CN109856254B - Method for establishing ethyl acetate part HPLC fingerprint spectrum of longan leaf - Google Patents
Method for establishing ethyl acetate part HPLC fingerprint spectrum of longan leaf Download PDFInfo
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Abstract
The invention discloses a method for establishing an HPLC fingerprint of an ethyl acetate part of longan leaves and a fingerprint thereof. The method comprises the following steps: preparing a test solution; preparing a reference solution containing ethyl gallate, astragalin and quercetin; and (5) measuring by adopting HPLC to obtain a fingerprint. The method has the advantages of rapidness, accuracy, high precision, good repeatability and stability, rich chromatographic peak information, good separation effect and the like. The HPLC standard fingerprint spectrum of the ethyl acetate part of the longan leaves established by the invention can effectively represent the quality of the longan leaves, particularly the quality of the ethyl acetate part of the longan leaves, further improve the standard of the longan leaves, perfect the quality evaluation system of the longan leaves, promote the standardization of traditional Chinese medicines, provide theoretical and practical bases for the comprehensive and effective control of the quality of the longan leaves and provide accurate and reliable bases for the safety and the effectiveness of clinical medication.
Description
Technical Field
The invention discloses a quality control method of a Chinese medicinal material, and particularly relates to a construction method of a longan leaf ethyl acetate part high performance liquid chromatography fingerprint and a standard fingerprint.
Background
Longyan ye, a Chinese herbal medicine with the national features of Guangxi, was first recorded in Dian nan materia medica legend. Is leaf or tender bud of longan of Sapindaceae, and is mainly produced in Guangxi province, guangdong province, fujian province, taiwan province, and other provinces, and cultivated in Hainan province, yunnan province, sichuan province, guizhou province, etc. It is sweet, bland and mild in nature and taste, and has the effects of relieving exterior syndrome, clearing heat, promoting diuresis and removing toxic substances. It is mainly used for treating common cold, fever, malaria, furuncle, eczema, etc. Longan leaf mainly contains tannin, phenolic acid, flavone and volatile oil. At present, few domestic researches on chemical components of longan leaves are reported, and the research is mainly focused on the aspect of flavonoid components. The inventor has carried out systematic chemical composition research on longan leaves and found that the main active ingredient of longan leaves is a flavone component. The inventor finds that the effective part of the longan leaves for resisting the type II diabetes is mainly the ethyl acetate part by researching the pharmacological action of different extraction parts of the longan leaves on the type II diabetes mice. In addition, the research results of the hypoglycemic effect of the longan leaves by the inventor show that the lupeol, ethyl gallate, kaempferol, luteolin, quercetin, astragalin and other components in the ethyl acetate part extracted and separated from the longan leaves have strong inhibition effect on alpha-glucosidase, and the inventor also conducts content determination method research on the active components and determines the content determination method for simultaneously determining the 5 components.
The traditional Chinese medicine fingerprint spectrum refers to a chromatogram or a spectrogram which can mark chemical characteristics of certain traditional Chinese medicinal materials or traditional Chinese medicine preparations by adopting a certain analysis means after the traditional Chinese medicinal materials or the traditional Chinese medicine preparations are properly processed. The method can comprehensively reflect the types and the quantity of chemical components contained in the medicinal materials, effectively reflect the integrity and the comprehensive action of the traditional Chinese medicine components, and has been widely applied to the aspects of traditional Chinese medicine analysis and identification, quality control and the like due to the characteristics of rapidness, accuracy and the like. The fingerprint spectrum reflects the integral characteristics of the samples, and the integral difference and the degree of affinity and sparseness among the samples can be reflected by comparing the fingerprint spectra. The inventor researches the HPLC fingerprint of the longan leaf medicinal material, determines the HPLC fingerprint determination method and the standard fingerprint thereof, and obtains 11 common peaks comprising 3 components of quercetin, luteolin and kaempferide. The inventor also researches fingerprints of longan leaves in different producing areas by using Fourier infrared spectroscopy (FTIR) technology, determines an FTIR fingerprint determination method and obtains an infrared standard fingerprint with 5 common peaks. In order to further improve the standard of the longan leaf medicinal material, promote the standardization of the traditional Chinese medicine, ensure and improve the quality of the longan leaf medicinal material and provide accurate and reliable basis for the safety and the effectiveness of clinical medication, the inventor researches the fingerprint of the ethyl acetate part which is the effective part of the longan leaf for resisting the type II diabetes on the basis of the previous research on the longan leaf, and no relevant report is found at present.
