CN106568856A - Fingerprint detection method for detecting effective part of lithocarpus polysachyus rehd leaf - Google Patents
Fingerprint detection method for detecting effective part of lithocarpus polysachyus rehd leaf Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention discloses a fingerprint detection method for detecting effective part of lithocarpus polysachyus rehd leaf and relates to a method for quality control of traditional Chinese medicines. The fingerprint detection method comprises the following steps: preparation of a control solution; preparation of a test solution; use of a chromatographic column with octadecyl silane as the filler; chromatographic conditions: gradient elution is adopted; a mobile phase is a gradient eluant compose of methanol and 0.1% formic acid and at the column temperature of 25 DEG C; UV detection wavelength is 285 nm; and time is 140 min; and high performance liquid chromatography for fingerprint. The method of the invention can be effectively used for quality control of lithocarpus polysachyus rehd leaf.
Description
Technical field
The present invention relates to Chinese medicine extract and its method of quality control, and in particular to the fingerprint image of Pasania cuspidata leaf effective-part
The foundation of spectrum detection method.
Background technology
Pasania cuspidata, from Fagaceae Lithocarpus Lithocarpus polystachyus (Wall.) Rehd., You Mingduo
Fringe stone Ke, multiple ear lithocarpus；Because leaf chew it is pleasantly sweet, it is therefore named for Folium hydrangeae strigosae, be referred to as sweet tea in Guizhou, be referred to as great Ye in Guangdong thick
Son.Each provinces and regions are distributed on the south China the Changjiang river, especially most wide with Wuyi Mountain distribution, additionally, in resource-savings such as osmanthus, Hunan, Anhui most
It is abundant, wherein the about hundreds of tons of resource storage in Guangxi (osmanthus).In Guangdong, cloud, river, Fujian etc., resource-saving amount is taken second place.India, it is safe
Also there is a little distribution in state.
Pasania cuspidata be it is a kind of have medicine, tea, the medicine food dual purpose plant of sugared three kinds of functions concurrently, its tender leaf that often takes among the people is made tea, tea
Coloured gold Huang is bright-coloured, slightly pained during taste is sweet, and lasting taste, ancients are usually used in clearing away summer-heat and quench one's thirst.Containing up in Pasania cuspidata leaf
10% high sugariness, non-sugar sweetener low in calories, its sweet ingredient is mainly dihydrochalcone-type phlorhizin, 3- hydroxyl root bark
Glycosides, Trilobatin etc..
Pasania cuspidata leaf has the functions such as blood sugar lowering, lipid-loweringing, antioxidation, and modern study shows, its abundant flavonoid being rich in
Composition is its principle active component.Further to improve the quality standard of Pasania cuspidata, while unstable to explore early stage pharmacological evaluation
The reason for determining, this experiment launches the research of the finger printing to its effective ingredient.
The content of the invention
The purpose of the present invention is to set up the detection method of Pasania cuspidata leaf effective-part HPLC finger printing.
The technical scheme for being adopted to achieve the object of the present invention：The effective portion of Pasania cuspidata leaf is set up with high performance liquid chromatography
The fingerprint atlas detection method of position, specifically includes following steps：
1. the preparation of reference substance solution：Precision weighs 3-OH phlorhizin reference substances, adds 50% ethanol to be dissolved, and is made into
Concentration is the solution of 0.50mg/mL, used as reference substance solution；
2. the preparation of need testing solution：Pasania cuspidata leaf medical material is taken, powder is beaten, and crosses No. two sieves, accurately weigh 5.00g, placed
In 125mL conical flask with cover, the ethanol for measuring 40mL50% is added thereto, and in being positioned over thermostat water bath, 80 DEG C are heated back
Stream extracts 60min, continuous to extract three times, finally by three extracting solution mix homogeneously, lets cool, and filters, by filtrate constant volume in 250mL
In measuring bottle, take in right amount, cross 0.45 μm of microporous filter membrane, subsequent filtrate is placed in liquid phase bottle, obtains final product；
3. chromatographic condition：With octadecylsilane chemically bonded silica as filler, using gradient elution, mobile phase is first to chromatographic column
The gradient eluent that alcohol is constituted with formic acid, column temperature is 20～30 DEG C；Ultraviolet detection wavelength：200～400nm；
4. determine：Precision draws need testing solution injection high performance liquid chromatograph, according to high effective liquid chromatography for measuring, obtains
Finger printing.
