CN114152705B - HPLC fingerprint quality evaluation method for rhizoma atractylodis stem and leaf - Google Patents

Abstract

The invention relates to the field of agricultural biotechnology, and specifically relates to a method for quality evaluation of HPLC fingerprints of Atractylodes stems and leaves extracts. This application uses high-performance liquid chromatography to establish a quality control method for Atractylodes stems and leaves. By examining the effects of wavelength, mobile phase, modifier, column temperature, flow rate, and analysis time on the quantity and separation of chemical components in Atractylodes stems and leaves, it is determined High-performance liquid chromatography analysis method of Atractylodes stems and leaves. The precision, stability and repeatability of the established liquid chromatography analysis method were further verified. Similarity analysis and chemometric methods were used to evaluate the relationship between HPLC fingerprints and antibacterial activity. The spectrum-effect relationship was finally determined to determine the batch-to-batch quality control points of Atractylodes stems and leaves, laying a quality foundation for the application of Atractylodes stems and leaves.

Description

Method for quality evaluation of HPLC fingerprints of Atractylodes stems and leaves

Technical field

The invention relates to the field of agricultural biotechnology, and specifically relates to a method for quality evaluation of HPLC fingerprints of Atractylodes stems and leaves.

Background technique

Traditional Chinese medicine has the effects of strengthening the body, preventing and curing diseases not only for humans but also for animals, so its market demand is growing rapidly. In addition, the complexity of traditional Chinese medicine ingredients has brought about tremendous changes, which has promoted the development of traditional Chinese medicine quality control and spectral efficacy evaluation technologies and methods. Fingerprint analysis is a recognized and effective method for quality control of traditional Chinese medicines. Traditional Chinese medicine fingerprint analysis is widely used in the standardized production process of Chinese medicinal materials. The quality standards for traditional Chinese medicines formulated in the Chinese Pharmacopoeia not only conform to the characteristics of traditional Chinese medicines, but the use of traditional Chinese medicine fingerprints to control the quality of traditional Chinese medicines has been internationally recognized. At the same time, high-performance liquid chromatography plays a vital role in the qualitative and quantitative determination of active ingredients in Chinese herbal medicines due to its advantages of high efficiency, high sensitivity, high speed, and high pressure.

Atractylodes is the rhizome part of Atractylodes lancea (Thunb.) DC. or A. chinensis (DC.) Koidz. Modern pharmacological research shows that as a medicinal material, Atractylodes has unique chemical composition and significant pharmacological activity. It has anti-ulcer, anti-arrhythmia, anti-inflammatory, antibacterial, hepatoprotective, blood pressure lowering and other effects, and is widely used. Atractylodes stems and leaves, as non-medicinal parts of Atractylodes, are discarded as garbage, resulting in a serious waste of resources. Research shows that Atractylodes stems and leaves have anti-inflammatory, antibacterial and antioxidant effects. To promote the application of Atractylodes stems and leaves, it is urgent to establish standards first. Therefore, it is particularly important to formulate standards for Atractylodes stems and leaves.

The chromatographic peaks in HPLC chromatograms are affected by many factors, such as detection wavelength, mobile phase flow rate, proportion of each component of the mixed mobile phase, column temperature, etc., which will affect the HPLC fingerprint of Atractylodes stems and leaves, especially the components of the mixed mobile phase. The proportion has a greater impact and is uncertain. Any change in chromatographic conditions means that the overall chromatographic pattern changes, making it difficult to establish HPLC fingerprints.

Contents of the invention

The purpose of the present invention is to provide a method for quality evaluation of HPLC fingerprints of Atractylodes stems and leaves.

The HPLC fingerprint quality evaluation method of Atractylodes stems and leaves according to the present invention includes the following steps:

Prepare a stem and leaf extract solution of the Atractylodes stem and leaf sample to be tested and a stem and leaf extract solution of the Atractylodes stem and leaf standard;

The extract solutions obtained above were subjected to HPLC chromatography analysis, where the conditions for HPLC chromatography analysis were:

The mobile phase is methanol-0.1vol% trifluoroacetic acid system, the flow rate is 1mL/min, the column temperature is 30°C, the detection wavelength is 260nm, and the analysis time is 40min.

