CN108896673A - A kind of content assaying method of longleaf campanumoea root Content of Chlorogenic Acid, luteolin and apiolin - Google Patents
A kind of content assaying method of longleaf campanumoea root Content of Chlorogenic Acid, luteolin and apiolin Download PDFInfo
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Abstract
The invention discloses a kind of longleaf campanumoea root medicinal material Content of Chlorogenic Acid, the content assaying method of luteolin and apiolin, the content assaying method is：Chromatographic condition：Chromatographic column：Pntulips BP-C18(4.6mm × 250mm, 2.5 μm)；Mobile phase：Mobile phase A is acetonitrile, and Mobile phase B is methanol, and mobile phase C is 0.1% phosphate aqueous solution, gradient elution；Detection wavelength：330~350nm；Column temperature：20~30 DEG C；Flow velocity：0.8~1.2mLmin‑1；Sample volume：5~15 μ L；The preparation of reference substance solution；The preparation of test solution；The measurement of HPLC method.The present invention uses HPLC method, by the optimization to extracting method and chromatographic condition, establishes the content that active constituent in method measurement longleaf campanumoea root medicinal material is commented in a survey more, provides Research foundation for formulation, the rational exploitation and utilization resource etc. of the quality of medicinal material standard.The measuring method is accurate, and high sensitivity is reproducible, as a result reliably, provides foundation for the control of longleaf campanumoea root quality of medicinal material and evaluation.
Description
Technical field
The present invention relates to a kind of longleaf campanumoea root medicinal material Content of Chlorogenic Acid, the content assaying method of luteolin and apiolin, belong to
The field of drug technology.
Background technique
Chinese medicine multicomponent, multiple target point characteristic determine that traditional Chinese medicine quality control mode must be that multicomponent is quantitative and multi objective
Monitoring.Method is commented to be widely used in the quality controling research of Chinese medicine and Chinese materia medica preparation currently, a survey, " one surveys comments more "
(QAMS) method is that modern development gets up and a kind of widely applied analytical technology and method, i.e., using in active chemical
Functional relation and proportionate relationship, 1 ingredient (reference substance can person cheap and easy to get) be measured, to realize to multiple ingredient (reference substances
Without or be difficult to the person of obtaining) synchronization monitoring, keep medicine quality evaluated easier and save the cost, the matter of Chinese medicine can be objectively evaluated
Amount.
Longleaf campanumoea root derives from the long impeller clock of Campanulaceae CampanuLaceae money Panthera Campanumoea perennial plant
The dry root of careless Campanumoea lancifolia (Roxb.) Kurz, also known as " red fruit ginseng ", " mountain water chestnut ", " abacus fruit " etc..
Longleaf campanumoea root medicinal material is version in 2003《Guizhou Province's Chinese medicine, Ethnic crude drugs quality standard》The new amendments of revision, it is sweet in flavor and micro-
Hardship, it is mild-natured, have moistening lung to arrest cough, qi-regulating, qi-restoratives, stasis-dispelling and pain-killing and other effects, be mainly used for treat traumatic injury, deficiency of vital energy and acking in strength,
The diseases such as intestinal colic, pulmonary tuberculosis cough, hernia.Its root is civil frequently as nourishing herbal medicine, and tender leaf can be used as vegetables and stir-fry and eat, and fruit can be made
It is edible for fruit, there is certain health-care efficacy.It is analyzed by Literature Consult, relatively to the research report of longleaf campanumoea root medicinal material at present
It is few, it is concentrated mainly on the research of its chemical component and antioxidant activity, still in blank in terms of quality control, is unfavorable for controlling
The quality of medicinal material and its prescribed preparation, not can guarantee the safety and validity of clinical application, and limit to longleaf campanumoea root into
The development and utilization of one step.
Summary of the invention
Present invention aims at provide the assay of a kind of longleaf campanumoea root medicinal material Content of Chlorogenic Acid, luteolin and apiolin
Method.The present invention uses HPLC method, by the optimization to extracting method and chromatographic condition, establishes a survey and method is commented to measure longleaf campanumoea root more
The content of active constituent in medicinal material provides Research foundation for formulation, the rational exploitation and utilization resource etc. of the quality of medicinal material standard.Institute
It is accurate to state measuring method, high sensitivity is reproducible, as a result reliably, provides foundation for the control of longleaf campanumoea root quality of medicinal material and evaluation.
