Construction method of HPLC fingerprint of small leaf three-point gold

Abstract

The application discloses a method for constructing a HPLC fingerprint of small-leaf three-point gold, and belongs to the technical field of medicinal material analysis. The method comprises the following technical steps: (1) preparation of a test sample solution; (2) preparation of a mixed control sample solution; (3) high performance liquid chromatography detection: the test sample solution and the mixed control sample solution are injected into a high performance liquid chromatograph for detection, and a chromatogram is recorded; (4) establishment of a fingerprint: the chromatogram is introduced into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, common peaks are matched, a control fingerprint is generated, and the common peaks are identified. The method has the advantages of good precision, repeatability and stability, etc., can comprehensively reflect the overall characteristics of the small-leaf three-point gold, rapidly, comprehensively and accurately evaluates the overall quality of the small-leaf three-point gold, and effectively guarantees the overall quality of medicinal materials.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicinal material analysis, and particularly relates to a construction method of HPLC fingerprint of Desmodium microphyllum. BACKGROUND

[0002] Desmodium microphyllum is the whole grass of Desmodium microphyllum (Thunb.) DC. in the Leguminosae family, also known as Banjiawo, Biansicao, and Xiaoyeshangludou. Desmodium microphyllum is produced in each province and region south of the Yangtze River, west to Yunnan and Tibet, and east to Taiwan. In Guangxi, it is mainly distributed in the northern part of Guilin, including Lingchuan County, Longsheng County, Yangshuo County, Ziyuan County, and Guangyang County. Desmodium microphyllum is a commonly used Yao medicinal material in Guangxi, and its Yao name is Yeshutugan. Desmodium microphyllum has a bitter and cool taste, and has the effects of clearing heat and dampness, relieving cough and asthma, and detoxifying. It is mainly used to treat snakebite and the like. Desmodium microphyllum contains various chemical components such as saccharides, flavonoids, phenylpropanoids, alkaloids, coumarins, terpenes, and volatile oils. Among them, the flavonoid components include catechin, prunus mume glycoside, isoprunus mume glycoside, and luteolin. Prunus mume glycoside has anti-inflammatory, analgesic, antiviral, and antibacterial pharmacological effects, isoprunus mume glycoside has anti-inflammatory, analgesic, antioxidant, and antiviral activities, and luteolin has anti-inflammatory pharmacological effects. It is a compound with relatively wide pharmacological activity and medicinal value.

[0003] At present, the research on Desmodium microphyllum in the literature focuses on the chemical components and pharmacological effects, and there is less research on the overall quality control, which makes it difficult to ensure the controllability and stability of the quality. As an important means of quality control of traditional Chinese medicine prescriptions, the fingerprint has the characteristics of large information quantity and strong characteristic, can reflect the overall characteristics of traditional Chinese medicine, and can reflect the action characteristics of multiple components of traditional Chinese medicine. High-performance liquid chromatography (HPLC) has the characteristics of high sensitivity, high selectivity, high efficiency, fast analysis speed, wide application range, and the like. Moreover, the method is not limited by the properties such as volatility and thermal stability of the sample, and most components in the sample can be analyzed and detected, so that the complex components in the traditional Chinese medicinal materials can be well separated, and the method is suitable for the construction of the fingerprint of traditional Chinese medicinal materials. SUMMARY

[0004] In view of the above problems, the application provides a construction method of HPLC fingerprint of Desmodium microphyllum, which can control the quality of Desmodium microphyllum from the overall characteristics, and provides a scientific basis for the quality control of medicinal materials.

[0005] The application is implemented by the following technical solutions.