At the detection wavelength of 260nm, column temperature of 30°C, and flow rate of 0.8mL/min, use methanol-0.1vol% trifluoroacetic acid solution as the mobile phase to perform gradient elution. The injection volume is 10μL. The gradient elution procedure is: elution gradient 0～15min, 5% methanol～35% 0.1% trifluoroacetic acid-water, 15～25min, 35% methanol～45% 0.1% trifluoroacetic acid-water, 25～30min, 45% methanol～60% 0.1 % trifluoroacetic acid-water; 30~40min, 60% methanol~95% 0.1% trifluoroacetic acid-water;

Compare the HPLC chromatograms of the stem and leaf extract solution of the Atractylodes stem and leaf sample to be tested and the stem and leaf extract solution of the Atractylodes stem and leaf standard. If the similarity of the HPLC chromatogram peak pattern of the stem and leaf extract solution of the leaf standard is ≥0.998, the Atractylodes stem and leaf sample to be tested is judged to be a qualified product.

According to the HPLC fingerprint quality evaluation method of Atractylodes stems and leaves extract of the present invention, the extract solution of Atractylodes stems and leaves is prepared through the following steps:

Grind the stems and leaves of Atractylodes into powder and pass through a 40-mesh screen;

Add the ethanol-water solution to the sieved powder;

Then ultrasonic extraction, centrifugation to collect the supernatant, then evaporation and concentration, and freeze-drying into powder;

Dissolve the powder in water.

According to the HPLC fingerprint quality evaluation method of Atractylodes stem and leaf extract of the present invention, the solid-liquid ratio of the sieved powder to the ethanol-aqueous solution is 1:40.

According to the HPLC fingerprint quality evaluation method of Atractylodes stems and leaves extract of the present invention, ultrasonic extraction is carried out at 500W for 30 minutes.

According to the HPLC fingerprint quality evaluation method of Atractylodes stem and leaf extract of the present invention, the supernatant is concentrated by rotary evaporation at 45°C, vacuum freeze-dried into powder, and stored at 4°C for later use.

This application uses high-performance liquid chromatography to establish a quality control method for Atractylodes stems and leaves. By examining the effects of wavelength, mobile phase, modifier, column temperature, flow rate, and analysis time on the quantity and separation of chemical components in Atractylodes stems and leaves, it is determined High-performance liquid chromatography analysis method of Atractylodes stems and leaves. The precision, stability and repeatability of the established liquid chromatography analysis method were further verified. Similarity analysis and chemometric methods were used to evaluate the relationship between HPLC fingerprints and antibacterial activity. The spectrum-effect relationship was finally determined to determine the batch-to-batch quality control points of Atractylodes stems and leaves, laying a quality foundation for the application of Atractylodes stems and leaves.

Description of the drawings

Figure 1 shows the separation effect of Atractylodes stems and leaves extract at 260nm wavelength;

Figure 2 shows the separation effect of Atractylodes stems and leaves extract at 340nm wavelength;

Figure 3 shows the full wavelength scanning results of Atractylodes stems and leaves extract;

Figure 4 shows the separation effect of atractylodes stems and leaves extract by methanol-water system;

Figure 5 shows the separation effect of acetonitrile-water system on Atractylodes stems and leaves extract;

Figure 6 shows the separation effect of methanol-water-formic acid modifier on Atractylodes stems and leaves extract;

Figure 7 shows the separation effect of methanol-water-acetic acid modifier on Atractylodes stems and leaves extract;

Figure 8 shows the separation effect of methanol-water-phosphoric acid modifier on Atractylodes stems and leaves extract;

Figure 9 shows the separation effect of methanol-water-trifluoroacetic acid modifier on Atractylodes stems and leaves extract;

Figure 10 The effect of elution procedure 1 on the separation effect of Atractylodes stems and leaves extract;

Figure 11 shows the effect of elution procedure 2 on the separation effect of Atractylodes stems and leaves extract;

Figure 12 shows the effect of elution procedure 3 on the separation effect of Atractylodes stems and leaves extract;

Figure 13 shows the effect of elution procedure 4 on the separation effect of Atractylodes stems and leaves extract;

Figure 14 shows the effect of column temperature 25°C on the separation effect of Atractylodes stems and leaves extract;

Figure 15 shows the effect of column temperature 30°C on the separation effect of Atractylodes stems and leaves extract;

Figure 16 shows the effect of column temperature 35°C on the separation effect of Atractylodes stems and leaves extract;

Figure 17 shows the effect of flow rate 0.8mL/min on the separation effect of Atractylodes stems and leaves extract;