The above background disclosure is only provided to assist understanding of the inventive concept and technical solutions of the present invention, which do not necessarily belong to the prior art of the present patent application, and should not be used to evaluate the novelty and inventive step of the present application in the case that there is no clear evidence that the above content has been disclosed at the filing date of the present patent application.
Disclosure of Invention
In order to further improve the standard of the longan leaf, promote the standardization of traditional Chinese medicines, ensure and improve the quality of the longan leaf and provide accurate and reliable basis for the safety and the effectiveness of clinical medication, the invention provides the establishment method of the HPLC fingerprint spectrum of the ethyl acetate part of the longan leaf and the standard fingerprint spectrum of the ethyl acetate part of the longan leaf obtained by the method.
The technical scheme adopted by the invention is as follows:
an establishment method of an ethyl acetate part HPLC fingerprint spectrum of longan leaves comprises the following steps:
s1, preparation of a test solution: 2g of coarse longan leaf powder is precisely weighed and placed in a conical flask with a plug, 20ml of 95% ethanol is added firstly for ultrasonic treatment for 1h, then 20ml of 50% ethanol is added for ultrasonic treatment for 30min, the mixture is filtered, filtrate is evaporated to dryness, water is added to the filtrate to be suspended in a separating funnel, an equal amount of petroleum ether is added for repeated extraction until an extracted petroleum ether layer is colorless, a lower-layer solution is taken, an equal amount of ethyl acetate is added for repeated extraction until an extracted ethyl acetate layer is colorless, the ethyl acetate layers are combined and evaporated to dryness, 2ml of methanol is precisely added, shaking is carried out uniformly, filtering is carried out, and a subsequent filtrate is taken as a sample solution;
s2, preparing a reference substance solution: precisely weighing ethyl gallate, astragalin and quercetin reference substances, and adding methanol to obtain mixed solution with concentration of each reference substance of 0.1 mg/ml;
s3, chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler for the chromatographic column; the ultraviolet detection wavelength is 280nm; the flow rate is 1.0ml/min; the column temperature is 30 ℃; the mobile phase A is methanol, the mobile phase B is 0.2% phosphoric acid solution, and the gradient elution procedure is as follows: 0 to 50min,15% → 26% A,50 to 120min,26% → 45% A,120 to 150min,45% A;
s4, determination: precisely sucking 10 μ l of each of the reference solution and the sample solution, respectively injecting into a high performance liquid chromatograph, measuring according to the step S3, and recording the chromatogram within 150min to obtain the fingerprint chromatogram.
Further, in the method for establishing an HPLC fingerprint of an ethyl acetate fraction of longan leaf, the method for preparing the reference substance solution in step S2 is preferably: precisely weighing ethyl gallate, astragalin and quercetin control substances 1mg respectively, placing in 10ml volumetric flask, adding methanol to dilute to scale, and shaking to obtain mixed solution of each control substance.
Further, in the method for establishing the HPLC fingerprint spectrum of the ethyl acetate fraction of longan leaf, the chromatographic column using octadecylsilane chemically bonded silica as a filler is preferably: thermo C 18 Column, 250 mm. Times.4.6 mm, particle size 5 μm.
The HPLC fingerprint of the ethyl acetate part of longan leaf obtained by the above method has 12 common fingerprint peaks, wherein the 5 th peak is gallic acid ethyl ester, the 11 th peak is astragalin, and the 12 th peak is quercetin.
The HPLC fingerprint spectrum of the ethyl acetate part of the longan leaf is applied to the authenticity identification and the base source identification of the longan leaf, preferably applied to the detection of the longan leaf extract and the preparation thereof, and most preferably applied to the detection of the ethyl acetate part of the longan leaf and the preparation thereof.
The invention has the beneficial effects that:
1. the invention establishes the establishment method of the longan leaf ethyl acetate part fingerprint, can obtain the HPLC fingerprint of the longan leaf ethyl acetate part by the method, can effectively represent the quality of the longan leaves, particularly the quality of the longan leaf ethyl acetate part, further improves the longan leaf medicinal material standard, perfects the longan leaf quality evaluation system, promotes the standardization of traditional Chinese medicines, provides theoretical and practical basis for comprehensively and effectively controlling the quality of longan leaves, and provides accurate and reliable basis for the safety and the effectiveness of clinical medication.
2. The method has the advantages of rapidness, accuracy, high precision, good repeatability and stability, rich chromatographic peak information, good separation effect and the like.