Further preferred method can be implemented by following step：
1. the preparation of reference substance solution：Precision weighs 3-OH phlorhizin 10.05mg, adds 50% ethanol to be dissolved, fixed
In being dissolved in 10mL volumetric flasks, precision pipettes the above-mentioned solution of 1mL, and in 10mL volumetric flasks, be made into concentration is 50% ethanol constant volume
The solution of 0.1005mg/mL；Precision weighs phlorhizin 5.00mg, adds 50% ethanol to be dissolved, and constant volume is in 10mL volumetric flasks
In, the solution that concentration is 0.50mg/mL is made into, obtain final product；
2. the preparation of need testing solution：The Pasania cuspidata leaf medical material that lot number is 20140815 is taken, powder is beaten, and crosses No. two sieves, it is accurate
5.00g is really weighed, in being positioned over 125mL conical flask with cover, the ethanol for measuring 40mL50% is added thereto, and is positioned over water bath with thermostatic control
In pot, 80 DEG C of heating and refluxing extractions 60min are continuous to extract three times, finally by three extracting solution mix homogeneously, let cool, and filter, will
Filtrate constant volume takes in right amount in 250mL measuring bottles, crosses 0.45 μm of microporous filter membrane, and subsequent filtrate is placed in liquid phase bottle, obtains final product；
3. chromatographic condition：Chromatographic column is with octadecylsilane chemically bonded silica as filler；Using gradient elution, mobile phase is first
The gradient eluent of alcohol and 0.1%～2.0% aqueous formic acid composition, column temperature is 20～30 DEG C；Ultraviolet detection wavelength be 200～
400nm；
4. determine：Precision draws need testing solution injection high performance liquid chromatograph, according to high effective liquid chromatography for measuring, obtains
Finger printing.
The optimal detection method of the present invention can be implemented by following step：
3. mobile phase is the gradient eluent of methanol and 0.1% aqueous formic acid composition to step in chromatographic condition, and column temperature is
25 DEG C, Detection wavelength is 254nm, and flow velocity is 0.8mL/min；Analysis time is 140min.
Step gradient elution 3. described in chromatographic condition, gradient elution program is carried out with the configuration of following volumetric concentration：
When 0 minute, mobile phase A is 18% methanol solution, 0.1% aqueous formic acid that Mobile phase B is 82%；
When 20 minutes, mobile phase A is 35% methanol solution, 0.1% aqueous formic acid that Mobile phase B is 65%；
When 60 minutes, mobile phase A is 35% methanol solution, 0.1% aqueous formic acid that Mobile phase B is 65%；
When 120 minutes, mobile phase A is 60% methanol solution, 0.1% aqueous formic acid that Mobile phase B is 40%；
When 140 minutes, mobile phase A is 60% methanol solution, 0.1% aqueous formic acid that Mobile phase B is 40%.
Specifically see the table below：
The detection method of Pasania cuspidata leaf effective-part finger printing, specifically includes following steps：
1. the preparation of reference substance solution：Precision weighs 3-OH phlorhizin 10.05mg, adds 50% ethanol to be dissolved, fixed
In being dissolved in 10mL volumetric flasks, precision pipettes the above-mentioned solution of 1mL, and in 10mL volumetric flasks, be made into concentration is 50% ethanol constant volume
The solution of 0.1005mg/mL；Precision weighs phlorhizin 5.00mg, adds 50% ethanol to be dissolved, and constant volume is in 10mL volumetric flasks
In, the solution that concentration is 0.50mg/mL is made into, obtain final product；
2. the preparation of need testing solution：The Pasania cuspidata leaf medical material that lot number is 20140815 is taken, powder is beaten, and crosses No. two sieves, it is accurate
5.00g is really weighed, in being positioned over 125mL conical flask with cover, the ethanol for measuring 40mL50% is added thereto, and is positioned over water bath with thermostatic control
In pot, 80 DEG C of heating and refluxing extractions 60min are continuous to extract three times, finally by three extracting solution mix homogeneously, let cool, and filter, will
Filtrate constant volume takes in right amount in 250mL measuring bottles, crosses 0.45 μm of microporous filter membrane, and subsequent filtrate is placed in liquid phase bottle, obtains final product；
3. chromatographic condition：Chromatographic column is with octadecylsilane chemically bonded silica as filler；Using gradient elution, mobile phase is first
The gradient eluent of alcohol and 0.1%～2.0% aqueous formic acid composition, column temperature is 20～30 DEG C；Ultraviolet detection wavelength be 200～
400nm；
4. determine：Precision draws need testing solution injection high performance liquid chromatograph, according to high effective liquid chromatography for measuring, obtains
Finger printing.