Figure 18 shows the effect of flow rate 1.0mL/min on the separation effect of Atractylodes stems and leaves extract;

Figure 19 shows the effect of flow rate 1.2mL/min on the separation effect of Atractylodes stems and leaves extract;

Figure 20 shows the characteristic fingerprint of Atractylodes stems and leaves extract;

Figure 21 is an overlay of 10 batches of Atractylodes stem and leaf extract samples in HPLC fingerprints;

Figure 22 is a cluster diagram of 10 batches of Atractylodes stem and leaf extracts;

Figure 23 shows principal component analysis;

Figure 24 shows the regression coefficient (PLS) of the antibacterial activity of Atractylodes stems and leaves extract;

Figure 25 shows the antibacterial activity VIP value (PLS) of Atractylodes stem and leaf extract.

Detailed ways

Example 1

1. Reagents

High-purity water was obtained using a Milliporemilli-Q water purification system (Billerica, MA, USA). Analytical grade ethanol was purchased from Beijing Chemical Factory (Beijing, China). Analytical grade phosphoric acid, formic acid, and trifluoroacetic acid were purchased from Tianjin Aijer Technology Co., Ltd., China. Chromatographic grade methanol and acetonitrile were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

2. Atractylodes stems and leaves

Atractylodes stem and leaf specimens were collected in Inner Mongolia in September 2020.

3. Sample preparation

Grind the stems and leaves of Atractylodes into powder and pass through a 40-mesh sieve. Accurately weigh 20g of the crushed and sieved sample, add 800mL of 50% (v/v) ethanol-water solution (material-to-liquid ratio is 1:40), and perform ultrasonic extraction at 500W for 30 minutes. After centrifugation at 5000 rpm for 10 min, the supernatant was collected, concentrated by rotary evaporation at 45°C, vacuum freeze-dried into powder, and stored at 4°C for later use.

4. Sample solution preparation

Take the fine powder of Atractylodes stems and leaves extract, accurately weigh 100mg, place it in a 15mL centrifuge tube, add 10mL of ultrapure water precisely, vortex to dissolve, filter through a 0.22μm filter, and take the filtrate for subsequent chromatographic analysis.

5. Instrument conditions

All chromatographic analyzes were performed using a Shimadzu high-performance liquid chromatography system, consisting of an LC-20AD pump, an autosampler (SIL-20A), a system controller (CBM-20A), a column greenhouse (CT0-20A), and UV-visible diode array detection. It is composed of device (SPD-M20A230v). An Agilent ZORBAXSB-C18 column was used for the study (4.6 × 250 mm, 5 μm; Agilent Technologies, Santa Clara, CA, USA). MTT was measured using a microplate reader (Multiskan ^TM SkyHigh, ThermoFisher Scientific, MA, USA).

Example 2 Optimization of High Performance Liquid Chromatography Conditions

Basic optimization of separation conditions of Atractylodes stems and leaves extract. The injection volume was set to 10 μL throughout the experiment.

2.1 Selection of detection wavelength

The complexity of the extract components. After comparing the separation effects of Atractylodes stems and leaves extracts at different wavelengths (Figure 1, Figure 2) and performing a full-wavelength scan at a wavelength of 200 to 600 nm (Figure 3), the results show that there are some components near the wavelength of 260 nm. The maximum absorption peak, the absorption peak signal is strong, and the number of peaks obtained is large, so 260nm is selected as the optimal detection wavelength.

2.2 Selection of mobile phase system

After determining the optimal detection wavelength, the effects of acetonitrile-water and methanol-water mobile phase systems on the separation of Atractylodes stems and leaves extracts were compared. The results are shown in Figures 4 and 5. At a wavelength of 260nm, the acetonitrile-water and methanol-water systems The separation effect and analysis time of Atractylodes stems and leaves extracts are similar, but the number of peaks obtained in the methanol-water system is one more than that of the acetonitrile-water system, so methanol-water was selected as the mobile phase for the analysis of Atractylodes stems and leaves extracts.

2.3 Selection of modifiers

Under the conditions of determining the wavelength and mobile phase, the effect of the modifier on the separation effect of the Atractylodes stems and leaves extract was investigated. The results are shown in Figure 6-9. When the modifiers are formic acid, acetic acid and phosphoric acid, compared with the modifier Compared with the mobile phase system of trifluoroacetic acid, the number of peaks is smaller and the retention time is longer. The trifluoroacetic acid mobile phase system as the modifier has the best separation effect, with as many as 91 peaks, and the absorption peak signal is obvious and the peak signal retention time is short. Therefore, the mobile phase system of methanol-water-0.1vol% trifluoroacetic acid was selected as the mobile phase system for chromatographic analysis of Atractylodes stems and leaves extracts.

