CN104007198B - A kind of glossy ganoderma emperor's preparation HPLC standard finger-print and construction method thereof and application - Google Patents
A kind of glossy ganoderma emperor's preparation HPLC standard finger-print and construction method thereof and application Download PDFInfo
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Abstract
The invention discloses glossy ganoderma emperor preparation HPLC standard finger-print and construction method thereof and application, this construction method, comprise the preparation of need testing solution and reference substance solution, adopt linear gradient elution, mobile phase A is acetonitrile, Mobile phase B is 0.05 ~ 0.15% (v/v) phosphate aqueous solution, determined wavelength is 252nm, column temperature is 25 ~ 35 DEG C, flow velocity is 1.0mL/min, measure with high performance liquid chromatography, obtain glossy ganoderma emperor preparation finger, using the common characteristic peak of at least 14 batches of finger-prints as the characteristic peak of standard finger-print; Standard finger-print has 40 characteristic peaks, belong to glossy ganoderma, the bighead atractylodes rhizome, sealwort, the seed of Chinese dodder, the fruit of Chinese magnoliavine, Radix Glycyrrhizae, the fruit of Chinese wolfberry characteristic peak have 20,3,3,5,1,5,2 respectively.The inventive method and standard finger-print can comprehensively, the total quality of accurate evaluation Lingzhu ', be conducive to the quality and the drug effect that ensure Lingzhu '.
Description
Technical field
The invention belongs to Pharmaceutical Analysis technical field, relate to a kind of analyzing detecting method of glossy ganoderma emperor preparation, be specifically related to a kind of glossy ganoderma emperor preparation HPLC standard finger-print and construction method thereof and application.
Background technology
Medicine finger-print means that Chinese medicine material medicinal material, medicine materical crude slice, middle product, finished product etc. are after suitably processing, and adopt certain analysis means, the collection of illustrative plates that can indicate the total peak of its feature obtained.Medicine finger-print can disclose and reflect globality and the characteristic of medicine inherent quality objectively, in order to evaluate the authenticity of medicine, validity, stability and consistance.The clinical efficacy of medicine is not by the effect of single-activity composition or the simple addition of various active composition effect, and compound preparation is all the more so.Be quality index with known some or several active component or effective constituent, being judged the quality of drug quality by qualitative and quantitative analysis, is obviously incomplete.The evaluation of drug quality needs comprehensive, macroscopic view, nonlinear quality evaluation system, and medicine finger-print is exactly a kind of quality assessment and the control model that can adapt to this feature preferably.In the world, as many countries such as Japan, Germany, the U.S., Canada, India, to some traditional medicines, crude drug and herbal medicine, all using the content one of of finger-print as quality control standard.The European Community also claims in the guide of herbal quality, and it is inadequate for depending merely on the stability measuring certain effective constituent investigation quality, because herbal medicine and preparation thereof take entirety as active substance.The World Health Organization (WHO) (WHO) evaluates in governing principle at herbal medicine in 1996 and specifies: if the active component of herbal medicine is failed to understand, feature spectrogram can be provided to compose, to prove the consistent of product quality.Therefore finger-print is one of medicine modernization and international gordian technique.
Glossy ganoderma emperor capsule is according to Traditional Chinese medical theory, by modern study achievement and practical experience, take glossy ganoderma as primary raw material, the auxiliary health products refined with the bighead atractylodes rhizome, sealwort, the fruit of Chinese wolfberry, Poria cocos, the tuber of dwarf lilyturf, the seed of Chinese dodder, the fruit of Chinese magnoliavine, coix seed, Radix Glycyrrhizae, prove through animal experiment, this product has immunological regulation, delay senility and the health-care effect of auxiliary Tumor suppression.Glossy ganoderma emperor preparation has the pharmacological action of develop immunitypty, is not directly act on cancer, but can assist Tumor suppression.It is by using modern high technology, extracts red ganoderma antineoplastic effective composition, and theoretical according to compound Chinese prescription, chooses multiple Chinese traditional herbs scientific compatibility and makes, can assist Tumor suppression, lower the trouble cancer chance of cancer people at highest risk.
Current glossy ganoderma emperor preparation still lacks method of quality control comprehensively and effectively, quality standard imperfection, is difficult to overall monitor finished product, the quality of middle product and the stability of production technology.Ling Yuanyu etc. are at the document assay of polysaccharide " in the glossy ganoderma emperor capsule " basic unit's J Chinese, 1997,11 (3): 40 report the content that anthrone method measures glossy ganoderma emperor capsule polysaccharide, but are only confined to the detection of individual components, can not reflect the characteristic of this product comprehensively.
Summary of the invention
In order to solve the problem, the present invention establish a kind of rationally, the standard finger-print of accurate evaluation glossy ganoderma emperor preparation globality quality, and the construction method of this finger-print.
The object of the present invention is to provide a kind of glossy ganoderma emperor preparation HPLC standard finger-print.
Another object of the present invention is to the construction method that glossy ganoderma emperor preparation HPLC standard finger-print is provided.
Another object of the present invention is the application providing glossy ganoderma emperor preparation HPLC standard finger-print.
The technical solution used in the present invention is:
A kind of glossy ganoderma emperor preparation HPLC standard finger-print, has 40 common characteristic peaks, is numbered 1 to 40 respectively in this finger-print, with numbering 16 characteristic peak for reference peak, the characteristic peak relative retention time being numbered 1 to 40 respectively is: 0.095 ~ 0.099min, 0.109 ~ 0.113min, 0.121 ~ 0.125min, 0.165 ~ 0.169min, 0.199 ~ 0.205min, 0.245 ~ 0.247min, 0.368 ~ 0.372min, 0.387 ~ 0.393min, 0.434 ~ 0.438min, 0.549 ~ 0.551min, 0.603 ~ 0.605min, 0.873 ~ 0.875min, 0.889 ~ 0.891min, 0.897 ~ 0.899min, 0.915 ~ 0.917min, 1.000min, 1.065 ~ 1.067min, 1.121 ~ 1.123min, 1.155 ~ 1.157min, 1.181 ~ 1.183min, 1.361 ~ 1.363min, 1.395 ~ 1.397min, 1.447 ~ 1.449min, 1.458 ~ 1.460min, 1.489 ~ 1.493min, 1.507 ~ 1.511min, 1.576 ~ 1.580min, 1.612 ~ 1.615min, 1.629 ~ 1.633min, 1.682 ~ 1.686min, 1.696 ~ 1.700min, 1.706 ~ 1.710min, 1.764 ~ 1.768min, 1.803 ~ 1.805min, 1.835 ~ 1.839min, 1.853 ~ 1.857min, 1.866 ~ 1.868min, 1.876 ~ 1.878min, 1.886 ~ 1.890min, 1.930 ~ 1.932min.
