CN109507312A - A kind of identification method of Cortex Phellodendri and its application - Google Patents
A kind of identification method of Cortex Phellodendri and its application Download PDFInfo
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Abstract
The invention discloses a kind of identification methods of Cortex Phellodendri, the identification method is prepared into test solution with Cortex Phellodendri, using the solution for being dissolved with Berberine hydrochloride reference substance, hydrochloric acid phellodendrine reference substance, magnoflorine reference substance as reference substance solution, the liquid chromatogram of test solution and control solution is obtained respectively by high performance liquid chromatograph measurement；Obtained liquid chromatogram is imported into similarity evaluation analysis, analysis obtains Cortex Phellodendri finger-print, the Cortex Phellodendri finger-print data are handled using hierarchical cluster analysis, principal component analysis or partial least squares analysis, to carry out taxonomic history to Cortex Phellodendri or its processed product.The present invention determines Cortex Phellodendri index components content on the basis of establishing Cortex Phellodendri finger-print, in conjunction with different Chemical Pattern Recognition methods, Cortex Phellodendri and its processed product, adulterant CORTEX PHELLODENDRI AMURENE effectively can be differentiated and be distinguished.
Description
Technical field
The present invention relates to Pharmaceutical Analysis fields, and in particular to a kind of identification method of Cortex Phellodendri and its application.
Background technique
Cortex Phellodendri, Chinese medicine name, the dry tree for being rutaceae wampee Phellodendron chinense Schneid.
Skin.First recorded in Shennong's Herbal, practises and claim " CORTEX PHELLODENDRI CHINENSE ", main product is in Sichuan, Guizhou, Hubei and other places.Chief active in Cortex Phellodendri
Ingredient is the alkaloid compounds such as Berberine hydrochloride and hydrochloric acid phellodendrine.Cortex Phellodendri taste bitter and cold, there is heat-clearing and damp-drying drug, and purging intense heat removes
It the effect of steaming, detoxification sore treatment, is widely used as traditional Chinese medicine in market circulation and clinical treatment.Currently, Cortex Phellodendri in the market
Specification, grade classification are unclear, and do not seek unity of standard, mostly still using the experiences such as the place of production and traditional " meat thickness color depth " into
Row is distinguished, and modern science intension is lacked.There is the phenomenon that pretending to be Cortex Phellodendri to circulate with adulterant CORTEX PHELLODENDRI AMURENE, often to ensure clinical application peace
Entirely, hidden danger is effectively brought.The Cortex Phellodendri quality control that it is therefore desirable to establish a set of science, reasonable, strong operability, practicability are good
With grade evaluation method, for specification Market of Chinese Materia Medica, realize that high quality and favourable price, guarantee clinical application provide strong support.
After traditional Chinese medicine fingerprint refers to that Chinese medicine or Chinese materia medica preparation are appropriately processed, using certain analysis means, obtain
The chromatogram or spectrogram that can indicate its chemical feature.It is a kind of establishes on the basis of chemical composition of Chinese materia medica system research
Synthesis, quantifiable identification of means, can effectively embody the globality and comprehensive function of traditional Chinese medicine ingredients, can provide abundant
Authentication information more comprehensively reflects the type and quantity of contained chemical component in Chinese medicine and its preparation, and then to drug quality
Carry out whole description and evaluation.But there is also such as baseline drifts, chromatography overlap of peaks, background noise for finger-print research
The chromatography FAQs such as high and low signal-signal-to-noise ratio limit finger-print in traditional Chinese medicine quality control to varying degrees
With the application in evaluation.Chemical Pattern Recognition passes through statistics or mathematical method, the shape of measured value and system to chemical system
Connection is established between state, can be used for solving the FAQs in traditional Chinese medicine fingerprint, and can provide a variety of analysis recognition methods,
The fast accurate classification for realizing similar medicinal material, the places of origin of raw materials, it is true and false identify, control of product quality and analysis, quality identification etc.
Aspect plays an important role.However, can lead to Chemical Pattern Recognition inaccuracy if the data of finger-print are not comprehensive.
Therefore, in order to preferably control Cortex Phellodendri quality, guarantee clinical efficacy, the matter of thoroughly evaluating Cortex Phellodendri need to be established
Amount control method.
Summary of the invention
It is the general introduction to the theme being described in detail herein below.This general introduction is not the protection model in order to limit claim
It encloses.
The present invention provides a kind of identification methods of Cortex Phellodendri, and processing great amount of samples can be analyzed with the short time by this method,
Cortex Phellodendri and its processed product, adulterant CORTEX PHELLODENDRI AMURENE are effectively differentiated and distinguished, for the control of Cortex Phellodendri quality and the division of Quality Grade standard
Providing method foundation；Solve the problems, such as that Cortex Phellodendri detection method can not detect and control its quality on the whole.
The present invention provides a kind of identification method of Cortex Phellodendri, the identification method the following steps are included:
(1) test solution the preparation of test solution: is prepared into Cortex Phellodendri；
(2) preparation of reference substance solution: to be dissolved with Berberine hydrochloride reference substance, hydrochloric acid phellodendrine reference substance, magnolia
The solution of alkali reference substance is reference substance solution；
(3) using the test solution of HPLC difference determination step (1) and the reference substance solution of step (2), and respectively
To the liquid chromatogram of the test solution and the reference substance solution；
(4) liquid chromatogram for obtaining step (3) imports similarity evaluation analysis, obtains
Cortex Phellodendri finger-print；
(5) hierarchical cluster analysis (Hierarchical Cluster Analysis, abbreviation HCA), principal component analysis are used
(Principal Components Analysis, abbreviation PCA) or partial least squares analysis (Partial Least
Squares-Discrimination Analysis, abbreviation PLS-DA) the obtained Cortex Phellodendri finger-print data of processing step (4),
To carry out taxonomic history to Cortex Phellodendri or its processed product.
In embodiments of the invention, the sample of the Cortex Phellodendri is collected from Cortex Phellodendri and its processed product of different sources.