[0006] A construction method of HPLC fingerprint of Desmodium microphyllum, comprising the following steps:

[0007] (1) Preparation of test solution: accurately weigh the sample powder of small leaf three-point gold, add 50% ethanol, weigh, ultrasonic treatment, cool, make up the weight loss, shake well, filter, centrifuge the filtrate, take the supernatant, and obtain the test solution;

[0008] (2) Preparation of mixed control solution: accurately weigh a certain amount of protocatechuic acid, isohispidin, hispidin and luteolin control samples respectively, add methanol to constant volume, shake well to obtain control sample stock solution, accurately take a certain amount of control sample stock solution, add methanol to constant volume, shake well to obtain mixed control solution;

[0009] (3) High performance liquid chromatography detection: inject the test solution and mixed control solution into the high performance liquid chromatograph for detection to obtain the test solution chromatogram and mixed control solution chromatogram respectively;

[0010] (4) Fingerprint establishment: import the test solution chromatogram into the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, match the common peaks, generate the control fingerprint, take hispidin chromatographic peak as the reference peak, calculate the relative retention time and relative peak area of each common peak, and identify the common peaks by comparing the retention time of the test solution chromatogram and the mixed control solution chromatogram.

[0011] Further, in step (3), the conditions of high performance liquid chromatography are as follows: the chromatographic column is SHIMADZU shim-pack VP-ODS, 4.6mmx250mm, 5μm type chromatographic column; the mobile phase is acetonitrile-0.1% formic acid for gradient elution, wherein acetonitrile is mobile phase A and 0.1% formic acid is mobile phase B; the detection wavelength adopts the conversion wavelength method; the flow rate is 0.8-1.0mL / min; the column temperature is 20-22℃; and the injection amount is 10-12μL.

[0012] Further, the gradient elution program is as follows: 0-12min, 8% A; 12-14min, 8-16% A; 14-35min, 16% A; 35-50min, 16-18% A; 50-70min, 18-40% A; 70-75min, 40% A.

[0013] Further, the conversion wavelength method is as follows: 0-18min, 260nm; 18-75min, 350nm.

[0014] Further, in step (2), the mass concentrations of protocatechuic acid, isohispidin, hispidin and luteolin in the control sample stock solution are 0.4040, 1.0180, 0.4113 and 0.2071mg / mL respectively.

[0015] Further, in step (4), the matching common peaks are corrected by the median method and matched with Mark peaks, with the time window width being 0.1, taking the sample S4 of Lophatherum gracile Brongn as a reference.

[0016] Further, in step (4), the control fingerprint comprises 9 common peaks, wherein the peak 1 corresponds to protocatechuic acid, the peak 4 corresponds to isohispidin, the peak 6 corresponds to hispidin, and the peak 9 corresponds to luteolin.

[0017] Further, in step (4), the relative retention time of the common peaks is as follows: the peak 1 is 0.401-0.411, the peak 2 is 0.575-0.588, the peak 3 is 0.863-0.880, the peak 4 is 0.911-0.923, the peak 5 is 0.962-0.966, the peak 6 is 1.000, the peak 7 is 1.423-1.457, the peak 8 is 1.436-1.485, and the peak 9 is 1.813-1.850.

[0018] Further, in step (4), the relative peak area of the common peaks is as follows: the peak 1 is 0.016-0.321, the peak 2 is 0.183-0.451, the peak 3 is 0.293-1.979, the peak 4 is 2.382-3.559, the peak 5 is 1.169-2.966, the peak 6 is 1.000, the peak 7 is 0.014-0.291, the peak 8 is 0.114-0.710, and the peak 9 is 0.026-0.871.

[0019] Further, in step (4), the similarity value of the sample of Lophatherum gracile Brongn and the control fingerprint is greater than 0.9.

[0020] Compared with the prior art, the application has the following advantages and beneficial effects:

[0021] 1. The HPLC fingerprint of Lophatherum gracile Brongn can accurately extract and separate the components in the medicinal material, has good precision, repeatability and stability, and can comprehensively reflect the overall characteristics of Lophatherum gracile Brongn, so as to quickly, comprehensively and accurately evaluate the overall quality of Lophatherum gracile Brongn and effectively ensure the overall quality of the medicinal material.

[0022] 2. The method for establishing the HPLC fingerprint of Lophatherum gracile Brongn is simple in operation, easy to realize the chromatographic conditions, high in detection efficiency, and has good accuracy and precision.

