CN105334273B - Detection method of anisetree bark - Google Patents
Detection method of anisetree bark Download PDFInfo
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- CN105334273B CN105334273B CN201510843038.XA CN201510843038A CN105334273B CN 105334273 B CN105334273 B CN 105334273B CN 201510843038 A CN201510843038 A CN 201510843038A CN 105334273 B CN105334273 B CN 105334273B
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Abstract
The invention discloses a detection method of anisetree bark. The detection method is characterized in that quercitrin, rutin, S1-3 and magnolol are taken as index components; the index components in the anisetree bark are analyzed by adopting high performance liquid chromatography, so that the detection on the quality of the anisetree bark is realized. According to the detection method disclosed by the invention, the quercitrin, the rutin, the S1-3 and the magnolol are taken as the index components; a detection method and a quality identification method for the quality of the anisetree bark are established by using the high performance liquid chromatography; detection results of contents of various components are accurate; the detection method is high in precision, is good in stability and repeatability, and is more effective for guaranteeing the quality of medicinal materials; the uniformity and the stability of the quality of traditional Chinese medicine anisetree bark can be effectively guaranteed.
Description
Technical field
The present invention relates to a kind of detection method of Cortex Illicii, belongs to technical field of medicine quality control.
Background technology
Chinese medicine Cortex Illicii is that being dried for anistree platymiscium Cortex Illicii Illicium difengpi K.I.B et K.I.M. is set
Skin, is that Chinese Pharmacopoeia includes kind (belonging to strong medicine kind originally), with expelling wind and removing dampness, the curative effect of promoting the circulation of QI to relieve pain, in Guangxi among the peoplely
Maple skin is mainly used in treating the diseases such as rheumatic arthritis, lumbar muscle strain and traumatic injury.The plant growing, in In Limestone Area, is wide
A kind of peculiar Chinese crude drug on western karst mountain, belongs to national II grade of Top-rated protected wild plants, and NATURAL DISTRIBUTION is in the southwest in Guangxi
The karst area of portion, middle part and the northwestward, wherein with Longzhou, Jingxi, all pacify, Mashan distribution it is most.According to the literature, at present from
In Cortex Illicii, isolated chemical composition mainly has the structure types such as Phenylpropanoid Glycosides, lignanoid, flavone, phenolic acid, terpenoid, sterol,
Wherein based on Phenylpropanoid Glycosides, lignanoid and flavone, and these type compounds show preferable antiinflammatory in pharmacological evaluation
Activity.In view of the result of study of medication curative effect among the people and the modern aspect such as material base and pharmacology, has been developed that various with Cortex Illici difengpi
Skin is for principal agent or is aided with preparation made by related medical material, such as：FENGSHI GUANJIEYAN PIAN, osmanthus dragon ointment, wind and cold are double from turning piece, treating rheumatism
Safe piece, chase after wind Shu Jing blood circulation promoting slices etc., it is seen that Chinese medicine Cortex Illicii has broad application prospects on field of medicaments.But Cortex Illicii
Because special growing environment is continuous worsening, unordered excavation year after year causes Cortex Illicii to be faced with the resource problem of sternness, thus results in
Crude drug source is chaotic, and phenomenon of adulterating happens occasionally, so that once there is the adulterant of " Guilin Cortex Illicii ", sends out in Clinical practice
Raw intoxication accident.Current this situation is caused, main cause is the absence of quality of medicinal material control method and pattern, at 2010 editions《In
State's pharmacopeia》Also only make the requirement that morphologic observation and chemistry differentiate to Cortex Illicii.In order to solve this problem, it is exactly to set up a kind of letter
Single, fast and effectively analysis method, the true and false of realization difference Chinese medicine Cortex Illicii and evaluation quality quality.
The essence of Chinese medicine is the complex system that various chemical substances are combined according to specific relative amount ratio, if
Under the operation sequence of specification, will obtain using appropriate extraction separation method that chemical composition is specific, feature and content it is relative
Stable Chinese medicine characteristic constituents total extract, while go to characterize its relative the Nomenclature Composition and Structure of Complexes using modern analysis means,
This repeatability with height and distinctive analysis method are highly suitable for the quality control of medical material.Have not yet to see logical
The analysis of high pressure lipuid chromatography (HPLC) centering medicine Cortex Illicii multiple key coastituents is crossed to realize the phase of the detection method of its quality of medicinal material
Close report.