Further, be above-mentionedly numbered in 40 common characteristic peaks of 1 to 40:
The characteristic peak belonging to glossy ganoderma is numbered 1,2,3,21,22,23,25,26,27,28,29,30,31,32,33,34,35,37,38,40 and has 20 altogether, and wherein No. 31 peaks are ganoderic acid A;
The characteristic peak belonging to the seed of Chinese dodder is numbered 9,10,14,15,16 and has 5 altogether, and wherein No. 14 peaks are Hyperoside, No. 15 peaks are isoquercitrin, No. 16 peaks are astragalin;
The characteristic peak belonging to Radix Glycyrrhizae is numbered 12,13,18,19,36 and has 5 altogether, and wherein No. 12 peaks are liquiritin, No. 36 peaks are glycyrrhizic acid;
The total peak belonging to sealwort is numbered 4,5,6 and has 3 altogether;
The characteristic peak belonging to the bighead atractylodes rhizome is numbered 10,17,24 and has 3 altogether;
The characteristic peak belonging to the fruit of Chinese magnoliavine is numbered 39 and has 1 altogether, and No. 39 peaks are schizandrin.
The characteristic peak belonging to the fruit of Chinese wolfberry is numbered 9,11 and has 2 altogether.
Further, above-mentioned glossy ganoderma emperor preparation is made up of glossy ganoderma, the bighead atractylodes rhizome, coix seed, sealwort, the fruit of Chinese wolfberry, Poria cocos, the tuber of dwarf lilyturf, the seed of Chinese dodder, the fruit of Chinese magnoliavine, Radix Glycyrrhizae 10 taste medicinal material.
A construction method for glossy ganoderma emperor preparation HPLC standard finger-print, comprises the following steps:
1) glossy ganoderma emperor preparation need testing solution is prepared;
2) prepare reference substance solution: precision takes ganoderic acid A, liquiritin, ammonium glycyrrhetate, Hyperoside, isoquercitrin, astragalin, schizandrin, add methyl alcohol and dissolve, make the reference substance solution that concentration is known respectively;
3) foundation of the HPLC finger-print of glossy ganoderma emperor preparation: draw reference substance solution and need testing solution respectively, sample introduction, measure with high performance liquid chromatograph, linear gradient elution program is as following table:
With numbering 16 characteristic peak for the retention time with reference to peak is for 1, calculate the relative retention time of each characteristic peak of test sample;
4) standard finger-print of glossy ganoderma emperor preparation HPLC: the finger-print measuring at least 10 batches of glossy ganoderma emperor formulation samples need testing solutions, the finger-print be made up of common characteristic peak in each finger-print is the standard finger-print of glossy ganoderma emperor preparation HPLC.
Further, step 1) in the concrete operations of preparation glossy ganoderma emperor preparation need testing solution be: precision takes glossy ganoderma emperor capsule 's content, be placed in the conical flask of band plug, add methyl alcohol, the concentration of glossy ganoderma emperor preparation is made to be 0.25 ~ 1.0g/mL, close plug, weighed weight, ultrasonic process 40 ~ 60min, leave standstill and let cool, weighed weight again, the weight of less loss is made up with methyl alcohol, shake up, filter, discard just filtrate, precision measures subsequent filtrate, put evaporate to dryness in evaporating dish, residue methyl alcohol dissolves, and with 1 ~ 2mL volumetric flask constant volume, shake up, filter, get subsequent filtrate, .
Further, step 3) described in the condition of high performance liquid chromatograph when measuring be mobile phase A be acetonitrile, Mobile phase B to be percent by volume be 0.05 ~ 0.15% phosphate aqueous solution, column temperature is 25 ~ 35 DEG C, flow velocity is 1.0mL/min, and determined wavelength is 252nm.
Further, step 4) described in the similarity of the HPLC finger-print of each batch of glossy ganoderma emperor formulation samples and the standard finger-print of glossy ganoderma emperor preparation HPLC should be greater than 0.9.
The invention has the beneficial effects as follows:
1) the present invention sets up glossy ganoderma emperor preparation HPLC standard finger-print, represent most chemical composition in glossy ganoderma emperor finished dosage form, effectively can characterize the quality of glossy ganoderma emperor finished dosage form, thus realize detecting the chemical composition of glossy ganoderma emperor preparation maximum possible, be conducive to the overall monitor to end product quality.
2) the present invention sets up glossy ganoderma emperor preparation HPLC standard finger-print integrally, focus on the tandem that each forms fingerprint characteristic peak, more holistic facial feature, both the one-sidedness judging glossy ganoderma emperor preparation total quality because only measuring one, two chemical composition had been avoided, the present invention be more comprehensively, the quality of accurate evaluation glossy ganoderma emperor preparation provides new method, will more ensure the quality of ginseng-astragalus blood-sugar lowering preparation and clinical curative effect thereof.
3) the present invention has that method is easy, good stability, precision are high, favorable reproducibility, is applicable to the global quality control of glossy ganoderma emperor preparation.
4) finger-print that the present invention sets up can express the feature of this product, gives the raw medicinal material that all common characteristic peaks belong to respectively, and gives the concrete material wherein representated by 7 common characteristic peaks, have good specificity.