3. The method treats the longan leaves as a whole, can find out the slight difference among different medicinal materials by comparing the common peaks, can comprehensively evaluate the quality of the longan leaves, particularly the quality of the ethyl acetate part, realizes the base source identification of the longan leaves, and is suitable for identifying and controlling the authenticity, the producing area and the quality of the longan leaves.
Drawings
FIG. 1 is a control HPLC chromatogram
In the figure: 5: 4, gallic acid ethyl ester; 11: astragalin; 12: quercetin.
FIG. 2 is a HPLC chromatogram of a sample
In the figure: 5: 4, gallic acid ethyl ester; 11: astragalin; 12: quercetin.
FIG. 3 is HPLC chromatogram stack of ethyl acetate parts of 10 different longan leaf medicinal materials in different producing areas.
FIG. 4 is a graph showing the matching of ethyl acetate parts of 10 different longan leaf herbs in different producing areas.
FIG. 5 shows fingerprint chromatogram of folium longan.
FIG. 6 is a graph showing the results of the cluster analysis of ethyl acetate fractions from 10 batches of longyan leaf herbs.
FIG. 7 is an analysis chart of the ethyl acetate fraction of 10 batches of longyan leaf medicinal materials.
Detailed Description
The invention is further described with reference to specific examples, without limiting the scope of protection and the scope of application of the invention:
the invention provides a method for establishing an HPLC fingerprint spectrum of an ethyl acetate part of longan leaves, which comprises the following steps:
s1, preparation of a test solution: 2g of coarse longan leaf powder is precisely weighed and placed in a conical flask with a plug, 20ml of 95% ethanol is added firstly for ultrasonic treatment for 1h, then 20ml of 50% ethanol is added for ultrasonic treatment for 30min, the mixture is filtered, filtrate is evaporated to dryness, water is added to the filtrate to be suspended in a separating funnel, an equal amount of petroleum ether is added for repeated extraction until an extracted petroleum ether layer is colorless, a lower-layer solution is taken, an equal amount of ethyl acetate is added for repeated extraction until an extracted ethyl acetate layer is colorless, the ethyl acetate layers are combined and evaporated to dryness, 2ml of methanol is precisely added, shaking is carried out uniformly, filtering is carried out, and a subsequent filtrate is taken as a sample solution.
S2, preparing a reference solution: accurately weighing ethyl gallate, astragalin and quercetin control, and adding methanol to obtain mixed solution with concentration of 0.1 mg/ml. Preferably, 1mg of each of ethyl gallate, astragalin and quercetin control substances is precisely weighed, placed in a 10ml volumetric flask, diluted to the scale with methanol, and shaken up to obtain the mixed solution of each control substance.
S3, chromatographic conditions: the chromatographic column uses octadecylsilane chemically bonded silica as filler, preferably Thermo C 18 Column (250 mm. Times.4.6 mm, particle size 5 μm); the ultraviolet detection wavelength is 280nm; the flow rate is 1.0ml/min; the column temperature is 30 ℃; the mobile phase A is methanol, the mobile phase B is 0.2% phosphoric acid solution, and the gradient elution procedure is as follows: 0 to 50min,15% → 26% A,50 to 120min,26% → 45% A,120 to 150min,45% A.
S4, determination: precisely sucking 10 μ l of each of the reference solution and the sample solution, respectively injecting into a high performance liquid chromatograph, measuring according to the step S3, and recording the chromatogram within 150min to obtain the fingerprint chromatogram.
The obtained longan leaf ethyl acetate part HPLC fingerprint has 12 common fingerprint peaks, wherein the peak No. 5 is ethyl gallate, the peak No. 11 is astragalin, and the peak No. 12 is quercetin.
1 Experimental materials, instruments and reagents
1.1 materials
1.1.1 medicinal materials
The folium longan medicinal material is leaf or tender bud of longan (Dimocarpus longan Lour.) of Sapindaceae, and the folium longan medicinal material used in the experiment is collected in each production place of Guangxi province. The sample was identified as leaves of longan, a sapindaceae plant, by professor Teng Jianbei, auxiliary professor, of the pharmaceutical institute of Guangxi traditional Chinese medicine university, and is shown in Table 1.
TABLE 1 sources of folium longan of Guangxi different producing areas
1.1.2 reagents and controls
Reagent: methanol, acetonitrile (chromatographically pure, fisher science world company, USA), methanol (Beijing chemical plant; analytical grade), ethanol (Beijing chemical plant; analytical grade), water as ultrapure water, and other reagents as analytical grade.