The content of the invention
The technical problem to be solved in the present invention is to provide the inspection of a kind of high accuracy, stability and reproducible Cortex Illicii
Survey method.
The detection method of the Cortex Illicii that the present invention is provided, is with Quercitroside, rutin, S1-3 [4-O- (2'-hydroxy-l'-
Hydroxymethylethyl) dihydroconiferyl alcohol p-hydroxybenzoyl-gluco side] and it is thick
Plain phenol is analyzed so as to realize to These parameters composition in Cortex Illicii using high performance liquid chromatography as index composition
Detection to Cortex Illici difengpi cortex amount, specifically includes following steps：
(1) preparation of reference substance solution
With Quercitroside, rutin, S1-3 honokiols as reference substance, it is 60～80% ethanol or volume fraction with volume fraction
Mixed reference substance solution is obtained for the dissolving preparation of 60～80% methanol, wherein, the concentration of Quercitroside is in mixed reference substance solution
2.00×10-2～6.00 × 10-1Mg/ml, the concentration of rutin is 6.60 × 10-3～1.98 × 10-1The concentration of mg/ml, S1-3 is
4.10×10-3～1.23 × 10-1Mg/ml, the concentration of magnolol is 6.00 × 10-3～1.80 × 10-1mg/ml；
(2) preparation of need testing solution
Take Cortex Illicii, crush, with volume fraction be 60～80% ethanol or volume fraction be 60～80% methanol be extraction
Solvent is extracted, and the Cortex Illicii with the amount ratio of Extraction solvent is：1g：35～45ml, after the completion of extraction, institute is worked as in cooling
Extract weight less than during the gross weight of Cortex Illicii and Extraction solvent, add Extraction solvent before extracting until weight just with
Before extracting, Cortex Illicii is equal with the gross weight of Extraction solvent, filters, obtains need testing solution；
(3) chromatographic condition
C18 chromatographic columns, flow velocity：0.5～1ml/min, column temperature：20～30 DEG C, wavelength：254nm or 280nm, mobile phase is by A
Liquid and B liquid composition, wherein A liquid are acetonitrile or methanol, and B liquid is that volume fraction is 0.1% aqueous formic acid or volume fraction is
0.1% phosphate aqueous solution, gradient elution, elution program are as follows：
During 0min, A liquid：B liquid=5～10%：95～90%；
During 30min, A liquid：B liquid=15～20%：85～80%；
During 40min, A liquid：B liquid=20～25%：80～75%；
During 65min, A liquid：B liquid=45～50%：55～50%；
During 85min, A liquid：B liquid=75～80%：25～20%；
(4) determine
It is accurate to draw a certain amount of above-mentioned mixed reference substance solution and need testing solution, high performance liquid chromatograph is injected separately into,
It is measured according to above-mentioned chromatographic condition.
In the step of above-mentioned method of quality control (1), it is preferred to use volume fraction is 65～75% ethanol or volume fraction
Mixed reference substance solution is obtained for the dissolving preparation of 65～75% methanol, is more preferably adopted with volume fraction as 70% ethanol or volume
Fraction is that the dissolving preparation of 70% methanol obtains mixed reference substance solution.
In the step of above-mentioned method of quality control (1), in the mixed reference substance solution of preparation, the concentration of Quercitroside is preferably controlled
Make 8.00 × 10-2～4.00 × 10-1Mg/ml, more preferably 2.00 × 10-1mg/ml；The concentration of rutin is preferably controlled in
1.32×10-2～1.32 × 10-1Mg/ml, more preferably 6.60 × 10-2mg/ml；The concentration of S1-3 is preferably controlled in 8.20 ×
10-3～6.56 × 10-2Mg/ml, more preferably 4.10 × 10-2mg/ml；The concentration of magnolol is preferably controlled in 6.00 × 10-3~
1.5×10-1Mg/ml, more preferably 7.20 × 10-2mg/ml。
In the step of above-mentioned method of quality control (2), it is 65～75% ethanol or body that Extraction solvent is preferably volume fraction
Fraction is 65～75% methanol, and more preferably volume fraction is that 70% ethanol or volume fraction are 70% methanol.In the step
Extraction solvent it is preferably identical with the solvent for preparing mixed reference substance solution in step (1).