In identification method embodiment of the invention, wherein the preparation of the test solution of the step (1) includes:
Cortex Phellodendri is smashed it through into No. 3 pharmacopeia sieves, it is accurately weighed, it is placed in 10mL volumetric flask, addition volume fraction is 70%-100% first
Alcohol solution (contains 0.1 volume % hydrochloric acid, here, the methanol aqueous solution is first when volume fraction is 100% methanol aqueous solution
Alcohol), it is settled to scale, weighed weight is ultrasonically treated 30min-50min, is cooled to room temperature, is 70%-100% with volume fraction
Methanol aqueous solution (contains 0.1 volume % hydrochloric acid, here, the methanol aqueous solution is first when volume fraction is 100% methanol aqueous solution
Alcohol) weight of supplying loss, it shakes up, centrifuge 4000r/min-6000r/min is centrifuged 5min-15min, takes supernatant, mistake
0.22 μm of miillpore filter, takes subsequent filtrate as test solution；Preferably, step (1) test solution
Preparation includes: that Cortex Phellodendri is smashed it through No. 3 pharmacopeia sieves, takes phellodendron powder 0.2g, accurately weighed, is placed in 10mL volumetric flask, adds
Enter methanol (containing 0.1 volume % hydrochloric acid), be settled to scale, weighed weight is ultrasonically treated 40min, is cooled to room temperature, uses methanol
The weight that (containing 0.1 volume % hydrochloric acid) supplies loss, shakes up, and centrifuge 4000r/min is centrifuged 10min, takes supernatant, crosses 0.22
μm miillpore filter, take subsequent filtrate as test solution.
In identification method embodiment of the invention, wherein the preparation of the reference substance solution of the step (2) includes:
Precision weighs Berberine hydrochloride reference substance, hydrochloric acid phellodendrine reference substance, magnoflorine reference substance, and solubilizer is made every 1mL and contains
The reference substance solution of the 400 μ g of μ g~600 Berberine hydrochlorides, the 50 μ g hydrochloric acid of μ g~100 phellodendrines, the 20 μ g magnoflorines of μ g~50.
Preferably, the preparation of step (2) reference substance solution includes: that precision weighs Berberine hydrochloride reference substance, hydrochloric acid Huang
Cypress alkali reference substance, magnoflorine reference substance add methanol (containing 0.1 volume % hydrochloric acid) that every 1mL is made and contain 500 μ g hydrochloric acid barberries
The reference substance solution of alkali, 80 μ g hydrochloric acid phellodendrines, 30 μ g magnoflorines.
In identification method embodiment of the invention, wherein the step (3) includes setting HPLC chromatogram condition: color
Spectrum column uses octadecylsilane chemically bonded silica for filler, and 25 DEG C -35 DEG C of column temperature, flow velocity 0.8mL/min-1.2mL/min, inspection
Wavelength 250nm-300nm is surveyed, and opens DAD detection；Using acetonitrile as mobile phase A, with volume fraction for 0.06%-0.2% phosphoric acid
Aqueous solution is Mobile phase B, gradient elution in the range of volume ratio is 5~100:95~0；
It is accurate respectively to draw the 5 μ L of μ L~20 of test solution and control solution, high performance liquid chromatograph is injected, is measured,
Respectively obtain the liquid chromatogram of test solution and control solution.
Preferably, the HPLC chromatogram condition of the step (3) includes: that chromatographic column is bonded using octadecylsilane
Silica gel is filler,C18 (4.6mm × 150mm, 5.0 μm) be chromatographic column, 30 DEG C of column temperature, flow velocity 1.0mL/
Min, Detection wavelength 254nm, and open DAD detection；It is water-soluble with the phosphoric acid that volume fraction is 0.2% using acetonitrile as mobile phase A
Liquid is Mobile phase B, carries out gradient elution, mobile phase A, the variation of the ratio of B are 0~20min, and A:B is 5%:95% → 10%:
90%；20~50min, A:B are 10%:90% → 20%:80%；50~60min, A:B are 20%:80% → 25%:75%；
60~75min, A:B are 25%:75% → 100%:0%；
It is accurate respectively to draw 5 μ L of test solution and control solution, high performance liquid chromatograph is injected, is measured, respectively
To the liquid chromatogram of test solution and control solution.
In identification method embodiment of the invention, wherein the chromatographic fingerprints of Chinese materia medica phase that the step (4) uses
It is 2004A editions like degree evaluation system.The step (4) includes: using similarity evaluation 2004A
Version, the data importing for the liquid chromatogram that step (3) is obtained, Supplements, Data Matching, analysis obtain Cortex Phellodendri finger-print.
In identification method embodiment of the invention, wherein the Cortex Phellodendri finger-print that the step (4) obtains include and
Corresponding No. 5 peaks of hydrochloric acid phellodendrine, corresponding No. 6 peaks of magnoflorine, corresponding No. 15 peaks (peak S) of Berberine hydrochloride, it is opposite
Retention time is respectively 0.382,0.396,1.000.
In identification method embodiment of the invention, wherein the Cortex Phellodendri finger-print that the step (4) obtains shares
Peak further includes that No. 1 peak that relative retention time is 0.140, No. 2 peaks, the relative retention times that relative retention time is 0.276 are
When 0.297 No. 3 peaks, No. 4 peaks that relative retention time is 0.356, No. 7 peaks that relative retention time is 0.450, opposite reservation
Between for 0.607 No. 8 peaks, No. 9 peaks that relative retention time is 0.638, No. 10 peaks that relative retention time is 0.672, opposite
No. 11 peaks that retention time is 0.716, No. 12 peaks that relative retention time is 0.817, No. 13 that relative retention time is 0.888
No. 14 peaks that peak, relative retention time are 0.919, No. 16 peaks that relative retention time is 1.168.
In identification method embodiment of the invention, wherein Cortex Phellodendri finger-print such as Fig. 3 that the step (4) obtains
It is shown.
In identification method embodiment of the invention, wherein the taxonomic history of the step (5) includes but unlimited
In difference CORTEX PHELLODENDRI CHINENSE and CORTEX PHELLODENDRI AMURENE, distinguishes different processed products or distinguish different appearance characters.