There are 10 features to have peak in the finger printing, wherein No. 3 chromatographic peaks are 3-OH phlorhizin, highest peak is No. 6
Chromatographic peak, is also that, with reference to peak phlorhizin, collection of illustrative plates total length is 140min, specific as follows：
According to having 10 features to have peak in present invention gained Pasania cuspidata leaf effective-part finger printing, wherein No. 3 chromatographs
Peak is 3-OH phlorhizin, and highest peak is No. 6 chromatographic peaks, is also that collection of illustrative plates total length is 140min, wherein unimodal with reference to peak phlorhizin
The super total peak area 2% of area has 7, and they are 1,3,5,6,7,8, No. 9 peaks, wherein the super total peak area 10% of unimodal area
There is 1, they are No. 6 chromatographic peaks, wherein the super total peak area 15% of unimodal area has 1, they are No. 6 chromatographic peaks, wherein
Unimodal area super total peak area 20% has 1, and they are No. 6 chromatographic peaks.
It is specific as follows：
No. 1 peak, Average residence time RT is 33.20min, and RSD is 0.41%, and peak area is that 292467.2, RSD is
34.25%；
No. 2 peaks, Average residence time RT is 38.35min, and RSD is 0.35%, and peak area is that 159295.1, RSD is
48.19%；
No. 3 peaks, Average residence time RT is 40.84min, and RSD is 0.90%, and peak area is that 551159.5, RSD is
26.29%；
No. 4 peaks, Average residence time RT is 46.65min, and RSD is 0.92%, and peak area is that 166383.2, RSD is
24.90%；
No. 5 peaks, Average residence time RT is 48.64min, and RSD is 0.65%, and peak area is that 669754.3, RSD is
43.54%；
No. 6 peaks, Average residence time RT is 61.09min, and RSD is 0.76%, and peak area is that 6032428.7, RSD is
34.22%；
No. 7 peaks, Average residence time RT is 68.70min, and RSD is 0.65%, and peak area is that 555096.1, RSD is
32.46%；
No. 8 peaks, Average residence time RT is 72.24min, and RSD is 0.66%, and peak area is that 212800.3, RSD is
44.16%；
No. 9 peaks, Average residence time RT is 90.31min, and RSD is 0.45%, and peak area is that 503140.4, RSD is
32.30%；
No. 10 peaks, Average residence time RT is 98.52min, and RSD is 0.32%, and peak area is that 105901.2, RSD is
34.59%.
It is solvent peak that RT is the peak occurred before 5min.
The principle of the present invention is to find that the effective ingredient of Pasania cuspidata leaf is main from the composition Study of Pasania cuspidata leaf effective-part
For dihydrochalcone glycosides compound, such compound has many biological activitys.Set up Pasania cuspidata leaf effective-part
HPLC fingerprint atlas detection methods, the finger printing of its effective site can be effective from the existing Pasania cuspidata leaf in the overall looks upper body of chromatograph
Component content situation.Being determined with this method can obtain identical, close fingerprint image from the Pasania cuspidata leaf medical material collected on the market
Spectrum.
Beneficial effects of the present invention are as follows：
(1) method provided by the present invention can provide reference to carry out further pharmacology pharmacodynamic experiment, be further
Develop Pasania cuspidata resource and scientific basis is provided.
(2) finger printing focuses on the tandem mutual relation that each constitutes fingerprint characteristic peak, focuses on overall facial feature,
Both avoided and judged the one-sidedness of Pasania cuspidata leaf effective-part composition total quality because determining individualized Xuecheng point, reduced again
The probability for processing is thought for requisite quality.
(3) present invention has easy, stable method, precision height, high repeatability and other advantages.