Comparison products: ethyl gallate (homemade in laboratory, purity > 99%); quercetin (China food and drug testing research institute, lot number: 100081-201610 for content determination); astragalin (Shanghai Huo pharmaceutical science and technology Co., ltd., lot number: 170326, purity > 99%); luteolin (Shanghai Huo pharmaceutical science and technology Co., ltd., batch No.: 170626, purity > 99%); kaempferol (Shanghai Huo pharmaceutical science and technology Co., ltd., batch No.: 170517, purity > 99%).
1.2 instruments
Agilent1260 high performance liquid chromatograph (Agilent technologies, inc., USA); KQ-500DA ultrasonic apparatus (ultrasonic apparatus Co., ltd., kunshan city); SQP electronic balance (sandisco scientific instruments (beijing) limited); high speed universal mill (Tester instruments, inc., tianjin). Millipore silicon-185 ultrapure water meter (Millipore, USA);
2 selection of chromatographic conditions
In order to better reflect the chemical component information of the ethyl acetate part of the longan leaves in the high-performance liquid fingerprint chromatography, an effective chromatographic separation method and detection conditions must be determined. Thus, this experiment examined several factors that affect sample separation: selecting chromatographic column, selecting mobile phase, selecting detection wavelength and selecting column temperature and flow rate, wherein the longan leaf medicinal material is extracted from all producing areas in Guangxi by the same preparation method. As set forth below:
2.1 selection of chromatography columns
The following three chromatographic columns using octadecylsilane chemically bonded silica as packing were selected for comparison in this experiment. (1) Thermo C18 column (4.6 mm. Times.150mm, 5 μm); (2) Agilent C18 (4.6 mm. Times.250mm, 5 μm); (3) Kromasil C18 (4.6 mm. Times.250mm, 5 μm). The experimental results show that in three chromatographic columns using octadecylsilane chemically bonded silica as packing, each peak has better resolution and a stable base line, wherein in a Thermo C18 column (250 mm multiplied by 4.6mm,5 mu m), each peak has the best resolution and the stable base line.
2.2 selection of mobile phase System
The test paper examines that different solvents such as methanol-water, acetonitrile-water, methanol-0.2% phosphoric acid, acetonitrile-0.2% phosphoric acid and the like are respectively adopted to carry out detection by adopting an isocratic elution method, and the result shows that chromatographic peaks in a chromatogram are fewer during isocratic elution, the chromatographic peaks are seriously overlapped and are not separated, and the separation effect is poor. Therefore, the system is adopted for gradient elution, and the HPLC (high performance liquid chromatography) spectrum of the ethyl acetate part of the longan leaves is investigated. Experiments show that the separation of each peak is poor and the peaks are seriously overlapped in an acetonitrile-water system and an acetonitrile-0.2% phosphoric acid system. The chromatographic pattern of the methanol-water system and the methanol-0.2 percent phosphoric acid system has better peak shape, more peak signals and better separation. However, the baseline shift phenomenon of the chromatographic peak of the methanol-water system is considered, so the methanol-0.2 percent phosphoric acid system is adopted for gradient elution.
2.3 selection of elution procedure
Comparison of isocratic and gradient elution is performed herein using a methanol-0.2% phosphoric acid solution system, respectively. Experiments show that the chromatographic peak is less in the chromatogram when isocratic elution is carried out, the peak appearance time is longer, the chromatographic peak cannot be eluted, and the method is not suitable for collecting the general chemical information fingerprint of the ethyl acetate part of the longan leaves. Therefore, in order to ensure that the chromatogram can generally summarize the chemical information of the medicinal materials, each chemical component has a peak as much as possible, and the analysis time is appropriate, a gradient elution procedure is determined.
In the experiment, phosphoric acid is added to improve the separation degree of chromatographic peaks and the peak shape, the influence of 0.1%, 0.2% and 0.4% phosphoric acid solution on the separation degree and the peak shape of chromatographic peaks is examined, and the effect of adding 0.2% phosphoric acid solution on improving the separation degree of chromatographic peaks of samples is found to be better, the separation degree and the peak shape are not improved by increasing the concentration of acid, and the concentration of added phosphoric acid is controlled to be 0.2% in consideration of the pH value which can be borne by a chromatographic column.
A methanol-0.2% phosphoric acid solution system is used for comparing different elution procedures, and a better elution procedure is selected, which is shown in table 2, so that each peak in the map can be better separated, the base line is relatively stable, the peak-producing time is proper, and the peak shapes are uniform.
