle active component in complete detection Rhizoma Anemones flaccidae medical material, Thus construction feature collection of illustrative plates, the total quality evaluation for Rhizoma Anemones flaccidae medical material provides effective ways, is ground dark glaucoma further The quality assurance of wet peace capsule provides material base.
Accompanying drawing explanation
Fig. 1 is the separation process figure of Rhizoma Anemones flaccidae main chemical compositions in embodiment 1；Fig. 2 is in embodiment 1 The high resolution mass spectrum figure of anhuienoside E；Fig. 3 is the nuclear magnetic resonance, NMR of the anhuienoside E in embodiment 1 Hydrogen spectrogram；Fig. 4 is DEPT figure and the carbon-13 nmr spectra figure of the anhuienoside E in embodiment 1；Figure 5 is the high resolution mass spectrum figure of the glycoside St-14a in embodiment 1；Fig. 6 is the glycoside in embodiment 1 The hydrogen nuclear magnetic resonance spectrogram of St-14a；Fig. 7 is DEPT figure and the core of the glycoside St-14a in embodiment 1 Magnetic resonance carbon spectrogram；Fig. 8 is the high resolution mass spectrum figure of the hemsgiganoside B in embodiment 1；Fig. 9 is The hydrogen nuclear magnetic resonance spectrogram of the hemsgiganoside B in embodiment 1；Figure 10 is in embodiment 1 DEPT figure and the carbon-13 nmr spectra figure of hemsgiganoside B；Figure 11 is in embodiment 1 The high resolution mass spectrum figure of flaccidoside II；Figure 12 is the nuclear magnetic resonance, NMR of the flaccidoside II in embodiment 1 Hydrogen spectrogram；Figure 13 is DEPT figure and the carbon-13 nmr spectra figure of the flaccidoside II in embodiment 1；Figure 14 is the high resolution mass spectrum figure of the hederasaponin B in embodiment 1；Figure 15 is in embodiment 1 The hydrogen nuclear magnetic resonance spectrogram of hederasaponin B；Figure 16 is the hederasaponin B in embodiment 1 DEPT figure and carbon-13 nmr spectra figure；Figure 17 is the test sample in embodiment 2 and reference substance solution chromatographic peak Stacking chart；Figure 18 is the need testing solution (A) in embodiment 2 and mixed mark solution (B) mirror image figure and each chromatographic peak Ultraviolet spectrogram；Figure 19 is need testing solution (A) in embodiment 2 and mixed mark solution (B) mirror image figure and each Structural formula corresponding to chromatographic peak；Figure 20 is the total ion current of the Rhizoma Anemones flaccidae medical material need testing solution in embodiment 2 Figure；Figure 21 is 17 batches of different sources Rhizoma Anemones flaccidae medical material characteristic spectrum coupling figures in embodiment 2；Figure 22 is real Execute the Rhizoma Anemones flaccidae medical material HPLC in example 2 and compare characteristic spectrum；
Detailed description of the invention
Below in conjunction with the drawings and specific embodiments, the present invention is further elaborated.
Test sample is originated: 17 batches of Rhizoma Anemones flaccidae (Anemone flaccida Fr.Schmidt) medical materials all pick up from Hubei governor Yang Xian, height above sea level is positioned at 1500～1800 meters, is shown in Table 1.
1 17 batches of Rhizoma Anemones flaccidae crude drug sources of table
Instrument: Bruker AV-400 type nuclear magnetic resonance chemical analyser；Agilent 6210ESI/TOF mass spectrograph； Agilent 1260 analytical type high performance liquid chromatograph (G1311C quaternary pump, G1329B automatic sampler, G1316A column oven, G1314F UV-detector, Agilent chromatographic work station)；Agilent 6210LC/MSD TOF mass spectrograph；Electronic analytical balance (sensibility reciprocal: 0.1mg；0.01mg, Sartorius company of Germany)； KQ-3000E type ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd., power 300W, frequency 40 KHz)；Direct-Q5 type ultrapure water machine (Millipore company of the U.S.)；Microporous filter membrane (rise experiment and set by Tianjin, Tianjin Standby company limited).