(4) the method can quickly and accurately differentiate that the true and false of product is good and bad.
Description of the drawings
The HPLC finger printing of Fig. 1 Pasania cuspidata leaf effective-parts.
The effective site characteristic fingerprint pattern of 10 batches of Pasania cuspidata leaves of Fig. 2.
The finger printing of Fig. 3 Pasania cuspidata leaf effective-part HPLC common patterns.
Specific embodiment
In order to be able to further appreciate that feature, technological means and the specific purposes for being reached, the function of the present invention, parsing is originally
The advantage of invention and spirit.By being further understood to the detailed description of the present invention below in conjunction with accompanying drawing and concrete mode.
Embodiment one：The finger printing of detection Pasania cuspidata leaf effective-part
1. instrument and reagent
1.1 instrument FZ-230 type Universalpulverizers (herd III and hundred happy machine tool plants in Wenling city)；A ten thousandth
SartoriusBP110s analyses electronic balance (German sartorius companies)；Ten a ten thousandth Sartorius CP225D are analyzed
Electronic balance (German sartorius companies)；HWS24 type electric-heated thermostatic water baths (the permanent Science and Technology Ltd. in Shanghai one)；DK-
8AD electric-heated thermostatic water baths (Shanghai precision instrumentation company limited)；SHZ-D (III) circulating water type vacuum pump (Henan Gongyi
Yu Hua instrument plants of city)；(LC-20AT efficient liquid phase pumps, SIL-20A enters Japanese Shimadzu Shimadzu high performance liquid chromatograph automatically
Sample device, SPD-20A diode array detector, CTO-20A column ovens)；Performance liquid chromatographic column：Phenomenex luna
C18 chromatographic columns (250mm × 4.6mm, 5 μm), Dikma Diamonsil C18 chromatographic columns (250mm × 4.6mm, 5 μm),
Thermo Hypersil Gold C18 chromatographic columns (250mm × 4.6mm, 5 μm), Kromasil (C18 250mm × 4.6mm, 5 μ
M) post；
1.2 reagent analysis methanol, acetonitrile (chromatographic grade, Merck KGaA company), (AG, Tianjin is big for formic acid, acetic acid
Luxuriant chemical reagent factory), phosphoric acid (AG, the huge chemical reagent factory of new east station of Guangzhou), happy precious pure water.
2. high performance liquid chromatography
2.1 C18 reversed phase chromatographic column Kromasil (C18 250mm × 4.6mm, 5 μm)；Flow phase system be methanol-
0.1% formic acid system, concrete gradient condition such as following table；Column temperature is 25 DEG C,；Flow velocity is 0.8mL/min；Detection wavelength is 254nm,
Sampling volume is 25 μ L；Acquisition time is 140min；
Gradient elution program such as following table：
Condition of gradient elution
The preparation of 2.2 reference substance solution：Precision weighs 3-OH phlorhizin 10.05mg, adds 50% ethanol to be dissolved, fixed
In being dissolved in 10mL volumetric flasks, precision pipettes the above-mentioned solution of 1mL, and in 10mL volumetric flasks, be made into concentration is 50% ethanol constant volume
The solution of 0.1005mg/mL；Precision weighs phlorhizin 5.00mg, adds 50% ethanol to be dissolved, and constant volume is in 10mL volumetric flasks
In, the solution that concentration is 0.50mg/mL is made into, obtain final product；
The preparation of 2.3 need testing solutions：The Pasania cuspidata leaf medical material that lot number is 20140815 is taken, powder is beaten, and crosses No. two sieves, it is accurate
5.00g is really weighed, in being positioned over 125mL conical flask with cover, the ethanol for measuring 40mL50% is added thereto, and is positioned over water bath with thermostatic control
In pot, 80 DEG C of heating and refluxing extractions 60min are continuous to extract three times, finally by three extracting solution mix homogeneously, let cool, and filter, will
Filtrate constant volume takes in right amount in 250mL measuring bottles, crosses 0.45 μm of microporous filter membrane, and subsequent filtrate is placed in liquid phase bottle, obtains final product；
2.4 determine：Precision draws need testing solution injection high performance liquid chromatograph, according to high effective liquid chromatography for measuring, obtains
To finger printing, such as Fig. 1.
