Method for constructing fingerprint spectrum of standard decoction of radix fici simplicissimae decoction pieces

Abstract

The invention discloses a construction method of a standard decoction fingerprint spectrum of radix fici simplicissimae decoction pieces, and relates to the technical field of traditional Chinese medicines. The construction method comprises the following steps: adding psoralen and bergapten into a methanol aqueous solution with the volume fraction of 50% to prepare a reference substance solution; the method comprises the following steps: respectively carrying out water extraction treatment on batches of radix fici simplicissimae to obtain different batches of standard decoction, respectively adding methanol into the different batches of standard decoction until the volume fraction of methanol is 50%, and then centrifuging to take supernatant to obtain different batches of test solution; and respectively performing high performance liquid chromatography detection on the different batches of test sample solutions to obtain fingerprint spectrums, and calibrating characteristic peaks by using the reference substance solution. By utilizing the construction method, the fingerprint spectrum of the radix fici simplicissimae decoction piece standard decoction can be accurately constructed, so that reference and basis are provided for overall quality control and evaluation of the radix fici simplicissimae decoction piece standard decoction and formula granules.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, and in particular to a method for constructing a fingerprint spectrum of a standard decoction of *Ficus hirta* (a type of medicinal herb). Background Technology

[0002] Five-finger fig (Ficus hirta Vahl) is the dried root of the Ficus hirta Vahl plant, belonging to the Moraceae family. It has a sweet taste and neutral properties, and is believed to invigorate the spleen and replenish qi, promote qi circulation and eliminate dampness, and relax muscles and tendons. Five-finger fig is a common medicinal and edible herb in the Lingnan region and was included in the 1977 edition of the Chinese Pharmacopoeia. Modern research shows that five-finger fig mainly contains various chemical components such as phenylpropanoids, flavonoids, terpenes, sterols, and volatile oils. Furthermore, psoralen is readily detectable, can enter the bloodstream unchanged, and has hepatoprotective effects; the anti-aging and anti-inflammatory effects of bergamot lactone are consistent with the pharmacological effects of five-finger fig.

[0003] Standardized decoctions of traditional Chinese medicine (TCM) are single-herb decoctions prepared using modern extraction methods and standardized processes, guided by TCM theory and based on clinical application. They can be used to standardize clinical medication, ensuring accuracy and dosage consistency. Standardized decoctions include both quantitative studies (such as yield, indicative components, and their transfer rates) and qualitative studies (such as fingerprinting or characteristic spectra), providing a new model for the overall quality control and evaluation of TCM. They have been widely applied in the research of TCM formula granules and classic prescription preparations. Given the lack of reported research on standardized decoctions of *Ficus hirta* (a type of herb), this invention, referencing the *Technical Requirements for Treatment Control and Standard Formulation of TCM Formula Granules* (hereinafter referred to as the *Technical Requirements*), *Management Standards for TCM Decoction Rooms in Medical Institutions* (hereinafter referred to as the *Management Standards*), and *Research Strategies for Standardized Decoctions of TCM*, prepares a standardized decoction of *Ficus hirta* and establishes its quality standards, aiming to provide reference and basis for the quality research and evaluation of *Ficus hirta* formula granules and related preparations. Summary of the Invention

[0004] The purpose of this invention is to provide a method for constructing a fingerprint spectrum of a standard decoction of *Ficus hirta* (a type of herb) to solve the problems existing in the prior art. This method can accurately construct a fingerprint spectrum of a standard decoction of *Ficus hirta*, thereby providing a reference and basis for the overall quality control and evaluation of standard decoctions and granules of *Ficus hirta*.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a method for constructing a fingerprint spectrum of a standard decoction of five-finger peach slices, comprising the following steps:

[0007] A reference solution was prepared by adding psoralen and bergamot lactone to a 50% (v / v) methanol aqueous solution.

[0008] After water extraction of batches of five-finger peach, different batches of standard decoctions were obtained. Methanol was added to the different batches of standard decoctions until the methanol volume fraction was 50%. After centrifugation, the supernatant was collected to obtain different batches of test sample solutions.

[0009] The fingerprint spectra of different batches of test solutions were obtained by high performance liquid chromatography, and the characteristic peaks were identified using the reference solution.