TABLE 2 gradient elution conditions
2.4 selection of detection wavelength
The detection wavelength is used for obtaining more chemical component information for the fingerprint, and the characteristics of the ethyl acetate part of the longan leaves are generally summarized. Chromatograms of absorption wavelengths under a full wavelength scan are examined herein.
The experimental result shows that the spectrum measured at 280nm shows more peak information and better signals, the peak shape of each obtained spectrum is better, the base line is stable, and the detection wavelength at 280nm is comprehensively considered and selected.
2.5 selection of column temperature and flow Rate
The column temperature is one of the important parameters of high performance liquid chromatography, and the change of the column temperature can cause the retention time shift of chromatographic peaks and influence the separation effect of each component. In the experiment, different column temperatures of 25 ℃,30 ℃ and 35 ℃ are set for comparison, and under the condition that other chromatographic conditions are the same and at the temperature of 30 ℃, the separation effect of each peak is the best, so that the column temperature is determined to be 30 ℃. Chromatograms at different flow rates of 0.8ml/min,1.0ml/min,1.2ml/min are also examined herein, and the results show that it is good to use 1.0 ml/min.
2.6 determining the chromatographic conditions of the fingerprint
And (3) chromatographic column: thermo C18 column (250 mm. Times.4.6 mm,5 μm);
mobile phase: methanol-0.2% phosphoric acid, gradient elution procedure as shown in table 2;
the detection wavelength is as follows: 280nm;
flow rate: 1.0ml/min;
column temperature: 30 ℃;
sample injection amount: 10 mu l of the mixture;
theoretical plate number: calculated by gallic acid ethyl ester, is not lower than 5000.
3 preparation method and methodological Studies
3.1 determination of sample preparation method
This experiment compared 3 different extraction methods, all using ultrasound extraction.
2g of coarse longan leaf powder is precisely weighed and placed in a conical flask with a plug, 20ml of 95% ethanol is added firstly for ultrasonic treatment for 1h, then 20ml of 50% ethanol is added for ultrasonic treatment for 30min, the mixture is filtered, filtrate is evaporated to dryness, water is added to the filtrate to be suspended in a separating funnel, an equal amount of petroleum ether is added for repeated extraction until an extracted petroleum ether layer is colorless, a lower-layer solution is taken, an equal amount of ethyl acetate is added for repeated extraction until an extracted ethyl acetate layer is colorless, the ethyl acetate layers are combined and evaporated to dryness, 2ml of methanol is precisely added, shaking is carried out uniformly, filtering is carried out, and a subsequent filtrate is taken as a sample solution.
Preparation of test solution 1: taking 2g of coarse longan leaf powder, precisely weighing, placing in a conical flask with a plug, firstly adding 20ml of 95% ethanol, carrying out ultrasonic treatment for 1h, then adding 20ml of 50% ethanol, carrying out ultrasonic treatment for 30min, filtering, evaporating the filtrate, adding water, suspending in a separating funnel, adding an equal amount of petroleum ether, repeatedly extracting until an extracted petroleum ether layer is colorless, taking a lower-layer solution, then adding an equal amount of ethyl acetate, repeatedly extracting until an extracted ethyl acetate layer is colorless, combining the ethyl acetate layers, drying by distillation, precisely adding 2ml of methanol, shaking uniformly, filtering, and taking a subsequent filtrate as a sample solution.
Preparation of test solution 2: taking about 2g of coarse longan leaf powder, accurately weighing, placing in a conical flask with a plug, adding 20ml of 95% ethanol, carrying out ultrasonic treatment for 1h, filtering, evaporating the filtrate to dryness, adding water, suspending in a separating funnel, adding equal amount of ethyl acetate, repeatedly extracting until an extracted ethyl acetate layer is colorless, combining the ethyl acetate layers, evaporating to dryness, accurately adding 2ml of methanol, shaking uniformly, filtering, and taking a subsequent filtrate as a sample solution.
Preparation of test solution 3: taking about 2g of coarse powder of longan leaf, precisely weighing, placing in a conical flask with a plug, adding ethyl acetate solution, performing ultrasonic treatment for 1h, filtering, evaporating the filtrate to dryness, precisely adding 2ml of methanol, shaking up, filtering, and taking the subsequent filtrate as a test solution.
As a result, in the case of preparation 1 of the sample solution, the number of chromatographic peaks is large, and the separation is easy, and the separation effect of each peak is good.