In order to solve the above technical problems, the present invention adopts the following technical scheme that realization：A kind of longleaf campanumoea root medicinal material Content of Chlorogenic Acid,
The content assaying method of luteolin and apiolin, the content assaying method are：
Chromatographic condition：Chromatographic column：Pntulips BP-C18(4.6mm × 250mm, 2.5 μm)；Mobile phase：Mobile phase A is second
Nitrile, Mobile phase B are methanol, and mobile phase C is 0.1% phosphate aqueous solution, gradient elution；Detection wavelength：330~350nm；Column temperature：
20~30 DEG C；Flow velocity：0.8~1.2mLmin-1；Sample volume：5~15 μ L；
The preparation of chlorogenic acid reference substance stock solution：Chlorogenic acid reference substance is taken, 50% methanol is added to dissolve and mass concentration is made
For 1.4046~1.4054mLmin-1Solution；
The preparation of luteolin reference substance stock solution：Luteolin reference substance is taken, 50% methanol is added to dissolve and quality is made
Concentration is 0.2015~0.2025mLmin-1Solution；
The preparation of apiolin reference substance stock solution：Apiolin reference substance is taken, 50% methanol is added to dissolve and mass concentration is made
For 0.2055~0.2065mLmin-1Solution；
The preparation of mixed reference substance solution：Draw above-mentioned each reference substance stock solution 2.0mL, 2.5mL, 0.5mL respectively, set with
In the volumetric flask of 5mL, constant volume, obtaining chlorogenic acid, luteolin, apiolin concentration is respectively 0.5620mLmin-1、
0.1010mL·min-1And 0.02060mLmin-1Mixed reference substance solution；
The preparation of test solution：0.8~1.2g of medicinal powder is taken, it is accurately weighed, it is placed in 50mL conical flask, precision adds
Enter 45~55% 10~15mL of methanol solution, after ultrasonic extraction, taking-up is put to room temperature, filtration, and subsequent filtrate crosses 0.45 μm of micropore filter
Film is to get test solution；
Assay：Precision draws test solution, injects liquid chromatograph, measures by above-mentioned chromatographic condition, measures peak
Area value simultaneously calculates content.
In the content assaying method of longleaf campanumoea root medicinal material Content of Chlorogenic Acid above-mentioned, luteolin and apiolin, gradient elution journey
Sequence is：0~5min, 2%~12%A, 3%~8%B, 95%~80%C；5~15min, 12%~6%A, 8%~14%B,
80%~80%C；15~20min, 6%~16%A, 14%~10%B, 80%~74%C；20~25min, 16%~22%
A, 10%~8%B, 74%~70%C；25~35min, 22%~34%A, 8%~16%B, 70%~50%C；35~
40min, 34%~0%A, 16%~100%B, 50%~0%C；40~45min, 0%A, 100%B, 0%C.
In the content assaying method of longleaf campanumoea root medicinal material Content of Chlorogenic Acid above-mentioned, luteolin and apiolin, the ultrasound
Extraction is：50~70min of ultrasonic extraction at power 100W, frequency 40Hz.
In the content assaying method of longleaf campanumoea root medicinal material Content of Chlorogenic Acid above-mentioned, luteolin and apiolin, the detection wave
It is long：340nm；Column temperature：25℃；Flow velocity：1mL·min-1。
It is described for examination in the content assaying method of longleaf campanumoea root medicinal material Content of Chlorogenic Acid above-mentioned, luteolin and apiolin
Product solution is prepared：Medicinal powder 1.0g is taken, it is accurately weighed, it is placed in 50mL conical flask, 50% methanol solution is added in precision
10mL, after ultrasonic extraction, taking-up is put to room temperature, filtration, and subsequent filtrate crosses 0.45 μm of miillpore filter to get test solution.