[0010] Furthermore, the preparation method of the standard decoction includes: taking slices of five-finger peach, soaking them in 8 times their weight of water for 30 minutes, boiling and decocting for 30 minutes, filtering to obtain a first filtrate and a residue, adding 6 times their weight of water to the residue, boiling and decocting for another 20 minutes, filtering to obtain a second filtrate, combining the first filtrate and the second filtrate, and concentrating under reduced pressure to obtain the standard decoction.

[0011] Furthermore, the high-performance liquid chromatography detection employs... XB-C18 chromatographic column.

[0012] Furthermore, the mobile phase for the high-performance liquid chromatography detection is methanol-0.1% phosphoric acid aqueous solution, wherein methanol is phase A and 0.1% phosphoric acid aqueous solution is phase B.

[0013] Furthermore, the gradient elution procedure for the high-performance liquid chromatography detection is as follows:

[0014] 0–10 min, 30% A phase;

[0015] 10–30 min, 30%–50% A phase;

[0016] 30~55min, 50%~95% A phase;

[0017] 55–60 min, 95% A phase.

[0018] Furthermore, the flow rate for the high-performance liquid chromatography detection is 1 mL / min.

[0019] Furthermore, the detection wavelength of the high-performance liquid chromatography is 245 nm.

[0020] Furthermore, the column temperature for the high-performance liquid chromatography detection is 30°C.

[0021] The present invention discloses the following technical effects:

[0022] This invention selected 19 batches of *Ficus hirta* (a type of medicinal herb) from different origins and used a combination of qualitative analysis using high-performance liquid chromatography (HPLC) and quantitative analysis using psoralen and bergamot lactone to study their standard decoctions, establishing a quality evaluation standard. The extract yield, psoralen content, and bergamot lactone content of the standard decoctions ranged from 4.87% to 12.37%, 15.39% to 28.59%, and 12.61% to 23.41%, respectively. The HPLC fingerprint identified eight common peaks and designated two others. Using the fingerprint constructed in this invention as a quality evaluation standard is convenient and reliable, providing a reference and basis for the overall quality control and evaluation of *Ficus hirta* standard decoctions and granules. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The figures are HPLC chromatograms of the reference solution (A) and the test solution (B); in A and B, 1 represents psoralen; 2 represents bergamot lactone.

[0025] Figure 2 Fingerprint spectrum (A) and control spectrum (B) of the standard decoction of five-finger peach slices. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] In the following examples, all percentage concentrations of the solution are volume fractions.

[0032] Example 1

[0033] 1. Instruments and Materials

[0034] 1.1 Instruments

[0035] Waters 2695 high-performance liquid chromatograph (Waters Corporation, USA) XB-C18 chromatographic column [Yuexu Technology (Shanghai) Co., Ltd.], Direct-Q5UV ultrapure water system (Guangxi Nanning Bomei Biotechnology Co., Ltd.), ME204E electronic analytical balance (Mettler-Toledo Instruments Ltd.), SB-1100 EYELA water bath (Tokyo RIKEN Co., Ltd.), N-1100 EYELA rotary evaporator (Tokyo RIKEN Co., Ltd.), Hangfang split-type Chinese medicine decoction pot (4L, Shenzhen Yihu Baiyin Industrial Co., Ltd.), KQ-500E ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), DHG-9140A electric thermostatic drying oven (Shanghai Qixin Science Co., Ltd.).

[0036] 1.2 Test Drugs

[0037] Psoralen reference standard (purity >98%, RP220515, Chengdu Medtronic Technology Co., Ltd.), bergapten reference standard (purity >98%, HR21513B1, Chengdu Medtronic Technology Co., Ltd.); chromatographic methanol [Thermo Fisher Scientific (China) Co., Ltd.], and phosphate was analytical grade. This invention collected 19 batches of processed *Ficus hirta* root slices, which were identified by Professor Zhu Yilin of the School of Pharmacy, Guangxi University of Traditional Chinese Medicine, as processed products of the dried root of *Ficus hirta* Vahl, a plant of the Moraceae family. Specific sample information is shown in Table 1.

