CN102809616B - Mastic medicine fingerprint establishing method and standard fingerprint - Google Patents
Mastic medicine fingerprint establishing method and standard fingerprint Download PDFInfo
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- CN102809616B CN102809616B CN2012102974432A CN201210297443A CN102809616B CN 102809616 B CN102809616 B CN 102809616B CN 2012102974432 A CN2012102974432 A CN 2012102974432A CN 201210297443 A CN201210297443 A CN 201210297443A CN 102809616 B CN102809616 B CN 102809616B
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Abstract
The invention provides a mastic medicine high performance liquid chromatography fingerprint establishing method and a standard fingerprint. The method comprises the following steps: firstly, taking mastic medicine powder and adding the mastic medicine powder in a 95 % methanol aqueous solution, carrying out ultrasonic extraction, filtering liquid supernatant by a millipore filter and taking the filtrate as a sample solution; secondly, carrying out high performance liquid chromatography, wherein a chromatographic column is Fortis-C18 (5 mum, 250*4.6mm) and a mobile phase is 1% glacial acetic acid aqueous solution-methanol; adopting a gradient elution mode, wherein column temperature is 30 DEG C, a detector is an evaporative light scattering detector, drift tube temperature is 81 DEG C, and gas flow rate is 2.1 L/min; absorbing the sample solution for sample introduction and obtaining an HPLC-ELSD (high performance liquid chromatography-evaporative light scattering detector) fingerprint of the mastic medicine; comparing the HPLC-ELSD fingerprint of 12 groups of mastic medicine from different sources, and determining the common fingerprint characteristic to obtain the standard fingerprint. The quality of the mastic medicine can be effectively monitored by use of the standard fingerprint.
Description
Technical field
The present invention relates to the method for building up of Fingerprint of Chinese medicine materia, the method for building up of frankincense medicinal material HPLC-ELSD finger-print especially, and the resulting frankincense medicinal material of method standard finger-print thus.Belong to the Pharmaceutical Analysis technical field.
Background technology
Traditional Chinese medicine fingerprint refers in particular to some Chinese crude drug or Chinese medicine preparation after suitably processing, and adopts certain analysis means, the chromatogram that can indicate its chemical feature or the spectrogram that obtain.Fingerprint pattern technology is the important means of estimating traditional Chinese medicine quality, is conducive to realize the modernization of Chinese medicine.FDA (Food and Drug Adminstration) is in " FDA is about the guide of vegetable products " formulated in 1996, and requiring provides corresponding finger-print to product and autonomic drug product in the middle of plant material, autonomic drug.The World Health Organization (WHO) also stipulates in herbal medicine in 1996 is estimated governing principle, if the active component of herbal medicine is indefinite, can provide chromatographic fingerprinting to prove the consistent of product quality.China national drug regulatory department has issued " technical requirement (provisional) of the finger-print research of traditional Chinese medicine " in 2000, explicitly call for the traditional Chinese medicine of newly declaring and the traditional Chinese medicine gone on the market are carried out to the finger-print standard.
The traditional Chinese medicine fingerprint technology has related to numerous methods, comprises the spectroscopic methodologies such as thin layer chromatography scanning (TLCS), high performance liquid chromatography (HPLC), vapor-phase chromatography (GC) and high performance capillary electrophoresis (HPCE) isochrome spectrometry and ultraviolet spectroscopy (UV), infra-red sepectrometry (IR), mass spectroscopy (MS), nuclear magnetic resonance method (NMR) and X-ray diffraction method.Wherein chromatography is that main stream approach, especially HPLC, TLCS and GC have become three kinds of generally acknowledged conventional analysis means.The traditional Chinese medicine ingredients overwhelming majority can carry out analyzing and testing on high performance liquid chromatograph, so high performance liquid chromatography has become the prefered method of traditional Chinese medicine fingerprint technology.