Inventor has carried out a large amount of experiment, is part Experiment research below
1. content assaying method of experimental example is investigated
1 instrument and reagent
1.1 instrument
1100 type of Agilent and 1260 type high performance liquid chromatographs (Agilent company of the U.S.)；Shimadzu LC-2030 type is high
Effect liquid phase chromatogram instrument (Japanese Shimadzu Corporation)；(U.S.'s match is silent to fly public affairs to 3000 type high performance liquid chromatograph of Thermo UItiMate
Department)；AG135 type electronic analytical balance (Mettler-Toledo company of Switzerland)；(Shanghai Guan Te is super for SG8200HFT type Ultrasound Instrument
Sound Instrument Ltd.)；202-1A type Constant Temp. Oven (Tianjin Stettlen Instrument Ltd.)；DK-98-II type water
Bath (Tianjin Stettlen Instrument Ltd.)；Chromatographic column：Agilent HC-C18(4.6mm × 250mm, 5 μm)；Agilent
ZORBAX XDB-C18(4.6mm × 250mm, 5 μm)；Agilent ZORBAX Extend-C18(4.6mm × 250mm, 5 μm)；
Aglent ZORBAXSB-C18(4.6mm × 150mm, 5 μm)；Agilent ZORBAXSB-CN (4.6mm × 250mm, 5 μm)；
Pntulips BP-C18(4.6mm × 250mm, 5 μm) etc..
1.2 reagent
Chlorogenic acid reference substance (lot number：110753-201716, content is in terms of 99.3%), luteolin reference substance (lot number：
111520-201605；Content is in terms of 99.6%), apiolin (lot number：111901-201603；Content is in terms of 99.2%) it is purchased from
National Institute for Food and Drugs Control；Methanol (Tianjin Kermel Chemical Reagent Co., Ltd., chromatographically pure), acetonitrile (Tianjin
Ke Miou chemical reagent Co., Ltd, chromatographically pure), phosphoric acid (Chongqing Chuan Dong Chemical Co., Ltd. analyzes pure), experimental water is attached most importance to
Distilled water, remaining reagent are that analysis is pure；
17 batches of longleaf campanumoea root medicinal materials pick up from Guizhou Longli, Du Shan, Kaiyang, Mount Fanjing and Guangxi province melt water by seminar respectively
Etc. ground, through Guiyang College of Traditional Chinese Medicine's Sun Qing culture and education award precise Identification be long impeller clock grass C.lancifolia (Roxb.) Kurz.
2 methods and result
2.1 chromatographic condition
Chromatographic column is Pntulips BP-C18(4.6mm × 250mm, 5 μm)；Mobile phase is acetonitrile (A)-methanol (B)-
0.1% phosphate aqueous solution (C), elution program is shown in Table 1.Flow velocity is 1.0mLmin-1；Detection wave is 340nm；Column temperature is 25 DEG C；
Sample volume is 10 μ L.
1 mobile phase elution program of table
The preparation of 2.2 reference substance solutions
It takes chlorogenic acid reference substance, luteolin reference substance, apiolin reference substance appropriate, adds 50% methanol to dissolve and matter is made
Measuring concentration is respectively 1.4050mgmL-1、0.2020mg·mL-1、0.2060mg·mL-1Reference substance stock solution；It inhales respectively again
Each reference substance stock solution 2.0mL, 2.5mL, 0.5mL are taken, is set in the volumetric flask with 5mL, constant volume, chlorogenic acid, luteolin, celery are obtained
Dish element concentration is respectively 0.5620mgmL-1、0.1010mg·mL-1、0.02060mg·mL-1Mixed reference substance solution.
2.3 the preparation of test solution
Medicinal powder 1.0g is taken, it is accurately weighed, it is placed in 50mL conical flask, 50% methanol solution 10mL is added in precision, surpasses
Sound extracts (100W, 40Hz) 60min, and taking-up is put to room temperature, and filtration, it is molten to get test sample that subsequent filtrate crosses 0.45 μm of miillpore filter
Liquid.
