CN105334273A - Quality control method of anisetree bark - Google Patents
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Abstract
The invention discloses a quality control method of anisetree bark. The quality control 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 quality control 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 the method for quality control of anisetree bark, belong to technical field of medicine quality control.
Background technology
Chinese medicine anisetree bark is the dry bark of Illicium plant anisetree bark IlliciumdifengpiK.I.BetK.I.M., for Chinese Pharmacopoeia includes kind (belonging to strong medicine kind originally), have and dispel rheumatism, the curative effect of promoting qi circulation and relieving pain, in Guangxi, anisetree bark among the people is mainly used in the diseases such as treatment rheumathritis, lumbar muscle strain and traumatic injury.This plant growth is in In Limestone Area; it is a kind of peculiar Chinese crude drug in EXPLOITATION AND UTILIZATION IN GUANGXI KARST; belong to country's II grade of Top-rated protected wild plants, NATURAL DISTRIBUTION in the west and south in Guangxi, the karst area of middle part and the northwestward, wherein with Longzhou, Jingxi, all pacify, Mashan distribution is maximum.According to the literature, from anisetree bark, be separated the chemical composition obtained mainly contains the structure types such as Phenylpropanoid Glycosides, lignanoid, flavones, phenolic acid, terpene, sterol at present, wherein based on Phenylpropanoid Glycosides, lignanoid and flavones, and these type compounds show good anti-inflammatory activity in pharmacological evaluation.In view of the result of study of medication curative effect among the people and the aspect such as modern material base and pharmacology, develop and multiplely taken anisetree bark as main ingredient or be aided with the preparation that relevant medicinal material makes, as two in: FENGSHI GUANJIEYAN PIAN, osmanthus dragon ointment, chill from turning sheet, beater wheel mill, chase after wind and relax through blood circulation promoting slice etc., visible Chinese medicine anisetree bark has broad application prospects on field of medicaments.But anisetree bark because of special growing environment continuous worsening, year after year unorderedly to excavate, cause anisetree bark to be faced with severe resource problem, therefore cause crude drug source chaotic, phenomenon of adulterating happens occasionally, so that once occurs the adulterant of " Guilin anisetree bark ", in Clinical practice, intoxication accident occurs.Cause current this situation, main cause lacks quality of medicinal material control method and pattern, also only does the requirement of morphologic observation and chemistry discriminating at 2010 editions " Chinese Pharmacopoeias " to anisetree bark.In order to address this problem, set up exactly one simply, analytical approach fast and effectively, realize the true and false of difference Chinese medicine anisetree bark and the quality of evaluation quality.
The essence of Chinese medicine is the complex system of number of chemical material according to specific relative content ratio combination, if under the running program of specification, adopt suitable extraction separation method by concrete for acquisition chemical composition, feature with content metastable Chinese medicine characteristic constituents total extract, utilize modern analysis means to go to characterize its relative the Nomenclature Composition and Structure of Complexes simultaneously, this there is height reappearance and distinctive analytical approach be highly suitable for the quality control of medicinal material.To have not yet to see by high pressure lipuid chromatography (HPLC) the analysis of Chinese medicine anisetree bark multiple key coastituents to realize the relevant report of the detection method of its quality of medicinal material.
Summary of the invention
The technical problem to be solved in the present invention is to provide that a kind of accuracy is high, the method for quality control of stability and reproducible anisetree bark.