Frankincense is the gum resin of olive subject plant Ka Shi Boswellia carterii, has the effect of regulating qi and activating blood, analgesic therapy, detumescence, myogenic, clinically is mainly used in controlling stagnation of QI-blood, trusted subordinate's pain, carbuncle pyogenic infections from tumour or sore, traumatic injury, arthralgia due to wind-dampness, dysmenorrhoea, postpartum blood stasis shouting pain etc.Frankincense ingredient complexity, principal ingredient resinous 60%～70%, natural gum 27%～35%, volatile oil 3%～8%, the principal ingredient of resin is for free α-masticinic acid, beta boswellic acid, in conjunction with boswellic acid, olibanoresene, natural gum is calcium salt and magnesium salts, western Radix Astragali gluing element and the amaroid of arabitic acid, and volatile oil contains firpene, amphene, sabinene, elemene, racemization-citrene etc.Due to the impact of the factors such as climate, the place of production, also there is some difference for different frankincense medicinal material ingredients.
" Chinese crude drug " 34(6) in 2011 " based on the frankincense evaluation of medical materials' quality researchs of principal component analysis (PCA) and similarity analysis " delivered, adopt respectively high performance liquid chromatograph-UV-detector, gas chromatograph, mass spectrometer to detect volatile ingredient and involatile constituent in the frankincense medicinal material, instrument is more, complex operation, and need to be by volatilization position and non-volatile position separate detection, testing cost is obviously very high.In addition, mobile phase is used acetonitrile, and acetonitrile belongs to medium malicious class reagent, have simultaneously the danger of mutagenicity and genotoxicity, and price is higher.And exist end in the frankincense medicinal material is absorbed to the shortcoming that the composition response is low and the drift of gradient elution base line is serious.Therefore, study a kind of easy operating, measure the method control accurately the frankincense quality of medicinal material and seem particularly important.
Summary of the invention
The method for building up that the purpose of this invention is to provide a kind of frankincense medicinal materials fingerprint, and the frankincense medicinal material standard finger-print that obtains of method thus.The present invention, by the research to frankincense medicinal material efficient liquid-phase chromatograph finger print atlas, has proposed a kind of quality of medicinal material of frankincense preferably control method, and the quality control of frankincense medicinal material is improved and science more.
The method for building up of frankincense medicinal material efficient liquid-phase chromatograph finger print atlas of the present invention comprises the following steps:
(1) preparation of need testing solution: precision takes medicinal powder 5g, puts in tool plug container, adds 90% methyl alcohol 200ml, and ultrasonic concussion 45min gets supernatant after cooling with 0.45 μ m filtering with microporous membrane, and filtrate is as need testing solution;
(2) making of finger-print: the chromatographic resolution column packing is 5 μ m Fortis-C
18, 250 * 4.6mm; Mobile phase is 1% glacial acetic acid aqueous solution-methyl alcohol; Adopt gradient elution mode 0min → 10min → 20min → 35min → 45min → 70min → 80min → 100min → 110min → 125min, methyl alcohol 60% → 60% → 75% → 85% → 85% → 88% → 90% → 95% → 100% → 100%; Flow velocity 1.0ml/min; 30 ℃ of column temperatures; Detecting device is evaporative light-scattering detector, 85 ℃ of drift tube temperatures, gas flow rate 2.3L/min; Sample size is 20 μ l; Record the chromatogram of 140 minutes, obtain the finger-print of frankincense medicinal material;
(3) determining of standard finger-print: determined 15 common characteristic peaks, take No. 3 peaks of object of reference is that 11-carbonyl-beta-acetyl masticinic acid peak is the R peak, calculate the relative retention time at each common characteristic peak and R peak, its relative retention time should setting ± 5% in, setting is: 0.801,0.813,1.000,1.039,1.096,1.188,1.225,1.297,1.354,1.468,1.538,1.690,1.715,1.856,1.919, these common characteristic peaks have formed the fingerprint characteristic of frankincense medicinal material, as the standard finger-print of frankincense medicinal material.