2.4 Selection of elution program

Under the conditions of determining the wavelength, mobile phase and modifier, the effect of the elution procedure (table) on the separation effect of Atractylodes stems and leaves extract was examined. The results are shown in Figure 10-13 and Table 1-4. Elution procedure 1 The largest number of chromatographic peaks was obtained, but the analysis time was also the longest; although the elution program 3 obtained the smallest number of chromatographic peaks, the analysis time was the shortest, with only 5 less chromatographic peaks with very low content than elution program 1. It does not affect the overall evaluation of the chemical components of Atractylodes stems and leaves extract, so elution procedure 3 was selected as the final elution procedure of Atractylodes stems and leaves extract.

Table 1 Elution procedure 1

Table 2 Elution procedure 2

Table 3 Elution procedure 3

Table 4 Elution procedure 4

2.5 Selection of column temperature

Further optimize the HPLC conditions and select different column temperatures for separation. The results show that compared with 25°C and 35°C (Figures 14 and 16), when the column temperature is set to 30°C (Figure 15), more chromatographic peaks and stronger peak signals can be obtained, and the separation effect is better.

2.6 Selection of flow rate

Flow rate is one of the important factors affecting the establishment of fingerprints of Atractylodes stems and leaves extracts. The effect of flow rate on the separation effect of Atractylodes stems and leaves extract was investigated. The results are shown in Figure 17-19. The flow rate of 0.8mL/min separated the largest number of peaks and the shortest retention time, so the flow rate was determined to be 0.8mL/min.

In summary, the optimal HPLC conditions for Atractylodes stems and leaves extract were established: using an Agilent ZORBAXSB-C18 (4.6×250mm, 5μm) chromatographic column, with a detection wavelength of 260nm, a column temperature of 30°C, and a flow rate of 0.8mL/min, with methanol ( A) -0.1vol% trifluoroacetic acid solution is used as the mobile phase, gradient elution is performed, and the injection volume is 10 μL. The gradient elution program is: elution gradient 0 to 15 minutes, 5% to 35% (A); 15 to 25 minutes, 35% to 45% (A); 25 to 30 minutes, 45% to 60% (A); 30 to 40min, 60%~95%(A).

Example 3 Stability, repeatability and accuracy of high performance liquid chromatography detection method of Atractylodes stems and leaves extract

According to the technical solution of the present application, the high-performance liquid chromatography analysis conditions of Atractylodes stem and leaf extract were optimized, and the obtained chromatogram was used as the standard characteristic fingerprint of Atractylodes stem and leaf extract (Figure 20). With high resolution and reasonable peak height, it is considered a common peak for sample identification. Fingerprint parameters based on chromatographic intensity, peak shape and stability were evaluated for 19 peaks that appeared in all samples within 40 minutes of detection.

Peak No. 10 with stable content was used as the reference peak (S), and the remaining 18 peaks were selected as the characteristic peaks with higher absorption intensity of Atractylodes stem and leaf extract to evaluate their related differences in the retention time field. The formulas of RRT and RPA are RRT=RTpeak/RTpeak10 and RPA=PApeak/PApeak10 respectively.

Prepare a solution of Atractylodes stems and leaves extract, measure 6 times continuously according to the optimal chromatographic conditions, record the peak area and peak time, and calculate the RSD of the relative retention time of each common peak <0.57%, and the RSD of the relative peak area <2.57%, indicating that The instrument precision is good.

Under the optimal chromatographic conditions, the extract solution was injected for detection at 0, 1, 2, 4, 6, 8, 10, and 12 h respectively. The solution was kept at room temperature. The RSD of the 19 shared peaks was <0.87% and the RSD of the relative peak area was <3.90%. The results showed that the extract solution was stable within 12 hours.

Six batches of Atractylodes stem and leaf extract were prepared at one time and evaluated under optimal chromatographic conditions. The RSD of the 19 shared peaks was <0.34% and the RSD of the relative peak area was <4.33%. The results show that this method has good repeatability and significant effect.