In the step of above-mentioned method of quality control (2), when extracting to Cortex Illicii, the mode of extraction can be prior art
In traditional extraction mode, such as extraction, heating extraction, reflux, extract, supersound extraction etc..It is preferred that using supersound extraction (work frequency
Rate is preferably 30～100KHz), the time of extraction is usually 0.5～2h, generally selects 1h.
In the step of above-mentioned method of quality control (3), in gradient elution, preferred elution program is as follows：
During 0min, A liquid：B liquid=10%：90%；
During 30min, A liquid：B liquid=18%：82%；
During 40min, A liquid：B liquid=24%：76%；
During 65min, A liquid：B liquid=45%：55%；
During 85min, A liquid：B liquid=75%：25%.
In the step of above-mentioned method of quality control (3), sample size determines as needed, usually 10 μ L or 20 μ L.
Detected as stated above, in measurement result, Cortex Illicii medical material presses dry product calculating, is not less than containing Quercitroside
0.2%, 0.018% is not less than containing rutin, is not less than 0.017% containing S1-3, be not less than 0.007% containing magnolol.
On the basis of the studies above, standard finger-print can also be further formulated, afterwards by Cortex Illicii testing sample
Finger printing is compared with the standard finger-print formulated, by calculating the common absorption that both similarity, identification have
Differentiating the quality of Cortex Illicii, wherein testing sample finger printing is compared the quantity at peak with the standard finger-print formulated, its phase
More than 0.88 be should be like degree, usually 0.9～1.0.
Compared with prior art, it is of the invention using Quercitroside, rutin, S1-3 honokiols as index composition, using height
Effect liquid phase chromatogram method sets up the detection method and Quality identification method to Cortex Illici difengpi cortex amount, and each component content testing result is accurate,
The precision of detection method is high, stability and reproducible, to ensure medical material quality more effectively, can effectively guarantee Chinese medicine ground
The stable homogeneous of maple cortex amount.
The following is applicant have determined that the experimental section of optimum chromatographic condition：
1. instrument, reagent chemicals：
Agilent1200 type high performance liquid chromatographs (vacuum on-line degassing system, quaternary pump, automatic sampler, VWD inspections
Survey device, Agilent company of the U.S.)；LC/MS-IT-TOF System mass spectrographs, (Shimadzu, Tokyo, Japan)；XS205 types
Electronic analytical balance (prunus mume (sieb.) sieb.et zucc. Teller-support benefit instrument Shanghai company limited)；KQ-300E type ultrasonic cleaners (city of Kunshan's ultrasound
Instrument Ltd.).
Reference substance Quercitroside, rutin, S1-3 [4-O- (2'-hydroxy-l'-hydroxymethylethyl)
Dihydroconiferyl alcohol p-hydroxybenzoyl-glucoside], magnolol be Guangxi Zhuang Autonomous Region
Guangxi Plant Inst. of the Chinese Academy of Sciences isolated (purity is calculated with area normalization method more than 98%, HPLC detections)；Second
Nitrile is chromatographically pure, and water is ultra-pure water, and it is pure that remaining reagent (such as methanol, ethanol, formic acid etc.) is analysis.
2 experimental techniques
2.1 chromatographic condition
1200 type high pressure liquid chromatographs of Agilent, Agilent ZORBAX SB-C18(5 μm, 4.6x250mm), stream
Speed：1ml/min, column temperature：30 DEG C, wavelength：254nm, sample size：20 μ L, mobile phase：Volume fraction is 0.1% formic acid (B
Liquid)-acetonitrile (A liquid), gradient elution, gradient elution program are as shown in table 1.
Table 1：Condition of gradient elution
The preparation of 2.2 solution
2.2.1 the preparation of reference substance solution
Precision weighs reference substance Quercitroside 20mg, rutin 6.6mg, S1-3 4.1mg honokiol 6.0mg, is placed in 10ml appearances
The EtOH Sonicate dissolving of appropriate volume fraction 70%, constant volume in measuring bottle, is added to shake up, wherein
The concentration of Quercitroside is 2.00mg/ml, and the concentration of rutin is 0.66mg/ml, and the concentration of S1-3 is 0.41mg/ml, magnolol
Concentration is 0.60mg/ml.