The present invention has following significant advantage and purposes:
(1) the frankincense medicinal material is done to as a whole treating, focus on front and back order and mutual relationship that each forms the fingerprint characteristic peak, focus on whole facial feature, avoided judging because only measuring wherein one or two chemical composition the one-sidedness of frankincense medicinal material total quality, reduced as the artificial possibility of processing of requisite quality.By comparing its total peak, the nuance between different medicinal materials can be found out, for quality complete, accurate evaluation frankincense medicinal material provides new reference standard, thereby the quality of frankincense medicinal material can better be controlled;
(2) the present invention have that assay method is easy and simple to handle, precision is high, good stability, favorable reproducibility, the characteristics that are easy to grasp;
(3) the mobile phase preparation is simple, and chromatographic condition is easily realized;
(4) be applicable to discriminating and the control to the frankincense medicinal material true and false, the place of production and quality.
The accompanying drawing explanation
Fig. 1 is the standard finger-print of frankincense medicinal material provided by the invention, in figure, has from left to right marked respectively No. 1 to 15, its common characteristic peak;
Fig. 2 is the HPLC-ELSD finger-print of 12 batches of frankincense medicinal materials of separate sources.
Embodiment
To those skilled in the art, according to technology contents disclosed by the invention, those skilled in the art will very clear other embodiment of the present invention, and the embodiment of the present invention is only as example.In the situation that do not violate purport of the present invention and scope, can carry out various changes and improvements to the present invention, but as long as use method of quality control of the present invention, all within protection domain of the present invention.
Embodiment mono-: the finger-print that detects the frankincense medicinal material
(1) instrument and reagent
Instrument: Shimadzu LC-20A high performance liquid chromatograph comprises LC-20AT quaternary gradient pump, DGU-20A
5Online degasser, CTO-10AS column oven, SIL-20A automatic sampler, LCsolution workstation; Alltech2000ES type evaporative light-scattering detector; HT-300 type ultrasonator (Ji Ninghai rises ultrasonic electronic equipment company limited); Electronic balance (Sai Duolisi scientific instrument company limited).
Reagent: methyl alcohol (chromatographically pure, U.S. TEDIA company), glacial acetic acid (analyze pure, Tianjin BASF chemical industry company limited), Wahaha Pure Water.
(2) preparation of need testing solution: precision takes medicinal powder 5g, puts in tool plug container, adds 90% methyl alcohol 200ml, and ultrasonic concussion 45min gets supernatant with 0.45 μ m filtering with microporous membrane, and filtrate is as need testing solution.
(3) efficient liquid phase chromatographic analysis: chromatographic column filler 5 μ m Fortis-C
18, 520 * 4.6mm; Mobile phase 1% glacial acetic acid aqueous solution-methyl alcohol; Adopt gradient elution mode 0min → 10min → 20min → 35min → 45min → 70min → 80min → 100min → 110min → 125min, methyl alcohol 60% → 60% → 75% → 85% → 85% → 88% → 90% → 95% → 100% → 100%; Flow velocity 1.0ml/min; 30 ℃ of column temperatures; Detecting device is evaporative light-scattering detector, 85 ℃ of drift tube temperatures, gas flow rate 2.3L/min; Sample size is 20 μ l.
(4) measure: accurate need testing solution and each 20 μ l injection liquid chromatographies of reference substance solution drawn, according to high effective liquid chromatography for measuring, record the chromatogram of 140 minutes, obtain the finger-print of frankincense medicinal material, as shown in Figure 1.