The method of this application has stability, repeatability and accuracy, and the similarity of the above results proves the effectiveness of the method. This method can be used to obtain relatively stable extracts of Atractylodes stems and leaves, which can be used as the basis for subsequent functional experiments.

3.1 Similarity analysis of Atractylodes stems and leaves extracts

Similarity has been widely recognized as an evaluation index for traditional Chinese medicine fingerprints. This application uses the data of 19 common peaks in the fingerprint spectrum for quality control, and uses the similarity evaluation method for quality control. In order to determine the consistency of each batch of medicinal materials, HPLC fingerprint analysis was performed on 10 batches of Atractylodes stem and leaf extracts. Prepare 10 batches of Atractylodes stem and leaf extracts and prepare them into solutions. Inject samples for measurement according to optimal chromatographic conditions, record the chromatograms, and import the chromatographic data at 260 nm into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2004A) provided by the Chinese Pharmacopoeia Commission. version)", perform multi-point calibration and chromatographic peak matching, and obtain the overlay spectrum and control spectrum (Figure 21) after full peak matching. It is easy to calculate the similarity value between the fingerprint patterns of each sample (Table 5). It was found that the similarity of 10 batches of samples was high, all ≥0.998 (Table 5).

3.2 Antibacterial effect

MTT assay was used to evaluate the inhibitory effect of Atractylodes stems and leaves extract on Escherichia coli. Test the inhibitory effect of 10 batches of Atractylodes stems and leaves extract (10mg/mL) on Escherichia coli. As shown in Table 5, the inhibition rate of 10 batches of Atractylodes stems and leaves extract against E. coli was relatively stable, with no statistical difference. The results showed that 10 batches of Atractylodes stems and leaves extract had a stable inhibitory effect on E. coli, indicating that good quality control can ensure the stability of the biological activity of Atractylodes stems and leaves between batches.

Table 510 Fingerprint similarity and antibacterial rate of atractylodes stem and leaf extracts from batches

3.3 Chemometric analysis

3.3.1 Hierarchical cluster analysis

Using the peak areas of 19 common peaks in 10 batches of Atractylodes stem and leaf extracts as variables, a 10×19-order original data matrix was obtained. SPSS23.0 software was used, the inter-group connection method was used, and the Euclidean square distance was used as the metric for clustering. Class analysis, the results are shown in Figure 22. When the classification distance is 15, 10 batches of Atractylodes stem and leaf extracts can be clustered into 3 categories, S2, S3, S5~S7, and S9 are clustered into category A, S1, S4, and S10 are clustered into category B, and S8 alone is a category C sample. . This clustering result indicates that the samples may not have been collected at the same time.

3.3.2 Principal component analysis

PCA transforms multiple variables into a few comprehensive variables (i.e. principal components), which is an effective statistical method for dimensionality reduction. This method eliminates the correlation between evaluation indicators and helps to describe the relative status of samples more objectively. , which is of great significance for the comprehensive evaluation of the quality of Atractylodes stems and leaves extracts. SPSS23.0 statistical software was used to form a matrix of the relative peak areas of 19 peaks in 10 batches of samples. After the data was factor analyzed by the software, 6 principal components were extracted, and the PCA eigenvalues and contribution rates were calculated. As can be seen from Table 6, the eigenvalues of the first six principal components are all >1, indicating that the first six principal components play a leading role in the quality evaluation of Atractylodes stem and leaf extracts and are the iconic components that cause classification differences among samples. The cumulative contribution rate of the six principal components reached 93.546% (>90%), which can objectively reflect the comprehensive quality of Atractylodes stems and leaves extract. The first six principal components were selected for analysis. In addition, the common peak area was imported into SIMCA10.0 software to draw the principal component analysis score chart 23. 10 batches of Atractylodes stem and leaf extracts were divided into 3 categories, which was basically consistent with the cluster analysis results.