5) the present invention adopts the detection method of finger-print, focus on contiguity and the globality of characteristic peak, reflect internal characteristics and the quality of compound preparation comprehensively, avoid unicity and the one-sidedness of glossy ganoderma emperor quality of the pharmaceutical preparations control, rationally evaluate the quality of medicine exactly, ensure the validity of preparation; And may be used for the qualitative detection of raw medicinal material, intermediate and finished product preparation, the quality of comprehensive control medicinal material, applied widely.
Accompanying drawing explanation
Fig. 1 is that the HPLC of glossy ganoderma emperor capsule at 252nm wavelength place is with reference to finger-print;
Fig. 2 mixes target HPLC finger-print at 252nm wavelength place;
Fig. 3 is that a HPLC fingerprint similarity evaluation result schematic diagram criticized by glossy ganoderma emperor capsule;
Fig. 4 is glossy ganoderma emperor capsule fingerprint pattern glossy ganoderma composition synergy collection of illustrative plates;
Fig. 5 is glossy ganoderma emperor capsule fingerprint pattern seed of Chinese dodder composition synergy collection of illustrative plates;
Fig. 6 is glossy ganoderma emperor capsule fingerprint pattern licorice ingredient synergy collection of illustrative plates;
Fig. 7 is glossy ganoderma emperor capsule fingerprint pattern sealwort composition synergy collection of illustrative plates;
Fig. 8 is glossy ganoderma emperor capsule fingerprint pattern bighead atractylodes rhizome composition synergy collection of illustrative plates;
Fig. 9 is glossy ganoderma emperor capsule fingerprint pattern Chemical composition of Wuweizi synergy collection of illustrative plates;
Figure 10 is glossy ganoderma emperor capsule fingerprint pattern fruit of Chinese wolfberry composition synergy collection of illustrative plates;
Figure 11 is glossy ganoderma emperor capsule precision test similarity evaluation result schematic diagram;
Figure 12 is glossy ganoderma emperor capsule stability test similarity evaluation result schematic diagram;
Figure 13 is glossy ganoderma emperor capsule replica test similarity evaluation result schematic diagram;
Figure 14 is the finger-print of glossy ganoderma emperor capsule at 252nm wavelength place in embodiment 2;
Figure 15 is the finger-print of glossy ganoderma emperor capsule at 252nm wavelength place in embodiment 3;
Figure 16 is the finger-print of glossy ganoderma emperor capsule at 252nm wavelength place in embodiment 4;
Figure 17 is the HPLC reference finger-print that glossy ganoderma emperor is dusted at 252nm wavelength place;
Figure 18 is HPLC fingerprint similarity evaluation result schematic diagram between glossy ganoderma emperor is dusted batch.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but is not limited thereto.
Embodiment 1:
1, instrument, reagent
Instrument: Agilent1200 high performance liquid chromatograph (quaternary pump, column oven, DAD detecting device and Agilent1200 chromatographic work station), Agilent company of the U.S.; The special ultrapure water machine in laboratory, Chongqing profit enlightening modern water technical equipment company limited; BP211D type analysis balance, German sartorius company; HWS24 type electric-heated thermostatic water bath, the permanent Science and Technology Ltd. in Shanghai one; KQ-400KPE ultrasonic washing instrument, Kunshan Ultrasonic Instruments Co., Ltd..
Reference substance:
Glossy ganoderma emperor capsule: unlimited pole (China) company limited product is capsule.Glossy ganoderma emperor preparation is mainly made up of medicinal materials such as glossy ganoderma, the bighead atractylodes rhizome, sealwort, the fruit of Chinese wolfberry, Poria cocos, the tuber of dwarf lilyturf, the seed of Chinese dodder, the fruit of Chinese magnoliavine, coix seed, Radix Glycyrrhizaes.
Contrast raw medicinal material: glossy ganoderma, the bighead atractylodes rhizome, sealwort, the seed of Chinese dodder, the fruit of Chinese magnoliavine, Radix Glycyrrhizae, the fruit of Chinese wolfberry provide by unlimited pole (China) company limited that (Poria cocos in glossy ganoderma emperor finished dosage form, coix seed, the chromatographic peak area of the tuber of dwarf lilyturf are very little, can ignore, therefore raw medicinal material can be contrasted without it).
2, the condition determination of high performance liquid chromatography
Chromatographic column: Kromasil100-5C
18(4.6 × 250mm i.d., 5 μm); Mobile phase: take acetonitrile as mobile phase A take percent by volume as the phosphate aqueous solution of 0.1% is Mobile phase B; Type of elution is: linear gradient elution; Determined wavelength is: 252nm; Column temperature: 30 DEG C; Flow velocity: 1.0mL/min.Linear gradient elution program is as shown in table 1 below.
Table 1 gradient elution program
3, the foundation of HPLC standard finger-print
1) preparation of test sample and reference substance
The preparation of glossy ganoderma emperor need testing solution: get glossy ganoderma emperor capsule 's content 2.0g, accurately weighed, be placed in the conical flask of band plug, precision adds methyl alcohol 50mL, close plug, weighed weight, ultrasonic process 50 minutes (power 500W, frequency 40KHz), leave standstill and let cool, more weighed weight, the weight of less loss is made up with methyl alcohol, shake up, filter, precision measures subsequent filtrate 25mL, put evaporate to dryness in evaporating dish, residue methyl alcohol dissolves, and puts 2mL volumetric flask, adds methyl alcohol to scale, shake up, filter, get subsequent filtrate, obtain glossy ganoderma emperor capsule test sample;
The preparation of reference substance solution: precision takes appropriate ganoderic acid A, liquiritin, ammonium glycyrrhetate, Hyperoside, isoquercitrin, astragalin, schizandrin, adds methyl alcohol and dissolves, make corresponding reference substance solution respectively; The concentration of each reference substance solution is respectively: ganoderic acid A concentration is 0.480mg/mL, liquiritin concentration is 0.345mg/mL, ammonium glycyrrhetate concentration is 0.109mg/mL, Hyperoside concentration is 0.0556mg/mL, isoquercitrin concentration is 0.054mg/mL, astragalin concentration is 0.113mg/mL, schizandrin concentration is 0.056mg/mL.