Reagent: trifluoroacetic acid aqueous solution (Merck company of Germany), Chromatographic Pure Methanol (Merck company of Germany), water is Self-control ultra-pure water, other reagent are analytical pure.
The preparation of main chemical compositions in embodiment 1 Rhizoma Anemones flaccidae medical material, purification and Structural Identification
As it is shown in figure 1, take Rhizoma Anemones flaccidae toatal saponins 110g, after 300mL water dissolution, it is processed good to be splined on MCI aperture resin column, successively by water, 15%, 30%, 50%, 70% and 90% methanol-eluted fractions, collects 70% Methanol-eluted fractions position, is evaporated to do, and obtains dry extract (39.36g).Extractum dissolves with 50% methanol, loading In ODS post, 60%～95% methanol elution gradient, wherein 60% methanol-eluted fractions position is through Sephadex LH-20 Gel column chromatography and preparation HPLC separate [Cosmosil 5C18-MS-Ⅱ(20mm×250mm,5μm) Chromatographic column, flowing is 30% acetonitrile-0.01% trifluoroacetic acid aqueous solution mutually, and flow velocity is 6.0mL min-1], obtain Compound 2 (300mg) and 3 (200mg), 65% methanol-eluted fractions position separates [Cosmosil through preparation HPLC 5C18-MS-II (20mm × 250mm, 5 μm) chromatographic column, flowing is 32% acetonitrile mutually, and flow velocity is 6.0 mL·min-1], obtain compound 1 (320mg).70% methanol-eluted fractions position separates through preparation HPLC [Cosmosil 5C18-MS-II (20mm × 250mm, 5 μm) chromatographic column, flowing is 45% ethanol mutually, flow velocity For 6.0mL min-1], obtain compound 4 (500mg) and 5 (200mg).
The chromatographic condition of described preparation HPLC is as follows:
Chromatographic column: Cosmosil 5C18-MS-Ⅱ(20mm×250mm,5μm)；Column temperature: 30 DEG C；Detection Wavelength: 210nm；Flow velocity: 6mL min-1；Sample size: 0.2mL.
Using method of spectroscopy to identify the structure of each compound, each physicochemical data is as follows:
Compound 1:anhuienoside E, amorphous white powder, its chemical constitution is as follows:
HR-ESI-MS m/z 1233.6274[M-H]-, molecular formula is C60H98O26, high resolution mass spectrum figure such as figure Shown in 2.
1H NMR(400MHz,pyridine-d5)δH: 6.57 (1H, br s, H-1 "), 6.25 (1H, d, J=7.4 Hz, H-1 " '), 5.86 (1H, br s, H-1 " " '), 5.43 (1H, br s, H-12), 5.01 (1H, d, J=7.2Hz, H-1 " "), 4.96 (1H, d, J=7.0Hz, H-1'), 3.37 (1H, dd, J=11.5,3.6Hz, H-3), 1.27 (3H, s,H3-23), 1.26 (3H, s, H3-27), 1.21 (3H, s, H3-24), 1.10 (3H, s, H3-26), 0.91 (6H, s, H3-29,30), 0.88 (3H, s, H3-25).The proton nmr spectra of anhuienoside E is as shown in Figure 3.
13C NMR(100MHz,pyridine-d5) data are shown in Table 2.The carbon-13 nmr spectra of anhuienoside E As shown in Figure 4.
List of references: YE W C, ZHANG Q W, ZHAO S X, et al.Four New oleanane Saponin from Anemone anhuiensis[J].Chemical and Pharmaceutical Bulletin,2001,49(5)；632-634.
Compound 2:glycoside St-14a, white amorphous powder, its chemical constitution is as follows:
HR-ESI-MS m/z 1101.5488[M-H]-, molecular formula is C54H86O23, glycoside St-14a's High resolution mass spectrum figure is as shown in Figure 5.