The method of quality control of anisetree bark provided by the invention, using quercitin, rutin, S1-3 [4-O-(2'-hydroxy-l'-hydroxymethylethyl) dihydroconiferylalcoholp-hydroxybenzoyl-glucoside] honokiol as index composition, adopt high performance liquid chromatography analyze These parameters composition in anisetree bark thus realize the detection of maple cortex amount over the ground, specifically comprise the following steps:
(1) preparation of reference substance solution
With quercitin, rutin, S1-3 honokiol for reference substance, by volume fraction, to be 60 ~ 80% ethanol or volume fraction be that 60 ~ 80% methyl alcohol dissolve preparations obtains mixing reference substance solution, and wherein, in mixing reference substance solution, the concentration of quercitin 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
Get anisetree bark, pulverize, with volume fraction to be 60 ~ 80% ethanol or volume fraction be 60 ~ 80% methyl alcohol for Extraction solvent extracts, the amount ratio of described anisetree bark and Extraction solvent is: 1g:35 ~ 45ml, after having extracted, and cooling, when extract obtained weight is less than the general assembly (TW) extracting front anisetree bark and Extraction solvent, add Extraction solvent until weight is just equal with the general assembly (TW) of Extraction solvent with anisetree bark before extraction, filter, obtain need testing solution;
(3) chromatographic condition
C18 chromatographic column, flow velocity: 0.5 ~ 1ml/min, column temperature: 20 ~ 30 DEG C, wavelength: 254nm or 280nm, mobile phase is made up of A liquid and B liquid, and wherein A liquid is acetonitrile or methyl alcohol, and B liquid is volume fraction be 0.1% aqueous formic acid or volume fraction is 0.1% phosphate aqueous solution, gradient elution, elution program is 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) measure
The a certain amount of above-mentioned mixing reference substance solution of accurate absorption and need testing solution, inject high performance liquid chromatograph respectively, measure according to above-mentioned chromatographic condition.
In the step (1) of above-mentioned method of quality control, preferred employing volume fraction is 65 ~ 75% ethanol or volume fraction is that 65 ~ 75% methyl alcohol dissolving preparations obtain mixing reference substance solution, and more preferably employing volume fraction is 70% ethanol or volume fraction is that 70% methyl alcohol dissolving preparation obtains mixing reference substance solution.
In the step (1) of above-mentioned method of quality control, in the mixing reference substance solution of preparation, the concentration of quercitin preferably controls 8.00 × 10
-2~ 4.00 × 10
-1mg/ml, is more preferably 2.00 × 10
-1mg/ml; The concentration of rutin preferably controls 1.32 × 10
-2~ 1.32 × 10
-1mg/ml, is more preferably 6.60 × 10
-2mg/ml; The concentration of S1-3 preferably controls 8.20 × 10
-3~ 6.56 × 10
-2mg/ml, is more preferably 4.10 × 10
-2mg/ml; The concentration of magnolol preferably controls 6.00 × 10
-3~ 1.5 × 10
-1mg/ml, is more preferably 7.20 × 10
-2mg/ml.
In the step (2) of above-mentioned method of quality control, it be 65 ~ 75% ethanol or volume fraction is 65 ~ 75% methyl alcohol that Extraction solvent is preferably volume fraction, and to be more preferably volume fraction be 70% ethanol or volume fraction is 70% methyl alcohol.Preferably to prepare the solvent mixing reference substance solution identical with step (1) for Extraction solvent in this step.
In the step (2) of above-mentioned method of quality control, when extracting anisetree bark, the mode of extraction can be traditional extraction mode of the prior art, as lixiviate, heating extraction, refluxing extraction, ultrasonic extraction etc.The ultrasonic extraction of preferred employing (frequency of operation is preferably 30 ~ 100KHz), the time of extraction is generally 0.5 ~ 2h, usually selects 1h.
In the step (3) of above-mentioned method of quality control, when 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 (3) of above-mentioned method of quality control, sample size is determined as required, is generally 10 μ L or 20 μ L.
Detect as stated above, in measurement result, anisetree bark medicinal material is pressed dry product and is calculated, and is not less than 0.2%, is not less than 0.018% containing rutin, be not less than 0.017% containing S1-3, be not less than 0.007% containing magnolol containing quercitin.
On the basis of above-mentioned research, standard finger-print can also be formulated further, afterwards the standard finger-print of anisetree bark testing sample finger-print and formulation is compared, by calculating similarity, the quantity of the common absorption peak that both identification has is to differentiate the quality of anisetree bark, the wherein standard finger-print comparison of testing sample finger-print and formulation, its similarity should be more than 0.88, is generally 0.9 ~ 1.0.