2) foundation of glossy ganoderma emperor HPLC finger-print
Accurate absorption reference substance solution, each 5 μ L of glossy ganoderma emperor capsule need testing solution respectively, sample introduction, measures with high performance liquid chromatograph, obtains chromatogram.The relatively finger-print of test sample and the finger-print of reference substance, determine the chromatographic peak of each main Chinese medicinal materials composition, namely obtain glossy ganoderma emperor capsule HPLC finger-print, see accompanying drawing 1, can find out in glossy ganoderma emperor capsule HPLC finger-print have 1 ~ 40 totally 40 characteristic peaks from accompanying drawing 1.With numbering 16 characteristic peak for the retention time with reference to peak is for 1, calculate the relative retention time of test sample 40 characteristic peaks.
Carry out qualitative to the principal character peak in accompanying drawing 1, by glossy ganoderma emperor capsule HPLC finger-print and reference substance finger-print are compared, determine that No. 12 peaks are liquiritin, No. 14 peaks are Hyperoside, No. 15 peaks are isoquercitrin, No. 16 peaks are astragalin, No. 31 peaks are ganoderic acid A, No. 36 peaks are glycyrrhizic acid; No. 39 peaks are schizandrin, mixed target finger-print as shown in Figure 2.
3) foundation of glossy ganoderma emperor capsule HPLC standard finger-print
Whether representative in order to determine the glossy ganoderma emperor capsule HPLC finger-print shown in accompanying drawing 1 further, with the finger-print in accompanying drawing 1 for reference fingerprint, separately get the glossy ganoderma emperor capsule of 14 batches of different lot numbers, prepare glossy ganoderma emperor capsule need testing solution after the same method, and measured by high performance liquid chromatograph, " similarity evaluation 2004A version " software is adopted to carry out data processing: using 14 batches and control group finger-print as sample set, the comparison diagram between finger-print is generated through software systems, as accompanying drawing 3, 40 characteristic peaks in visible accompanying drawing 1 finger-print are the common characteristic peak of glossy ganoderma emperor capsule, in addition, calculate the similarity of these 14 batches of collection of illustrative plates and accompanying drawing 1 collection of illustrative plates, evaluate batch between stability, the similarity that result shows between this 14 batch sample and accompanying drawing 1 is good, all more than 0.96, as shown in table 2, illustrate that accompanying drawing 1 is representative, be glossy ganoderma emperor capsule HPLC standard finger-print.
The similarity of finger-print between the glossy ganoderma emperor capsule sample of table 2 different lot number
According to above result, calculate each common characteristic peak relative retention time in glossy ganoderma emperor's capsule HPLC standard finger-print (Fig. 1 and Fig. 3), adopt peak 16 for calculating other common characteristic peak relative retention times respectively with reference to peak (S), the relative retention time being numbered the common characteristic peak of 1 to 40 respectively is: 0.095 ~ 0.099min, 0.109 ~ 0.113min, 0.121 ~ 0.125min, 0.165 ~ 0.169min, 0.199 ~ 0.205min, 0.245 ~ 0.247min, 0.368 ~ 0.372min, 0.387 ~ 0.393min, 0.434 ~ 0.438min, 0.549 ~ 0.551min, 0.603 ~ 0.605min, 0.873 ~ 0.875min, 0.889 ~ 0.891min, 0.897 ~ 0.899min, 0.915 ~ 0.917min, 1.000min, 1.065 ~ 1.067min, 1.121 ~ 1.123min, 1.155 ~ 1.157min, 1.181 ~ 1.183min, 1.361 ~ 1.363min, 1.395 ~ 1.397min, 1.447 ~ 1.449min, 1.458 ~ 1.460min, 1.489 ~ 1.493min, 1.507 ~ 1.511min, 1.576 ~ 1.580min, 1.612 ~ 1.615min, 1.629 ~ 1.633min, 1.682 ~ 1.686min, 1.696 ~ 1.700min, 1.706 ~ 1.710min, 1.764 ~ 1.768min, 1.803 ~ 1.805min, 1.835 ~ 1.839min, 1.853 ~ 1.857min, 1.866 ~ 1.868min, 1.876 ~ 1.878min, 1.886 ~ 1.890min, 1.930 ~ 1.932min.
4, the ownership of each characteristic peak in glossy ganoderma emperor capsule HPLC standard finger-print
In order to determine in accompanying drawing 1 40 characteristic peaks belong to which kind of composition in raw medicinal material respectively, carry out following experiment further.
1) preparation of raw medicinal material need testing solution: respectively by glossy ganoderma, the bighead atractylodes rhizome, sealwort, the fruit of Chinese wolfberry, the seed of Chinese dodder, the fruit of Chinese magnoliavine, this seven tastes raw medicinal material of Radix Glycyrrhizae, obtain after the middle product of corresponding raw medicinal material according to the prescription proportioning of each taste medicine to formulation and technology, put in band plug conical flask, precision adds methyl alcohol 50mL, close plug, weighed weight, ultrasonic process 50 minutes (power 500W, frequency 40KHz), leave standstill and let cool, weighed weight again, the weight of less loss is made up with methyl alcohol, shake up, filter, discard just filtrate, precision measures subsequent filtrate 25mL, put evaporate to dryness in evaporating dish, residue methyl alcohol dissolves, put 2mL volumetric flask, add methyl alcohol to scale, shake up, filter, get subsequent filtrate, obtain,
Above-mentioned raw materials medicinal material glossy ganoderma, the bighead atractylodes rhizome, sealwort, the fruit of Chinese wolfberry, the seed of Chinese dodder, the fruit of Chinese magnoliavine, Radix Glycyrrhizae provide by unlimited pole (China) company limited.