Embodiment two：The finger printing of the effective site of 10 batches of Pasania cuspidata leaves
The batch of Pasania cuspidata medical material 10 is taken, Pasania cuspidata leaf effective-part is prepared and by the condition of embodiment one by the method for embodiment one
Detected, obtained the HPLC collection of illustrative plates of 10 batches of samples.By the comparison of 10 batches of HPLC collection of illustrative plates, similarity evaluation is carried out, determine that it is special
Levy total peak：
There are 10 features to have peak in finger printing, now by the retention time of collection of illustrative plates, the meansigma methodss of peak area and total peak
Area summing, it is specific as follows：
No. 1 peak, Average residence time RT is 33.20min, and RSD is 0.41%, and peak area is that 292467.2, RSD is
34.25%；
No. 2 peaks, Average residence time RT is 38.35min, and RSD is 0.35%, and peak area is that 159295.1, RSD is
48.19%；
No. 3 peaks, Average residence time RT is 40.84min, and RSD is 0.90%, and peak area is that 551159.5, RSD is
26.29%；
No. 4 peaks, Average residence time RT is 46.65min, and RSD is 0.92%, and peak area is that 166383.2, RSD is
24.90%；
No. 5 peaks, Average residence time RT is 48.64min, and RSD is 0.65%, and peak area is that 669754.3, RSD is
43.54%；
No. 6 peaks, Average residence time RT is 61.09min, and RSD is 0.76%, and peak area is that 6032428.7, RSD is
34.22%；
No. 7 peaks, Average residence time RT is 68.70min, and RSD is 0.65%, and peak area is that 555096.1, RSD is
32.46%；
No. 8 peaks, Average residence time RT is 72.24min, and RSD is 0.66%, and peak area is that 212800.3, RSD is
44.16%；
No. 9 peaks, Average residence time RT is 90.31min, and RSD is 0.45%, and peak area is that 503140.4, RSD is
32.30%；
No. 10 peaks, Average residence time RT is 98.52min, and RSD is 0.32%, and peak area is that 105901.2, RSD is
34.59%.
It is solvent peak that RT is the peak occurred before 5min.
There are 10 features to have peak in the finger printing of the Pasania cuspidata leaf effective-part, wherein No. 3 chromatographic peaks are 3-OH
Phlorhizin, highest peak is No. 6 chromatographic peaks, is also that collection of illustrative plates total length is 140min, wherein unimodal area is super total with reference to peak phlorhizin
Peak area 2% has 7, and they are 1,3,5,6,7,8, No. 9 peaks, wherein the super total peak area 10% of unimodal area has 1, it
Be No. 6 chromatographic peaks, wherein the super total peak area 15% of unimodal area has 1, they are No. 6 chromatographic peaks, wherein unimodal area
Super total peak area 20% has 1, and they are No. 6 chromatographic peaks.
Similarity evaluation：The chromatogram of 10 batches of Pasania cuspidata leaf effective-parts is imported into " chromatographic fingerprints of Chinese materia medica similarity
Evaluation system (Chinese Pharmacopoeia committee 2004A versions) ", numerical selection peak (phlorhizin) is used as with reference to peak, such as Fig. 2, it is determined that 10 altogether
There is peak.Control collection of illustrative plates R is automatically generated using median method Auto-matching, each medical material similarity is obtained.10 batch Pasania cuspidatas it is similar
Degree is more than 0.90.It is shown in Table 1.
The similarity of the HPLC finger printing of 1 10 batches of Pasania cuspidata leaf medical material effective site of table
The finger printing of the Pasania cuspidata leaf effective-part HPLC common patterns set up by said method is as shown in Figure 3.
Embodiment described above only expresses some embodiments of the present invention, in the embodiment disclosed in this and all sights
Point, should be considered to illustrate the present invention, and its description is more concrete and detailed, but therefore can not be interpreted as to model of the present invention
The restriction enclosed.It should be pointed out that for the person of ordinary skill of the art, in the premise without departing from present inventive concept
Under, some deformations and improvement can also be gone out, these belong to protection scope of the present invention.Therefore protection scope of the present invention should be with
Claim is defined, and covers legal equivalents.