[0023] 3, The HPLC fingerprint of small leaf three-point gold established by the application determines 9 common peaks and identifies 4 components, which are protocatechuic acid (peak 1), isohispidin (peak 4), hispidin (peak 6) and luteolin (peak 9); the similarity of the fingerprint of 12 batches of small leaf three-point gold is above 0.9, which can provide a reference for the overall quality evaluation of small leaf three-point gold.

[0024] 4, The application also performs similarity evaluation on the HPLC fingerprint of small leaf three-point gold established by the application, and analyzes the fingerprint data by combining cluster analysis, principal component analysis and partial least squares-discriminant analysis, to comprehensively and objectively evaluate the quality differences of different batches of small leaf three-point gold and provide data reference for further improving the overall quality control of small leaf three-point gold. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 It is the HPLC superimposed chromatogram of 12 batches of small leaf three-point gold samples in Example 1.

[0026] Figure 2 It is the chromatogram of the mixed reference solution in Example 1.

[0027] Figure 3 It is the control fingerprint of small leaf three-point gold samples in Example 1.

[0028] Figure 4 It is the cluster analysis tree diagram of 12 batches of small leaf three-point gold samples in Example 1.

[0029] Figure 5 It is the scatter plot of 12 batches of small leaf three-point gold samples in Example 1.

[0030] Figure 6 It is the PCA-X score plot of 12 batches of small leaf three-point gold samples in Example 1.

[0031] Figure 7 It is the displacement retention plot of 12 batches of small leaf three-point gold samples in Example 1.

[0032] Figure 8 It is the OPLS-DA score plot of 12 batches of small leaf three-point gold samples in Example 1.

[0033] Figure 9 It is the VIP plot of OPLS-DA of 12 batches of small leaf three-point gold samples in Example 1. DETAILED DESCRIPTION

[0034] The application will be further described in detail through the following examples, which are only used to illustrate the application and do not limit the protection scope of the application.

[0035] Establishment of HPLC fingerprint of Desmodium microphyllum

[0036] 1 Experimental instruments, materials and reagents

[0037] 1.1 Instruments

[0038] Agilent 1260 type high performance liquid chromatograph (including four element pump, automatic sampler, column temperature box, ultraviolet detector, Agilent company of America); ME204 / 02 electronic balance (Mettler-Toledo instrument (Shanghai) Co., Ltd.); XSR205DU / A analytical balance (Mettler-Toledo instrument (Shanghai) Co., Ltd.); KQ-5200B ultrasonic cleaner (Kunshan ultrasonic instrument Co., Ltd. of Jiangsu province); Milli-Q Reference ultrapure water instrument (Millipore company of America). Chromatographic column: (1) SHIMADZU shim-pack VP-ODS (4.6mm×250mm, 5μm); (2) Waters: HSS T3 (4.6mm×250mm, 5μm); (3) Thermo: SCIENTIFIC Acclaim C18 (4.6mm×250mm, 5μm).

[0039] 1.2 Drugs and reagents

[0040] Protocatechuic acid reference substance (Chengdu Mai Desheng Technology Co., Ltd., batch number: RP190605, purity: 99.99%); Isoastragalin reference substance (Chengdu Mai Desheng Technology Co., Ltd., batch number: RP200522, purity: 99.90%); Sophoran reference substance (Chengdu Mai Desheng Technology Co., Ltd., RP190301, purity: 99.82%); Luteolin reference substance (Chengdu Mai Desheng Technology Co., Ltd., batch number: RP210105, purity: 99.17%); Acetonitrile chromatographically pure (Fisher company of America). Different batches of Desmodium microphyllum are shown in table 1. The medicinal materials were identified as the whole grass of Desmodium microphyllum (Thunb.) DC. by Professor Wei Song and Zhu Yilin of Guangxi University of Chinese Medicine.