2) preparation of negative need testing solution: get scarce glossy ganoderma sample respectively, lack bighead atractylodes rhizome sample, lack sealwort sample, lack seed of Chinese dodder sample, lack the sample tuber of dwarf lilyturf, lack fruit of Chinese wolfberry sample, lack Radix Glycyrrhizae sample, lack fruit of Chinese magnoliavine sample appropriate (as, lacking glossy ganoderma sample refers to compared with glossy ganoderma emperor capsule, not containing glossy ganoderma, other all the same with glossy ganoderma emperor), after being lacked the middle product of taste medicinal material accordingly according to formulation and technology, put in band plug conical flask, precision adds methyl alcohol 50mL, close plug, weighed weight, ultrasonic process 50 minutes (power 500W, frequency 40KHz), leave standstill and let cool, weighed weight again, the weight of less loss is made up with methyl alcohol, shake up, filter, discard just filtrate, precision measures subsequent filtrate 25mL, put evaporate to dryness in evaporating dish, residue methyl alcohol dissolves, put 2mL volumetric flask, add methyl alcohol to scale, shake up, filter, get subsequent filtrate, obtain.
3) characteristic peak analysis:
Accurate absorption glossy ganoderma emperor capsule need testing solution, each raw medicinal material need testing solution, each 5 μ L of negative need testing solution respectively, sample introduction, measures with high performance liquid chromatograph, as shown in accompanying drawing 4 ~ 10, wherein:
The characteristic peak belonging to glossy ganoderma is numbered 1,2,3,21,22,23,25,26,27,28,29,30,31,32,33,34,35,37,38,40 and has 20 altogether, wherein No. 31 peaks are ganoderic acid A, as shown in Figure 4, " lack glossy ganoderma " in figure and represent negative test sample, it is not except containing except glossy ganoderma, and other compositions are all the same with glossy ganoderma emperor capsule test sample;
The characteristic peak belonging to the seed of Chinese dodder is numbered 9,10,14,15,16 and has 5 altogether, wherein No. 14 peaks are Hyperoside, No. 15 peaks are isoquercitrin, No. 16 peaks are astragalin, as shown in Figure 5, " lack the seed of Chinese dodder " in figure and represent negative test sample, except not containing except the seed of Chinese dodder, other compositions are all the same with glossy ganoderma emperor capsule test sample;
The characteristic peak belonging to Radix Glycyrrhizae is numbered 12,13,18,19,36 and has 5 altogether, wherein No. 12 peaks are liquiritin, No. 36 peaks are glycyrrhizic acid, as shown in Figure 6, " lack Radix Glycyrrhizae " and represent negative test sample in figure, except not containing except Radix Glycyrrhizae, other compositions are all the same with glossy ganoderma emperor capsule test sample;
The total peak belonging to sealwort is numbered 4,5,6 and has 3 altogether, and as shown in Figure 7, " lack sealwort " and represent negative test sample in figure, it is not except containing except sealwort, and other compositions are all the same with glossy ganoderma emperor capsule test sample;
The characteristic peak belonging to the bighead atractylodes rhizome is numbered 10,17,24 and has 3 altogether, and as shown in Figure 8, " lack the bighead atractylodes rhizome " and represent negative test sample in figure, it is not except containing except the bighead atractylodes rhizome, and other compositions are all the same with glossy ganoderma emperor capsule test sample;
The characteristic peak belonging to the fruit of Chinese magnoliavine is numbered 39 and has 1 altogether, and No. 39 peaks are schizandrin, and as shown in Figure 9, " lack the fruit of Chinese magnoliavine " and represent negative test sample in figure, except not containing except the fruit of Chinese magnoliavine, other compositions are all the same with glossy ganoderma emperor capsule test sample;
The characteristic peak belonging to the fruit of Chinese wolfberry is numbered 9,11 and has 2 altogether, and as shown in Figure 10, " lack the fruit of Chinese wolfberry " and represent negative test sample in figure, except not containing except the fruit of Chinese wolfberry, other compositions are all the same with glossy ganoderma emperor capsule test sample.
Except 7,8, No. 20 total peaks fail to sort out, other all belong to corresponding raw medicinal material, and wherein No. 9 peaks are the common characteristic peak of the seed of Chinese dodder, the fruit of Chinese wolfberry two kinds of medicinal materials, and No. 10 peaks are the common characteristic peak of the bighead atractylodes rhizome, the seed of Chinese dodder two kinds of medicinal materials.
5, precision detects
Get same glossy ganoderma emperor capsule solution, continuous sample introduction 6 times, obtain finger-print (S1 ~ S6), as accompanying drawing 11 (wherein reference fingerprint obtained for 6 times for the continuous sample introduction in accompanying drawing 11); Adopt " similarity evaluation 2004A version " software to evaluate, result display instrument precision is good, and similarity is all greater than 0.9, as shown in table 3.
Table 3 glossy ganoderma emperor capsule fingerprint pattern precision test similarity result
6, Detection of Stability
Get with a collection of glossy ganoderma emperor capsule solution, respectively at 0h (S1), 4h (S2), 8h (S3), 12h (S4), 24h (S5), 48h (S6) sample introduction, record finger-print, as accompanying drawing 12 (wherein reference fingerprint obtained for 6 sample introductions in accompanying drawing 12); Adopt " similarity evaluation 2004A version " software to evaluate, result show sample has good stability, and similarity is all greater than 0.9, as shown in table 4.
Table 4 glossy ganoderma emperor capsule fingerprint pattern stability test similarity result
7, repeatability detects
Get with a collection of glossy ganoderma emperor capsule 6 parts, 6 parts of test samples are prepared according to medicinal material sample solution preparation method, respectively sample introduction, records finger-print (S1 ~ S6), as accompanying drawing 13 (wherein reference fingerprint obtained for 6 sample introductions in accompanying drawing 13); Adopt " similarity evaluation 2004A version " software to evaluate, result show sample repeatability is good, and similarity is all greater than 0.9, as shown in table 5.
Table 5 glossy ganoderma emperor capsule fingerprint pattern replica test similarity result.