3.2 preparation of control solutions
Taking ethyl gallate, astragalin, quercetin, luteolin and kaempferol as reference substances, precisely weighing the ethyl gallate, astragalin, quercetin, luteolin and kaempferol respectively at 1mg, placing the reference substances in a 10ml volumetric flask, adding methanol to dilute the reference substances to a scale, shaking the reference substances uniformly, precisely absorbing 10 mul of the reference substances, injecting the reference substances into a high performance liquid chromatograph, and recording the chromatogram, wherein the retention time of chromatographic peaks of the ethyl gallate, astragalin, quercetin, luteolin and kaempferol is consistent with that of No. 5, no. 11 and No. 12 peaks in a sample chromatogram, and the peak patterns are not obvious under the condition of 280nm because the absorption wavelength of the luteolin and the kaempferol in the sample is about 360nm, so the reference substances are not taken as common peak marks, and the No. 5 peak (ethyl gallate) is taken as a reference peak.
3.3 precision test
Taking about 2g of dry powder of a longan leaf medicinal material produced in Sterculia urensis in Guangxi, precisely weighing, preparing the same test sample according to the test sample preparation method 1 under 3.1 items, continuously feeding samples for 6 times under 2.6 items of chromatographic conditions, and recording a chromatogram, wherein the result shows that the RSD value of the relative retention time of each chromatographic peak is between 0.0660 and 0.1095 percent; the relative peak area RSD value of each chromatographic peak is between 0.5064% and 1.6139%, which shows that the precision of the instrument is good, and the results are shown in tables 3-4.
TABLE 3 fingerprint precision test-relative retention time of ethyl acetate part of folium longan medicinal material
TABLE 4 fingerprint precision test-relative peak area of ethyl acetate part of folium longan medicinal material
3.4 repeatability experiments
Taking about 2g of 6 parts of powder of longan leaves produced in Sterculia in Guangxi, precisely weighing, preparing according to the test sample preparation method 1 under item 3.1, measuring under chromatographic conditions of item 2.6, recording a chromatogram, and inspecting the repeatability of the experimental method. The result shows that the relative retention time RSD value of each chromatographic peak in 6 samples is between 0.0926% and 0.3409%, the relative peak area RSD value of each chromatographic peak is between 0.5029% and 1.5290%, and the experimental repeatability is good. The results are shown in tables 5 to 6.
TABLE 5 repeatability test of ethyl acetate fingerprint of folium longan medicinal material-relative retention time
TABLE 6 repeat test of ethyl acetate part fingerprint of folium longan medicinal material-relative peak area
3.5 stability test
Taking a medicinal material of longan leaves produced in Sterculia urensis in Guangxi province, preparing 1 sample under 3.1 items to prepare the same sample solution, detecting for 0, 5, 10, 15, 20 and 25 hours respectively according to fingerprint chromatography conditions under 2.6 items, and inspecting the stability of the sample solution. The result shows that the relative retention time RSD value of each chromatographic peak is between 0.1356 and 0.3142 percent, the relative peak area RSD value of each chromatographic peak is between 0.5240 and 1.6663 percent, and the stability of the test sample in 25 hours is good. The results are shown in tables 7 to 8.
TABLE 7 stability test of ethyl acetate part fingerprint of folium longan medicinal Material-relative retention time
TABLE 8 fingerprint stability test-relative peak area for ethyl acetate part of folium longan medicinal material
4 establishment of fingerprint and technical parameters
4.1 blank test
In order to examine the interference of the flow relative to the sample analysis, 10 mu l of methanol is precisely absorbed under the chromatographic condition of 2.6 finger prints, and the methanol is injected into a high performance liquid chromatograph, and a chromatogram is recorded. The results show that almost no impurities in the mobile phase interfere with the sample analysis.
4.2 extended Wash test
Sampling the sample solution, analyzing according to 2.6 chromatographic conditions, gradient eluting for 150min, wherein the organic phase proportion in the mobile phase gradually rises to 100% from 150min, recording chromatogram, and almost no other chromatographic peak appears after 150 min.
4.3 reference chromatographic Peak establishment
The technical requirements for the research of the fingerprint spectrum of the traditional Chinese medicine injection stipulate that a reference object must be established for establishing the fingerprint spectrum. An appropriate reference substance should be selected as a reference substance according to the nature of the chemical components contained in the sample. The ethyl gallate in the experimental study is a known effective component in the longan leaves, and the retention time of the No. 5 chromatographic peak in the fingerprint spectrum of the ethyl gallate is moderate. Therefore, ethyl gallate (No. 5 peak) is selected as a reference peak, and the relative retention time and the relative peak area are calculated, so that the requirements of the fingerprint are met. And respectively sampling the mixed reference substance solution and the test solution according to the fingerprint chromatogram condition of 2.6 items, wherein the chromatogram figures are shown in figure 1 and figure 2.