Compared with prior art, the present invention is using quercitin, rutin, S1-3 honokiol as index composition, high performance liquid chromatography is utilized to set up detection method and the Quality identification method of maple cortex amount over the ground, each component content testing result is accurate, the precision of detection method is high, stability and reproducible, to ensureing that the quality of medicinal material is more effective, the stable homogeneous of Chinese medicine anisetree bark quality effectively can be guaranteed.
Below the experimental section that applicant determines optimum chromatographic condition:
1. instrument, reagent chemicals:
Agilent1200 type high performance liquid chromatograph (the online degas system of vacuum, quaternary pump, automatic sampler, VWD detecting device, Agilent company of the U.S.); LC/MS-IT-TOFSystem mass spectrometer, (Shimadzu, Tokyo, Japan); XS205 type electronic analytical balance (plum Teller-Tuo benefit instrument Shanghai company limited); KQ-300E type ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.).
Reference substance quercitin, rutin, S1-3 [4-O-(2'-hydroxy-l'-hydroxymethylethyl) dihydroconiferylalcoholp-hydroxybenzoyl-glucoside], magnolol are Guangxi Plant Inst., Chinese Academy of Sciences, Guangxi Zhuang Autonomous and are separated and obtain that (purity is greater than 98%, HPLC detects, and calculates by area normalization method); Acetonitrile is chromatographically pure, and water is ultrapure water, and it is pure that all the other reagent (as methyl alcohol, ethanol, formic acid etc.) are analysis.
2 experimental techniques
2.1 chromatographic condition
Agilent1200 type high pressure liquid chromatograph, AgilentZORBAXSB-C
18(5 μm, 4.6x250mm), flow velocity: 1ml/min, column temperature: 30 DEG C, wavelength: 254nm, sample size: 20 μ L, mobile phase: volume fraction is formic acid (B the liquid)-acetonitrile (A liquid) of 0.1%, and gradient elution, gradient elution program is 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 takes reference substance quercitin 20mg, rutin 6.6mg, S1-34.1mg honokiol 6.0mg, be placed in 10ml volumetric flask, the EtOH Sonicate adding appropriate volume mark 70% dissolves, constant volume, shake up and namely obtain mixing reference substance solution, wherein the concentration of quercitin is 2.00mg/ml, and the concentration of rutin is 0.66mg/ml, the concentration of S1-3 is 0.41mg/ml, and the concentration of magnolol is 0.60mg/ml.
2.2.2 the preparation of need testing solution
Dry anisetree bark (through Guangxi Plant Inst., Chinese Academy of Sciences, Guangxi Zhuang Autonomous Tang Hui researcher qualification, being defined as Illicium plant anisetree bark IlliciumdifengpiK.I.BetK.I.M.), pulverized 40 eye mesh screens.
Precision take 0.5g powder proceed to put plug conical flask in, add the ethanolic solution 20ml of volume fraction 70%, weigh, ultrasonic extraction 1h, the ethanol of volume fraction 70% is added wherein after cooling, until the weight of whole system with carry out extracting being equal in weight of front system, filter, obtain need testing solution.
3. the foundation of chromatographic condition and optimization:
3.1 the selection of the selected and wavelength of index composition
The selected of index composition should be representative, and in test liquid, content is higher, and ultraviolet region is inhaled comparatively strong, and degree of separation is better easy to identify.Early stage Chinese medicine anisetree bark LC/MS to analyze and on the separation and purification of chemical composition and Structural Identification working foundation, selected quercitin, rutin, S1-3 honokiol are as index composition (MS data as described in Table 2), reason has following 3 points: in 4 compound structures that (1) is selected, quercitin and rutin are flavonoid glycoside, S1-3 is phenylpropyl alcohol glycoside, magnolol is then lignanoids, is all the characteristic chemical constituents in Chinese medicine anisetree bark, and has anti-inflammatory activity.(2) ultraviolet absorption group is contained in index constituent structure.(3) in need testing solution, the higher and degree of separation of content is better, is easy to identify (as shown in Figure 1).