Embodiment 2:
1, instrument, reagent are with embodiment 1.
2, the condition determination of high performance liquid chromatography
Chromatographic column: Kromasil100-5C
18(4.6 × 250mm i.d., 5 μm); Mobile phase: take acetonitrile as mobile phase A take percent by volume as the phosphate aqueous solution of 0.15% is Mobile phase B; Determined wavelength is: 252nm; Column temperature: 30 DEG C; Flow velocity: 1.0mL/min.Adopt the gradient elution mode identical with embodiment 1:
3, the foundation of HPLC finger-print
1) preparation of test sample and reference substance
The preparation of glossy ganoderma emperor need testing solution: get glossy ganoderma emperor capsule 's content 0.5g, accurately weighed, be placed in the conical flask of band plug, precision adds methyl alcohol 30mL, close plug, weighed weight, ultrasonic process 30 minutes (power 500W, frequency 40KHz), leaves standstill and lets cool, weighed weight again, make up the weight of less loss with methyl alcohol, shake up, filter, discard just filtrate, precision measures subsequent filtrate 15mL, puts evaporate to dryness in evaporating dish, and residue methyl alcohol dissolves, put 1mL volumetric flask, add methyl alcohol to scale, shake up, filter, get subsequent filtrate, obtain glossy ganoderma emperor capsule test sample.
The preparation of reference substance solution is with embodiment 1.
2) finger-print is measured
Accurate draw reference substance solution 5 μ L, accurately draw glossy ganoderma emperor capsule need testing solution 10 μ L, sample introduction respectively, measures with high performance liquid chromatograph, obtains chromatogram, as accompanying drawing 14, peak type and accompanying drawing 1 basically identical.
Embodiment 3:
1, instrument, reagent are with embodiment 1.
2, the condition determination of high performance liquid chromatography
Chromatographic column: Kromasil100-5C
18(4.6 × 250mm i.d., 5 μm); Mobile phase: take acetonitrile as mobile phase A take percent by volume as the phosphate aqueous solution of 0.05% is Mobile phase B; Determined wavelength is: 252nm; Column temperature: 25 DEG C; Flow velocity: 1.0mL/min.Adopt the gradient elution mode identical with embodiment 1.
3, the foundation of HPLC finger-print
1) preparation of test sample and reference substance
The preparation of glossy ganoderma emperor need testing solution: get glossy ganoderma emperor capsule 's content 2.0g, accurately weighed, be placed in the conical flask of band plug, precision adds methyl alcohol 40mL, close plug, weighed weight, ultrasonic process 60 minutes (power 500W, frequency 40KHz), leaves standstill and lets cool, weighed weight again, make up the weight of less loss with methyl alcohol, shake up, filter, discard just filtrate, precision measures subsequent filtrate 20mL, puts evaporate to dryness in evaporating dish, and residue methyl alcohol dissolves, put 1mL volumetric flask, add methyl alcohol to scale, shake up, filter, get subsequent filtrate, obtain glossy ganoderma emperor capsule test sample.
The preparation of reference substance solution is with embodiment 1.
2) finger-print is measured
Respectively accurately draw reference substance solution and each 5 μ L of glossy ganoderma emperor capsule need testing solution, sample introduction, measure with high performance liquid chromatograph, obtain chromatogram, as accompanying drawing 15, peak type and accompanying drawing 1 basically identical.
Embodiment 4:
1, instrument, reagent are with embodiment 1.
2, the condition determination of high performance liquid chromatography
Chromatographic column: Kromasil100-5C
18(4.6 × 250mm i.d., 5 μm); Mobile phase: take acetonitrile as mobile phase A take percent by volume as the phosphate aqueous solution of 0.1% is Mobile phase B; Determined wavelength is: 252nm; Column temperature: 35 DEG C; Flow velocity: 1.0mL/min.Adopt the gradient elution mode identical with embodiment 1.
3, the foundation of HPLC finger-print
1) preparation of test sample and reference substance
The preparation of glossy ganoderma emperor need testing solution: get glossy ganoderma emperor capsule 's content 1.0g, accurately weighed, be placed in the conical flask of band plug, precision adds methyl alcohol 40mL, close plug, weighed weight, ultrasonic process 40 minutes (power 500W, frequency 40KHz), leaves standstill and lets cool, weighed weight again, make up the weight of less loss with methyl alcohol, shake up, filter, discard just filtrate, precision measures subsequent filtrate 20mL, puts evaporate to dryness in evaporating dish, and residue methyl alcohol dissolves, put 1mL volumetric flask, add methyl alcohol to scale, shake up, filter, get subsequent filtrate, obtain glossy ganoderma emperor capsule test sample.
The preparation of reference substance solution is with embodiment 1.
2) finger-print is measured
Accurate draw reference substance solution 5 μ L, accurately draw glossy ganoderma emperor capsule need testing solution 10 μ L, sample introduction respectively, measures with high performance liquid chromatograph, obtains chromatogram, as accompanying drawing 16, peak type and accompanying drawing 1 basically identical.
Embodiment 5: glossy ganoderma emperor is dusted the foundation of HPLC standard finger-print
1, instrument, reagent: except test article is that glossy ganoderma emperor is dusted (the middle product of glossy ganoderma emperor capsule product), (China) company limited provides by unlimited pole, and other are with embodiment 1.
2, the condition determination of high performance liquid chromatography: with embodiment 1.
3, the foundation of HPLC finger-print
1) preparation of test sample and reference substance
Glossy ganoderma emperor is dusted the preparation of need testing solution: get glossy ganoderma emperor and to dust 1.6g (suitable glossy ganoderma emperor capsule 's content 2.0g), accurately weighed, be placed in the conical flask of band plug, precision adds methyl alcohol 50mL, close plug, weighed weight, ultrasonic process 50 minutes (power 500W, frequency 40KHz), leave standstill and let cool, weighed weight again, the weight of less loss is made up with methyl alcohol, shake up, filter, precision measures subsequent filtrate 25mL, put evaporate to dryness in evaporating dish, residue methyl alcohol dissolves, put 2mL volumetric flask, add methyl alcohol to scale, shake up, filter, get subsequent filtrate, obtain glossy ganoderma emperor to dust test sample,
The preparation of reference substance solution: with embodiment 1.