2.2.2 the preparation of need testing solution
Dry Cortex Illicii (Jing Guangxi Plant Inst., Chinese Academy of Sciences, Guangxi Zhuang Autonomous Tang Hui researcher identifies,
It is defined as anistree platymiscium Cortex Illicii Illicium difengpi K.I.B et K.I.M.), crushed 40 eye mesh screens.
Precision weighs 0.5g powder and proceeds to put fills in the ethanol solution 20ml for adding volume fraction 70% in conical flask, weighs,
Supersound extraction 1h, adds the ethanol of volume fraction 70% thereto after cooling, until whole system weight with extracted before
Being equal in weight for system, filters, obtains final product need testing solution.
3. the foundation and optimization of chromatographic condition：
The selection of the selected and wavelength of 3.1 index compositions
Selecting for index composition should be representative, and in test liquid, content is higher, and ultra-violet (UV) band inhales relatively strong, and separating degree
It is preferably readily identified.Early stage Chinese medicine Cortex Illicii LC/MS analysis and chemical composition isolate and purify and Structural Identification work base
On plinth, select Quercitroside, rutin, S1-3 honokiols as index composition (MS data are as described in Table 2), reason have with
Lower 3 points：(1) in 4 selected compound structures, Quercitroside and rutin are flavonoid glycoside, and S1-3 is phenylpropyl alcohol glycoside, magnolol
It is then lignanoids, is the characteristic chemical constituent in Chinese medicine Cortex Illicii, and there is anti-inflammatory activity.(2) index constituent structure
In contain ultraviolet absorption group.(3) in need testing solution, content is higher and separating degree is preferable, it is easy to recognize (as shown in Figure 1).
By three-dimensional of DAD (diode array detector) the detector recording need testing solution in the range of 195～400nm
Map analysis, it is determined that the index composition selected is respectively provided with preferably absorption in 254nm and 280nm, selects using 254nm as chromatograph
The Detection wavelength of condition.
Table 2：Index composition MS data
The selection of 3.2 eluting solvents and chromatographic column
Analyzed by literature survey and index constituent structure, in reversed-phase high-performance liquid chromatography, the change containing phenolic hydroxyl group
Compound, can partly dissociate in water, and the hydroxyl not dissociated is stronger with fixed phase separation, so as to cause hangover, it is therefore desirable to plus
Enter buffer to overcome conditions of streaking, therefore from 0.1% formic acid of volume fraction as buffer, test result indicate that volume fraction
0.1% formic acid/acetonitrile has preferable separating degree as mobile phase to the composition in test sample.
Agilent ZORBAX SB-C have been selected in test18(5μm,4.6x 250mm)、phenomenex Luna C18(5μ
M, 4.6x 250mm) and Reprosil-pur Basic C18(5 μm, 4.6x 250mm) chromatographic column, with 0.1% first of volume fraction
Acid/acetonitrile preferably can be separated as flowing relative indicatrix composition, show C18The chromatographic column of filler is to need testing solution
There is preferable separating degree.
3.3 test samples extract the selection of solution
This test is preferably based on following two aspects from the ethanol that volume fraction is 70% as Extraction solvent：One side
The property of face, methanol and ethanol is similar, can efficiently extract the material in Chinese medicine Cortex Illicii, and ethanol has no toxic side effect；Separately
On the one hand, in 4 index compositions, magnolol belongs to aglycon composition, and polarity is less, and other three belong to methods of glycosides, polarity compared with
Greatly.In order to be able to extract index composition as far as possible completely simultaneously, investigate volume fraction and be respectively 50%, 70%, 90%,
Extraction effect of 100% ethanol as Extraction solvent, as a result shows the need testing solution that 70% ethanol solution of volume fraction is extracted
In 4 index component content highests, as a result as described in Table 3.