In identification method embodiment of the invention, wherein the hierarchical cluster analysis that the step (5) uses is no prison
The pattern recognition model superintended and directed, the principal component analysis perhaps used is unsupervised pattern recognition model or uses partially minimum
Two multiply analysis to there is the pattern recognition model of supervision.
In identification method embodiment of the invention, wherein the hierarchical cluster analysis that the step (5) uses is no prison
The pattern recognition model superintended and directed shows cluster result with system clustering tree, for distinguishing CORTEX PHELLODENDRI CHINENSE and CORTEX PHELLODENDRI AMURENE, distinguishing different processings
Product distinguish different appearance characters.
In identification method embodiment of the invention, wherein the principal component analysis that the step (5) uses is unsupervised
Pattern recognition model, cluster result is shown with shot chart, for distinguish CORTEX PHELLODENDRI CHINENSE and CORTEX PHELLODENDRI AMURENE, distinguish different processed products or
Distinguish different appearance characters.
In identification method embodiment of the invention, wherein the partial least squares analysis that the step (5) uses is has
The pattern recognition model of supervision, rendering model 3D figure and VIP figure (variable importance projection), for distinguishing CORTEX PHELLODENDRI CHINENSE and closing yellow
Cypress distinguishes different processed products or distinguishes different appearance characters；Each variable can also be weighed for the importance shadow of discernment
It rings, finds out chemical markers, divide providing method foundation for the control of Cortex Phellodendri quality and its grade evaluation.
In identification method embodiment of the invention, wherein the step (5) can include according to above-mentioned three kinds simultaneously
Pattern-recognition as a result, the reliability of result is mutually authenticated, to improve the accuracy of model.
Second aspect, the present invention provides the identification method of above-mentioned Cortex Phellodendri differentiate and/or distinguish CORTEX PHELLODENDRI CHINENSE and CORTEX PHELLODENDRI AMURENE,
Differentiate and/or distinguish Cortex Phellodendri and processes or differentiate and/or distinguish the application in the appearance character of Cortex Phellodendri or above-mentioned Cortex Phellodendri
The identification of application or above-mentioned Cortex Phellodendri of the identification method in the control of Cortex Phellodendri quality or the division of Cortex Phellodendri Quality Grade standard
Method judges the application in not in the Cortex Phellodendri true and false.
The technical effect that the present invention obtains includes at least following one or more:
(1) the Cortex Phellodendri finger-print obtained with the Cortex Phellodendri of different sources can represent most of effective component of Cortex Phellodendri, energy
Enough quality for effectively characterizing Cortex Phellodendri.
(2) treated as a whole with the finger-print of Cortex Phellodendri, focus on the fingerprint characteristic peak and phase of various chemical components
Mutual relation focuses on the whole facial feature of Cortex Phellodendri, avoids and only measures one, two index chemical component and determine Cortex Phellodendri quality
One-sidedness, and a possibility that reduce artificial subjective judgement and cause error.
(3) it is an advantage of the invention that in conjunction with Chemical Pattern Recognition, the essence that chemically metric data infers material class belongs to
Property, substance is identified and sorted out.Cortex Phellodendri and its adulterant CORTEX PHELLODENDRI AMURENE obviously gather Chemical Pattern Recognition as the result is shown is two
It should be noted that differentiation when class, clinical practice and formulation application；Obviously gather in display Cortex Phellodendri sample for two classes, a kind of Cortex Phellodendri removes thick bolt
Skin or thick cork are less, and Cortex Phellodendri thickness is no more than 0.5mm, and a kind of Cortex Phellodendri does not go thick cork or thick cork more, and Cortex Phellodendri thickness is super
Cross 1.5mm；Display Cortex Phellodendri and its processed product stir-baked CORTEX PHELLODENDRI with salt solution, Cortex Phellodendri charcoal can be distinguished obviously.Prompt size heterogeneity and medicinal material net system to process,
Process it is significant for the Quality Grade establishment of standard of Cortex Phellodendri, prompt the thin and thick of thick cork, Berberine hydrochloride in Cortex Phellodendri,
Hydrochloric acid Cortex Phellodendri alkali content can be used as Cortex Phellodendri classification standard classification foundation.
(4) identification method of a kind of Cortex Phellodendri that the present invention establishes, reflected comprehensively by finger-print Cortex Phellodendri intrinsic chemical at
The type and quantity divided, and then react the quality of Cortex Phellodendri；By Chemical Pattern Recognition to the measured value and system shape of chemical system
The feature of sample is found out in the connection established between state, and then sample is identified and sorted out.The two use in conjunction to Cortex Phellodendri not
The analysis and predictive ability of same specification, processed product, true and false product are Cortex Phellodendri medicinal material, the quality evaluation of medicine materical crude slice and its processed product and true
Puppet, which identifies, provides new method, establishes comprehensive quality standard for Cortex Phellodendri, clinical rational drug use provides reference.
Other features and advantages of the present invention will be illustrated in the following description, also, partly becomes from specification
It obtains it is clear that understand through the implementation of the invention.The objectives and other advantages of the invention can be by specification, right
Specifically noted structure is achieved and obtained in claim and attached drawing.
Detailed description of the invention
Attached drawing is used to provide to further understand technical solution of the present invention, and constitutes part of specification, with this
The embodiment of application technical solution for explaining the present invention together, does not constitute the limitation to technical solution of the present invention.