[0038] Table 1. Sample Information of Five-Finger Peach Root Slices

[0039]

[0040] 2. Methods and Results

[0041] 2.1 Preparation of Standard Decoction

[0042] The standard decoction of *Ficus hirta* slices was prepared in accordance with the "Management Specifications" and "Technical Requirements". 100g of each batch of *Ficus hirta* slices was accurately weighed and soaked in 8 times its weight of water for 30 minutes; after boiling, it was decocted for 30 minutes, filtered while hot to obtain the first filtrate and the residue. The residue was then added to 6 times its weight of water, boiled, and decocted for another 20 minutes, filtered while hot to obtain the second filtrate. The first and second filtrates were combined and concentrated under reduced pressure to 500mL to obtain the final product.

[0043] 2.2 High Performance Liquid Chromatography (HPLC) Conditions

[0044] chromatographic column is XB-C18 column (4.6 mm × 250 mm, 5 μm); methanol as mobile phase A, 0.1% phosphoric acid solution as mobile phase B; elution gradient: 0–10 min, 30% A; 10–30 min, 30%–50% A; 30–55 min, 50%–95% A; 55–60 min, 95% A; column temperature: 30 °C; flow rate: 1 mL / min. -1 The detection wavelength was 245 nm; the injection volume was 20 μL. The chromatograms of the reference solution and the test solution under these conditions are shown below. Figure 1 .

[0045] 2.2.1 Preparation of reference solution

[0046] Accurately weigh psoralen and bergamot lactone, and add 50% methanol aqueous solution to prepare a solution containing 574.00 μg of psoralen and 124.00 μg of bergamot lactone per 1 mL.

[0047] 2.2.2 Preparation of the test solution

[0048] Accurately measure an appropriate amount of the standard decoction of *Ficus hirta* under item "2.1", add methanol to a volume fraction of 50%, and heat at 14000 r·min. -1 Centrifuge for 8 minutes and collect the supernatant.

[0049] 2.3 Methodological Examination

[0050] 2.3.1 Examination of Linear Range

[0051] Accurately measure the reference solution and dilute it with 50% methanol aqueous solution to prepare a series of reference solution concentrations. Inject the solutions according to the chromatographic conditions in section "2.2". Plot a standard curve with the reference solution concentration (X, μg / mL) as the abscissa and the peak area (Y) as the ordinate to obtain the linear regression equation and correlation coefficient. The results showed that the regression equation for psoralen was Y = 83011.85X - 563477.32, R... 2 =0.9995; the regression equation for bergamot lactone is Y = 71551.56X - 177025.25, R0 2 =0.9991, psoralen and bergamot lactone ranged from 17.22 to 574.00 μg·mL, respectively. -1 and 3.72–124.00 μg·mL -1 The linear relationship is good within the range.

[0052] 2.3.2 Precision Test

[0053] Take the test solution (S16) and inject it 6 times consecutively under the chromatographic conditions in section “2.2”. Record the chromatogram. The RSDs of the peak areas of psoralen and bergamot lactone are 0.27% and 0.49%, respectively, indicating that the instrument has good precision.

[0054] 2.3.3 Repeatability Test

[0055] Six test solutions were prepared from the same batch of standard decoction (S16) according to the method in section "2.2.2". The solutions were injected separately according to the chromatographic conditions in section "2.2". The RSDs of the peak areas of psoralen and bergamot lactone were 0.43% and 0.48%, respectively, indicating that the method has good repeatability.

[0056] 2.3.4 Stability Test

[0057] Take the test solution (S16) and inject it at 0h, 2h, 8h, 12h, 18h and 24h according to the chromatographic conditions under section “2.2”. The RSD of the peak areas of psoralen and bergamot lactone were 0.31% and 0.44%, respectively, indicating that the test solution had good stability within 24h.

[0058] 2.3.5 Spiking Recovery Test

[0059] Six portions of the standard decoction (S16) with known contents of psoralen and bergamot lactone were taken, and the reference solution was added to 100% of the contents to be measured. The test solution was prepared according to the method in section "2.2.2", and the sample was injected according to the chromatographic conditions in section "2.2". The average recovery rate and RSD of psoralen were 100.45% and 0.58%, respectively, and the average recovery rate and RSD of bergamot lactone were 102.81% and 4.44%, respectively, indicating that the method has good accuracy. The specific results are shown in Table 2.