4.4 fingerprint collection of longan leaves in each production area
Taking about 1g of 10 batches of longan leaf medicinal material powder in each production place of Guangxi, precisely weighing, preparing according to a preparation method of a test sample under item 3.1, measuring according to chromatographic conditions under item 2.6, recording HPLC chromatograms of 10 batches of longan leaf medicinal material injection, establishing a longan leaf medicinal material ethyl acetate part fingerprint according to technical requirements of traditional Chinese medicine fingerprint research, and establishing an HPLC chromatogram for 10 batches of longan leaf medicinal material ethyl acetate part HPLC chromatograms, wherein the chromatogram is shown in figure 3.
4.5 derivation of longan leaf chromatogram and calculation of similarity
Integrating HPLC chromatograms obtained under the same chromatographic condition, converting the integrated HPLC chromatograms into an AIA format, and introducing the integrated HPLC chromatograms into software for data analysis and research according to the following flow:
opening software → import atlas file (AIA) format → reference atlas with atlas No. 2 → control atlas generation method (median) and time window width (drift range 0.5 min) → automatic matching spectral peak → generation of control fingerprint atlas → performing data matching and calculating similarity → exporting.
The similarity of 10 imported maps is shown in tables 9 and 10, the data matching is shown in table 11, and the longan leaf matching maps of different origins are shown in fig. 4.
TABLE 9 calculation results of HPLC fingerprint similarity of ethyl acetate part of folium longan medicinal material
TABLE 10 calculation results of HPLC fingerprint similarity of ethyl acetate part of longan leaf medicinal materials in batches
By applying software of ' traditional Chinese medicine chromatogram fingerprint similarity evaluation system 2012 ' and by investigating ethyl acetate parts of longan leaf medicinal materials in different producing areas through high performance liquid chromatogram, the similarity of HPLC (high performance liquid chromatogram) spectrums of 10 batches of Guangxi longan leaf medicinal materials at the ethyl acetate parts is more than 0.9 under the detection wavelength of 280nm, and the specification of ' technical requirements (provisional) for researching traditional Chinese medicine injection fingerprints ' 2012 ' is met.
4.6 determination of fingerprint and common peaks
The experiment is regulated according to the technical requirements (temporary) of the national issued traditional Chinese medicine injection fingerprint spectrum research, and the chromatography must calibrate the common fingerprint peak of the traditional Chinese medicine by adopting relative retention time according to the detection result of more than 10 batches of samples.
The experiment has assisted software of 'traditional Chinese medicine chromatogram fingerprint similarity evaluation system 2012 edition' issued by the State pharmacopoeia Committee to automatically match relevant parameters of the fingerprint, demarcates the common fingerprint peak of the medicinal materials, and simultaneously gives a comparison fingerprint and the like, thereby avoiding the blindness of manual peak judgment and ensuring the justness and objectivity of the determination of the common peak.
The software is adopted for automatic matching, the relevant parameters are shown in a table 11, and 12 chromatographic peaks with the matching number of 10 producing areas are calibrated to be common fingerprint peaks. Selecting longan leaf medicinal material atlas with number 1 as reference atlas, taking median method as generation method of comparison fingerprint atlas, setting time window width to 0.1min, extracting common mode of longan leaf medicinal material, and establishing comparison fingerprint atlas as shown in figure 5.
By comparison we calibrated 12 common fingerprint peaks for samples from different sources. Ethyl gallate is active ingredient of folium longan, and 5 # in fingerprint is ethyl gallate absorption peak as reference peak S, and other characteristic peaks are labeled sequentially with Arabic numerals 1, 2, 3 \8230, N, as shown in FIG. 5. The retention time and peak area of the reference peak are taken as 1, and the relative peak area and relative retention time of the common fingerprint peak are calculated in tables 12 to 14. The chromatographic method established in the experiment can ensure that ethyl acetate parts of different batches of the longan leaf medicinal material have basically consistent chromatographic behaviors, but the contents of various chemical components among the medicinal materials are greatly different under the influence of various factors.
TABLE 12 common peak relative retention times for the bulk of longan leaf drug samples
TABLE 13 common peak relative peak area of longyan leaf drug samples
5 Cluster analysis
And (3) applying SPSS22.0 software, adopting an inter-group average connection method, and selecting the cosine of an included angle as a measure to perform clustering analysis. 10 samples were classified into 4 categories, where S3, S9, S5, and S7 are 1 st category, S2 and S4 are 2 nd category, S1 and S10 are 3 rd category, and S6 and S8 are 4 th category, as shown in FIG. 6.