1H NMR(400MHz,pyridine-d5)δH: 6.07 (1H, d, J=7.6Hz, H-1 "), 5.69 (1H, Br s, H-1 " "), 5.25 (1H, br s, H-12), 4.93 (1H, d, J=7.8Hz, H-1 " '), 4.89 (1H, d, J=7.5 Hz, H-1'), 3.27 (1H, dd, J=11.3,3.2Hz, H-3), 1.19 (3H, s, H3-23), 1.14 (3H, s, H3-27), 0.92 (3H, s, H3-26), 0.88 (3H, s, H3-24), 0.79 (3H, s, H3-29), 0.76 (3H, s, H3-30), 0.73 (3H, s, H3-25).The proton nmr spectra of glycoside St-14a is as shown in Figure 6.
13C NMR(100MHz,pyridine-d5) data are shown in Table 2.The nuclear magnetic resonance, NMR carbon of glycoside St-14a Spectrum is as shown in Figure 7.
List of references: Han Lintao, Huang Fang. flaccid anemone rhizome triterpenoid saponins research [J]. Chinese crude drug, 2009,32 (7): 1059-1062.
Compound 3:hemsgiganoside B, white amorphous powder, its chemical structural formula is as follows:
HR-ESI-MS m/z 955.4901[M-H]-, molecular formula is C48H76O19, hemsgiganoside B's High resolution mass spectrum figure is as shown in Figure 8.
1H NMR(400MHz,pyridine-d5)δH: 6.20 (1H, d, J=8.1Hz, H-1 "), 5.36 (1H, br S, H-12), 5.02 (1H, d, J=7.7Hz, H-1 " '), 5.00 (1H, d, J=7.9Hz, H-1'), 3.36 (1H, dd, J=11.5,4.0Hz, H-3), 1.28 (3H, s, H-23), 1.24 (3H, s, H3-27), 1.05 (3H, s, H3-26), 0.97(3H,s,H3-24), 0.86 (3H, s, H3-29), 0.85 (3H, s, H3-30), 0.82 (3H, s H3-25)。 The nucleus magnetic hydrogen spectrum data of hemsgiganoside B are as shown in Figure 9.
13C NMR(100MHz,pyridine-d5) data are shown in Table 2.The nuclear-magnetism carbon spectrum number of hemsgiganoside B According to as shown in Figure 10.
List of references: CHEN Y, CHIU M H, GU K, et al.Cucurbitacin and triterpenoid glycosides from Hemsleya giganthy[J].Chinese Chemical Letters,2003,14(5):475-478.
Compound 4:flaccidoside II, white amorphous powder, its chemical structural formula is as follows:
HR-ESI-MS m/z 1203.6172[M-H]-, molecular formula is C59H96O25, the height of flaccidoside II Resolution mass spectra is as shown in figure 11.
1H NMR(400MHz,pyridine-d5)δH: 6.55 (1H, br s, H-1 "), 6.26 (1H, d, J=8.0 Hz, H-1 " '), 5.88 (1H, br s, H-1 " " '), 5.39 (1H, br s, H-12), 5.01 (1H, d, J=7.8Hz, H-1 " "), 4.84 (1H, d, J=7.2Hz, H-1'), 3.32 (1H, dd, J=11.6,3.7Hz, H-3), 1.26 (3H, s,H3-23), 1.25 (3H, s, H3-27), 1.21 (3H, s, H3-26), 1.11 (3H, s, H3-24), 0.90 (9H, s, H3-25,29,30).The nucleus magnetic hydrogen spectrum data of flaccidoside II are as shown in figure 12.
13C NMR(100MHz,pyridine-d5) data are shown in Table 2.The nuclear-magnetism carbon modal data of flaccidoside II As shown in figure 13.
List of references:
[1] Fan Chunlin, Wang Ying, Wang Guiyang, etc. from Rhizoma Anemones flaccidae medical material, prepare W simultaneously1And W3Method: China, 201310488046.8[P].2015-7-1.
[2]Zhao L,Chen W M,Fang Q C.Two New Oleanane Saponins from Anemone flaccida[J]. Planta Medica,1991,57(6):572-574.
Compound 5:hederasaponin B, white amorphous powder, its chemical structural formula is as follows:
HR-ESI-MS m/z 1203.6174[M-H]-, molecular formula is C59H96O25.Hederasaponin B's High resolution mass spectrum figure is as shown in figure 14.