Fig. 1 is the HPLC chromatogram of 3 kinds of reference substances；
In Fig. 1, A: hydrochloric acid phellodendrine reference substance, B: magnoflorine reference substance, C: Berberine hydrochloride reference substance；
Fig. 2 is the common pattern map of the Cortex Phellodendri measured in the embodiment of the present invention 1；
In Fig. 2, No. 5 peaks: hydrochloric acid phellodendrine, No. 6 peaks: magnoflorine, No. 15 peaks: Berberine hydrochloride；
Fig. 3 is that 20 batch Cortex Phellodendri finger-prints are superimposed map in the embodiment of the present invention 1；
Fig. 4 is Cortex Phellodendri-CORTEX PHELLODENDRI AMURENE health product HCA dendrogram in the embodiment of the present invention 3；
Fig. 5 is Cortex Phellodendri health product-processed product stir-baked CORTEX PHELLODENDRI with salt solution, Cortex Phellodendri charcoal HCA dendrogram in the embodiment of the present invention 3；
Fig. 6 is Cortex Phellodendri-CORTEX PHELLODENDRI AMURENE health product PCA shot chart in the embodiment of the present invention 3；
Fig. 7 is Cortex Phellodendri health product-processed product stir-baked CORTEX PHELLODENDRI with salt solution, Cortex Phellodendri charcoal PCA shot chart in the embodiment of the present invention 3；
Fig. 8 is Cortex Phellodendri in the embodiment of the present invention 3-CORTEX PHELLODENDRI AMURENE health product PLS-DA model 3D figure；
Fig. 9 is Cortex Phellodendri health product PLS-DA model VIP figure in the embodiment of the present invention 3.
Specific embodiment
It, hereinafter will be to the embodiment of the present invention for the purposes, technical schemes and advantages of the application are more clearly understood
It is described in detail.It should be noted that in the absence of conflict, the features in the embodiments and the embodiments of the present application can
With mutual any combination.
The instrument and reagent that embodiment is used are as follows:
1, instrument and reagent
1.1 instrument
Ten a ten thousandth balances (Sartorius BT125D, Sai Duolisi scientific instrument Co., Ltd)；Desk type high speed from
Scheming (TG16-WS, Changsha Xiang Yi centrifuge Instrument Ltd.)；High performance liquid chromatograph (LC-10A, Japanese Shimadzu Corporation,
DAD detector)；Ultrasonic cleaner (KH3200B, Kunshan He Chuan ultrasonic instrument Co., Ltd)；Medicinal herb grinder (HC-150,
Yellow city high-speed multifunctional pulverizer).
1.2 reagent
Hydrochloric acid phellodendrine reference substance (lot number: 170307, content > 98%), magnoflorine reference substance (lot number: 170104, contain
Amount > 98%), Berberine hydrochloride reference substance (lot number: 161223, content > 98%), the above reference substance is purchased from Hiroad standing grain medicine
Science and Technology Ltd..
Chromatography methanol, acetonitrile are purchased from Sigma Co., USA, and chromatography phosphoric acid and hydrochloric acid are purchased from Tianjin Ke Miou chemical reagent and have
Limit company.Water used is Watson distilled water.
The Cortex Phellodendri sample for collecting 20 batches altogether, is identified as rutaceae wampee Phellodendron
The dry bark of chinense Schneid..For sample, this essential information is shown in Table 1 to the Cortex Phellodendri of 20 batches.
1 Cortex Phellodendri of table is for this essential information of sample
The Cortex Phellodendri processed product sample for having collected 10 batches altogether, is identified as stir-baked CORTEX PHELLODENDRI with salt solution and Cortex Phellodendri charcoal.The salt of 7 batches is yellow
For sample, this essential information is shown in Table 2 to the Cortex Phellodendri charcoal of cypress and 3 batches.
2 Cortex Phellodendri processed product of table is for this essential information of sample
The CORTEX PHELLODENDRI AMURENE sample for having collected 5 batches altogether is identified as Rutaceae cork tree Phellodendron amurense
Rupr. dry bark.For sample, this essential information is shown in Table 3 to the CORTEX PHELLODENDRI AMURENE of 5 batches.
3 CORTEX PHELLODENDRI AMURENE of table is for this essential information of sample
The method for building up and methodological study of 1 Cortex Phellodendri finger-print of embodiment
1, the method for building up of Cortex Phellodendri finger-print
The method for building up of Cortex Phellodendri finger-print, comprising the following steps:
(1) preparation of test solution: taking 20 batch different sources Cortex Phellodendris of the above table 1 to smash it through No. 3 pharmacopeia sieves,
Phellodendron powder 0.2g is taken, it is accurately weighed, it is placed in 10mL volumetric flask, methanol (containing 0.1% hydrochloric acid) is added, is settled to scale, claims
Determine weight, be ultrasonically treated 40min, be cooled to room temperature, the weight for supplying loss with methanol (containing 0.1% hydrochloric acid) shakes up, centrifuge
4000r/min is centrifuged 10min, takes supernatant, crosses 0.22 μm of miillpore filter, takes subsequent filtrate as test solution；
(2) preparation of reference substance solution: precision weighs Berberine hydrochloride reference substance, hydrochloric acid phellodendrine reference substance, magnolia
Alkali reference substance adds methanol (containing 0.1% hydrochloric acid) that every 1mL is made and contains 500 μ g Berberine hydrochlorides, 80 μ g hydrochloric acid phellodendrines, 30 μ g
The reference substance solution of magnoflorine；
(3) HPLC chromatogram condition: chromatographic column uses octadecylsilane chemically bonded silica for filler, C18
(4.6mm × 150mm, 5.0 μm) is chromatographic column, 30 DEG C of column temperature, flow velocity 1.0mL/min, Detection wavelength 254nm, and open
DAD detection.It is 0.2% phosphate aqueous solution as Mobile phase B using volume fraction using acetonitrile as mobile phase A, carries out gradient elution, stream
The ratio variation of dynamic phase A, B is 0~20min, and A:B is 5%:95% → 10%:90%；20~50min, A:B 10%:90%
→ 20%:80%；50~60min, A:B are 20%:80% → 25%:75%；60~75min, A:B be 25%:75% →
100%:0%；
(4) accurate respectively to draw 5 μ L of test solution and control solution, high performance liquid chromatograph is injected, is measured, respectively
Obtain the liquid chromatogram of test solution and control solution；
(5) chromatograms are imported into fingerprint similarity evaluation software (chromatographic fingerprints of Chinese materia medica similarity evaluation system
System 2004A editions) analysis, using SC5 medicinal material map as referring to map, choosing " time window width " is 0.1min, is used
Average method calculates, Supplements, Data Matching, generates superposition chromatogram (see Fig. 3) and control map (see figure
2).It will
The finger-print of the 20 batches of Cortex Phellodendris and the reference fingerprint of generation carry out similarity analysis, as the result is shown 20 batches of Cortex Phellodendris
Fingerprint
Map is all larger than 0.981 with the similarity for compareing map, is shown in Table 4.