Table 3：Impact of the different volumes fraction ethanol to 4 kinds of index components peak areas
The investigation of 3.4 extraction times
Precision weighs above-mentioned 3 parts identified of the Chinese medicine Difengpi Barks of 0.5g respectively, proceeds to respectively and puts in plug conical flask, point
Not Jia Ru volume fraction be 70% ethanol solution 20ml, weigh, respectively supersound extraction 0.5h, 1h, 2h, are mended after cooling thereto
Plus the ethanol of volume fraction 70%, until whole system weight with carry out extracting being equal in weight for front system, filter, note respectively
Enter high performance liquid chromatograph to be analyzed, as a result as described in Table 4.As a result the later 4 index composition transfers of 2h are shown not
Greatly, considering extraction time is ultimately determined to 1h.
Table 4：Extraction time is investigated
4. system suitability and methodological study
4.1 index compositions are demarcated and number of theoretical plate
By above-mentioned chromatographic condition respectively by mixed mark (i.e. mixed reference substance solution, similarly hereinafter) and the injection of Cortex Illicii need testing solution
High performance liquid chromatograph is analyzed, and chromatogram is as shown in Fig. 2 and parameter ingredient Quercetin theoretical plate in the chromatography column
Number, as a result as described in Table 5.
Table 5：Index composition number of theoretical plate in the chromatography column
| Quercitroside (sample) | Quercitroside (mixed mark) | |
| TR(min) | 36.219 | 36.104 |
| W(h/2)(min) | 0.43 | 0.45 |
| Number of theoretical plate | 39304.82 | 35661.15 |
4.2 Precision Experiment
Need testing solution is taken, by above-mentioned chromatographic condition, continuous sample introduction 5 times, 20 μ L of sample size, chromatogram are as shown in Figure 3.Knot
Fruit is as described in Table 6.4 kinds of index composition RSD<2%, show that precision is good.
Table 6：Precision Experiment result
4.3 stability experiment
By above-mentioned chromatographic condition, need testing solution is carried out point respectively at 20 μ L of 0h, 3h, 6h, 9h, 12h sample introduction after the production
Analysis, the RSD of 4 kinds of index compositions<2%, chromatogram is as shown in Figure 4.As a result as described in Table 7, as a result show that test sample exists
It is stable in 12h.
Table 7：Stability experiment result
4.4 repeated experiment
5 parts of samples are prepared by need testing solution preparation method, by above-mentioned chromatographic condition, 20 μ L of sample introduction are analyzed respectively,
Chromatogram is as shown in Figure 5.4 kinds of index composition RSD<4%, as a result show the method favorable reproducibility.As a result it is as shown in table 8.
Table 8：Repeated experiment result
4.5 reference substance standard curves are set up
Mixing reference substance mother solution is prepared by the preparation method of reference substance solution, mixing reference substance is drawn successively with liquid-transfering gun molten
10,20,40,80,100,120,160,200,250,300 μ L of liquid, are separately added into the ethanol of volume fraction 70% quantitatively to 1ml,
The mixed reference substance solution for obtaining final product variable concentrations is shaken up, high liquid chromatograph of liquid is injected separately into, is surveyed by above-mentioned chromatographic condition
Fixed, with peak area as vertical coordinate, sample size is abscissa, sets up reference substance standard curve, and measurement result is as described in Table 9.
9 reference substance standard curve of table
| Reference substance | Regression equation | R2 | The range of linearity (ng) |
| Rutin | Y=1.2275x-13.25 | 0.9999 | 66～1980 |
| S1-3 | Y=1.3123x-13.544 | 0.9994 | 41～1230 |
| Quercitroside | Y=4.0887x-78.441 | 0.9999 | 200～6000 |
| Magnolol | Y=1.3981x-1.9871 | 0.9999 | 60～1800 |
Description of the drawings
HPLC-UV chromatograms of the Fig. 1 for need testing solution；
Fig. 2 is the HPLC-UV chromatograms of mixed reference substance solution and need testing solution；
Fig. 3 is the HPLC-UV chromatograms of need testing solution continuous sample introduction 5 times in Precision Experiment；
Fig. 4 is HPLC-UV chromatogram of the need testing solution in 5 different times in stability experiment；
Fig. 5 is the HPLC-UV chromatograms of 5 parts of need testing solutions in repeated experiment；
Fig. 6 is the HPLC-UV chromatograms of Cortex Illicii adulterant on the market and Illicium jiadifengpi；
Fig. 7 is the Cortex Illicii HPLC-UV chromatograms of 10 batch separate sources；
Fig. 8 is Cortex Illicii HPLC-UV finger printing.