[0041] Table 1 Different batches of Desmodium microphyllum

[0042]

[0043] 2 Experimental methods and results

[0044] 2.1 Chromatographic conditions

[0045] Column: SHIMADZU shim-pack VP-ODS (4.6 mm x 250 mm, 5 μm); gradient elution with acetonitrile-0.1% formic acid as mobile phase; detection wavelength used the conversion wavelength method; flow rate: 0.8 mL / min; column temperature: 20 °C; injection volume: 10 μL.

[0046] Table 2 Elution procedure

[0047]

[0048]

[0049] Table 3 Detection wavelength

[0050]

[0051] 2.2 Preparation of mixed reference solution

[0052] Take appropriate amount of protocatechuic acid, isohispidin, hispidin, luteolin reference substance respectively, accurately weigh, respectively placed in 25, 10, 25, 50 mL volumetric flask, add methanol to constant volume, shake, respectively get the reference stock solution of mass concentration of 0.4040, 1.0180, 0.4113, 0.2071 mg / mL. In turn, accurately take the above-mentioned stock solution into the same 10 mL volumetric flask, add methanol to constant volume, shake, get mixed reference solution.

[0053] 2.3 Preparation of test solution

[0054] Take about 1 g of the product powder (through No. 4 sieve), accurately weigh, place in a stoppered conical flask, accurately add 50% ethanol 25 mL, weigh, ultrasonic treat for 30 min, cool, make up the weight loss, shake, filter, centrifuge (13000 r / min, 10 min), take the supernatant, get it.

[0055] 2.4 Methodological investigation of Xiaoye San Dian Jin HPLC fingerprint

[0056] 2.4.1 Precision test

[0057] Prepare the test solution according to "2.3", continuously inject 6 times according to the chromatographic conditions under "2.1", record the retention time and peak area of each of the 9 common peaks in the spectrum. Take hispidin chromatographic peak (No. 6) as the reference peak, calculate the relative retention time and relative peak area of the 9 common peaks in the fingerprint. The results show that the retention time RSD of each chromatographic peak of Xiaoye San Dian Jin is less than 0.76%; the peak area RSD of each peak is less than 2.13%, indicating that the precision of the instrument is good. The results are shown in Tables 4 and 5.

[0058] Table 4 Fingerprint precision relative retention time results (n = 6)

[0059]

[0060]

[0061] Table 5 Fingerprint precision relative peak area results (n = 6)

[0062]

[0063] 2.4.2 Reproducibility test

[0064] Take 6 samples from the same batch (S4), prepare the test sample solution according to item 2.3, inject under the chromatographic conditions of item 2.1, and record the retention time and peak area of each of the 9 common peaks in the spectrum. Take the spectrum peak of astragalin (No. 6) as the reference peak, calculate the relative retention time and relative peak area of the 9 common peaks in the fingerprint, and the results show that the retention time RSD of each chromatographic peak of small leaf three-point gold is <0.35%; the peak area RSD of each peak is <2.62%, indicating good reproducibility, and the results are shown in Tables 6 and 7.

[0065] Table 6 Fingerprint reproducibility relative retention time results (n = 6)

[0066]

[0067]

[0068] Table 7 Fingerprint reproducibility relative peak area results (n = 6)

[0069]

[0070] 2.4.3 Stability test

[0071] Take the S4 batch sample, prepare the test sample solution according to item 2.3, and analyze at 0, 2, 4, 8, 10, 12, 24h, respectively, under the chromatographic conditions of item 2.1, record the retention time and peak area of each of the 9 common peaks in the spectrum. Take the spectrum peak of astragalin (No. 6) as the reference peak, calculate the relative retention time and relative peak area of the 9 common peaks in the fingerprint, and the results show that the retention time RSD of each chromatographic peak of small leaf three-point gold is <0.38%; the peak area RSD of each peak is <3.81%, indicating good stability, and the results are shown in Tables 8 and 9.