The fingerprint similarity of 4 20 batches of Cortex Phellodendris of table
It is compared with 3 kinds of reference substances, determines that the peak 20.173min is hydrochloric acid phellodendrine (No. 5 peaks), the peak 20.948min is wood
Magnoflorine (No. 6 peaks), the peak 52.846min are Berberine hydrochloride (No. 15 peaks).Wherein choose separating degree it is preferable, peak area it is larger and
Stable Berberine hydrochloride is referring to peak (S).Determine that 16 share altogether with the HPLC finger-print that Berberine hydrochloride is reference peak
Peak., for 1, to calculate the relative retention time at other each shared peaks referring to the relative retention time at peak, the results are shown in Table 5.
The fingerprint of 5 20 batches of Cortex Phellodendris of table shares peak relative retention time
2, fingerprint spectrum method is investigated
2.1 precision test
Cortex Phellodendri (SC5) is taken, test solution is prepared by the preparation method of the test solution of embodiment 1, by embodiment 1
6 needle of chromatographic condition continuous sample introduction obtains 6 chromatograms.It is to calculate each shared peak and referring to peak referring to peak with Berberine hydrochloride peak
Relative peak area and relative retention time, and calculate RSD value, the results are shown in Table 6 and table 7.
6 Cortex Phellodendri finger-print precision test of table investigates result (each shared peak relative peak area)
7 Cortex Phellodendri finger-print precision test of table investigates result (each shared peak relative retention time)
By table 6 and table 7 it is found that relative peak area RSD% < 5% of each shared chromatographic peak, relative retention time RSD% <
2%, illustrate that this method precision is good.
2.2 repetitive test
Cortex Phellodendri (SC5) is taken, weighs 6 parts in parallel, prepares 6 parts of test samples by the preparation method of the test solution of embodiment 1
Solution distinguishes sample introduction by chromatographic condition in embodiment 1, obtains 6 chromatograms.It is to be calculated each referring to peak with Berberine hydrochloride peak
Shared peak and the relative peak area and relative retention time referring to peak, and RSD value is calculated, it the results are shown in Table 8 and table 9.
8 Cortex Phellodendri finger-print repetitive test of table investigates result (each shared peak relative peak area)
9 Cortex Phellodendri finger-print repetitive test of table investigates result (each shared peak relative retention time)
By table 8 and table 9 it is found that relative peak area RSD% < 5% of each shared chromatographic peak, relative retention time RSD% <
2%, illustrate that this method repeatability is good.
2.3 stability test
Cortex Phellodendri (SC5) is taken, 6 parts of test solutions are prepared by the preparation method of the test solution of embodiment 1, by embodiment
Chromatographic condition in 1 obtains 6 chromatograms respectively at 0h, 2h, 4h, 8h, 12h, for 24 hours condition sample introduction.It is with Berberine hydrochloride peak
Referring to peak, each shared peak and the relative peak area and relative retention time referring to peak are calculated, and calculates RSD value, the results are shown in Table 10
With table 11.
Result (each shared peak relative peak area) is investigated in 10 Cortex Phellodendri finger-print stability test of table
Result (each shared peak relative retention time) is investigated in 11 Cortex Phellodendri finger-print stability test of table
By table 10 and table 11 it is found that relative peak area RSD% < 5% of each shared chromatographic peak, relative retention time RSD% <
2%, illustrate test solution interior for 24 hours basicly stable.
The multi-wavelength of 2 Cortex Phellodendri finger-print of embodiment quantifies detection and the methodological study of ingredient
1, multi-wavelength quantifies the detection of ingredient
Hydrochloric acid phellodendrine, magnoflorine, 3 kinds of pharmacodynamics index ingredients of Berberine hydrochloride retention time be respectively as follows:
20.173min,20.948min,52.846min.It opens DAD detector and carries out full wavelength scanner, under the conditions of comparing different wave length
The peak area of each pharmacodynamics index ingredient obtains the most suitable Detection wavelength of each index components, i.e., detects hydrochloric acid under 284nm wavelength condition
The content of phellodendrine detects the content of magnoflorine under 268nm wavelength condition, and hydrochloric acid barberry is detected under 265nm wavelength condition
The content of alkali.
2, multi-wavelength quantifies component detection method investigation
2.1 linear investigations
The hydrochloric phellodendrine of precision measurement, magnoflorine, Berberine hydrochloride reference substance solution are appropriate, dilute, are made into step by step
A series of reference substance solution of concentration, sample introduction is analyzed, respectively at measuring under wavelength 284nm, 268nm, 265nm and record peak face
Product.Using peak area as ordinate (Y), concentration (X) is abscissa, the regression equation of the phellodendrine acquired: Y=5E+06X-
1468.2 correlation coefficient r=0.9998；The range of linearity: 0.00205mgmL-1~0.082mgmL-1；Magnoflorine returns
Return equation: Y=1E+07X-1880, correlation coefficient r=0.9998；The range of linearity: 0.0007mgmL-1~0.028mgmL-
1；The regression equation of Berberine hydrochloride: Y=2E+07X-40531, correlation coefficient r=0.9998；The range of linearity: 0.015mg
ML-1~0.6mgmL-1.
2.2 precision test
Reference substance solution is prepared by the preparation method of the reference substance solution of embodiment 1, it is continuous by chromatographic condition in embodiment 1
6 needle of sample introduction obtains 6 chromatograms.The peak area and retention time of each characteristic peak are calculated, and calculates RSD value, the results are shown in Table 12 Hes
Table 13.