Specific embodiment
With reference to specific embodiment, the present invention is described in further detail, to more fully understand present disclosure, but
The present invention is not limited to following examples.
Embodiment 1
(1) preparation of reference substance solution
Precision weighs reference substance Quercitroside 20mg, rutin 6.6mg, S1-3 4.1mg honokiol 6.0mg, is placed in 10ml appearances
In measuring bottle, add the EtOH Sonicate dissolving of appropriate volume fraction 70%, constant volume to shake up, that is, obtain mixed reference substance solution, wherein
The concentration of Quercitroside is 2.00mg/ml, and the concentration of rutin is 0.66mg/ml, and the concentration of S1-3 is 0.41mg/ml, magnolol
Concentration is 0.60mg/ml.
(2) preparation of need testing solution
Take the Guangxi Liang Ge places of production (Evalution In Duan County (abbreviation Du'an of Guangxi), Guangxi Hechi Tiane County (abbreviation Guangxi Tiane))
Chinese medicine Cortex Illicii, Jing Guangxi Plant Inst., Chinese Academy of Sciences, Guangxi Zhuang Autonomous Tang Hui researcher identification, be defined as
Anistree platymiscium Cortex Illicii Illicium difengpi K.I.B et K.I.M., the sample in the above-mentioned two place of production are designated as respectively
1# test samples and 2# test samples, crushed 40 eye mesh screens respectively.Precision weighs 0.5g 1# test sample powder and proceeds to puts plug conical flask
In, the ethanol solution 20ml of volume fraction 70% being added, is weighed, supersound extraction 1h adds volume fraction thereto after cooling
70% ethanol, until whole system weight with carry out extracting being equal in weight for front system, filter, obtain final product 1# test samples molten
Liquid.2# need testing solutions are obtained in the same way.
(3) chromatographic condition：
Agilent ZORBAX SB-C18(5 μm, 4.6x 250mm), flow velocity：1ml/min, column temperature：30 DEG C, wavelength：
254nm, sample size：20 μ L, mobile phase are made up of A liquid and B liquid, and wherein A liquid is acetonitrile, and it is 0.1% formic acid that B liquid is volume fraction
Aqueous solution, gradient elution, elution program are as follows：
During 0min, A liquid：B liquid=10%：90%；
During 30min, A liquid：B liquid=18%：82%；
During 40min, A liquid：B liquid=24%：76%；
During 65min, A liquid：B liquid=45%：55%；
During 85min, A liquid：B liquid=75%：25%.
(4) determine
1# need testing solutions and 2# need testing solutions obtained in 20 μ L steps (2) of accurate absorption, are injected separately into efficient liquid phase
Chromatograph, is measured according to step (3) chromatographic condition, each sample parallel assay 3 times, measurement result such as table 10 below institute
Show.
10 Cortex Illicii assay result of table
(5) sample for being denoted as Cortex Illicii on the market and Illicium jiadifengpi sample analysis compare
Take the sample for being denoted as Cortex Illicii and Illicium jiadifengpi sample (each Cortex Illicii sample provenance such as table of market purchase
Shown in 11), the need testing solution as obtained in above-mentioned steps (2) is accurate respectively to draw 20 μ L need testing solutions, injects efficient liquid phase
Chromatograph, is measured according to above-mentioned steps (3) chromatographic condition, as a result sees Fig. 6.Can be very by HPLC-UV chromatograms
Above-mentioned bought Cortex Illicii is clearly seen widely different with the authentic Cortex Illicii through identification, thus Cortex Illicii can be accredited as
Adulterant.It can be seen that, the HPLC chromatogram method set up by the present invention can be with the true and false of quick discriminating sample.