Table 13: Characteristic values and contribution rates of principal components of Atractylodes stems and leaves extract

main ingredient Eigenvalues Contribution rate % Cumulative contribution rate 1 7.888 41.513 41.513 2 3.207 16.881 58.394 3 2.129 11.206 69.601 4 1.907 10.035 79.635 5 1.470 7.735 87.37 6 1.173 6.175 93.546

3.3.3 Spectrum-effect relationship

Combining chromatographic fingerprints with efficacy evaluation can discover and identify relevant marker components of traditional Chinese medicine efficacy. The study of the relationship between spectrum and effect transforms the "visual" nature of traditional Chinese medicine into the consistency between "spectrum" and "effect". The BCA model was used to establish the spectral efficiency relationship between 19 common peak area data and the inhibition rate of E. coli. The correlation coefficient is shown in Figure 24. P1, P3, P5, P11, P13, P14, P15, P16 and P19 are effective in inhibiting E. coli. positive effects. In the PLSR model, the peak areas of the 19 common peaks are defined as independent variables, and the dependent variable is the antibacterial rate. Obtain VIP values from SIMCA software (version 14.1). As shown in Figure 25, using VIP>1 as the screening criterion, P1, P5, P3, P6 and P4 are the main marker components that affect the inhibition of E. coli.

The therapeutic effects of most Chinese medicines are the result of the combined action of different chemical components of Chinese medicines. This application establishes the HPLC fingerprint of Atractylodes stems and leaves extract, which fully reflects the types and quantities of the chemical components of Atractylodes stems and leaves extracts. The similarity of the chromatographic fingerprints of 10 batches of extracts is as high as 0.998, and the distribution between peaks is stable and consistent, indicating that Atractylodes stem and leaf extracts from the same origin have sufficient similarity in chemical composition to prove their stable quality. At the same time, the spectrum-effect relationship between quality control and antibacterial effect of Atractylodes stems and leaves extracts was evaluated using chemometric methods. In summary, the HPLC fingerprint quality evaluation method of Atractylodes stems and leaves extracts established in this application has certain reference significance for further improving the quality standards of Atractylodes stems and leaves extracts.

Claims (5)

1. A method for quality evaluation of HPLC fingerprints of Atractylodes stems and leaves, characterized in that the method includes the following steps: (1) Prepare the stem and leaf extract solution of the Atractylodes stem and leaf sample to be tested and the stem and leaf extract solution of the Atractylodes stem and leaf standard; (2) Use a 4.6 × 250 mm, 5 µm, ZORBAX SB-C18 chromatographic column to conduct HPLC chromatographic analysis of the extract solution obtained in step (1). The conditions for HPLC chromatographic analysis are: The mobile phase is a methanol-0.1vol% trifluoroacetic acid system, the flow rate is 1mL/min, the column temperature is 30°C, the detection wavelength is 260nm, the analysis time is 40min, the detection wavelength is 260nm, the column temperature is 30°C, the flow rate is 0.8 mL/min, and methanol is used. -0.1vol% trifluoroacetic acid solution was used as the mobile phase, and gradient elution was performed. The injection volume was 10 µL. The gradient elution program was: elution gradient 0 ~ 15min, 5% ~ 35% methanol, 15 ~ 25 min, 35 % ~ 45% methanol, 25 ~ 30 min, 45% ~ 60% methanol, 30 ~ 40 min, 60% ~ 95% methanol; (3) Compare the HPLC chromatograms of the stem and leaf extract solution of the Atractylodes stem and leaf sample to be tested and the stem and leaf extract solution of the Atractylodes stem and leaf standard, and the HPLC chromatographic peak pattern of the stem and leaf extract solution of the Atractylodes stem and leaf sample to be tested. If the HPLC chromatogram peak pattern of the stem and leaf extract solution of the Atractylodes stems and leaves standard product is the same and the similarity is ≥0.998, then the Atractylodes stems and leaves sample to be tested is judged to be a qualified product. 2. The HPLC fingerprint quality evaluation method of Atractylodes stems and leaves according to claim 1, characterized in that the extract solution of Atractylodes stems and leaves is prepared through the following steps: Grind the stems and leaves of Atractylodes into powder and pass through a 40-mesh screen; Add the ethanol-water solution to the sieved powder; Then ultrasonic extraction, centrifugation to collect the supernatant, then evaporation and concentration, and freeze-drying into powder; Dissolve the powder in water. 3. Atractylodes stems and leaves HPLC fingerprint quality evaluation method according to claim 2, characterized in that the material-liquid ratio of the sieved powder to the ethanol-aqueous solution is 1:40. 4. Atractylodes stems and leaves HPLC fingerprint quality evaluation method according to claim 2, characterized in that ultrasonic extraction is carried out at 500 W for 30 min. 5. The HPLC fingerprint quality evaluation method of Atractylodes stems and leaves according to claim 2, characterized in that the supernatant is concentrated by rotary evaporation at 45°C.