Claims (5)
1. a kind of fingerprint atlas detection method of Pasania cuspidata leaf effective-part, it is comprised the following steps：
（1）The preparation of reference substance solution：Precision weighs 3-OH phlorhizin 10.05mg, adds 50% ethanol to be dissolved, constant volume in
In 10mL volumetric flasks, precision pipettes the above-mentioned solution of 1mL, and in 10mL volumetric flasks, be made into concentration is 50% ethanol constant volume
The solution of 0.1005mg/mL；Precision weighs phlorhizin 5.00mg, adds 50% ethanol to be dissolved, and constant volume is in 10mL volumetric flasks
In, the solution that concentration is 0.50mg/mL is made into, obtain final product；
（2）The preparation of need testing solution：The Pasania cuspidata leaf medical material that lot number is 20140815 is taken, powder is beaten, and crosses No. two sieves, accurately claimed
5.00g is taken, in being positioned over 125mL conical flask with cover, the ethanol for measuring 40mL50% is added thereto, and is positioned over thermostat water bath
In, 80 DEG C of heating and refluxing extractions 60min are continuous to extract three times, finally by three extracting solution mix homogeneously, let cool, and filter, will
Filtrate constant volume takes in right amount in 250mL measuring bottles, crosses 0.45 μm of microporous filter membrane, and subsequent filtrate is placed in liquid phase bottle, obtains final product；
（3）Chromatographic condition：With octadecylsilane chemically bonded silica as filler, using gradient elution, mobile phase is methanol to chromatographic column
With the gradient eluent of 0.1% formic acid composition, column temperature is 25 DEG C；Ultraviolet detection wavelength is 254nm；
（4）Determine：Precision draws need testing solution injection high performance liquid chromatograph, according to high effective liquid chromatography for measuring, is referred to
Stricture of vagina collection of illustrative plates.
2. the fingerprint atlas detection method of a kind of Pasania cuspidata leaf effective-part according to claim 1, it is characterised in that：
The preparation of step (1) reference substance solution, using precision 3-OH phlorhizin 10.05mg are weighed, and add 50% ethanol to enter
Row dissolving, in 10mL volumetric flasks, precision pipettes the above-mentioned solution of 1mL to constant volume, adds 50% ethanol constant volume in 10mL volumetric flasks,
It is made into the solution that concentration is 0.1005mg/mL；Precision weighs phlorhizin 5.00mg, adds 50% ethanol to be dissolved, constant volume in
In 10mL volumetric flasks, the solution that concentration is 0.50mg/mL is made into, is obtained final product；
The preparation of step (2) need testing solution, adopts the Pasania cuspidata leaf medical material for taking lot number for 20140815, beats powder, and mistake
No. two sieves, accurately weigh 5.00g, and in being positioned over 125mL conical flask with cover, the ethanol for measuring 40mL50% is added thereto, and place
In thermostat water bath, 80 DEG C of heating and refluxing extractions 60min are continuous to extract three times, finally by three extracting solution mix homogeneously, put
It is cold, filter, by filtrate constant volume in 250mL measuring bottles, take in right amount, 0.45 μm of microporous filter membrane is crossed, in liquid phase bottle, i.e., subsequent filtrate is placed in
；
Step (3) chromatographic condition：Chromatographic column is adopted with octadecylsilane chemically bonded silica as filler；Using gradient elution,
Mobile phase is the gradient elution that methanol and 0.1% aqueous formic acid are constituted；Column temperature is 25 DEG C；Ultraviolet detection wavelength 254nm；Stream
Speed：0.8mL/min；Time：140min；
The step (4) determines：The μ L of need testing solution 25 injection high performance liquid chromatographs are drawn using accurate, according to high-efficient liquid phase color
Spectrometry is determined, and obtains finger printing.
3. the fingerprint atlas detection method of a kind of Pasania cuspidata leaf effective-part according to claim 1, it is characterised in that：
Mobile phase is the gradient eluent that methanol and 0.1% aqueous formic acid are constituted in step (3) chromatographic condition；Column temperature is
25℃；Ultraviolet detection wavelength 254nm；Flow velocity：0.8mL/min；Analysis time：140min.