2.4 specificities investigate test
Precision draws above-mentioned mixed reference substance solution and each 10 μ L of test solution, injects high performance liquid chromatograph, presses
Chromatographic condition is measured under " 2.1 " item, records chromatogram.The result shows that 1 in test sample map, the reservation of 2, No. 3 chromatographic peaks
Time is consistent with mixed reference substance solution Content of Chlorogenic Acid, luteolin, the retention time of apiolin chromatographic peak, and UV spectrogram is bent
Line is identical, and it is good that the purity factor is followed successively by 999.916,999.891,999.155 peak purities, and it is good to show that this method has
Specificity, the result is shown in Figure 1.
The drafting of 2.5 standard curves
Precision draws the single reference substance solution of each ingredient under " 2.2 " item, is diluted to the reference substance of series of concentrations respectively
Solution (chlorogenic acid：0.3513mg·mL-1、0.1756mg·mL-1、0.08780mg·mL-1、0.04390mg·mL-1、
0.02200mg·mL-1；Luteolin：0.1010mg·mL-1、0.05050mg·mL-1、0.02525mg·mL-1、
0.01262mg·mL-1、0.006312mg·mL-1；Apiolin：0.04120mg·mL-1、0.01648mg·mL-1、
0.006592mg·mL-1、0.002637mg·mL-1、0.0005274mg·mL-1), it is surveyed by chromatographic condition under " 2.1 " item
It is fixed, it is measured in parallel 3 times, using reference substance sample volume as abscissa (x), peak area average value is ordinate (y), and it is bent to draw standard
Line carries out linear regression, obtains the regression equation and related coefficient of chlorogenic acid, luteolin, apiolin, the results are shown in Table 2 and Fig. 2.
3 components regression equations, related coefficient and the range of linearity (n=5) in 2 longleaf campanumoea root medicinal material of table
The measurement of 2.6 ingredient correction factors to be measured
According to the chromatographic condition under " 2.1 " item, mixed reference substance solution under " 2.2 " item is taken, precision draws 2 μ L, 4 μ L, 6 μ L, 8
μ L, 10 μ L, 12 μ L injection high performance liquid chromatograph are measured, using chlorogenic acid as internal reference object, according to correction factor calculation formula
fsi=fs/fi=(As×Ci)/(Ai×Cs), A in the formulas、CsThe respectively peak area and concentration of internal reference object；Ai、CiRespectively
The peak area and concentration of ingredient to be measured；fsiFor the relative correction factor of ingredient to be measured.Calculate separately chlorogenic acid to luteolin and
Relative correction factor (the f of apiolinS/BFor the relative correction factor of luteolin, fS/CFor the relative correction factor of apiolin),
Its RSD is respectively less than 5.0% (being shown in Table 3).
3 luteolin of table, apiolin correction factor calculated result (n=6)
2.7 precision test
Precision draws 10 μ L of mixed reference substance solution under " 2.2 " item, according to the chromatographic condition under " 2.1 " item, continuous sample introduction 6
It is secondary, measure peak area value, calculate the RSD value of peak area, chlorogenic acid, luteolin, apiolin RSD value be respectively 0.26%,
0.74%, 0.26%, respectively less than 2.0%, show that instrument precision is good.It the results are shown in Table 4 and Fig. 3.
4 Precision test result of table (n=6)
2.8 repetitive test
Take 6 parts, every part of about 1.0g of same longleaf campanumoea root sample (No. Z7), it is accurately weighed, by the preparation of " 2.3 " item method for examination
Liquid is measured by chromatographic condition under " 2.1 " item, is calculated chlorogenic acid, luteolin, apiolin average mass fraction and is respectively
0.59%, 0.097%, 0.020%, RSD value is respectively 2.6%, 1.4%, 1.4%, the results showed that this method repeatability is good
It is good.It the results are shown in Table 5 and Fig. 4.
5 repetitive test result (n=6) of table
2.9 stability test
Precision drew same test solution (No. Z7), respectively at 0,3,6,8,12,24 hour injection high performance liquid chromatography
Instrument is analyzed, and by the peak area value for measuring chlorogenic acid, luteolin, apiolin under " 2.1 " item in accordance with the law, and calculates three kinds of ingredients
Content, RSD value is respectively 0.67%, 1.5%, 1.9%, shows that test solution is more stable in 48h.It the results are shown in Table 6
And Fig. 5.