12 Cortex Phellodendri multi-wavelength of table quantifies composition detection precision test and investigates result (characteristic peak peak area)
* 5 (hydrochloric acid phellodendrines), 6 (magnoflorines), 15 (Berberine hydrochlorides)
13 Cortex Phellodendri multi-wavelength of table quantifies composition detection precision test and investigates result (characteristic peak retention time)
* 5 (hydrochloric acid phellodendrines), 6 (magnoflorines), 15 (Berberine hydrochlorides)
By table 12 and table 13 it is found that peak area RSD% < 5%, retention time RSD% < 2% of each characteristic peak, illustrate the party
Method precision is good.
2.3 repetitive test
By the preparation method of the reference substance solution of embodiment 1,6 parts of reference substance solutions are prepared in parallel, by chromatography in embodiment 1
Condition distinguishes sample introduction, obtains 6 chromatograms.The peak area and retention time of each characteristic peak are calculated, and calculates RSD value, is as a result seen
Table 14 and table 15.
14 Cortex Phellodendri multi-wavelength of table quantifies composition detection repetitive test and investigates result (characteristic peak peak area)
* 5 (hydrochloric acid phellodendrines), 6 (magnoflorines), 15 (Berberine hydrochlorides)
15 Cortex Phellodendri multi-wavelength of table quantifies composition detection repetitive test and investigates result (characteristic peak retention time)
* 5 (hydrochloric acid phellodendrines), 6 (magnoflorines), 15 (Berberine hydrochlorides)
By table 14 and table 15 it is found that peak area RSD% < 5%, retention time RSD% < 2% of each characteristic peak, illustrate the party
Method precision is good.
2.4 stability test
By the preparation method of the reference substance solution of embodiment 1,6 parts of reference substance solutions are prepared in parallel, by chromatography in embodiment 1
Condition obtains 6 chromatograms respectively at 0h, 2h, 4h, 8h, 12h, for 24 hours condition sample introduction.Calculate it is each altogether characteristic peak peak area and
Retention time, and RSD value is calculated, it the results are shown in Table 16 and table 17.
16 Cortex Phellodendri multi-wavelength of table quantifies composition detection stability test and investigates result (characteristic peak peak area)
* 5 (hydrochloric acid phellodendrines), 6 (magnoflorines), 15 (Berberine hydrochlorides)
17 Cortex Phellodendri multi-wavelength of table quantifies composition detection stability test and investigates result (characteristic peak retention time)
* 5 (hydrochloric acid phellodendrines), 6 (magnoflorines), 15 (Berberine hydrochlorides)
By table 16 and table 17 it is found that peak area RSD% < 5%, retention time RSD% < 2% of each characteristic peak, illustrate to compare
Product solution is interior for 24 hours basicly stable.
The test of 2.5 sample recovery rates
Weigh 6 parts of the Cortex Phellodendri medicinal powder (SC5) of known content, respectively it is accurate be added hydrochloric phellodendrine, magnoflorine,
Berberine hydrochloride reference substance solution is appropriate, prepares 6 parts of test solutions by the preparation method of the test solution of embodiment 1, presses
Chromatographic condition in embodiment 1, sample introduction, obtains 6 chromatograms respectively.Calculate hydrochloric acid phellodendrine, magnoflorine, Berberine hydrochloride
Sample recovery rate, and RSD value is calculated, it the results are shown in Table 18.
18 characteristic peak sample recovery rate result of table
As shown in Table 18, the sample recovery rate of each characteristic peak illustrates that this method sample recovery rate meets in 98%-105%
Test requirements document.
3, the measurement of index components content
The Cortex Phellodendri for taking 20 batch different sources prepares 20 parts for examination by the preparation method of the test solution of embodiment 1
Product solution distinguishes sample introduction by chromatographic condition in embodiment 1, the content of hydrochloric acid phellodendrine, 268nm is detected under 284nm wavelength condition
The content that magnoflorine is detected under wavelength condition, detects the content of Berberine hydrochloride under 265nm wavelength condition,
Record the peak area of each index components, substitute into equation of linear regression and calculate content, each sample duplicate measurements twice,
It is averaged.Calculate hydrochloric acid phellodendrine, magnoflorine, content of berberine hydrochloride in different sources Cortex Phellodendri.It the results are shown in Table 19.
Index components content measurement result in 19 20 batch Cortex Phellodendris of table
As shown in Table 19, there are notable differences for the Cortex Phellodendri index components content of different sources.
The foundation of 3 Cortex Phellodendri Chemical Pattern Recognition method of embodiment
1, identification of the hierarchical cluster analysis (HCA) to Cortex Phellodendri
Using Waters empower work station data management function, Cortex Phellodendri and each Component peak area of CORTEX PHELLODENDRI AMURENE, guarantor are obtained
Stay the relevant informations such as time.The data matrix of acquisition is imported in SPSS 19.0, using average connection pearson correlation (group
It is interior) to Cortex Phellodendri sample progress hierarchical cluster analysis, dendrogram is as shown in Figure 4 for connection.When distance scale about 22 when, Cortex Phellodendri with
CORTEX PHELLODENDRI AMURENE can be distinguished significantly, and CORTEX PHELLODENDRI AMURENE gathers for a kind of S3, and Cortex Phellodendri gathers for two class S1 and S2.By to Cortex Phellodendri sample shape, thickness,
Color, quality carry out investigation discovery: S1 sample is mostly the more person of thick cork, and quality is coarse, and maximum gauge can reach the left side 1.5mm
It is right；S2 sample is mostly the less person of thick cork, and quality is more smooth, and thickness is no more than 0.5mm.
Coupled between group using Euclidean poly- to Cortex Phellodendri health product-processed product (stir-baked CORTEX PHELLODENDRI with salt solution and Cortex Phellodendri charcoal) progress sequence
Alanysis, dendrogram are as shown in Figure 5.When distance scale about 19 when, Cortex Phellodendri health product and stir-baked CORTEX PHELLODENDRI with salt solution, Cortex Phellodendri charcoal can significant areas
Point.Wherein S1 is Cortex Phellodendri health product；S2 and S3 difference stir-baked CORTEX PHELLODENDRI with salt solution and Cortex Phellodendri charcoal.