By the three-dimensional plot analysis of DAD (diode array detector) detector recording need testing solution within the scope of 195 ~ 400nm, determine that the index composition selected all has good absorption at 254nm and 280nm, select the determined wavelength using 254nm as chromatographic 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, compound containing phenolic hydroxyl group, in water can partial solution from, and the hydroxyl do not dissociated and Stationary liquid effect are comparatively strong, thus cause hangover, therefore need to add damping fluid to overcome conditions of streaking, therefore select volume fraction 0.1% formic acid as damping fluid, experimental result shows that volume fraction 0.1% formic acid/acetonitrile has better degree of separation as mobile phase to the composition in test sample.
AgilentZORBAXSB-C has been selected in test
18(5 μm, 4.6x250mm), phenomenexLunaC
18(5 μm, 4.6x250mm) and Reprosil-purBasicC
18(5 μm, 4.6x250mm) chromatographic column, all better can be separated as flowing relative indicatrix composition using volume fraction 0.1% formic acid/acetonitrile, be shown C
18the chromatographic column of filler has good degree of separation to need testing solution.
3.3 test samples extract the selection of solution
This test preferably select volume fraction be 70% ethanol as Extraction solvent based on following two aspects: on the one hand, methyl alcohol is similar with the character of ethanol, all effectively can extract the material in Chinese medicine anisetree bark, and ethanol has no side effect; On the other hand, in 4 index compositions, magnolol belongs to aglycon composition, and polarity is less, and other three belong to methods of glycosides, and polarity is larger.In order to can index composition be extracted as far as possible completely simultaneously, investigate volume fraction and be respectively 50%, 70%, 90%, 100% ethanol is as the extraction effect of Extraction solvent, result shows that in the need testing solution that volume fraction 70% ethanolic solution extracts, 4 index component contents are the highest, and result as described in Table 3.
Table 3: different volumes mark ethanol is on the impact of 4 kinds of index components peak areas
The investigation of 3.4 extraction times
Precision takes the above-mentioned Chinese medicine Difengpi Bark 3 parts through qualification of 0.5g respectively, proceed to respectively and put in plug conical flask, add the ethanolic solution 20ml that volume fraction is 70% respectively, weigh, ultrasonic extraction 0.5h, 1h, 2h, add the ethanol of volume fraction 70% wherein after cooling respectively, until the weight of whole system with carry out extracting being equal in weight of front system, filter, inject high performance liquid chromatograph respectively and analyze, result as described in Table 4.Result shows that 4 index composition transfer are little after 2h, considers extraction time to be finally defined as 1h.
Table 4: extraction time is investigated
4. system suitability and methodological study
4.1 index compositions are demarcated and number of theoretical plate
Mark will be mixed respectively by above-mentioned chromatographic condition and (namely mix reference substance solution, lower same) and the analysis of anisetree bark need testing solution injection high performance liquid chromatograph, as shown in Figure 2, and parameter ingredient Quercetin number of theoretical plate in the chromatography column, result as described in Table 5 for chromatogram.
Table 5: index composition number of theoretical plate in the chromatography column
| Quercitin (sample) | Quercitin (mixed mark) | |
| T R(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
Get need testing solution, by above-mentioned chromatographic condition, continuous sample introduction 5 times, sample size 20 μ L, chromatogram as shown in Figure 3.Result 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 analyzed respectively at 0h, 3h, 6h, 9h, 12h sample introduction 20 μ L after the production, and the RSD<2% of 4 kinds of index compositions, chromatogram as shown in Figure 4.As described in Table 7, result shows that test sample is stable in 12h to result.
Table 7: stability experiment result
4.4 repeated experiment
Prepare 5 increment product by need testing solution preparation method, by above-mentioned chromatographic condition, sample introduction 20 μ L analyzes respectively, and chromatogram as shown in Figure 5.4 kinds of index composition RSD<4%, result shows the method favorable reproducibility.Result is as shown in table 8.