6 principal component analysis
The principal component analysis is a multivariate statistical analysis method which converts a plurality of indexes into a few irrelevant comprehensive indexes and classifies the comprehensive indexes according to a certain rule. The analysis method can reduce the index dimension, concentrate index information and simplify complex problems, thereby enabling problem analysis to be more visual and effective. The SPSS22.0 is adopted for principal component analysis in the experiment, and most information in the original data can be explained by 1-2 principal components under most conditions, so that the distribution of the original data can be projected in a two-dimensional plane or a three-dimensional space. Compared with clustering analysis, the principal component analysis can provide visual distribution conditions of samples, and is suitable for exploration analysis in an unsupervised mode. From tables 14 and 15, it can be seen that the number of principal components is two, and the cumulative value is greater than 95%, which indicates that two principal components are enough to explain most of the variance in the data.
TABLE 14 Total variation coefficient Table
TABLE 15 scoring table for the first and second main components of ethyl acetate part of longan leaf crude drug in 10 batches
A PCA plane score chart is established by referring to the first and second main component scores of peak areas of 10 batches of longan leaf ethyl acetate parts in different producing areas, as shown in FIG. 7, the internal correlation performance of 10 batches of samples can be better represented, so that the samples can be classified, and as can be seen from the chart, the longan leaf medicinal material ethyl acetate parts in 10 different producing areas in Guangxi can be divided into four categories, wherein S3, S9, S5 and S7 are the 1 st category, S2 and S4 are the 2 nd category, S1 and S10 are the 3 rd category, S6 and S8 are the 4 th category, and the result is consistent with the cluster analysis result and the similarity evaluation result.
Claims (4)
1. A method for establishing an HPLC fingerprint spectrum of an ethyl acetate part of longan leaves is characterized by comprising the following steps:
s1, preparation of a test solution: taking 2g of coarse longan leaf powder, precisely weighing, placing the coarse longan leaf powder in a conical flask with a plug, firstly adding 20ml of 95% ethanol, carrying out ultrasonic treatment for 1h, then adding 20ml of 50% ethanol, carrying out ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, adding water, suspending the filtrate in a separating funnel, adding an equal amount of petroleum ether, repeatedly extracting until an extracted petroleum ether layer is colorless, taking a lower-layer solution, then adding an equal amount of ethyl acetate, repeatedly extracting until an extracted ethyl acetate layer is colorless, combining the ethyl acetate layers, drying by distillation, precisely adding 2ml of methanol, shaking uniformly, filtering, and taking a subsequent filtrate as a sample solution;
s2, preparing a reference solution: precisely weighing ethyl gallate, astragalin and quercetin each 1mg, placing in 10ml volumetric flask, adding methanol to dilute to scale, and shaking to obtain mixed solution of each reference;
s3, chromatographic conditions: the chromatographic column is Thermo C 18 Column, 250mm × 4.6mm, particle size 5 μm; the ultraviolet detection wavelength is 280nm; the flow rate is 1.0ml/min; the column temperature is 30 ℃; the mobile phase A is methanol, the mobile phase B is 0.2% phosphoric acid solution, and the gradient elution procedure is as follows: 0 to 50min,15% → 26% A,50 to 120min,26% → 45% A,120 to 150min,45% A;
s4, determination: precisely sucking 10 μ l of each of the reference solution and the sample solution, respectively injecting into a high performance liquid chromatograph, measuring according to the method of step S3, and recording the chromatogram within 150min to obtain fingerprint chromatogram, wherein the fingerprint chromatogram has 12 common fingerprint peaks, wherein the peak No. 5 is ethyl gallate, the peak No. 11 is astragalin, and the peak No. 12 is quercetin.
2. The method for establishing an ethyl acetate fraction HPLC fingerprint of longan leaf as recited in claim 1, wherein the fingerprint obtained by said method is used for true-false identification and basic source identification of longan leaf.
3. The method for establishing an ethyl acetate fraction HPLC fingerprint of longan leaf according to claim 1, wherein the fingerprint obtained by the establishing method is used for detecting longan leaf extract and preparation thereof.
4. The method for establishing an HPLC fingerprint of an ethyl acetate part of longan leaf according to claim 1, wherein the fingerprint obtained by the establishing method is applied to detection of an ethyl acetate extract of longan leaf and a preparation thereof.
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