[0072] Table 8 Fingerprint stability relative retention time results (n = 7)

[0073]

[0074] Table 9 Relative peak area results of fingerprint stability (n = 7)

[0075]

[0076]

[0077] 2.5 Establishment of HPLC fingerprint of Folium Microcos Paniculatae

[0078] 2.5.1 Blank test

[0079] In order to investigate the interference of solvent on sample analysis, 50% ethanol was injected under the chromatographic conditions in item "2.1", and the chromatogram was recorded. The results showed that there was no impurity interference in the solvent.

[0080] 2.5.2 Extension of flushing test

[0081] The test sample solution was injected, and the gradient elution was performed for 120 min under the chromatographic conditions in item "2.1". No other chromatographic peaks appeared after 75 min of observation.

[0082] 2.5.3 Establishment of fingerprint

[0083] The experimental data were analyzed and evaluated by the computer software of "Similarity Evaluation System of Chromatographic Fingerprint of Traditional Chinese Medicine 2012.1 Edition" issued by the National Pharmacopoeia Committee. With S4 as the reference, the chromatographic peaks of each fingerprint were corrected by the median method and matched with Mark peaks, and the time window width was 0.1. The superimposed chromatogram generated is shown in Figure 1 . By comparing with the chromatogram of the mixed reference substance Figure 2 , 4 common chromatographic peaks were identified, which were protocatechuic acid (peak 1), isohispidin (peak 4), hispidin (peak 6), and luteolin (peak 9).

[0084] 2.5.4 Identification of common peaks

[0085] Nine representative common peaks were selected as the common peaks of the fingerprint by Mark peak matching. The relative retention time and relative peak area of the common peaks of 12 batches of Folium Microcos Paniculatae are shown in Table 10 and Table 11. The results showed that the relative retention time RSD of 12 batches of Folium Microcos Paniculatae was less than 0.84%, which met the "Technical Requirements for Research on Fingerprint of Traditional Chinese Medicine Injection"; the maximum RSD of relative peak area was 138.77%, indicating that the content of chemical components in different batches of Folium Microcos Paniculatae was quite different. It may be due to the influence of factors such as harvesting time, harvesting area, climate, environment, etc.

[0086] Table 10 Relative retention time results of common peaks of 12 batches of Folium Microcos Paniculatae

[0087]

[0088]

[0089] Table 11 Relative peak area results of 12 batches of Folium Tetracentri

[0090]

[0091] 2.5.5 Similarity evaluation

[0092] The software of Similarity Evaluation System of Chromatographic Fingerprint of Traditional Chinese Medicine 2012.1 version was used. The time window was set as 0.1 min, and the similarity of 12 batches of Folium Tetracentri was calculated by the median method. The software generated results are shown in Table 12, the similarity results are shown in Table 13, and the similarity values of 12 batches of Folium Tetracentri and the control fingerprint are greater than 0.9, as shown in Figure 3 . The results showed that 12 batches of Folium Tetracentri had good similarity with the control fingerprint.

[0093] Table 12 Similarity calculation results

[0094]

[0095]

[0096] Table 13 Similarity results

[0097]

[0098] 2.5.6 Cluster analysis (CA)

[0099] With 9 common peak areas of 12 batches of samples as variables, SPSS23.0 statistical software was used for cluster analysis, between-groups linkage was used, and squared euclidean distance was used as the measure of samples for systematic cluster analysis, and the cluster dendrogram was obtained, as shown in Figure 4 When the squared euclidean distance was 10, 12 batches of Folium Tetracentri samples were roughly divided into 2 categories, I was S6 and S8, and II was S1-S5, S7, S9-S12. It showed that the content of different batches of Folium Tetracentri had certain differences.