Table 8: repeated experiment result
4.5 reference substance typical curves are set up
Mixing reference substance mother liquor is prepared by the preparation method of reference substance solution, mixing reference substance solution 10,20,40,80,100,120,160,200,250,300 μ L is drawn successively with liquid-transfering gun, add the ethanol of volume fraction 70% respectively quantitatively to 1ml, shake up and obtain the mixing reference substance solution of variable concentrations, inject high liquid liquid chromatograph respectively, measure by above-mentioned chromatographic condition, take peak area as ordinate, sample size is horizontal ordinate, set up reference substance typical curve, measurement result as described in Table 9.
Table 9 reference substance typical curve
| Reference substance | Regression equation | R 2 | 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 |
| Quercitin | y＝4.0887x-78.441 | 0.9999 | 200～6000 |
| Magnolol | y＝1.3981x-1.9871 | 0.9999 | 60～1800 |
Accompanying drawing explanation
Fig. 1 is the HPLC-UV chromatogram of need testing solution;
Fig. 2 is the HPLC-UV chromatogram of mixing reference substance solution and need testing solution;
Fig. 3 is the HPLC-UV chromatogram of need testing solution continuous sample introduction 5 times in Precision Experiment;
Fig. 4 is the HPLC-UV chromatogram of need testing solution in 5 different times in stability experiment;
Fig. 5 is the HPLC-UV chromatogram of 5 parts of need testing solutions in repeated experiment;
Fig. 6 is the HPLC-UV chromatogram of anisetree bark adulterant and the Illicium jiadifengpi that market is sold;
Fig. 7 is the anisetree bark HPLC-UV chromatogram of 10 batches of separate sources;
Fig. 8 is anisetree bark HPLC-UV finger-print.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail, and to understand content of the present invention better, but the present invention is not limited to following examples.
Embodiment 1
(1) preparation of reference substance solution
Precision takes reference substance quercitin 20mg, rutin 6.6mg, S1-34.1mg honokiol 6.0mg, be placed in 10ml volumetric flask, the EtOH Sonicate adding appropriate volume mark 70% dissolves, constant volume, shakes up, and namely obtains mixing reference substance solution, wherein the concentration of quercitin is 2.00mg/ml, the concentration of rutin is the concentration of 0.66mg/ml, S1-3 is 0.41mg/ml, and the concentration of magnolol is 0.60mg/ml.
(2) preparation of need testing solution
Get the Chinese medicine anisetree bark of the Liang Ge place of production, Guangxi (Evalution In Duan County (abbreviation Du'an of Guangxi), Tiane County, Guangxi Hechi (being called for short Tiane, Guangxi)), identify through Guangxi Plant Inst., Chinese Academy of Sciences, Guangxi Zhuang Autonomous Tang Hui researcher, all be defined as Illicium plant anisetree bark IlliciumdifengpiK.I.BetK.I.M., the sample in above-mentioned two places of production is designated as 1# test sample and 2# test sample respectively, pulverizes 40 eye mesh screens respectively.Precision take 0.5g1# test sample powder proceed to put plug conical flask in, add the ethanolic solution 20ml of volume fraction 70%, weigh, ultrasonic extraction 1h, the ethanol of volume fraction 70% is added wherein after cooling, until the weight of whole system with carry out extracting being equal in weight of front system, filter, obtain 1# need testing solution.2# need testing solution is obtained by same method.
(3) chromatographic condition:
AgilentZORBAXSB-C
18(5 μm, 4.6x250mm), flow velocity: 1ml/min, column temperature: 30 DEG C, wavelength: 254nm, sample size: 20 μ L, mobile phase is made up of A liquid and B liquid, and wherein A liquid is acetonitrile, B liquid is volume fraction is 0.1% aqueous formic acid, gradient elution, and 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%.