11 Cortex Illicii of table (purchase) is originated
| Sample | The place of production |
| It is denoted as the sample of Cortex Illicii | Guilin |
| It is denoted as the sample of Cortex Illicii | Hebei |
| Illicium jiadifengpi sample | Guangxi resource |
Cortex Illicii HPLC-UV finger printing is set up and technical parameter
1st, the demarcation of characteristic peak
Accurate 10 batches of need testing solutions (the various Cortex Illicii samples for drawing 20 μ L separate sources as obtained in abovementioned steps (2)
Product source is shown in Table 12, the equal Guangxi Plant Inst., Chinese Academy of Sciences, Guangxi Zhuang Autonomous Tang Hui researcher mirror of each Cortex Illicii sample
It is fixed, it is defined as anistree platymiscium Cortex Illicii Illicium difengpiK.I.B et K.I.M.), it is injected separately into efficient liquid phase
Chromatograph, is measured according to abovementioned steps (3) chromatographic condition.According to given by 10 batch test sample HPLC-UV collection of illustrative plates
Relevant parameter, the chromatographic peak of Cortex Illicii all the components all occurs in 85min, and wherein Quercitroside is its main component, product
Divide percent maximum, therefore Quercitroside is selected as reference peak.
Reference peak Quercitroside is numbered S, other characteristic peaks are numbered 1,2 successively ... N.
Table 12：The source of 10 batch Cortex Illiciis and identification
| Collecting location | Plant name | Numbering |
| Du'an of Guangxi | Illicium difengpi | S1 |
| Du'an of Guangxi | Illicium difengpi | S2 |
| Mashan Guangxi | Illicium difengpi | S3 |
| Mashan Guangxi | Illicium difengpi | S4 |
| Mashan Guangxi | Illicium difengpi | S5 |
| Guangxi Tiane | Illicium difengpi | S6 |
| Guangxi Tiane | Illicium difengpi | S7 |
| Guangxi Tiane | Illicium difengpi | S8 |
| Guangxi Xincheng | Illicium difengpi | S9 |
| Longzhou | Illicium difengpi | S10 |
2nd, characteristic peak relative retention time and integration relative ratio
According to the relevant parameter that 10 batch test sample HPLC-UV collection of illustrative plates are given, compare each test sample chromatogram (see Fig. 7),
Wherein 18 peaks are that 10 batch test samples have, it is thus determined that this 18 peaks are total fingerprint peakses.It is with reference to peak with Quercitroside
(S), rutin (7), S1-3 (9) honokiol (16) are characterized peak, set up Cortex Illicii HPLC-UV finger printing (result such as Fig. 8 institutes
Show), each relative retention time at total peak and the ratio of relative peak area are shown in Table 13 and table 14.
Table 13：The relative retention time at the total peak of 10 batch Cortex Illiciis
Table 14：The relative peak area at the total peak of 10 batch Cortex Illiciis
3rd, fingerprint similarity
Employing fingerprint collection of illustrative plates similarity evaluation software carries out similarity meter to the Cortex Illicii medicinal materials fingerprint in each place of production
Calculate.The similarity for obtaining different sources Cortex Illicii and compareing spectrogram.As a result except No. 9 similarities it is slightly biased it is low in addition to, the ground in other places of production
Maple skin similarity the results are shown in Table 15 more than 90%.