1H NMR(400MHz,pyridine-d5)δH: 6.26 (1H, d, J=8.2Hz, H-1 " '), 6.13 (1H, br S, H-1 "), 5.87 (1H, br s, H-1 " " '), 5.43 (1H, br s, H-12), 5.01 (1H, d, J=8.1Hz, H-1 " "), 4.93 (1H, d, J=5.1Hz, H-1'), 3.26 (1H, dd, J=12.0,3.6Hz, H-3), 1.27 (3H, s,H3-23), 1.19 (3H, s, H3-27), 1.12 (3H, s, H3-26), 1.09 (3H, s, H3-24), 0.92 (9H, s, H3-25,29,30).Hederasaponin B hydrogen nuclear magnetic resonance spectrogram is as shown in figure 15.
13C NMR(100MHz,pyridine-d5) data are shown in Table 2.Hederasaponin B carbon-13 nmr spectra figure As shown in figure 16.
List of references:
[1] Fan Chunlin, Wang Ying, Wang Guiyang, etc. from Rhizoma Anemones flaccidae medical material, prepare W simultaneously1And W3Method: China, 201310488046.8[P].2015-7-1.
[2]SANO K,SANADA S,IDA Y,et al.Studies on the constituents of the bark of Kalopanax pictus Nakai[J].Chemical and Pharmaceutical Bulletin,1991,39(4):865-870.
Table 2 compound 1-5 carbon modal data (100MHz, pyridine-d5)
Embodiment 2 Rhizoma Anemones flaccidae medical material characteristic spectrum construction method
(1) preparation of reference substance solution
It is appropriate that precision weighs each reference substance, adds 30% acetonitrile and is respectively prepared anhuienoside E (0.25mg mL-1)、 glycoside St-14a(0.25mg·mL-1)、hemsgiganoside B(0.25mg·mL-1)、flaccidoside Ⅱ(0.5mg·mL-1)、hederasaponin B(0.25mg·mL-1) reference substance solution, filter, to obtain final product.
Table 3 reference substance purity
Note: S is object of reference peak
(2) preparation of need testing solution
Take Rhizoma Anemones flaccidae medical material sample powder about 0.5g, accurately weighed, put in tool plug conical flask, accurate addition 75% Methanol 50mL, weighs, and supersound extraction lets cool, and supplies the weight of less loss, shakes up, with 0.22 μm filter membrane Filter, take subsequent filtrate, to obtain final product.
(3) foundation of HPLC-UV chromatographic condition
The need testing solution of step (2) is carried out high effective liquid chromatography for measuring, and chromatographic condition is:
Agilent 1260 type high performance liquid chromatograph；Chromatographic column: Phenomenex Luna C18(4.6mm× 250mm,5μm)；Flowing phase: acetonitrile-0.01% trifluoroacetic acid solution (30:70)；Detection wavelength: 210nm； Flow velocity: 1.0mL min-1；Column temperature: 30 DEG C；Sample size: 10 μ L.
(4) use reference substance that chromatographic peak is confirmed
Use above-mentioned chromatographic condition detection Rhizoma Anemones flaccidae medical material need testing solution and reference substance solution, and use diode battle array Row detector (DAD) carries out full wavelength scanner at 190～400nm, records chromatogram, and contrast test sample is molten Liquid and the retention time of each chromatographic peak of reference substance solution and characteristic ultraviolet absorption.Test sample is main with reference substance solution Chromatographic peak stacking chart as shown in figure 17, need testing solution (A) and mixed mark solution (B) mirror image figure and each chromatographic peak Ultraviolet spectrogram is as shown in figure 18.
Characteristic ultraviolet absorption according to main chromatographic peak each in reference substance and need testing solution and retention time, to respectively Chromatographic peak carries out chemistry and points out, and need testing solution (A) is marked corresponding to solution (B) mirror image figure and each chromatographic peak with mixed Structural formula is as shown in figure 19.
(5) use HPLC-ESI-MS technology that chromatographic peak is confirmed
Mass Spectrometry Conditions: ion source: ESI (negative ion mode)；Dryer temperature: 325 DEG C；Dry gas stream speed: 9L·min-1；Capillary voltage: 4.0kV；Scanning karyoplasmic ratio scope: 100～1500；Automatically reference ion: 119.0363 and 1033.9881.