[0060] Table 2. Recovery results of psoralen and bergamot lactone (n=6)

[0061]

[0062]

[0063] 2.5 Sample determination and transfer rate

[0064] The test solutions of each batch of Five-Finger Peach Standard Decoction were prepared according to the method in section “2.2.2”. The samples were injected according to the chromatographic conditions in section “2.2” to determine the contents of psoralen and bergamot lactone. The transfer rate was calculated according to formula (1). The specific results are shown in Table 3.

[0065] Transfer rate (%) = Content of index components in decoction / Content of index components in sliced ​​medicinal materials × 100% (1)

[0066] 2.6 Extract yield

[0067] Take 10 mL of the standard decoction precisely, place it in a constant-weight evaporating dish, evaporate it to dryness in a water bath, dry it at 105℃ for 3 hours, cool it in a desiccator for 30 minutes, weigh it, and calculate the yield of the extract according to formula (2). The results show that the yield of the standard decoction of five-finger peach slices ranges from 6.01% to 10.75%, with an average yield of 8.62% and a standard deviation (SD) of 1.25. The specific results are shown in Table 3.

[0068] Extract yield (%) = (weight of solids in 10mL standard decoction × 50mL) / weight of medicinal slices × 100% (2)

[0069] Table 3. Determination values ​​of index components in standard decoctions of Five-Finger Peach (n=2)

[0070]

[0071]

[0072] Note: a represents the data that was previously measured in the early stages of this invention; b represents the data obtained after removing extreme values.

[0073] 2.7 Feature Map

[0074] 2.7.1 Chromatographic conditions

[0075] The chromatographic conditions, preparation of reference solutions, and preparation of test solutions are the same as those in section “2.2”.

[0076] 2.7.2 Methodological Examination

[0077] According to the National Drug Standards Working Manual (Fourth Edition), similarity evaluation can be used for methodological investigation, so this method was adopted in this investigation.

[0078] Take the test solution (S16) and inject it 6 times consecutively under the chromatographic conditions in section “2.2”. Record the chromatogram and import the chromatogram into the “Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (Version 2012.13)” for evaluation. The similarity is >0.98, which meets the technical requirements of the characteristic chromatogram, indicating that the instrument has good precision.

[0079] Weigh out 6 portions of the same batch of standard decoction (S16) and prepare test solutions according to the method in section "2.2.2". Inject the solutions according to the chromatographic conditions in section "2.2" and record the chromatograms. Evaluate the chromatograms according to the above method. The similarity of all chromatograms is >0.98, which meets the technical requirements of characteristic chromatograms, indicating that the method has good repeatability.

[0080] Take the test solution and inject it at 0h, 2h, 8h, 12h, 18h and 24h according to the chromatographic conditions under section “2.2”. Record the chromatograms and evaluate the chromatograms according to the above method. The similarity is 0.98, which meets the technical requirements of the characteristic chromatogram, indicating that the sample is stable within 24h.

[0081] 2.7.3 Fingerprint pattern establishment and similarity evaluation

[0082] Take each batch of standard decoction, prepare the test solution according to the method in section "2.2.2", inject the sample according to the chromatographic conditions in section "2.2", and record the chromatograms. Import the chromatograms into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (Version 2012.13)" for evaluation. Using chromatogram S1 as the reference chromatogram, the median method was used, and the time window was set to 0.1 to establish the superimposed chromatogram and the control chromatogram of the standard decoction of *Ficus hirta* (see...). Figure 2 The similarity was calculated. Results showed that eight common peaks were identified in the standard decoctions of *Ficus hirta* slices from various batches. By comparing with the reference standard, peaks 6 and 7 were identified as psoralen and bergamot lactone, respectively. (See details...) Figure 2 The average similarity between each batch of samples and the control spectrum was above 0.95, indicating that the chemical composition of each batch of standard decoction was highly consistent. The specific similarity evaluation results are shown in Table 4.