Table 15：The fingerprint similarity result of calculation of 10 batch Cortex Illiciis
| Sample | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | Reference fingerprint |
| Similarity | 1 | 0.99 | 0.93 | 0.92 | 0.91 | 0.91 | 0.92 | 0.92 | 0.88 | 0.90 | 0.97 |
Claims (6)
1. a kind of detection method of Cortex Illicii, it is characterised in that：Using Quercitroside, rutin, S1-3 honokiols as index into
Point, these index compositions in Cortex Illicii are analyzed so as to realize the inspection to Cortex Illici difengpi cortex amount using high performance liquid chromatography
Survey, specifically include following steps：
(1) preparation of reference substance solution
With Quercitroside, rutin, S1-3 honokiols as reference substance, it is that 60～80% ethanol or volume fraction are 60 with volume fraction
The dissolving preparation of～80% methanol obtains mixed reference substance solution, and wherein, in mixed reference substance solution, the concentration of Quercitroside is 2.00
×10-2～6.00 × 10-1Mg/ml, the concentration of rutin is 6.60 × 10-3～1.98 × 10-1The concentration of mg/ml, S1-3 is 4.10
×10-3～1.23 × 10-1Mg/ml, the concentration of magnolol is 6.00 × 10-3～1.80 × 10-1mg/ml；
(2) preparation of need testing solution
Take Cortex Illicii, crush, with volume fraction be 60～80% ethanol or volume fraction be 60～80% methanol as Extraction solvent
Extracted, the Cortex Illicii with the amount ratio of Extraction solvent is：1g：35～45ml, after the completion of extraction, cooling, when gained is carried
Take the weight of thing less than Cortex Illicii before extracting and Extraction solvent gross weight when, add Extraction solvent until weight just with extraction
Front Cortex Illicii is equal with the gross weight of Extraction solvent, filters, obtains need testing solution；
(3) chromatographic condition
C18 chromatographic columns, flow velocity：0.5～1ml/min, column temperature：20～30 DEG C, wavelength：254nm or 280nm, mobile phase by A liquid and
B liquid is constituted, and wherein A liquid is acetonitrile or methanol, and it is that 0.1% aqueous formic acid or volume fraction are 0.1% phosphorus that B liquid is volume fraction
Aqueous acid, gradient elution, elution program are as follows：
During 0min, A liquid：B liquid=5～10%：95～90%；
During 30min, A liquid：B liquid=15～20%：85～80%；
During 40min, A liquid：B liquid=20～25%：80～75%；
During 65min, A liquid：B liquid=45～50%：55～50%；
During 85min, A liquid：B liquid=75～80%：25～20%；
(4) determine
It is accurate to draw a certain amount of above-mentioned mixed reference substance solution and need testing solution, high performance liquid chromatograph is injected separately into, according to
Above-mentioned chromatographic condition is measured.
2. the detection method of Cortex Illicii according to claim 1, it is characterised in that：In step (1), it is 65 with volume fraction
～75% ethanol or volume fraction are that the dissolving preparation of 65～75% methanol obtains mixed reference substance solution.
3. the detection method of Cortex Illicii according to claim 1, it is characterised in that：In step (1), mixed reference substance solution
The concentration of middle Quercitroside is 8.00 × 10-2～4.00 × 10-1Mg/ml, the concentration of rutin is 1.32 × 10-2～1.32 × 10- 1The concentration of mg/ml, S1-3 is 8.20 × 10-3～6.56 × 10-2Mg/ml, the concentration of magnolol is 6.00 × 10-3～1.50 ×
10-1mg/ml。
4. the detection method of Cortex Illicii according to claim 1, it is characterised in that：In step (2), Extraction solvent is volume
Fraction is that 65～75% ethanol or volume fraction are 65～75% methanol.
5. the detection method of the Cortex Illicii according to any one of Claims 1 to 4, it is characterised in that：In step (3),
During gradient elution, elution program is as follows：
During 0min, A liquid：B liquid=10%：90%；
During 30min, A liquid：B liquid=18%：82%；
During 40min, A liquid：B liquid=24%：76%；
During 65min, A liquid：B liquid=45%：55%；
During 85min, A liquid：B liquid=75%：25%.
6. the detection method of the Cortex Illicii according to any one of Claims 1 to 4, it is characterised in that：
In step (1), it is that 70% ethanol or volume fraction obtain mixing reference substance for the dissolving preparation of 70% methanol with volume fraction
Solution；
In step (1), in mixed reference substance solution, the concentration of Quercitroside is 2.00 × 10-1Mg/ml, the concentration of rutin is 6.60 ×
10-2The concentration of mg/ml, S1-3 is 4.10 × 10-2Mg/ml, the concentration of magnolol is 7.20 × 10-2mg/ml；
In step (2), it is that 70% ethanol or volume fraction are 70% methanol that Extraction solvent is volume fraction；
In step (2), the Cortex Illicii with the amount ratio of Extraction solvent is：1g：40ml；
In step (3), in gradient elution, elution program is as follows：
During 0min, A liquid：B liquid=10%：90%；
During 30min, A liquid：B liquid=18%：82%；
During 40min, A liquid：B liquid=24%：76%；
During 65min, A liquid：B liquid=45%：55%；
During 85min, A liquid：B liquid=75%：25%.
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