2) glossy ganoderma emperor is dusted the foundation of HPLC finger-print
Accurately respectively draw reference substance solution and glossy ganoderma emperor and to dust each 5 μ L of need testing solution, sample introduction, measures with high performance liquid chromatograph, obtains chromatogram.Relatively the finger-print of test sample and the finger-print of reference substance, determine the chromatographic peak of each medicinal ingredient.With the peak area of the chromatographic peak relative retention time of reference substance for 1, calculate the relative peak area in corresponding retention time of test sample, namely obtain glossy ganoderma emperor to dust HPLC finger-print, see accompanying drawing 17, can find out that from accompanying drawing 17 glossy ganoderma emperor is dusted and to be had 1 ~ 40 totally 40 characteristic peaks in HPLC finger-print.
Precision draws reference substance solution respectively, glossy ganoderma emperor dusts each 5 μ L of need testing solution, sample introduction, measures, obtain chromatogram with high performance liquid chromatograph.The relatively finger-print of test sample and the finger-print of reference substance, determine the chromatographic peak of main each medicinal ingredient, namely obtain glossy ganoderma emperor to dust HPLC finger-print, see accompanying drawing 17, can find out that from accompanying drawing 17 glossy ganoderma emperor is dusted and to be had 1 ~ 40 totally 40 characteristic peaks in HPLC finger-print.With numbering 16 characteristic peak for the retention time with reference to peak is for 1, calculate the relative retention time of test sample 40 characteristic peaks.
Carry out qualitative to the main peaks in accompanying drawing 17, by glossy ganoderma emperor being dusted, HPLC finger-print and reference substance finger-print compare, and determine that No. 12 peaks are liquiritin, No. 14 peaks are Hyperoside, No. 15 peaks are isoquercitrin, No. 16 peaks are astragalin, No. 31 peaks are ganoderic acid A, No. 36 peaks are glycyrrhizic acid; No. 39 peaks are schizandrin, mixed target finger-print as shown in Figure 2.
3) glossy ganoderma emperor is dusted the foundation of HPLC standard finger-print
Representative in order to determine the HPLC finger-print that dusts of the glossy ganoderma emperor shown in accompanying drawing 17 further, with the finger-print in accompanying drawing 17 for control group, the glossy ganoderma emperor separately getting 10 batches of different lot numbers is dusted, prepare glossy ganoderma emperor after the same method to dust need testing solution, and measured by high performance liquid chromatograph, " similarity evaluation 2004A version " software is adopted to carry out data processing: using these 10 batches and control group finger-print as sample set, the comparison diagram between finger-print is generated through software systems, as accompanying drawing 18, be common characteristic peak from 40 characteristic peaks accompanying drawing 17 finger-print as seen, in addition, calculate the similarity of these 10 batches of collection of illustrative plates and accompanying drawing 17 collection of illustrative plates, evaluate batch between stability, the similarity that result shows between this 10 batch sample and accompanying drawing 17 is good, all more than 0.94, as shown in table 6, illustrate that accompanying drawing 17 is representative, can be used as glossy ganoderma emperor and to dust HPLC standard finger-print.
The glossy ganoderma emperor of the different lot number of table 6 is dusted the similarity of sample room finger-print
According to above result, calculate glossy ganoderma emperor to dust each common characteristic peak relative retention time in HPLC standard finger-print (Figure 17 and Figure 18), adopt peak 16 for calculating other common characteristic peak relative retention times respectively with reference to peak (S), the common characteristic peak relative retention time being numbered 1 to 40 respectively is: 0.085 ~ 0.089min, 0.096 ~ 0.102min, 0.106 ~ 0.110min, 0.145 ~ 0.149min, 0.175 ~ 0.179min, 0.213 ~ 0.215min, 0.321 ~ 0.325min, 0.339 ~ 0.343min, 0.378 ~ 0.382min, 0.476 ~ 0.478min, 0.523 ~ 0.525min, 0.756 ~ 0.758min, 0.769 ~ 0.771min, 0.776 ~ 0.780min, 0.792 ~ 0.794min, 0.864 ~ 0.868min, 0.920 ~ 0.924min, 0.968 ~ 0.972min, 1.000min, 1.021 ~ 1.023min, 1.177 ~ 1.179min, 1.206 ~ 1.208min, 1.250 ~ 1.252min, 1.260 ~ 1.262min, 1.288 ~ 1.290min, 1.303 ~ 1.305min, 1.363 ~ 1.365min, 1.392 ~ 1.394min, 1.408 ~ 1.410min, 1.454 ~ 1.456min, 1.466 ~ 1.468min, 1.475 ~ 1.477min, 1.526 ~ 1.528min, 1.558 ~ 1.560min, 1.587 ~ 1.589min, 1.602 ~ 1.604min, 1.613 ~ 1.615min, 1.621 ~ 1.623mm, 1.630 ~ 1.632min, 1.668 ~ 1.670min.
The explanation of said determination result, the standard finger-print of the HPLC standard finger-print that the middle product glossy ganoderma emperor of glossy ganoderma emperor capsule product is dusted and glossy ganoderma emperor capsule is basically identical, illustrate that the glossy ganoderma emperor capsule HPLC fingerprint spectrum method that the present invention sets up not only may be used for controlling finished product (glossy ganoderma emperor capsule), the quality control of middle product (glossy ganoderma emperor dust) can also be used for.