[0100] 2.5.7 Principal component analysis (PCA)

[0101] With 9 common peak areas of 12 batches of samples as variables, SPSS23.0 statistical software was used for principal component analysis. With the principle of eigenvalue greater than 1 for extraction, 2 principal components were extracted, and the results are shown in Table 14, and the scatter plot is shown in Figure 5The cumulative contribution rate was 84.866%, indicating that the two principal components contained 84.866% of the information of the nine common components, and could reflect the main characteristics of the samples and had good representativeness; in the broken stone graph, the curve was steep when the characteristic value was greater than 1, and tended to be flat when the characteristic value was less than 1, indicating that the extraction of two principal components was reasonable. According to the principal component loading matrix results, it was known that the quality difference of small leaf Sanjiaojin was the result of the joint action of multiple components, as shown in Table 15. The information of the first principal component was mainly derived from chromatographic peaks 2, 4, 5, 6 and 8, and the information of the second principal component was mainly derived from chromatographic peaks 1 and 9. The two extracted principal components were Y1 and Y2. The linear combination expression obtained by analysis was:

[0102] Y1 = 0.057X1 + 0.387X2 + 0.295X3 + 0.434X4 + 0.422X5 + 0.437X6 + 0.235X7 + 0.373X8 - 0.086X9;

[0103] Y2 = 0.555X1 + 0.206X2 - 0.454X3 - 0.015X4 - 0.066X5 + 0.009X6 + 0.299X7 + 0.089X8 + 0.584X9.

[0104] The 12 batches of small leaf Sanjiaojin samples were brought into the expression for linear calculation, and the principal component Y1 and Y2 scores were obtained, and then brought into the comprehensive evaluation function Y = 0.57484Y1 + 0.27382Y2 to calculate the comprehensive score value, as shown in Table 16. The higher the score, the higher the comprehensive quality of the sample. The comprehensive score of S2 batch sample was the highest, and the comprehensive quality was the best.

[0105] The principal component score graph of 12 batches of small leaf Sanjiaojin samples was drawn by using SIMCA-P 14.1 software, as shown in Figure 6 The 12 batches of different batches of small leaf Sanjiaojin samples were obviously distributed in the left and right two regions, and the samples could be divided into two categories, which was consistent with the clustering analysis result.

[0106] Table 14 Characteristic value, contribution rate and cumulative contribution rate of PCA

[0107]

[0108] Table 15 Principal component loading matrix

[0109]

[0110]

[0111] Table 16 Principal component scores and comprehensive scores

[0112]

[0113] 2.5.8 Partial Least Squares-Discriminant Analysis (OPLS-DA)

[0114] The peak areas of 9 common peaks in 12 batches of small leaf three-point gold medicinal materials were introduced into the SIMCA 14.1 multivariate statistical software as variables for OPLS-DA analysis. Under the model, R 2 X = 0.81, R 2 Y = 0.834, Q 2 = 0.692, indicating that the model has good prediction ability; through 200 permutation tests, the results show that R 2 and Q 2 are both > 0.5, indicating that the model has good fitting degree, and there is no overfitting phenomenon, and the permutation test results are shown in Figure 7 . The OPLS-DA score matrix diagram is shown in Figure 8 , the OPLS-DA results are consistent with the CA and PCA results, further verifying the reliability of the analysis results. In order to clarify the substances causing the quality differences of small leaf three-point gold from different producing areas, combined with the variable importance projection (VIP) normal distribution diagram, see Figure 9 , the variables with greater contribution are screened out with the standard of VIP value > 1, which are peaks 6 (sophorin), 4 (isosophorin), 2, 5 and 8, among which the VIP value of sophorin is 1.1866 and the VIP value of isosophorin is 1.19779. It is indicated that the two components play an important role in distinguishing small leaf three-point gold from different producing areas and different batches, and are the main marker components of small leaf three-point gold.