6 stability test result (n=7) of table
2.10 sample recovery rate is tested
6 parts, every part of about 0.5g of longleaf campanumoea root medicinal powder (No. Z7) of known content are taken, it is accurately weighed, it is accurate respectively to be added
Chlorogenic acid, luteolin, apiolin reference substance solution are appropriate, by legal system available test sample solution below " 2.3 " item, press " 2.1 " item
Lower chromatographic condition measures respectively, calculate separately chlorogenic acid, luteolin, apiolin average recovery rate be followed successively by 96.00%,
96.34%, 95.93%, RSD value is followed successively by 1.5%, 2.4%, 2.5%, shows that the accuracy of this method measurement result is high.As a result
It is shown in Table 7 and Fig. 6.
7 sample recovery rate test result (n=6) of table
Influence of the 2.11 different instruments and chromatographic column to relative correction factor
The high performance liquid chromatograph of 4 kinds of models and the C of 3 kinds of different models have been investigated in 4 laboratories in this experiment18Chromatography
Column.It is accurate respectively to draw the 10 μ L of mixing reference substance prepared under " 2.2 " item, it is measured, calculates by chromatographic condition under " 2.1 " item
The relative correction factor of luteolin, apiolin, respectively between 0.578~0.617,0.471~0.551, different model instrument
Relative correction factor RSD value obtained by device and chromatographic column is respectively 2.2% and 3.8%, shows different model instrument and different model
Chromatographic column influences the relative correction factor of each ingredient without conspicuousness, the results are shown in Table 8.
The positional parameter of 2.12 component chromatographic peaks to be measured is investigated
It establishes QAMS method purpose and is that utilization is to be measured using a kind of reference substance (content that can measure other ingredients to be measured)
To the retention time difference or relative retention time of chlorogenic acid as localization criteria, this experiment investigates 4 for ingredient luteolin, apiolin
The instrument of different model and retention time difference and relative retention time under chromatographic column in a laboratory, the experimental results showed that different
Model instrument and chromatographic column are smaller to the fluctuation of each ingredient retention time difference, and RSD value is respectively 1.9% and 3.0%, when retaining relatively
Between differ greatly, RSD value is respectively 6.3% and 5.4%, therefore using the legal position luteolin of retention time difference, celery plain color
Spectral peak the results are shown in Table 8.
8 relative correction factor of table, retention time difference and relative retention time investigate result (n=3)
2.13 sample measures
Longleaf campanumoea root medicinal material sample is taken, 21 batches are amounted to, respectively by legal system available test sample solution below " 2.3 " item, is pressed
Chromatographic condition under " 2.1 " item, sample introduction measurement, measure chlorogenic acid, luteolin, apiolin peak area, be respectively adopted ESM method and
QAMS method, calculates the content of luteolin and apiolin in dry product longleaf campanumoea root medicinal material, and compares two methods measurement result
Compared with the results are shown in Table 9 and Fig. 5.
9 21 batch longleaf campanumoea root medicinal material sample measurement result (n=3) of table
Note：Not detect the ingredient
3 conclusions
The content of two kinds of luteolin, apiolin ingredients in the longleaf campanumoea root medicinal material that this experiment calculates ESM method and QAMS method
It is compared, carries out independent samples t test result P>0.05, show the amount of the resulting two kinds of ingredients of two kinds of measuring methods without significant
As a result accurately and reliably sex differernce further demonstrates QAMS method measurement longleaf campanumoea root medicinal material Content of Chlorogenic Acid, luteolin, apiolin
Content be it is feasible, each ingredient with its retention time difference positioning, retention time difference/relative correction of luteolin and apiolin
The factor is respectively 24.02/0.604,26.71/0.525.The content of three kinds of ingredients in QAMS method and ESM method measurement longleaf campanumoea root medicinal material
There was no significant difference, therefore, longleaf campanumoea root medicinal material Content of Chlorogenic Acid that this experiment is established, luteolin, apiolin assay
Method is easy quickly, accurate feasible, can be used for measuring the content of longleaf campanumoea root medicinal material Content of Chlorogenic Acid, luteolin, apiolin, is it
Certain experiment basis is established in the research of quality control standard.