[0083] Table 4 Similarity Results of Fingerprint Chromatograms of Standard Decoctions of 19 Batches of Ficus hirta Vahl Slices

[0084]

[0085]

[0086] 3 Result Analysis

[0087] The authenticity and representativeness of the origin of traditional Chinese medicine slices are the primary conditions in the research process of standard decoctions. Given that there is no clear authentic production area for Ficus hirta Vahl, and it is not included in the Chinese Pharmacopoeia 2020 Edition, in this invention, according to the relevant requirements of the "Technical Requirements" and the "Administrative Specifications", 19 batches of samples from 2 main production areas of Ficus hirta Vahl were selected for the research of standard decoctions, and each batch of Ficus hirta Vahl slices met the various regulations of the quality standard of Ficus hirta Vahl slices previously formulated by the research group. Since the standard decoction has lost the original character identification features of the slices and it is impossible to comprehensively control its quality only from the qualitative and quantitative aspects of the index components, in this invention, the overall qualitative analysis of the HPLC fingerprint chromatogram is combined with the extract yield of the slices and the quantitative analysis of the index components to comprehensively control the quality of the standard decoction of Ficus hirta Vahl slices from the perspectives of overall chemical profile qualitative analysis and quantitative analysis of index components, so as to provide reference for the research and development of Ficus hirta Vahl formula granules and their compound preparations.

[0088] 3.1 Preparation of Standard Decoction

[0089] Both the "Administrative Specifications" and the "Technical Requirements" recommend that the amount of water added during the decoction of slices should be appropriate with the water covering the drug surface by 2 - 5 cm. In the article "Research Strategy for Standard Decoctions of Traditional Chinese Medicine Slices", it is recommended that for whole herb slices, 12 times the amount of water is added for the first decoction and 10 times the amount of water for the second decoction. This invention found that when 8 times the amount of water is added to 100 g of Ficus hirta Vahl slices, the water covers the drug surface by 3 - 4 cm; when 6 times the amount of water is added, the water covers the drug surface by 2 - 3 cm. To make full use of the slices, therefore, based on the principle of its upper limit of water addition amount, that is, 8 times and 6 times the amount of water are added to 100 g of Ficus hirta Vahl slices respectively to prepare its standard decoction.

[0090] 3.2 Selection of Mobile Phase

[0091] When determining the HPLC mobile phase in this invention, the organic phase (methanol, acetonitrile) and the aqueous phase (water, formic acid water, acetic acid water, phosphoric acid water with different ratios) were respectively optimized. The results showed that when acetonitrile was used as the organic phase, there were many and miscellaneous peaks and the separation was difficult, so methanol was selected as the organic phase; when phosphoric acid aqueous solution was used as the aqueous phase, its separation effect was better than that of formic acid aqueous solution and acetic acid aqueous solution, and there was no obvious difference in the separation effect of 0.1% and 0.2% phosphoric acid water on each peak of the test sample, so finally methanol - 0.1% phosphoric acid water was selected as the HPLC mobile phase.

[0092] 3.3 Preparation of Test Sample

[0093] When performing HPLC analysis on the test sample, the organic phase used is a methanol solution, while the standard decoction is an aqueous extract. If the sample is injected directly, the hydrophilic components will precipitate in the methanol, resulting in precipitation in the column and affecting column life. Therefore, when preparing the test sample, methanol should be added to make the alcohol content 50%, and then the precipitate should be removed by centrifugation. The supernatant should then be injected to extend the column life, and the HPLC chromatogram of the prepared test sample solution will be optimal.