Claims (7)
1. a construction method for glossy ganoderma emperor preparation HPLC standard finger-print, is characterized in that: comprise the following steps:
1) glossy ganoderma emperor preparation need testing solution is prepared;
2) prepare reference substance solution: precision takes ganoderic acid A, liquiritin, ammonium glycyrrhetate, Hyperoside, isoquercitrin, astragalin, schizandrin, add methyl alcohol and dissolve, make the reference substance solution that concentration is known respectively;
3) foundation of the HPLC finger-print of glossy ganoderma emperor preparation: draw reference substance solution and need testing solution respectively, sample introduction, measure with high performance liquid chromatograph, linear gradient elution program is as following table:
With numbering 16 characteristic peak for the retention time with reference to peak is for 1, calculate the relative retention time of each characteristic peak of test sample;
4) standard finger-print of glossy ganoderma emperor preparation HPLC: the finger-print measuring at least 10 batches of glossy ganoderma emperor formulation samples need testing solutions, the finger-print be made up of common characteristic peak in each finger-print is the standard finger-print of glossy ganoderma emperor preparation HPLC;
Above-mentioned mobile phase A is acetonitrile, Mobile phase B to be percent by volume be 0.05 ~ 0.15% phosphate aqueous solution.
2. the construction method of a kind of glossy ganoderma emperor preparation HPLC standard finger-print according to claim 1, it is characterized in that: the concrete operations preparing glossy ganoderma emperor preparation need testing solution in step 1) are: precision takes glossy ganoderma emperor capsule 's content, be placed in the conical flask of band plug, add methyl alcohol, the concentration of glossy ganoderma emperor preparation is made to be 0.25 ~ 1.0g/mL, close plug, weighed weight, ultrasonic process 40 ~ 60min, leave standstill and let cool, weighed weight again, the weight of less loss is made up with methyl alcohol, shake up, filter, discard just filtrate, precision measures subsequent filtrate, put evaporate to dryness in evaporating dish, residue methyl alcohol dissolves, and with 1 ~ 2 mL volumetric flask constant volume, shake up, filter, get subsequent filtrate, .
3. the construction method of a kind of glossy ganoderma emperor preparation HPLC standard finger-print according to claim 1, it is characterized in that: column temperature when high performance liquid chromatograph measures described in step 3) is 25 ~ 35 DEG C, flow velocity is 1.0mL/min, and determined wavelength is 252nm.
4. the construction method of a kind of glossy ganoderma emperor preparation HPLC standard finger-print according to claim 1, is characterized in that: the similarity of the HPLC finger-print of each batch of glossy ganoderma emperor formulation samples described in step 4) and the standard finger-print of glossy ganoderma emperor preparation HPLC should be greater than 0.9.
5. the construction method of a kind of glossy ganoderma emperor preparation HPLC standard finger-print according to claim 1, it is characterized in that: in the HPLC standard finger-print that the method builds, have 40 common characteristic peaks, be numbered 1 to 40 respectively, with numbering 16 characteristic peak for reference peak, the characteristic peak relative retention time being numbered 1 to 40 respectively is: 0.095 ~ 0.099 min, 0.109 ~ 0.113 min, 0.121 ~ 0.125 min, 0.165 ~ 0.169 min, 0.199 ~ 0.205 min, 0.245 ~ 0.247 min, 0.368 ~ 0.372 min, 0.387 ~ 0.393 min, 0.434 ~ 0.438 min, 0.549 ~ 0.551 min, 0.603 ~ 0.605 min, 0.873 ~ 0.875 min, 0.889 ~ 0.891 min, 0.897 ~ 0.899 min, 0.915 ~ 0.917 min, 1.000 min, 1.065 ~ 1.067 min, 1.121 ~ 1.123 min, 1.155 ~ 1.157 min, 1.181 ~ 1.183 min, 1.361 ~ 1.363 min, 1.395 ~ 1.397 min, 1.447 ~ 1.449 min, 1.458 ~ 1.460 min, 1.489 ~ 1.493 min, 1.507 ~ 1.511 min, 1.576 ~ 1.580 min, 1.612 ~ 1.615 min, 1.629 ~ 1.633 min, 1.682 ~ 1.686 min, 1.696 ~ 1.700 min, 1.706 ~ 1.710 min, 1.764 ~ 1.768 min, 1.803 ~ 1.805 min, 1.835 ~ 1.839 min, 1.853 ~ 1.857 min, 1.866 ~ 1.868 min, 1.876 ~ 1.878 min, 1.886 ~ 1.890 min, 1.930 ~ 1.932 min.
6. the construction method of a kind of glossy ganoderma emperor preparation HPLC standard finger-print according to claim 5, is characterized in that: described numbering is respectively in 40 common characteristic peaks of 1 to 40:
The characteristic peak belonging to glossy ganoderma is numbered 1,2,3,21,22,23,25,26,27,28,29,30,31,32,33,34,35,37,38,40 and has 20 altogether, and wherein No. 31 peaks are ganoderic acid A;
The characteristic peak belonging to the seed of Chinese dodder is numbered 9,10,14,15,16 and has 5 altogether, and wherein No. 14 peaks are Hyperoside, No. 15 peaks are isoquercitrin, No. 16 peaks are astragalin;
The characteristic peak belonging to Radix Glycyrrhizae is numbered 12,13,18,19,36 and has 5 altogether, and wherein No. 12 peaks are liquiritin, No. 36 peaks are glycyrrhizic acid;
The total peak belonging to sealwort is numbered 4,5,6 and has 3 altogether;
The characteristic peak belonging to the bighead atractylodes rhizome is numbered 10,17,24 and has 3 altogether;
The characteristic peak belonging to the fruit of Chinese magnoliavine is numbered 39 and has 1 altogether, and No. 39 peaks are schizandrin;
The characteristic peak belonging to the fruit of Chinese wolfberry is numbered 9,11 and has 2 altogether.
7. the construction method of a kind of glossy ganoderma emperor preparation HPLC standard finger-print according to claim 5 or 6, is characterized in that: described glossy ganoderma emperor preparation is made up of glossy ganoderma, the bighead atractylodes rhizome, sealwort, the fruit of Chinese wolfberry, Poria cocos, the tuber of dwarf lilyturf, the seed of Chinese dodder, the fruit of Chinese magnoliavine, coix seed, Radix Glycyrrhizae 10 taste medicinal material.
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