Invention uses HPLC method, by the optimization to extracting method and chromatographic condition, establishes a survey and comments method more
The content for measuring active constituent in longleaf campanumoea root medicinal material, provides for formulation, the rational exploitation and utilization resource etc. of the quality of medicinal material standard
Research foundation.The measuring method is accurate, and high sensitivity is reproducible, as a result reliably, controls and comments for longleaf campanumoea root quality of medicinal material
Valence provides foundation.
Detailed description of the invention
Fig. 1 is that specificity investigates HPLC chromatogram and UV spectrogram (wherein a：Reference substance solution；b：Test solution；c：It is empty
White solution；1- chlorogenic acid chromatographic peak and UV spectrogram；2- luteolin chromatographic peak and UV spectrogram；3- apiolin chromatographic peak and UV
Spectrogram)；
Fig. 2 is chlorogenic acid, luteolin, apiolin canonical plotting；It (a) is chlorogenic acid canonical plotting；It (b) is sweet-scented osmanthus
Careless element canonical plotting；It (c) is apiolin canonical plotting；
Fig. 3 is precision test HPLC chromatogram stacking chart；
Fig. 4 is repetitive test HPLC chromatogram stacking chart；
Fig. 5 is stability test HPLC chromatogram stacking chart；
Fig. 6 is sample recovery rate test HPLC chromatogram stacking chart；
Fig. 7 is 21 crowdes of longleaf campanumoea root medicinal material sample HPLC chromatogram stacking charts.
Below with reference to embodiment, the present invention is further illustrated.
Specific embodiment
Embodiment 1：
A kind of content assaying method of longleaf campanumoea root medicinal material Content of Chlorogenic Acid, luteolin and apiolin：
Chromatographic condition：Pntulips BP-C18Column (4.6mm × 250mm, 2.5 μm)；Mobile phase：Mobile phase A is acetonitrile, stream
Dynamic phase B is methanol, and mobile phase C is 0.1% phosphate aqueous solution, gradient elution；Detection wavelength：340nm；Column temperature：25℃；Flow velocity：
1.0mL·min-1；Sample volume：10μL；Gradient elution program elution program is：0~5min, 2%~12%A, 3%~8%B,
95%~80%C；5~15min, 12%~6%A, 8%~14%B, 80%~80%C；15~20min, 6%~16%A,
14%~10%B, 80%~74%C；20~25min, 16%~22%A, 10%~8%B, 74%~70%C；25~
35min, 22%~34%A, 8%~16%B, 70%~50%C；35~40min, 34%~0%A, 16%~100%B,
50%~0%C；40~45min, 0%A, 100%B, 0%C.
The preparation of chlorogenic acid reference substance stock solution：Chlorogenic acid reference substance is taken, 50% methanol is added to dissolve and mass concentration is made
For the solution of 1.405mg/mL；
The preparation of luteolin reference substance stock solution：Luteolin reference substance is taken, 50% methanol is added to dissolve and quality is made
Concentration is the solution of 0.2020mg/mL；
The preparation of apiolin reference substance stock solution：Apiolin reference substance is taken, 50% methanol is added to dissolve and mass concentration is made
For the solution of 0.2060mgmL；
The preparation of mixed reference substance solution：Draw above-mentioned each reference substance stock solution 2.0mL, 2.5mL, 0.5mL respectively, set with
In the volumetric flask of 5mL, constant volume, obtaining chlorogenic acid, luteolin, apiolin concentration is respectively 0.5620mg/mL, 0.1010mg/mL
With the mixed reference substance solution of 0.02060mg/mL；
The preparation of test solution：Medicinal powder 1.0g is taken, it is accurately weighed, it is placed in 50mL conical flask, precision is added
50% methanol solution 10mL, after ultrasonic extraction, taking-up is put to room temperature, filtration, and subsequent filtrate crosses 0.45 μm of miillpore filter to get for examination
Product solution；
The measurement of HPLC method：Reference substance solution is drawn respectively and test solution distinguishes sample introduction, liquid chromatograph is injected, by upper
Chromatographic condition measurement is stated, peak area value is measured and calculates content.