Embodiment bis-: the finger-print that detects 12 batches of frankincense medicinal materials of separate sources
(1) get 12 batches of frankincense medicinal materials of separate sources, detect by the condition of embodiment mono-, obtain the HPLC-ELSD finger-print of 12 batch samples, as shown in Figure 2.The comparison of the HPLC-ELSD finger-print by 12 batches of frankincense medicinal materials, carry out similarity evaluation, 15 common characteristic peaks have been determined altogether, take No. 3 peaks of object of reference is that 11-carbonyl-beta-acetyl masticinic acid peak is the R peak, calculate the relative retention time at each common characteristic peak and R peak, its relative retention time should setting ± 5% in, setting is: 0.801(peak 1), 0.813(peak 2), 1.000(peak R), 1.039(peak 4), 1.096(peak 5), 1.188(peak 6), 1.225(peak 7), 1.297(peak 8), 1.354(peak 9), 1.468(peak 10), 1.538(peak 11), 1.690(peak 12), 1.715(peak 13), 1.856(peak 14), 1.919(peak 15), these common characteristic peaks have formed the fingerprint characteristic of frankincense medicinal material, can be used as the standard finger-print of frankincense medicinal material.
(2) similarity evaluation: " similarity evaluation " software (2004A version) that adopts Chinese Pharmacopoeia Commission to recommend is estimated collection of illustrative plates, and similarity evaluation the results are shown in Table 1.
Table 1HPLC-ELSD fingerprint similarity evaluation result
The test sample finger-print calculates through " similarity evaluation " software, and similarity is greater than 0.900.
Claims (1)
1. the method for building up of a frankincense medicinal material high performance liquid chromatography finger-print comprises the following steps:
(1) preparation of need testing solution: precision takes medicinal powder 5g, puts in tool plug container, adds 90% methyl alcohol 200ml, and ultrasonic concussion 45min gets supernatant after cooling with 0.45 μ m filtering with microporous membrane, and filtrate is as need testing solution;
(2) making of finger-print: the chromatographic resolution column packing is 5 μ m Fortis-C18,250 * 4.6mm; Mobile phase is 1% glacial acetic acid aqueous solution-methyl alcohol; Adopt gradient elution mode 0min → 10min → 20min → 35min → 45min → 70min → 80min → 100min → 110min → 125min, methyl alcohol 60% → 60% → 75% → 85% → 85% → 88% → 90% → 95% → 100% → 100%; Flow velocity 1.0ml/min; 30 ℃ of column temperatures; Detecting device is evaporative light-scattering detector, 85 ℃ of drift tube temperatures, gas flow rate 2.3L/min; Sample size is 20 μ l; Record the chromatogram of 140 minutes, obtain the finger-print of frankincense medicinal material;
(3) determining of standard finger-print: determined 15 common characteristic peaks, take No. 3 peaks of object of reference is that 11-carbonyl-beta-acetyl masticinic acid peak is the R peak, calculate the relative retention time at each common characteristic peak and R peak, its relative retention time should setting ± 5% in, setting is: 0.801,0.813,1.000,1.039,1.096,1.188,1.225,1.297,1.354,1.468,1.538,1.690,1.715,1.856,1.919, these common characteristic peaks have formed the fingerprint characteristic of frankincense medicinal material, as the standard finger-print of frankincense medicinal material.
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| CN101380362A (en) * | 2008-10-24 | 2009-03-11 | 大连美罗中药厂有限公司 | Quality control method of volatile ingredients fingerprint of bone knitting medicine of traumatology |
| CN101721475A (en) * | 2008-12-11 | 2010-06-09 | 承德颈复康药业集团有限公司 | Quality control method for Yaotongning capsules |
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Denomination of invention: Mastic medicine fingerprint establishing method and standard fingerprint Effective date of registration: 20181213 Granted publication date: 20131127 Pledgee: CITIC Bank, Limited by Share Ltd, Zibo branch Pledgor: Qidu Pharmaceutical Co., Ltd., Shandong Prov. Registration number: 2018370000224 |
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Date of cancellation: 20200426 Granted publication date: 20131127 Pledgee: CITIC Bank, Limited by Share Ltd, Zibo branch Pledgor: SHANDONG QIDU PHARMACEUTICAL Co.,Ltd. Registration number: 2018370000224 |