Taking Rhizoma Anemones flaccidae medical material need testing solution to detect under above-mentioned chromatographic condition and Mass Spectrometry Conditions, Rhizoma Anemones flaccidae medical material supplies The total ion current figure of test sample solution is as shown in figure 20.Retention time and the molecular formula at each total peak are as shown in table 4.
Table 4 has retention time and the molecular formula at peak
(6) foundation of characteristic spectrum
Use " similarity evaluation (2004A version) " software to 17 batches of ground Radixs Linderae Material need testing solution chromatographic peak is analyzed, and uses median method, and time window 0.1, with No. 4 peak flaccidoside II is object of reference peak, and the HPLC generating Rhizoma Anemones flaccidae medical material after Supplements Auto-matching compares characteristic spectrum.17 Criticize different sources Rhizoma Anemones flaccidae medical material characteristic spectrum coupling figure as shown in figure 21, determine 5 common characteristic peaks altogether.Ground Radix Linderae material HPLC comparison characteristic spectrum is as shown in figure 22.
The assay method of embodiment 3 Rhizoma Anemones flaccidae medical material characteristic spectrum
(1) preparation of need testing solution
Take Rhizoma Anemones flaccidae medical material sample powder about 0.5g, accurately weighed, put in tool plug conical flask, accurate addition 75% Methanol 50mL, close plug, weighed weight, supersound process 40 minutes, let cool, the most weighed weight, with 75% Methanol supplies the weight of less loss, shakes up, and filters with 0.22 μm filter membrane, takes subsequent filtrate, obtain need testing solution.
(2) high effective liquid chromatography for measuring
The need testing solution of step (1) is carried out high effective liquid chromatography for measuring, and chromatographic condition is:
Agilent 1260 type high performance liquid chromatograph；Chromatographic column: Cosmosil 5C18-MS-Ⅱ(4.6mm×250 mm,5μm)；Flowing phase: acetonitrile-0.01% trifluoroacetic acid solution (30:70)；Detection wavelength: 205nm；Stream Speed: 1.1mL min-1；Column temperature: 35 DEG C；Sample size: 5 μ L.
Using said method detection Rhizoma Anemones flaccidae medical material need testing solution, obtain chromatogram, other steps are with embodiment 2.
The assay method of embodiment 4 Rhizoma Anemones flaccidae medical material characteristic spectrum
(1) preparation of need testing solution
Take Rhizoma Anemones flaccidae medical material sample powder about 0.5g, accurately weighed, put in tool plug conical flask, accurate addition 75% Methanol 50mL, close plug, weighed weight, supersound process 40 minutes, let cool, the most weighed weight, with 75% Methanol supplies the weight of less loss, shakes up, and filters with 0.22 μm filter membrane, takes subsequent filtrate, obtain need testing solution.
(2) high effective liquid chromatography for measuring
The need testing solution of step (1) is carried out high effective liquid chromatography for measuring, and chromatographic condition is:
Agilent 1260 type high performance liquid chromatograph；Chromatographic column: Ultimate XB-C18(4.6mm×250mm, 5μm)；Flowing phase: acetonitrile-0.01% trifluoroacetic acid solution (30:70)；Detection wavelength: 200nm；Flow velocity: 1.2mL·min-1；Column temperature: 40 DEG C；Sample size: 15 μ L.
Using said method detection Rhizoma Anemones flaccidae medical material need testing solution, obtain chromatogram, other steps are with embodiment 2.
The assay method of embodiment 5 Rhizoma Anemones flaccidae medical material characteristic spectrum
(1) preparation of need testing solution
Take Rhizoma Anemones flaccidae medical material sample powder about 0.5g, accurately weighed, put in tool plug conical flask, accurate addition 75% Methanol 50mL, close plug, weighed weight, supersound process 40 minutes, let cool, the most weighed weight, with 75% Methanol supplies the weight of less loss, shakes up, and filters with 0.22 μm filter membrane, takes subsequent filtrate, obtain need testing solution.