(4) measure
The 1# need testing solution that accurate absorption 20 μ L steps (2) are obtained and 2# need testing solution, inject high performance liquid chromatograph respectively, measure according to step (3) described chromatographic condition, each sample replicate determination 3 times, measurement result as described in Table 10.
Table 10 anisetree bark assay result
(5) sample being denoted as anisetree bark market sold and Illicium jiadifengpi sample analysis compare
Get the sample being denoted as anisetree bark and Illicium jiadifengpi sample (each anisetree bark sample provenance is as shown in table 11) bought in market, by the need testing solution that above-mentioned steps (2) is obtained, accurate absorption 20 μ L need testing solutions respectively, inject high performance liquid chromatograph, measure according to above-mentioned steps (3) described chromatographic condition, the results are shown in Figure 6.Very clearly can finding out that above-mentioned bought anisetree bark is widely different with the authentic anisetree bark through identifying by HPLC-UV chromatogram, thus can be accredited as anisetree bark adulterant.Visible, the HPLC chromatographic process set up by the present invention can differentiate the true and false of sample fast.
Table 11 anisetree bark (purchase) is originated
| Sample | The place of production |
| Be denoted as the sample of anisetree bark | Guilin |
| Be denoted as the sample of anisetree bark | Hebei |
| Illicium jiadifengpi sample | Guangxi resource |
Anisetree bark HPLC-UV finger-print is set up and technical parameter
1, the demarcation of characteristic peak
(various anisetree bark sample source is in table 12 for 10 batches of need testing solutions of the separate sources that the accurate 20 μ L of absorption obtain by abovementioned steps (2), the equal Guangxi Plant Inst., Chinese Academy of Sciences, Guangxi Zhuang Autonomous Tang Hui researcher qualification of each anisetree bark sample, all be defined as Illicium plant anisetree bark IlliciumdifengpiK.I.BetK.I.M.), inject high performance liquid chromatograph respectively, measure according to abovementioned steps (3) described chromatographic condition.According to the correlation parameter given by 10 batches of test sample HPLC-UV collection of illustrative plates, the chromatographic peak of anisetree bark all the components all occurs in 85min, and wherein quercitin is its principal ingredient, and integration number percent is maximum, therefore selects quercitin as reference peak.
Reference peak quercitin label is S, and other characteristic peaks successively label are 1,2 ... N.
The source of table 12:10 batch of anisetree bark and qualification
| 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 |
| Tiane, Guangxi | Illicium difengpi | S6 |
| Tiane, Guangxi | Illicium difengpi | S7 |
| Tiane, Guangxi | Illicium difengpi | S8 |
| Xincheng, Guangxi | Illicium difengpi | S9 |
| Longzhou | Illicium difengpi | S10 |
2, characteristic peak relative retention time and integration relative ratio
According to the correlation parameter that 10 batches of test sample HPLC-UV collection of illustrative plates provide, more each test sample chromatogram (see Fig. 7), wherein 18 peaks are that 10 batches of test samples have, and therefore determine that these 18 peaks are for total fingerprint peaks.Be with reference to peak (S) with quercitin, rutin (7), S1-3 (9) honokiol (16) are characteristic peak, set up anisetree bark HPLC-UV finger-print (result as shown in Figure 8), the relative retention time at each total peak and the ratio of relative peak area are in table 13 and table 14.
Show the relative retention time that 13:10 batch of anisetree bark has peak
Show the relative peak area that 14:10 batch of anisetree bark has peak
3, fingerprint similarity
The anisetree bark medicinal materials fingerprint of employing fingerprint collection of illustrative plates similarity evaluation software to each place of production carries out Similarity Measure.Obtain Different sources anisetree bark and the similarity contrasting spectrogram.Result except No. 9 similarities slightly biased low except, the anisetree bark similarity in other places of production, all more than 90%, the results are shown in Table 15.