2, identification of the principal component analysis (PCA) to Cortex Phellodendri
Different sources Cortex Phellodendri and CORTEX PHELLODENDRI AMURENE health product pca model are initially set up using Simca-p11.5.Shot chart such as Fig. 6 institute
Show, as can be seen from the figure sample gathers for three classes, and wherein S3 is CORTEX PHELLODENDRI AMURENE, and S1, S2 are Cortex Phellodendri.Cortex Phellodendri is identified as two classes, warp
Cross with its appearance character comparative analysis, find the thick cork in S1 class Cortex Phellodendri surface it is more, quality is thicker；And the sample that S2 class includes is yellow
The thick cork in cypress surface is less, and quality is relatively thin.The thin and thick of prompt cork can be used as the weight for dividing Cortex Phellodendri Quality Grade standard
It will foundation.In pca model, Cross gain modulation Q2(cum)=0.418.According to the characteristic values such as Cross gain modulation selection most preferably at
Divide number, obtains principal component, contribution rate of accumulative total R2X (cum)=0.727, precision is preferable.It, can according to the pca model established
Obviously to distinguish CORTEX PHELLODENDRI AMURENE and Cortex Phellodendri.Differentiation and identification to medicinal material also have important meaning.
Next establishes CORTEX PHELLODENDRI CHINENSE health product and processed product pca model.Shot chart is as shown in Figure 7.It can be seen from the figure that being built
Cortex Phellodendri health product and stir-baked CORTEX PHELLODENDRI with salt solution and Cortex Phellodendri charcoal significantly can be divided into three classes by vertical pca model, be differentiation and the mirror of processed product
A new method is not provided.
3, identification of the partial least squares analysis (PLS-DA) to Cortex Phellodendri
In order to preferably find chemical markers, the PLS-DA model for having supervision is established to CORTEX PHELLODENDRI AMURENE, Cortex Phellodendri.Fig. 8 is
PLS-DA model 3D figure.Wherein the part S3 is CORTEX PHELLODENDRI AMURENE, other samples are Cortex Phellodendri；Cortex Phellodendri is obviously divided into two classes, with pca model
Result it is consistent, demonstrate the reliability of result.
It is influenced to weigh each variable for the importance of discernment, depicts VIP figure (variable importance projection) (figure
2-5), variable and X and Y importance associated are explained.It is shown in figure, the VIP value of variable is greater than 1, this meaning
Taste these variables play an important role in difference.Wherein chemical component 10,11 has maximum VIP value, passes through
It is Berberine hydrochloride that standard items, which point out No. 10 peaks, is the characteristic chemical constituent of Cortex Phellodendri, chemical component 11 is not possible to effective with standard items
It points out.It proves PLS-DA models fitting data and predicts new data well.In PLS-DA model, Cross gain modulation Q2
(cum)=0.962.Optimal components number is selected according to characteristic values such as Cross gain modulations, obtains principal component, contribution rate of accumulative total
R2X (cum)=0.999, R2Y (cum)=0.406, shows that precision is better.As a result prompting Berberine hydrochloride in Cortex Phellodendri is to Huang
Cypress distinguishes the variable to play an important role, can be used as the foundation of Cortex Phellodendri classification standard classification.
The above test results show that a kind of evaluation method of Cortex Phellodendri finger-print-Chemical Pattern Recognition provided by the invention,
From finger-print this whole angle, three kinds of Chemical Pattern Recognition technologies of system research are to the certified products of Cortex Phellodendri, adulterant, big gun
The analysis and predictive ability of product provide new method for the quality evaluation of Cortex Phellodendri and its processed product and true and false identification, are Cortex Phellodendri
Medicinal material, the prepared slice quality of different size control and the division providing method foundation of Quality Grade standard, is clinical application and medicine
The differentiation use of Cortex Phellodendri is offered reference in industry.Research is found: there is some difference for different sources Cortex Phellodendri index components content, is
Cortex Phellodendri quality is connected with the place of production to be provided a certain basis；Cortex Phellodendri is identified as two classes in Cortex Phellodendri sample, and a kind of thick cork is more,
Quality is coarse, and maximum gauge can reach 1.5mm or so, and the less person of a kind of thick cork, quality is more smooth, and thickness is no more than 0.5mm；
Important chemical markers Berberine hydrochloride is obtained by PLS-DA model.Prompt CORTEX PHELLODENDRI AMURENE differs greatly with Cortex Phellodendri, does not advocate
It is mixed.Prompt that the thin and thick of thick cork, content of berberine hydrochloride can be used as Cortex Phellodendri classification standard classification foundation in Cortex Phellodendri.
Although embodiment disclosed by the application is as above, the content only for ease of understanding the application and use
Embodiment is not limited to the application.Technical staff in any the application fields, is taken off not departing from the application
Under the premise of the spirit and scope of dew, any modification and variation, but the application can be carried out in the form and details of implementation
Scope of patent protection, still should be subject to the scope of the claims as defined in the appended claims.
Claims (10)
1. a kind of identification method of Cortex Phellodendri, the identification method the following steps are included:
(1) test solution the preparation of test solution: is prepared into Cortex Phellodendri；
(2) preparation of reference substance solution: to be dissolved with Berberine hydrochloride reference substance, hydrochloric acid phellodendrine reference substance, magnoflorine pair
Solution according to product is reference substance solution；
(3) using the test solution of HPLC difference determination step (1) and the reference substance solution of step (2), and institute is respectively obtained
State the liquid chromatogram of test solution and the reference substance solution；
(4) liquid chromatogram for obtaining step (3) imports similarity evaluation analysis, obtains Cortex Phellodendri
Finger-print；
(5) the Cortex Phellodendri fingerprint obtained using hierarchical cluster analysis, principal component analysis or partial least squares analysis processing step (4)
Spectrum data, to carry out taxonomic history to Cortex Phellodendri or its processed product.