(2) high effective liquid chromatography for measuring
The need testing solution of step (1) is carried out high effective liquid chromatography for measuring, and chromatographic condition is:
Ultimate 3000 type high performance liquid chromatograph；Chromatographic column: Phenomenex Luna C18(4.6mm× 250mm,5μm)；Flowing phase: acetonitrile-0.01% trifluoroacetic acid solution (30:70)；Detection wavelength: 215nm； Flow velocity: 0.8mL min-1；Column temperature: 20 DEG C；Sample size: 20 μ L.
Using said method detection Rhizoma Anemones flaccidae medical material need testing solution, obtain chromatogram, other steps are with embodiment 2.
The assay method of embodiment 6 Rhizoma Anemones flaccidae medical material characteristic spectrum
(1) preparation of need testing solution
Take Rhizoma Anemones flaccidae medical material sample powder about 0.5g, accurately weighed, put in tool plug conical flask, accurate addition 75% Methanol 50mL, close plug, weighed weight, supersound process 40 minutes, let cool, the most weighed weight, with 75% Methanol supplies the weight of less loss, shakes up, and filters with 0.22 μm filter membrane, takes subsequent filtrate, obtain need testing solution.
(2) high effective liquid chromatography for measuring
The need testing solution of step (1) is carried out high effective liquid chromatography for measuring, and chromatographic condition is:
Ultimate 3000 type high performance liquid chromatograph；Chromatographic column: Cosmosil 5C18-MS-Ⅱ(4.6mm× 250mm,5μm)；Flowing phase: acetonitrile-0.01% trifluoroacetic acid solution (30:70)；Detection wavelength: 220nm； Flow velocity: 0.9mL min-1；Column temperature: 25 DEG C；Sample size: 20 μ L.
Using said method detection Rhizoma Anemones flaccidae medical material need testing solution, obtain chromatogram, other steps are with embodiment 2.
The assay method of embodiment 7 Rhizoma Anemones flaccidae medical material characteristic spectrum
(1) preparation of need testing solution
Take Rhizoma Anemones flaccidae medical material sample powder about 0.5g, accurately weighed, put in tool plug conical flask, accurate addition 75% Methanol 50mL, close plug, weighed weight, supersound process 40 minutes, let cool, the most weighed weight, with 75% Methanol supplies the weight of less loss, shakes up, and filters with 0.22 μm filter membrane, takes subsequent filtrate, obtain need testing solution.
(2) high effective liquid chromatography for measuring
The need testing solution of step (1) is carried out high effective liquid chromatography for measuring, and chromatographic condition is:
Ultimate 3000 type high performance liquid chromatograph；Chromatographic column: Ultimate XB-C18(4.6mm×250mm, 5μm)；Flowing phase: acetonitrile-0.01% trifluoroacetic acid solution (30:70)；Detection wavelength: 210nm；Flow velocity: 1.2mL·min-1；Column temperature: 40 DEG C；Sample size: 5 μ L.
Using said method detection Rhizoma Anemones flaccidae medical material need testing solution, obtain chromatogram, other steps are with embodiment 2.
The checking of embodiment 8 Rhizoma Anemones flaccidae medical material characteristic spectrum method for building up
1, instrument precision test
Take Rhizoma Anemones flaccidae medical material sample S2, prepare need testing solution by step (2) in embodiment 2, use embodiment 2 The chromatographic condition of middle step (3) detects, continuous sample introduction 6 times, records chromatogram.With No. 4 peak (flaccidoside II) it is object of reference peak (S), calculates relative retention time and the relative peak area of each chromatographic peak, the results are shown in Table 5 Hes Table 6.
The relative retention time result of calculation (n=6) of table 5 instrument precision test
Note: S is object of reference peak
The relative peak area result of calculation (n=6) of table 6 instrument precision test
Note: S is object of reference peak
Result shows, in need testing solution, the RSD value of the relative retention time at each total peak is less than 0.08%, The RSD value of relative peak area is less than 0.61%, shows that the precision of instrument is good.With first time sample introduction gained Collection of illustrative plates as reference, use " similarity evaluation 2004A version " software to enter Row is analyzed, and calculates the similarity of 6 gained characteristic spectrums of sample introduction, and result shows, similarity is 1.000, Similarity is good, is shown in Table 7.