Claims (5)
1. the content assaying method of a kind of longleaf campanumoea root medicinal material Content of Chlorogenic Acid, luteolin and apiolin, it is characterised in that：It is described to contain
Quantity measuring method is：
Chromatographic condition：Chromatographic column：Pntulips BP-C18(4.6mm × 250mm, 2.5 μm)；Mobile phase：Mobile phase A is acetonitrile,
Mobile phase B is methanol, and mobile phase C is 0.1% phosphate aqueous solution, gradient elution；Detection wavelength：330~350nm；Column temperature：20~
30℃；Flow velocity：0.8~1.2mLmin-1；Sample volume：5~15 μ L；
The preparation of chlorogenic acid reference substance stock solution：Chlorogenic acid reference substance is taken, adds 50% methanol to dissolve and mass concentration is made and be
1.4046~1.4054mLmin-1Solution；
The preparation of luteolin reference substance stock solution：Luteolin reference substance is taken, 50% methanol is added to dissolve and mass concentration is made
For 0.2015~0.2025mLmin-1Solution；
The preparation of apiolin reference substance stock solution：Apiolin reference substance is taken, adds 50% methanol to dissolve and mass concentration is made and be
0.2055~0.2065mLmin-1Solution；
The preparation of mixed reference substance solution：Above-mentioned each reference substance stock solution 2.0mL, 2.5mL, 0.5mL are drawn respectively, are set and 5mL
Volumetric flask in, constant volume, obtaining chlorogenic acid, luteolin, apiolin concentration is respectively 0.5620mLmin-1、0.1010mL·
min-1And 0.02060mLmin-1Mixed reference substance solution；
The preparation of test solution：0.8~1.2g of medicinal powder is taken, it is accurately weighed, it is placed in 50mL conical flask, precision is added 45
After~55% 10~15mL of methanol solution, 50~60min of ultrasonic extraction, taking-up is put to room temperature, filtration, subsequent filtrate cross 0.45 μm it is micro-
Hole filter membrane is to get test solution；
Assay：Precision draws test solution, injects liquid chromatograph, measures by above-mentioned chromatographic condition, measures peak area
It is worth and calculates content.
2. the content assaying method of longleaf campanumoea root medicinal material Content of Chlorogenic Acid as described in claim 1, luteolin and apiolin, special
Sign is：Gradient elution program is：0~5min, 2%~12%A, 3%~8%B, 95%~80%C；5~15min, 12%
~6%A, 8%~14%B, 80%~80%C；15~20min, 6%~16%A, 14%~10%B, 80%~74%C；20
~25min, 16%~22%A, 10%~8%B, 74%~70%C；25~35min, 22%~34%A, 8%~16%B,
70%~50%C；35~40min, 34%~0%A, 16%~100%B, 50%~0%C；40~45min, 0%A, 100%
B, 0%C.
3. the content assaying method of longleaf campanumoea root medicinal material Content of Chlorogenic Acid as described in claim 1, luteolin and apiolin, special
Sign is：The ultrasonic extraction is：The ultrasonic extraction 60min at power 100W, frequency 40Hz.
4. the content assaying method of longleaf campanumoea root medicinal material Content of Chlorogenic Acid as described in claim 1, luteolin and apiolin, special
Sign is：The Detection wavelength：340nm；Column temperature：25℃；Flow velocity：1mL·min-1。
5. the content assaying method of longleaf campanumoea root medicinal material Content of Chlorogenic Acid as described in claim 1, luteolin and apiolin, special
Sign is：The test solution is prepared：Medicinal powder 1.0g is taken, it is accurately weighed, it is placed in 50mL conical flask, it is accurate
Be added 50% methanol solution 10mL, after ultrasonic extraction, taking-up is put to room temperature, filtration, subsequent filtrate cross 0.45 μm of miillpore filter to get
Test solution.
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