2. identification method according to claim 1, wherein the preparation of the test solution of the step (1) includes: will be yellow
Cypress smashes it through No. 3 pharmacopeia sieves, accurately weighed, is placed in 10mL volumetric flask, the methanol that volume fraction is 70%-100% is added
Aqueous solution, is settled to scale, and weighed weight is ultrasonically treated 30-50min, is cooled to room temperature, is 70%-100% with volume fraction
Methanol aqueous solution supply the weight of loss, shake up, centrifuge 4000r/min-6000r/min is centrifuged 5min-15min, takes
Clear liquid crosses 0.22 μm of miillpore filter, takes subsequent filtrate as test solution；The 70%-100% methanol aqueous solution contains 0.1
Volume % hydrochloric acid；Alternatively,
The preparation of step (1) test solution includes: that Cortex Phellodendri is smashed it through No. 3 pharmacopeia sieves, takes phellodendron powder 0.2g, essence
It is close weighed, it is placed in 10mL volumetric flask, methanol is added, be settled to scale, weighed weight is ultrasonically treated 40min, is cooled to room
Temperature is supplied the weight of loss with methanol, is shaken up, and centrifuge 4000r/min is centrifuged 10min, takes supernatant, crosses 0.22 μm of micropore
Filter membrane takes subsequent filtrate as test solution；Here, the methanol contains 0.1 volume % hydrochloric acid.
3. identification method according to claim 1, wherein the preparation of the reference substance solution of the step (2) includes: precision
Berberine hydrochloride reference substance, hydrochloric acid phellodendrine reference substance, magnoflorine reference substance are weighed, solubilizer is made every 1mL and contains 400 μ g
The reference substance solution of~600 μ g Berberine hydrochlorides, the 50 μ g hydrochloric acid of μ g~100 phellodendrines, the 20 μ g magnoflorines of μ g~50；Or
The preparation of step (2) reference substance solution includes: that precision weighs Berberine hydrochloride reference substance, the control of hydrochloric acid phellodendrine
Product, magnoflorine reference substance add methanol that every 1mL is made and contain 500 μ g Berberine hydrochlorides, 80 μ g hydrochloric acid phellodendrines, 30 μ g lily magnolias
The reference substance solution of flower alkali；Here, the methanol contains 0.1 weight % hydrochloric acid.
4. identification method according to claim 1, wherein the step (3) includes setting HPLC chromatogram condition: chromatographic column
It uses octadecylsilane chemically bonded silica for filler, 25 DEG C -35 DEG C of column temperature, flow velocity 0.8mL/min-1.2mL/min, detects wave
Long 250nm-300nm, and open DAD detection；It is the phosphoric acid water of 0.06%-0.2% with volume fraction using acetonitrile as mobile phase A
Solution is Mobile phase B, gradient elution in the range of volume ratio is 5~100:95~0；
It is accurate respectively to draw 5~20 μ L of test solution and control solution, high performance liquid chromatograph is injected, is measured, respectively
To the liquid chromatogram of test solution and control solution.
5. identification method according to claim 4, wherein the step (3) includes setting HPLC chromatogram condition: chromatographic column
Use octadecylsilane chemically bonded silica for filler,C18 (4.6mm × 150mm, 5.0 μm) is chromatographic column,
30 DEG C of column temperature, flow velocity 1.0mL/min, Detection wavelength 254nm, and open DAD detection；Using acetonitrile as mobile phase A, with volume
The phosphate aqueous solution that score is 0.2% is Mobile phase B, carries out gradient elution, and mobile phase A, the variation of the ratio of B are 0~20min,
A:B is 5%:95% → 10%:90%；20~50min, A:B are 10%:90% → 20%:80%；50~60min, A:
B is 20%:80% → 25%:75%；60~75min, A:B are 25%:75% → 100%:0%；
It is accurate respectively to draw 5 μ L of test solution and control solution, high performance liquid chromatograph is injected, measurement respectively obtains confession
The liquid chromatogram of test sample solution and reference substance solution.
6. identification method according to any one of claims 1-5, wherein the step (4) includes: using Chinese medicine chromatography
Fingerprint similarity evaluation system 2004A editions, the data importing for the liquid chromatogram that step (3) is obtained, Supplements, data
Matching, analysis obtain Cortex Phellodendri finger-print.
7. identification method according to claim 6, wherein the Cortex Phellodendri finger-print that the step (4) obtains includes and salt
Corresponding No. 5 peaks of sour phellodendrine, corresponding No. 6 peaks of magnoflorine, corresponding No. 15 peaks of Berberine hydrochloride, relative retention time
Respectively 0.382,0.396,1.000.
8. identification method according to claim 7, wherein the shared peak for the Cortex Phellodendri finger-print that the step (4) obtains
It further include that No. 1 peak that relative retention time is 0.140, No. 2 peaks, the relative retention times that relative retention time is 0.276 are
When 0.297 No. 3 peaks, No. 4 peaks that relative retention time is 0.356, No. 7 peaks that relative retention time is 0.450, opposite reservation
Between for 0.607 No. 8 peaks, No. 9 peaks that relative retention time is 0.638, No. 10 peaks that relative retention time is 0.672, opposite
No. 11 peaks that retention time is 0.716, No. 12 peaks that relative retention time is 0.817, No. 13 that relative retention time is 0.888
No. 14 peaks that peak, relative retention time are 0.919, No. 16 peaks that relative retention time is 1.168.
9. identification method according to claim 1, wherein the taxonomic history of the step (5) includes that difference river is yellow
Cypress and CORTEX PHELLODENDRI AMURENE distinguish different processed products or distinguish different appearance characters.
10. identification method of any of claims 1-9 differentiate and/or distinguish CORTEX PHELLODENDRI CHINENSE and CORTEX PHELLODENDRI AMURENE, differentiate and/
Or distinguish Cortex Phellodendri process or differentiate and/or distinguish Cortex Phellodendri appearance character in application or above-mentioned Cortex Phellodendri identification method
The identification method of application or above-mentioned Cortex Phellodendri in the control of Cortex Phellodendri quality or the division of Cortex Phellodendri Quality Grade standard is in Huang
Application in the tasting not of the cypress true and false.
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