The Similarity Measure result (n=6) of table 7 instrument precision test
2, method replica test
Take Rhizoma Anemones flaccidae medical material sample S2, by step (2) parallel preparation Rhizoma Anemones flaccidae medical material need testing solution 6 in embodiment 2 Part, use the chromatographic condition of step (3) in embodiment 2 to detect, record chromatogram.With No. 4 peaks (flaccidoside II) is object of reference peak (S), calculates relative retention time and the relative peak area of each chromatographic peak, sees Table 8 and table 9.
The relative retention time result of calculation (n=6) of table 8 replica test
Note: S is object of reference peak
The relative peak area result of calculation (n=6) of table 9 replica test
Note: S is object of reference peak
Result shows, the RSD value of the relative retention time at each total peak is less than 0.11%, relative peak area RSD value is less than 1.60%, shows that the repeatability of method is good.Using the collection of illustrative plates of first time sample introduction gained as reference, Use " similarity evaluation 2004A version " software to be analyzed, calculate 6 parts The similarity of need testing solution gained characteristic spectrum, result shows, similarity is 1.000, and similarity is good, It is shown in Table 10.
The Similarity Measure result (n=6) of table 10 replica test
3, sample stability test
Take sample S2, by the method preparation Rhizoma Anemones flaccidae medical material need testing solution of step (2) in embodiment 2, use real Execute 0 after preparation of the chromatographic condition of step (3) in example 2,2,4,6,8,12,24,36,48h Detect, record chromatogram.It is object of reference peak (S) with No. 4 peaks (flaccidoside II), calculates each chromatographic peak Relative retention time and relative peak area, be shown in Table 11 and table 12.
The relative retention time result of calculation (n=9) of table 11 stability test
Note: S is object of reference peak
The relative peak area result of calculation (n=9) of table 12 stability test
Note: S is object of reference peak
Result shows, the RSD value of the relative retention time at each total peak is less than 0.15%, relative peak area RSD value is less than 0.36%, shows that need testing solution is good at 48h internal stability.With 0h sample introduction gained Collection of illustrative plates, as reference, uses " similarity evaluation 2004A version " software to carry out Analyzing, calculate the similarity of need testing solution gained characteristic spectrum, result shows, similarity is 1.000, Similarity is good, is shown in Table 13.
The Similarity Measure result (n=9) of table 13 stability test
The determination of embodiment 9 Rhizoma Anemones flaccidae medical material characteristic spectrum each chromatographic peak relative retention time
Different high performance liquid chromatograph (Agilent 1260 high-efficient liquid phase color is used according to embodiment 2～embodiment 7 Spectrometer and Ultimate 3000 high performance liquid chromatograph) and different chromatographic column [Phenomenex Luna C18(4.6 mm×250mm,5μm)、Cosmosil 5C18-MS-II (4.6mm × 250mm, 5 μm) and Ultimate XB-C18(4.6mm × 250mm, 5 μm)] each chromatographic peak of being measured and object of reference peak (flaccidoside II) Relative retention time value, average as each characteristic peak in Rhizoma Anemones flaccidae medical material characteristic spectrum and object of reference peak Relative retention time value between (flaccidoside II), as shown in table 14.
Table 14 each chromatographic peak relative retention time
Note: S is object of reference peak
5 total peaks of characteristic spectrum are confirmed, with No. 4 peaks by reference substance and HPLC-MS technology Flaccidoside II is object of reference peak (S), the relative retention time of characteristic peak 1～5 be respectively 0.46 (peak 1, Anhuienoside E), 0.53 (peak 2, glycoside St-14a), 0.66 (peak 3, hemsgiganoside B), 1.00 (peak 4, flaccidoside II) and 1.07 (peak 5, hederasaponin B), and specify the relative guarantor at 5 total peaks Stay the time should setting ± 10% within.