[0115] The above only describes the preferred embodiments of the present application and should not be used to limit the present application, and any modifications, equivalent replacements, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for constructing an HPLC fingerprint of *Gynostemma pentaphyllum*, characterized in that, Includes the following steps: (1) Preparation of test solution: Accurately weigh the powder of *Gynostemma pentaphyllum* sample, add 50% ethanol, weigh, sonicate, cool, make up the weight loss, shake well, filter, centrifuge the filtrate, take the supernatant to obtain the test solution; (2) Preparation of mixed reference solution: Weigh appropriate amounts of protocatechuic acid, isoharmonic acid, harmonic acid and luteolin reference standards respectively, add methanol to make up to volume, shake well to obtain reference stock solution, accurately measure appropriate amount of reference stock solution, add methanol to make up to volume to obtain mixed reference solution. (3) High performance liquid chromatography detection: Take the test solution and the mixed reference solution, inject them into the high performance liquid chromatograph for detection, and record the chromatograms of the test solution and the mixed reference solution respectively; The high-performance liquid chromatography (HPLC) conditions were as follows: the column was a SHIMADZU shim-pack VP-ODS, 4.6 mm × 250 mm, 5 μm model column; the mobile phase was acetonitrile-0.1% formic acid, with gradient elution, wherein acetonitrile was mobile phase A and 0.1% formic acid was mobile phase B; the detection wavelength was determined using a wavelength switching method; the flow rate was 0.8–1.0 mL / min; the column temperature was 20–22 °C; and the injection volume was 10–12 μL. The gradient elution program is as follows: 0–12 min, 8% A; 12–14 min, 8–16% A; 14–35 min, 16% A; 35–50 min, 16–18% A; 50–70 min, 18–40% A; 70–75 min, 40% A. The wavelength conversion method is specifically as follows: 0–18 min, 260 nm; 18–75 min, 350 nm; (4) Establishment of fingerprint spectrum: The chromatogram of the test sample solution is imported into the similarity evaluation system of chromatographic fingerprint spectrum of traditional Chinese medicine, the common peaks are matched, the reference fingerprint spectrum is generated, and the chromatographic peak of purslane is used as the reference peak. The relative retention time and relative peak area of ​​each common peak are calculated, and the common peaks are identified by comparing the retention time of the chromatogram of the test sample solution and the chromatogram of the mixed reference solution.

2. The method for constructing the HPLC fingerprint of *Gynostemma pentaphyllum* according to claim 1, characterized in that, In step (2), the mass concentrations of protocatechuic acid, isoharbitis glycoside, harbitis glycoside and luteolin in the reference stock solution are 0.4040, 1.0180, 0.4113 and 0.2071 mg / mL, respectively.

3. The method for constructing the HPLC fingerprint of *Gynostemma pentaphyllum* according to claim 1, characterized in that, In step (4), the matching common peak is based on the sample number S4 of *Gynostemma pentaphyllum*. The median method is used to perform multi-point correction and Mark peak matching on the chromatographic peaks of each fingerprint spectrum, with a time window width of 0.

1.

4. The method for constructing the HPLC fingerprint of *Gynostemma pentaphyllum* according to claim 1, characterized in that, In step (4), the reference fingerprint spectrum includes 9 common peaks, of which peak 1 corresponds to protocatechuic acid, peak 4 corresponds to isoharmonin, peak 6 corresponds to harmonin, and peak 9 corresponds to luteolin.

5. The method for constructing the HPLC fingerprint of *Gynostemma pentaphyllum* according to claim 1, characterized in that, In step (4), the relative retention times of the common peaks are: peak 1 0.401~0.411, peak 2 0.575~0.588, peak 3 0.863~0.880, peak 4 0.911~0.923, peak 5 0.962~0.966, peak 6 1.000, peak 7 1.423~1.457, peak 8 1.436~1.485, and peak 9 1.813~1.

850.

6. The method for constructing the HPLC fingerprint of *Gynostemma pentaphyllum* according to claim 1, characterized in that, In step (4), the relative peak areas of the common peaks are: peak 1 0.016~0.321, peak 2 0.183~0.451, peak 3 0.293~1.979, peak 4 2.382~3.559, peak 5 1.169~2.966, peak 6 1.000, peak 7 0.014~0.291, peak 8 0.114~0.710, and peak 9 0.026~0.

871.

7. The method for constructing the HPLC fingerprint of *Gynostemma pentaphyllum* according to claim 1, characterized in that, In step (4), the similarity value between the small leaf three-point gold sample and the control fingerprint spectrum is greater than 0.9.

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