The fingerprint similarity result of calculation of table 15:10 batch of anisetree bark
| 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. the method for quality control of anisetree bark, it is characterized in that: using quercitin, rutin, S1-3 honokiol as index composition, adopt high performance liquid chromatography analyze these index compositions in anisetree bark thus realize the detection of maple cortex amount over the ground, specifically comprise the following steps:
(1) preparation of reference substance solution
With quercitin, rutin, S1-3 honokiol for reference substance, by volume fraction, to be 60 ~ 80% ethanol or volume fraction be that 60 ~ 80% methyl alcohol dissolve preparations obtains mixing reference substance solution, and wherein, in mixing reference substance solution, the concentration of quercitin 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
Get anisetree bark, pulverize, with volume fraction to be 60 ~ 80% ethanol or volume fraction be 60 ~ 80% methyl alcohol for Extraction solvent extracts, the amount ratio of described anisetree bark and Extraction solvent is: 1g:35 ~ 45ml, after having extracted, and cooling, when extract obtained weight is less than the general assembly (TW) extracting front anisetree bark and Extraction solvent, add Extraction solvent until weight is just equal with the general assembly (TW) of Extraction solvent with anisetree bark before extraction, filter, obtain need testing solution;
(3) chromatographic condition
C18 chromatographic column, flow velocity: 0.5 ~ 1ml/min, column temperature: 20 ~ 30 DEG C, wavelength: 254nm or 280nm, mobile phase is made up of A liquid and B liquid, and wherein A liquid is acetonitrile or methyl alcohol, and B liquid is volume fraction be 0.1% aqueous formic acid or volume fraction is 0.1% phosphate aqueous solution, gradient elution, elution program is 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) measure
The a certain amount of above-mentioned mixing reference substance solution of accurate absorption and need testing solution, inject high performance liquid chromatograph respectively, measure according to above-mentioned chromatographic condition.
2. the method for quality control of anisetree bark according to claim 1, is characterized in that: in step (1), and by volume fraction, to be 65 ~ 75% ethanol or volume fraction be that 65 ~ 75% methyl alcohol dissolve preparations obtains mixing reference substance solution.
3. the method for quality control of anisetree bark according to claim 1, is characterized in that: in step (1), and in mixing reference substance solution, the concentration of quercitin 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 method for quality control of anisetree bark according to claim 1, is characterized in that: in step (2), and Extraction solvent is volume fraction be 65 ~ 75% ethanol or volume fraction is 65 ~ 75% methyl alcohol.
5. the method for quality control of the anisetree bark according to any one of Claims 1 to 4, is characterized in that: in step (3), when 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 method for quality control of the anisetree bark according to any one of Claims 1 to 4, is characterized in that:
In step (1), by volume fraction, to be 70% ethanol or volume fraction be that 70% methyl alcohol dissolves preparation obtains mixing reference substance solution;
In step (1), in mixing reference substance solution, the concentration of quercitin 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), Extraction solvent is volume fraction be 70% ethanol or volume fraction is 70% methyl alcohol;
In step (2), the amount ratio of described anisetree bark and Extraction solvent is: 1g:40ml;
In step (3), when 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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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105816524A (en) * | 2016-04-18 | 2016-08-03 | 广西壮族自治区中国科学院广西植物研究所 | Anti-inflammatory active cortex illicii extract as well as preparation method and application thereof |
| CN111579701A (en) * | 2020-05-19 | 2020-08-25 | 河南科高中标检测技术有限公司 | Standard fingerprint spectrum establishment method for camelina sativa seeds and method for detecting rutin content |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105816524A (en) * | 2016-04-18 | 2016-08-03 | 广西壮族自治区中国科学院广西植物研究所 | Anti-inflammatory active cortex illicii extract as well as preparation method and application thereof |
| CN105816524B (en) * | 2016-04-18 | 2020-04-03 | 广西壮族自治区中国科学院广西植物研究所 | Cortex illicii anti-inflammatory active extract and preparation method and application thereof |
| CN111579701A (en) * | 2020-05-19 | 2020-08-25 | 河南科高中标检测技术有限公司 | Standard fingerprint spectrum establishment method for camelina sativa seeds and method for detecting rutin content |
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