[0094] 3.4 Standardization of Standard Decoctions

[0095] The "Technical Requirements" specify that the ranges for the standard decoction's extract yield, indicative component content, and transfer rate can be determined by adding or subtracting three times the SD from the mean (referred to as Method 1), or by using 70% to 130% of the mean (referred to as Method 2). Table 3 shows that, calculated using Method 1, the standard decoction's extract yield is 4.9% to 12.4%, and the psoralen and bergamot lactone contents range from 109.3 μg·g⁻¹. -1 ~791.5 μg·g -1 and 4.3 μg·g -1 ~175.0 μg·g -1 The transfer rates of psoralen and bergamot lactone ranged from -0.9% to 43.0% and 0.5% to 35.0%, respectively; the yield of the standard decoction calculated according to Method 2 was 8.2% to 11.2%, and the contents of psoralen and bergamot lactone ranged from 416.3 μg·g. -1 ~585.5 μg·g -1 and 81.1 μg·g -1 ~116.6 μg·g -1 The transfer rates of psoralen and bergamot lactone ranged from 18.9% to 27.4% and 16.1% to 23.1%, respectively. Given that this invention only studied 19 batches of samples, a relatively small number, there was a risk of the evaluation indicators being too narrow. Therefore, the wider ranges were adopted to establish permissible ranges, namely, the extract yield ranged from 4.9% to 12.4%, and the psoralen and bergamot lactone contents in the standard decoction ranged from 109.3 μg·g⁻¹, respectively. -1 ~791.5 μg·g -1 and 4.3 μg·g -1 ~175.0 μg·g -1 Based on the combined methods and the transfer rates of 19 batches of standard decoctions of *Ficus hirta* (a type of medicinal herb), the range of bergamot lactone transfer rate in the standard decoctions was determined to be 8.0%–35.0%. However, the range of psoralen transfer rate in the standard decoctions calculated using Method 1 resulted in a negative value, which is not conventional. Therefore, a combined approach using both methods was adopted, and the range was determined to be 10.0%–35.0%. Specific results are shown in Table 3.

[0096] In summary, this invention selected 19 batches of *Ficus hirta* (a type of medicinal herb) from different origins and used a combination of qualitative analysis via HPLC characteristic chromatograms and quantitative analysis of psoralen and bergamot lactone to study their standard decoctions, establishing a quality evaluation standard. The extract yield, psoralen content, and bergamot lactone content of the standard decoctions ranged from 4.87% to 12.37%, 15.39% to 28.59%, and 12.61% to 23.41%, respectively. The HPLC fingerprint chromatograms identified eight common peaks and designated two others. The quality evaluation standard established in this invention is convenient and reliable, providing a reference and basis for the overall quality control and evaluation of *Ficus hirta* standard decoctions and granules.

[0097] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for constructing a fingerprint spectrum of a standard decoction of *Ficus hirta* (a type of medicinal herb), characterized in that, Includes the following steps: A reference solution was prepared by adding psoralen and bergamot lactone to a 50% (v / v) methanol aqueous solution. After water extraction of batches of five-finger peach, different batches of standard decoctions were obtained. Methanol was added to the different batches of standard decoctions until the methanol volume fraction was 50%. After centrifugation, the supernatant was collected to obtain different batches of test sample solutions. The fingerprint spectra of different batches of test solutions were obtained by high performance liquid chromatography, and the characteristic peaks were identified using the reference solution.

2. The construction method according to claim 1, characterized in that, The preparation method of the standard decoction includes: taking slices of five-finger peach, soaking them in 8 times their weight of water for 30 minutes, boiling and simmering for 30 minutes, filtering to obtain a first filtrate and a residue, adding 6 times their weight of water to the residue, boiling and simmering for another 20 minutes, filtering to obtain a second filtrate, combining the first filtrate and the second filtrate, and concentrating under reduced pressure to obtain the standard decoction.

3. The construction method according to claim 1, characterized in that, The high-performance liquid chromatography detection method is employed. XB-C18 chromatographic column.

4. The construction method according to claim 1, characterized in that, The mobile phase for the high-performance liquid chromatography detection is methanol-0.1% phosphoric acid aqueous solution, wherein methanol is phase A and 0.1% phosphoric acid aqueous solution is phase B.

5. The construction method according to claim 4, characterized in that, The gradient elution procedure for high-performance liquid chromatography detection is as follows: 0–10 min, 30% A phase; 10–30 min, 30%–50% A phase; 30~55min, 50%~95% A phase; 55–60 min, 95% A phase.

6. The construction method according to claim 1, characterized in that, The flow rate for the high-performance liquid chromatography detection is 1 mL / min.

7. The construction method according to claim 1, characterized in that, The detection wavelength for the high-performance liquid chromatography is 245 nm.

8. The construction method according to claim 1, characterized in that, The column temperature for the high-performance liquid chromatography detection was 30°C.

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