4. a kind of fingerprint atlas detection method of the Pasania cuspidata leaf effective-part according to Claims 2 or 3, its feature exists
In：
The step（3）Gradient elution described in chromatographic condition, gradient elution program is carried out with the configuration of following volumetric concentration：
When 0 minute, mobile phase A is 18% methanol solution, 0.1% aqueous formic acid that Mobile phase B is 82%；
When 20 minutes, mobile phase A is 35% methanol solution, 0.1% aqueous formic acid that Mobile phase B is 65%；
When 60 minutes, mobile phase A is 35% methanol solution, 0.1% aqueous formic acid that Mobile phase B is 65%；
When 120 minutes, mobile phase A is 60% methanol solution, 0.1% aqueous formic acid that Mobile phase B is 40%；
When 140 minutes, mobile phase A is 60% methanol solution, 0.1% aqueous formic acid that Mobile phase B is 40%.
5. a kind of fingerprint atlas detection method of Pasania cuspidata leaf effective-part, it is characterised in that：
The foundation of Pasania cuspidata leaf effective-part finger printing, specifically includes following steps：
（1）The preparation of reference substance solution：Precision weighs 3-OH phlorhizin 10.05mg, adds 50% ethanol to be dissolved, constant volume in
In 10mL volumetric flasks, precision pipettes the above-mentioned solution of 1mL, and in 10mL volumetric flasks, be made into concentration is 50% ethanol constant volume
The solution of 0.1005mg/mL；Precision weighs phlorhizin 5.00mg, adds 50% ethanol to be dissolved, and constant volume is in 10mL volumetric flasks
In, the solution that concentration is 0.50 mg/mL is made into, obtain final product；
（2）The preparation of need testing solution：The Pasania cuspidata leaf medical material that lot number is 20140815 is taken, powder is beaten, and crosses No. two sieves, accurately claimed
5.00g is taken, in being positioned over 125mL conical flask with cover, the ethanol for measuring 40mL50% is added thereto, and is positioned over thermostat water bath
In, 80 DEG C of heating and refluxing extractions 60min are continuous to extract three times, finally by three extracting solution mix homogeneously, let cool, and filter, will
Filtrate constant volume takes in right amount in 250mL measuring bottles, crosses 0.45 μm of microporous filter membrane, and subsequent filtrate is placed in liquid phase bottle, obtains final product；
（3）Chromatographic condition：With octadecylsilane chemically bonded silica as filler, using gradient elution, mobile phase is methanol to chromatographic column
With the gradient eluent of 0.1% formic acid composition, column temperature is 25 DEG C；Ultraviolet detection wavelength is 285nm；
（4）Determine：Precision draws need testing solution injection high performance liquid chromatograph, according to high effective liquid chromatography for measuring, is referred to
Stricture of vagina collection of illustrative plates；
There are 10 features to have peak in the finger printing, wherein No. 3 chromatographic peaks are 3-OH phlorhizin, highest peak is No. 6 chromatographs
Peak is phlorhizin reference peak, and collection of illustrative plates total length is 140min, specific as follows：
No. 1 peak, Average residence time RT is 33.20min, and RSD is 0.41%, and it is 34.25% that peak area is 292467.2, RSD；
No. 2 peaks, Average residence time RT is 38.35min, and RSD is 0.35%, and it is 48.19% that peak area is 159295.1, RSD；
No. 3 peaks, Average residence time RT is 40.84min, and RSD is 0.90%, and it is 26.29% that peak area is 551159.5, RSD；
No. 4 peaks, Average residence time RT is 46.65min, and RSD is 0.92%, and it is 24.90% that peak area is 166383.2, RSD；
No. 5 peaks, Average residence time RT is 48.64min, and RSD is 0.65%, and it is 43.54% that peak area is 669754.3, RSD；
No. 6 peaks, Average residence time RT is 61.09min, and RSD is 0.76%, and it is 34.22% that peak area is 6032428.7, RSD；
No. 7 peaks, Average residence time RT is 68.70min, and RSD is 0.65%, and it is 32.46% that peak area is 555096.1, RSD；
No. 8 peaks, Average residence time RT is 72.24min, and RSD is 0.66%, and it is 44.16% that peak area is 212800.3, RSD；
No. 9 peaks, Average residence time RT is 90.31min, and RSD is 0.45%, and it is 32.30% that peak area is 503140.4, RSD；
No. 10 peaks, Average residence time RT is 98.52min, and RSD is 0.32%, and it is 34.59% that peak area is 105901.2, RSD.
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