The investigation of embodiment 10 Rhizoma Anemones flaccidae medical material characteristic spectrum similarity
1, the mensuration of 17 batches of Rhizoma Anemones flaccidae medical materials and the acquisition of HPLC characteristic spectrum
17 batches of Rhizoma Anemones flaccidae medical materials are provided by Guangzhou Kanghe Pharmaceutical Co., Ltd., according to the step (2) in embodiment 2 Preparing need testing solution, the chromatographic condition of the step (3) in employing embodiment 2 is to 17 batches of Rhizoma Anemones flaccidae medical material test samples Solution is measured, and records chromatogram.
2, Rhizoma Anemones flaccidae medical material HPLC characteristic spectrum similarity is investigated
Use " similarity evaluation 2004A version " software to 17 batches of Rhizoma Anemones flaccidae medical materials Need testing solution HPLC chromatogram is analyzed, and calculates the similar of every batch of Rhizoma Anemones flaccidae medical material HPLC characteristic spectrum Degree, result is as shown in Table 15.
As shown in Table 19,17 batches of Rhizoma Anemones flaccidae medical material HPLC characteristic spectrums remove individual with the similarity compareing characteristic spectrum Outward, similarity is all higher than 0.94 to other batch (S4), shows the method for building up weight of Rhizoma Anemones flaccidae medical material HPLC characteristic spectrum Renaturation is good, and similarity is high.

Claims (3)

1. Rhizoma Anemones flaccidae medical material HPLC-UV characteristic spectrum construction method, it is characterised in that comprise the following steps:
(1) preparation of reference substance solution
Precision weighs drying under reduced pressure to the anhuienoside E of constant weight, glycoside St-14a, hemsgiganoside B, flaccidoside II, hederasaponin B reference substance are appropriate, and add 30% acetonitrile making concentration is 0.2～0.5 mg·mL-1Mixing reference substance solution, filter, to obtain final product；
(2) preparation of need testing solution
Take Rhizoma Anemones flaccidae medical material sample powder about 0.5g, accurately weighed, put in tool plug conical flask, accurate addition 50～100% Methanol, weighs, and supersound extraction lets cool, and supplies the weight of less loss, shakes up, and filters with 0.22 μm filter membrane, Take subsequent filtrate, to obtain final product；
(3) foundation of HPLC-UV chromatographic condition
Chromatographic column: octadecylsilane chemically bonded silica post；
Flowing phase: acetonitrile-0.01% trifluoroacetic acid solution (30:70)；
Detection wavelength: 200～220nm；
Flow velocity: 0.8～1.2mL min-1；
Column temperature: 20～50 DEG C；
Sample size: 2～20 μ L；
(4) structure of characteristic spectrum
Precision draws need testing solution and reference substance solution respectively, injects high performance liquid chromatograph and is measured, note Record chromatogram, uses " similarity evaluation (2004A version) " software to institute's Dry Sack Spectrogram processes, and obtains the comparison characteristic spectrum of Rhizoma Anemones flaccidae medical material, uses the method calculating relative retention time true Surely the identification at peak is had.
Rhizoma Anemones flaccidae medical material HPLC-UV characteristic spectrum construction method the most according to claim 1, its feature exists In, step (3) described chromatographic column is Phenomenex Luna C18Post, specification is 4.6mm × 250mm, 5 μm, or Cosmosil 5C18-MS-II post, specification is 4.6mm × 250mm, 5 μm, or Ultimate XB-C18Post, specification is 4.6mm × 250mm, 5 μm.
Rhizoma Anemones flaccidae medical material HPLC-UV characteristic spectrum construction method the most according to claim 1, its feature exists In, step (4) described Rhizoma Anemones flaccidae medical material characteristic spectrum has 5 total peaks, wherein, with No. 4 peak flaccidoside II is object of reference peak, and peak 1 is anhuienoside E, relative retention time 0.46；Peak 2 is glycoside St-14a, Relative retention time 0.53；Peak 3 is hemsgiganoside B, relative retention time 0.66；Peak 4 is Flaccidoside II, relative retention time 1.00；Peak 5 is hederasaponin B: relative retention time 1.07； The relative retention time of each chromatographic peak should setting ± 10% within.
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